WO2012109735A1 - Methods of preparing cyclic amino acid molecules using arylboronic or arylborinic acid catalysts - Google Patents

Abstract

Macrocyclization of amino acids or linear peptides upon reaction with amphoteric amino aldehydes and isocyanides in the presence of a catalyst selected from an arylboronic acid or an aryl borinic acid is provided.

Description

METHODS OF PREPARING CYCLIC AMINO ACID MOLECULES USING ARYLBORONIC OR ARYLBORINIC ACID CATALYSTS

FIELD The present invention relates to methods of preparing cyclic amino acid molecules, and in particular the macrocyclization of amino acids or linear peptides upon reaction with amphoteric amino aldehydes and isocyanides in the presence of arylboronic or arylborinic acid catalysts.

BACKGROUND Peptides control a vast range of intra- and intercellular processes.^1,2 In contrast to linear peptides, cyclic variants are more resistant to both exo- and endoproteases,³'⁴ which explains the potential of this class of molecules as therapeutics and as molecular probes in chemical biology.⁵'⁶'⁷ Peptide macrocycles have shown remarkable capacity for functional fine-tuning. Once the amino acid sequence involved in target binding is known, high levels of specificity can be attained by adjusting the peptide conformation. For example, Cilengitide, a cyclic pentapeptide containing an RGD fragment, inhibits angiogenesis by targeting a_vl¾ receptors on the surfaces of cancer cells.⁸ A simple change from a penta- to a hexapeptide macrocycle equipped with the RGD fragment switches the selectivity from the <¾, ¾ receptor towards the <¾^/3₃ receptor. Amino acid residues, the main constituents of peptide macrocycles, participate in "native" interactions with protein targets.⁹ Interrogation of protein-protein interactions using these cyclic molecules is arguably their most attractive application.¹⁰ Of particular significance are the compounds that mimic secondary structures in various contexts, giving rise to a multitude of closely related, low energy peptide conformers. The resulting molecules can effectively represent different regions of protein loops and grooves, accurately recreating inter-surface binding elements.¹¹'¹²'¹³ Cyclic peptides can be difficult to prepare using traditional synthetic methods. This is because the ground state E geometry of the amide bond makes it challenging to attain the ring-like conformation conducive to cyclization. We have been interested in developing general strategies for constraining linear peptides into their macrocyclic forms. Recently, we resorted to amphoteric aziridine aldehydes

Nucleophilic

and Ugi four-component condensation to achieve this goal. ' While this method is effective, there remains a need for further synthetic methods for the preparation of cyclic amino acid molecules.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a process to produce a cyclic amino acid molecule comprising reacting an amino acid molecule, having an amino terminus and a carboxyl terminus, with:

(i) an isocyanide, and

(ii) a compound having formula (la) and/or (lb):

(!a) (lb) wherein: n = 0 or 1 , and R_\, R₂, R₃, R4 and R5 are independently selected from H; lower alkyl; alkenyl; heterocycle; cyckoalkyl; esters of the formula -C(0)OR* wherein R* is selected from alkyl and aryl; amides of the formula - C(0)NR^**R^***, wherein R^** and R^*** are independently selected from alkyl and aryl; -CH₂C(0)R, wherein R is selected from -OH, lower alkyl, aryl, -lower alkyl-aryl, or -NR_aR_b, where R_a and R_b are independently selected from H, lower alkyl, aryl or -lower alkyl-aryl; -C(0)R_c, wherein R_c is selected from lower alkyl, aryl or -lower alkyl-aryl; or -lower alkyl-OR_d, wherein R_d is a suitable protecting group or OH group; all of which are optionally substituted at one or more substitutable positions with one or more suitable substituents; and the aldehyde component thereof may optionally be in its bisulfite adduct form; and the amino acid molecule is an amino acid, a linear peptide or a salt of the foregoing, provided that if the amino acid molecule is a linear peptide, the compound comprises an azindine chiral center proximal to the aldehyde with matching stereochemistry to the carbon atom proximal to the amino terminus of the peptide, in the presence of a catalyst selected from an arylboronic acid or an arylborinic acid. In another aspect, the invention provides for the use of an arylboronic acid catalyst or an arylborinic acid catalyst for catalyzing the cyclization of an amino acid molecule.

In still another aspect, the invention provides a process to produce a mixture of cyclic amino acid molecules comprising reacting a mixture of amino acid molecules, each having an amino terminus and a carboxy terminus, with:

(i) an isocyanide, and a compound having formula (la) and/or (lb):

(la) (lb) wherein: n = 0 or 1, and Ri, R2, R3, R4 and R5 are independently selected from H; lower alkyl; alkenyl; heterocycle; cyckoalkyl; esters of the formula -C(0)OR* wherein R* is selected from alkyl and aryl; amides of the formula -

C(0)NR R , wherein R and R are independently selected from alkyl and aryl; -CH₂C(0)R, wherein R is selected from -OH, lower alkyl, aryl, -lower alkyl-aryl, or -NR_aRb, where R_a and R are independently selected from H, lower alkyl, aryl or -lower alkyl-aryl; -C(0)Rc, wherein Rc is selected from lower alkyl, aryl or -lower alkyl-aryl; or -lower alkyl-ORd, wherein d is a suitable protecting group or OH group; all of which are optionally substituted at one or more substitutable positions with one or more suitable substituents; and the aldehyde component thereof may optionally be in its bisulfite adduct form; and the mixture of amino acid molecules comprises (a) amino acids; (b) linear peptides; (c) salts of the foregoing; or (d) mixtures of (a) - (c); provided that if the mixture of amino acid molecules comprises one or more linear peptides, the compound comprises an aziridine chiral center proximal to the aldehyde with matching stereochemistry to the carbon atom proximal to the amino terminus of each of the one or more linear peptides, in the presence of a catalyst selected from an arylboronic acid or an arylborinic acid. In still yet another aspect, the invention provides a cyclic amino acid molecule of formula (Ila):

amino acid molecule

wherein, n = 0 or 1, and Ri, R₂, R₃, R-t and R₅ are independently selected from H; lower alkyl; aryl; heteroaryl; alkenyl; heterocycle; esters of the formula -C(0)OR* wherein R* is selected from alkyl and aryl; amides of the formula - C(0)NR R , wherein R and R are independently selected from alkyl and aryl; -CH₂C(0)R, wherein R is selected from -OH, lower alkyl, aryl, - loweralkyl-aryl, or -NR_aR_b, where R_a and R_b are independently selected from H, lower alkyl, aryl or -loweralkyl-aryl; -C(0)R_c, wherein Rc is selected from lower alkyl, aryl or -lower alkyl-aryl; or -lower alkyl-OR_d, wherein R_d is a suitable protecting group or OH group; all of which are optionally substituted at one or more substitutable positions with one or more suitable substituents, bonds [a] and [b] are syn to each other;

R' is an amino acid side chain of the amino terminus amino acid; R" is an optionally substituted amide; and the amino acid molecule is an amino acid, a linear peptide, or a salt of the foregoing, wherein N' is the nitrogen at the amino terminus end of the amino acid molecule and C is the carbon at the carboxy terminus end of the amino acid molecule, and provided that if the amino acid molecule is a linear peptide, bonds [a] and [c] are anti to each other, and

R^z is H.

BRIEF DESCRIPTION OF THE FIGURES:

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

FIG. 1 illustrates CID/MS² fragmentation of compound 12. Compound 12 was sequenced following CID/MS² fragmentation. BioAnalyst tagged the full peptide as the 19th highest scoring tag. [12+H]+ HRMS = 884.5050 (for fragmentation ion list and another example, see Example 3 and Table 4). FIG. 2 illustrates the CID/MS² spectra for the hydrolysis product 14 of cyclic peptide PpYVNVP (13).

FIG. 3 illustrates MS analysis of the crude reaction mixture after subjecting a mixture of seven linear peptides of varying length (peptide = PL, FA, LGF, GLGF, LGPF, GLGLF, GLFGF) to macrocyclization conditions. MS of the crude reaction mixture indicated formation of monomeric respective cyclic peptides (cp-peptide) exclusive of heteromeric linear or cyclic side products.

FIG. 4 provides a legend of the compounds prepared by the processes of the invention. FIG. 5 shows theΉ and ^l3C NMR spectra for cyclic product 5a. FIG. 6 shows the COSY NMR spectrum for cyclic product 5a. FIG. 7 shows the Ή and ¹³C NMR spectra for cyclic product 5b. FIG. 8 shows the COSY NMR spectrum for cyclic product 5b. FIG. 9 shows the Ή and ¹³C NMR spectra for cyclic product 5c. FIG. 10 shows the COSY and HSQC NMR spectra for cyclic product 5c.

FIG. 11 shows the H and COSY NMR spectra for cyclic product 5d. FIG. 12 shows the H and COSY NMR spectra for cyclic product 5e. FIG. 13 shows the ³C NMR spectrum for cyclic product 5e. FIG. 14 shows the H and COSY NMR spectra for cyclic product 5f. FIG. 15 shows the H and ¹³C NMR spectra for cyclic product 5g. FIG. 16 shows the H and ¹³C NMR spectra for cyclic product 5h.

1 "

FIG. 17 shows the H and C NMR spectra for cyclic product 5i. FIG. 18 shows the H and ¹³C NMR spectra for cyclic product 5j.

FIG. 19 shows the H and ¹³C NMR spectra for cyclic product 5k.

FIG. 20 shows the H and COSY NMR spectra for cyclic product 7a.

FIG. 21 shows the C NMR spectrum for cyclic product 7a. FIG. 22 shows the H and ^l3C NMR spectra for cyclic product 7b.

FIG. 23 shows the H and ¹³C NMR spectra for cyclic product 7c.

FIG. 24 shows the H and C NMR spectra for cyclic product 7d.

FIG. 25 shows the H and ¹³C NMR spectra for cyclic product 7e.

FIG. 26 shows the 'H and ¹³C NMR spectra for cyclic product 7f. DETAILED DESCRIPTION

There is described herein a one-step process that provides cyclic peptides in high yields and selectivities while evading the problems typically encountered during traditional cyclization reactions. The late stage diversification of macrocyclic molecules can now be achieved in a seamless fashion. The products of the macrocyclization are equipped with specific modification sites, which enable late-stage structural modification of cyclic peptides. The post cyclization diversification has been a historic challenge for macrocycle libraries in both biotechnology and pharmaceutical industries. The present macrocyclization approach solves this problem, among others.

Here we report that readily available arylboronic acids provide dramatic rate acceleration during peptide macrocyclization. This novel catalytic process is especially effective in delivering peptide macrocycles from primary amine-terminated linear precursors. The reaction remains selective even when applied to a mixture of linear peptides. Upon site-selective hydrolysis at low pH, the macrocycles can be readily sequenced using MS . More particularly, the invention provides a process to produce a cyclic amino acid molecule comprising reacting an amino acid molecule, having an amino terminus and a carboxyl terminus, with:

(i) an isocyanide, and

(ii) a compound having formula (la) and/or (lb):

(la) (lb) wherein: n = 0 or 1 , and Ri, R₂, R₃, R4 and R₅ are independently selected from H; lower alkyl; alkenyl; heterocycle; cyckoalkyl; esters of the formula -C(0)OR* wherein R* is selected from alkyl and aryl; amides of the formula -

C(0)NR R , wherein R and R are independently selected from alkyl and aryl; -CH₂C(0)R, wherein R is selected from -OH, lower alkyl, aryl, -lower alkyl-aryl, or -NR_aR_b, where R_a and R_b are independently selected from H, lower alkyl, aryl or -lower alkyl-aryl; -C(0)R_c, wherein R_c is selected from lower alkyl, aryl or -lower alkyl-aryl; or -lower alkyl-OR_d, wherein R_d is a suitable protecting group or OH group; all of which are optionally substituted at one or more substitutable positions with one or more suitable substituents; and the aldehyde component thereof may optionally be in its bisulfite adduct form; and the amino acid molecule is an amino acid, a linear peptide or a salt of the foregoing, provided that if the amino acid molecule is a linear peptide, the compound comprises an aziridine chiral center proximal to the aldehyde with matching stereochemistry to the carbon atom proximal to the amino terminus of the peptide, in the presence of a catalyst selected from an arylboronic acid or an arylborinic acid.

Compounds of formulae (la) and (lb) are described in PCT Application No. PCT/CA2007/001882, filed October 22, 2007 (published as WO/2008/046232 on April 24, 2008) to Andrei K. Yudin et al., the contents of which are hereby incorporated by reference in its entirety.

In one embodiment, Ri is CH₂OH. In another embodiment, any one of Ri - R5 is H. In still yet another embodiment, n=0 and Ri - R₃ is H. In another embodiment, n=0 and R₂ and R₃ is H. In another embodiment, R_t is CH₂OTBDMS, CH₂OH or CH^Pr.

In one embodiment, the amino acid molecule is a linear peptide. In another embodiment, the linear peptide is a primary amine-terminated linear peptide. In still yet another embodiment, the amino terminus amino acid of the linear peptide is selected from the group consisting of proline and an amino acid with an amino group substituted with NHBn, NHCH₂CH₂S0₂Ph or NHCH₂CH₂CN.

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. In one embodiment, the amino acid molecule is an alpha-amino acid. In another embodiment, the amino acid molecule is a beta-amino acid. In still yet another embodiment, the amino acid molecule is a gamma-amino acid.

In one embodiment, the isocyanide used in the processes disclosed herein is selected from the group consisting of: (S)-(-)-a-Methylbenzyl isocyanide; 1,1,3,3,- Tetramethylbutyl isocyanide; 1-Pentyl isocyanide; 2,6-Dimethylphenyl isocyanide; 2- Morpholinoethyl isocyanide; 2-Naphthyl isocyanide; 2-Pentyl isocyanide; 4- Methoxyphenyl isocyanide; Benzyl isocyanide; Cutyl isocyanide; Cyclohexyl isocyanide; Isopropyl isocyanide; p-Toluenesulfonylmethyl isocyanide; Phenyl isocyanide dichloride; tert-Butyl isocyanide; (Trimethylsilyl)methyl isocyanide; 1H- Benzotriazol-l-ylmethyl isocyanide; 2-Chloro-6-methylphenyl isocyanide; Di-tert- butyl 2-isocyanosuccinate; tert-Butyl 2-isocyano-3-methylbutyrate; tert-Butyl 2- isocyano-3-phenylpropionate; tert-Butyl 2-isocyanopropionate; and tert-Butyl 3- isocyanopropionate. In one embodiment, the isocyanide is tert-Butyl isocyanide. In another embodiment, the catalyst is an arylboronic acid. In still yet another embodiment, the arylboronic acid is phenylboro ic acid or 5-indolylboronic acid.

In another embodiment, the process is conducted in a non-nucleophilic reaction medium, for example in trifluoroethanol or in HFIP mixed with water. In another embodiment, the amino acid molecule is an amino acid and the process is conducted in water.

In another embodiment, the amino acid molecule is a peptide between 2 and 30 amino acids in length.

In one embodiment, the concentration of the amino acid molecule is at least at 0.002M. In another embodiment, the concentration of the amino acid molecule is between 0.002M and 0.5M. In other embodiments, the concentration of the amino acid molecule is at least 0.1M, or is around 0.2M.

In another embodiment, the invention provides for the use of an arylboronic acid catalyst or an arylborinic acid catalyst for catalyzing the cyclization of an amino acid molecule. In one embodiment the cyclization occurs by the processes disclosed herein. In yet another embodiment, the invention provides a process to produce a mixture of cyclic amino acid molecules comprising reacting a mixture of amino acid molecules, each having an amino terminus and a carboxy terminus, with:

(i) an isocyanide, and

(ii) a compound having formula (la) and/or (lb):

(la) (lb) wherein: n = 0 or 1, and Rj, R₂, R₃, and R₅ are independently selected from H; lower alkyl; alkenyl; heterocycle; cyckoalkyl; esters of the formula -C(0)OR^* wherein R^* is selected from alkyl and aryl; amides of the formula -

C(0)NR R , wherein R and R are independently selected from alkyl and aryl; -CH₂C(0)R, wherein R is selected from -OH, lower alkyl, aryl, -lower alkyl-aryl, or -NR_aRb, where R_a and R_b are independently selected from H, lower alkyl, aryl or -lower alkyl-aryl; -C(0)R_c, wherein Rc is selected from lower alkyl, aryl or -lower alkyl-aryl; or -lower alkyl-ORa, wherein Rj is a suitable protecting group or OH group; all of which are optionally substituted at one or more substitutable positions with one or more suitable substituents; and the aldehyde component thereof may optionally be in its bisulfite adduct form; and the mixture of amino acid molecules comprises (a) amino acids; (b) linear peptides; (c) salts of the foregoing; or (d) mixtures of (a) - (c); provided that if the mixture of amino acid molecules comprises one or more linear peptides, the compound comprises an aziridine chiral center proximal to the aldehyde with matching stereochemistry to the carbon atom proximal to the amino terminus of each of the one or more linear peptides, in the presence of a catalyst selected from an arylboronic acid or an arylborinic acid.

In one embodiment, the process further comprises isolating the mixture of cyclic peptides. In another embodiment, the process further comprises purifying the mixture of cyclic peptides. In still yet another embodiment, the process further comprises characterizing the mixture of cyclic peptides via subjecting the mixture of cyclic peptides to hydrolysis, followed by CID/MS² analysis.

Arylboronic and arylborinic acids are known to those of skill in the art. Arylboronic and arylborinic acid catalysts that may be used in the processes of the invention include, but are not limited to, B(Ph)₂(OH), and other boron reagents described in Chemfiles (Sigma-Aldrich Product Directory) "Boron Reagents for Suzuki Coupling" (2007) Volume 7, Number 7: arylboronic acids (page 4-17). N-heteroarylboronic acids (page 17-18, Chemfiles, supra), and S-heteroarylboronic acids (page 18, Chemfiles, supra) may also find utility in the processes of the invention.. Alkylboronic acids (page 3, Chemfiles, supra), and alkenylboronic acids (page 4, Chemfiles, supra) may also find utility in the processes of the invention.

More particularly, the arylboronic acids may be selected from:



In another aspect, there is provided a cyclic amino acid molecule prepared using the process described herein.

One would understand that cyclization, in some cases, may require and would include protecting certain peptide or amino acid side chains in manner known to a person skilled in the art. In another aspect, there is provided a cyclic amino acid molecule of formula (II): amino acid molecule

n = 0 or 1, and R_\, R₂, R₃, R4 and R5 are independently selected from H; lower alkyl; aryl; heteroaryl; alkenyl; heterocycle; esters of the formula -C(0)OR* wherein R* is selected from alkyl and aryl; amides of the formula - C(0)NR R , wherein R and R are independently selected from alkyl and aryl; -CH₂C(0)R, wherein R is selected from -OH, lower alkyl, aryl, - loweralkyl-aryl, or -NR_aR_b, where Ra and R_b are independently selected from H, lower alkyl, aryl or -loweralkyl-aryl; -C(0)Rc, wherein R_c is selected from lower alkyl, aryl or -lower alkyl-aryl; or -lower alkyl-OR_d, wherein Rj is a suitable protecting group or OH group; all of which are optionally substituted at one or more substitutable positions with one or more suitable substituents, bonds [a] and [b] are syn to each other;

R' is an amino acid side chain of the amino terminus amino acid; R" is an optionally substituted amide; and the amino acid molecule is an amino acid, a linear peptide, or a salt of the foregoing, wherein N' is the nitrogen at the amino terminus end of the amino acid molecule and C is the carbon at the carboxy terminus end of the amino acid molecule, and provided that if the amino acid molecule is a linear peptide, bonds [a] and [c] are anti to each other.

In one embodiment, any one of R] - R₅ is H. Preferably, n=0 and R₂ and R₃ is H or R - R₃ is H.

In a particular embodiment Ri is CH₂OTBDMS, CH₂OH, or CH₂'Pr. In one embodiment, the amino acid molecule is a linear peptide.

In another embodiment, the cyclic amino acid has the formula (Ila):

amino acid molecule

wherein n, Ri-R₅, R', R", C, N', and bonds [a], [b], and [c] are as defined above, and

R^z is selected from H, Bn, -CH₂C¾S0₂Ph or -CH₂CH₂CN. In another embodiment, R^z and R' combine to form a 5-membered cycloalkyl ring. Preferably, in one embodiment, R is H. In another embodiment, the amino terminus amino acid of the linear peptide is selected from the group consisting of proline and an amino acid with an amino group substituted with NHBn, NHCH₂CH₂S0₂Ph or NHCH₂CH₂CN.

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, beta-amino acid or gamma- amino acid.

As noted above, R" is an optionally substituted amide, such as -C(0)(NHR"'), wherein R'" may be selected from H, alkyl, aryl, cycloalkyl, and the like, all of which may be optionally substituted at one or more substitutable positions with one or more suitable substituents. In one embodiment R" is tert-Butyl amide. As used herein, the term "amino acid molecule" is meant to include single amino acids and also peptides.

As used herein, the term "amino acid" refers to molecules containing an amine group, a carboxylic acid group and a side chain that varies. Amino acid is meant to include not only the twenty amino acids commonly found in proteins but also non-standard amino acids and unnatural amino acid derivatives known to those of skill in the art, and therefore includes, but is not limited to, alpha, beta and gamma amino acids. Peptides are polymers of at least two amino acids and may include standard, nonstandard, and unnatural amino acids.

The term "suitable substituent" as used in the context of the present invention is meant to include independently H; hydroxyl; cyano; alkyl, such as lower alkyl, such as methyl, ethyl, propyl, n-butyl, t-butyl, hexyl and the like; alkoxy, such as lower alkoxy such as methoxy, ethoxy, and the like; aryloxy, such as phenoxy and the like; vinyl; alkenyl, such as hexenyl and the like; alkynyl; formyl; haloalkyl, such as lower haloalkyl which includes CF₃, CC1₃ and the like; halide; aryl, such as phenyl and napthyl; heteroaryl, such as thienyl and furanyl and the like; amide such as C(0)NR_aRt_>, where R_a and R_b are independently selected from lower alkyl, aryl or benzyl, and the like; acyl, such as C(0)-C₆H5, and the like; ester such as -C(0)OCH₃ the like; ethers and thioethers, such as O-Bn and the like; thioalkoxy; phosphino; and -NRJR_b, where R_a and R_b are independently selected from lower alkyl, aryl or benzyl, and the like. It is to be understood that a suitable substituent as used in the context of the present invention is meant to denote a substituent that does not interfere with the formation of the desired product by the processes of the present invention.

As used in the context of the present invention, the term "lower alkyl" as used herein either alone or in combination with another substituent means acyclic, straight or branched chain alkyl substituent containing from one to six carbons and includes for example, methyl, ethyl, 1-methylethyl, 1-methylpropyl, 2-methylpropyl, and the like. A similar use of the term is to be understood for "lower alkoxy", "lower thioalkyl", "lower alkenyl" and the like in respect of the number of carbon atoms. For example, "lower alkoxy" as used herein includes methoxy, ethoxy, /-butoxy.

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

The term "aryl" as used herein, either alone or in combination with another substituent, means an aromatic monocyclic system or an aromatic polycyclic system. For example, the term "aryl" includes a phenyl or a napthyl ring, and may also include larger aromatic polycyclic systems, such as fluorescent (eg. anthracene) or radioactive labels and their derivatives.

The term "heteroaryl" as used herein, either alone or in combination with another substituent means a 5, 6, or 7-membered unsaturated heterocycle containing from one to 4 heteroatoms selected from nitrogen, oxygen, and sulphur and which form an aromatic system. The term "heteroaryl" also includes a polycyclic aromatic system comprising a 5, 6, or 7-membered unsaturated heterocycle containing from one to 4 heteroatoms selected from nitrogen, oxygen, and sulphur.

The term "cycloalkyl" as used herein, either alone or in combination with another substituent, means a cycloalkyl substituent that includes for example, but is not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.

The term "cycloalkyl-alkyl-" as used herein means an alkyl radical to which a cycloalkyl radical is directly linked; and includes, but is not limited to, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, 1-cyclopentylethyl, 2-cyclopentylethyl, cyclohexylmethyl, 1-cyclohexylethyl and 2-cyclohexylethyl. A similar use of the "alkyl" or "lower alkyl" terms is to be understood for aryl-alkyl-, aryl-loweralkyl- (eg. benzyl), -lower alkyl-alkenyl (eg. allyl), heteroaryl-alkyl-, and the like as used herein. For example, the term "aryl-alkyl-" means an alkyl radical, to which an aryl is bonded. Examples of aryl-alkyl- include, but are not limited to, benzyl (phenylmethyl), 1-phenylethyl, 2-phenylethyl and phenylpropyl.

As used herein, the term "heterocycle", either alone or in combination with another radical, means a monovalent radical derived by removal of a hydrogen from a three- to seven-membered saturated or unsaturated (including aromatic) heterocycle containing from 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, homo- piperazine, 1,4-dioxane, 4-morpholine, 4-thiomorpholine, pyridine, pyridine-N-oxide or pyrimidine, and the like. The term "alkenyl", as used herein, either alone or in combination with another radical, is intended to mean an unsaturated, acyclic straight chain radical containing two or more carbon atoms, at least two of which are bonded to each other by a double bond. Examples of such radicals 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, acyclic straight chain radical containing two or more carbon atoms, at least two of which are bonded to each other by a triple bond. Examples of such radicals include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, and 1-butynyl.

The term "alkoxy" as used herein, either alone or in combination with another radical, means the radical -0-(Ci_-n)alkyl wherein alkyl is as defined above containing 1 or more carbon atoms, and includes for example methoxy, ethoxy, propoxy, 1-methylethoxy, butoxy and 1,1-dimethylethoxy. Where n is 1 to 6, the term "lower alkoxy" applies, as noted above, whereas the term "alkoxy" encompasses "lower alkoxy" as well as alkoxy groups where n is greater than 6 (for example, n = 7 to 10). The term "aryloxy" as used herein alone or in combination with another radical means -O-aryl, wherein aryl is defined as noted above. A peptide is a polymer of two or more amino acids.

Here, we report a serendipitous discovery of arylboronic acid-catalyzed macrocyclization. Using this chemistry, primary amine terminated peptides can be rapidly converted into aziridine-containing macrocycles under mild reaction conditions. In contrast to other cyclic peptides, our molecules can be easily sequenced using MS². These findings will not only enable rapid construction of macrocyclic peptide libraries from readily accessible linear inputs but should also lead to the development of other macrocylizations catalyzed by arylboronic acids.

Cyclic amino acid molecules and methods of preparing the same are described in PCT Application No. PCT/CA2010/000408, filed March 16, 2010 (published as WO/2010/105363 on September 23, 2010) to Andrei K. Yudin et al., the contents of which are hereby incorporated by reference in its entirety. Referring to Scheme 1 below, iminium ions such as 1 are key intermediates en route to peptide macrocycles under the Ugi reaction conditions. As part of a mechanistic investigation, we attempted to intercept these transient iminium ions with phenylboronic acid. Scheme 1. An attempted interception of the iminium ion intermediate with phenylboronic acid

Our original plan was to characterize the proposed borono-Mannich product 3. We chose proline as the cyclization substrate, however, when we reacted it with the aziridine aldehyde reagent and i-butyl isocyanide in trifluoroethanol, we detected no traces of the borono-Mannich adduct in the reaction mixture. While the elements of phenylboronic acid were not incorporated into the product, this additive dramatically accelerated the macrocyclization process, cutting the reaction time from hours down to several minutes. The product was isolated free of boron impurities upon removal of the catalyst from the reaction mixture using silica-bound carbonate.

Amino acids and challenging medium sized peptides were subjected to the reaction conditions (Table 1). Table 1. Representative substrates.

5a-k

Reaction time

Entry³ Ri Linear Peptide % Yield^b'^c No catalyst PhB(OH)₂

1 H Pro 5a 98 1.5 h 15 min

2 H Pro-Leu 5b 76 4 h 30 min

3 CH2CH(CH3)2 Pro-Leu 5c 74 4 h 50 min

4 H Pro-Gly-Leu-Gly-Phe 5d 82 6 h 20 min

5 CH2CH(CH3)2 Pro-Gly-Leu-Gly-Phe 5e 84 4 h 55 min

6 H Pro-Ala-Asn-Phe-Leu-Val-His 5f 83 4 h 25 min

7 CH₂OTBDMS N-Bn-Phe-Ala^d 5g 85 4 h 50 min

8 CH₂OTBDMS Pro-Gly-Leu 5h 87 6 h 50 min

9 CH₂OTBDMS Pro-Arg(pfb)-Gly-Asp(t-Bu)-Ala 5i 83 8 h 50 min

10 CH₂OTBDMS Pro-Thr(t-Bu)-Met-Lys(t-Bu)-Ala 5j 80 9 h 40 min

11 CH₂OTBD S Pro-Gly-His-Tyr(t-Bu)-Ala 5k 79 6 h 45 min

¹ Reactions were performed at room temperature using 0.2 mmol of isocyanide and peptide, and 0.1 mmol of aziridine aldehyde dimer in TFE (0.2 M). % Isolated yield from PhB(OH)₂ catalyzed reaction (for screening of boronic acid catalysts, see Scheme 2 and Table 2). ^c Diastereoselectivity of >20: 1 was confirmed by Ή NMR analysis of crude reaction mixture. ^d N-Bn terminated peptide.

When compared to the boronic acid-free protocol, the reaction times decreased by 5-13 fold in all cases and the products 5a-k were formed in good yields with excellent diastereoselectivities (>20: 1) (see Example 2 for further details). Several arylboronic acids were screened for their efficiency to catalyze macrocyclization of H-Phe-Ala-OH. Substitutents on the phenyl ring (F, OH, S0₂Me) led to moderate effect on the reaction. 4-Pyridinylboronic acid and 2- (dimethylamino)pyridine-5-boronic acid resulted in poor conversions. In the first case this was largely due to unreactive aminal (peptide and aziridine aldehyde) formation. Phenylboronic acid and 5-indolylboronic acid were found to catalyze the reaction efficiently to give cyclic peptide in 84% and 86% yield, respectively.

Scheme 2. Catalyst screening.

, . yⁱe (AminahCP 1 :1) 4 h, 34% yield

Table 2. Catalyst loading.

Entry PhB(OH)₂ (x mol%) Reaction time (min)

1 100 30

2 50 30

3 20 30

4 10 30

5 5 45

6 0 240

Structurally, boronic acids are tnvalent boron-containing compounds that possess one alkyl substituent and two hydroxyl groups to fill the remaining valences on the boron atom. With only six valence electrons and a consequent deficiency of two electrons, the sp -hybridized boron atom possesses a vacant p orbital. This low-energy orbital is orthogonal to the three substituents that are oriented in a trigonal planar geometry.

Table 3. ^ΠΒ NMR of substrate-catalyst complexes.

Entry ⁿB nuclei Chemical shift ⁿB coordination dinate

4 PhB(OH)₂-Serine dimer 7.30 Tetracoordinate

"B NMR [400 MHz, Toluene-d₈:MeOH-d4 (9:1)] δ (ppm); Tricoordinate: -25-30 ppm; Tetracoordinate: ~10 ppm (from literature).

NMR analysis shows that all three substrates (aziridine aldehyde dimer, amino acid and isocyanide) coordinate to phenylboronic acid. The extent of coordination by donation of lone pair into the empty p-orbital of boron is reflected in ^UB chemical shift of the complex. When the reaction mixture was followed by NMR, boronic acid complex with aziridine aldehyde dimer and isocyanide was observed. Scheme 3. Aziridine aldehyde dimer activation.

The serendipitous discovery of boronic acid-promoted rate acceleration of the peptide macrocyclization was particularly advantageous in the processes for converting primary amine-terminated linear peptides into macrocycles (Example 2, compounds 7a-g; Scheme 4). Scheme 4. Primary amine terminated peptides in macrocyclization. Ri = CH₂OH.

7a-g

0.5 h, 79% yield 1 h, 68% yield 2 h, 72% yield

7e 7f 7g

Reactions were performed at room temperature using 0.2 mmol of isocyanide and amino acid, and 0.1 mmol of amino aldehyde dimer in TFE (0.2 M). The isolated yields are shown above. Diastereoselectivity of >20:1 was confirmed by ^!H NMR analysis of crude reaction mixture.

We were wary of the possibility that, even if formed, our products could be unstable due to transannular attack of the secondary amine of 10 at the aziridine amide (Scheme 5). Scheme 5. Proposed mechanism for the catalytic effect of phenylboronic acid.

10 Contrary to our suspicion, when aziridine aldehyde 6 was reacted with H₂N-Phe-Ala- OH and t-butyl isocyanide in the presence of phenylboronic acid, the macrocycle 7a was formed exclusively and isolated in 84% yield. To explore the scope of this new process, linear peptides of varying lengths were subjected to the catalytic conditions (for further details, see Example 2). The macrocyclization outcompeted both the borono-Mannich and Passerini reactions, as well as the intermolecular coupling. In the proposed mechanism of this new catalytic process, the boronic acid plays a two-fold role by catalyzing formation of the iminium ion and by mediating the assembly of the putative macrocyclization intermediate 8. In support of the proposed route, a sodium complex of the arylboronic acid - iminium ion complex 8 was detected by ESI-MS.

As a test for this catalytic system, we wanted to see if a mixture of linear peptides could be cyclized selectively without cross-reactivity or oligomenzation. A collection of linear peptides was subjected to the macrocyclization conditions at 0.2 M concentration. Seven peptides in total (peptide = PL, FA, LGF, GLGF, LGPF, GLGLF, GLFGF) were macrocyclized to form monomelic respective cyclic peptides (cp- peptide) with no observed heteromeric linear or cyclic side products as confirmed by MS of the crude reaction mixture (Scheme 6; Figure 3).

Scheme 6. Macrocyclization of a mixture of cyclic peptides and identification through hydrolysis-CID/MS². ent

The ability to convert linear peptide "space" into its cyclic counterpart can be a valuable tool, but must rely on the ability to deconvolute the results. Techniques such as AS-MS (affinity selection-mass spectrometry) have been used to obtain hits from mixtures of compounds without the labour of assay development.¹⁷ Whereas linear peptides can be identified by a combination of molecular weight and CID/MS², cyclic peptides lack a free N-terminus and cannot be readily sequenced.¹⁸ Additionally, the molecules of cyclic peptides can fragment at multiple positions, complicating interpretation of the MS² spectra. The cyclic topology also rules out Edman degradation for sequence elucidation. Elaborate protocols to overcome these limitations must be engineered.¹⁹'²⁰

We have found that our aziridine-containing macrocycles can be readily sequenced, owing to a chemically distinct aziridine amide bond produced during macrocyclization. When we subjected our products to the aqueous medium at pH 2, site-selective hydrolysis of the aziridine amide took place instead of the aziridine ring-opening (Scheme 7).¹⁴

Scheme 7. Chemically distinct aziridine amide bond in cyclic peptide provides hard and soft electrophilic centers for chemoselective reactions.

The hydrolysis product is composed of the linear peptide segment and the isocyanide- derived "tag" at the N-terminus. Whereas the cyclized peptides do not yield informative sequence ions with CID/MS , the linearized versions exhibit distinct a, b and/or y ions. We envisioned this site-specific linearization as a tool to enable facile sequencing of peptides using MS².

The cyclic peptide ANFLVH 11 was linearized into compound 12 (Scheme 8, and Example 3 below). Scheme 8. Site-selective hydrolysis at pH 2 - linearization of ANFLVH 11 into compound 12

11

This peptide fragmented readily with CID/MS²; initially by a loss of the cyclization tag and subsequently to characteristic b and y ions (Figure l).²¹ BioAnalyst²² was used to annotate the spectra for sequence information using the most common 20 natural amino acids. The highest scoring tags corresponded to partially tagged internal sequences and the highest scoring full sequence was the correct peptide sequence.

Similarly, the cyclic peptide PpYVNVP 13 was linearized into compound 14 (see Figure 2, Scheme 9, and Example 3 below).

Scheme 9. Site-selective hydrolysis at pH 2 - linearization of PpYVNVP 13 into compound 14.

13 In both cases, the products 12 and 14 were purified by flash column chromatography before characterization by CID/MS². Fragment ions lists were collected at an intensity cutoff of 4 and 0.15 counts, for 12 and 14 respectively. Masses were determined to four decimal places with an external calibration. Sequencing was performed in tag mode with a mass tolerance of 0.1 and 0.06 Da for 12 and 14 respectively. The screened amino acid library was composed of the 20 most common natural amino acids. pY was added to the amino acid library for 14. The software was not programmed to assign the cyclization tags, due to their prompt loss during fragmentation. The calculated error for 12 was within 10 ppm and within 15 ppm for 14.

The ability to site-specifically cleave and sequence aziridine containing macrocycles is significant in that it will allow for mixture decoding following screening without peptide tagging or specialized protocols.

In summary, we report the discovery of dramatic rate acceleration during macrocyclization of linear peptides. This novel process utilizes readily available aziridine aldehydes and arylboronic acid catalysts. Upon completion, the catalyst can be readily removed from the reaction mixture using solid phase extraction, leaving behind no boron impurities. The reaction works rapidly with primary amine-terminated peptides to furnish peptide macrocycles with high levels of stereoselectivity and without cyclodimerization or oligomerization. The aziridine amide-directed ring scission enables straightforward sequencing of peptide macrocycles.

The following examples are illustrative of various aspects of the invention, and do not limit the broad aspects of the invention as disclosed herein. EXAMPLES

Materials and Methods

General Information: Anhydrous toluene and dimethylformamide (DMF) were purchased and used as received. Tetrahydrofuran (THF) was distilled from sodium benzophenone ketyl under argon. All other solvents including TFE (2,2,2,- trifluoroethanol) and HFIP (l,l,l,3,3,3-hexafluoro-2-isopropanol) were of reagent grade quality. Melting points were obtained on a MelTemp melting-point apparatus and are uncorrected.

Chromatography: Flash column chromatography was carried out using Silicycle 230- 400 mesh silica gel. Thin-layer chromatography (TLC) was performed on Macherey Nagel pre-coated glass backed TLC plates (SIL G/UV254, 0.25 mm) and visualized using a UV lamp (254 ran) and iodine stain. RP-HPLC was performed on a Waters Prep LC 4000 system with Waters 2487 dual λ absorbance detector with CI 8 semi- preparative column. Nuclear magnetic resonance spectra: 'H and ¹³C NMR spectra were recorded on Varian Mercury 400 or 500 MHz spectrometers. ^JH NMR spectra were referenced to TMS (0 ppm), CD₃OD (3.30 ppm) and ¹³C NMR spectra were referenced to CDC13 (77.2 ppm) and CD3OD (49.0 ppm). Cyclic peptides aggregate at concentrations higher than 0.5-3 mM. Peak multiplicities are designated by the following abbreviations: s, singlet; bs, broad singlet; d, doublet; t, triplet; q, quartet; m, multiplet; ds, doublet of singlets; dd, doublet of doublets; ddd, doublet of doublet of doublets; bt, broad triplet; td, triplet of doublets; tdd, triplet of doublets of doublets.

Mass Spectrometry: High-resolution mass spectra were obtained on a VG 70-250S (double focusing) mass spectrometer at 70 eV on a QStar XL (AB Sciex, Concord, ON, Canada) mass spectrometer with electrospray ionization (ESI) source, MS/MS and accurate mass capabilities. Low resolution mass spectra (ESI) were obtained at 60 eV, 70 eV and 100 eV. MS² Peptide Sequencing: Peptide fragmentation spectra were measured using a QStar XL (AB Sciex, Concord, ON, Canada) quadrupole time-of-flight mass spectrometer (Q-TOF-MS) operated in the positive ion mode. Peptide solutions were prepared in a 1 : 1 mixture of methanol and aqueous 0.1% formic acid to a concentration of ~1-10 μΜ and were infused at a flow rate of 5 min^"1 to the ESI source. The mass spectrometer is equipped with an HSID atmosphereic pressure interface (IONICS Mass Spectrometry Group Inc., Bolton, ON, Canada). Monoisotopically-resolved precursor ion populations were isolated in the mass-selective quadrupole (Ql) and fragmented in the collision cell (Q2) having an elevated pressure of N₂ collision gas. Product ion spectra were recorded for several minutes at a variety of collision voltages to optimize fragment ion intensities and signal-to-noise ratio. The peptide fragmentation spectra were analyzed using BioAnalyst v. 1.1 (AB Sciex).

EXAMPLE 1 - Synthetic routes toward aziridine aldehyde dimers (a) Synthesis of diethyl tartrate-derived aziridine aldehyde dimer Scheme 10: Synthetic route to aziridine aldehyde dimers S7 and S8.

S6 S5 S4

DIBAL DIBAL

Toluene Toluene

(S,S) Diethyl aziridine-2,3-dicarboxylate (S4)

The title compound was prepared using a literate method (see: Bruening, A.; Vicik, R.; Schirmeister, T. Tetrahedron: Asymmetry, 2003, 14, 330). In a round bottom flask equipped with Teflon coated magnetic stirring bar and a pressure equalizing addition funnel was placed L-diethyl-tartrate (SI, 17.73 g, 86 mmol). The reaction was cooled to 0 °C and then SOCl₂ (7.3 ml, 100 mmol) was added dropwise through the addition funnel over a period of 15 minutes. After the addition was complete, 20 drops of anhydrous DMF was added to the reaction mixture and the vessel was first allowed to warm to room temperature, and then it was heated at 50 °C for 30 minutes. The reaction was allowed to cool back to room temperature and N₂ was bubbled through the mixture for 1 hour in order to remove excess SOCl₂. The mixture was then concentrated using rotary evaporator at 50 °C to remove residual SOCl₂, then further concentrated under high vacuum to afford the cyclic sulfite S2 as pale yellow oil. The cyclic sulfite (21.67 g, 86 mmol) was then dissolved in 50 ml of anhydrous DMF. NaN₃ (16.77g, 258 mmol) was then added to the solution and the reaction was allowed to stir for 24 hours. 50 ml of CH₂C1₂ and 60 ml of water were then added to the reaction, and stirred for 2 hours. The aqueous phase was extracted three times with CH₂C1₂ (50 ml) and the collected organic phases were dried over Na₂S0₄ and concentrated under reduced pressure to afford the azido alcohol S3 in 95 % yield (18.87 g, 81.7 mmol) over two steps as a yellow oil, which was pure by NMR and carried over to the next step. The azido alcohol S3 (18.87 g, 81.7 mmol) was dissolved in 400 ml of anhydrous DMF and cooled to 0°C. PPh₃ (22.5 g, 85.79 mmol) was added in portions over a period of 30 minutes. The reaction vessel was then allowed to warm to room temperature and stirred at this temperature of 90 minutes. The reaction vessel was then warmed to 85 °C and stirred until completed by TLC (3: 1 Et₂0/hexanes, Rf = 0.34). The reaction was then concentrated under reduced pressure and purified by flash column chromatography (gradient 9: 1 - 7:3 hexanes/EtOAc) to afford the title compound S4 as a pale yellow oil in 79 % yield (12.1 g). Ή NMR (CDC1₃, 400MHz) 5: 4.30 (m, 4H), 2.87 (dd, J = 9.2 Hz, 3.2 Hz, 2H), 1.82 (bt, J= 9.2 Hz, 1H), 1.31 (dt, J = 10.4 Hz, 7.2 Hz, 6 H) ppm. ¹³C NMR (CDC1₃, 50MHz) δ: 170.6, 168.9, 62.4, 61.8, 36.3, 35.5, 14.2 ppm.

(S,S) 3-Hydroxymethylaziridine-2-carboxyIic acid ethyl ester (S5)

In a round bottom flask equipped with a magnetic stirring bar and a septum was placed S4 (1.87 g, 10 mmol) dissolved into 30 ml of EtOH. The vessel was cooled to 0 °C and NaBH₄ (302.6 mg, 8 mmol) was added slowly. The reaction mixture was allowed to stir at 0°C until the reaction was complete according to TLC (EtOAc, R_f = 0.66), which took approximately 2 hours. The reaction was quenched by the addition of pH 7 phosphate buffer, and extracted three times with CH₂C1₂ (10 ml). The combined organic layers were dried over Na₂S0₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (gradient 8:2 EtOAc/hexanes - 100 % EtOAc) to afford the title compound S5 in 84% yield as a pale yellow oil. Ή NMR (CDCI3, 200MHz) 5: 4.22 (q, J= 7.0 Hz, 2H), 3.82 (dd, J = 12.4 Hz, 2.8 Hz, 1H), 3.48 (dd, J =12 Hz, 4.8 Hz, 1H), 2.46 (m, 2H), 1.50 (bs, 1H), 1.31 (t, J = 7 .0 Hz, 3H) ppm. ¹³C NMR (CDC1₃, 50MHz) δ: 172.2, 62.0, 61.5, 39.8, 32.7, 14.3 ppm. HRMS (ESI) [M+H]⁺ calcd. for C₆H,₂N0₃ 146.0817, found 146.0820.

3-(tei-i-Butyldimethylsilanyloxymethyl)-aziridine-2-carboxyIic acid ethyl ester (S6)

In a flame dried flask equipped with a magnetic stirring bar and a rubber septum with a N₂ inlet was added S5 (296 mg, 2.04 mmol) and 12 ml of CH₂C1₂. The reaction vessel was cooled to 0 °C then TBDMSC1 (377 mg, 2.50 mmol) and DMAP (623 mg, 5.1 mmol) was added. The reaction was allowed to stir for 1 hour at 0 °C followed by stirring at room temperature until the reaction was complete according to TLC (R_f = 0.65; 7:3 hexanes/EtOAc). The reaction was diluted with CH₂C1₂ (5 ml) and water (10 ml) was added. The organic layer was extracted three times with CH₂C1₂ (10 ml), and the combined organic layers were washed first with saturated NaHC0₃, then water, then brine and dried over solid Na₂S0₄. The mixture was filtered and dried under reduced pressure to afford a pale yellow oil, which was subjected to flash column chromatography (8:2 hexanes/EtOAc) to afford the title compound S6 as a thick colourless oil in 99 % yield. H NMR (CDC1₃, 400MHz) δ: 4.21 (q, J = 3.6 Hz, 2H), 3.66 (dd, J = 11.2 Hz, 5.2 Hz, 1H), 3.56 (dd, 10.8 Hz, 4.8 Hz, 1H), 2.42 (m, 2H), 1.37 (bt, 1H), 1.29 (t, J = 7.2 Hz, 3H) ppm. ¹³C NMR (CDCI₃, 100MHz) δ: 172.6, 64.8, 61.7, 40.3, 33.7, 26.1, 18.5, 14.4, -5.1 ppm. HRMS (ESI) [M+H]⁺ calcd. for Ci₂H₂₅N0₃Si 260.1676, found 260.1675.

(2R,4R,5S,6S)-6-(hydroxymethyl)-2-((2S,3S)-3-(hydroxymethyl)aziridia-2-yl)-3- oxa-l-azabicyclo[3.1.0]hexan-4-ol (S7) In a flame dried 100 ml Schlenk tube equipped with a magnetic stirring bar was placed

55 (223.5 mg, 1.54 mmol) in 6 ml of toluene under nitrogen atmosphere. The solution was cooled to -78 °C and a 1.5 M solution of DIBAL in toluene (2.2 ml, 3.3 mmol) was added dropwise along the wall of the vessel. Once the addition was complete, the reaction was allowed to stir at -78 °C for 5 hours at which point TLC showed disappearance of the starting material. MeOH (0.5 ml) was slowly added along the wall of the vessel at -78 °C. The reaction mixture was then allowed to stir for 30 minutes while warming to room temperature. Saturated aqueous Na₂S0₄ (0.5 ml) was used to initiate precipitation of aluminum salts, which were filtered off after stirring for an additional 30 minutes. The filtrate was concentrated under reduced pressure. The resulting clear oil was pure by NMR analysis and was used in subsequent transformations.

6-(tei^*i-Butyldimethylsilanyloxymethyl)-2-[3-(tert-butyIdimethylsilanyloxymethyI)- aziridin-2-yI]-3-oxa-l-azabicyclo[3.1.0]hexan-4-ol (S8)

In a flame dried 100 ml Schlenk tube equipped with a magnetic stirring bar was placed

56 (400 mg, 1.54 mmol) in 6 ml of toluene under nitrogen atmosphere. The solution was cooled to -78 °C and a 1.5 M solution of DIBAL in toluene (2.2 ml, 3.3 mmol) was added dropwise along the wall of the vessel. Once the addition was complete, the reaction was allowed to stir at -78 °C for another hour at which point TLC showed disappearance of the starting material. MeOH (0.5 ml) was slowly added along the wall of the vessel at -78 °C. The reaction mixture was then allowed to stir for 30 minutes while warming to room temperature. Saturated Na₂S0₄ was then added and the solution was allowed to stir for another 15 minutes. The reaction was then filtered and water and ether were added. The organic layer was extracted three times, washed with brine, dried over Na₂S0₄ and concentrated under reduced pressure. The resulting clear oil S8 was pure by NMR analysis and was used in subsequent transformations. TLC (7:3, hexanes/EtOAc R_f = 0 - 0.55 streaking. Ή NMR (CDC1₃, 400MHz) δ: 8.20 - 8.05 (bs, 1H) 5.29 (bs, 1H), 4.95 (s, 1H), 3.86 - 3.85 (m, 2H), 3.65 (dd, J= 1 1.2 Hz, 6 Hz, 1H), 3.56 (dd, J = 11.6 Hz, 5.6 Hz, 1H), 2.53 (d, J = 2.8 Hz, 1H), 2.39 (bs, 1H), 2.13 (bs, 1H), 1.65 (sextet, J = 2.8 Hz), 1.20 (bs, 1H), 0.89 (s, 9H), 0.87 (s, 9H), 0.07 (ds, 6 H), 0.04 (ds, 6H) ppm ¹ C NMR (CDC1₃, 100 MHz) δ: 96.5, 94.7, 64.0, 58.4, 48.5, 40.3, 34.0, 33.5, 26.1, 26.0, 18.6, 18.5, -4.9, -5.0, -5.3, -5.4 ppm. HRMS (ESI) [MH]⁺ calcd. For C₂₀H43N₂O₄Si₂ 431.2755, found 431.2749.

(b) Synthesis of leucine-derived aziridine aldehyde dimer

Scheme 11: Synthesis of amino aldehyde dimer SI 3.

N-Boc-D-leucinol (S10)

To a solution of S9 (23.1 g, 100 mmol) in 100 ml of THF at -10 °C was added N- methyl morpholine (11 ml, 100 mmol) followed by isobutyl chloroformate (13.1 ml, 100 mmol). After stirring at -10 °C for 5 minutes the reaction was filtered (Biichner funnel, coarse glass frit) to remove the precipitate and the filtrate was then cooled back to -10 °C. NaBKU (5.67 g, 150 mmol) dissolved in 50 ml of water was then added to the reaction mixture over 5 minutes. After the addition was complete, the reaction was diluted with water and the reaction was extracted three times with EtOAc. The organic extracts were dried over Na₂S0₄, filtered, and concentrated under reduced pressure to afford 18.8 g (87 %) of S10 as a colourless oil, which was used in the next step without further purification.

(i?)-N-Boc 2-isobutylaziridine (Sll) To a solution of S10 (12.5 g, 58 mmol) dissolved in 1000 ml of Et₂0 was added finely ground KOH (13.02 g, 232 mmol) and tosyl chloride (13.27 g, 69.6 mmol). The reaction mixture was then heated to reflux for 16 hours. The reaction was then cooled to room temperature and water was added to the mixture and the organic phase was extracted three times with Et₂0 (250 ml). The combined organic phases were dried over Na₂S0₄, filtered and concentrated under reduced pressure to afford a slightly yellow oil, which was subjected to Kugelrohr distillation to afford Sll in 8.77 g (76 %) as a colourless oil.

(2S,3R)-tert-butyl 3-isobutylaziridine-2-carboxylate (S12) (see: Hodgson and co- workers Angew. Chem. Int. Ed. 2007, 46, 2245 - 2248)

In a flame dried flask equipped with a magnetic stirring bar and a rubber septum was added 2,2,6,6-tetramethyl piperidine (5.06 ml, 30 mmol) in 150 ml of anhydrous THF. The mixture was cooled to -78 °C and then «-BuLi (1.6 M in hexanes, 18.80 ml, 30 mmol) was added dropwise along the wall. The reaction was then allowed to warm to room temperature and stirred for 30 minutes. The reaction was then cooled down to - 78 °C and Sll (1.99 g, 10 mmol) was added along the wall of the flask over a period of ten minutes. The reaction was stirred for two hours at -78 °C. Saturated aqueous NE CI (5 ml) was added to quench the reaction, and the mixture was allowed to warm to room temperature. The mixture was extracted three times with EtOAc (10 ml), and the extracts were dried over Na₂S0₄, filtered and concentrated under reduced pressure. The product was then subject to flash column chromatography (20 % EtOAc in hexanes) to yield the title compound S12 as a colourless oil in 70% yield. R_f = 0.15 (20 % EtOAc in hexanes).

(l^j^^S^ -i-e-isobutyH-iilS^Ri-S-isobutylaziridin-l-y -a-oxa-l- azabicyclo[3.1.0]hexaii-4-ol (S13)

To a flame dried flask equipped with a magnetic stirring rod and a rubber septum was added S12 (2.94 g, 14.75 mmol) and 50 ml of anhydrous toluene. The reaction was cooled to -78 °C after which DIBAL (1.5 M in toluene, 19.67 ml, 29.5 mmol) was added along the wall of the vessel over a period of 45 minutes. The reaction was stirred at -78 °C for an additional two hours and then methanol was added along the wall of the vessel over a period of 30 minutes. The reaction was allowed to warm to room temperature and then saturated aqueous Na₂S0₄ (3 ml) was added to the mixture. After stirring for 15 minutes, the white precipitate was filtered, and the filter cake was washed with EtOAc (Note: a gel may form at this point, which can be emulsified using EtOAc prior to filtering). Water was added to the filtrate from which the product was extracted three times with EtOAc (20 ml). The combined organic layers were dried over Na₂S0 , filtered, and then concentrated under reduced pressure to afford S13 as a pale yellow solid (1.61 g, 86 % yield). Mp = 64 °C (hexanes) R_f = 0.38 (50 % EtOAc in hexanes) ^!H NMR (300 MHz, CDC1₃) δ: 7.94 (s, 1H), 5.26 (s, 1H), 4.94 (s, 1H), 2.41 (d, J = 2.8, 1H), 2.1 1 (s, 1H), 1.98 (d, J = 3.4, 1H), 1.76 (dtd, J = 2.2, 6.8, 8.8, 3H), 1.47 - 1.19 (m, 6H), 1.02 - 0.87 (m, 15H), 0.51 (s, 1H) ppm. ¹³C NMR (75 MHz, CDCI3) δ: 96.54, 94.45, 50.64, 43.15, 40.24, 38.95, 38.02, 32.99, 27.69, 27.22, 23.02, 22.96, 22.57, 22.51 ppm.

(c) Synthesis of serine-derived aziridine aldehyde dimer

Scheme 12: Synthesis of amino aldehyde dimer SI 6.

S14 S15 S16

(5)-butyl aziridine-2-carboxylate (S15)

In a 50 ml separatory funnel was added a suspension of 780 mg (4.84 mmol) of serine butyl ester/HCl salt in CH₂C1₂ (10 ml). Concentrated NH₄OH (5 ml) was then added and the mixture was shaken for 1 minute. The organic layer was extracted 5 times using CH₂C1₂ (5 ml) and the combined organic layers were dried over Na₂S0₄ and concentrated under reduced pressure to yield free base S14 as a colourless oil. The oil was dissolved in 25 ml (0.2 M solution) of CH2CI2, and the mixture was cooled to 0 °C with the aid of an ice-bath. 1.27 g (4.84 mmol) of PPh₃ was then added at 0 °C in three equal portions over 5 minutes. Keeping the bath at 0 °C, DIAD (953 μΐ, 4.84 mmol) was added drop-wise over a period of 15 minutes with vigorous stirring. The reaction was maintained at 0 °C for another 45 minutes after the addition of DIAD, and then allowed to warm to room temperature (-20 °C) and stirred for a further 16 hours. When the reaction was complete as monitored by TLC (I₂ stain), the mixture was concentrated, then Et₂0 was added and the solution was cooled to 0 °C for 1 hour. The precipitated OPPh₃ was filtered off, and the filtrate was concentrated at 25 °C under reduced pressure. The crude oil was subjected to Kugelrohr distillation to afford S15 in 81 % yield. For a 65 mmol scale reaction, S15 was isolated in 70 % yield.

(l^ZR^H-^-aziridin- -y -S-o a-S-aza-bicycIoIS.l.Olhe an-Z-ol (S16)

To a flame dried Schlenk tube equipped with a magnetic stirring bar, a rubber septum, and a N₂ inlet was added aziridine ester S15 (10 mmol) dissolved in 25 ml of anhydrous toluene. The reaction flask Was cooled to -78 °C, at which point 13 ml of a 1.5 M solution of DIBAL in toluene was added slowly via syringe to the reaction mixture over a period of 30 minutes. The reaction was allowed to stir for 5 hours or until completed by TLC. MeOH was added via syringe to the reaction mixture over a period of 15 minutes while maintaining a temperature of -78 °C. After the addition of MeOH, the reaction mixture was allowed to warm to room temperature and stirred for 30 minutes. Saturated aqueous Na₂S0₄ (0.5 ml) was used to initiate precipitation of aluminum salts, which were filtered off after stirring for an additional 30 minutes. The filtrate was concentrated under reduced pressure to yield a thick clear oil. Precipitation of the product was achieved upon addition of Et₂0 and EtOAc to afford S16 as a white solid in 76 % yield. Mp = 80 °C (EtOAc). R_f = 0.32 (20 % water in MeCN), mp = ^!H NMR (CDC1₃ with D₂0 present, 400MHz) δ: 5.27 (s, 1H), 4.92 (s, 1 H), 2.62 (dd, J = 5.2 Hz, 3.2 Hz, 1H), 2.47 - 2.43 (m, 1H), 1.86 (d, J = 6.4 Hz, 1H), 1.76 (d, J = 5.2 Hz, 1H), 1.70 (dd, J= 5.2 Hz, 3.6 Hz, 1H), 1.26 (d, J = 3.2 Hz, lH) ppm. ¹³C NMR (CDCI3, 100 MHz) δ: 96.6, 94.8, 44.0, 31.6, 28.0, 21.3 ppm.

EXAMPLE 2 -Boronic acid catalyzed synthesis of piperazinones and cyclic peptides

In a screw-cap vial equipped with a magnetic stirring bar was added peptide (0.2 mmol) and 1 ml of TFE and stirred until homogeneous solution has been obtained. Aziridine aldehyde dimer (0.1 mmol), boronic acid (0.02 mmol) and isocyanide (0.2 mmol) were then added sequentially and the resulting mixture was stirred for the time specified in the corresponding tables or schemes. Reactions were monitored by ESI-MS at 60 eV and/or TLC analysis. After completion of reaction, 1 ml of water and 1 ml of Et₂0 were added and the mixture was shaken vigorously and then cooled on ice. The resulting precipitate was filtered and washed with hexanes and cold Et₂0 (1 ml) to afford the cyclic peptide. For products that are water soluble or do not precipitate, the reaction mixture was concentrated under reduced pressure and then triturated with Et₂0 and hexanes (0.2 ml). The crude cyclic peptide was dissolved in CH₂C1₂ and incubated (3 times) with carbonate resin (1 equiv.) for 30 min to remove the boronic acid. The product isolated was analytically pure in several cases. Some of the cyclic peptides were further purified by flash column chromatography or RP-HPLC.

(c) Compound Characterization:

Please refer to FIG. 4 for a legend of the compounds referenced herein.

Please refer to FIGS. 5-26 for NMR spectra for compounds 5a-k and compounds 7a-f.

Connectivity and stereochemistry assignment - The relative stereochemistry of cyclic peptides was established by correlating the methine region of each Ή NMR spectrum with that of piperazinone (for X-ray crystal structure analysis of piperazinone, see Ref. 14). For a representative larger cycle 5c, formation of aziridine amide was confirmed by typical C=0 chemical shift of 175.3 ppm in CD₃OD. Magnetization transfer between protons on adjacent atoms and non-adjacent atoms was observed by COSY and TOCSY respectively. The heteronuclear experiments, HSQC and HMBC, were done for complete mapping of carbon and hydrogen connectivity. Finally, the stereochemistry was established by NOESY where NOE between H^a-H^e, H^a-H^f and H^e- H were observed.

c 5c (3aS,8R,8aS)-N-(tert-butyl)-3-oxooctahydroazirino[l,2-a]pyrrolo[l,2-d]pyrazine- 8-carboxamide (5a)

^!H NMR (CDCI3, 400 MHz) δ: 6.24 (bs, 1H), 3.42 (d, J = 6.2 Hz, 1H), 3.13 (dd, J = 9.5, 5.2 Hz, 1H), 3.05 (dd, J = 10.7, 4.6 Hz, 1H), 2.98 (t, J = 8.6 Hz, 1H), 2.41 (d, J = 4.8 Hz, 1H), 2.23 (d, J = 3.9 Hz, 1H), 2.21 (m, 1H), 2.13 (m, 2H), 1.86 (m, 2H), 1.35 (s, 9H) ppm. ¹³C NMR (CDCI3, 100 MHz) 6: 183.4, 169.0, 64.5, 63.2, 54.5, 51.4, 37.0, 30.5, 28.9, 22.0, 21.9 ppm. MS (ESI) [MH]⁺ calcd. 252.3, found 252.1

(4S,6aS,llR,llaS)-N-tert-butyl-4-isobutyl-3,6-dioxodecahydro-lH-azirino[l,2- a]pyrrolo[l,2-d][l,4,7]triazonine-ll-carboxamide (5b)

Ή NMR (CDCI3, 400 MHz) δ: 7.68 (bs, 1H), 6.70 (bs, 1H), 4.75 (t, J = 7.6 Hz, 1H), 4.35 (bs, 1H), 4.27 (dd, J = 14.3, 7.7 Hz, 1H), 4.10 (t, J = 8.6 Hz, 1H), 3.37 (dd, J = 9.1, 6.8 Hz, 1H), 3.18 (d, J = 5.7 Hz, 1H), 2.97 (bd, J = 8.5 Hz, 1H), 2.44 (m, 2H), 1.90 (m, 2H), 1.76 (m, 2H), 1.66 (m, 2H), 1.26 (s, 9H), 0.93 (d, J = 6.6 Hz, 3H), 0.91 (J = 6.6 Hz, 3H) ppm; ¹³C NMR (CDCI3, 100 MHz) δ: 173.6, 162.0, 155.4, 73.7, 62.2, 61.0, 58.0, 54.3, 52.6, 51.3, 50.8, 39.9, 29.9, 28.8, 26.1, 23.4, 23.0 ppm. MS (ESI) [MH]⁺ calcd. 365.5, found 365.2

(lR,4S,6aS,llR,llaS)-N-tert-butyl-l,4-diisobutyl-3,6-dioxodecahydro-lH- azirino[l,2-a]pyrrolo[l,2-d] [l,4,7]triazonine-ll-carboxamide (5c)

^!H NMR (CD₃OD, 400 MHz) 5: 4.51 (dt, J= 10.1, 1.9 Hz, 1H), 4.12 (dd, J= 8.6, 5.8 Hz, 1H), 3.82 (dd, J = 8.5, 5.5 Hz, 1H), 3.35 (d, J = 5.5 Hz, 1H), 3.26 (m, 2H), 2.44 (m, 2H), 1.92 (m, 3H), 1.86 (m, 1H), 1.80 (m, 3H), 1.66 (m, 1H), 1.56 (dd, J= 10.7, 9.0 Hz, 1H), 1.29 (s, 9H), 1.03 (d, J= 6.5 Hz, 3H), 1.00 (d, J= 6.4 Hz, 3H), 0.97 (d, J= 6.6 Hz, 3H), 0.95 (d, J= 6.6 Hz, 3H) ppm; ¹³C NMR (CD₃OD, 100 MHz) δ: 175.3, 165.3, 158.6, 86.1, 63.5, 61.7, 59.5, 59.3, 55.4, 51.5, 42.8, 41.0, 30.1, 27.3, 27.1 , 26.5, 24.0, 23.4, 22.4 (2 peaks), 21.9 ppm. MS (ESI) [MH]⁺calcd. 3H40N4O3 421.6, found 421.3.

(4S,10S,15aS,20R,20aS)-4-benzyl-N-(tert-butyl)-10-isobutyI-3,6,9,12,15- pentaoxoicosahy droazirino [1 ,2-a] pyrrolo [1 ,2- d] [1,4,7,10,13, 16]hexaazacyclooctadecine-20-carboxamide (5d)

Ή NMR (CDCI₃, 400 MHz) δ: 8.33 (bs, 1H), 8.17 (d, J = 9.1 Hz, 1H), 8.03 (d, J = 7.4 Hz, 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 = 8.1 , 5.0 Hz, 1H), 3.80 (bs, 1H), 2.48 (dd, J = 3.6, 1.8 Hz, 2H), 2.16 (m, 2H), 1.62-1.57 (m, 8H), 1.48 (m, 2H), 1.42 (m, 2H), 1.35 (m, 2H), 1.20 (s, 9H), 0.81 (m, 6H) ppm. MS (ESI) [MH]⁺ calcd. 626.8, found 626.3 and 313.7 for [M+2H]²⁺/2

(li?,laS,2R,6aS,12S,18S)-18-benzyI-N-tert-butyl-l,12-diisobutyl-7,10,13,16,19- pentaoxoicosahy droazirino [ 1 ,2-a] pyrrolo [ 1 ,2- d] [1,4,7,10,13, 16]hexaazacyclooctadecine-2-carboxamide (5e)

Ή NMR (CDCI3, 500 MHz) δ: 7.82 (bs, NH), 7.40 - 7.20 (m, 5Η), 7.18 (bs, NH), 7.05 (bs, NH), 6.81 (bs, 2 NH), 4.64 (q, J = 6.8 Hz, 1H), 4.13 (dd, J = 16.0, 7.6 Hz, 1H), 4.10 - 3.90 (m, 2H), 3.57 (dd, J= 16.0, 4.8 Hz, 1H), 3.50 - 3.39 (m, 2H), 3.26 (dd, J = 14.0, 5.6 Hz, 1H), 3.12 (dd, J = 14.0, 7.2 Hz, 1H), 3.12 - 3.08 (m, 1H), 3.00 - 2.92 (m, 2H), 2.63 (t, J = 3.6 Hz, 1H), 2.28 - 2.18 (m, 1H), 1.90 - 1.70 (m, 2H), 1.37 - 1.25 (m, 9H), 1.10 - 0.88 (m, 12H) ppm. ¹³C NMR (CDCI₃, 125 MHz) δ: 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 ppm. MS (ESI) [MH]⁺ calcd. 682.4, found 682.4 (4S,7S,10S,13S,16S,19S,21aS,26R,26aS)-4-((lH-imidazol-4-yl)methyl)-16-(2- amino-2-oxoethyl)-13-benzyl-N-(tert-butyl)-10-isobutyl-7-isopropyl-19-methyl- 3,6,9,12,15,18,21-heptaoxohexacosahydroazirino[l,2-a]pyrrolo[l,2- d] [1,4,7,1043, 16,19,22] octaaza-cyclotetracosine-26-carboxamide (5f) Ή NMR (DMSO-d₆, 400 MHz) 6: 9.63 (bs, 1H), 8.96 (s, 1H), 8.39 (d, J= 8.1 Hz, 1H), 8.31 (d, J= 7.0 Hz, 1H), 8.19 (d, J= 7.8 Hz, 1H), 8.03 (d, J= 8.0 Hz, 1H), 7.79 (s, 1H), 7.63 (d, J = 9.0 Hz, 1H), 7.42 (bs, 1H), 7.38 (s, 1H), 7.35 (s, 1H), 7.21 (m, 5H), 6.99 (bs, 1H), 4.56 (m, 2H), 4.47 (m, 2H), 4.31 (m, 2H), 4.14 (dd, J = 8.6, 6.3 Hz, 1H), 4.13 (m, 1H), 3.16 (dd, J = 14.5, 3.7 Hz, 1H), 3.06 (dd, J = 13.5, 3.5 Hz, 1H), 2.98 (dd, J = 14.9, 9.9 Hz, 1H), 2.78 (dd, J= 14.2, 9.2 Hz, 2H), 2.65 (m, 1H) 2.39 (m, 2H), 2.22 (d, J = 9.8 Hz, 1H), 2.05 (m, 2H), 1.84 (m, 1H), 1.76 (d, J = 6.9 Hz, 1H), 1.48-1.61 (m, 4H), 1.42 (m, 2H), 1.30 (d, J = 6.8 Hz, 3H), 1.26 (s, 9H), 0.88 (d, J = 6.5 Hz, 3H), 0.82 (d, J = 6.5 Hz, 3H), 0.81 (d, J = 6.7 Hz, 3H),0.68 (d, J = 6.8 Hz, 3H) ppm. MS (ESI) [MH]⁺ calcd. 934.1, found 934.3 and 467.3 for [M+2H]²⁺/2. Cyclic peptide 5f was purified by RP-HPLC, gradient elution with water/acetonitrile (3% to 60% acetonitrile over 45 min) mixture buffered with 0.1 % TFA on a CI 8 semi-preparative column.

(3S,6S,8R,9S,10S)-6,7-dibenzyl-N-(tert-butyl)-10-(((tert- butyldimethylsilyl)oxy)methyl)-3-methyl-2,5-dioxo-l,4,7- triazabicyclo[7.1.0]decane-8-carboxamide (5g)

Ή NMR (CDCI₃, 300 MHz) δ: 7.41-7.24 (m, 10H), 4.21 (q, J = 8.8 Hz, 1H), 3.90 (s, 2H), 3.74-3.58 (m, 4H), 3.65 (dd, J = 11.4, 5.9 Hz, 1H), 2.57 (d, J = 2.8 Hz, 1H), 2.44 (d, J = 2.8 Hz, 1H), 2.17 (s, 1H), 1.38 (d, J = 12.4 Hz, 3H), 1.33 (s, 9H), 0.92 (s, 9H), 0.11 (s, 3H), 0.08 (s, 3H) ppm. ¹³C NMR (CD₃OD, 75 MHz) δ: 174.5, 167.2, 165.5, 129.7, 129.3, 129.2, 128.9, 128.2, 127.5, 126.5, 122.8, 63.9, 58.4, 54.3, 48.5, 40.4, 34.0, 33.5, 30.9, 29.8, 28.9, 26.1, 25.9, 18.6, 18.5, -5.0, -5.3 ppm. MS (ESI) [MH]⁺ calcd. 607.9, found 607.4 (15,45,9aS 4i? 4a-S)-N-tert-butyl-l-((tert-butyldimethylsilyloxy)methyl)-4- isobutyl-3,6,9-trioxotetradecahydroazirino[l,2-a]pyrrolo[l,2- d] [l,4,7,10]tetraazacyclododecine-14-carboxamide (5h) *H NMR (CD3OD, 400 MHz) δ: 4.55 (ddd, J= 8.0, 3.2, 1.6 Hz, 1H), 4.32 (dd, J= 10.4, 4.0 Hz, 1H), 4.21 (dd, J = 14.0, 6.0 Hz, 1H), 4.15 - 4.05 (m, 3H), 3.91 (dd, J = 12.0, 3.2 Hz, 1H), 3.41 Hz (dd, J = 6.0 Hz, 1 H), 3.36 - 3.28 (m, 1H), 2.50 - 2.30 (m, 2H), 2.00 - 1.50 (m, 7H), 1.31 (s, 9 H), 0.98 - 0.92 (m, 6H), 0.92 (s, 9 H), 0.13 (s, 3H), 0.11 (s, 3H) ppm. ¹³C NMR (CD₃OD, 100 MHz) δ: 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, -6.5 ppm. MS (ESI) [MH]⁺ calcd. 566.4, found 566.4

tert-butyl-2-((lS,4S,7S,13S,l5aS,20R,20aS)-20-(tert-butyIcarbamoyl)-l-(((tert butyldimethylsilyl)oxy)methyl)-4-methyl-3,6,9,12,15-pentaoxo-13-(3-(3-((2,2,4,6,7- pentametbyl-2,3-dihydrobenzofuran-5- yl)sulfonyl)guanidino)propyl)icosahydroazirino [1 ,2-a] pyr rolo [1 ,2- d][l,4,7,10,13,16]hexaazacyclooctadecin-7-yl)acetate (5i)

^!H NMR (CDC1₃, 400 MHz) δ: 7.84 (d, J = 9.7 Hz, 2H), 7.58 (dd, J = 18.1 , 9.1 Hz, 2H), 7.07 (s, 1H), 6.41 (s, 2H), 5.68 (s, 1H), 4.85 (dq, J= 13.7, 6.8 Hz, 1H), 4.70 - 4.58 (m, 1H), 4.35 (q, J = 8.1 Hz, 1H), 4.17 - 4.07 (m, 1H), 3.97 (dd, J = 14.2, 7.5 Hz, 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 = 2.3 Hz, 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) ppm. ¹³C NMR (CDCI₃, 100 MHz) δ: 187.1, 176.1 , 174.5, 171.5, 171.0, 169.1, 166.9, 158.8, 156.4, 138.6, 133.7, 132.6, 124.7, 1 17.6, 105.2, 86.5, 82.6, 63.9, 63.8, 60.8, 57.1, 51.7, 51.3, 50.4, 49.6, 45.5, 43.5, 40.4, 37.6, 36.0, 30.3, 29.2, 28.8, 28.2, 26.1 , 24.6, 19.5, 18.7, 18.2, 17.3, 12.7, -5.1, -5.4 ppm. MS (ESI) [MH]⁺ calcd. 1 104.6, found 1 104.6

tert-butyl (4-((lS,4S,75,105,13S,15a5,20R,20aS)-13-((R)-l-(tert-butoxy)ethyl)-20- (tert-butylcarbamoyl)-l-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-10-(2- (methylthio)ethyl)-3,6,9,12,15-pentaoxoicosahydroazirino[l,2-a]pyrrolo[l,2d]

[1,4,7,10,13,16] hexaazacyclooctadecin-7-yl)butyl)carbamate (5j)

Ή NMR (CDC1₃, 400 MHz) δ: 7.83 (d, J = 8.1 Hz, 1H), 7.77 (s, 1H), 7.50 (d, J = 21.3 Hz, 1H), 7.35 (m, 2H), 7.08 (dd, J = 15.7, 7.5 Hz, 1H), 6.90 (s, 1H), 6.82 (s, 1H), 4.98 (d, J = 27.6 Hz, 1H), 4.84 - 4.67 (m, lH), 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 = 6.3 Hz, 1H), 3.45 - 3.28 (m, 2H), 3.27 - 2.99 (m, 6H), 3.00 - 2.86 (m, 2H), 2.84 (s, 1H), 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 = 4.7 Hz, 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 = 10.5 Hz, 4H), 0.93 - 0.82 (m, 14H), 0.06 (s, 3H), 0.05 (s, 3H) ppm. ¹³C NMR (CDCI3, 100 MHz) δ: 183.0, 175.2, 171.9, 170.8, 170.4, 168.9, 156.4, 77.4, 75.7, 66.3, 64.4, 63.9, 61.8, 57.5, 56.6, 54.3, 42.7, 40.4, 31.2, 31.0, 30.1, 29.9, 29.4, 28.9, 28.7, 26.0, 24.9, 24.2, 18.9, 18.5, 17.9, 15.5, -5.3, -5.3 ppm. MS (ESI) [MH]⁺ calcd. 983.6, found 983.6

(lS,7S,10S,15aS,20i-,20aS)-10-((lH-imidazol-5-yl)methyI)-7-(4-(tert- butoxy)benzyl)-N-(tert-butyl)-l-(((tert-butyldimethylsilyl)oxy)methyl)-3,6,9,12,15- pentaoxoicosahydroazir.no [1 ,2-a]pyrrolo [1 ,2- d] [l,4,7,10,13,16]hexaazacyclooctadecine-20-carboxamide (5k)

Ή NMR (CDCI₃, 400 MHz) 8: 9.92 (s, 1H), 7.49 (d, J = 4.8 Hz, 2H), 7.39 - 7.27 (m, 11H), 7.17 - 7.02 (m, 11H), 6.78 (d, J = 8.4 Hz, 2H), 6.69 (s, 1H), 4.48 (p, J = 6.9 Hz, 1H), 4.42 - 4.18 (m, 2H), 4.06 - 3.99 (m, 1H), 3.91 (dd, J = 11.4, 3.8 Hz, 1H), 3.78 (dd, J = 11.3, 4.4 Hz, 1H), 3.61 (dd, J = 9.8, 4.8 Hz, 1H), 3.48 - 3.37 (m, 1H), 3.32 - 2.99 (m, 8H), 2.83 (dd, J = 6.8, 3.4 Hz, 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) ppm. ¹³C NMR (CDC1₃, 100 MHz) δ: 184.3, 175.2, 172.6, 170.7, 169.5, 168.1, 154.5, 142.3, 138.2, 136.8, 131.9, 130.0, 129.9, 128.4, 128.4, 120.1, 77.4, 63.5, 62.2, 61.6, 57.8, 55.0, 52.2, 51.2, 48.7, 43.4, 42.5, 41.8, 36.4, 31.8, 31.0, 29.9, 29.8, 29.4, 29.1, 28.7, 26.1, 24.5, 19.8, 18.6, -5.2 ppm. MS (ESI) [MH]⁺ calcd. 866.5, found 866.5

(3S,6S,8R,9R,10S)-6-benzyl-N-(tert-butyl)-10-(hydroxymethyl)-3-methyl-2,5- dioxo-l,4,7-triazabicyclo[7.1.0]decane-8-carboxamide (7a) H NMR (CD₃OD, 400 MHz) δ: 7.72-7.59 (m, 2H), 7.29 (m, 3H), 4.92 (s, 1H), 4.38 (dd, J = 14.8, 7.6 Hz, 1H), 4.01 (dd, J = 11.7, 5.0 Hz, 1H), 3.26 (dd, J = 9.1, 3.9 Hz, 2H), 3.02 (dd, J= 13.8, 3.8 Hz, 1H), 2.71 (dd, J=13.8, 9.3 Hz, 1H), 2.59 (d, J= 5.2 Hz, 1H), 2.25 (m, 1H), 2.00 (d, J = 7.6 Hz, 3H), 1.19 (s, 9H) ppm. ¹³C NMR (CD₃OD, 100 MHz) δ: 176.0, 174.0, 173.1, 138.8, 134.5, 128.6, 128.0, 66.1, 64.1, 61.5, 52.0, 40.9, 38.0, 36.8, 34.3, 28.8, 17.3 ppm. MS (ESI) [MH]⁺ calcd. 403.5, found 403.3

(3S,9S,llR,12R,13S)-3-benzyl-N-(tert-butyl)-13-(hydroxymethyl)-9-isobutyl-2,5,8- trioxo-l,4,7,10-tetraazabicyclo[10.1.0]tridecane-ll-carboxamide (7b)

Ή NMR (CD₃OD, 400 MHz) δ: 7.60 (m, 2H), 7.23 (m, 3H), 5.03 (s, 1H), 4.69-4.58 (m, 2H), 4.20 (m, 1H), 4.04 (dd, J = 18.3, 9.2 Hz, 1H), 3.57 (d, J = 4.7 Hz, 2H), 2.54- 2.41 (m, 3H), 2.35 (t, J = 8.2 Hz, 1H), 1.72 (dd, J = 13.3, 6.7 Hz, 2H), 1.62 (m, 1H), 1.35 (s, 9H), 0.96 (d, J = 6.7 Hz, 3H), 0.90 (d, J = 6.7 Hz, 3H) ppm. ^l3C NMR (CD₃OD, 100 MHz) δ: 177.7, 173.5, 173.4, 172.9, 134.5, 133.2, 129.7, 129.2, 70.4, 69.9, 69.1, 62.9, 50.8, 40.6, 33.7, 33.5, 31.9, 31.7, 28.8, 23.7, 22.1 ppm. MS (ESI) [MH]⁺ calcd. 502.6, found 502.2 (lS,laR,2R,4S,12aS,15S)-15-benzyI-N-(tert-butyl)-l-(hydroxymethyl)-4-isobutyl-

5,8,13,16-tetraoxohexadecahydro-lH-azirino[l,2-g]pyrrolo[l,2- a] [l,4,7,10,13]pentaazacycIopentadecine-2-carboxamide (7c)

Ή NMR (CD₃OD, 400 MHz) δ: 7.56 (m, 2H), 7.23 (m, 3H), 5.07 (s, 1H), 4.62 (dd, J = 13.6, 6.9 Hz, 1H), 4.48-4.34 (m, 3H), 4.20 (dd, J = 13.9, 6.7 Hz, 1H), 3.56 (m, 2H), 3.20 (m, 1H), 3.06 (m, 2H), 2.48 (m, 3H), 2.02 (m, 2H), 1.88 (m, 2H), 1.76 (m, 2H), 1.65 (s, 1H), 1.35 (s, 9H), 0.95 (d, J = 6.8 Hz, 3H), 0.90 (d, J = 6.9 Hz, 3H) ppm. ¹³C NMR (CD₃OD, 100 MHz) δ: 177.8, 173.4, 173.1, 173.0, 172.9, 139.3, 134.5, 129.6, 129.2, 70.1, 69.9, 69.8, 62.8, 58.3, 56.9, 52.9, 42.6, 42.2, 40.6, 39.0, 33.8, 33.5, 28.9, 26.1, 23.7 ,23.0, 22.3 ppm. MS (ESI) [MH]⁺ calcd. 599.7, found 599.3

(3S,9S,14R,15R,16S)-3-benzyl-N-(tert-butyl)-16-(hydroxymethyl)-9-isobutyl-

2,5,8,ll-tetraoxo-l,4,7,10,13-pentaazabicyclo[13.1.0]hexadecane-14-carboxamide

(7d) Ή NMR (CD₃OD, 400 MHz) δ: 7.59 (m, 2H), 7.21 (m, 3H), 5.03 (s, 1H), 4.64 (m, 1H), 4.49-4.39 (m, 1H), 3.56 (m, 2H), 3.20 (m, 2H), 3.03 (m, 2H), 2.99 (m, 1H), 2.51- 2.41 (m, 3H), 2.02 (m, 2H), 1.62 (m, 1H), 1.35 (s, 9H), 0.95 (d, J = 6.7 Hz, 3H), 0.90 (d, J = 6.7 Hz, 3H) ppm. ¹³C NMR (CD₃OD, 100 MHz) δ: 177.7, 173.6, 173.4, 173.0, 172.9, 134.5, 133.2, 130.4, 129.2, 70.1, 69.8, 69.4, 62.9, 52.9, 50.8, 40.6, 39.6, 39.2, 38.4, 33.5, 28.9, 25.9, 23.5 ppm. MS (ESI) [MH]⁺ calcd. 559.7, found 559.5

(3S,6S,12S,17R,18R,19S)-3-benzyl-N-(tert-butyl)-19-(hydroxymethyl)-6,12- diisobutyl-2,5,8,ll,14-pentaoxo-l,4,7,10 3,16-hexaazabicyclo[16.1.0]nonadecane- 17-carboxamide (7e)

Ή NMR (CD₃OD, 400 MHz) δ: 7.57 (m, 2H), 7.25 (m, 3H), 4.63 (m, 1H), 4.44-4.33 (m, 2H), 4.01 (dd, J = 18.2, 9.1 Hz, 1H), 3.55 (bs, 2H), 3.30 (bs, 2H), 3.19 (m, 2H), 3.02 (m, 2H), 2.50-2.44 (m, 3H), 2.34 (t, J = 8.1 Hz, 1H), 2.00 (m, 2H), 1.64 (m, 2H), 1.34 (s, 9H), 0.98-0.91 (m, 9H), 0.89 (d, J = 6.8 Hz, 3H) ppm. ¹³C NMR (CD₃OD, 100 MHz) δ: 180.1, 177.7, 174.7, 173.5, 172.8, 171.2, 139.2, 138.6, 134.5, 133.2, 70.4, 70.1, 69.9, 69.7, 62.8, 52.9, 50.8, 45.7, 43.4, 41.9, 41.0, 40.6, 33.5, 28.9, 25.8, 23.5, 21.8 ppm. MS (ESI) [MH]⁺ calcd. 672.8, found 672.3

(3S,9S,12S,17R,18R,19S)-3,9-dibenzyl-N-(tert-butyl)-19-(hydroxymethyl)-12- isobutyl-2,5,8,ll,14-pentaoxo-l,4,7,10,13,16-hexaazabicyclo[16.1.0]nonadecane-17- carboxamide (7f) ^!H NMR (CD₃OD, 400 MHz) δ: 7.57 (m, 4H), 7.43-7.03 (m, 6H), 5.01 (d, J = 13.2 Hz, 1H), 4.69-4.56 (m, 2H), 4.49 (dd, J = 13.6, 8.8 Hz, 1H), 4.20 (dd, J= 14.2, 7.1 Hz, 1H),

4.03 (dd, J = 18.4, 9.2 Hz, 1H), 3.56 (d, J = 4.9 Hz, 1H), 3.42 (t, J = 6.8 Hz, 1H), 3.20 (m, 2H), 2.85 (s, 1H), 2.37-2.31 (m, 3H), 2.34 (t, J = 8.2 Hz, 1H), 2.06-1.97 (m, 2H), 1.71 (dd, J = 13.3, 6.6 Hz, 1H), 1.59 (m, 1H), 1.34 (s, 9H), 0.94 (d, J = 6.7 Hz, 3H), 0.89 (d, J = 6.7 Hz, 3H) ppm. ¹³C NMR (CD₃OD, 100 MHz) δ: 180.8, 177.8, 176.6, 174.7, 173.4, 170.4, 134.5, 133.2, 130.4, 129.5, 129.3, 129.2, 128.5, 127.3, 70.1, 69.9, 69.8, 69.5, 62.9, 57.4, 56.1, 52.9, 50.8, 40.8, 33.5, 31.9, 31.7, 28.9, 25.9, 23.4 ppm. MS (ESI) [MH]⁺ calcd. 706.8, found 706.5

EXAMPLE 3 - characterization of cyclic compounds by CID/MS²

Method of hydrolysis of cyclic peptides:

In a screw-cap vial equipped with a magnetic stirring bar was added cyclic peptide (0.1 mmol) and 1 ml of 0.1% TFA in H₂0. The reaction was allowed to stir for 5 hours or until completed by ESI-MS at 60 eV and/or TLC analysis. The reaction mixture was concentrated in vacuo. Precipitation of the product was achieved upon addition of Et₂0 to afford hydrolyzed product as analytically pure white solid.

Sequencing of cyclic compounds 12 and 14 by CID/MS²: In both cases, the products 12 and 14 were purified by flash column chromatography before characterization by CID/MS . Fragment ions lists were collected at an intensity cutoff of 4 and 0.15 counts, for 12 and 14 respectively. Masses were determined to four decimal places with an external calibration.

Table 4. Fragment ion list for 12 and 14

221.1295 12.8871 155.1135 0.1800

228.1713 5.0914 156.0983 0.6733

237.1365 4.8602 171.1086 2.9867

238.0840 4.0753 186.1205 0.1800

254.1623 5.3763 197.0891 0.4867

255.1462 24.1452 201.1 197 2.2467

297.1520 3.9570 214.1 155 3.4267

322.1893 5.5000 216.0377 0.2933

333.1583 23.6075 269.1936 0.1822

335.1732 9.9247 270.1784 3.3178

365.2313 9.6344 304.6264 0.4378

368.2313 8.6720 313.0933 1.7156

370.2102 7.5538 341.0847 3.7556

375.2035 7.0860 343.0977 0.2133

383.1755 5.9624 369.2461 0.1933

418.2442 4.0591 375.6612 0.2200

427.1632 5.9409 412.1579 0.8822

444.2212 6.5215 424.6863 0.191 1

446.2416 18.8226 425.1806 0.5022

464.2971 4.2634 433.6921 7.8822 482.2430 18.6613 440.1549 3.0956

500.2615 6.5538 452.7122 0.1511

515.3011 13.6559 453.2088 0.2889

517.2975 5.9086 457.1568 0.1911

528.2839 4.0161 461.7249 14.9178

540.2490 4.8011 467.1256 0.1556

545.3089 10.9570 485.1379 0.4022

577.3796 3.9731 509.1777 0.2578

595.3278 11.2043 526.2022 0.3778

613.3359 5.2097 537.1747 0.2467

629.3420 7.4409 539.1952 0.1511

630.3604 5.7473 554.1969 5.5778

682.3682 10.1505 556.2144 0.2267

699.3981 20.6129 582.3659 0.2000

700.3831 47.7473 584.2047 0.2778

701.3971 3.7742 608.2439 0.2822

729.4295 7.6882 625.2677 1.7333

734.3657 4.6774 636.2411 0.3089

765.4047 4.2634 653.2658 4.8844

793.4040 19.1667 825.3993 0.1533 811.4172 14.0000

831.4520 5.5161

838.4945 8.0215

848.4816 19.4355

849.4675 22.4462

866.4946 241.4247

867.4817 41.0591

884.5050 202.8763

885.5018 14.3065

Table 5. Tagged MS sequences of linearized cyclic peptides

(339.2913)VL(314.1384) 80.62 P(806.3858) 79.09

(369.2992)P(399.2302) 80.35 (439.1472)NV(251.1800) 78.27

(369.2992)FL(236.1304) 80.30 (200.1293)P(606.2565) 77.84

(339.2913)VA(356.1853) 80.28 (510.2992)V(294.0709) 77.79

(184.2176)AN(496.2845) 80.26 (170.1011)V(634.2691) 77.65

(255.2585)NFL(236.1282) 80.09 (553.1894)VP(154.1280) 77.62

(184.2176)ANF(349.2161) 79.91 (170.1011)W(535.2006) 77.06

(184.2176)ANP(399.2318) 79.86 (198.0960)V(606.2741) 76.97

(367.2239)F(351.2898) 79.78 PM(675.3453) 76.63

(255.2585)NF(349.2123) 79.75 PpY(563.3561) 76.51

(184.2176)ANFLVH(0.0047) 79.72 PT(705.3381) 76.47

(440.3794)F(278.1343) 79.58 (241.1846)P(565.2011) 76.38

(629.4541)VH(0.0007) 79.55 (340.0771)VNV(251.1817) 76.36

(255.2585)NP(399.2280) 79.51 (439.1472)N(350.2484) 76.10

(728.5232)H(0.0000) 79.51 PpYVNVP(154.1236) 76.08

(369.2992)FLVH(0.0031) 79.34 (439.1472)NVP(154.1272) 75.03

(516.3688)LVH(0.0020) 79.00 TV(703.3224) 75.02

(369.2992)V(397.2145) 78.92 LS(703.3224) 75.00

(483.3833)L(269.1148) 78.65 (170.1011)WN(421.1577) 74.37

(255.2585)NFLVH(0.0009) 78.22 (340.0771)VNVP(154.1289) 74.27

(255.2585)NV(397.2123) 78.22 PTV(606.2697) 74.24

(417.2368)P(351.2926) 77.94 (340.0771)VN(350.2501) 74.19

(184.2176)ANV(397.2161) 77.80 (340.0771)V(464.2930) 74.06 (468.3787)K(269.1084) 77.80 VT(703.3224) 73.94

(384.3384)A(410.2067) 77.79 PpYVNV(251.1764) 73.88

(501.4239)Q(236.0996) 77.67 (553.1894)H(213.1902) 73.78

(440.3794)R(269.1016) 77.58 (198.0960)A(634.3054) 73.11

(369.2992)VK(269.1195) 77.53 (142.0711)A(690.3303) 73.11

(415.2140)V(351.2998) 77.27 PpYV(464.2877) 73.07

(466.3559)Y(236.1629) 76.84 (297.1658)A(535.2356) 73.00

(384.3384) V(382.1754) 76.20 LGW(535.1962) 72.67

(255.2585)NVK(269.1174) 76.15 (413.2790)PV(294.0383) 72.66

As noted above, sequencing was performed in tag mode with a mass tolerance of 0.1 and 0.06 Da for 12 and 14 respectively. The screened amino acid library was composed of the 20 most common natural amino acids. pY was added to the amino acid library for 14. The software was not programmed to assign the cyclization tags, due to their prompt loss during fragmentation.

Table 6. Calculated error in most significant fragment ions

Υ4 515.298 515.301 -0.0033 6.4 ys 629.341 629.342 -0.0012 1.9

Fimmonium 120.081 120.082 -0.0008 7

Hjmmonium 1 10.071 1 10.071 -0.0001 1

[14+2H]^Z+ 461 .726 461 .725 0.0010 2.1 a₂ 3 13.095 313.093 0.0015 4.8 a₃ 412.163 412.158 0.0053 12.9 a₄ 526.206 526.202 0.0039 7.4

14 s 625.275 625.268 0.0068 10.9

b₂ 341.09 341.085 0.0050 14.7 b₃ 440.158 440.155 0.0032 7.3 b₄ 554.201 554.197 0.0041 7.4 b₅ 653.27 653.266 0.0037 5.7

The calculated error for 12 was within 10 ppm and within 15 ppm for 14.

Although preferred embodiments of the 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. (1) Nomura, D.K., Dix, M.M. & Cravatt, B.F. Nat. Rev. Cancer 10, 630-638 (2010).

(2) Bromley, E.H.C., Channon, K., Moutevelis, E. & Woolfson, D.N. ACS

Chem. Biol. 3, 38-50 (2008).

(3) Fletcher, J.M., Morton, C.J., Zwar, R.A., Murray, S.S., O'Leary, P.D. & Hughes, R.A. J. Biol. Chem. 283, 33375-3383 (2008).

(4) Tyndall, J.D.A., Nail, T. & Fairlie, D.P. Chem. Rev. 105, 973-999 (2005).

(5) Frye S.V. Nat. Chem. Biol. 6, 159-161 (2010).

(6) Kwon, Y-U., Reddy, M.M. & Kodadek, T. Chem. Commun. 44, 5704-5706 (2008).

(7) Agarwal, T., Roy, S., Chakraborty, T.K. & Maiti, S. Biochemistry 49, 8388- 8397 (2010).

(8) Haubner, R., Schmitt, W., Holzemann, G., Goodman, S.L., Jonczk, A. & Kessler, H. J. Am. Chem. Soc. 118, 7881-7891 (1996).

(9) London, N., Movshovitz-Attias, D. & Schueler-Furman O. Structure 18,

188-199 (2010).

(10) Driggers, E.M, Hale, S.P., Lee, J. & Terrett, N.K. Nat. Rev. Drug Discov. 7, 608-624 (2008).

(11) Wells J.A. & McClendon C.L. Nature 450, 1001-1009 (2007).

(12) Schafmeister, C.E., Po, J. & Verdine G.L. J. Am. Chem. Soc. 122, 5891- 5892 (2000). (13) Stewart, M.L., Fire, E., Keating, A.E. & Walensky, L.D. Nat. Chem. Biol. 6, 595-601(2010).

(14) Hili, R., Rai, V. & Yudin, A.K. J. Am. Chem. Soc. 132, 2889-2891 (2010);

WO/2010/105363 (published September 23, 2010). (15) Jebrail, M., Ng, A.H.C., Rai, V., Hili, R., Yudin, A.K. & Wheeler, A.R.

Angew. Chem. Int. Ed. in press (2010).

(16) Petasis, N.A., Zavialov, LA. J. Am. Chem. Soc. 119, 445-446 (1997).

(17) Annis, D.A., Nazef, N., Chuang, C.-C, Scott, M.P. & Nash, H.M. J. Am.

Chem. Soc. 126, 15495-15503 (2004). (18) Kelly, M.A., McLellan, T.J. & Rosner, P.J. Anal. Chem. 74, 1-9 (2002).

(19) Joo, S.H., Xiao, Q., Ling, Y., Gopishetty, B. & Pei, D. J. Am. Chem. Soc.

128, 13000-13009 (2006).

(20) Li, S., Marthandan, N., Bowerman, D., Garner, H.R. & Kodadek, T. Chem.

Commun. 581-583 (2005). (21) Hunt D.F., Yates III J.R. & Shabanowitz J. Proc. Nat. Acad. Sci. U.S.A. 83,

6233-6237 (1986).

(22) BioAnalyst v. 1.1 (AB Sciex, Foster City, CA, USA).

Additional references relating to materials and methods may be found in the Exampli section.

Claims

CLAIMS:

1. A process to produce a cyclic amino acid molecule comprising reacting amino acid molecule, having an amino terminus and a carboxyl terminus, with:

(i) an isocyanide, and

(ii) a compound having formula (la) and/or (lb):

(la) (lb) wherein: n = 0 or 1, and R_ls R₂, R₃, R4 and R₅ are independently selected from H; lower alkyl; alkenyl; heterocycle; cyckoalkyl; esters of the formula -C(0)OR^* wherein R^* is selected from alkyl and aryl; amides of the formula -

C(0)NR R , wherein R and R are independently selected from alkyl and aryl; -CH₂C(0)R, wherein R is selected from -OH, lower alkyl, aryl, -lower alkyl-aryl, or -NR_aRb, where R_a and R_b are independently selected from H, lower alkyl, aryl or -lower alkyl-aryl; -C(0)R_c, wherein R^ is selected from lower alkyl, aryl or -lower alkyl-aryl; or -lower alkyl-OR_d, wherein R_d is a suitable protecting group or OH group; all of which are optionally substituted at one or more substitutable positions with one or more suitable substituents; and the aldehyde component thereof may optionally be in its bisulfite adduct form; and the amino acid molecule is an amino acid, a linear peptide or a salt of the foregoing, provided that if the amino acid molecule is a linear peptide, the compound comprises an aziridine chiral center proximal to the aldehyde with matching stereochemistry to the carbon atom proximal to the amino terminus of the peptide, in the presence of a catalyst selected from an arylboronic acid or an arylborinic acid.

2. The process of claim 1 , wherein R_\ is CH₂OH.

3. The process of claim 1 , wherein any one of Ri - R5 is H.

4. The process of claim 1, wherein n=0 and Ri - R₃ is H.

5. The process of claim 1, wherein n=0 and R₂ and R₃ is H.

6. The process of claim 5, wherein Ri is CH₂OTBDMS, CH₂OH or CK^Pr.

7. The process of any one of claims 1-6, wherein the amino acid molecule is a linear peptide.

8. The process of claim 7, wherein the linear peptide is a primary amine- terminated linear peptide.

9. The process of claim 7, wherein the amino terminus amino acid of the linear peptide is selected from the group consisting of proline and an amino acid with an amino group substituted with NHBn, NHCH₂CH₂S0₂Ph or NHCH₂CH₂CN.

10. The process of any one of claims 1-6 wherein 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, histidme, isoleucine, leucine, lysine, methionine, phenylalanine, proline, selenocysteine, serine, tyrosine, threonine, tryptophan and valine.

11. The process of any one of claims 1-6 wherein the amino acid molecule is an alpha-amino acid.

12. The process of any one of claims 1-6 wherein the amino acid molecule is a beta- amino acid.

13. The process of any one of claims 1-6 wherein the amino acid molecule is a gamma-amino acid.

14. The process of any one of claims 1-13 wherein the isocyanide is selected from the group consisting of: (S)-(-)-a-Methylbenzyl isocyanide; 1,1,3,3,-Tetramethylbutyl isocyanide; 1-Pentyl isocyanide; 2,6-Dimethylphenyl isocyanide; 2-Morpholinoethyl isocyanide; 2-Naphthyl isocyanide; 2-Pentyl isocyanide; 4-Methoxyphenyl isocyanide; Benzyl isocyanide; Cutyl isocyanide; Cyclohexyl isocyanide; Isopropyl isocyanide; p- Toluenesulfonylmethyl isocyanide; Phenyl isocyanide dichloride; tert-Butyl isocyanide; (Trimethylsilyl)methyl isocyanide; lH-Benzotriazol-l-ylmethyl isocyanide; 2-Chloro- 6-methylphenyl isocyanide; Di-tert-butyl 2-isocyanosuccinate; tert-Butyl 2-isocyano-3- methylbutyrate; tert-Butyl 2-isocyano-3-phenylpropionate; tert-Butyl 2- isocyanopropionate; and tert-Butyl 3-isocyanopropionate.

15. The process of any one of claims 1-13 wherein the isocyanide is tert-Butyl isocyanide.

16. The process of any one of claims 1-15, wherein the catalyst is an arylboronic acid.

17. The process of claim 16, wherein the arylboronic acid is phenylboronic acid or 5- indolylboronic acid.

18. The process of any one of claims 1-17 wherein the process is conducted in a non-nucleophilic reaction medium.

19. The process of claim 18 wherein the non-nucleophilic reaction medium is trifluoroethanol.

20. The process of claim 18 wherein the non-nucleophilic reaction medium is HFIP mixed with water.

21. The process of any one of claims 1-17, wherein the amino acid molecule is an amino acid and the process is conducted in water.

22. The process of any one of claims 1-9 wherein the peptide is between 2 and 30 amino acids in length.

23. The process of any one of claims 1-22, wherein the concentration of the amino acid molecule is at least at 0.002M.

24. The process of any one of claims 1-22, wherein the concentration of the amino acid molecule is between 0.002M and 0.5M.

25. The process of any one of claims 1-22, wherein the concentration of the amino acid molecule is at least 0.1M.

26. The process of any one of claims 1-22, wherein the concentration of the amino acid molecule is around 0.2M.

27. Use of an arylboronic acid catalyst or an arylbonnic acid catalyst for catalyzing the cyclization of an amino acid molecule.

28. The use of claim 27, wherein the cyclization occurs by the process of any one of claims 1 to 26.

29. A process to produce a mixture of cyclic amino acid molecules comprising reacting a mixture of amino acid molecules, each having an amino terminus and a carboxy terminus, with:

(i) an isocyanide, and

(ii) a compound having formula (la) and/or (lb):

(ia) (lb) wherein: n = 0 or 1, and R_{l s} R₂, R₃, R4 and R5 are independently selected from H; lower alkyl; alkenyl; heterocycle; cyckoalkyl; esters of the formula -C(0)OR* wherein R* is selected from alkyl and aryl; amides of the formula - C(0)NR R , wherein R and R are independently selected from alkyl and aryl; -CH₂C(0)R, wherein R is selected from -OH, lower alkyl, aryl, -lower alkyl-aryl, or -NR_aRb, where R_a and R¾ are independently selected from H, lower alkyl, aryl or -lower alkyl-aryl; -C(0)Rc, wherein Rc is selected from lower alkyl, aryl or -lower alkyl-aryl; or -lower alkyl-ORd, wherein Rd is a suitable protecting group or OH group; all of which are optionally substituted at one or more substitutable positions with one or more suitable substituents; and the aldehyde component thereof may optionally be in its bisulfite adduct form; and the mixture of amino acid molecules comprises (a) amino acids; (b) linear peptides; (c) salts of the foregoing; or (d) mixtures of (a) - (c); provided that if the mixture of amino acid molecules comprises one or more linear peptides, the compound comprises an aziridine chiral center proximal to the aldehyde with matching stereochemistry to the carbon atom proximal to the amino terminus of each of the one or more linear peptides, in the presence of a catalyst selected from an arylboronic acid or an arylborinic acid.

30. The process of claim 29, further comprising isolating the mixture of cyclic peptides.

31. The process of claim 30, further comprising purifying the mixture of cyclic peptides.

32. The process of claim 30 or claim 31, further comprising characterizing the mixture of cyclic peptides via subjecting the mixture of cyclic peptides to hydrolysis, followed by CID/MS² analysis.

33. A cyclic amino acid molecule of formula (Ha):

amino acid molecule

wherein, n = 0 or 1, and Rj, R₂, R₃, R4 and R5 are independently selected from H; lower alkyl; aryl; heteroaryl; alkenyl; heterocycle; esters of the formula -C(0)OR* wherein R* is selected from alkyl and aryl; amides of the formula - C(0)NR R , wherein R and R are independently selected from alkyl and aryl; -CH₂C(0)R, wherein R is selected from -OH, lower alkyl, aryl, - loweralkyl-aryl, or -NR_aRb, where Ra and Rb are independently selected from H, lower alkyl, aryl or -loweralkyl-aryl; -C(0)Rc, wherein Rc is selected from lower alkyl, aryl or -lower alkyl-aryl; or -lower alkyl-ORd, wherein d is a suitable protecting group or OH group; all of which are optionally substituted at one or more substitutable positions with one or more suitable substituents, bonds [a] and [b] are syn to each other;

R' is an amino acid side chain of the amino terminus amino acid; R' ' is an optionally substituted amide; and the amino acid molecule is an amino acid, a linear peptide, or a salt of the foregoing, wherein N' is the nitrogen at the amino terminus end of the amino acid molecule and C is the carbon at the carboxy terminus end of the amino acid molecule, and provided that if the amino acid molecule is a linear peptide, bonds [a] and [c] are anti to each other, and

R^z is H.