Patent Publication Number: US-2020299675-A1

Title: Methods and Compositions for Display of Macrocyclic Peptides

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation-in-part of U.S. patent application Ser. No. 15/107,387, filed Dec. 23, 2014, which claims priority to and the benefit of U.S. provisional patent application Ser. No. 61/920,181, entitled Methods and Compositions for Ribosomal Synthesis of Macrocyclic Peptides, filed Dec. 23, 2013, which is incorporated herein by reference in its entirety. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
     The disclosed invention was made with government support under Grant No. CA187502 and Grant No. GM134076 from the National Institutes of Health. The government has rights in this invention. 
    
    
     1. TECHNICAL FIELD 
     The present invention relates to methods and compositions for generating macrocyclic peptides from genetically encoded, ribosomally produced polypeptide precursors. The invention also relates to methods and compositions for the display of genetically encoded macrocyclic peptides on a biological surface; to nucleic acid molecules, polypeptides, and methods for generating combinatorial libraries of macrocyclic peptides displayed on a biological surface and their use for selecting macrocyclic peptides with a desired property 
     2. BACKGROUND 
     Peptides molecules represent valuable tools for investigating biological systems, studying the binding and activity properties of biomolecules (e.g., enzymes, cell receptors, antibodies, kinases), exploring the etiopathological causes of diseases, and for validating pharmacological targets. Peptides are also attractive ligands for targeting protein-protein interactions and modulating the function of biological molecules such as enzymes and nucleic acids. The synthesis of combinatorial libraries of small peptides followed by screening of these chemical libraries in biological assays can enable the identification of compounds that exhibit a variety of biological and pharmacological properties. Bioactive peptides identified in this manner can constitute valuable lead compounds or facilitate the development of lead compounds towards the discovery of new drugs. 
     While many peptides exhibit interesting biological activity, linear peptides do not generally represent suitable pharmacological agents as they are generally only poorly adsorbed, do not cross biological membranes readily, and are prone to proteolytic degradation. In addition, linear peptides fail to bind proteins that recognize discontinuous epitopes. The use of molecular constraints to restrict the conformational freedom of the molecule backbone can be used to overcome these limitations. In many cases, conformationally constrained peptides exhibit enhanced enzymatic stability (Fairlie, Tyndall et al. 2000; Wang, Liao et al. 2005), membrane permeability (Walensky, Kung et al. 2004; Rezai, Bock et al. 2006; Rezai, Yu et al. 2006), and protein binding affinity (Tang, Yuan et al. 1999; Dias, Fasan et al. 2006) and selectivity (Henchey, Porter et al. 2010), compared to their linear counterparts. Constraints that lock-in the active conformation of a peptide molecule can result in increased affinity due to the reduced conformational entropy loss upon binding to the receptor. Many bioactive and therapeutically relevant peptides isolated from natural sources occur indeed in cyclized form or contain intramolecular bridges that reduce the conformational flexibility of these molecules (e.g., immunosuppressant cyclosporin A, antitumor dolastatin 3 and diazonamide A, anti-HIV luzopeptin E2, and the antimicrobial vancomycin). Since macrocyclic peptides constitute promising molecular scaffolds for the development of bioactive compounds and therapeutic agents (Katsara, Tselios et al. 2006; Driggers, Hale et al. 2008; Obrecht, Robinson et al. 2009; Marsault and Peterson 2011), methods for generating macrocyclic peptides and combinatorial libraries thereof, are of high synthetic value and practical utility, in particular in the context of drug discovery. 
     While cyclic peptides can be prepared synthetically via a variety of known methods (White and Yudin 2011), the possibility to generate macrocyclic peptides starting from genetically encoded polypeptide precursors offers several advantages (Frost, Smith et al. 2013; Smith, Frost et al. 2013). Among these, there are: (a) the high combinatorial potential inherent to the ribosomal synthesis of genetically encoded polypeptides, which can enable the production of very large collections of peptide sequences (10 8 -1010 members or higher) in a cost- and time-effective manner; (b) the possibility to link these peptide libraries to powerful, high-throughput screening platforms such as phage display, mRNA display, or yeast display, in order to identify peptide ligands with the desired property (e.g., high binding affinity toward a target protein); (c) the ease by which these chemical libraries can be deconvoluted in order to identify the library members of interest (i.e. via sequencing of the peptide-encoding DNA or RNA sequence). 
     Phage display constitutes a particularly powerful and versatile technique for the creation of large (e.g., up to 10 9  members) combinatorial libraries of genetically encoded polypeptides and the enrichment of peptide ligands for a protein of interest (Smith and Petrenko 1997; Sidhu, Lowman et al. 2000; Ladner, Sato et al. 2004). This methodology relies on the fusion of a genetically randomized polypeptide sequence to a coat protein of a bacteriophage (e.g., M13 phage), resulting in the display of the polypeptide on the surface of the phage particle, which also contains the DNA encoding for it. Upon ‘panning’ of the phage display peptide library against a target protein immobilized on a plate or resin bead, library members capable of binding the target protein can be isolated and their structure be elucidated by DNA sequencing. Using this technique, peptide libraries comprising up to 10 8 -10 9  members can be generated and screened to identify peptides with the desired protein binding affinity and selectivity (Smith and Petrenko 1997; Sidhu, Lowman et al. 2000; Ladner, Sato et al. 2004). This technique has been applied to generate and screen combinatorial libraries of linear peptides, also incorporating non-canonical amino acids (Feng, Tsao et al. 2004; Liu, Mack et al. 2008), from which linear peptide ligands for a target molecule of interest could be identified (Smith and Petrenko 1997; Sidhu, Lowman et al. 2000; Ladner, Sato et al. 2004). 
     In addition to phage display, other display techniques such as yeast display (Boder, Raeeszadeh-Sarmazdeh et al. 2012; Bosma, Rink et al. 2019; Linciano, Pluda et al. 2019) and bacterial display (Samuelson, Gunneriusson et al. 2002; Lofblom 2011; Shivange and Daugherty 2015), in which a polypeptide is genetically fused to an outer membrane protein of the yeast or bacterial cell, has enabled the screening of large combinatorial libraries of linear peptides (e.g., 10 6  to 10 9  members) from which peptide ligands for a target molecule of interest could be identified (Shivange and Daugherty 2015; Linciano, Pluda et al. 2019). As noted above, however, linear peptides suffer several limitations as affinity ligands or drug candidates for the development of new therapeutics. 
     Various methods have been developed for producing biological libraries of conformationally constrained peptides (Frost, Smith et al. 2013; Smith, Frost et al. 2013). For example, libraries of disulfide-constrained cyclic peptides have been prepared using phage display and fusing randomized polypeptide sequences flanked by two cysteines to a phage particle as described, e.g., in U.S. Pat. No. 7,235,626. Disulfide bridges are however potentially reactive and this chemical linkage is unstable under reducing conditions or in a reductive environment such as the intracellular milieu. Alternatively, ribosomally produced peptides have also been constrained through the use of cysteine- or amine-reactive cross-linking agents (Millward, Takahashi et al. 2005; Seebeck and Szostak 2006; Heinis, Rutherford et al. 2009; Schlippe, Hartman et al. 2012). A drawback of these methods is the risk of producing multiple undesired products via reaction of the cross-linking agents with multiple sites within the randomized peptide sequence or the carrier protein in a display system. In addition, these methods do not allow for the formation of macrocyclic peptides inside the polypeptide-producing cell host. Other methods have been described that are useful for preparing head-to-tail cyclic peptides by using natural (i.e., naturally occurring) or engineered (i.e., non-naturally occurring, artificial or synthetic) split inteins, as described in U.S. Pat. Nos. 7,354,756, 7,252,952 and 7,105,341. An advantage of these strategies is the possibility to couple the intracellular formation of cyclic peptide libraries with an cell-based reporter or selection system, which can facilitate the identification of functional peptide ligands (Horswill, Savinov et al. 2004; Cheng, Naumann et al. 2007; Naumann, Tavassoli et al. 2008; Young, Young et al. 2011). However, the peptide cyclization efficiency was found to be highly dependent on the peptide sequence (Scott, Abel-Santos et al. 2001). In addition, only head-to-tail cyclic peptides can be obtained through these strategies, which limits the extent of structural diversity of the ligand libraries generated through these methods. Finally, methods have also been reported for generating cyclic peptides through the enzymatic modification of linear peptide precursors (Hamamoto, Sisido et al. 2011; Touati, Angelini et al. 2011). However, the need for exogenous reagents and/or enzyme catalysts for mediating peptide cyclization and, in some cases, moderate cyclization efficiency limit the scope and utility of these approaches toward the generation and screening of cyclic peptide libraries. 
     Thus, while being highly desirable in the art, none of the aforementioned methods allows for the display of genetically encoded macrocyclic peptides that are cyclized through non-reducible linkages without the assistance of a chemical or enzymatic cyclization procedure. The methods and compositions described herein provide a solution to this need, enabling the display of genetically encoded macrocyclic peptides constrained by one or more non-reducible thioether bridge(s) on an outer biological surface of a phage particle or host cell. These methods can be applied to screen large combinatorial libraries of macrocyclic peptides in a high-throughput manner, from which macrocyclic peptides with a desired function can be selected and identified. 
     Citation or identification of any reference in Section 2, or in any other section of this application, shall not be considered an admission that such reference is available as prior art to the present invention. 
     3. SUMMARY 
     In some embodiments, the invention relates to a method for displaying a macrocyclic peptide on an outer biological surface, the method comprising: 
     a. providing an artificial nucleic acid molecule encoding for a polypeptide of structure: 
       (AA) m -Z-(AA) n -Cys-(AA) p   (I)
 
       or 
       (AA) m -Cys-(AA) n -Z-(AA) p   (II)
 
       or 
       (AA) m -Cys-(AA) n -Z2-(AA) o -Cys-(AA) p   (V)
         wherein:   i. (AA) m  is an N-terminal amino acid or peptide sequence,   ii. Z is a non-canonical amino acid carrying a side-chain functional group FG 1 , FG 1  being a functional group selected from the group consisting of —(CH 2 ) n X, where X is F, Cl, Br, or I and n is an integer number from 1 to 10; —C(O)CH 2 X, where X is F, Cl, Br, or I; —CH(R′)X, where X is F, Cl, Br, or I; —C(O)CH(R′)X, where X is F, Cl, Br, or I; —OCH 2 CH 2 X, where X is F, Cl, Br, or I; —C(O)CH═C═C(R′)(R″); —SO 2 C(R′)═C(R′)(R″); —C(O)C(R′)═C(R′)(R″); —C(R′)═C(R′)C(O)OR′; —C(R′)═C(R′)C(O)N(R′)(R″); —C(R′)═C(R′)—CN; —C(R′)═C(R′)—NO 2 ; —C≡C—C(O)OR′; —C≡C—C(O)N(R′)(R″); unsubstituted or substituted oxirane; unsubstituted or substituted aziridine; 1,2-oxathiolane 2,2-dioxide; 4-fluoro-1,2-oxathiolane 2,2-dioxide; and 4,4-difluoro-1,2-oxathiolane 2,2-dioxide, where each R′ and R″ is independently H, an aliphatic, a substituted aliphatic, an aryl, or a substituted aryl group.   iii. Z2 is a non-canonical amino acid carrying two side-chain functional groups FG 1  and FG 2 , wherein each of FG 1  and FG 2  is a functional group independently selected from the group consisting of —(CH 2 ) n X, where X is F, Cl, Br, or I and n is an integer number from 1 to 10; —C(O)CH 2 X, where X is F, Cl, Br, or I; —CH(R′)X, where X is F, Cl, Br, or I; —C(O)CH(R′)X, where X is F, Cl, Br, or I; —OCH 2 CH 2 X, where X is F, Cl, Br, or I; —C(O)CH═C═C(R′)(R″); —SO 2 C(R′)═C(R′)(R″); —C(O)C(R′)═C(R′)(R″); —C(R′)═C(R′)C(O)OR′; —C(R′)═C(R′)C(O)N(R′)(R″); —C(R′)═C(R′)—CN; —C(R′)═C(R′)—NO 2 ; —C≡C—C(O)OR′; —C≡C—C(O)N(R′)(R″); unsubstituted or substituted oxirane; unsubstituted or substituted aziridine; 1,2-oxathiolane 2,2-dioxide; 4-fluoro-1,2-oxathiolane 2,2-dioxide; and 4,4-difluoro-1,2-oxathiolane 2,2-dioxide, where each R′ and R″ is independently H, an aliphatic, a substituted aliphatic, an aryl, or a substituted aryl group,   iv. (AA) n  is a target peptide sequence,   v. (AA) o  is a second target peptide sequence,   vi. (AA) p  is a C-terminal amino acid or peptide sequence; and   vii. wherein at least one of (AA) p  and (AA) m  comprises an amino acid sequence of a polypeptide, or fragment thereof, for presentation of the macrocyclic peptide on an outer surface of a cell or phage particle,
 
b. introducing the nucleic acid molecule into an expression system and expressing the nucleic acid molecule in the expression system, thereby producing the polypeptide; and
 
c. allowing the functional group FG 1 , and whenever present, FG 2 , to react with the side-chain sulfhydryl group (—SH) of the cysteine (Cys) residue(s), thereby producing the macrocyclic peptide.
       

     In some embodiments, Z is an amino acid of structure: 
     
       
         
         
             
             
         
       
     
     wherein FG 1  is a functional group selected from the group consisting of —(CH 2 ) n X, where X is F, Cl, Br, or I and n is an integer number from 1 to 10; —C(O)CH 2 X, where X is F, Cl, Br, or I; —CH(R′)X, where X is F, Cl, Br, or I; —C(O)CH(R′)X, where X is F, Cl, Br, or I; —OCH 2 CH 2 X, where X is F, Cl, Br, or I; —C(O)CH═C═C(R′)(R″); —SO 2 C(R′)═C(R′)(R″); —C(O)C(R′)═C(R′)(R″); —C(R′)═C(R′)C(O)OR′; —C(R′)═C(R′)C(O)N(R′)(R″); —C(R′)═C(R′)—CN; —C(R′)═C(R′)—NO 2 ; —C≡C—C(O)OR′; —C≡C—C(O)N(R′)(R″); unsubstituted or substituted oxirane, unsubstituted or substituted aziridine; 1,2-oxathiolane 2,2-dioxide; 4-fluoro-1,2-oxathiolane 2,2-dioxide; and 4,4-difluoro-1,2-oxathiolane 2,2-dioxide; where each R′ and R″ is independently H, an aliphatic, a substituted aliphatic, an aryl, or a substituted aryl group; 
     wherein Y is a linker group selected from the group consisting of aliphatic, aryl, substituted aliphatic, substituted aryl, heteroatom-containing aliphatic, heteroatom-containing aryl, substituted heteroatom-containing aliphatic, substituted heteroatom-containing aryl, alkoxy, and aryloxy groups. 
     In some embodiments, Z is an amino acid of structure (IV) and Y is a linker group selected from the group consisting of C 1 -C 24  alkyl, C 1 -C 24  substituted alkyl, C 1 -C 24  substituted heteroatom-containing alkyl, C 1 -C 24  substituted heteroatom-containing alkyl, C 2 -C 24  alkenyl, C 2 -C 24  substituted alkenyl, C 2 -C 24  substituted heteroatom-containing alkenyl, C 2 -C 24  substituted heteroatom-containing alkenyl, C 5 -C 24  aryl, C 5 -C 24  substituted aryl, C 5 -C 24  substituted heteroatom-containing aryl, C 5 -C 24  substituted heteroatom-containing aryl, C 1 -C 24  alkoxy, and C 5 -C 24  aryloxy groups. 
     In some embodiments, Y is a linker group of —CH 2 —C 6 H 4 —, —CH 2 —C 6 H 4 —O—, —CH 2 —C 6 H 4 —NH—, —(CH 2 ) 4 —, —(CH 2 ) 4 NH—, —(CH 2 ) 4 NHC(O)—, or —(CH 2 ) 4 NHC(O)O—. 
     In some embodiments, the amino acid Z is 4-(2-bromoethoxy)-phenylalanine, 3-(2-bromoethoxy)-phenylalanine, 4-(2-chloroethoxy)-phenylalanine, 4-(4-bromobutoxy)-phenylalanine, 4-(4-chlorobutoxy)-phenylalanine, 3-(4-bromobutoxy)-phenylalanine, 3-(4-bromobutoxy)-phenylalanine, 3-(2-chloroethoxy)-phenylalanine, 4-(1-bromoethyl)-phenylalanine, 3-(1-bromoethyl)-phenylalanine, 4-(aziridin-1-yl)-phenylalanine, 3-(aziridin-1-yl)-phenylalanine, 4-acrylamido-phenylalanine, 3-acrylamido-phenylalanine, 4-(2-fluoro-acetamido)-phenylalanine, 3-(2-fluoro-acetamido)-phenylalanine, 4-(2-chloro-acetamido)-phenylalanine, 3-(2-chloro-acetamido)-phenylalanine, 4-(2-bromo-acetamido)-phenylalanine, 3-(2-bromo-acetamido)-phenylalanine, 4-(acrylamido)-phenylalanine, 3-(acrylamido)-phenylalanine, 4-(vinylsulfonamido)-phenylalanine, 3-(vinylsulfonamido)-phenylalanine, 3-(2-fluoro-acetyl)-phenylalanine, 4-(2-fluoro-acetyl)-phenylalanine, N ε -((2-bromoethoxy)carbonyl)-lysine, N ε -((2-chloroethoxy)carbonyl)-lysine, N ε -(buta-2,3-dienoyl)-lysine, N ε -acryl-lysine, N ε -crotonyl-lysine, N ε -(2-fluoro-acetyl)-lysine, N ε -(2-chloro-acetyl)-lysine, N ε -(2-bromoacetyl)-lysine, or N ε -vinylsulfonyl-lysine. 
     In some embodiments, Z2 is an amino acid of structure: 
     
       
         
         
             
             
         
       
     
     wherein each of FG 1  and FG 2  is a functional group independently selected from —(CH 2 ) n X, where X is F, Cl, Br, or I and n is an integer number from 1 to 10; —C(O)CH 2 X, where X is F, Cl, Br, or I; —CH(R′)X, where X is F, Cl, Br, or I; —C(O)CH(R′)X, where X is F, Cl, Br, or I; —OCH 2 CH 2 X, where X is F, Cl, Br, or I; —C(O)CH═C═C(R′)(R″); —SO 2 C(R′)═C(R′)(R″); —C(O)C(R′)═C(R′)(R″); —C(R′)═C(R′)C(O)OR′; —C(R′)═C(R′)C(O)N(R′)(R″); —C(R′)═C(R′)—CN; —C(R′)═C(R′)—NO 2 , —C≡C—C(O)OR′; —C≡C—C(O)N(R′)(R″); unsubstituted or substituted oxirane; unsubstituted or substituted aziridine; 1,2-oxathiolane 2,2-dioxide; 4-fluoro-1,2-oxathiolane 2,2-dioxide; and 4,4-difluoro-1,2-oxathiolane 2,2-dioxide, where each R′ and R″ is independently H, an aliphatic, a substituted aliphatic, an aryl, or a substituted aryl group; 
     wherein Y 2 , Y 3 , and L are linker groups selected from aliphatic, aryl, substituted aliphatic, substituted aryl, heteroatom-containing aliphatic, heteroatom-containing aryl, substituted heteroatom-containing aliphatic, substituted heteroatom-containing aryl, alkoxy, and aryloxy groups. 
     In some embodiments, Z2 is an amino acid of structure (VI) and Y 2  is a linker group selected from C 1 -C 24  alkyl, C 1 -C 24  substituted alkyl, C 1 -C 24  substituted heteroatom-containing alkyl, C 1 -C 24  substituted heteroatom-containing alkyl, C 2 -C 24  alkenyl, C 2 -C 24  substituted alkenyl, C 2 -C 24  substituted heteroatom-containing alkenyl, C 2 -C 24  substituted heteroatom-containing alkenyl, C 5 -C 24  aryl, C 5 -C 24  substituted aryl, C 5 -C 24  substituted heteroatom-containing aryl, C 5 -C 24  substituted heteroatom-containing aryl, C 1 -C 24  alkoxy, and C 5 -C 24  aryloxy groups. 
     In some embodiments, Y is a linker group selected from —CH 2 —C 6 H 4 —, —CH 2 —C 6 H 4 —O—, —CH 2 —C 6 H 4 —NH—, —CH 2 —C 6 H 4 —OCH 2 —, —(CH 2 ) 4 NH—, —(CH 2 ) 4 NHC(O)—, —(CH 2 ) 4 NHC(O)O—, —(CH 2 ) 4 NHC(O)OCH 2 —, 
     
       
         
         
             
             
         
       
     
     In some embodiments, the amino acid Z2 is 3,5-bis(2-bromoethoxy)-phenylalanine, 3,5-bis(2-chloroethoxy)-phenylalanine, 3,5-bis(4-bromobutoxy)-phenylalanine, 3,5-bis(4-chlorobutoxy)-phenylalanine, 3,5-bis(1-bromoethyl)-phenylalanine, 3,5-bis(4-acrylamido)-phenylalanine, 3,5-bis(2-chloro-acetamido)-phenylalanine, 3,5-bis(2-bromo-acetamido)-phenylalanine, 3,5-bis(vinylsulfonamido)-phenylalanine, 3,5-bis(aziridin-1-yl)-phenylalanine, 3,5-bis-acrylamido-phenylalanine, 3,5-bis(2-fluoro-acetamido)-phenylalanine, 3,5-bis(2-fluoro-acetyl)-phenylalanine, 4-((1,3-dibromopropan-2-yl)oxy)-phenylalanine, 4-((1,3-dichloropropan-2-yl)oxy)-phenylalanine, N ε -(((1,3-dibromopropan-2-yl)oxy)carbonyl)-lysine, N ε -(((1,3-dichloropropan-2-yl)oxy)carbonyl)-lysine, 4-(2,3-dibromopropoxy)-phenylalanine, 3-(2,3-dibromopropoxy)-phenylalanine, 4-(2,3-dichloropropoxy)-phenylalanine, 3-(2,3-dichloropropoxy)-phenylalanine, N ε -((2,3-dibromopropoxy)carbonyl)-lysine, N ε -((2,3-dichloropropoxy)carbonyl)-lysine, N ε -bis-(acryl)-lysine, N ε -bis-(crotonyl)-lysine, N ε -bis-(2-fluoro-acetyl)-lysine, N ε -bis-(2-chloro-acetyl)-lysine, N ε -bis-(2-bromoacetyl)-lysine, or N ε -bis-(vinylsulfonyl)-lysine. 
     In some embodiments, the codon encoding for Z or Z2 is an amber stop codon TAG, an ochre stop codon TAA, an opal stop codon TGA, or a four base codon. 
     In some embodiments, the expression system comprises an aminoacyl-tRNA synthetase polypeptide or an engineered variant thereof that is at least 90% identical to SEQ ID NO:77, 78, 79, or 80; and a transfer RNA molecule encoded by a polynucleotide that is at least 90% identical to SEQ ID NO:101, 105, 109, 113, or 117. 
     In some embodiments, the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:77 comprises an amino acid substitution at position: X32, X63, X65, X70, X107, X108, X109, X155, X158, X159, X160, X161, X162, X163, X164, X167, or X286 of SEQ ID NO:77. 
     In some embodiments, the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:78 comprises an amino acid substitution at position: X302, X305, X306, X309, X346, X348, X364, X384, X401, X405, or X417 of SEQ ID NO:78. 
     In some embodiments, the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:79 comprises an amino acid substitution at position: X76, X266, X270, X271, X273, X274, X313, X315, at X349 of SEQ ID NO:79. 
     In some embodiments, the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:80 comprises an amino acid substitution at position: X37, X182, X183, X186, or X265 of SEQ. ID NO. 80. 
     In some embodiments, the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:77 comprises at least one of the features of: X32 is Tyr, Leu, Ala, Gly, Thr, His, Glu, Val, or Gln; X65 is Leu, His, Tyr, Val, Ser, Thr, Gly, or Glu; X67 is Ala or Gly; X70 is His, Ala, Cys, or Ser; X107 is Glu, Pro, Asn, or Thr; X108 is Phe, Trp, Ala, Ser, Arg, Gly, Tyr, His, Trp, or Glu; X109 is Gln, Met, Asp, Lys, Glu, Pro, His, Gly, Met, or Leu; X155 is Gln, Glu, or Gly; X158 is Asp, Gly, Glu, Ala, Pro, Thr, Ser, or Val; X159 is Ile, Cys, Pro, Leu, Ser, Trp, His, or Ala; X160 is His or Gln; X161 is Tyr or Gly; X162 is Leu, Arg, Ala, Gln, Gly, Lys, Ser, Glu, Tyr, or His; X163 is Gly or Asp; X164 is Val or Ala; X167 is Ala or Val; X286 is Asp or Arg. 
     In some embodiments, the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:78 comprises at least one of the features of: X302 is Ala or Thr; X305 is Leu or Met; X306 is Tyr, Ala, Met, Ile, Leu, Thr, Gly; X309 is Leu, Ala, Pro, Ser, or Arg; X346 is Asn, Ala, Ser, or Val; X348 is Cys, Ala, Thr, Leu, Lys, Met, or Trp; X364 is Thr or Lys; X384 is Tyr or Phe; X405 is Ile or Arg; X401 is Val or Leu; or X417 is Trp, Thr, or Leu. 
     In some embodiments, the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:79 comprises at least one of the features of: X76 is Asp or Gly; X266 is Leu, Val, or Met; X270 is Leu or Ile; X271 is Tyr, Phe, Leu, Met, or Ala; X274 is Leu, Ala, Met, or Gly; X313 is Cys, Phe, Ala, Val, or Ile; X315 is Met or Phe; or X349 is Tyr, Phe, or Trp. 
     In some embodiments, the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:80 comprises at least one of the features of: X37 is Tyr, Ile, Gly, Val, Leu, Thr, or Ser; X182 is Asp, Gly, Ser, or Thr; X183 is Phe, Met, Tyr, or Ala; X186 is Leu, Ala, Met, or Val; or X265 is Asp or Arg. 
     In some embodiments, the expression system comprises an aminoacyl-tRNA synthetase of SEQ ID NO: 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 209, 210, 211, 212 or 213; and a transfer RNA molecule encoded by a polynucleotide of SEQ ID NO:101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120. 
     In some embodiments, the presentation peptide or fragment thereof comprised within the N-terminal tail polypeptide, (AA) m , comprises at least one polypeptide sequence of a T7 phage protein 10A (SEQ ID NO: 138), T7 phage protein 10B (SEQ ID NO: 139),  E. coli  NlpA (SEQ ID NO:140),  E. coli  OmpC (SEQ ID NO:141),  E. coli  FadL (SEQ ID NO:142),  E. coli  Lpp-OmpA (SEQ ID NO:143),  E. coli  PgsA (SEQ ID NO: 144),  E. coli  EaeA (SEQ ID NO:145),  S. cerevisiae  Aga2p (SEQ ID NO: 146),  S. cerevisiae  Flolp (SEQ ID NO: 147),  S. cerevisiae  Cwplp (SEQ ID NO:217),  S. cerevisiae  Cwp2p (SEQ ID NO:218),  S. cerevisiae  Tiplp (SEQ ID NO:219),  S. cerevisiae  Sedlp (SEQ ID NO:220),  S. cerevisiae  YCR89w (SEQ ID NO:221),  S. cerevisiae  Tirl (SEQ ID NO:222), human NF-κB p50 protein (SEQ ID NO: 148), M13 phage coat protein pIII leader sequence (SEQ ID NO: 149), M13 phage coat protein pVIII leader sequence (SEQ ID NO:150), M13 phage protein pVI (SEQ ID NO:151), M13 phage protein pIII (SEQ ID NO:154), Snap-tag (SEQ ID NO:152), Clip-Tag (SEQ ID NO:153), a barcode sequence, a pelB leader sequence (SEQ ID NO:216), or engineered variants thereof. 
     In some embodiments, the presentation peptide or fragment thereof comprised within the C-terminal tail polypeptide, (AA) p , comprises at least one polypeptide sequence of a M13 phage coat protein pIII (SEQ ID NO:154), M13 phage coat protein pVIII (SEQ ID NO:155), M13 phage coat protein pIX (SEQ ID NO:214), M13 phage coat protein pVII (SEQ ID NO:215), RepA protein (SED ID NO: 156),  S. cerevisiae  Agalp (SEQ ID NO:157), Snap-tag (SEQ ID NO:152), Clip-Tag (SEQ ID NO:153), P2A protein (SED ID NO: 158), a barcode sequence, or engineered variants thereof. 
     In some embodiments, the outer biological surface is selected from a phage surface or a cell surface. 
     In some embodiments, the phage is a bacteriophage. In some embodiments, the bacteriophage is a M13 phage. 
     In some embodiments, the expression system comprises a helper phage. 
     In some embodiments, the host cell is a bacterial, a yeast, a insect, or a mammalian cell. In some embodiments, the yeast cell is  Saccharomyces cerevisiae . In some embodiments, the bacterial cell is  Escherichia coli.    
     In some embodiments, at least one of polypeptides (AA) n , (AA) o , (AA) m , or (AA) p , is fully or partially genetically randomized so that a plurality of macrocyclic peptides is obtained upon a thioether bond-forming reaction between the cysteine (Cys) residue and the side-chain functional group FG 1  in Z or between the cysteine (Cys) residues and the side-chain functional groups FG 1  and FG 2  in Z2, and so that each macrocyclic peptide is displayed on the outer surface of a host organism containing the nucleic acid molecule encoding for the macrocyclic peptide. 
     In some embodiments, the method comprises fully or partially randomizing any of polypeptides (AA) n , (AA) o , (AA) m , or (AA) p , wherein, upon a thioether bond-forming reaction between the cysteine (Cys) residue and the side-chain functional group FG 1  in Z or between the cysteine (Cys) residues and the side-chain functional groups FG 1  and FG 2  in Z2, a macrocyclic peptide display library is produced. 
     In some embodiments, the invention relates to a macrocyclic peptide library display system, comprising at least one artificial nucleic acid molecule encoding for a polypeptide of structure: 
       (AA) m -Z-(AA) n -Cys-(AA) p   (I)
 
       or 
       (AA) m -Cys-(AA) n -Z-(AA) p   (II)
 
       or 
       (AA) m -Cys-(AA) n -Z2-(AA) o -Cys-(AA) p   (V)
         wherein:   i. (AA) m  is an N-terminal amino acid or peptide sequence,   ii. Z is a non-canonical amino acid carrying a side-chain functional group FG 1 , FG 1  being a functional group selected from the group consisting of —(CH 2 ) n X, where X is F, Cl, Br, or I and n is an integer number from 1 to 10; —C(O)CH 2 X, where X is F, Cl, Br, or I; —CH(R′)X, where X is F, Cl, Br, or I; —C(O)CH(R′)X, where X is F, Cl, Br, or I; —OCH 2 CH 2 X, where X is F, Cl, Br, or I; —C(O)CH═C═C(R′)(R″); —SO 2 C(R′)═C(R′)(R″); —C(O)C(R′)═C(R′)(R″); —C(R′)═C(R′)C(O)OR′; —C(R′)═C(R′)C(O)N(R′)(R″); —C(R′)═C(R′)—CN; —C(R′)═C(R′)—NO 2 ; —C≡C—C(O)OR′; —C≡C—C(O)N(R′)(R″); unsubstituted or substituted oxirane; unsubstituted or substituted aziridine; 1,2-oxathiolane 2,2-dioxide; 4-fluoro-1,2-oxathiolane 2,2-dioxide; and 4,4-difluoro-1,2-oxathiolane 2,2-dioxide, where each R′ and R″ is independently H, an aliphatic, a substituted aliphatic, an aryl, or a substituted aryl group.   iii. Z2 is a non-canonical amino acid carrying two side-chain functional groups FG 1  and FG 2 , wherein each of FG 1  and FG 2  is a functional group independently selected from the group consisting of —(CH 2 ) n X, where X is F, Cl, Br, or I and n is an integer number from 1 to 10; —C(O)CH 2 X, where X is F, Cl, Br, or I; —CH(R′)X, where X is F, Cl, Br, or I; —C(O)CH(R′)X, where X is F, Cl, Br, or I; —OCH 2 CH 2 X, where X is F, Cl, Br, or I; —C(O)CH═C═C(R′)(R″); —SO 2 C(R′)═C(R′)(R″); —C(O)C(R′)═C(R′)(R″); —C(R′)═C(R′)C(O)OR′; —C(R′)═C(R′)C(O)N(R′)(R″); —C(R′)═C(R′)—CN; —C(R′)═C(R′)—NO 2 ; —C≡C—C(O)OR′; —C≡C—C(O)N(R′)(R″); unsubstituted or substituted oxirane; unsubstituted or substituted aziridine; 1,2-oxathiolane 2,2-dioxide; 4-fluoro-1,2-oxathiolane 2,2-dioxide; and 4,4-difluoro-1,2-oxathiolane 2,2-dioxide, where each R′ and R″ is independently H, an aliphatic, a substituted aliphatic, an aryl, or a substituted aryl group,   iv. (AA) n  is a target peptide sequence,   v. (AA) o  is a second target peptide sequence,   vi. (AA) p  is a C-terminal amino acid or peptide sequence; and   vii. wherein at least one of (AA) p  and (AA) m  comprises an amino acid sequence of a polypeptide, or fragment thereof, for presentation of the macrocyclic peptide on an outer surface of a cell or phage particle, wherein the functional group FG 1 , and whenever present, FG 2 , react with the side-chain sulfhydryl group (—SH) of the cysteine (Cys) residue(s), thereby producing the macrocyclic peptide.       

     In some embodiments, Z is an amino acid of structure: 
     
       
         
         
             
             
         
       
     
     wherein FG 1  is a functional group selected from the group consisting of —(CH 2 )X, where X is F, Cl, Br, or I and n is an integer number from 1 to 10; —C(O)CH 2 X, where X is F, Cl, Br, or I; —CH(R′)X, where X is F, Cl, Br, or I; —C(O)CH(R′)X, where X is F, Cl, Br, or I; —OCH 2 CH 2 X, where X is F, Cl, Br, or I; —C(O)CH═C═C(R′)(R″); —SO 2 C(R′)═C(R′)(R″); —C(O)C(R′)═C(R′)(R″); —C(R′)═C(R′)C(O)OR′; —C(R′)═C(R′)C(O)N(R′)(R″); —C(R′)═C(R′)—CN; —C(R′)═C(R′)—NO 2 ; —C≡C—C(O)OR′; —C≡C—C(O)N(R′)(R″); unsubstituted or substituted oxirane, unsubstituted or substituted aziridine; 1,2-oxathiolane 2,2-dioxide; 4-fluoro-1,2-oxathiolane 2,2-dioxide; and 4,4-difluoro-1,2-oxathiolane 2,2-dioxide; where each R′ and R″ is independently H, an aliphatic, a substituted aliphatic, an aryl, or a substituted aryl group; 
     wherein Y is a linker group selected from the group consisting of aliphatic, aryl, substituted aliphatic, substituted aryl, heteroatom-containing aliphatic, heteroatom-containing aryl, substituted heteroatom-containing aliphatic, substituted heteroatom-containing aryl, alkoxy, and aryloxy groups. 
     In some embodiments, Z is an amino acid of structure (IV) and Y is a linker group selected from the group consisting of C 1 -C 24  alkyl, C 1 -C 24  substituted alkyl, C 1 -C 24  substituted heteroatom-containing alkyl, C 1 -C 24  substituted heteroatom-containing alkyl, C 2 -C 24  alkenyl, C 2 -C 24  substituted alkenyl, C 2 -C 24  substituted heteroatom-containing alkenyl, C 2 -C 24  substituted heteroatom-containing alkenyl, C 5 -C 24  aryl, C 5 -C 24  substituted aryl, C 5 -C 24  substituted heteroatom-containing aryl, C 5 -C 24  substituted heteroatom-containing aryl, C 1 -C 24  alkoxy, and C 5 -C 24  aryloxy groups. 
     In some embodiments, Y is a linker group of —CH 2 —C 6 H 4 —, —CH 2 —C 6 H 4 —O—, —CH 2 —C 6 H 4 —NH—, —(CH 2 ) 4 —, —(CH 2 ) 4 NH—, —(CH 2 ) 4 NHC(O)—, or —(CH 2 ) 4 NHC(O)O—. 
     In some embodiments, the amino acid Z is 4-(2-bromoethoxy)-phenylalanine, 3-(2-bromoethoxy)-phenylalanine, 4-(2-chloroethoxy)-phenylalanine, 4-(4-bromobutoxy)-phenylalanine, 4-(4-chlorobutoxy)-phenylalanine, 3-(4-bromobutoxy)-phenylalanine, 3-(4-bromobutoxy)-phenylalanine, 3-(2-chloroethoxy)-phenylalanine, 4-(1-bromoethyl)-phenylalanine, 3-(1-bromoethyl)-phenylalanine, 4-(aziridin-1-yl)-phenylalanine, 3-(aziridin-1-yl)-phenylalanine, 4-acrylamido-phenylalanine, 3-acrylamido-phenylalanine, 4-(2-fluoro-acetamido)-phenylalanine, 3-(2-fluoro-acetamido)-phenylalanine, 4-(2-chloro-acetamido)-phenylalanine, 3-(2-chloro-acetamido)-phenylalanine, 4-(2-bromo-acetamido)-phenylalanine, 3-(2-bromo-acetamido)-phenylalanine, 4-(acrylamido)-phenylalanine, 3-(acrylamido)-phenylalanine, 4-(vinylsulfonamido)-phenylalanine, 3-(vinylsulfonamido)-phenylalanine, 3-(2-fluoro-acetyl)-phenylalanine, 4-(2-fluoro-acetyl)-phenylalanine, N ε -((2-bromoethoxy)carbonyl)-lysine, N ε -((2-chloroethoxy)carbonyl)-lysine, N ε -(buta-2,3-dienoyl)-lysine, N ε -acryl-lysine, N ε -crotonyl-lysine, N ε -(2-fluoro-acetyl)-lysine, N ε -(2-chloro-acetyl)-lysine, N ε -(2-bromoacetyl)-lysine, or N ε -vinylsulfonyl-lysine. 
     In some embodiments, Z2 is an amino acid of structure: 
     
       
         
         
             
             
         
       
     
     wherein each of FG 1  and FG 2  is a functional group independently selected from —(CH 2 ) n X, where X is F, Cl, Br, or I and n is an integer number from 1 to 10; —C(O)CH 2 X, where X is F, Cl, Br, or I; —CH(R′)X, where X is F, Cl, Br, or I; —C(O)CH(R′)X, where X is F, Cl, Br, or I; —OCH 2 CH 2 X, where X is F, Cl, Br, or I; —C(O)CH═C═C(R′)(R″); —SO 2 C(R′)═C(R′)(R″); —C(O)C(R′)═C(R′)(R″); —C(R′)═C(R′)C(O)OR′; —C(R′)═C(R′)C(R′)N(R′)(R″); —C(R′)═C(R′)—CN; —C(R′)═C(R′)—NO 2 , —C—C(O)OR′; C≡C—C(O)N(R′)(R″); unsubstituted or substituted oxirane; unsubstituted or substituted aziridine; 1,2-oxathiolane 2,2-dioxide; 4-fluoro-1,2-oxathiolane 2,2-dioxide; and 4,4-difluoro-1,2-oxathiolane 2,2-dioxide, where each R′ and R″ is independently H, an aliphatic, a substituted aliphatic, an aryl, or a substituted aryl group; 
     wherein Y 2 , Y 3 , and L are linker groups selected from aliphatic, aryl, substituted aliphatic, substituted aryl, heteroatom-containing aliphatic, heteroatom-containing aryl, substituted heteroatom-containing aliphatic, substituted heteroatom-containing aryl, alkoxy, and aryloxy groups. 
     In some embodiments, Z2 is an amino acid of structure (VI) and Y 2  is a linker group selected from C 1 -C 24  alkyl, C 1 -C 24  substituted alkyl, C 1 -C 24  substituted heteroatom-containing alkyl, C 1 -C 24  substituted heteroatom-containing alkyl, C 2 -C 24  alkenyl, C 2 -C 24  substituted alkenyl, C 2 -C 24  substituted heteroatom-containing alkenyl, C 2 -C 24  substituted heteroatom-containing alkenyl, C 5 -C 24  aryl, C 5 -C 24  substituted aryl, C 5 -C 24  substituted heteroatom-containing aryl, C 5 -C 24  substituted heteroatom-containing aryl, C 1 -C 24  alkoxy, and C 5 -C 24  aryloxy groups. 
     In some embodiments, Y is a linker group selected from —CH 2 —C 6 H 4 —, —CH 2 —C 6 H 4 —O—, —CH 2 —C 6 H 4 —NH—, —CH 2 —C 6 H 4 —OCH 2 —, —(CH 2 ) 4 NH—, —(CH 2 ) 4 NHC(O)—, —(CH 2 ) 4 NHC(O)O—, —(CH 2 ) 4 NHC(O)OCH 2 —, 
     
       
         
         
             
             
         
       
     
     In some embodiments, the amino acid Z2 is 3,5-bis(2-bromoethoxy)-phenylalanine, 3,5-bis(2-chloroethoxy)-phenylalanine, 3,5-bis(4-bromobutoxy)-phenylalanine, 3,5-bis(4-chlorobutoxy)-phenylalanine, 3,5-bis(1-bromoethyl)-phenylalanine, 3,5-bis(4-acrylamido)-phenylalanine, 3,5-bis(2-chloro-acetamido)-phenylalanine, 3,5-bis(2-bromo-acetamido)-phenylalanine, 3,5-bis(vinylsulfonamido)-phenylalanine, 3,5-bis(aziridin-1-yl)-phenylalanine, 3,5-bis-acrylamido-phenylalanine, 3,5-bis(2-fluoro-acetamido)-phenylalanine, 3,5-bis(2-fluoro-acetyl)-phenylalanine, 4-((1,3-dibromopropan-2-yl)oxy)-phenylalanine, 4-((1,3-dichloropropan-2-yl)oxy)-phenylalanine, N ε -(((1,3-dibromopropan-2-yl)oxy)carbonyl)-lysine, N ε -(((1,3-dichloropropan-2-yl)oxy)carbonyl)-lysine, 4-(2,3-dibromopropoxy)-phenylalanine, 3-(2,3-dibromopropoxy)-phenylalanine, 4-(2,3-dichloropropoxy)-phenylalanine, 3-(2,3-dichloropropoxy)-phenylalanine, N ε -((2,3-dibromopropoxy)carbonyl)-lysine, N ε -((2,3-dichloropropoxy)carbonyl)-lysine, N ε -bis-(acryl)-lysine, N ε -bis-(crotonyl)-lysine, N ε -bis-(2-fluoro-acetyl)-lysine, N ε -bis-(2-chloro-acetyl)-lysine, N ε -bis-(2-bromoacetyl)-lysine, or N ε -bis-(vinylsulfonyl)-lysine. 
     In some embodiments, the codon encoding for Z or Z2 is an amber stop codon TAG, an ochre stop codon TAA, an opal stop codon TGA, or a four base codon. 
     In some embodiments, the expression system comprises an aminoacyl-tRNA synthetase polypeptide or an engineered variant thereof that is at least 90% identical to SEQ ID NO:77, 78, 79, or 80; and a transfer RNA molecule encoded by a polynucleotide that is at least 90% identical to SEQ ID NO:101, 105, 109, 113, or 117. 
     In some embodiments, the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:77 comprises an amino acid substitution at position: X32, X63, X65, X70, X107, X108, X109, X155, X158, X159, X160, X161, X162, X163, X164, X167, or X286 of SEQ ID NO:77. 
     In some embodiments, the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:78 comprises an amino acid substitution at position: X302, X305, X306, X309, X346, X348, X364, X384, X401, X405, or X417 of SEQ ID NO:78. 
     In some embodiments, the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:79 comprises an amino acid substitution at position: X76, X266, X270, X271, X273, X274, X313, X315, at X349 of SEQ ID NO:79. 
     In some embodiments, the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:80 comprises an amino acid substitution at position: X37, X182, X183, X186, or X265 of SEQ. ID NO. 80. 
     In some embodiments, the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:77 comprises at least one of the features of: X32 is Tyr, Leu, Ala, Gly, Thr, His, Glu, Val, or Gln; X65 is Leu, His, Tyr, Val, Ser, Thr, Gly, or Glu; X67 is Ala or Gly; X70 is His, Ala, Cys, or Ser; X107 is Glu, Pro, Asn, or Thr; X108 is Phe, Trp, Ala, Ser, Arg, Gly, Tyr, His, Trp, or Glu; X109 is Gln, Met, Asp, Lys, Glu, Pro, His, Gly, Met, or Leu; X155 is Gln, Glu, or Gly; X158 is Asp, Gly, Glu, Ala, Pro, Thr, Ser, or Val; X159 is Ile, Cys, Pro, Leu, Ser, Trp, His, or Ala; X160 is His or Gln; X161 is Tyr or Gly; X162 is Leu, Arg, Ala, Gin, Gly, Lys, Ser, Glu, Tyr, or His; X163 is Gly or Asp; X164 is Val or Ala; X167 is Ala or Val; X286 is Asp or Arg. 
     In some embodiments, the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:78 comprises at least one of the features of: X302 is Ala or Thr; X305 is Leu or Met; X306 is Tyr, Ala, Met, Ile, Leu, Thr, Gly; X309 is Leu, Ala, Pro, Ser, or Arg; X346 is Asn, Ala, Ser, or Val; X348 is Cys, Ala, Thr, Leu, Lys, Met, or Trp; X364 is Thr or Lys; X384 is Tyr or Phe; X405 is Ile or Arg; X401 is Val or Leu; or X417 is Trp, Thr, or Leu. 
     In some embodiments, the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:79 comprises at least one of the features of: X76 is Asp or Gly; X266 is Leu, Val, or Met; X270 is Leu or Ile; X271 is Tyr, Phe, Leu, Met, or Ala; X274 is Leu, Ala, Met, or Gly; X313 is Cys, Phe, Ala, Val, or Ile; X315 is Met or Phe; or X349 is Tyr, Phe, or Trp. 
     In some embodiments, the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:80 comprises at least one of the features of: X37 is Tyr, Ile, Gly, Val, Leu, Thr, or Ser; X182 is Asp, Gly, Ser, or Thr; X183 is Phe, Met, Tyr, or Ala; X186 is Leu, Ala, Met, or Val; or X265 is Asp or Arg. 
     In some embodiments, the expression system comprises an aminoacyl-tRNA synthetase of SEQ ID NO: 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 209, 210, 211, 212 or 213; and a transfer RNA molecule encoded by a polynucleotide of SEQ ID NO:101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120. 
     In some embodiments, the presentation peptide or fragment thereof comprised within the N-terminal tail polypeptide, (AA) m , comprises at least one polypeptide sequence of a T7 phage protein 10A (SEQ ID NO: 138), T7 phage protein 10B (SEQ ID NO: 139),  E. coli  NlpA (SEQ ID NO:140),  E. coli  OmpC (SEQ ID NO:141),  E. coli  FadL (SEQ ID NO:142),  E. coli  Lpp-OmpA (SEQ ID NO:143),  E. coli  PgsA (SEQ ID NO: 144),  E. coli  EaeA (SEQ ID NO:145),  S. cerevisiae  Aga2p (SEQ ID NO: 146),  S. cerevisiae  Flolp (SEQ ID NO: 147),  S. cerevisiae  Cwplp (SEQ ID NO:217),  S. cerevisiae  Cwp2p (SEQ ID NO:218),  S. cerevisiae  Tiplp (SEQ ID NO:219),  S. cerevisiae  Sedlp (SEQ ID NO:220),  S. cerevisiae  YCR89w (SEQ ID NO:221),  S. cerevisiae  Tirl (SEQ ID NO:222), human NF-κB p50 protein (SEQ ID NO: 148), M13 phage coat protein pIII leader sequence (SEQ ID NO: 149), M13 phage coat protein pVIII leader sequence (SEQ ID NO:150), M13 phage protein pVI (SEQ ID NO:151), M13 phage protein pIII (SEQ ID NO:154), Snap-tag (SEQ ID NO:152), Clip-Tag (SEQ ID NO:153), a barcode sequence, a pelB leader sequence (SEQ ID NO:216), or engineered variants thereof. 
     In some embodiments, the presentation peptide or fragment thereof comprised within the C-terminal tail polypeptide, (AA) p , comprises at least one polypeptide sequence of a M13 phage coat protein pIII (SEQ ID NO:154), M13 phage coat protein pVIII (SEQ ID NO:155), M13 phage coat protein pIX (SEQ ID NO:214), M13 phage coat protein pVII (SEQ ID NO:215), RepA protein (SED ID NO: 156),  S. cerevisiae  Agalp (SEQ ID NO:157), Snap-tag (SEQ ID NO:152), Clip-Tag (SEQ ID NO:153), P2A protein (SED ID NO: 158), a barcode sequence, or engineered variants thereof. 
     In some embodiments, the outer biological surface is selected from a phage surface or a cell surface. 
     In some embodiments, the phage is a bacteriophage. In some embodiments, the bacteriophage is a M13 phage. 
     In some embodiments, the expression system comprises a helper phage. 
     In some embodiments, the host cell is a bacterial, a yeast, a insect, or a mammalian cell. In some embodiments, the yeast cell is  Saccharomyces cerevisiae . In some embodiments, the bacterial cell is  Escherichia coli.    
     In some embodiments, at least one of polypeptides (AA) n , (AA) o , (AA) m , or (AA) p , is fully or partially genetically randomized so that a plurality of macrocyclic peptides is obtained upon a thioether bond-forming reaction between the cysteine (Cys) residue and the side-chain functional group FG 1  in Z or between the cysteine (Cys) residues and the side-chain functional groups FG 1  and FG 2  in Z2, and so that each macrocyclic peptide is displayed on the outer surface of a host organism containing the nucleic acid molecule encoding for the macrocyclic peptide. 
     In some embodiments, the method comprises fully or partially randomizing any of polypeptides (AA) n , (AA) o , (AA) m , or (AA) p , wherein, upon a thioether bond-forming reaction between the cysteine (Cys) residue and the side-chain functional group FG 1  in Z or between the cysteine (Cys) residues and the side-chain functional groups FG 1  and FG 2  in Z2, a macrocyclic peptide display library is produced. 
     In some embodiments, the system comprises at least 10 4  artificial nucleic acid molecules encoding unique macrocyclic peptides. 
     In some embodiments, the invention relates to a method of generating a macrocyclic peptide library display system, comprising: providing at least one artificial nucleic acid molecule encoding for a polypeptide of structure: 
       (AA) m -Z-(AA) n -Cys-(AA) p   (I)
 
       or 
       (AA) m -Cys-(AA) n -Z-(AA) p   (II)
 
       or 
       (AA) m -Cys-(AA) n -Z2-(AA) o -Cys-(AA) p   (V)
         wherein:   i. (AA) m  is an N-terminal amino acid or peptide sequence,   ii. Z is a non-canonical amino acid carrying a side-chain functional group FG 1 , FG 1  being a functional group selected from the group consisting of —(CH 2 ) n X, where X is F, Cl, Br, or I and n is an integer number from 1 to 10; —C(O)CH 2 X, where X is F, Cl, Br, or I; —CH(R′)X, where X is F, Cl, Br, or I; —C(O)CH(R′)X, where X is F, Cl, Br, or I; —OCH 2 CH 2 X, where X is F, Cl, Br, or I; —C(O)CH═C═C(R′)(R″); —SO 2 C(R′)═C(R′)(R″); —C(O)C(R′)═C(R′)(R″); —C(R′)═C(R′)C(O)OR′; —C(R′)═C(R′)C(O)N(R′)(R″); —C(R′)═C(R′)—CN; —C(R′)═C(R′)—NO 2 ; —C≡C—C(O)OR′; —C≡C—C(O)N(R′)(R″); unsubstituted or substituted oxirane; unsubstituted or substituted aziridine; 1,2-oxathiolane 2,2-dioxide; 4-fluoro-1,2-oxathiolane 2,2-dioxide; and 4,4-difluoro-1,2-oxathiolane 2,2-dioxide, where each R′ and R″ is independently H, an aliphatic, a substituted aliphatic, an aryl, or a substituted aryl group.   iii. Z2 is a non-canonical amino acid carrying two side-chain functional groups FG 1  and FG 2 , wherein each of FG 1  and FG 2  is a functional group independently selected from the group consisting of —(CH 2 ) n X, where X is F, Cl, Br, or I and n is an integer number from 1 to 10; —C(O)CH 2 X, where X is F, Cl, Br, or I; —CH(R′)X, where X is F, Cl, Br, or I; —C(O)CH(R′)X, where X is F, Cl, Br, or I; —OCH 2 CH 2 X, where X is F, Cl, Br, or I; —C(O)CH═C═C(R′)(R″); —SO 2 C(R′)═C(R′)(R″); —C(O)C(R′)═C(R′)(R″); —C(R′)═C(R′)C(O)OR′; —C(R′)═C(R′)C(O)N(R′)(R″); —C(R′)═C(R′)—CN; —C(R′)═C(R′)—NO 2 ; —C≡C—C(O)OR′; —C≡C—C(O)N(R′)(R″); unsubstituted or substituted oxirane; unsubstituted or substituted aziridine; 1,2-oxathiolane 2,2-dioxide; 4-fluoro-1,2-oxathiolane 2,2-dioxide; and 4,4-difluoro-1,2-oxathiolane 2,2-dioxide, where each R′ and R″ is independently H, an aliphatic, a substituted aliphatic, an aryl, or a substituted aryl group,   iv. (AA) n  is a target peptide sequence,   v. (AA) o  is a second target peptide sequence,   vi. (AA) p  is a C-terminal amino acid or peptide sequence, and   vii. wherein at least one of (AA) p  and (AA) m  comprises an amino acid sequence of a polypeptide, or fragment thereof, for presentation of the macrocyclic peptide on an outer surface of a cell or phage particle;   fully or partially randomizing at least one of polypeptides (AA) n , (AA) o , (AA) m , and (AA) p , to generate a plurality of unique macrocyclic peptide encoding nucleic acid molecules; introducing the plurality of nucleic acid molecules into a suitable expression system that allows for the incorporation of the non-canonical amino acid Z or Z2 into the polypeptide; expressing the nucleic acid molecule in said expression system, thereby producing the polypeptide; and allowing the functional group FG 1 , and whenever present, FG 2 , to react with the side-chain sulfhydryl group (—SH) of the cysteine (Cys) residue(s), thereby producing a plurality of display macrocyclic peptides anchored on the outer biological surface of a host organism, wherein each host organism contains the nucleic acid molecule encoding for the macrocyclic peptide displayed on its outer biological surface.       

     In some embodiments, Z is an amino acid of structure: 
     
       
         
         
             
             
         
       
     
     wherein FG 1  is a functional group selected from the group consisting of —(CH 2 ) n X, where X is F, Cl, Br, or I and n is an integer number from 1 to 10; —C(O)CH 2 X, where X is F, Cl, Br, or I; —CH(R′)X, where X is F, Cl, Br, or I; —C(O)CH(R′)X, where X is F, Cl, Br, or I; —OCH 2 CH 2 X, where X is F, Cl, Br, or I; —C(O)CH═C═C(R′)(R″); —SO 2 C(R′)═C(R′)(R″); —C(O)C(R′)═C(R′)(R″); —C(R′)═C(R′)C(O)OR′; —C(R′)═C(R′)C(O)N(R′)(R″); —C(R′)═C(R′)—CN; —C(R′)═C(R′)—NO 2 ; —C≡C—C(O)OR′; —C≡C—C(O)N(R′)(R″); unsubstituted or substituted oxirane, unsubstituted or substituted aziridine; 1,2-oxathiolane 2,2-dioxide; 4-fluoro-1,2-oxathiolane 2,2-dioxide; and 4,4-difluoro-1,2-oxathiolane 2,2-dioxide; where each R′ and R″ is independently H, an aliphatic, a substituted aliphatic, an aryl, or a substituted aryl group; 
     wherein Y is a linker group selected from the group consisting of aliphatic, aryl, substituted aliphatic, substituted aryl, heteroatom-containing aliphatic, heteroatom-containing aryl, substituted heteroatom-containing aliphatic, substituted heteroatom-containing aryl, alkoxy, and aryloxy groups. 
     In some embodiments, Z is an amino acid of structure (IV) and Y is a linker group selected from the group consisting of C 1 -C 24  alkyl, C 1 -C 24  substituted alkyl, C 1 -C 24  substituted heteroatom-containing alkyl, C 1 -C 24  substituted heteroatom-containing alkyl, C 2 -C 24  alkenyl, C 2 -C 24  substituted alkenyl, C 2 -C 24  substituted heteroatom-containing alkenyl, C 2 -C 24  substituted heteroatom-containing alkenyl, C 5 -C 24  aryl, C 5 -C 24  substituted aryl, C 5 -C 24  substituted heteroatom-containing aryl, C 5 -C 24  substituted heteroatom-containing aryl, C 1 -C 24  alkoxy, and C 5 -C 24  aryloxy groups. 
     In some embodiments, Y is a linker group of —CH 2 —C 6 H 4 —, —CH 2 —C 6 H 4 —O—, —CH 2 —C 6 H 4 —NH—, —(CH 2 ) 4 —, —(CH 2 ) 4 NH—, —(CH 2 ) 4 NHC(O)—, or —(CH 2 ) 4 NHC(O)O—. 
     In some embodiments, the amino acid Z is 4-(2-bromoethoxy)-phenylalanine, 3-(2-bromoethoxy)-phenylalanine, 4-(2-chloroethoxy)-phenylalanine, 4-(4-bromobutoxy)-phenylalanine, 4-(4-chlorobutoxy)-phenylalanine, 3-(4-bromobutoxy)-phenylalanine, 3-(4-bromobutoxy)-phenylalanine, 3-(2-chloroethoxy)-phenylalanine, 4-(1-bromoethyl)-phenylalanine, 3-(1-bromoethyl)-phenylalanine, 4-(aziridin-1-yl)-phenylalanine, 3-(aziridin-1-yl)-phenylalanine, 4-acrylamido-phenylalanine, 3-acrylamido-phenylalanine, 4-(2-fluoro-acetamido)-phenylalanine, 3-(2-fluoro-acetamido)-phenylalanine, 4-(2-chloro-acetamido)-phenylalanine, 3-(2-chloro-acetamido)-phenylalanine, 4-(2-bromo-acetamido)-phenylalanine, 3-(2-bromo-acetamido)-phenylalanine, 4-(acrylamido)-phenylalanine, 3-(acrylamido)-phenylalanine, 4-(vinylsulfonamido)-phenylalanine, 3-(vinylsulfonamido)-phenylalanine, 3-(2-fluoro-acetyl)-phenylalanine, 4-(2-fluoro-acetyl)-phenylalanine, N ε -((2-bromoethoxy)carbonyl)-lysine, N ε -((2-chloroethoxy)carbonyl)-lysine, N ε -(buta-2,3-dienoyl)-lysine, N ε -acryl-lysine, N ε -crotonyl-lysine, N ε -(2-fluoro-acetyl)-lysine, N ε -(2-chloro-acetyl)-lysine, N ε -(2-bromoacetyl)-lysine, or N ε -vinylsulfonyl-lysine. 
     In some embodiments, Z2 is an amino acid of structure: 
     
       
         
         
             
             
         
       
     
     wherein each of FG 1  and FG 2  is a functional group independently selected from —(CH 2 ) n X, where X is F, Cl, Br, or I and n is an integer number from 1 to 10; —C(O)CH 2 X, where X is F, Cl, Br, or I; —CH(R′)X, where X is F, Cl, Br, or I; —C(O)CH(R′)X, where X is F, Cl, Br, or I; —OCH 2 CH 2 X, where X is F, Cl, Br, or I; —C(O)CH═C═C(R′)(R″); —SO 2 C(R′)═C(R′)(R″); —C(O)C(R′)═C(R′)(R″); —C(R′)═C(R′)C(O)OR′; —C(R′)═C(R′)C(O)N(R′)(R″); —C(R′)═C(R′)—CN; —C(R′)═C(R′)—NO 2 , —C≡C—C(O)OR′; —C≡C—C(O)N(R′)(R″); unsubstituted or substituted oxirane; unsubstituted or substituted aziridine; 1,2-oxathiolane 2,2-dioxide; 4-fluoro-1,2-oxathiolane 2,2-dioxide; and 4,4-difluoro-1,2-oxathiolane 2,2-dioxide, where each R′ and R″ is independently H, an aliphatic, a substituted aliphatic, an aryl, or a substituted aryl group; 
     wherein Y 2 , Y 3 , and L are linker groups selected from aliphatic, aryl, substituted aliphatic, substituted aryl, heteroatom-containing aliphatic, heteroatom-containing aryl, substituted heteroatom-containing aliphatic, substituted heteroatom-containing aryl, alkoxy, and aryloxy groups. 
     In some embodiments, Z2 is an amino acid of structure (VI) and Y 2  is a linker group selected from C 1 -C 24  alkyl, C 1 -C 24  substituted alkyl, C 1 -C 24  substituted heteroatom-containing alkyl, C 1 -C 24  substituted heteroatom-containing alkyl, C 2 -C 24  alkenyl, C 2 -C 24  substituted alkenyl, C 2 -C 24  substituted heteroatom-containing alkenyl, C 2 -C 24  substituted heteroatom-containing alkenyl, C 5 -C 24  aryl, C 5 -C 24  substituted aryl, C 5 -C 24  substituted heteroatom-containing aryl, C 5 -C 24  substituted heteroatom-containing aryl, C 1 -C 24  alkoxy, and C 5 -C 24  aryloxy groups. 
     In some embodiments, Y is a linker group selected from —CH 2 —C 6 H 4 —, —CH 2 —C 6 H 4 —O—, —CH 2 —C 6 H 4 —NH—, —CH 2 —C 6 H 4 —OCH 2 —, —(CH 2 ) 4 NH—, —(CH 2 ) 4 NHC(O)—, —(CH 2 ) 4 NHC(O)O—, —(CH 2 ) 4 NHC(O)OCH 2 —, 
     
       
         
         
             
             
         
       
     
     In some embodiments, the amino acid Z2 is 3,5-bis(2-bromoethoxy)-phenylalanine, 3,5-bis(2-chloroethoxy)-phenylalanine, 3,5-bis(4-bromobutoxy)-phenylalanine, 3,5-bis(4-chlorobutoxy)-phenylalanine, 3,5-bis(1-bromoethyl)-phenylalanine, 3,5-bis(4-acrylamido)-phenylalanine, 3,5-bis(2-chloro-acetamido)-phenylalanine, 3,5-bis(2-bromo-acetamido)-phenylalanine, 3,5-bis(vinylsulfonamido)-phenylalanine, 3,5-bis(aziridin-1-yl)-phenylalanine, 3,5-bis-acrylamido-phenylalanine, 3,5-bis(2-fluoro-acetamido)-phenylalanine, 3,5-bis(2-fluoro-acetyl)-phenylalanine, 4-((1,3-dibromopropan-2-yl)oxy)-phenylalanine, 4-((1,3-dichloropropan-2-yl)oxy)-phenylalanine, N ε -(((1,3-dibromopropan-2-yl)oxy)carbonyl)-lysine, N ε -(((1,3-dichloropropan-2-yl)oxy)carbonyl)-lysine, 4-(2,3-dibromopropoxy)-phenylalanine, 3-(2,3-dibromopropoxy)-phenylalanine, 4-(2,3-dichloropropoxy)-phenylalanine, 3-(2,3-dichloropropoxy)-phenylalanine, N ε -((2,3-dibromopropoxy)carbonyl)-lysine, N ε -((2,3-dichloropropoxy)carbonyl)-lysine, N ε -bis-(acryl)-lysine, N-bis-(crotonyl)-lysine, N ε -bis-(2-fluoro-acetyl)-lysine, N ε -bis-(2-chloro-acetyl)-lysine, N-bis-(2-bromoacetyl)-lysine, or N ε -bis-(vinylsulfonyl)-lysine. 
     In some embodiments, the codon encoding for Z or Z2 is an amber stop codon TAG, an ochre stop codon TAA, an opal stop codon TGA, or a four base codon. 
     In some embodiments, the expression system comprises an aminoacyl-tRNA synthetase polypeptide or an engineered variant thereof that is at least 90% identical to SEQ ID NO:77, 78, 79, or 80; and a transfer RNA molecule encoded by a polynucleotide that is at least 90% identical to SEQ ID NO:101, 105, 109, 113, or 117. 
     In some embodiments, the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:77 comprises an amino acid substitution at position: X32, X63, X65, X70, X107, X108, X109, X155, X158, X159, X160, X161, X162, X163, X164, X167, or X286 of SEQ ID NO:77. 
     In some embodiments, the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:78 comprises an amino acid substitution at position: X302, X305, X306, X309, X346, X348, X364, X384, X401, X405, or X417 of SEQ ID NO:78. 
     In some embodiments, the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:79 comprises an amino acid substitution at position: X76, X266, X270, X271, X273, X274, X313, X315, at X349 of SEQ ID NO:79. 
     In some embodiments, the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:80 comprises an amino acid substitution at position: X37, X182, X183, X186, or X265 of SEQ. ID NO. 80. 
     In some embodiments, the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:77 comprises at least one of the features of: X32 is Tyr, Leu, Ala, Gly, Thr, His, Glu, Val, or Gln; X65 is Leu, His, Tyr, Val, Ser, Thr, Gly, or Glu; X67 is Ala or Gly; X70 is His, Ala, Cys, or Ser; X107 is Glu, Pro, Asn, or Thr; X108 is Phe, Trp, Ala, Ser, Arg, Gly, Tyr, His, Trp, or Glu; X109 is Gln, Met, Asp, Lys, Glu, Pro, His, Gly, Met, or Leu; X155 is Gln, Glu, or Gly; X158 is Asp, Gly, Glu, Ala, Pro, Thr, Ser, or Val; X159 is Ile, Cys, Pro, Leu, Ser, Trp, His, or Ala; X160 is His or Gln; X161 is Tyr or Gly; X162 is Leu, Arg, Ala, Gln, Gly, Lys, Ser, Glu, Tyr, or His; X163 is Gly or Asp; X164 is Val or Ala; X167 is Ala or Val; X286 is Asp or Arg. 
     In some embodiments, the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:78 comprises at least one of the features of: X302 is Ala or Thr; X305 is Leu or Met; X306 is Tyr, Ala, Met, Ile, Leu, Thr, Gly; X309 is Leu, Ala, Pro, Ser, or Arg; X346 is Asn, Ala, Ser, or Val; X348 is Cys, Ala, Thr, Leu, Lys, Met, or Trp; X364 is Thr or Lys; X384 is Tyr or Phe; X405 is Ile or Arg; X401 is Val or Leu; or X417 is Trp, Thr, or Leu. 
     In some embodiments, the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:79 comprises at least one of the features of: X76 is Asp or Gly; X266 is Leu, Val, or Met; X270 is Leu or Ile; X271 is Tyr, Phe, Leu, Met, or Ala; X274 is Leu, Ala, Met, or Gly; X313 is Cys, Phe, Ala, Val, or Ile; X315 is Met or Phe; or X349 is Tyr, Phe, or Trp. 
     In some embodiments, the engineered variant of the aminoacyl-tRNA synthetase polypeptide of SEQ ID NO:80 comprises at least one of the features of: X37 is Tyr, Ile, Gly, Val, Leu, Thr, or Ser; X182 is Asp, Gly, Ser, or Thr; X183 is Phe, Met, Tyr, or Ala; X186 is Leu, Ala, Met, or Val; or X265 is Asp or Arg. 
     In some embodiments, the expression system comprises an aminoacyl-tRNA synthetase of SEQ ID NO: 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 209, 210, 211, 212 or 213; and a transfer RNA molecule encoded by a polynucleotide of SEQ ID NO:101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120. 
     In some embodiments, the presentation peptide or fragment thereof comprised within the N-terminal tail polypeptide, (AA) m , comprises at least one polypeptide sequence of a T7 phage protein 10A (SEQ ID NO: 138), T7 phage protein 10B (SEQ ID NO: 139),  E. coli  NlpA (SEQ ID NO:140),  E. coli  OmpC (SEQ ID NO:141),  E. coli  FadL (SEQ ID NO:142),  E. coli  Lpp-OmpA (SEQ ID NO:143),  E. coli  PgsA (SEQ ID NO: 144),  E. coli  EaeA (SEQ ID NO:145),  S. cerevisiae  Aga2p (SEQ ID NO: 146),  S. cerevisiae  Flolp (SEQ ID NO: 147),  S. cerevisiae  Cwplp (SEQ ID NO:217),  S. cerevisiae  Cwp2p (SEQ ID NO:218),  S. cerevisiae  Tiplp (SEQ ID NO:219),  S. cerevisiae  Sedlp (SEQ ID NO:220),  S. cerevisiae  YCR89w (SEQ ID NO:221),  S. cerevisiae  Tirl (SEQ ID NO:222), human NF-κB p50 protein (SEQ ID NO: 148), M13 phage coat protein pIII leader sequence (SEQ ID NO: 149), M13 phage coat protein pVIII leader sequence (SEQ ID NO:150), M13 phage protein pVI (SEQ ID NO:151), M13 phage protein pIII (SEQ ID NO:154), Snap-tag (SEQ ID NO:152), Clip-Tag (SEQ ID NO:153), a barcode sequence, a pelB leader sequence (SEQ ID NO:216), or engineered variants thereof. 
     In some embodiments, the presentation peptide or fragment thereof comprised within the C-terminal tail polypeptide, (AA) p , comprises at least one polypeptide sequence of a M13 phage coat protein pIII (SEQ ID NO:154), M13 phage coat protein pVIII (SEQ ID NO:155), M13 phage coat protein pIX (SEQ ID NO:214), M13 phage coat protein pVII (SEQ ID NO:215), RepA protein (SED ID NO: 156),  S. cerevisiae  Agalp (SEQ ID NO:157), Snap-tag (SEQ ID NO:152), Clip-Tag (SEQ ID NO:153), P2A protein (SED ID NO: 158), a barcode sequence, or engineered variants thereof. 
     In some embodiments, the outer biological surface is selected from a phage surface or a cell surface. 
     In some embodiments, the phage is a bacteriophage. In some embodiments, the bacteriophage is a M13 phage. 
     In some embodiments, the expression system comprises a helper phage. 
     In some embodiments, the host display organism is a bacterial, a yeast, a insect, or a mammalian cell. In some embodiments, the yeast cell is  Saccharomyces cerevisiae . In some embodiments, the bacterial cell is  Escherichia coli.    
     In some embodiments, at least one of polypeptides (AA) n , (AA) o , (AA) m , or (AA) p , is fully or partially genetically randomized so that a plurality of macrocyclic peptides is obtained upon a thioether bond-forming reaction between the cysteine (Cys) residue and the side-chain functional group FG 1  in Z or between the cysteine (Cys) residues and the side-chain functional groups FG 1  and FG 2  in Z2, and so that each macrocyclic peptide is displayed on the outer surface of a host organism containing the nucleic acid molecule encoding for the macrocyclic peptide. 
     In some embodiments, the method comprises fully or partially randomizing any of polypeptides (AA) n , (AA) o , (AA) m , or (AA) p , wherein, upon a thioether bond-forming reaction between the cysteine (Cys) residue and the side-chain functional group FG 1  in Z or between the cysteine (Cys) residues and the side-chain functional groups FG 1  and FG 2  in Z2, a macrocyclic peptide display library is produced. 
     In some embodiments, the macrocylic display library comprises at least 10 4  artificial nucleic acid molecules encoding unique macrocyclic peptides. 
     In some embodiments, the nucleic acid molecule comprises two pre-defined barcode nucleotide sequences, one located upstream and one located downstream of the nucleic acid sequence encoding for the macrocyclic peptide, wherein this set of barcode nucleotide sequences correspond to the non-canonical amino acid Z or Z2 incorporated into the display macrocyclic peptides. In some embodiments, the barcode nucleotide sequences are used to propagate and/or identify members of the display macrocyclic peptide library containing a specific non-canonical amino acid Z or Z2. In some embodiments, the barcode sequence is located within a non-coding region of the vector comprising the gene for the expression of the macrocyclic peptide. In some embodiments, the barcode sequence is located within a coding region (i.e., open reading frame) of the vector comprising the gene for the expression of the macrocyclic peptide. In some embodiments, different barcode sequences located within a coding region encode for different amino acid sequences. In some embodiments, different barcode sequences located within a coding region encode for the same amino acid sequence. 
     In some embodiments, the display library is a library of polycyclic peptides generated by fusing together two or more copies of the portion of the nucleic acid molecule encoding for -Z-(AA) n -Cys-, -Cys-(AA) n -Z-, and/or -Cys-(AA) n -Z2-(AA). 
     In some embodiments, the invention relates to a method for obtaining a macrocyclic peptide having a desired property, this method comprising,
         i. contacting a macrocyclic peptide library display system with a target for screening; and   ii. selecting the member(s) of the display macrocyclic peptide library that has/have the desired property.       

     In some embodiments, the display macrocyclic peptide library is anchored on the outer biological surface of a phage. In some embodiments, the phage is a M13 phage. 
     In some embodiments, the display macrocyclic peptide library is anchored on the outer biological surface of a cell. In some embodiments, the cell is  Saccharomyces cerevisiae  or  Escherichia coli.    
     In some embodiments, the selection procedure is an affinity selection procedure. In some embodiments, the selection procedure is a sorting procedure using fluorescence-activated cell sorting. 
    
    
     
       4. BRIEF DESCRIPTION OF THE DRAWINGS 
       Embodiments are described herein with reference to the accompanying drawings, in which similar reference characters denote similar elements throughout the several views. It is to be understood that in some instances, various aspects of the embodiments may be shown exaggerated or enlarged to facilitate an understanding of the invention. 
         FIGS. 1A-B . Schematic representation of two general methods for making macrocyclic peptides from ribosomally produced precursor polypeptides of general formula (I) (panel A) or general formula (II) (panel B). W corresponds to the linker group resulting from the bond-forming reaction between the functional group FG 1  and the cysteine residue. 
         FIGS. 2A-B . Schematic representation of a variation of the general methods of  FIGS. 1A-B , wherein an intein protein is comprised within the C-terminal tail of a precursor polypeptide of general formula (I) (panel A) or of general formula (II) (panel B). W corresponds to the linker group resulting from the bond-forming reaction between the functional group FG 1  and the cysteine residue. 
         FIGS. 3A-B . Schematic representation of another variation of the general methods of  FIGS. 1A-B , wherein an intein protein is comprised within the N-terminal tail of a precursor polypeptide of general formula (I) (panel A) or of general formula (II) (panel B). W corresponds to the linker group resulting from the bond-forming reaction between the functional group FG 1  and the cysteine residue. 
         FIGS. 4A-B . Schematic representation of another variation of the general methods of  FIGS. 1A-B , wherein the C- and N-domains of a split intein is comprised within the N-terminal tail and C-terminal tail, respectively, of a precursor polypeptide of general formula (I) (panel A) or of general formula (II) (panel B). W corresponds to the linker group resulting from the bond-forming reaction between the functional group FG 1  and the cysteine residue. 
         FIG. 5 . Synthetic routes for the synthesis of the cysteine-reactive unnatural amino acids p-2beF, 2becK, and p-1beF. 
         FIG. 6 . Synthetic routes for the synthesis of the cysteine-reactive unnatural amino acids 2cecK, bdnK, and OdbpY. 
         FIGS. 7A-B . Fluorescence-based assay for screening of AARS/tRNA pairs. The graphs indicate the relative efficiency of incorporation of the unnatural amino acid p-2beF (A) and 2becK (B) into the reporter protein YFP(TAG) by different amber stop codon suppressor AARS/tRNA pairs. 
         FIG. 8 . Strategy for ribosomal synthesis of thioether-bridged macrocyclic peptides via p-2beF-mediated cyclization. The linear precursor polypeptide comprises an N-terminal tail (N-term), the unnatural amino acid p-2beF, a variable target sequence containing the reactive cysteine (black circle) and GyrA intein. Depending on the nature of the ‘I-1’ residue, the macrocyclic peptide can be released in vitro via thiol-induced Intein splicing (path A) or directly in vivo (path B). 
         FIGS. 9A-B . Dependence of macrocyclization efficiency on relative position of the Cys residue with respect to the unnatural amino acid ‘Z’. (A) Percentage of cyclization for the different p-2beF-containing constructs as determined by LCMS after in vitro splicing of the GyrA intein. (B) (Percentage of cyclization for the different 2becK- and 2cecK-containing constructs as determined by LCMS after in vitro splicing of the GyrA intein. In each case, proteins were isolated after expression in  E. coli  for 12 hours at 27° C. (see Examples for details). 
         FIGS. 10-15 . Representative examples of macrocyclic peptides produced from p-2beF-containing precursor polypeptides according to the methods disclosed herein. Each figure describes the sequence of the precursor polypeptide, the chemical structure of the macrocyclic peptide product, and the MS/MS spectrum and LC-MS extracted-ion chromatogram (inset) of the macrocyclic peptide. 
         FIG. 16 . Representative MS/MS spectrum corresponding to the macrocyclic peptide obtained from construct 12mer-Z6C(2-beF). The assignment of the a and b fragments is indicated. 
         FIGS. 17 a - d   . Deconvoluted LC-MS mass spectra of proteins isolated after benzyl mercaptan-induced splicing of purified construct (a) 12mer-Z1C, (b) 12mer-Z4C, (c) 10mer-C6Z, and (d) 10mer-C8Z. 
         FIGS. 18-24 . Representative examples of macrocyclic peptides produced from 2becK-, 2cecK, p-1beF-, and bdnK-containing precursor polypeptides according to the methods disclosed herein. Each figure describes the sequence of the precursor polypeptide, the chemical structure of the macrocyclic peptide product, and the MS/MS spectrum and LC-MS extracted-ion chromatogram (inset) of the macrocyclic peptide. 
         FIGS. 25-27 . Macrocyclic peptides isolated via streptavidin-affinity chromatography from bacterial lysate. Each figure describes the sequence of the precursor polypeptide, the chemical structure of the macrocyclic peptide product, and the MS/MS spectrum and LC-MS extracted-ion chromatogram (inset) of the macrocyclic peptide. 
         FIGS. 28-32 . Bicyclic peptides isolated via streptavidin-affinity chromatography from bacterial lysate. Each figure describes the sequence of the precursor polypeptide, the chemical structure of the bicyclic peptide product, and the MS/MS spectrum and LC-MS extracted-ion chromatogram (inset) of the bicyclic peptide. 
         FIGS. 33 a - d   . Deconvoluted LC-MS mass spectra of proteins isolated from the cell lysate using Ni-NTA beads: (a) Strep1-Z5C(p-2beF) construct, (b) Strep2-Z7C(p-2beF) construct; and using chitin beads: (c) cStrep3(C)-Z3C(p-2beF) construct, (d) cStrep3(S)-Z3C(p-2beF) construct 
         FIGS. 34-35 . Representative examples of macrocyclic peptides produced from p-2beF-containing precursor polypeptides of general formula (II). Each figure describes the sequence of the precursor polypeptide, the chemical structure of the macrocyclic peptide product, and the MS/MS spectrum and LC-MS extracted-ion chromatogram (inset) of the macrocyclic peptide. 
         FIG. 36 . Representative example of a polycyclic peptide produced from a precursor polypeptide containing two Cys/Z pairs, where Z is p-2beF. The figure describes the sequence of the precursor polypeptide, the chemical structure of the polycyclic peptide product, and the MS/MS spectrum and LC-MS extracted-ion chromatogram (inset) of the macrocyclic peptide. 
         FIGS. 37A-B . Schematic representation of the general methods for making polycyclic peptides from ribosomally produced precursor polypeptides of general formula (V) containing a bifunctional cysteine-reactive amino acid (Z2) of general formula (VI) (panel A) or (VII) (panel B). W 1  and W 2  correspond to the linker groups resulting from the bond-forming reaction between the cysteine residues and functional group FG 1  and FG 2 , respectively. 
         FIG. 38 . Representative example of a polycyclic peptide produced from a precursor polypeptide containing two cysteines and a bifunctional cysteine-reactive amino acid (ObdpY). The figure describes the sequence of the precursor polypeptide, the chemical structure of the polycyclic peptide product, and the MS/MS spectrum and LC-MS extracted-ion chromatogram (inset) of the macrocyclic peptide. 
         FIGS. 39A-B . Competitive binding assay for measuring streptavidin binding affinity of HPQ-containing cyclic and bicyclic peptides. (A) Schematic illustration of the in-solution inhibition assay. IC 50  values are obtained from the dose-dependent decrease in horseradish peroxidase (HRP) activity at increasing concentration of the cyclic or bicyclic streptavidin-binding peptide. (B) Inhibition curve. 
         FIG. 40 . Synthetic routes for the synthesis of the cysteine-reactive unnatural amino acids p-VsaF and pAaF. 
         FIG. 41 . Synthetic routes for the synthesis of the cysteine-reactive unnatural amino acids p-CaaF and O4bbeY. 
         FIG. 42 . Schematic overview of three general macrocyclic peptide display systems according to the methods disclosed herein. Specifically, the figure illustrates a representative example of the display of macrocyclic peptides from polypeptides of general formula (I) in phage display, bacterial display, plasmid display, and yeast display format. W corresponds to the linker group resulting from the bond-forming reaction between the functional group FG 1  and the cysteine residue. 
         FIG. 43  depicts a schematic overview of the macrocyclic peptide phage display system (MOrPH-PhD). A macrocycle precursor sequence (MPS) is fused to the N-terminal end of the M13 pII protein encoded by a pSEX-based phagemid vector. Spontaneous, post-translational peptide cyclization is mediated by the cysteine-reactive O2beY introduced via amber stop codon suppression with an orthogonal AARS/tRNA pair. Phage production in the presence of a helper phage (M13K07ΔpIII) results in M13 phage particles displaying the thioether-bridged macrocycles on the pIII coat protein. The phage-displayed peptide macrocycle library is panned and enriched against an immobilized target, followed by hit deconvolution via DNA sequencing of the MPS encoding gene contained in the bacteriophage. 
         FIG. 44A - FIG. 44C  depict representative results demonstrating a display of O2beY-containing peptide on M13 phages.  FIG. 44A  depicts an incorporation of cysteine-reactive O2beY and cysteine-unreactive OpgY into a linear nonapeptide (NB9)N-terminally fused to the M13 phage coat protein pIII.  FIG. 44B  depicts a plaque forming units (p.f.u.) generated in the absence and presence of either non-canonical amino acid from  E. coli  cells expressing the polyspecific O2beY-RS synthetase, as determined by the phage titer assay.  FIG. 44C  depicts a selective recovery of O2beY-displaying phages over OpgY-displaying ones using streptavidin-coated beads after phage exposure to biotin-conjugated cysteine reagent (Biot-Cys). 
         FIG. 45A - FIG. 45C  depict the affinity selection of streptavidin binding peptide macrocycles using methods disclosed herein.  FIG. 42A  depicts libraries of semi-randomized O2beY-linked peptide macrocycles (X=NNK codon) displayed on phages and hit sequences identified by deep sequencing (relative abundance=n/54,000 sequences) after panning of the macrocyclic peptide phage display library against streptavidin-coated magnetic beads. K D  values correspond to the corresponding FLAG-macrocycle-CBD constructs in purified form.  FIG. 45B  depicts binding curves for selected peptide macrocycle hits as determined using a direct binding assay with plate-immobilized streptavidin and HRP-conjugated anti-FLAG antibody for detection of the bound peptide. CBD alone shows no detectable binding to streptavidin.  FIG. 45D  depicts phage enrichment over the four rounds of affinity selection and amplification as determined via the phage titer assay. 
         FIG. 46A - FIG. 46C  depict the selection of macrocyclic peptide inhibitors of the Keap1/Nrf2 interaction using methods disclosed herein.  FIG. 46A  depicts libraries of semi-randomized O2beY-linked peptide macrocycles (X=NNK codon) displayed on phages and hit sequences identified by deep sequencing (relative abundance=n/56,000 sequences) after library panning against immobilized Keap1 Kelch domain (KKD). K D  values correspond to the FLAG-tagged CBD-fused peptide macrocycles.  FIG. 46B  depicts binding curves for selected macrocyclic peptides as determined using a direct binding assay with plate-immobilized KKD and HRP-conjugated anti-FLAG antibody for detection of the bound peptide.  FIG. 46C  depicts results from a competition assay in which binding of FLAG-KKD-m1 to immobilized KKD is inhibited by a Nrf2-derived peptide (IC 50 =2.8±0.1 nM). 
         FIG. 47A - FIG. 47B  depicts the selection of Keap1 binding macrocyclic peptides from a naïve macrocyclic peptide phage display library.  FIG. 47A  depicts the naïve library of i/i+6-linked macrocyclic peptides (X=NNK; Y*=O2beY) and the sequences and binding affinity for selected macrocyclic Keap1-binding peptides isolated after panning the macrocyclic peptide display library against immobilized Keap1 Kelch Domain (K D D).  FIG. 47B  depicts the binding curves binding curves for selected macrocyclic peptides as determined using a direct binding assay with plate-immobilized KKD and HRP-conjugated anti-FLAG antibody for detection of the bound peptide. 
         FIG. 48A - FIG. 48C . MALDI-TOF MS spectra for purified streptavidin-( FIG. 48A ) and Keap1-( FIG. 48B-C ) binding macrocyclic peptides isolated using the macrocyclic peptide phage display system. Calc.=calculated; Obs=observed. 
         FIG. 49A - FIG. 49B  illustrates the identification of a suitable orthogonal AARS/tRNA pairs for the incorporation of the non-canonical amino acids pCaaF, pVsaF, pAaF, and O4bbY described in  FIGS. 40 and 41  using a fluorescence-based assay.  FIG. 49A  depicts the relative efficiency of incorporation of the unnatural amino acid pCaaF, pVsaF, and pAaF into the reporter protein YFP(TAG) via amber codon suppression using the Mj-TyrRS42/Mj-tRNA Tyr CUA pair (left panel) or the Mj-VsF-RS/Mj-tRNA Tyr   CUA  pair (right panel).  FIG. 49A  depicts the relative efficiency of incorporation of the unnatural amino acid O4bbY into the reporter protein YFP(TAG) using different amber stop codon suppressor AARS/tRNA pairs. These results indicated that both O2beY-RS and pBzF2-RS are suitable for genetic incorporation of this unnatural amino acid into a recombinant protein. 
         FIG. 50A - FIG. 50B  illustrates the generation of macrocyclic peptides using the non-natural amino acids pCaaF, pVsaF, pAaF, and O4bbY according to the methods disclosed herein.  FIG. 50A  depicts the macrocyclization efficiency of a series of precursor polypeptides with varying interdistance (i/i+1, i/i+2, . . . , i/i+20) and orientation of the Cys/Z pair using O4bbY (top panel) and pCaaF (bottom panel) as the Z residue. The target sequences in the precursor polypeptides are described in Table 1.  FIG. 50A  depicts the macrocyclization efficiency of the same series of precursor polypeptides using pAaF (top panel) and pVsaF (bottom panel) as the Z residue. Percentage of cyclization was determined by LCMS after proteolytic cleavage (Factor Xa) of a C-terminal CBD tag. In each case, proteins were isolated after expression in  E. coli  for 12 hours at 27° C. and purified using Ni-affinity chromatography using a His tag fused to the C-terminus of the CBD tag.  FIG. 50A - FIG. 50B  illustrates the generation of macrocyclic peptides using the non-natural amino acids pCaaF, pVsaF, pAaF, and O4bbY according to the methods 
         FIG. 51  illustrates the selective amplification of phagemid vectors containing four different sets of barcodes (BC1-BC4) using barcode-specific primers. The figure illustrates agarose gel analysis of PCR products obtained from 4 different set of barcode-specific primers in the presence of the different barcoded phagemids (BC1, BC2, BC3, BC4) as the template. The target PCR product is 1,390 bp long. 
         FIG. 52A - FIG. 52B  illustrates the propagation of a naïve macrocyclic peptide phage display library (-Z-(Xaa) 5 -Cys-) generated using the Z amino acids O4bbY, pAaF, and pCaaF over two rounds of infection/amplification in  E. coli  TOP10F′ cells.  FIG. 52A  depicts the relative amount of cysteine-containing sequences within the randomized region of the displayed peptide after first and second round of panning in the absence (R1, R2) and in the presence (TCEP R1, TCEP R2) of phage treatment with TCEP prior to next step of infection/amplication in  E. coli .  FIG. 52A  depicts top ten sequences of the TCEP treated libraries after the second panning round. *=amber stop codon. The libraries were barcoded with a non-silent barcode within the linker region preceding the randomized sequence (Ala=O4bbY; Gly=pAaF; Thr=pCaaF) enabling multiplexed deconvolution of the library panning results. 
     
    
    
     5. DETAILED DESCRIPTION 
     For clarity of disclosure, and not by way of limitation, the detailed description is divided into the subsections set forth below. 
     5.1 Definitions 
     Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. 
     The singular forms “a,” “an,” and “the” used herein include plural referents unless the content clearly dictates otherwise. 
     The term “plurality” refers to two or more referents unless the content clearly dictates otherwise. The term “at least one” refers to one or more referents. 
     The term “functional group” as used herein refers to a contiguous group of atoms that, together, may undergo a chemical reaction under certain reaction conditions. Examples of functional groups are, among many others, —OH, —NH 2 , —SH, —(C═O)—, —N 3 , —C≡CH. 
     The term “aliphatic” or “aliphatic group” as used herein means a straight or branched C 1-15  hydrocarbon chain that is completely saturated or that contains at least one unit of unsaturation, or a monocyclic C 3-8  hydrocarbon, or bicyclic C 8-12  hydrocarbon that is completely saturated or that contains at least one unit of unsaturation, but which is not aromatic (also referred to herein as “cycloalkyl”). For example, suitable aliphatic groups include, but are not limited to, linear or branched alkyl, alkenyl, alkynyl groups or hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkynyl)alkyl. The alkyl, alkenyl, or alkynyl group may be linear, branched, or cyclic and may contain up to 15, up to 8, or up to 5 carbon atoms. Alkyl groups include, but are not limited to, methyl, ethyl, propyl, cyclopropyl, butyl, cyclobutyl, pentyl, and cyclopentyl groups. Alkenyl groups include, but are not limited to, propenyl, butenyl, and pentenyl groups. Alkynyl groups include, but are not limited to, propynyl, butynyl, and pentynyl groups. 
     The term “aryl” and “aryl group” as used herein refers to an aromatic substituent containing a single aromatic or multiple aromatic rings that are fused together, directly linked, or indirectly linked (such as linked through a methylene or an ethylene moiety). An aryl group may contain from 5 to 24 carbon atoms, 5 to 18 carbon atoms, or 5 to 14 carbon atoms. 
     The terms “heteroatom” means nitrogen, oxygen, or sulphur, and includes, but is not limited to, any oxidized forms of nitrogen and sulfur, and the quaternized form of any basic nitrogen. Heteroatom further includes, but is not limited to, Se, Si, or P. 
     The term “heteroaryl” as used herein refer to an aryl group in which at least one carbon atom is replaced with a heteroatom. In various embodiments, a heteroaryl group is a 5- to 18-membered, a 5- to 14-membered, or a 5- to 10-membered aromatic ring system containing at least one heteroatom selected from the group consisting of oxygen, sulphur, and nitrogen atoms. Heteroaryl groups include, but are not limited to, pyridyl, pyrrolyl, furyl, thienyl, indolyl, isoindolyl, indolizinyl, imidazolyl, pyridonyl, pyrimidyl, pyrazinyl, oxazolyl, thiazolyl, purinyl, quinolinyl, isoquinolinyl, benzofuranyl, and benzoxazolyl groups. 
     A heterocyclic group may be any monocyclic or polycyclic ring system which contains at least one heteroatom and may be unsaturated or partially or fully saturated. The term “heterocyclic” thus includes, but is not limited to, heteroaryl groups as defined above as well as non-aromatic heterocyclic groups. In various embodiments, a heterocyclic group is a 3- to 18-membered, a 3- to 14-membered, or a 3- to 10-membered, ring system containing at least one heteroatom selected from the group consisting of oxygen, sulphur, and nitrogen atoms. Heterocyclic groups include, but are not limited to, the specific heteroaryl groups listed above as well as pyranyl, piperidinyl, pyrrolidinyl, dioaxanyl, piperazinyl, morpholinyl, thiomorpholinyl, morpholinosulfonyl, tetrahydroisoquinolinyl, and tetrahydrofuranyl groups. 
     A halogen atom may be a fluorine, chlorine, bromine, or iodine atom. 
     By “optionally substituted”, it is intended that in the any of the chemical groups listed above (e.g., alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl, heteroaryl, heterocyclic, triazolyl groups), at least one of the hydrogen atoms is optionally replaced with an atom or chemical group other than hydrogen. Specific examples of such substituents include, but are not limited to, halogen atoms, hydroxyl (—OH), sulfhydryl (—SH), substituted sulfhydryl, carbonyl (—CO—), carboxy (—COOH), amino (—NH 2 ), nitro (—NO 2 ), sulfo (—SO 2 —OH), cyano (—C≡N), thiocyanato (—S—C≡N), phosphono (—P(O)OH 2 ), alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl, heteroaryl, heterocyclic, alkylthiol, alkyloxy, alkylamino, arylthiol, aryloxy, or arylamino groups. Where “optionally substituted” modifies a series of groups separated by commas (e.g., “optionally substituted A, B, or C”; or “A, B, or C optionally substituted with”), it is intended that each of the groups (e.g., A, B, or C) is optionally substituted. 
     The term “heteroatom-containing aliphatic” as used herein refer to an aliphatic moiety where at least one carbon atom is replaced with a heteroatom, e.g., oxygen, nitrogen, sulphur, selenium, phosphorus, or silicon, and typically oxygen, nitrogen, or sulphur. 
     The terms “alkyl” and “alkyl group” as used herein refer to a linear, branched, or cyclic saturated hydrocarbon typically containing 1 to 24 carbon atoms, or 1 to 12 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl and the like. 
     The term “heteroatom-containing alkyl” as used herein refers to an alkyl moiety where at least one carbon atom is replaced with a heteroatom, e.g., oxygen, nitrogen, sulphur, phosphorus, or silicon, and typically oxygen, nitrogen, or sulphur. 
     The terms “alkenyl” and “alkenyl group” as used herein refer to a linear, branched, or cyclic hydrocarbon group of 2 to 24 carbon atoms, or of 2 to 12 carbon atoms, containing at least one double bond, such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, and the like. 
     The term “heteroatom-containing alkenyl” as used herein refer to an alkenyl moiety where at least one carbon atom is replaced with a heteroatom. 
     The terms “alkynyl” and “alkynyl group” as used herein refer to a linear, branched, or cyclic hydrocarbon group of 2 to 24 carbon atoms, or of 2 to 12 carbon atoms, containing at least one triple bond, such as ethynyl, n-propynyl, and the like. 
     The term “heteroatom-containing alkynyl” as used herein refer to an alkynyl moiety where at least one carbon atom is replaced with a heteroatom. 
     The term “heteroatom-containing aryl” as used herein refer to an aryl moiety where at least one carbon atom is replaced with a heteroatom. 
     The terms “alkoxy” and “alkoxy group” as used herein refer to an aliphatic group or a heteroatom-containing aliphatic group bound through a single, terminal ether linkage. In various embodiments, aryl alkoxy groups contain 1 to 24 carbon atoms, or contain 1 to 14 carbon atoms. 
     The terms “aryloxy” and “aryloxy group” as used herein refer to an aryl group or a heteroatom-containing aryl group bound through a single, terminal ether linkage. In various embodiments, aryloxy groups contain 5 to 24 carbon atoms, or contain 5 to 14 carbon atoms. 
     The term “substituent” refers to a contiguous group of atoms. Examples of “substituents” include, but are not limited to: alkoxy, aryloxy, alkyl, heteroatom-containing alkyl, alkenyl, heteroatom-containing alkenyl, alkynyl, heteroatom-containing alkynyl, aryl, heteroatom-containing aryl, alkoxy, heteroatom-containing alkoxy, aryloxy, heteroatom-containing aryloxy, halo, hydroxyl (—OH), sulfhydryl (—SH), substituted sulfhydryl, carbonyl (—CO—), thiocarbonyl, (—CS—), carboxy (—COOH), amino (—NH 2 ), substituted amino, nitro (—NO 2 ), nitroso (—NO), sulfo (—SO 2 —OH), cyano (—C≡N), cyanato (—O—C≡N), thiocyanato (—S—CN), formyl (—CO—H), thioformyl (—CS—H), phosphono (—P(O)OH 2 ), substituted phosphono, and phospho (—PO 2 ). 
     The term “contact” as used herein with reference to interactions of chemical units indicates that the chemical units are at a distance that allows short range non-covalent interactions (such as Van der Waals forces, hydrogen bonding, hydrophobic interactions, electrostatic interactions, dipole-dipole interactions) to dominate the interaction of the chemical units. For example, when a protein is ‘contacted’ with a chemical species, the protein is allowed to interact with the chemical species so that a reaction between the protein and the chemical species can occur. 
     The term “bioorthogonal” as used herein with reference to a reaction, reagent, or functional group, indicates that such reaction, reagent, or functional group does not exhibit significant or detectable reactivity towards biological molecules such as those present in a bacterial, yeast or mammalian cell. The biological molecules can be, e.g., proteins, nucleic acids, fatty acids, or cellular metabolites. 
     In general, the term “mutant” or “variant” as used herein with reference to a molecule such as polynucleotide or polypeptide, indicates that such molecule has been mutated from the molecule as it exists in nature. In particular, the term “mutate” and “mutation” as used herein indicates any modification of a nucleic acid and/or polypeptide which results in an altered nucleic acid or polypeptide. Mutations include, but are not limited to, any process or mechanism resulting in a mutant protein, enzyme, polynucleotide, or gene. A mutation can occur in a polynucleotide or gene sequence, by point mutations, deletions, or insertions of single or multiple nucleotide residues. A mutation in a polynucleotide includes, but is not limited to, mutations arising within a protein-encoding region of a gene as well as mutations in regions outside of a protein-encoding sequence, such as, but not limited to, regulatory or promoter sequences. A mutation in a coding polynucleotide such as a gene can be “silent”, i.e., not reflected in an amino acid alteration upon expression, leading to a “sequence-conservative” variant of the gene. A mutation in a polypeptide includes, but is not limited to, mutation in the polypeptide sequence and mutation resulting in a modified amino acid. Non-limiting examples of a modified amino acid include, but are not limited to, a glycosylated amino acid, a sulfated amino acid, a prenylated (e.g., farnesylated, geranylgeranylated) amino acid, an acetylated amino acid, an acylated amino acid, a PEGylated amino acid, a biotinylated amino acid, a carboxylated amino acid, a phosphorylated amino acid, and the like. 
     The term “engineer” refers to any manipulation of a molecule that result in a detectable change in the molecule, wherein the manipulation includes, but is not limited to, inserting a polynucleotide and/or polypeptide heterologous to the cell and mutating a polynucleotide and/or polypeptide native to the cell. 
     The term “nucleic acid molecule” as used herein refers to any chain of at least two nucleotides bonded in sequence. For example, a nucleic acid molecule can be a DNA or a RNA. 
     The term “peptide”, “polypeptide”, and “protein” as used herein refers to any chain of at least two amino acids bonded in sequence, regardless of length or post-translational modification. 
     The term “peptide-containing molecule” as used herein refers to a molecule that contains at least two amino acids. 
     The term “non-natural” and “unnatural” as used herein means being directly or indirectly made or caused to be made through human action. Thus, a “non-natural amino acid” is an amino acid that has been produced through human manipulation and does not occur in nature. The term “non-canonical amino acid” is equivalent in meaning to the terms “non-natural amino acid” or “unnatural amino acid”. 
     The term “cyclic” and “macrocyclic” as used herein means having constituent atoms forming a ring. Thus, a “macrocyclic peptide” is a peptide molecule that contains at least one ring formed by atoms comprised in the molecule. As such, the term “macrocyclic peptide” comprises peptides that contain at least two rings separated from each other via a polypeptide sequence (also referred to herein as “polycyclic peptides”) and peptides that contain at least two rings fused to each other (also referred to herein as “polycyclic peptides”). The term “macrocyclic peptide” also comprises peptides that contain two rings fused to each other (referred to herein also as “bicyclic peptides”). 
     The terms “cyclization” or “macrocyclization” as used herein refer to a process or reaction whereby a cyclic molecule is formed or is made to be formed. 
     The term “peptidic backbone” as used herein refers to a sequence of atoms corresponding to the main backbone of a natural protein. 
     The term “precursor polypeptide” or “polypeptide precursor” as used herein refers to a polypeptide that is capable of undergoing macrocyclization according to the methods disclosed herein. 
     The term “ribosomal polypeptide”, “ribosomally produced polypeptide” or “ribosomally derived polypeptide” as used herein refers to a polypeptide that is produced by action of a ribosome, and specifically, by the ribosomal translation of a messenger RNA encoding for such polypeptide. The ribosome can be a naturally occurring ribosome, e.g. a ribosome derived from an archea, procaryotic or eukaryotic organism, or an engineered (i.e., non-naturally occurring, artificial or synthetic) variant of a naturally occurring ribosome. 
     The term “intein” and “intein domain” as used herein refers to a naturally occurring or artificially constructed polypeptide sequence embedded within a precursor protein that can catalyze a splicing reaction during post-translational processing of the protein. The NEB Intein Registry (http://www.neb.com/neb/inteins.html) provides a list of known inteins. 
     The term “split intein” as used herein refers to an intein that has at least two separate components not fused to one another. 
     The term “splicing” as used herein refers to the process involving the cleavage of the main backbone of an intein-containing polypeptide by virtue of a reaction or process catalyzed by an intein or portions of an intein. “N-terminal splicing” refers to the cleavage of a polypeptide chain fused to the N-terminus of an intein, such reaction typically involving the scission of the thioester (or ester) bond formed via intein-catalyzed N→S (or N→O acyl) transfer, by action of a nucleophilic functional group or a chemical species containing a nucleophilic functional group. “C-terminal splicing” refers to the cleavage of a polypeptide chain fused to the C-terminus of an intein. “Self-splicing” as used herein refers to the process involving the cleavage of an intein from a polypeptide, within which the intein is embedded. “Trans-splicing” as used herein refers to a self-splicing process involving split inteins. 
     The term “affinity tag” as used herein refers to a polypeptide that is able to bind reversibly or irreversibly to an organic molecule, a metal ion, a protein, or a nucleic acid molecule. 
     The terms “vector” and “vector construct” as used herein refer to a vehicle by which a DNA or RNA sequence (e.g., a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g., transcription and translation) of the introduced sequence. A common type of vector is a “plasmid”, which generally is a self-contained molecule of double-stranded DNA that can be readily accept additional (foreign) DNA and which can readily introduced into a suitable host cell. A large number of vectors, including plasmid and fungal vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts. Non-limiting examples include, but are not limited to, pKK plasmids (Clonetech), pUC plasmids, pET plasmids (Novagen, Inc., Madison, Wis.), pRSET or pREP plasmids (Invitrogen, San Diego, Calif.), or pMAL plasmids (New England Biolabs, Beverly, Mass.), and many appropriate host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art. The terms “express” and “expression” refer to allowing or causing the information in a gene or DNA sequence to become manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence. A DNA sequence is expressed in or by a cell to form an “expression product” such as a protein. The expression product itself, e.g., the resulting protein, may also be said to be “expressed” by the cell. A polynucleotide or polypeptide is expressed recombinantly, for example, when it is expressed or produced in a foreign host cell under the control of a foreign or native promoter, or in a native host cell under the control of a foreign promoter. 
     The term “fused” as used herein means being connected through at least one covalent bond. The term “bound” as used herein means being connected through non-covalent interactions. Examples of non-covalent interactions are van der Waals, hydrogen bond, electrostatic, and hydrophobic interactions. Thus, a “DNA-binding peptide” refers to a peptide capable of connecting to a DNA molecule via non-covalent interactions. The term “tethered” as used herein means being connected through non-covalent interactions or through covalent bonds. Thus, a “polypeptide tethered to a solid support” refers to a polypeptide that is connected to a solid support (e.g., surface, resin bead) either via non-covalent interactions or through covalent bonds. 
     The term “virus”, “virion”, or “viral particle” refers to a virus consisting a protein coat containing nucleic acid useful for the assembly and/or replication of the phages in a host cell. The term “phage”, “bacteriophage” or “phage particle” refers to a virus consisting a protein coat containing nucleic acid useful for the assembly and/or replication of the phages in a bacterial cell. The nucleic acid may be DNA or RNA, either double or single stranded, linear or circular. Examples of bacteriophages included phage lamda and filamentous phages such as M13, fd and fl bacteriophages. During the assembly of the bacteriophages, the coat proteins may package different nucleic acid sequences, such nucleic acid sequences typically comprising a packaging signal. 
     The term “phagemid” refers to a plasmid having at least a bacteriophage origin of replication (e.g., fl) and optionally a bacterial origin of replication (e.g., ColE1). As disclosed herein, phagemids can include additional genetic elements, such as an intergenic region of a bacteriophage, a gene encoding a polypeptide capable of conferring antibiotic resistance (e.g., bla) to a recipient host organism, as well as genes encoding additional polypeptides or engineered polypeptides. The phagemid may form infectious or non-infectious phage particles. This term includes phagemids which contain a phage coat protein gene or fragment thereof fused to a gene encoding for a heterologous polypeptide gene such that the heterologous polypeptide is display on the surface of the phage particle. 
     The term “coat protein” refers to a protein or a fragment thereof which is present on the surface of a phage particle. In the case of filamentous bacteriophages, such as M13, fd and fl bacteriophages, coat proteins include pIII, pVI, pVII, pVIII, and pIX coat proteins. A coat protein is typically referred to as a “major coat protein” if present in more than 10 copies in the phage particle (e.g., pVIII), and as “minor coat protein” if present in less than 10 copies in the phage particle (e.g., pIII). 
     In the context of the present disclosure, the term “host display organism” refers to a virus, phage or cell displaying the macrocyclic peptide(s) on their outer surface. Host display viruses include, but are not limited to bacteriophages, insect viruses, plant viruses, and mammalian viruses. Host display cells include, but are not limited to, bacterial cells, including  Escherichia coli  or  Bacillus subtilis  cells, fungal cells, including yeast cells, plant cells, insect cells, and mammalian cells. 
     The term “library” refers to a collection or “plurality” of at least two particles or molecules which differ in at least part of their compositions, properties, and/or sequences. For example, a macrocyclic peptide library refers to a collection of macrocyclic peptides which differs in at least part of their compositions such as, for example, an amino acid residue. 
     In the context of the present application, the term “desired property” refers to a predetermined property which forms the basis for the screening and/or selection of a library, such as a library of macrocyclic peptides. Such properties include but are not limited to, binding to a target molecule, blocking the function of a target molecule, blocking or promoting the interaction between a target molecule and another molecule, activating or inhibiting a reaction mediated by a target molecule, and activating or inhibiting the activity of an enzyme or receptor. 5.2 Methods for producing macrocyclic peptides from ribosomal polypeptides 
     Methods and compositions are provided for making artificial macrocyclic peptides from genetically encoded, ribosomally produced artificial polypeptides. These methods are based on the use of artificial precursor polypeptides comprising (a) a non-canonical amino acid residue carrying a thiol-reactive functional group (referred to as FG 1 ); and (b) a cysteine residue that is positioned either upstream or downstream of the non-canonical amino acid in the polypeptide sequence. These methods are based on the ability of the FG 1 -bearing amino acid and cysteine residue to react with each other after ribosomal synthesis of the polypeptide, so that a macrocyclic peptide carrying a side-chain-to-side-chain covalent (thioether) linkage is formed. Schematic representations of these embodiments are provided in  FIGS. 1A-B . 
     Methods and compositions are also provided for making macrocyclic peptides from genetically encoded, ribosomally produced, intein-fused polypeptides. These methods are based on the use of artificial precursor polypeptides comprising (a) a non-canonical amino acid residue with a thiol-reactive functional group (referred to as FG 1 ); (b) a cysteine residue positioned upstream or downstream of the non-canonical amino acid within the polypeptide sequence; and (c) an intein protein positioned upstream or downstream of the non-canonical amino acid or of the cysteine residue within the polypeptide sequence. These methods exploit the ability of this non-canonical amino acid and cysteine residue to react with each other after ribosomal synthesis of the precursor polypeptide, so that a macrocyclic peptide carrying a side-chain-to-side-chain covalent (thioether) linkage is formed. These methods also exploit the ability of the intein to undergo N-terminal splicing, C-terminal splicing, or self-splicing, so that the macrocyclic peptide is released upon intein splicing. Schematic representations of these embodiments are provided in  FIGS. 2A-B  and  3 A-B. 
     Methods and compositions are also provided for making artificial macrocyclic peptides from genetically encoded, ribosomally produced, split intein-fused polypeptides. These methods are based on the use of artificial precursor polypeptides comprising (a) a non-canonical amino acid residue with a thiol-reactive functional group (referred to as FG 1 ); (b) a cysteine residue positioned upstream or downstream of the non-canonical amino acid within the polypeptide sequence; and (c) a split intein domain positioned upstream or downstream of the non-canonical amino acid or the cysteine residue within the polypeptide sequence. These methods exploit the ability of this non-canonical amino acid and cysteine residue to react with each other after ribosomal synthesis of the precursor polypeptide, so that a macrocyclic peptide carrying a side-chain-to-side-chain covalent (thioether) linkage is formed. These methods also exploit the ability of the split intein to undergo trans-splicing, so that the bicyclic peptide is released upon split intein trans-splicing. Schematic representations of these embodiments are provided in  FIGS. 4A-B . 
     Methods and compositions are also provided for making artificial macrocyclic peptides from genetically encoded, ribosomally produced, split intein-fused polypeptides. These methods are based on the use of artificial precursor polypeptides comprising (a) a non-canonical amino acid residue with two thiol-reactive functional groups (referred to as FG 1  and FG 2 ); (b) two cysteine residues positioned upstream and downstream of the non-canonical amino acid within the polypeptide sequence. These methods are based on the ability of the FG 1 /FG 2 -bearing amino acid to react with the two cysteine residues after ribosomal synthesis of the polypeptide, so that a bicyclic peptide carrying two side-chain-to-side-chain covalent (thioether) linkages is formed. Schematic representations of these embodiments are provided in  FIGS. 37A-B . 
     Artificial, engineered and recombinant nucleic acid molecules and peptide sequences (or amino acid sequences) for use in these methods are also provided. 
     Methods for Display of Macrocyclic Peptides 
     Methods and compositions are provided for the display of macrocyclic peptides on a outer biological surface of a viral particle or a cell. These methods are based on the use of artificial precursor polypeptides, which (i) comprise (a) a non-canonical amino acid residue carrying a thiol-reactive functional group (referred to as FG 1 ); and (b) a cysteine residue that is positioned either upstream or downstream of the non-canonical amino acid in the polypeptide sequence; and (ii) are fused to a presentation polypeptide, or fragment thereof, of a host viral particle or a host display cell. These methods are based on the ability of the FG 1 -bearing amino acid and cysteine residue to react with each other after ribosomal synthesis of the polypeptide, so that a macrocyclic peptide carrying a side-chain-to-side-chain covalent (thioether) linkage is formed. Fusion of this macrocyclic peptide to a presentation polypeptide of the host viral particle or cell further allows the display of the macrocyclic peptide on to the outer biological surface of the host viral particle or cell. This system further allows for the gene encoding for the macrocyclic peptide to be contained (or physically linked) to the viral particle or cell displaying the macrocyclic peptide, such that the sequence of the macrocyclic peptide can determined by sequencing of this gene. Schematic representations of these embodiments are provided in  FIGS. 40-43 . 
     In some embodiments, a method is provided for displaying a macrocyclic peptide on an outer biological surface, the method comprising: 
     a. providing a nucleic acid molecule encoding for a polypeptide of structure: 
       (AA) m -Z-(AA) n -Cys-(AA) p   (I)
 
       or 
       (AA) m -Cys-(AA) n -Z-(AA) p   (II)
         wherein:   i. (AA) m  is an N-terminal amino acid or peptide sequence,   ii. Z is a non-canonical amino acid carrying a side-chain functional group FG 1 , this FG 1  being a functional group selected from the group consisting of —(CH 2 ) n X, where X is F, Cl, Br, or I and n is an integer number from 1 to 10; —C(O)CH 2 X, where X is F, Cl, Br, or I; —CH(R′)X, where X is F, Cl, Br, or I; —C(O)CH(R′)X, where X is F, Cl, Br, or I; —OCH 2 CH 2 X, where X is F, Cl, Br, or I; —C(O)CH═C═C(R′)(R″), —SO 2 C(R′)═C(R′)(R″), —C(O)C(R′)═C(R′)(R″), —C(R′)═C(R′)C(O)OR′, —C(R′)═C(R′)C(O)N(R′)(R″), —C(R′)═C(R′)—CN, —C(R′)═C(R′)—NO 2 , —C≡C—C(O)OR′, —C≡C—C(O)N(R′)(R″), unsubstituted or substituted oxirane, unsubstituted or substituted aziridine, 1,2-oxathiolane 2,2-dioxide, 4-fluoro-1,2-oxathiolane 2,2-dioxide, and 4,4-difluoro-1,2-oxathiolane 2,2-dioxide, where each R′ and R″ is independently H, an aliphatic, a substituted aliphatic, an aryl, or a substituted aryl group.   iii. (AA) n  is a target peptide sequence,   iv. (AA) p  is a C-terminal amino acid or peptide sequence, and   vii. the polypeptide is fused to a presentation polypeptide or fragment thereof;
 
b. introducing the nucleic acid molecule into a suitable expression system that allows for the incorporation of the non-canonical amino acid Z into the polypeptide;
 
c. expressing the nucleic acid molecule in said expression system, thereby producing the polypeptide fused to the presentation polypeptide or fragment thereof; and
 
d. allowing the functional group FG 1 , and whenever present, FG 2 , to react with the side-chain sulfhydryl group (—SH) of the cysteine (Cys) residue(s), thereby producing the macrocyclic peptide anchored on the outer biological surface.
       

     In some embodiments, a method is provided for displaying a macrocyclic peptide on an outer biological surface, the method comprising: 
     a. providing a nucleic acid molecule encoding for a polypeptide of structure: 
       (AA) m -Cys-(AA) n -Z2-(AA) o -Cys-(AA) p   (V)
         wherein:   i. (AA) m  is an N-terminal amino acid or peptide sequence,   ii. Z2 is a non-canonical amino acid carrying two side-chain functional groups FG 1  and FG 2 , these FG 1  and FG 2  being a functional group independently selected from the group consisting of —(CH 2 ) n X, where X is F, Cl, Br, or I and n is an integer number from 1 to 10; —C(O)CH 2 X, where X is F, Cl, Br, or I; —CH(R′)X, where X is F, Cl, Br, or I; —C(O)CH(R′)X, where X is F, Cl, Br, or I; —OCH 2 CH 2 X, where X is F, Cl, Br, or I; —C(O)CH═C═C(R′)(R″), —SO 2 C(R′)═C(R′)(R″), —C(O)C(R′)═C(R′)(R″), —C(R′)═C(R′)C(O)OR′, —C(R′)═C(R′)C(O)N(R′)(R″), —C(R′)═C(R′)—CN, —C(R′)═C(R′)—NO 2 , —C≡C—C(O)OR′, —C≡C—C(O)N(R′)(R″), unsubstituted or substituted oxirane, unsubstituted or substituted aziridine, 1,2-oxathiolane 2,2-dioxide, 4-fluoro-1,2-oxathiolane 2,2-dioxide, and 4,4-difluoro-1,2-oxathiolane 2,2-dioxide, where each R′ and R″ is independently H, an aliphatic, a substituted aliphatic, an aryl, or a substituted aryl group.   iii. (AA) n  is a target peptide sequence,   iv. (AA) o  is a second target peptide sequence,   v. (AA) p  is a C-terminal amino acid or peptide sequence;   vii. the polypeptide is fused to a presentation polypeptide or fragment thereof;
 
b. introducing the nucleic acid molecule into a suitable expression system that allows for the incorporation of the non-canonical amino acid Z2 into the polypeptide;
 
c. expressing the nucleic acid molecule in said expression system, thereby producing the polypeptide fused to the presentation polypeptide or fragment thereof; and
 
d. allowing the functional group FG 1 , and whenever present, FG 2 , to react with the side-chain sulfhydryl group (—SH) of the cysteine (Cys) residue(s), thereby producing the macrocyclic peptide anchored on the outer biological surface.
       

     According to the method, (AA) m  is a N-terminal sequence comprising at least one amino acid, where AA corresponds to a generic amino acid residue and m corresponds to the number of amino acid residues composing such sequence. (AA) m  is also referred to as “N-terminal tail”. (AA) p  is a C-terminal sequence that has 0 or at least one amino acid, where AA corresponds to a generic amino acid residue and p corresponds to the number of amino acid residues composing such sequence. (AA) p  is also referred to as “C-terminal tail”. (AA) n (and (AA) o , when present) is a peptide sequence of variable length (also referred to as “target peptide sequence”), where AA corresponds to a generic amino acid residue and n corresponds to the number of amino acid residues composing such peptide sequence. Cys is a cysteine amino acid residue. Z is an amino acid that carries a side-chain functional group FG 1 , which can react with the side-chain sulfhydryl group (—SH) of the cysteine residue to form a stable thioether bond. 
     As disclosed herein, the ability of an artificial polypeptide of formula (I) or (II) (also referred herein to as “precursor polypeptide”) to produce a macrocyclic peptide is conferred by the ability of the nucleophilic sulfhydryl group carried by the cysteine residue to react intramolecularly with the electrophilic functional group FG 1  carried by the amino acid Z, thereby forming a covalent, inter-side-chain thioether bond. Depending on the nature of FG 1 , this reaction proceeds via a thiol-mediated nucleophilic substitution reaction, a thiol-mediated Michael-type addition reaction, or a radical thiol-ene or thiol-yne reaction. Whereas the electrophilic functional group FG 1  in the precursor polypeptide could in principle react intermolecularly with free cysteine or other thiol-containing molecules contained in the expression system (e.g. glutathione), it was discovered by the inventors that appropriate functional groups FG 1  can be found so that the desired intramolecular thioether-bond forming reaction occurs exclusively or preferentially over the undesired intermolecular side-reactions. This result can be achieved because of the spatial proximity between the nucleophilic cysteine residue and the electrophilic Z amino acid, resulting in an increased effective concentration of the reacting species (i.e. —SH and FG 1  groups, respectively) in the intramolecular settings as compared to the intermolecular settings, which in turn favors the intramolecular peptide cyclization reaction over undesired intermolecular reactions. Similar considerations can be made in the context of certain embodiments, wherein a precursor polypeptide of formula (V) along with a bifunctional cysteine-reactive amino acid capable of forming thioether bonds with two cysteine residues within the polypeptide (residue Z2) is used. 
     A first advantage of the methods described herein is that they provide a highly versatile approach for the display of structurally diverse artificial macrocyclic peptides. Indeed, they offer multiple opportunities toward the structural and functional diversification of these compounds, e.g., through variation of the length and composition of the target peptide sequence ((AA) n ), variation of the structure of the amino acid Z, variation of the position of the amino acid Z relative to the cysteine residue (e.g., precursor polypeptide (I) versus (II)), variation of the length and composition of the N-terminal tail ((AA) m ), and variation of the length and composition of the C-terminal tail ((AA) p ). Further structural diversification can be achieved by combining multiple Z/Cys pairs within the same precursor polypeptide or by using bifunctional cysteine-reactive amino acids (Z2) in order to obtain polycyclic and bicyclic peptides. Accordingly, and because of the genetically encoded and ribosomal nature of the precursor polypeptides and spontaneous (i.e., not chemically or enzymatically) formation of the thioether-bridge, the methods and compositions described herein can be used to produce vast libraries of structurally and functionally diverse macrocyclic peptides displayed on the outer surface of a viral particle or cell. In turn, these libraries can be screened to identify macrocyclic peptides that can bind to a target molecule (e.g., enzyme, protein, or nucleic acid), and/or inhibit or promote the interaction between the target molecule and another molecule(s) (e.g., protein-protein or protein-nucleic acid interactions) for a variety of applications, including drug discovery. 
     A second advantage of the methods disclosed herein is that they produce peptide molecules whose conformational flexibility is restrained by virtue of at least one intramolecular thioether linkage. As illustrated in Example 8, this feature can confer these molecules with advantageous properties such as, for example, enhanced binding affinity, increased stability against proteolysis, and/or more favorable membrane-crossing properties, as compared to linear peptides or peptides lacking the intramolecular thioether linkage. In addition, the thioether linkage is redox and chemically stable in biological milieu, including the intracellular environment. 
     A third advantage of the methods disclosed herein is that they allow for the preparation of genetically encoded macrocyclic peptides displayed on the outer surface of a variety of virus- and cell-based systems. Indeed, very large and structurally diverse libraries of macrocyclic peptides can be rapidly and cost-effectively produced utilizing precursor polypeptides in which the target peptide sequence ((AA) n ), N-terminal tail ((AA) m ), and/or C-terminal tail ((AA) m ), is partially or fully randomized genetically. According to the methods of the disclosed therein, this library of macrocyclic peptides is produced as fusions to a presentation polypeptide of a virus-, phage- or cell-based display system, such as phage display, yeast display, and bacterial display, and the like. In turn, this enables the production of a library of viral particles, phage particles, or cells, in which each member of the library displays a unique macrocyclic peptide (in single or multiple copies) and contains the gene encoding for it. This virus-, phage-, or cell-based display library of macrocyclic peptides can be subjected to high-throughput screening or selection procedures (e.g., affinity selection) and rapidly deconvoluted (e.g., via DNA sequencing) in order to identify macrocyclic peptides with a desired property. So, for example, the methods described herein allow one to generate vast and structurally diverse combinatorial libraries of macrocyclic peptides fused to a bacteriophage particle. As demonstrated in Example 10, these phage display libraries of macrocyclic peptides can be then ‘panned’ against a target molecule of interest in order to identify macrocyclic peptide binders or inhibitors of such molecule. 
     A fourth advantage of the methods described herein is that they allow for fusion of the macrocyclic peptide to the N-terminal end or the C-terminal end of a presentation polypeptide, or within the central region of a presentation polypeptide, making this macrocyclic peptide system compatible with a broad range of display formats, such as phage display, yeast display, and bacterial display. Further adding to this versatility, the methods described herein allows for the production of the macrocyclic peptides in any cell-based expression host, including bacterial, yeast, insect, or mammalian cells, or a cell-derived expression system such as a cell lysate. 
     A fifth advantage of the methods described herein is that they also enable the production of macrocyclic peptides inside a cell-based expression host such as a bacterial, yeast, insect, or mammalian cell. Intracellular production of the macrocyclic peptide can then be coupled to an (intra)cellular reporter system, phenotypic screen, or selection system, in order to identify a macrocyclic peptide capable of inhibiting or activating a certain cellular process, biomolecule, or enzymatic reaction linked to the reporter output, phenotype, or cell survival, respectively. 
     A sixth advantage of the methods disclosed herein is that the production of the macrocyclic peptides can be carried out under physiological conditions (e.g., in aqueous buffer, neutral pH, physiological temperature) and in complex biological media (e.g., inside a cell, in cell lysate) and in the presence of biological molecules (proteins, nucleic acids, cell metabolites) and biological material. One implication of this is that the production of macrocyclic peptides according to the methods disclosed herein can be coupled to one of the several techniques known in the art for the display and high-throughput screening of biological peptide libraries. 
     Because of the aforementioned advantageous features, the methods described herein can be useful to greatly accelerate and facilitate the discovery of bioactive peptide-based compounds as potential drug molecules and chemical probes or the identification of lead structures for the development of new chemical probes and drugs. 
     In some embodiments, Z is an amino acid of structure: 
     
       
         
         
             
             
         
       
     
     wherein FG 1  is a functional group selected from the group consisting of —(CH 2 ) n X, where X is F, Cl, Br, or I and n is an integer number from 1 to 10; —C(O)CH 2 X, where X is F, Cl, Br, or I; —CH(R′)X, where X is F, Cl, Br, or I; —C(O)CH(R′)X, where X is F, Cl, Br, or I; —OCH 2 CH 2 X, where X is F, Cl, Br, or I; —C(O)CH═C═C(R′)(R″), —SO 02 C(R′)═C(R′)(R″), —C(O)C(R′)═C(R′)(R″), —C(R′)═C(R′)C(O)OR′, —C(R′)═C(R′)C(O)N(R′)(R″), —C(R′)═C(R′)—CN, —C(R′)═C(R′)—NO 2 , —C≡C—C(O)OR′, —C≡C—C(O)N(R′)(R″), unsubstituted or substituted oxirane, unsubstituted or substituted aziridine, 1,2-oxathiolane 2,2-dioxide, 4-fluoro-1,2-oxathiolane 2,2-dioxide, and 4,4-difluoro-1,2-oxathiolane 2,2-dioxide, where each R′ and R″ is independently H, an aliphatic, a substituted aliphatic, an aryl, or a substituted aryl group; and 
     wherein Y is a linker group selected from the group consisting of aliphatic, aryl, substituted aliphatic, substituted aryl, heteroatom-containing aliphatic, heteroatom-containing aryl, substituted heteroatom-containing aliphatic, substituted heteroatom-containing aryl, alkoxy, and aryloxy groups. 
     In some embodiments, Z is an amino acid of structure (IV) wherein Y is a linker group selected from the group consisting of C 1 -C 24  alkyl, C 1 -C 24  substituted alkyl, C 1 -C 24  substituted heteroatom-containing alkyl, C 1 -C 24  substituted heteroatom-containing alkyl, C 2 -C 24  alkenyl, C 2 -C 24  substituted alkenyl, C 2 -C 24  substituted heteroatom-containing alkenyl, C 2 -C 24  substituted heteroatom-containing alkenyl, C 5 -C 24  aryl, C 5 -C 24  substituted aryl, C 5 -C 24  substituted heteroatom-containing aryl, C 5 -C 24  substituted heteroatom-containing aryl, C 1 -C 24  alkoxy, C 5 -C 24  aryloxy groups. 
     In some embodiments, Z is an amino acid of structure (IV) wherein Y is a linker group selected from —CH 2 —C 6 H 4 —, —CH 2 —C 6 H 4 —O—, —CH 2 —C 6 H 4 —NH—, —(CH 2 ) 4 —, —(CH 2 ) 4 NH—, —(CH 2 ) 4 NHC(O)—, and —(CH 2 ) 4 NHC(O)O—. 
     In specific embodiments, the amino acid Z is selected from the group consisting of 4-(2-bromoethoxy)-phenylalanine, 3-(2-bromoethoxy)-phenylalanine, 4-(2-chloroethoxy)-phenylalanine, 4-(4-bromobutoxy)-phenylalanine, 4-(4-chlorobutoxy)-phenylalanine, 3-(4-bromobutoxy)-phenylalanine, 3-(4-bromobutoxy)-phenylalanine, 3-(2-chloroethoxy)-phenylalanine, 4-(1-bromoethyl)-phenylalanine, 3-(1-bromoethyl)-phenylalanine, 4-(aziridin-1-yl)-phenylalanine, 3-(aziridin-1-yl)-phenylalanine, 4-acrylamido-phenylalanine, 3-acrylamido-phenylalanine, 4-(2-fluoro-acetamido)-phenylalanine, 3-(2-fluoro-acetamido)-phenylalanine, 4-(2-chloro-acetamido)-phenylalanine, 3-(2-chloro-acetamido)-phenylalanine, 4-(2-bromo-acetamido)-phenylalanine, 3-(2-bromo-acetamido)-phenylalanine, 4-(acrylamido)-phenylalanine, 3-(acrylamido)-phenylalanine, 4-(vinylsulfonamido)-phenylalanine, 3-(vinylsulfonamido)-phenylalanine, 3-(2-fluoro-acetyl)-phenylalanine, 4-(2-fluoro-acetyl)-phenylalanine, N ε -((2-bromoethoxy)carbonyl)-lysine, N ε -((2-chloroethoxy)carbonyl)-lysine, N ε -(buta-2,3-dienoyl)-lysine, N ε -acryl-lysine, N ε -crotonyl-lysine, N ε -(2-fluoro-acetyl)-lysine, N ε -(2-chloro-acetyl)-lysine, N ε -(2-bromoacetyl)-lysine, and N ε -vinylsulfonyl-lysine. 
     In some embodiments, Z2 is an amino acid of structure: 
     
       
         
         
             
             
         
       
     
     wherein FG 1  and FG 2  are a functional group independently selected from the group consisting of —(CH 2 ) n X, where X is F, Cl, Br, or I and n is an integer number from 1 to 10; —C(O)CH 2 X, where X is F, Cl, Br, or I; —CH(R′)X, where X is F, Cl, Br, or I; —C(O)CH(R′)X, where X is F, Cl, Br, or I; —OCH 2 CH 2 X, where X is F, Cl, Br, or I; —C(O)CH═C═C(R′)(R″), —SO 2 C(R′)═C(R′)(R″), —C(O)C(R′)═C(R′)(R″), —C(R′)═C(R′)C(O)OR′, —C(R′)═C(R′)C(O)N(R′)(R″), —C(R′)═C(R′)—CN, —C(R′)═C(R′)—NO 2 , —C≡C—C(O)OR′, —C≡C—C(O)N(R′)(R″), unsubstituted or substituted oxirane, unsubstituted or substituted aziridine, 1,2-oxathiolane 2,2-dioxide, 4-fluoro-1,2-oxathiolane 2,2-dioxide, and 4,4-difluoro-1,2-oxathiolane 2,2-dioxide, where each R′ and R″ is independently H, an aliphatic, a substituted aliphatic, an aryl, or a substituted aryl group; and 
     wherein Y 2 , Y 3 , and L are linker groups selected from the group consisting of aliphatic, aryl, substituted aliphatic, substituted aryl, heteroatom-containing aliphatic, heteroatom-containing aryl, substituted heteroatom-containing aliphatic, substituted heteroatom-containing aryl, alkoxy, aryloxy groups. 
     In some embodiments, Z2 is an amino acid of structure (VI) wherein Y 2  is a linker group selected from the group consisting of C 1 -C 24  alkyl, C 1 -C 24  substituted alkyl, C 1 -C 24  substituted heteroatom-containing alkyl, C 1 -C 24  substituted heteroatom-containing alkyl, C 2 -C 24  alkenyl, C 2 -C 24  substituted alkenyl, C 2 -C 24  substituted heteroatom-containing alkenyl, C 2 -C 24  substituted heteroatom-containing alkenyl, C 5 -C 24  aryl, C 5 -C 24  substituted aryl, C 5 -C 24  substituted heteroatom-containing aryl, C 5 -C 24  substituted heteroatom-containing aryl, C 1 -C 24  alkoxy, C 5 -C 24  aryloxy groups. 
     In some embodiments, Z2 is an amino acid of structure (VI) wherein Y 2  is a linker group selected from the group consisting of —CH 2 —C 6 H 4 —, —CH 2 —C 6 H 4 —O—, —CH 2 —C 6 H 4 —NH—, —CH 2 —C 6 H 4 —OCH 2 —, —(CH 2 ) 4 NH—, —(CH 2 ) 4 NHC(O)—, —(CH 2 ) 4 NHC(O)O—, —(CH 2 ) 4 NHC(O)OCH 2 —, 
     
       
         
         
             
             
         
       
     
     In specific embodiments, the amino acid Z2 is selected from the group consisting of 3,5-bis(2-bromoethoxy)-phenylalanine, 3,5-bis(2-chloroethoxy)-phenylalanine, 3,5-bis(4-bromobutoxy)-phenylalanine, 3,5-bis(4-chlorobutoxy)-phenylalanine, 3,5-bis(1-bromoethyl)-phenylalanine, 3,5-bis(4-acrylamido)-phenylalanine, 3,5-bis(2-chloro-acetamido)-phenylalanine, 3,5-bis(2-bromo-acetamido)-phenylalanine, 3,5-bis(vinylsulfonamido)-phenylalanine, 3,5-bis(aziridin-1-yl)-phenylalanine, 3,5-bis-acrylamido-phenylalanine, 3,5-bis(2-fluoro-acetamido)-phenylalanine, 3,5-bis(2-fluoro-acetyl)-phenylalanine, 4-((1,3-dibromopropan-2-yl)oxy)-phenylalanine, 4-((1,3-dichloropropan-2-yl)oxy)-phenylalanine, N ε -(((1,3-dibromopropan-2-yl)oxy)carbonyl)-lysine, N ε -(((1,3-dichloropropan-2-yl)oxy)carbonyl)-lysine, 4-(2,3-dibromopropoxy)-phenylalanine, 3-(2,3-dibromopropoxy)-phenylalanine, 4-(2,3-dichloropropoxy)-phenylalanine, 3-(2,3-dichloropropoxy)-phenylalanine, N ε -((2,3-dibromopropoxy)carbonyl)-lysine, N ε -((2,3-dichloropropoxy)carbonyl)-lysine, N ε -bis-(acryl)-lysine, N ε -bis-(crotonyl)-lysine, N ε -bis-(2-fluoro-acetyl)-lysine, N ε -bis-(2-chloro-acetyl)-lysine, N ε -bis-(2-bromoacetyl)-lysine, and N ε -bis-(vinylsulfonyl)-lysine. 
     Artificial nucleic acid molecules for use according to the methods provided herein include, but are not limited to, those that encode for a polypeptide of general formula (I), (II), or (V) as defined above. The codon encoding for the amino acid Z (or Z2) in these polypeptides can be one of the 61 sense codons of the standard genetic code, a stop codon (TAG, TAA, TGA), or a four-base frameshift codon (e.g., TAGA, AGGT, CGGG, GGGT, CTCT). In some embodiments, the codon encoding for the amino acid Z (or Z2) within the nucleotide sequence encoding for the precursor polypeptide of formula (I), (II) or (V) is an amber stop codon (TAG), an ochre stop codon (TAA), an opal stop codon (TGA), or a four-base frameshift codon (see Example 2). In other embodiments, the codon encoding for Z (or Z2) in the nucleotide sequence encoding for these precursor polypeptides is the amber stop codon, TAG, or the 4-base codon, TAGA. 
     The non-canonical amino acid Z (or Z2) can be introduced into the precursor polypeptide through direct incorporation during ribosomal synthesis of the precursor polypeptide, or generated post-translationally through enzymatic or chemical modification of the precursor polypeptide, or by a combination of these procedures. In some embodiments, the amino acid Z (or Z2) is introduced into the precursor polypeptide during ribosomal synthesis of the precursor polypeptide via either stop codon suppression or four-base frameshift codon suppression. In other embodiments, the amino acid Z (or Z2) is introduced into the precursor polypeptide during ribosomal synthesis of the precursor polypeptide via amber (TAG) stop codon suppression or via 4-base TAGA codon suppression. 
     Several methods are known in the art for introducing a non-canonical amino acid into a recombinant or in vitro translated artificial polypeptide, any of which can be applied for preparing artificial precursor polypeptides suitable for the methods disclosed herein. These art-known methods include, but are not limited to, methods for suppression of a stop codon or of a four-based frameshift codon with a non-canonical amino acid using engineered (i.e., non-naturally occurring, artificial or synthetic) tRNA/aminoacyl-tRNA synthetase (AARS) pairs (Wang, Xie et al. 2006; Wu and Schultz 2009; Liu and Schultz 2010; Fekner and Chan 2011; Lang and Chin 2014). Examples of tRNA/aminoacyl-tRNA synthetase (AARS) pairs used for this purpose include, but are not limited to, engineered variants of  Methanococcus jannaschii  AARS/tRNA pairs (e.g., TyrRS/tRNA TYr ), of  Saccharomyces cerevisiae  AARS/tRNA pairs (e.g., AspRS/tRNA Asp , GlnRS/tRNA Gln , TyrRS/tRNA Tyr , and PheRS/tRNA Phe ), of  Escherichia coli  AARS/tRNA pairs (e.g., TyrRS/tRNA TYr , LeuRS/tRNA Leu ), of  Methanosarcina mazei  AARS/tRNA pairs (PylRS/tRNA Pyl ), and of  Methanosarcina mazei  AARS/tRNA pairs (PylRS/tRNA Pyl ) (Wang, Xie et al. 2006; Wu and Schultz 2009; Liu and Schultz 2010; Fekner and Chan 2011; Lang and Chin 2014). Alternatively, natural or engineered four-codon suppressor tRNAs and their cognate aminoacyl-tRNA synthetases can be used for the same purpose (Anderson, Wu et al. 2004; Rodriguez, Lester et al. 2006; Neumann, Slusarczyk et al. 2010; Neumann, Wang et al. 2010). Alternatively, a non-canonical amino acid can be incorporated into a polypeptide using chemically (Dedkova, Fahmi et al. 2003) or enzymatically (Bessho, Hodgson et al. 2002; Hartman, Josephson et al. 2006) aminoacylated tRNA molecules and using a cell-free protein expression system in the presence of the aminoacylated tRNA molecules (Kourouklis, Murakami et al. 2005; Murakami, Ohta et al. 2006). Alternatively, a non-canonical amino acid can be incorporated into a polypeptide by exploiting the promiscuity of wild-type aminoacyl-tRNA synthetase enzymes using a cell-free protein expression system, in which one or more natural amino acids are replaced with structural analogs (Josephson, Hartman et al. 2005; Hartman, Josephson et al. 2007). Any of these methods can be used to introduce an unnatural amino acid of the type (III), (IV), (VI) or (VII) into the precursor polypeptide for the purpose of generating macrocyclic peptides according to the methods disclosed herein. 
     In some embodiments, the non-canonical amino acid Z (or Z2) is incorporated into the precursor polypeptide via stop codon or four-base codon suppression methods using an engineered AARS/tRNA pair derived from  Methanococcus jannaschii  tyrosyl-tRNA synthetase (MjTyrRS) and its cognate tRNA (MjtRNA Tyr ), an engineered AARS/tRNA pair derived from  Methanosarcina mazei  pyrrolysyl-tRNA synthetase (MmPylRS) and its cognate tRNA (tRNA Pyl ), an engineered AARS/tRNA pair derived from  Methanosarcina mazei  pyrrolysyl-tRNA synthetase (MmPylRS) and its cognate tRNA (tRNA Pyl ), or an engineered AARS/tRNA pair derived from  Escherichia coli  tyrosyl-tRNA synthetase (EcTyrRS) and its cognate tRNA (EctRNA Tyr ). 
     In the characterization of the aminoacyl-tRNA synthetase enzymes disclosed herein, these enzymes can be described in reference to the amino acid sequence of a naturally occurring aminoacyl-tRNA synthetase or another engineered aminoacyl-tRNA synthetase. As such, the amino acid residue is determined in the aminoacyl-tRNA synthetase enzyme beginning from the first amino acid after the initial methionine (M) residue (i.e., the first amino acid after the initial methionine M represents residue position 1). It will be understood that the initiating methionine residue may be removed by biological processing machinery such as in a host cell or in vitro translation system, to generate a mature protein lacking the initiating methionine residue. The amino acid residue position at which a particular amino acid or amino acid change is present is sometimes described herein as “Xn”, or “position n”, where n refers to the residue position. 
     In some embodiments, the stop codon/frameshift codon suppression system used for incorporating the amino acid Z (or Z2) into the precursor polypeptide comprises an engineered variant of  Methanococcus jannaschii  tRNA Tyr  as encoded by a nucleotide of sequence SEQ ID NO:: 101, 102, 103, or 104; and an engineered variant of  Methanococcus jannaschii  tyrosyl-tRNA synthetase (SEQ ID NO:: 77), said variant comprising an amino acid change at at least one of the following amino acid positions of SEQ ID NO:77: X32, X63, X65, X70, X107, X108, X109, X155, X158, X159, X160, X161, X162, X163, X164, X167, and X286. 
     In other embodiments, the stop codon/frameshift codon suppression system used for incorporating the amino acid Z (or Z2) into the precursor polypeptide consists of a  Methanococcus jannaschii  tRNA Tyr  variant selected from the group of tRNA molecules encoded by the nucleotide sequence of SEQ ID NOs: 101, 102, 103, and 104; and a  Methanococcus jannaschii  tyrosyl-tRNA synthetase variant selected from the group of polypeptides of SEQ ID NOs: 77, 81, 82, 83, 84, 85, 86, 87, 88, 89, and 90. 
     In some embodiments, the stop codon/frameshift codon suppression system used for incorporating the amino acid Z (or Z2) into the precursor polypeptide comprises an engineered variant of  Methanosarcina  species tRNA Pyl  or  Desulfitobacterium hafniense  tRNA Pyl  as encoded by a nucleotide of sequence SEQ ID NO:: 105, 106, 107, 108, 109, 110, 111, or 112; and an engineered variant of  Methanosarcina mazei  pyrrolysyl-tRNA synthetase (SEQ ID NO:: 78), said variant comprising an amino acid change at at least one of the following amino acid positions of SEQ ID NO:78: X302, X305, X306, X309, X346, X348, X364, X384, X401, X405, and X417. 
     In some embodiments, the stop codon/frameshift codon suppression system used for incorporating the amino acid Z (or Z2) into the precursor polypeptide comprises an engineered variant of  Methanosarcina  species tRNA Pyl  or  Desulfitobacterium hafniense  tRNA Pyl  as encoded by a nucleotide of sequence SEQ ID NO:: 105, 106, 107, 108, 109, 110, 111, or 112; and an engineered variant of  Methanosarcina barkeri  pyrrolysyl-tRNA synthetase (SEQ ID NO:: 79), said variant comprising an amino acid change at at least one of the following amino acid positions of SEQ ID NO:: 79: X76, X266, X270, X271, X273, X274, X313, X315, and X349. 
     In other embodiments, the stop codon/frameshift codon suppression system used for incorporating the amino acid Z (or Z2) into the precursor polypeptide consists of a tRNA Pyl variant selected from the group of tRNA molecules encoded by the nucleotide sequence of SEQ ID NO:: 105, 106, 107, 108, 109, 110, 111, and 112; and a pyrrolysyl-tRNA synthetase variant selected from the group of polypeptides of SEQ ID NOs: 78, 79, 91, 92, 93, 94, 95, and 96. 
     In some embodiments, the stop codon/frameshift codon suppression system used for incorporating the amino acid Z (or Z2) into the precursor polypeptide comprises an engineered variant of  Escherichia coli  tRNA Tyr  or  Bacillus stearothermophilus  tRNA Tyr  as encoded by a nucleotide of sequence SEQ ID NO:: 113, 114, 115, 116, 117, 118, 119, or 120; and an engineered variant of  Escherichia coli  tyrosyl-tRNA synthetase (SEQ ID NO:: 80), said variant comprising an amino acid change at at least one of the following amino acid positions of SEQ ID NO:: 80: X37, X182, X183, X186, and X265. 
     In other embodiments, the stop codon/frameshift codon suppression system used for incorporating the amino acid Z (or Z2) into the precursor polypeptide consists of a tRNA Tyr  variant selected from the group of tRNA molecules encoded by the nucleotide sequence of SEQ ID NO: 113, 114, 115, 116, 117, 118, 119, and 120; and a  E. coli  tyrosyl-tRNA synthetase variant selected from the group of polypeptides of SEQ ID NOs: 80, 97, 98, 99, 100. 
     In some embodiments, the aminoacyl-tRNA synthetase used for incorporating the amino acid Z (or Z2) into the precursor polypeptide can have additionally at least one amino acid residue differences at positions not specified by an X above as compared to the sequence SEQ ID NO:: 77, 78, 79, or 80. In some embodiments, the differences can be 1-2, 1-5, 1-10, 1-20, 1-30, 1-40, 1-50, 1-75, 1-100, 1-150, or 1-200 amino acid residue differences at other positions not defined by X above. 
     In some embodiments, the suppressor tRNA molecule used for incorporating the amino acid Z (or Z2) into the precursor polypeptide can have additionally at least one nucleotide difference as compared to the sequence encoded by the gene of SEQ ID NO:: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120. In some embodiments, the differences can be 1-2, 1-5, 1-10, 1-20, 1-30, 1-40, 1-50, or 1-60 nucleotide differences as compared to the sequences encoded by these genes. 
     In another embodiment of the method, the engineered variant of  Methanococcus jannaschii  tyrosyl-tRNA synthetase (SEQ ID NO:: 77) comprises at least one of the features selected from the group consisting of: X32 is Tyr, Leu, Ala, Gly, Thr, His, Glu, Val, or Gln; X65 is Leu, His, Tyr, Val, Ser, Thr, Gly, or Glu; X67 is Ala or Gly; X70 is His, Ala, Cys, or Ser; X107 is Glu, Pro, Asn, or Thr; X108 is Phe, Trp, Ala, Ser, Arg, Gly, Tyr, His, Trp, or Glu; X109 is Gln, Met, Asp, Lys, Glu, Pro, His, Gly, Met, or Leu; X155 is Gln, Glu, or Gly; X158 is Asp, Gly, Glu, Ala, Pro, Thr, Ser, or Val; X159 is Ile, Cys, Pro, Leu, Ser, Trp, His, or Ala; X160 is His or Gln; X161 is Tyr or Gly; X162 is Leu, Arg, Ala, Gln, Gly, Lys, Ser, Glu, Tyr, or His; X163 is Gly or Asp; X164 is Val or Ala; X167 is Ala or Val; X286 is Asp or Arg. 
     In another embodiment of the method, the engineered variant of  Methanosarcina mazei  pyrrolysyl-tRNA synthetase (SEQ ID NO:: 78) comprises at least one of the features selected from the group consisting of: X302 is Ala or Thr; X305 is Leu or Met; X306 is Tyr, Ala, Met, Ile, Leu, Thr, Gly; X309 is Leu, Ala, Pro, Ser, or Arg; X346 is Asn, Ala, Ser, or Val; X348 is Cys, Ala, Thr, Leu, Lys, Met, or Trp; X364 is Thr or Lys; X384 is Tyr or Phe; X405 is Ile or Arg; X401 is Val or Leu; X417 is Trp, Thr or Leu. 
     In another embodiment of the method, the engineered variant of  Methanosarcina barkeri  pyrrolysyl-tRNA synthetase (SEQ ID NO: 79) comprises at least one of the features selected from the group consisting of: X76 is Asp or Gly; X266 is Leu, Val, or Met; X270 is Leu or Ile; X271 is Tyr, Phe, Leu, Met, or Ala; X274 is Leu, Ala, Met, or Gly; X313 is Cys, Phe, Ala, Val, or Ile; X315 is Met or Phe; X349 is Tyr, Phe, or Trp. 
     In another embodiment of the method, the engineered variant of  Escherichia coli  tyrosyl-tRNA synthetase (SEQ ID NO: 80) comprises at least one of the features selected from the group consisting of: X37 is Tyr, Ile, Gly, Val, Leu, Thr, or Ser; X182 is Asp, Gly, Ser, or Thr; X183 is Phe, Met, Tyr, or Ala; X186 is Leu, Ala, Met, or Val; X265 is Asp or Arg. 
     An aspect of the methods disclosed herein is the identification and selection of a suitable aminoacyl-tRNA synthetase for incorporating an amino acid Z (or Z2) as defined above, into the artificial precursor polypeptide. Various methods are known in the art to evaluate and quantify the relative efficiency of a given wild-type or engineered aminoacyl-tRNA synthetase to incorporate a non-canonical amino acid into a protein (Young, Young et al. 2011). Any of these methods can be used to guide the identification and choice of a suitable aminoacyl-tRNA synthetase for incorporating a desired amino acid Z (or Z2) into the precursor polypeptide. For example, such efficiency can be measured via a fluorescence assay based on the expression of a reporter fluorescent protein (e.g. green fluorescent protein), whose encoding gene has been modified to contain a codon to be suppressed (e.g. amber stop codon). Expression of the reporter fluorescent protein is then induced in a suitable expression system (e.g. an  E. coli  or yeast cell) in the presence of the aminoacyl-tRNA synthetase to be tested, a cognate suppressor tRNA (e.g. amber stop codon suppressor tRNA), and the desired non-canonical amino acid. Under these conditions, the relative amount of the expressed (i.e. ribosomally produced) fluorescent protein is linked to the relative efficiency of the aminoacyl-tRNA synthetase to charge the cognate suppressor tRNA with the non-canonical amino acid, which can thus be quantified via fluorimetric means. A demonstration of how this procedure can be applied for selecting an aminoacyl-tRNA synthetase/suppressor tRNA pair for incorporating a desired amino acid Z (or Z2) into the precursor polypeptide is provided in Example 3. 
     If necessary, the ability of a given aminoacyl-tRNA synthetase/suppressor tRNA pair to incorporate a target non-canonical amino acid into a protein can be improved by means of rational design or directed evolution. While the fluorescence-based method described above can be used to screen several hundreds of engineered aminoacyl-tRNA synthetase variants and/or suppressor tRNA variants for this purpose, higher throughput procedures are also known in the art, which are, for example, based on selection systems (Wang, Xie et al. 2006; Wu and Schultz 2009; Liu and Schultz 2010; Fekner and Chan 2011). One such system involves introducing a library of mutated aminoacyl-tRNA synthetases and/or of mutated suppressor tRNAs into a suitable cell-based expression host (e.g.  E. coli  or yeast cells), whose survival under a suitable selective medium or growth conditions is dependent upon the functionality of the aminoacyl-tRNA synthetase/suppressor tRNA pair. This can be achieved, for example, by introducing a stop codon or four-base codon that is to be suppressed, into a gene encoding for a protein or enzyme essential for survival of the cell, such as a protein or enzyme conferring resistance to an antibiotic. In this case, the ability of the aminoacyl-tRNA synthetase/suppressor tRNA pair to incorporate the desired non-canonical amino acid into the selection marker protein is linked to the survival of the host, thereby enabling the rapid isolation of suitable aminoacyl-tRNA synthetase/suppressor tRNA pair(s) for the incorporation of a particular non-canonical amino acid from very large engineered libraries. The selectivity of these aminoacyl-tRNA synthetase/suppressor tRNA pair toward the desired non-canonical amino acid over the twenty natural amino acids can be further improved by iterative rounds of positive and negative selection as described in (Wang, Xie et al. 2006; Wu and Schultz 2009; Liu and Schultz 2010; Fekner and Chan 2011). Procedures such as those described above can be thus applied to generate and isolate an engineered aminoacyl-tRNA synthetase/suppressor tRNA pair suitable for incorporation of the amino acid Z as defined above, into the precursor polypeptide. 
     Engineered aminoacyl-tRNA synthetase/tRNA pairs for the incorporation of the amino acid Z (or Z2) into the precursor polypeptide can be prepared via mutagenesis of the polynucleotide encoding for the aminoacyl-tRNA synthetase enzymes of SEQ ID NOs: 77, 78, 79, 80, or an engineered variant thereof; and via mutagenesis of the tRNA-encoding polynucleotides of SEQ ID NOs: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, or an engineered variant thereof. Many mutagenesis methods are known in the art and these include, but are not limited to, site-directed mutagenesis, site-saturation mutagenesis, random mutagenesis, cassette-mutagenesis, DNA shuffling, homologous recombination, non-homologous recombination, site-directed recombination, and the like. Detailed description of art-known mutagenesis methods can be found, among other sources, in U.S. Pat. Nos. 5,605,793; 5,830,721; 5,834,252; WO 95/22625; WO 96/33207; WO 97/20078; WO 97/35966; WO 98/27230; WO 98/42832; WO 99/29902; WO 98/41653; WO 98/41622; WO 98/42727; WO 00/18906; WO 00/04190; WO 00/42561; WO 00/42560; WO 01/23401; WO 01/64864. 
     As described above, the engineered aminoacyl-tRNA synthetases and cognate suppressor tRNA obtained from mutagenesis of SEQ ID NO:77 to 80, and from mutagenesis of SEQ ID NO: 101 to 120, can be screened for identifying aminoacyl-tRNA synthetase/suppressor tRNA pairs being able, or having improved ability as compared to the corresponding wild-type enzyme/tRNA molecule, to incorporate the amino acid Z (or Z2) into the precursor polypeptide. 
     In some embodiments, the engineered aminoacyl-tRNA synthetase used in the method comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99% or more identical to the sequence SEQ ID NOs: 77, 78, 79, or 80. 
     In some embodiments, the engineered suppressor tRNA used in the method is encoded by a polynucleotide comprising a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99% or more identical to the sequence SEQ ID NOs: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120. 
     The target peptide sequence, (AA) n , in the precursor polypeptide of formula (I), (II) and (V) and the second target peptide sequence, (AA) o , in the precursor polypeptide of formula (V), can be a polypeptide comprising 1 to 1,000 amino acid residues. In some embodiments, (AA) n (and (AA) o ) consists of a polypeptide comprising 1 to 50 amino acid residues and, in other embodiments, (AA) n (and (AA) o ) consists of a polypeptide comprising 1 to 20 amino acid residues. 
     The N-terminal tail, (AA) m , in the precursor polypeptide of formula (I), (II), and (V) can be a polypeptide comprising 1 to 10,000 amino acid residues. In some embodiments, (AA) m  consists of a polypeptide comprising 1 to 1,000 amino acid residues and, in other embodiments, (AA) m  consists of a polypeptide comprising 1 to 600 amino acid residues. 
     The C-terminal tail, (AA) p , in the precursor polypeptide of formula (I), (II), and (V) may not be present, and when present, it can be a polypeptide comprising 1 to 10,000 amino acid residues. When present, (AA) m  consists, in some embodiments, of a polypeptide comprising 1 to 1,000 amino acid residues and, in other embodiments, (AA) m  consists of a polypeptide comprising 1 to 600 amino acid residues. 
     The N-terminal tail, (AA) m , the C-terminal tail, (AA) p , or both, in the precursor polypeptides of formula (I), (II), and (V) can comprise a polypeptide affinity tag, a DNA-binding polypeptide, a protein-binding polypeptide, an enzyme, a fluorescent protein, an intein protein, or a combination of these polypeptides. 
     Introduction of a polypeptide affinity tag within the N-terminal tail and/or C-terminal tail of the precursor polypeptide results in macrocyclic peptides fused to such polypeptide affinity tag. Such affinity tags can be useful for isolating, purifying, and/or immobilizing onto a solid support the macrocyclic peptides generated according to the methods disclosed herein. Accordingly, in some embodiments, the N-terminal tail, C-terminal tail, or both, of the precursor polypeptides comprise at least one polypeptide affinity tags selected from the group consisting of a polyarginine tag (e.g., RRRRR) (SEQ ID NO: 121), a polyhistidine tag (e.g., HHHHHH) (SEQ ID NO: 122), an Avi-Tag (SGLNDIFEAQKIEWHELEL) (SEQ ID NO: 123), a FLAG tag (DYKDDDDK) (SEQ ID NO: 124), a Strep-tag II (WSHPQFEK) (SEQ ID NO: 125), a c-myc tag (EQKLISEEDL) (SEQ ID NO: 126), a S tag (KETAAAKFERQHMDS) (SEQ ID NO: 127), a calmodulin-binding peptide (KRRWKKNFIAVSAANRFKKISSSGAL) (SEQ ID NO: 128), a streptavidin-binding peptide (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP) (SEQ ID NO: 129), a chitin-binding domain (SEQ ID NO:130), a glutathione S-transferase (GST; SEQ ID NO:131), a maltose-binding protein (MBP; SEQ ID NO:132), streptavidin (SEQ ID NO:133), and engineered variants thereof. These aspects are illustrated in Example 2. 
     The N-terminal tail, (AA) m , the C-terminal tail, (AA) p , or both, in the precursor polypeptides of formula (I), (II), and (V) can comprise a reporter protein or enzyme. This approach will result in the formation of macrocyclic peptides fused to a reporter protein or enzyme, which can be useful to facilitate the functional screening of said macrocyclic peptides. Accordingly, in some embodiments, the N-terminal tail, (AA) m  and/or the C-terminal tail, (AA) p , in the precursor polypeptides of formula (I), (II), and (V) comprise at least one polypeptide selected from the group consisting of green fluorescent protein (SEQ ID NO: 134), luciferase (SEQ ID NO: 135), alkaline phosphatase (SEQ ID NO: 136), and engineered variants thereof. 
     The N-terminal tail, (AA) m , the C-terminal tail, (AA) p , or both, in the precursor polypeptides of formula (I), (II), or (V) can comprise a protein or enzyme that is part of a display system such as, for example, a phage display (e.g. M13, T7, or lambda phage display), a yeast display, a bacterial display, a DNA display, a plasmid display, a CIS display, a ribosome display, or a mRNA display system. As mentioned above, this approach can be useful for generating large libraries of macrocyclic peptides which are physically linked to, or compartmentalized with the polynucleotide sequence that encodes for the corresponding precursor polypeptides. In turn, this approach can be useful toward isolating functional macrocyclic peptides that are able to bind, inhibit or activate a certain target biomolecule (e.g. protein, enzyme, DNA or RNA molecule) or target biomolecular interaction. 
     Accordingly, in some embodiments, the N-terminal tail, (AA) m , comprises a polypeptide selected from the group consisting of M13 phage coat protein pVI (SEQ ID NO:137), T7 phage protein 10A (SEQ ID NO:138), T7 phage protein 10B (SEQ ID NO:139),  E. coli  NlpA (SEQ ID NO: 140),  E. coli  OmpC (SEQ ID NO: 141),  E. coli  FadL (SEQ ID NO: 142),  E. coli  Lpp-OmpA (SEQ ID NO: 143),  E. coli  PgsA (SEQ ID NO: 144),  E. coli  EaeA (SEQ ID NO:145),  S. cerevisiae  Aga2p (SEQ ID NO:146),  S. cerevisiae  Flolp (SEQ ID NO: 147),  S. cerevisiae  Cwplp (SEQ ID NO:217),  S. cerevisiae  Cwp2p (SEQ ID NO:218),  S. cerevisiae  Tiplp (SEQ ID NO:219),  S. cerevisiae  Sedlp (SEQ ID NO:220),  S. cerevisiae  YCR89w (SEQ ID NO:221),  S. cerevisiae  Tirl (SEQ ID NO:222), human NF-κB p50 protein (SEQ ID NO: 148), M13 phage coat protein pIII leader sequence (SEQ ID NO: 149), M13 phage coat protein pVIII leader sequence (SEQ ID NO:150), M13 phage protein pVI (SEQ ID NO:151), Snap-tag (SEQ ID NO: 152), Clip-Tag (SEQ ID NO: 153), a pelB leader sequence (SEQ ID NO:216) and engineered variants thereof. 
     In other embodiments, the C-terminal tail, (AA) p , comprises a polypeptide selected from the group consisting of M13 phage coat protein pIII (SEQ ID NO: 154), M13 phage coat protein pVIII (SEQ ID NO:155), M13 phage coat protein pIX (SEQ ID NO:214), M13 phage coat protein pVII (SEQ ID NO:215), RepA protein (SED ID NO: 156),  S. cerevisiae  Agalp (SEQ ID NO:157), Snap-tag (SEQ ID NO:152), Clip-Tag (SEQ ID NO:153), P2A protein (SED ID NO: 158), and engineered variants thereof. 
     In other embodiments, the C-terminal tail, (AA) p , comprises a molecule selected from the group consisting of puromycin, puromycin analog, a puromycin-DNA conjugate, and a puromycin-RNA conjugate. 
     The N-terminal tail, (AA) m , the C-terminal tail, (AA) p , or both, can comprise a barcode sequence. In some embodiments, the barcode comprises a unique sequence that allows for identification of individual samples in a multiplexed assay. A “barcode”, as used herein, refers to a nucleotide sequence that serves as a means of identification for sequenced polynucleotides of the present invention. Barcodes of the present invention may comprise at least 4 random bases, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more bases in length. Alternatively, or in addition to the random nucleotides, the barcode may have three or more fixed bases, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more bases in length. In some embodiments, both random and fixed bases are used as barcodes. For example, a barcode can be composed of 5 random bases and 4 fixed bases. Methods for designing barcodes are known in the art. See, e.g., Bystrykh (2012) PLoS ONE, 7(5): e36852; Mir et al., (2013) PLoS ONE, 8(12): e82933. In some embodiments, the nucleic acid molecule encoding a macrocyclic polypeptide comprises at least two barcode sequences. In one embodiment, one of the at least two barcode sequences is located upstream of a gene encoding for a precursor polypeptide, and one of the at least two barcode sequences is located downstream of a gene encoding for a precursor polypeptide. 
     In some embodiments, the methods described herein allow one to generate combinatorial libraries of macrocyclic peptides that are fused to a N-terminal and/or C-terminal sequence. In some embodiments, the macrocyclic peptides are fused via a linker. In some embodiments, the linker is 1 to 30 or more amino acids in length and can be a small neutral polar or non-polar amino acid such as glycine, cysteine, serine, or threonine. An exemplary linker has an amino acid sequence Lys-Xaa-Xaa, wherein Xaa is a small neutral polar or nonpolar amino acid. In some embodiments, Xaa is glycine. In some embodiments, the linker sequence is flexible so as not to hold the macrocyclic peptide in a single rigid conformation. The linker sequence can be used, for example, to space the macrocyclic peptide from another domain. In some embodiments, the linker is cleavable, for example, to facilitate separation of a C-terminal tag from the macrocyclic peptide. In some embodiments, a protease cleavage site can be included between the macrocyclic peptide and another domain (e.g., a tag). Examples of such protease cleavage sites include, but are not limited to, Factor Xa and tobacco etch virus (TEV) protease cleavage sites. 
     The N-terminal tail, (AA) m , the C-terminal tail, (AA) p , or both, in the precursor polypeptides of formula (I), (II), or (V) can comprise an intein protein. Inteins are polypeptides that are found as in-frame insertions in various natural proteins and can undergo a self-catalyzed intramolecular rearrangement leading to self-excision (self-splicing) of the intein and ligation of the flanking polypeptides together. The mechanism of intein splicing is well known (Xu and Perler 1996; Paulus 2000) and it involves the formation of a (thio)ester bond at the junction between the intein and the polypeptide fused the N-terminus of the intein (commonly referred to as “N-extein”) by action of a catalytic cysteine or serine residue at the first position of the intein sequence. This reversible N(backbone)→S(side-chain) or a N(backbone)→O(side-chain) acyl transfer is followed by a trans(thio)esterification step whereby the N-extein acyl unit is transferred to the side-chain thiol/hydroxyl group of a cysteine, serine, or threonine residue at the first position of the polypeptide fused the C-terminus of the intein (“C-extein”). The last step of the intein self-splicing process involves cleavage of the peptide bond connecting the intein to the C-extein via an intramolecular transamidation reaction by action of a conserved catalytic asparagine residue at the C-terminal position of the intein sequence (Paulus 2000). 
     Knowledge of the splicing mechanism of intein has enabled the preparation of engineered inteins with altered splicing behavior (Perler 2005; Xu and Evans 2005; Elleuche and Poggeler 2010). For example, it is known that removal of the conserved asparagine residue at the C-terminus of the intein sequence can result in an engineered intein protein capable of only N-terminal splicing (i.e. cleavage of the peptide bond between the N-extein and the intein), which can occurs spontaneously (i.e. via hydrolysis of N-terminal (thio)ester bond) or upon incubation with a thiol reagent (e.g. thiophenol, benzylmercaptan, dithiothreitol, sodium 2-sulfanylethanesulfonate), depending on the nature of the intein and of the C-terminal amino acid(s) in the N-extein sequence. Similarly, removal of the conserved cysteine or serine residue at the N-terminus of the intein sequence can result in an engineered intein protein capable of only C-terminal splicing (i.e. cleavage of the peptide bond between the intein and C-extein), which can occurs spontaneously or promoted via a change in pH or temperature, depending on the nature of the intein and of the N-terminal amino acid(s) in the C-extein sequence. Furthermore, certain intein proteins occur as split inteins, having an N-domain and C-domain. Upon association of the N-domain with the C-domain, split inteins acquires the ability to self-splice according to a mechanism analogous to single-polypeptide intein proteins (Mootz 2009). As for the latter, the N-terminal cysteine or serine residue and C-terminal asparagine residue can be mutated, resulting in altered splicing behavior as described above (Perler 2005; Xu and Evans 2005; Mootz 2009; Elleuche and Poggeler 2010). 
     According to the methods described herein, introduction of a natural or engineered intein protein within the N-terminal tail, (AA) m , or C-terminal tail, (AA) p , of the precursor polypeptide of formula (I), (II), or (V) results in the formation of a macrocyclic peptide that is fused to either the C-terminus or the N-terminus, respectively, of such natural or engineered intein. This aspect enables one to control and modulate the release of the macrocyclic peptide from the intein-fused polypeptide based on the self-splicing and altered splicing behavior of natural and engineered intein proteins as summarized above. This aspect can be useful to facilitate the isolation and characterization of the macrocyclic peptide from a complex mixture such as, for example, the lysate of a cell expressing the precursor polypeptide or a cell-free translation system. This aspect can also be useful to facilitate the accumulation, and if desired, control the formation of a target macrocyclic peptide, prepared according the methods described herein, inside a cell-based expression host. In turn, this capability can facilitate the functional screening of in vivo (i.e. in-cell) produced macrocyclic peptide libraries, prepared according the methods disclosed herein, using an intracellular reporter system or a selection system as described above. These aspects are illustrated by Examples 4-8. 
     Nucleotide sequences encoding for intein proteins that can be used can be derived from naturally occurring inteins and engineered variants thereof. A rather comprehensive list of such inteins is provided by the Intein Registry (http://www.neb.com/neb/inteins.html). Inteins that can be used include, but are not limited to, any of the naturally occurring inteins from organisms belonging to the Eucarya, Eubacteria, and Archea. Among these, for example, inteins of the GyrA group (e.g., Mxe GyrA, Mfl GyrA, Mgo GyrA, Mkas GyrA, Mle-TN GyrA, Mma GyrA), DnaB group (e.g., Ssp DnaB, Mtu-CDC1551 DnaB, Mtu-H37Rv DnaB, Rma DnaB), RecA group (e.g., Mtu-H37Rv RecA, Mtu-So93 RecA), RIR1 group (e.g., Mth RIR1, Chy RIR1, Pfu RIR1-2, Ter RIR1-2, Pab RIR1-3), and Vma group (e.g., Sce Vma, Ctr Vma), intein Mxe GyrA (SEQ ID NO: 1) and the engineered ‘mini Ssp DnaB (‘eDnaB’, SEQ ID NO:2) can be used. 
     Intein proteins suitable in the methods described herein include, but are not limited to, engineered variants of natural inteins (or genetic fusion of split inteins), which have been modified by mutagenesis in order, for example, to prevent or minimize splicing at the N-terminal or C-terminal end of the intein. Examples of these modifications include, but are not limited to, mutation of the conserved cysteine or serine residue at the N-terminus of the intein (e.g., via substitution to an alanine) with the purpose, for example, of preventing cleavage at the N-terminus of the intein. Examples of these modifications include, but are not limited to, mutation of the conserved asparagine residue at the C-terminus of the intein (e.g., via substitution to an alanine) with the purpose, for example, of preventing cleavage at the C-terminus of the C-terminus of the intein. Examples of these modifications are provided in Example 2. Intein variants useful for the methods disclosed herein also include, but are not limited to, engineered inteins whose internal endonuclease domain, which is not essential for the splicing mechanism, is removed. For example, a variant of Ssp DnaB (‘eDnaB’, SEQ ID NO:2) lacking the internal endonuclease domain is used for the preparation of the precursor polypeptides. Inteins to be comprised in the precursor polypeptide can also be engineered with the purpose, for example, of altering the splicing properties of the intein in order to increase or reduce the splicing efficiency or in order to make the intein-catalyzed splicing process dependent upon variation of certain parameters such as pH or temperature. 
     Accordingly, in some embodiments, the N-terminal tail, (AA) m , the C-terminal tail, (AA) p , or both, in the precursor polypeptides of formula (I), (II), and (V) comprise an intein protein, or an engineered variant thereof. In some embodiments, the N-terminal tail, (AA) m , the C-terminal tail, (AA) p , or both, in the precursor polypeptides of formula (I), (II), and (V) comprise an intein protein selected from the group consisting of Mxe GyrA (SEQ ID NO: 1), eDnaB (SEQ ID NO:2), Hsp-NRC1 CDC21 (SEQ ID NO:3), Ceu ClpP (SEQ ID NO:4), Tag Pol-1 (SEQ ID NO:5), Tfu Pol-1 (SEQ ID NO:6), Tko Pol-1 (SEQ ID NO:7), Psp-GBD Pol (SEQ ID NO:8), Tag Pol-2 (SEQ ID NO:9), Thy Pol-1 (SEQ ID NO:10), Tko Pol-2 (SEQ ID NO:11), Tli Pol-1 (SEQ ID NO: 12), Tma Pol (SEQ ID NO: 13), Tsp-GE8 Pol-1 (SEQ ID NO:14), Tthi Pol (SEQ ID NO:15), Tag Pol-3 (SEQ ID NO:16), Tfu Pol-2 (SEQ ID NO:17), Thy Pol-2 (SEQ ID NO:18), Tli Pol-2 (SEQ ID NO:19), Tsp-GE8 Pol-2 (SEQ ID NO:20), Pab Pol-II (SEQ ID NO:21), Mtu-CDC1551 DnaB (SEQ ID NO:22), Mtu-H37Rv DnaB (SEQ ID NO:23), Rma DnaB (SEQ ID NO:24), Ter DnaE-1 (SEQ ID NO:25), Ssp GyrB (SEQ ID NO:26), Mfl GyrA (SEQ ID NO:27), Mgo GyrA (SEQ ID NO:28), Mkas GyrA (SEQ ID NO:29), Mle-TN GyrA (SEQ ID NO:30), Mma GyrA (SEQ ID NO:31), Ssp DnaX (SEQ ID NO:32), Pab Lon (SEQ ID NO:33), Mja PEP (SEQ ID NO:34), Afu-FRRO163 PRP8 (SEQ ID NO:35), Ani-FGSCA4 PRP8 (SEQ ID NO:36), Cne-A PRP8 (SEQ ID NO:37), Hca PRP8 (SEQ ID NO:38), Pch PRP8 (SEQ ID NO:39), Pex PRP8 (SEQ ID NO:40), Pvu PRP8 (SEQ ID NO:41), Mtu-H37Rv RecA (SEQ ID NO:42), Mtu-So93 RecA (SEQ ID NO:43), Mfl RecA (SEQ ID NO:44), Mle-TN RecA (SEQ ID NO:45), Nsp-PCC7120 RIR1 (SEQ ID NO:46), Ter RIR1-1 (SEQ ID NO:47), Pab RIR1-1 (SEQ ID NO:48), Pfu RIR1-1 (SEQ ID NO:49), Chy RIR1 (SEQ ID NO:50), Mth RIR1 (SEQ ID NO:51), Pab RIR1-3 (SEQ ID NO:52), Pfu RIR1-2 (SEQ ID NO:53), Ter RIR1-2 (SEQ ID NO:54), Ter RIR1-4 (SEQ ID NO:55), CIV RIR1 (SEQ ID NO:56), Ctr VMA (SEQ ID NO:57), Sce VMA (SEQ ID NO:58), Tac-ATCC25905 VMA (SEQ ID NO:59), Ssp DnaB (SEQ ID NO:60), engineered variants thereof, and engineered variants thereof wherein the N-terminal cysteine or serine residue of the engineered variant is mutated to any of the natural amino acid residues other than cysteine or serine, or wherein the C-terminal asparagine residue of the engineered variant is mutated to any of the natural amino acid residues other than asparagine. 
     In some embodiments, the N-terminal tail, (AA) m , the C-terminal tail, (AA) p , or both, in the precursor polypeptides of formula (I), (II), and (V) comprise the N-domain, C-domain, or both the N-domain and C-domain of a split intein, or an engineered variant thereof. In some embodiments, the N-terminal tail, (AA) m , the C-terminal tail, (AA) p , or both, in the precursor polypeptides of formula (I), (II), and (V) comprise the N-domain, C-domain, or both the N-domain and C-domain of a split intein selected from the group consisting of Ssp DnaE (SEQ ID NO:61-SEQ ID NO:62), Neq Pol (SEQ ID NO:63-SEQ ID NO:64), Asp DnaE (SEQ ID NO:65-SEQ ID NO:66), Npu-PCC73102 DnaE (SEQ ID NO:67-SEQ ID NO:68), Nsp-PCC7120 DnaE (SEQ ID NO:69-SEQ ID NO:70), Oli DnaE (SEQ ID NO:71-SEQ ID NO:72), Ssp-PCC7002 DnaE (SEQ ID NO:73-SEQ ID NO:74), Tvu DnaE (SEQ ID NO:75-SEQ ID NO:76), engineered variants thereof, and engineered variants wherein the N-terminal cysteine or serine residue of the split intein N-domain of the engineered variant is mutated to any of the natural amino acid residues other than cysteine or serine, or wherein the C-terminal asparagine residue of the split intein C-domain of the engineered variant is mutated to any of the natural amino acid residues other than asparagine. 
     In some embodiments, the N-terminal tail, (AA) m , in the precursor polypeptides of formula (I), (II), and (V) comprises the C-domain of a split intein and the C-terminal tail, (AA) p , of said precursor polypeptides comprises the corresponding N-domain of the split intein. In some embodiments, the N-terminal tail, (AA) m , in the precursor polypeptides of formula (I), (II), and (V) comprises the C-domain of a split intein selected from the group consisting of Ssp DnaE-c (SEQ ID NO:62), Neq Pol-c (SEQ ID NO:64), Asp DnaE-c (SEQ ID NO:66), Npu-PCC73102 DnaE-c (SEQ ID NO:68), Nsp-PCC7120 DnaE-c (SEQ ID NO: 70), Oli DnaE-c (SEQ ID NO:72), Ssp-PCC7002 DnaE-c (SEQ ID NO:74), Tvu DnaE-c (SEQ ID NO:76), and engineered variants thereof; and the C-terminal tail, (AA) p , comprises the corresponding N-domain of the split intein selected from the group consisting of Ssp DnaE-n (SEQ ID NO:61), Neq Pol-n (SEQ ID NO:63), Asp DnaE-n (SEQ ID NO:65), Npu-PCC73102 DnaE-n (SEQ ID NO:67), Nsp-PCC7120 DnaE-n (SEQ ID NO:69), Oli DnaE-n (SEQ ID NO:71), Ssp-PCC7002 DnaE-n (SEQ ID NO:73), Tvu DnaE-n (SEQ ID NO:75), and engineered variants thereof. 
     5.3 Polynucleotides and Host Cells for Expression of Precursor Polypeptides 
     In another aspect, polynucleotide molecules are provided encoding for precursor polypeptides of formula (I), (II), and (V) as defined above. Polynucleotide molecules are provided for encoding for the aminoacyl-tRNA synthetases and cognate tRNA molecules for the ribosomal incorporation of the amino acid Z into the precursor polypeptides of formula (I) and (II) and for the ribosomal incorporation of the amino acid Z2 into the precursor polypeptides of formula (V). Polynucleotide molecules are provided encoding for polypeptide sequences that can be introduced within the N-terminal tail ((AA) m ) or C-terminal tail ((AA) p ) of the precursor polypeptides of formula (I), (II) and (V), such as peptide and protein affinity tags, reporter proteins and enzymes, carrier proteins of a display system, and intein proteins, as described above. Since the correspondence of all the possible three-base codons to the various amino acids is known, providing the amino acid sequence of the polypeptide provides also a description of all the polynucleotide molecules encoding for such polypeptide. Thus, a person skilled in the art will be able, given a certain polypeptide sequence, to generate any number of different polynucleotides encoding for the same polypeptide. In some embodiments, the codons are selected to fit the host cell in which the polypeptide is being expressed. For example, codons used in bacteria can be used to express the polypeptide in a bacterial host. The polynucleotides may be linked to one or more regulatory sequences controlling the expression of the polypeptide-encoding gene to form a recombinant polynucleotide capable of expressing the polypeptide. 
     Numerous methods for making nucleic acids encoding for polypeptides having a predetermined or randomized sequence are known to those skilled in the art. For example, oligonucleotide primers having a predetermined or randomized sequence can be prepared chemically by solid phase synthesis using commercially available equipments and reagents. Polynucleotide molecules can then be synthesized and amplified using a polymerase chain reaction, digested via endonucleases, ligated together, and cloned into a vector according to standard molecular biology protocols known in the art (e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (Third Edition), Cold Spring Harbor Press, 2001). These methods, in combination with the mutagenesis methods mentioned above, can be used to generate polynucleotide molecules that encode for the aforementioned polypeptides as well as suitable vectors for the expression of these polypeptides in a host expression system. 
     The precursor polypeptides can be produced by introducing said polynucleotides into an expression vector, by introducing the resulting vectors into an expression host, and by inducing the expression of the encoded precursor polypeptides in the presence of the amino acid Z (or Z2) and, whenever necessary, also in the presence of a suitable stop codon or frameshift codon suppression system for mediating the incorporation of the amino acid Z (or Z2) into the precursor polypeptides. 
     Nucleic acid molecules can be incorporated into any one of a variety of expression vectors suitable for expressing a polypeptide. Suitable vectors include, but are not limited to, chromosomal, nonchromosomal, artificial and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, pseudorabies, adenovirus, adeno-associated viruses, retroviruses and many others. Any vector that transduces genetic material into a cell, and, if replication is desired, which is replicable and viable in the relevant host can be used. A large number of expression vectors and expression hosts are known in the art, and many of these are commercially available. A person skilled in the art will be able to select suitable expression vectors for a particular application, e.g., the type of expression host (e.g., in vitro systems, prokaryotic cells such as bacterial cells, and eukaryotic cells such as yeast, insect, or mammalian cells) and the expression conditions selected. 
     Expression hosts that may be used for the preparation of the precursor polypeptides and macrocyclic peptides include, but are not limited to, any systems that support the transcription, translation, and/or replication of a nucleic acid. In some embodiments, the expression host system is a cell or a cell lysate. Host cells for use in expressing the polypeptides encoded by the expression vector of this disclosure are well known in the art and include, but are not limited to, bacterial cells (e.g.,  Escherichia coli, Streptomyces  sp.,  Bacillus  sp.); fungal cells, including yeast cells (e.g.,  Saccharomyces cerevisiae, Pichia pastoris ); insect cells; plant cells; and animal cells, such as mammalian cells and human cells. Other expression systems include lysates of prokaryotic cells (e.g., bacterial cells) and lysates of eukaryotic cells (e.g., yeast, insect, or mammalian cells). In preferred embodiments, the expression host system is a cell. In some embodiments, the expression host system is the same as the host display organism. This is the case whenever the macrocyclic peptide is anchored on the outer biological surface of a cell, such as a bacterial cell, a yeast cell, an insect cell, or a mammalian cell. In other embodiments, the expression host system is different from the host display organism. This is the case whenever the macrocyclic peptide is anchored on the outer biological surface of a viral particle, such as a bacteriophage particle. In this case, the expression host system is a cell and the host display organism is the viral particle. 
     The choice of the expression vector, host expression system, and host display organism depends on the type of application intended for the methods disclosed herein and a person skilled in the art will be able to select a suitable expression host based on known features and application of the different expression hosts and host display organisms. 
     In some embodiments, the macrocyclic peptide is fused to a presentation polypeptide that is part of the outer surface of a viral particle, so that the macrocyclic peptide is produced as tethered to the outer surface of the viral particle. This method comprises providing a nucleic acid encoding for the precursor polypeptide, wherein the N- or C-terminal tail comprises the presentation polypeptide, or a fragment thereof, introducing the nucleic acid encoding for the macrocyclic peptide and for the viral particle into an expression host, allowing for the precursor polypeptide to be integrated into the viral particle, and allowing for the precursor polypeptide to undergo intramolecular cyclization via a bond-forming reaction between the side-chain sulfhydryl group of the cysteine and the FG 1  group of the amino acid Z (or between the cysteines and the FG 1  and FG 2  groups of the amino acid Z2), thereby producing the macrocyclic peptide as tethered to the outer surface of the viral particle. 
     In some embodiments, the macrocyclic peptide is fused to a presentation polypeptide that is part of the outer surface of a cell, so that the macrocyclic peptide is produced as tethered to the outer surface of the cell. This method comprises providing a nucleic acid encoding for the precursor polypeptide, wherein the N- or C-terminal tail comprises the presentation polypeptide, or a fragment thereof, introducing the nucleic acid into the cell-based host display organism, which also serves as the expression host, inducing the expression of the precursor polypeptide, allowing for the precursor polypeptide to be integrated into the outer surface of the cell, and allowing for the precursor polypeptide to undergo intramolecular cyclization via a bond-forming reaction between the side-chain sulfhydryl group of the cysteine and the FG 1  group of the amino acid Z (or between the cysteines and the FG 1  and FG 2  groups of the amino acid Z2), thereby producing the macrocyclic peptide as tethered to the outer surface of the cell. 
     In some embodiments, the formation of the macrocyclic peptides from the biosynthetic polypeptides as defined above is carried out within the cell-based expression host that produces the precursor polypeptides, so that the macrocyclic peptides are produced within this cell-based expression host. This method comprises providing a nucleic acid encoding for the precursor polypeptide, introducing the nucleic acid into the cell-based expression host, inducing the expression of the precursor polypeptide, allowing for the precursor polypeptide to undergo intramolecular cyclization via a bond-forming reaction between the side-chain sulfhydryl group of the cysteine and the FG 1  group of the amino acid Z (or between the cysteines and the FG 1  and FG 2  groups of the amino acid Z2), thereby producing the macrocyclic peptide inside the cell-based expression host. These aspects are illustrated in Examples 4 through 8. 
     In some embodiments, the formation of the macrocyclic peptides from the biosynthetic polypeptides as defined above is carried out on the surface of a cell or on a viral particle, so that the macrocyclic peptides are produced as tethered to a cell or a viral particle, respectively. This method comprises providing a nucleic acid encoding for the precursor polypeptide, wherein the N- or C-terminal tail comprises a polypeptide component of the cell membrane (e.g.  S. cerevisiae  membrane protein Aga2p) or of the viral particle (e.g. M13 phage pII protein), introducing the nucleic acid into the expression host, inducing the expression of the precursor polypeptide, allowing for the precursor polypeptide to be integrated into the cell membrane or viral particle, and allowing for the precursor polypeptide to undergo intramolecular cyclization via a bond-forming reaction between the side-chain sulfhydryl group of the cysteine and the FG 1  group of the amino acid Z (or between the cysteines and the FG 1  and FG 2  groups of the amino acid Z2), thereby producing the macrocyclic peptide as tethered to the membrane of the cell or to the viral particle. 
     In some embodiments, the formation of the macrocyclic peptides from the biosynthetic polypeptides as defined above is carried out within a cell-free expression system, so that the macrocyclic peptides are produced within this cell-free expression system. This method comprises providing a nucleic acid encoding for the precursor polypeptide, introducing the nucleic acid into the cell-free expression host, inducing the expression of the precursor polypeptide, allowing for the precursor polypeptide to undergo intramolecular cyclization via a bond-forming reaction between the side-chain sulfhydryl group of the cysteine and the FG 1  group of the amino acid Z (or between the cysteines and the FG 1  and FG 2  groups of the amino acid Z2), thereby producing the macrocyclic peptide within the cell-free expression host. 
     A method is also provided for making a library of macrocyclic peptides via cyclization of a plurality of precursor polypeptides of formula (I) or (II) that contain an heterogeneous peptide target sequence (AA) n , or an heterogeneous N-terminal tail (AA) m , or an heterogeneous C-terminal tail (AA) p , or a combination of these. This method comprises: (a) constructing a plurality of nucleic acid molecules encoding for a plurality of precursor polypeptides, said precursor polypeptides having an heterogeneous peptide target sequence (AA) n , or an heterogeneous N-terminal tail (AA) m , or an heterogeneous C-terminal tail (AA) p , or a combination of these; (b) introducing each of the plurality of said nucleic acid molecules into an expression vector, and introducing the resulting vectors into an expression host; (c) expressing the plurality of precursor polypeptides; (d) allowing for the precursor polypeptides to undergo intramolecular cyclization via a bond-forming reaction between the side-chain sulfhydryl group of the cysteine and the FG 1  group of the amino acid Z, thereby producing a plurality of macrocyclic peptides. 
     A method is also provided for making a library of macrocyclic peptides via cyclization of a plurality of precursor polypeptides of formula (V) that contain an heterogeneous peptide target sequence (AA) n , or an heterogeneous second peptide target sequence (AA) o , or an heterogeneous N-terminal tail (AA) m , or an heterogeneous C-terminal tail (AA) p , or a combination of these. This method comprises: (a) constructing a plurality of nucleic acid molecules encoding for a plurality of precursor polypeptides, said precursor polypeptides having an heterogeneous peptide target sequence (AA) n , or an heterogeneous second peptide target sequence (AA) o , or an heterogeneous N-terminal tail (AA) m , or an heterogeneous C-terminal tail (AA) p , or a combination of these; (b) introducing each of the plurality of said nucleic acid molecules into an expression vector, and introducing the resulting vectors into an expression host; (c) expressing the plurality of precursor polypeptides; (d) allowing for the precursor polypeptides to undergo intramolecular cyclization via a bond-forming reaction between the side-chain sulfhydryl group of the cysteines and the FG 1  and FG 2  group2 of the amino acid Z2, thereby producing a plurality of macrocyclic peptides. 
     In specific embodiments, each of the plurality of macrocyclic peptides prepared as described above is tethered to a cell component, to a cell membrane component, to a bacteriophage, to a viral particle, or to a DNA molecule, via a polypeptide comprised within the N-terminal tail or within the C-terminal tail of said macrocyclic peptide molecule. 
     Therefore, in some embodiments, a method is provided for making a display library of macrocyclic peptides via cyclization of a plurality of precursor polypeptides of formula (I) or (II), which contain an heterogeneous peptide target sequence (AA), or an heterogeneous N-terminal tail (AA) m , or an heterogeneous C-terminal tail (AA) p , or a combination of these, and which are fused to a presentation polypeptide. This method comprises: (a) constructing a plurality of nucleic acid molecules encoding for a plurality of precursor polypeptides fused to a presentation polypeptide, said precursor polypeptides having an heterogeneous peptide target sequence (AA) n , or an heterogeneous N-terminal tail (AA) m , or an heterogeneous C-terminal tail (AA) p , or a combination of these; (b) introducing each of the plurality of said nucleic acid molecules into an expression vector, and introducing the resulting vectors into an expression host; (c) expressing the plurality of precursor polypeptides, each of which is fused to the presentation polypeptide; (d) allowing for the precursor polypeptide to be integrated into the outer surface of the host display organism; and (e) allowing for the precursor polypeptides to undergo intramolecular cyclization via a bond-forming reaction between the side-chain sulfhydryl group of the cysteine and the FG 1  group of the amino acid Z, thereby producing a plurality of macrocyclic peptides, each of which is anchored to the outer biological surface of the host display organism. 
     A method is also provided for making a display library of macrocyclic peptides via cyclization of a plurality of precursor polypeptides of formula (V) that contain an heterogeneous peptide target sequence (AA) n , or an heterogeneous second peptide target sequence (AA) o , or an heterogeneous N-terminal tail (AA) m , or an heterogeneous C-terminal tail (AA) p , or a combination of these, and which are fused to a presentation polypeptide. This method comprises: (a) constructing a plurality of nucleic acid molecules encoding for a plurality of precursor polypeptides, said precursor polypeptides having an heterogeneous peptide target sequence (AA) n , or an heterogeneous second peptide target sequence (AA) o , or an heterogeneous N-terminal tail (AA) m , or an heterogeneous C-terminal tail (AA) p , or a combination of these; (b) introducing each of the plurality of said nucleic acid molecules into an expression vector, and introducing the resulting vectors into an expression host; (c) expressing the plurality of precursor polypeptides, each of which is fused to the presentation polypeptide; (d) allowing for the precursor polypeptide to be integrated into the outer surface of the host display organism; and (e) allowing for the precursor polypeptides to undergo intramolecular cyclization via a bond-forming reaction between the side-chain sulfhydryl group of the cysteines and the FG 1  and FG 2  group2 of the amino acid Z2, thereby producing a plurality of macrocyclic peptides, each of which is anchored to the outer biological surface of the host display organism. 
     In some embodiments, a macrocyclic peptide library display system is provided. This macrocyclic peptide library display system includes a plurality of macrocyclic peptides, each of which is produced as a fusion to a presentation polypeptide anchored to the outer biological surface of a host display organism according to the methods disclosed herein. In some embodiments, the host display organism is selected from a viral particle or a cell. In preferred embodiments, the host display organism is a bacteriophage particle. In other preferred embodiments, the host display organism is a yeast cell. In specific embodiments, each of the plurality of macrocyclic peptides prepared as described above is fused to an outer surface component selected from a group consisting of a cell component, a cell membrane component, a cell wall component, a viral coat protein, and a bacteriophage coat protein. In some embodiments, the nucleic acid molecule encoding for the library of macrocyclic peptides fused to the presentation polypeptide is expressed from an expression system selected from a group consisting of phagemid, a plasmid, a cosmid, and a chromosome. 
     Several methods of making polynucleotides encoding for heterogeneous peptide sequences are known in the art. These include, among many others, methods for site-directed mutagenesis (Botstein, D.; Shortle, D. Science (New York, N.Y., 1985, 229, 1193; Smith, M. Annual review of genetics, 1985, 19, 423; Dale, S. J.; Felix, I. R. Methods in molecular biology (Clifton, N.J., 1996, 57, 55; Ling, M. M.; Robinson, B. H. Analytical biochemistry, 1997, 254, 157), oligonucleotide-directed mutagenesis (Zoller, M. J. Current opinion in biotechnology, 1992, 3, 348; Zoller, M. J.; Smith, M. Methods Enzymol, 1983, 100, 468; Zoller, M. J.; Smith, M. Methods Enzymol, 1987, 154, 329), mutagenesis by total gene synthesis and cassette mutagenesis (Nambiar, K. P.; Stackhouse, J.; Stauffer, D. M.; Kennedy, W. P.; Eldredge, J. K.; Benner, S. A. Science (New York, N.Y., 1984, 223, 1299; Grundstrom, T.; Zenke, W. M.; Wintzerith, M.; Matthes, H. W.; Staub, A.; Chambon, P. Nucleic acids research, 1985, 13, 3305; Wells, J. A.; Vasser, M.; Powers, D. B. Gene, 1985, 34, 315), and the like. Additional methods are described in the following U.S. patents, PCT publications, and EPO publications: U.S. Pat. No. 5,605,793 “Methods for In vitro Recombination”, U.S. Pat. No. 5,830,721 “DNA Mutagenesis by Random Fragmentation and Reassembly”, WO 95/22625 “Mutagenesis by Random Fragmentation and Reassembly”, WO 96/33207 “End Complementary Polymerase Chain Reaction”, EP 752008 “DNA Mutagenesis by Random Fragmentation and Reassembly”, WO 98/27230 “Methods and Compositions for Polypeptide Engineering”, WO 00/00632, “Methods for Generating Highly Diverse Libraries”, WO 98/42832 “Recombination of Polynucleotide Sequences Using Random or Defined Primers”, WO 99/29902 “Method for Creating Polynucleotide and Polypeptide Sequences”. Any of these methods or modifications thereof can be utilized for generating nucleotide molecules that encode for precursor polypeptides of formula (I), (II), or (V) which are fused to presentation polypeptide and which contain an heterogeneous peptide target sequence (AA) n , an heterogeneous second peptide target sequence (AA) o , an heterogeneous N-terminal tail (AA) m , an heterogeneous C-terminal tail (AA) p , or a combination of these, for the purpose of generating a macrocyclic peptide library display system. 
     The compounds provided herein may contain one or more chiral centers. Accordingly, the compounds are intended to include, but not be limited to, racemic mixtures, diastereomers, enantiomers, and mixture enriched in at least one stereoisomer or a plurality of stereoisomers. When a group of substituents is disclosed herein, all the individual members of that group and all subgroups, including any isomers, enantiomers, and diastereomers are intended to be included in the disclosure. Additionally, all isotopic forms of the compounds disclosed herein are intended to be included in the disclosure. For example, it is understood that any one or more hydrogens in a molecule disclosed herein can be replaced with deuterium or tritium. 
     5.4 Methods for Screening and Selection of Display Libraries of Macrocyclic Peptides 
     A method is provided for obtaining a macrocyclic peptide having a desired property, this method comprising (i) providing the macrocyclic peptide library display system prepared according to the methods described above; and (ii) screening the display library of macrocyclic peptides and/or selecting from said library the member(s) that has/have the desired property. 
     In some embodiments, a method is provided for obtaining a macrocyclic peptide having a desired property, this method comprising, (i) providing a diverse collection of bacteriophage particles, wherein said bacteriophage particles display a macrocyclic peptide on their outer surface and contain the gene encoding for the macrocyclic peptide displayed on their outer surface; (ii) screening the phage display library of macrocyclic peptides and/or selecting from said library the member(s) that has/have the desired property. 
     In some embodiments, a library of polycyclic peptides is generated by fusing together two or more copies of the portion of the nucleic acid molecule encoding for -Z-(AA) n -Cys-, -Cys-(AA) n -Z-, and/or -Cys-(AA) n -Z2-(AA). 
     In some embodiments, a method is provided for obtaining a macrocyclic peptide having a desired property, this method comprising, (i) providing a diverse collection of cells, wherein said cells display a macrocyclic peptide on their outer surface and contain the gene encoding for the macrocyclic peptide displayed on their outer surface; (ii) screening the cell surface display library of macrocyclic peptides and/or selecting from said library the member(s) that has/have the desired property. 
     In some embodiments, the desired property of the macrocyclic peptide is binding to a target molecule. Other desired properties of the macrocyclic peptide include, but are not limited to, blocking the function of a target molecule, blocking or promoting the interaction between a target molecule and another molecule, activating or inhibiting a reaction mediated by a target molecule, activating or inhibiting the activity of an enzyme, and/or activating or inhibiting the activity of a receptor. 
     In some embodiments, a method is provided for obtaining a macrocyclic peptide capable of binding a target molecule of interest, this method comprising, (i) providing a diverse collection of bacteriophage particles, wherein said bacteriophage particles display a macrocyclic peptide on their outer surface and contain the gene encoding for the macrocyclic peptide displayed on their outer surface; (ii) selecting from the phage display library of macrocyclic peptides the member(s) that are capable of binding the target molecule, wherein this step further comprises: (i) contacting the diverse collection of bacteriophage particles with the target molecule; (ii) eluting bacteriophage particles not binding to the target molecule; and (iii) eluting bacteriophage particles binding to the target molecule. 
     In some embodiments, a method is provided for obtaining a macrocyclic peptide capable of binding a target molecule of interest, this method comprising, (i) providing a diverse collection of cells, wherein said cells display a macrocyclic peptide on their outer surface and contain the gene encoding for the macrocyclic peptide displayed on their outer surface; (ii) selecting from the cell surface display library of macrocyclic peptides the member(s) that are capable of binding the target molecule, wherein this step further comprises: (i) contacting the diverse collection of cells with the target molecule; (ii) eluting cells not binding to the target molecule; and (iii) eluting cells binding to the target molecule. 
     In some embodiments, the target molecule is immobilized on a solid support, said solid support consisting of a plate, a slide, a polymeric bead, a magnetic bead, and the like. 
     In some embodiments, a method is provided for obtaining a macrocyclic peptide capable of binding a target molecule of interest, this method comprising, (i) providing a diverse collection of cells, wherein said cells display a macrocyclic peptide on their outer surface and contain the gene encoding for the macrocyclic peptide displayed on their outer surface; (ii) selecting from the cell surface display library of macrocyclic peptides the member(s) that are capable of binding the target molecule, wherein this step further comprises: (i) contacting the diverse collection of cells with the target molecule conjugated to a fluorophore; (ii) sorting the cells binding the target molecule conjugated to a fluorophore. In some embodiments, the cell sorting procedures includes using fluorescence-activate cell sorting. 
     After the library screening or selection procedure, the structure of the ‘hit’ macrocyclic peptides can be readily determined based on sequencing of the gene encoding for the selected macrocyclic peptides and knowledge of the structure of the non-canonical amino acid utilized to generate the macrocyclic peptide display library. 
     In some embodiments, the macrocyclic peptide display library subjected to the screening or selection procedure is generated using a single type of non-canonical amino acid with structure Z (or Z2). In this case, the identity of the non-canonical amino acid in the selected ‘hits’ is predetermined. 
     In other embodiments, the macrocyclic peptide display library subjected to the screening or selection procedure consists of sub-libraries of macrocyclic peptides generated using two or more different types of non-canonical amino acids with structure Z (or Z2). This type of macrocyclic peptide display libraries are referred herein as “multiplexed macrocyclic peptide display libraries” and have the advantage of comprising a broader structural diversity compared to libraries generated using a single non-canonical amino acid Z (or Z2), and/or the advantage of streamlining the screening of different sets of macrocyclic peptide display libraries against a single target. In turn, these features can increase the likelihood of finding a macrocyclic peptide with a desired property. In the case of multiplexed macrocyclic peptide display libraries, after the library screening or selection procedure, it is highly desirable to be able to rapidly deconvolute the identity of the non-canonical amino acid contained in the selected ‘hits’. Accordingly, methods are provided for generating, screening, and decovoluting multiplexed macrocyclic peptide display libraries consisting of multiple macrocyclic peptide display libraries generated using different types of non-canonical aminoacids Z (or Z2). 
     In some embodiments, a method is provided for generating, screening, and deconvoluting a multiplexed macrocyclic peptide display library, this method comprising: (i) providing a multiplexed macrocyclic peptide display library, wherein each of the different non-canonical amino acid Z (or Z2) is encoded by a different codon compared to the codons used for the other non-canonical amino acids Z (or Z2); (ii) screening the multiplexed macrocyclic peptide display library and/or selecting from said library the member(s) that has/have the desired property. According to this method, the identify of the non-canonical amino acid Z (or Z2) in the ‘hits’ selected after the screening or selection procedure is determined based on the identity of the codon corresponding to said non-canonical amino acid in the gene encoding for the macrocyclic peptide. In preferred embodiments, the codon encoding for the amino acid Z (or Z2) in this type of multiplexed macrocyclic peptide display library is selected from a group consisting of the amber stop codon (TAG), the ochre stop codon (TAA), the opal stop codon (TGA), and a four-base frameshift codon. In preferred embodiments, the multiplexed macrocyclic peptide display library is prepared by pooling together multiple macrocyclic peptide display sub-libraries, each sub-library being generated separately using a different non-canonical amino acid Z (or Z2) and an appropriate expression system according to the methods of the invention. In other embodiments, the multiplexed macrocyclic peptide display library is prepared within the same expression system, wherein said expression system contains a translational machinery sufficient for orthogonal incorporation of multiple non-canonical amino acids in response to different codons. 
     In some embodiments, a method is provided for generating, screening, and deconvoluting a multiplexed macrocyclic peptide display library, this method comprising: (i) introducing a unique set of two nucleotide sequences (herein referred to as “barcode nucleotide sequences” or “barcode sequences”). In one embodiment, a barcode sequence can be included upstream, downstream or both of a gene encoding for the precursor polypeptide, each set of barcode sequences corresponding to a different non-canonical amino acid Z (or Z2); (ii) producing separately the barcoded macrocyclic peptide display library using the corresponding non-canonical amino acid Z (or Z2); (iii) pooling together the different barcoded macrocyclic peptide display libraries to generate a multiplexed macrocyclic peptide display library, wherein each sub-library is barcoded; (ii) screening the multiplexed macrocyclic peptide display library and/or selecting from said library the member(s) that has/have the desired property. According to this method, the identify of the non-canonical amino acid Z (or Z2) in the ‘hits’ selected after the screening or selection procedure is determined based on identity of the barcode nucleotide sequences comprised within the vector containing the gene encoding for the macrocyclic peptide. In addition to enabling rapid deconvolution of multiplexed macrocyclic peptide libraries, this method provides the advantage of enabling a high degree of multiplexing (i.e., simultaneous screening of &gt;10 or more sublibraries), since the same suppression technique (i.e. amber stop codon) can be used for incorporating the different non-canonical amino acids and thus generating the sublibraries. In addition, this method allows one to selectively amplify the subset of the library corresponding to a particular Z (or Z2). As multiple iterative rounds of selection and amplification are typically desirable during the library screening process, this method thus allows one to subject a multiplexed library through multiple rounds of selection and amplification. 
     According to the methods disclosed herein, in some embodiments, the host display organism also contains the gene encoding for the macrocyclic peptide displayed on its outer surface, thus providing a physical linkage between the display macrocyclic peptide and the gene encoding for its amino acid sequence. Since the non-canonical amino acid used for macrocyclization is defined by the operator, the overall structure of the displayed macrocyclic peptide is defined (or can be deconvoluted, e.g., after a library screening process) based on knowledge of the structure of the non-canonical amino acid and peptide sequence of the macrocyclic peptide. In some embodiments, the methods disclosed herein can be thus used for generating large and diverse display libraries of macrocyclic peptides which can be screened and readily deconvoluted to identify macrocyclic peptides with a desired property. In some embodiments, the macrocyclic peptides within a large library are linked to a barcode sequence, as described in detail elsewhere herein. 
     The terms and expression that are employed herein are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described and portions thereof, but it is recognized that various modifications are possible within the scope of the subject matter claimed herein. Thus, it should be understood that although various embodiments and optional features have been disclosed herein, modification and variation of the concepts herein disclosed may be resorted to those skilled in the art, and that such modifications and variations are considered to be encompassed by the appended claims. 
     Unless otherwise indicated, the disclosure is not limited to specific molecular structures, substituents, synthetic methods, reaction conditions, or the like, as such may vary. It is to be understood that the embodiments are not limited to particular compositions or biological systems, which can, of course, vary. 
     A skilled artisan will appreciate that starting materials, biological materials, reagents, synthetic methods, purification methods, analytical methods, assay methods, and biological methods other than those specifically exemplified can be employed in the practice of the methods and compositions disclosed herein. All art-known functional equivalents of any such materials and methods are intended to be included in the methods and compositions disclosed herein. 
     6. EXAMPLES 
     The following examples are offered by way of illustration and not by way of limitation. 
     6.1 Example 1: Synthesis of Cysteine-Reactive Unnatural Amino Acids 
     This example demonstrates the preparation of various cysteine-reactive unnatural amino acids, i.e., various Z and Z2 amino acids, which can be used for preparation of macrocyclic peptide molecules according to the general methods illustrated in  FIGS. 1A-B ,  2 A-B,  3 A-B,  4 A-B, and  37 A-B. 
     The unnatural amino acid 4-(2-bromoethoxy)-phenylalanine (1, p-2beF) was prepared according to the synthetic route provide in Scheme 1 of  FIG. 5 . The unnatural amino acid N ε -((2-bromoethoxy)carbonyl)-lysine (2, 2-becK) was prepared according to the synthetic route provide in Scheme 2 of  FIG. 5 . The unnatural amino acid 4-(1-bromoethyl)-phenylalanine (3, p-1beF) was prepared according to the synthetic route provide in Scheme 3 of  FIG. 5 . The unnatural amino acid N ε -((2-chloroethoxy)carbonyl)-lysine (4, 2-cecK) was prepared according to the synthetic route provide in Scheme 4 of  FIG. 6 . The unnatural amino acid N ε -(buta-2,3-dienoyl)-lysine (5, bdnK) was prepared according to the synthetic route provide in Scheme 5 of  FIG. 6 . The bifunctional unnatural amino acid O-(2,3-dibromoethyl)-tyrosine (6, OdbpY) was prepared according to the synthetic route provide in Scheme 6 of  FIG. 6 . A person skilled in the art would readily recognize that many other cysteine-reactive amino acids of general formula (III), (IV), (VI), or (VII) can be prepared in an analogous manner either through modification of naturally occurring amino acids (e.g., p-2beF, 2-becK, 2-cecK, bdnK, ObdpY) or via synthesis ex novo (e.g., p-1beF). 
     Experimental Details. 
     Synthesis of 4-(2-bromoethoxy)-phenylalanine (p-2beF) (1) 
     To a reaction flask containing N-tert-butoxycarbonyl-tyrosine (2 g, 7.1 mmol) and potassium carbonate (2.94 g, 21.3 mmol) in dry DMF (20 mL) dibromoethane (1.83 mL, 21.3 mmol) was added dropwise over 20 min. The reaction mixture was stirred at room temperature for 18 h after which the reaction mixture was filtered, diluted with 60 mL of water, acidified with acetic acid to pH 4 and extracted with 2×100 mL of EtOAc. Organic layers were combined and dried over sodium sulfate. The solvent was removed under reduced pressure yielding yellow oil as crude product which was purified by flash column chromatography using 10:9:1 hexane:EtOAc:HOAc acid as solvent system. Fractions of interest were combined and solvent removed under reduced pressure yielding N-Boc-4-(2-bromoethoxy)-phenylalanine as an off-white powder (2.3 g, 84%).  1 H NMR (400 MHz, CD 3 OD) δ 1.39 (s, 9H), 2.8-3.05 (m, 2H), 3.3 (t, 2H), 3.51 (t, 2H), 4.37 (t, 2H), 6.69 (d, 2H), 7.02 (d, 2H);  13 C NMR (125 MHz, CD 3 OD) δ 28.73, 29.49, 37.92, 56.82, 65.77, 80.69, 116.27, 128.84, 131.32, 157.39, 157.77, 173.414. MS (ESI) calculated for C 14 H 19 NO 5  [M]+: m/z 387.07, found 387.17. Purified N-Boc-4-(2-bromoethoxy)-phenylalanine was treated with 20 mL of 30% TFA/DCM to remove the N-terminal protection. Upon completed reaction (determined by TLC), the solvent was removed under reduced pressure, crude residue dissolved 2× in 10 mL of HOAc followed by solvent evaporation yielding the final product 1 as an off-white solid in quantitative yield (1.7 g).  1 H NMR (400 MHz, CD 3 OD) δ 3.05-3.25 (m, 2H), 3.58 (t, 2H), 4.28 (t, 1H), 4.51 (t, 2H), 6.77 (d, 2H), 7.09 (d, 2H);  13 C NMR (125 MHz, CD 3 OD) δ 29.1, 36.9, 55.35, 66.92, 116.92, 125.54, 131.59, 158.41, 169.93. MS (ESI) calculated for C 11 H 14 BrNO3 [M+H]+: m/z 288.02, found 288.51. 
     Synthesis of N ε -((2-bromoethoxy)carbonyl)-lysine (2becK) (2) 
     To a solution of N-tert-butoxycarbonyl-lysine (1 g, 4.06 mmol) and NaOH (162.4 mg, 4.06 mmol, 1 eq) dissolved in 20 mL of water 2-bromoethylchloroformate (0.435 mL, 4.06 mmol, 1 eq) and, separately, an additional equivalent of NaOH were added simultaneously dropwise over 30 min. The reaction mixture was stirred at room temperature for 18 h. Upon acidification with HOAc, the aqueous phase was extracted with EtOAc (3×80 mL). The combined organic phases were dried over sodium sulfate, solvent was removed under reduced pressure yielding yellow oil as crude product which was purified by flash column chromatography using 10:9:1 hexane:EtOAc:HOAc as solvent system. Fractions of interest were combined and solvent removed under reduced pressure yielding N-Boc-N ε -((2-bromoethoxy)carbonyl)-lysine as an off-white powder (1.1 g, 68%).  1 H NMR (400 MHz, CD 3 OD) (1.43 (s, 9H), 1.5 (m, 2H), 1.65 (m, 2H), 1.79 (m, 2H), 3.09 (t, 2H), 3.54 (t, 2H), 4.05 (t, 1H), 4.29 (t, 2H);  13 C NMR (125 MHz, CD 3 OD) δ 24.09, 28.78, 30.39, 30.47, 32.434, 41.44, 54.82, 65.51, 80.51, 158.15, 158.44, 176.24; MS (ESI) calculated for C 14 H 19 NO 5  [M+H]+: m/z 397.1, found 397.47. Purified N-Boc-N ζ -((2-bromoethoxy)carbonyl)-lysine was treated with 20 mL of 30% TFA/DCM to remove the N-terminal protection. Upon completed reaction (determined by TLC), the solvent was removed under reduced pressure, crude residue dissolved 2× in 10 mL of acetic acid followed by solvent evaporation yielding the final product 2 as an off-white solid in quantitative yield (0.82 g).  1 H NMR (400 MHz, CD 3 OD) δ 1.45 (m, 2H), 1.64 (m, 2H), 1.76 (m, 2H), 2.95 (t, 2H), 3.6 (t, 2H), 3.85 (t, 1H), 4.22 (t, 2H);  13 C NMR (100 MHz, CD 3 OD) δ 20.74, 23.16, 30.36, 31.16, 41.21, 53.86, 65.54, 158.52, 175.21; MS (ESI) calculated for C 11 H 14 BrNO3 [M+H]+: m/z 297.04, found 297.7. 
     Synthesis of 4-(1-bromoethyl)-phenylalanine (p-1beF) (3) 
     Solution of 4-acetylphenylalanine (0.5 g, 2.415 mmol), prepared as reported previously (Frost, Vitali et al. 2013), in methanol was placed in an ice bath followed by addition of triethylamine (0.51 mL, 3.63 mmol, 1.5 eq) and dropwise addition of di-tert-butyl dicarbonate (0.665 mL, 2.9 mmol, 1.2 eq) over 30 min. The reaction was left at room temperature for additional 3 h after which the solvent was removed in vacuo. The residue was redissolved in EtOAc and extracted with acidified water (pH 4). Organic phase was dried over sodium sulfate, solvent removed under reduced pressure and the crude yellow oil purified using flash column chromatography with 10:9:1 hexane:EtOAc:HOAc as solvent system. Fractions of interest were combined yielding N-Boc-4-acetylphenylalanine as a yellow powder (0.665 g, 90%) which was dissolved in MeOH, placed in an ice bath and treated with NaBH 4  (0.164 g, 4.34 mmol, 2 eq) for 3 h. Following aqueous workup, the crude product was dissolved in DCM, placed in an ice bath and PBr 3  (1 M solution in DCM) was added in portions (5.2 mL, 5.2 mmol, 2.4 eq) over 2 h. The reaction was warmed to reach room temperature and left stirring overnight. After workup, the aqueous layer was lyophilized and used as crude product 3 (0.382 g, 65%).  1 H NMR (400 MHz, CD 3 OD) (1.99 (d, 3H), 2.8-3.2 (m, 2H), 4.31 (t, 1H), 4.78 (q, 1H), 7.18 (d, 2H), 7.27 (d, 2H); MS (ESI) calculated for C 11 H 14 BrNO 2  [M+H]+: m/z 272.03, found 272.53. 
     Synthesis of N ε -((2-chloroethoxy)carbonyl)-lysine (2-cecK) (4) 
     To a solution of N-tert-butoxycarbonyl-lysine 1 (1 g, 4.06 mmol) and NaOH (162.4 mg, 4.06 mmol, 1 eq) dissolved in 20 mL of water 2-chloroethylchloroformate (0.419 mL, 4.06 mmol, 1 eq) and, separately, an additional equivalent of NaOH were added simultaneously dropwise over 30 min. The reaction mixture was stirred at room temperature for 10-12 h. Upon acidification with HOAc, the aqueous phase was extracted with EtOAc (3×80 mL). The combined organic phases were dried over sodium sulfate, solvent was removed under reduced pressure yielding yellow oil as crude product which was purified by flash column chromatography using 10:9:1 hexane:EtOAc:HOAc as solvent system. Fractions of interest were combined and solvent removed under reduced pressure yielding off-white powder as product (1.04 g, 75%). Purified product was treated with 20 mL of 30% TFA/DCM to remove the N-terminal Boc-protection. Upon completed reaction (determined by TLC), the solvent was removed under reduced pressure, yielding the final product 4 as off-white solid in quantitative yield (0.75 g).  1 H NMR (400 MHz, CD 3 OD) δ 1.45 (m, 2H), 1.64 (m, 2H), 1.76 (m, 2H), 2.95 (t, 2H), 3.6 (t, 2H), 3.85 (t, 1H), 4.22 (t, 2H). 
     Synthesis of N ε -(buta-2,3-dienoyl)-lysine (bdnK) (5) 
     3-butynoic acid was prepared by oxidation of 3-butyn-1-ol. About 20 mL of water was added to a 150 mL single neck RBF followed by 65% HNO 3  (45 μL, 0.66 mmol, 0.05 eq), Na 2 Cr 2 O 7  (40 mg, 0.132 mmol, 0.01 eq) and NaIO 4  (6.22 g, 29 mmol, 2.2 eq) and stirred vigorously on an ice bath. After 15 min 1 mL of 3-butyn-1-ol (1 eq, 13.2 mmol) dissolved in 5 mL of cold water was added dropwise over 30 min. The reaction was left stirring overnight followed by product extraction with diethyl ether. Solvent was evaporated to yield off-white/yellow solid (g, %). 1H NMR (400 MHz, CDCl3) δ 3.35 (d, 2H), 2.22 (t, 1H). 3-butynoic acid (0.436 g, 5.2 mmol, 1 eq) was dissolved in dry DCM and 1.5 eq of 2-chloro-1-methylpyridinium iodide was added (2.2 g). The reaction was stirred for 1 h at room temperature followed by dropwise addition of N α -tert-butoxycarbonyl-lysine (1.4 g, 5.72 mmol, 1.1 eq) and triethylamine (1.2 mL, 7.8 mmol, 1.5 eq). The reaction was monitored by TLC and upon completion (4-5 h) extracted with water. Organic layer was evaporated and the crude product was purified using flash column chromatography with 10:9:1 hexane:EtOAc:HOAc as solvent system. Fractions containing the desired product were pooled together and the solvent was removed under reduced pressure giving the desired product in 55% yield.  1 H NMR (400 MHz, CD 3 OD) δ 1.4 (s, 9H), 1.5 (m, 2H), 1.62 (m, 2H), 1.81 (m, 2H), 3.13 (t, 2H), 4.51 (m, 3H), 5.8 (m, 1H). The final Boc-deprotection was achieved using 20 mL of 30% TFA/DCM for 30 min followed by solvent removal resulting in product 5 (g).  1 H NMR (400 MHz, CD 3 OD) δ 1.48 (m, 2H), 1.63 (m, 2H), 1.82 (m, 2H), 3.12 (t, 2H), 4.21 (t, 1H), 4.51 (d, 2H), 5.8 (m, 1H). 
     Synthesis of O-(2,3-dibromoethyl)-tyrosine (OdbpY) (6) 
     To a reaction flask containing N α -tert-butoxycarbonyl-tyrosine (2 g, 7.1 mmol) and potassium carbonate (2.94 g, 21.3 mmol, 2 eq) in dry DMF (20 mL) 1,2,3-tribromopropane (0.915 mL, 7.82 mmol, 1.1 eq) was added dropwise over 20 min. The reaction mixture was stirred at room temperature for 8 h after which the reaction mixture was filtered, diluted with 60 mL of water, acidified with acetic acid to pH 4 and extracted with 2×100 mL of EtOAc. Organic layers were combined and dried over sodium sulfate. The solvent was removed under reduced pressure yielding yellow oil as crude product which was purified by flash column chromatography using 10:9:1 hexane:EtOAc:HOAc acid as solvent system. Fractions of interest were combined and solvent removed under reduced pressure yielding off-white powder as product (g, %).  1 H NMR (400 MHz, CD 3 OD) δ 1.41 (s, 9H), 2.81-3.07 (m, 2H), 3.6-3.81 (m, 2H), 4.21-4.43 (m, 3H), 4.61-4.72 (m, 1H), 6.71 (d, 2H), 7.04 (d, 2H). Purified product was treated with 20 mL of 30% TFA/DCM to remove the N-terminal protection. Upon completed reaction (determined by TLC), the solvent was removed under reduced pressure yielding the final product 6 as an off-white solid in quantitative yield (g).  1 H NMR (400 MHz, CD 3 OD) δ 2.81-3.07 (m, 2H), 3.6-3.81 (m, 2H), 4.12 (t, 1H), 4.21-4.43 (m, 2H), 4.61-4.72 (m, 1H), 6.71 (d, 2H), 7.04 (d, 2H). 
     6.2 Example 2: Polynucleotides for Expression of Precursor Polypeptides 
     This example demonstrates procedures for the construction of polynucleotide molecules for the expression of precursor polypeptides of the type (I), (II), or (V) according to the methods described herein. 
     To illustrate the various embodiments, a series of a plasmid-based vectors were prepared that encode for precursor polypeptides in different formats (Table 1) according to the macrocyclization methods schematically described in  FIGS. 1A-B ,  2 A-B,  3 A-B,  4 A-B and  37 A-B. Specifically, a first series of constructs (Entries 1-9 and 13-15, Table 1) were prepared for the expression of precursor polypeptides of general formula (I), in which (i) the N-terminal tail, (AA) m , consists of a Met-Gly dipeptide; (ii) the target peptide sequence, (AA) n , consists of 1- to 12-amino acid long polypeptides, some of which were designed to include a streptavidin-binding HPQ motif (Katz 1995; Naumann, Savinov et al. 2005) (Entries 13-15, Table 1); and (iii) the C-terminal tail, (AA) p , consists of a short (1 to 8 amino acid-long) polypeptide sequence C-terminally fused to Mxe GyrA intein (SEQ ID NO: 1). In these constructs, an amber stop codon was used to enable the introduction of the desired, cysteine-reactive unnatural amino acid Z, upstream of the peptide target sequence via amber stop codon suppression. Moreover, the C-terminal asparagine of Mxe GyrA intein was mutated to an alanine (N 198 A) to prevent C-terminal splicing and allow for the introduction of a polyhistidine affinity tag at the C-terminus of the polypeptide construct. These constructs were designed to demonstrate the general methods described in  FIGS. 1A and 2A . 
     A second series of constructs (Entries 10-12, Table 1) were prepared for the expression of precursor polypeptides of general formula (II), in which (i) the N-terminal tail, (AA) m , consists of a short (2 to 6 amino acid-long) polypeptide; (ii) the target peptide sequence, (AA) n , consists of a 3 to 7-amino acid long polypeptide; and (iii) the C-terminal tail, (AA) p , consists of the N 198 A variant of Mxe GyrA intein (SEQ ID NO: 1) followed by a polyhistidine tag. In these constructs, an amber stop codon was used to enable the introduction of the desired, cysteine-reactive unnatural amino acid Z, downstream of the peptide target sequence via amber stop codon suppression. These constructs were designed to probe the functionality of the general methods described in  FIGS. 1B and 2B . 
     A third series of constructs (Entries 16-20, Table 1) were prepared for the expression of precursor polypeptides of general formula (I), in which (i) the N-terminal tail, (AA) m , contains the C-domain of  Synechocystis  sp. DnaE split intein (SEQ ID NO:62); (ii) the C-terminal tail, (AA) p , contains the N-domain of  Synechocystis  sp. DnaE split intein (SEQ ID NO:61); and (iii) a streptavidin-binding HPQ motif (Naumann, Savinov et al. 2005) is included within (Entry 18-20, Table 1) or downstream of the target peptide sequence (AA) n (Entries 16-17, Table 1). In these constructs, an amber stop codon was used to enable the introduction of the desired, cysteine-reactive unnatural amino acid Z, upstream of the peptide target sequence. Furthermore, these constructs contain a CBD (cellulose binding domain) affinity tag fused to the C-terminal end of the split intein N-domain. These constructs were designed to probe the functionality of the general methods described in  FIGS. 4A-B . 
     An additional construct (Entry 21, Table 1) was prepared for the expression of a precursor polypeptide which carries two Cys/Z pairs comprising two different target peptide sequences (HPQF (SEQ ID NO:185) and NTSK (SEQ ID NO:186)) and being separated from each other by an intervening polypeptide sequence (ENLYFQS (SEQ ID NO: 187)). This construct is instrumental for demonstrating the possibility to generate polycyclic peptides using the methods disclosed herein. 
     Finally, a construct (Entry 22, Table 1) was prepared for the expression of a precursor polypeptide which carries a bifunctional cysteine-reactive amino acid (Z2) and two cysteine residues. This construct is instrumental for demonstrating the possibility to generate polycyclic peptides according to the general methods described in  FIGS. 37A-B . 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Precursor polypeptide constructs. a   
               
            
           
           
               
               
               
            
               
                 Entry 
                 Construct Name 
                 Peptide Sequence 
               
               
                   
               
               
                  1 
                 12mer-Z1C 
                 MG- ( Z )- C   GSKLAEYGT-(GyrA N198A )LEHHHHHH 
               
               
                   
                   
                 (SEQ ID NO: 159) 
               
               
                   
               
               
                  2 
                 12mer-Z2C 
                 MG- ( Z )- T   C   SKLAEYGT-(GyrA N198A )LEHHHHHH 
               
               
                   
                   
                 (SEQ ID NO: 160) 
               
               
                   
               
               
                  3 
                 12mer-Z3C 
                 MG- ( Z )- TG   C   KLAEYGT-(GyrA N198A )LEHHHHHH 
               
               
                   
                   
                 (SEQ ID NO: 161) 
               
               
                   
               
               
                  4 
                 12mer-Z4C 
                 MG- ( Z )- TGS   C   LAEYGT-(GyrA N198A )-LEHHHHHH 
               
               
                   
                   
                 (SEQ ID NO: 162) 
               
               
                   
               
               
                  5 
                 12mer-Z5C 
                 MG- ( Z )- TGSK   C   AEYGT-(GyrA N198A )LEHHHHHH 
               
               
                   
                   
                 (SEQ ID NO: 163) 
               
               
                   
               
               
                  6 
                 12mer-Z6C 
                 MG- ( Z )- TGSKL   C   EYGT-(GyrA N198A )LEHHHHHH 
               
               
                   
                   
                 (SEQ ID NO: 164) 
               
               
                   
               
               
                  7 
                 12mer-Z8C 
                 MG- ( Z )- TGSKLAE   C   GT-(GyrA N198A )LEHHHHHH 
               
               
                   
                   
                 (SEQ ID NO: 165) 
               
               
                   
               
               
                  8 
                 14mer-Z10C 
                 MG- ( Z )- TGSKYLNAE   C   GT-(GyrA N198A )LEHHHHHH 
               
               
                   
                   
                 (SEQ ID NO: 166) 
               
               
                   
               
               
                  9 
                 16mer-Z12C 
                 MG- ( Z )- TGSHKYLRNAE   C   GT-(GyrA N198A )LEHHHHHH 
               
               
                   
                   
                 (SEQ ID NO: 167) 
               
               
                   
               
               
                 10 
                 l0mer-C4Z 
                 MGSEAG   C   NIA- ( Z ) -(GyrA N198A )LEHHHHHH 
               
               
                   
                   
                 (SEQ ID NO: 168; SEQ ID NO: 169) 
               
               
                   
               
               
                 11 
                 l0mer-C6Z 
                 MGSE   C   GTNIA- ( Z ) -(GyrA N198A )LEHHHHHH 
               
               
                   
                   
                 (SEQ ID NO: 170; SEQ ID NO: 169) 
               
               
                   
               
               
                 12 
                 l0mer-C8Z 
                 MG   C   EAGTNIA- ( Z ) -(GyrA N198A )LEHHHHHH 
               
               
                   
                   
                 (SEQ ID NO: 171; SEQ ID NO: 169) 
               
               
                   
               
               
                 13 
                 Strep1-Z5C 
                 MG- ( Z )- HPQF   C   GD-(GyrA N198A )LEHHHHHH 
               
               
                   
                   
                 (SEQ ID NO: 172) 
               
               
                   
               
               
                 14 
                 Strep2-Z7C 
                 MG- ( Z )- HPQGPP   C   GD-(GyrA N198A )LEHHHHHH 
               
               
                   
                   
                 (SEQ ID NO: 173) 
               
               
                   
               
               
                 15 
                 Strep3-Z11C 
                 MG- ( Z )- FTNVHPQFAN   C   D-(GyrA N198A )LEHHHHHH 
               
               
                   
                   
                 (SEQ ID NO: 174) 
               
               
                   
               
               
                 16 
                 cStrep3(C)-Z3C 
                 (DnaE C )-C- ( Z )- TN   C   HPQFANA-(DnaE N )-(CBD) 
               
               
                   
                   
                 (SEQ ID NO: 175; SEQ ID NO: 176) 
               
               
                   
               
               
                 17 
                 cStrep3(S)-Z3C 
                 (DnaE C )-S- ( Z )- TN   C   HPQFANA-(DnaE N )-(CBD) 
               
               
                   
                   
                 (SEQ ID NO: 177; SEQ ID NO: 178) 
               
               
                   
               
               
                 18 
                 cStrep3(C)-Z8C 
                 (DnaE C )-C- ( Z )- TNVHPQF   C   NA-(DnaE N )-(CBD) 
               
               
                   
                   
                 (SEQ ID NO: 175; SEQ ID NO: 179) 
               
               
                   
               
               
                 19 
                 cStrep4(S)-Z8C 
                 (DnaE C )-S- ( Z )- TNVHPQF   C   NAKGDA-(DnaE N )-(CBD) 
               
               
                   
                   
                 (SEQ ID NO: 177; SEQ ID NO: 180) 
               
               
                   
               
               
                 20 
                 cStrep5(S)-Z8C 
                 (DnaE C )-S- ( Z )- TNVHPQF   C   NAKGDTQA-(DnaE N )-(CBD) 
               
               
                   
                   
                 (SEQ ID NO: 177; SEQ ID NO: 181) 
               
               
                   
               
               
                 21 
                 Strep6_Z4C7C4Z 
                 MG- ( Z )- HPQF   C   ENLYFQS   C   NTSK- ( Z ) -(GyrA N198A )LEHHHHHH 
               
               
                   
                   
                 (SEQ ID NO: 182; SEQ ID NO: 169) 
               
               
                   
               
               
                 22 
                 Strep7_C5Z4C 
                 MG   C   AYDSG- ( Z2 )- HPQF   C   GT-(GyrA N198A )LEHHHHHH 
               
               
                   
                   
                 (SEQ ID NO: 183; SEQ ID NO: 184) 
               
               
                   
               
               
                   a GyrA N190A  corresponds to the N190A variant of  Mycobacterium xenopi  GyrA (SEQ. ID NO: 1), CBD corresponds to the Chitin Binding Domain (CBD) of  Bacillus circulans  chitinase A1 (SEQ ID NO: 130), DnaE N  and DnaE C  correspond to the N-domain and C-domain, respectively, of  Synechocystis  sp. DnaE split intein (SEQ ID NOS: 61 and 62). The reactive amino acid residues involved in peptide macrocyclization (i.e., Cys and Z residues; Cys and Z2 residues) are highlighted in bold. 
               
            
           
         
       
     
     Experimental Details. 
     Cloning and plasmid construction. The plasmid vector pET22b(+) (Novagen) was used as cloning vector to prepare the plasmids for the expression of the precursor polypeptides of Entries 1-15 and 21-22 in Table 1. Briefly, synthetic oligonucleotides (Integrated DNA Technologies) were used for the PCR amplification of a gene encoding for N-terminal peptide and peptide target sequence fused to GyrA N198A  intein using a previously described GyrA-containing vector (pBP_MG6) (Smith, Vitali et al. 2011) as template. The resulting PCR product (ca. 0.6 Kbp) was digested with Nde I and Xho I and cloned into pET22b(+) to provide the plasmids for the expression of the precursor polypeptides of Entries 1-15 and 21-22 in Table 1. The cloning process placed the polypeptide-encoding gene under the control of an IPTG-inducible T7 promoter and introduced a poly-histidine tag at the C-terminus of the intein. Plasmids for the expression of the polypeptide constructs of Entries 16 through 20 of Table 1 were prepared in a similar manner but using pBAD plasmid (Life Technologies) as the cloning and expression vector. The genes encoding for DnaEN and DnaEc were amplified from Addgene plasmids pSFBAD09 and pJJDuet30. The sequences of the plasmid constructs were confirmed by DNA sequencing. 
     6.3 Example 3: Identification of tRNA/Aminoacyl-tRNA Synthetase Pairs for Incorporation of Cysteine-Reactive Amino Acids 
     This example illustrates how a suitable tRNA/aminoacyl-tRNA synthetase pair can be identified for the purpose of incorporating a desired cysteine-reactive, unnatural amino acid into a precursor polypeptide of general formula (I), (II), or (V) according to the methods disclosed herein. In particular, this example describes the identification of tRNA/aminoacyl-tRNA synthetase pairs for the incorporation of the unnatural amino acid 4-(2-bromoethoxy)-phenylalanine (p-2beF), N-((2-bromoethoxy)carbonyl)-lysine (2becK), 4-(1-bromoethyl)-phenylalanine (p-1beF), N ε -((2-chloroethoxy)carbonyl)-lysine (2cecK), N ε -(buta-2,3-dienoyl)-lysine (bdnK), and O-(2,3-dibromoethyl)-tyrosine (OdbpY), which were synthesized as described in Example 1. 
     A high-throughput fluorescence-based screen was applied to identify viable tRNA/aminoacyl-tRNA synthetase (AARS) pairs for the ribosomal incorporation of the unnatural amino acid p-2beF, 2becK, p-1beF, 2cecK, bdnK, or OdbpY, in response to an amber stop codon. In this assay,  E. coli  cells are co-transformed with two plasmids with compatible origins of replications and selection markers; one plasmid directs the expression of the tRNA/AARS pair to be tested, whereas the second plasmid contains a gene encoding for a variant of Yellow Fluorescence Protein (YFP), in which an amber stop codon (TAG) is introduced at the second position of the polypeptide sequence following the initial Met residue (called YFP(TAG)). The ability of the tRNA/AARS pair to suppress the amber stop codon with the unnatural amino acid of interest can be thus determined and quantified based on the relative expression of YFP as determined by fluorescence. Using this assay, a panel of engineered aminoacyl-tRNA synthetase (AARS) variants derived from  M. jannaschii  tyrosyl-tRNA synthetase (SEQ ID NO:77),  M. barkeri  pyrrolysyl-tRNA synthetase (SEQ ID NO:79), or  M. mazei  pyrrolysyl-tRNA synthetase (SEQ ID NO:78) in combination with their cognate amber stop codon suppressor tRNA (i.e., MjtRNA CUA   Tyr  (SEQ ID NO: 101) for Mj AARSs and Mm/MbtRNA CUA   Pyl  (SEQ ID NO:105) for the Mm and Mb AARSs) were tested for their ability to incorporate the target amino acids p-2beF, 2becK, p-1beF, 2cecK, bdnK, or OdbpY into the reporter YFP(TAG) protein. In a representative experiment, this panel of AARS enzymes included the known engineered AARSs Mj-pAcF-RS (SEQ ID NO:81), Mj-pAmF-RS (SEQ ID NO:87), Mb-CrtK-RS (SEQ ID NO:93), and Mm-pXF-RS (SEQ ID NO:91) (Young, Young et al. 2011)) as well as the newly engineered Mj-OpgY2-RS (SEQ ID NO: 85). The latter, which is derived from Mj-OpgY-RS (SEQ ID NO:84) (Deiters and Schultz 2005), carries an Ala32G mutation that was designed to facilitate the recognition of O-substituted tyrosine derivatives such as p-2beF and OdbpY based on the available crystal structure of the parent enzyme Mj-TyrRS (SEQ ID NO:77) (Kobayashi, Nureki et al. 2003). As illustrated by the representative data in  FIGS. 7A-B , the AARS/tRNA pair consisting of Mj-pOgY2-RS/MjtRNA CUA   Tyr  was found to enable the efficient incorporation of p-2beF ( FIG. 7A ), whereas the AARS/tRNA pair consisting of Mb-CrtK-RS/Mm/MbtRNA CUA   Pyl  was found to enable the efficient incorporation of 2becK into the reporter YFP(TAG) protein ( FIG. 7B ). Control experiments with no unnatural amino acid added to the culture medium show no or negligible expression of the reporter YFP protein, evidencing the discriminating selectivity of these AARS/tRNA pairs for the desired unnatural amino acid over the pool of natural amino acids (this property is referred here as “orthogonal reactivity” or simply “orthogonality” of the AARS/tRNA). 
     Using an analogous procedure, it was established that the Mj-pAcF-RS/MjtRNA CUA   Tyr  pair can enable efficient amber stop codon suppression with p-1beF; the Mb-CrtK-RS/Mm/MbtRNA CUA   Pyl  pair can enable efficient amber stop codon suppression with 2cecK or bdnK; and the Mj-pOgY2-RS/MjtRNA CUA   Tyr  pair can enable efficient amber stop codon suppression with OdbpY. These results provide an exemplary demonstration of viable procedures that can be used to identify suitable AARS/tRNA pairs for the ribosomal incorporation of cysteine-reactive unnatural amino acid into a polypeptide for the purpose of producing macrocyclic peptide according to methods disclosed herein and as illustrated in the following Examples. 
     Experimental Details. 
     YFP Expression Assay. 
     Competent BL21(DE3)  E. coli  were cotransformed with a pEVOL-based plasmid (Smith, Vitali et al. 2011) for the expression of the desired AARS/tRNA pair and a pET22-YFP (TAG) plasmid for the expression of the reporter YFP protein. After overnight growth at 37° C. in LB medium supplemented with chloramphenicol (25 μg/mL) and ampicillin (50 μg/mL), cell cultures were used to inoculate 96-well plates containing 0.9 mL of minimal (M9) media (25 μg/mL chloramphenicol, 50 μg/mL ampicillin, 1% glycerol) per well. At OD 600 =0.6, protein expression was induced with 0.05% L-arabinose and 1 mM IPTG. Test wells were supplemented with the desired unnatural amino acid (e.g., 4-(2-bromoethoxy)-phenylalanine (p-2beF) at 2 to 5 mM, whereas no amino acid was added to the negative control wells. Cultures were grown overnight at 27° C. and then diluted 1:100 with phosphate buffer (50 mM KPi (pH 7.5), 150 mM NaCl) into microtiter plates. Fluorescence intensity was measured using a Tecan Infinite 1000 multi-well plate reader (λ exc : 514 nm; λ em : 527 nm). 
     6.4 Example 4: Preparation and Isolation of Macrocyclic Peptides from p-2beF-Containing Precursor Polypeptides of General Formula (I) 
     This example demonstrates the formation and isolation of macrocyclic peptides produced via the cyclization of ribosomally derived precursor polypeptides of general formula (I) and containing the cysteine-reactive unnatural amino acid p-2beF. In particular, this example demonstrates certain embodiments as schematically described in  FIGS. 1A and 2A . 
     For these experiments, the precursor polypeptides corresponding to Entries 1 through 9 in Table 1 were expressed in BL21(DE3)  E. coli  cells containing a second, pEVOL-based plasmid for the co-expression of Mj-pOgY2-RS and MjtRNA CUA   Tyr . As described in Example 3, this AARS/tRNA pair was established to allow for the efficient ribosomal incorporation of p-2beF into a polypeptide in response to an amber stop codon. According to our strategy ( FIGS. 1A-B ), these precursor polypeptides were expected to undergo cyclization via a nucleophilic substitution reaction between the cysteine side-chain thiol group and the p-2beF side-chain bromoalkyl group flanking the target peptide sequence after ribosomal synthesis of the precursor polypeptides in the expression system ( E. coli ) ( FIG. 8 ). To establish the occurrence and efficiency of the cyclization, these proteins were isolated by Ni-affinity chromatography exploiting the C-terminal poly-histidine tag present in these constructs (Table 1). In all the aforementioned constructs, a Thr residue was placed at the site preceding the GyrA intein (“I-1 site”). This substitution minimizes premature hydrolysis of GyrA-fusion proteins during expression in  E. coli  (Frost, Vitali et al. 2013), thereby facilitating analysis of the target peptide sequences after chemically induced splicing of the intein from the purified proteins in vitro ( FIG. 8 , path A). This procedure would also permit the isolation of any product resulting from the unselective reaction of p-2beF with other nucleophiles in vivo (e.g., glutathione). Accordingly, after purification, the proteins were made react with benzyl mercaptan in order to release the desired macrocyclic peptide (in the form of C-terminal benzyl thioester or the corresponding C-terminal carboxylic acid after thioester hydrolysis) from the GyrA intein via thiol-induced splicing of the intein. The reaction mixtures were then analysed by LC-MS to detect and quantify the amount of the desired thioether-linked macrocyclic product as well as that of any uncyclized linear byproduct, as judged based on the peak areas in the corresponding extracted-ion chromatograms ( FIGS. 10-15 ). Uncyclized byproducts would appear as unmodified linear peptides or as linear adducts where the bromoalkyl group in p-2beF has undergone nucleophilic substitution with the benzyl mercaptan reagent during the in vitro reaction or with glutathione in vivo. 
     As summarized in  FIG. 9A , these studies revealed that peptide macrocyclization had occurred with very high efficiency (80-95%) across the constructs with Cys and p-2beF being separated by two to eight residues (i.e. Cys at Z+2 to Z+8). Increasing this distance (i.e., with Cys at Z+10 and Z+12, Entries 8-9 in Table 1) resulted in a decrease of the cyclic product (50-20%,  FIG. 9A ). Interestingly, cyclization could also be achieved also when the Cys was located immediately adjacent to the unnatural amino acid (Entry 1, Table 1), albeit at a lower extent (5%) as compared to the other constructs. This result can be rationalized based on the comparatively less favorable 14-membered macrocycle formed when the p-2beF/Cys pair are in a i/i+1 relationship. For each construct tested, the identity of the macrocyclic product could be further confirmed by analysis of the corresponding MS/MS fragmentation spectrum as illustrated in  FIG. 16 . 
     GyrA intein contains a Cys at its N-terminal end which is crucial for mediating protein splicing in the context of the application of the present methods for producing peptide macrocycles inside the cells (see Example 5). Since this residue is partially buried within the active site (Klabunde, Sharma et al. 1998), we did not expect it to readily react with p-2beF side chain. Notably, quantitative splicing of the GyrA moiety upon treatment of all the aforementioned contructs with benzyl mercaptan indicated that no reaction occurred between p-2beF and the catalytic Cys at the intein I+1 site (see representative results in  FIGS. 17 a - d   ). Furthermore, no adducts or dimers were observed for any of the constructs described above, including those undergoing only partial cyclization (i.e. Entries 8-9,  FIG. 9A ). Altogether, these results further highlight the high chemo- and regioselectivity of the macrocyclization reaction. 
     Experimental Details. 
     Protein Expression and Purification. 
     The protein constructs were expressed using BL21(DE3)  E. coli  cells co-transformed with a pET22-based vector for the expression of the precursor polypeptide and a pEVOL-based vector for the expression of the Mj-pOgY2-RS/MjtRNA CUA   Tyr  pair. Cultures of these cells were grown overnight in LB media (50 mg/L ampicillin; 25 mg/L chloramphenicol) and used to inoculate 0.2 L of minimal (M9) media containing the same concentration of antibiotics, 1% glycerol, and 1 mM p-2BeF. At OD 600 =0.6, IPTG (1 mM) and L-arabinose (0.05%) was added to the culture media to induce protein expression. Cultures were grown for 14 h at 27° C. and then harvested by centrifugation. Cell pellets were resuspended in 50 mM Tris, 300 mM NaCl, 20 mM imidazole buffer (pH 7.5) and cells were lysed by sonication. The cell lysate was loaded on a Ni-NTA affinity column and proteins were eluted with 50 mM Tris, 150 mM NaCl, 300 mM imidazole buffer (pH 7.5). Fractions were combined and concentrated followed by buffer exchange with potassium phosphate buffer (50 mM, 150 mM NaCl, pH 7.5). The identity of the isolated proteins was confirmed using MALDI-TOF MS and LC-MS. 
     Intein Splicing and MS Analysis. 
     Aliquots of the purified proteins (200 μM) were incubated with 15 mM benzylmercaptan, 20 mM TCEP in 50 mM phosphate buffer (pH 8). The identity of the target macrocycles was confirmed using MALDI-TOF MS and LC-MS analysis. LC-MS analyses were performed on Thermo Scientific LTQ Velos ESI/ion-trap mass spectrometer coupled to an Accela U-HPLC. Macrocycles were analyzed using Thermo Scientific HyPurity C4 column (particle size 5 m, 100×2.1 mm i.d.) and a linear gradient 5% to 95% ACN (with 0.1% formic acid) in water (with 0.1% formic acid) over 9 min. MALDI-TOF spectra were acquired on the Bruker Autoflex III mass spectrometer. 
     6.5 Example 5: In Vivo Production of Macrocyclic Peptides from p-2beF-Containing Precursor Polypeptides of General Formula (I) 
     This example further demonstrates the formation and isolation of macrocyclic peptides produced via the cyclization of ribosomally derived precursor polypeptides of general formula (I) and containing the cysteine-reactive unnatural amino acid p-2beF. In particular, this example provides a demonstration of the functionality of the methods described herein for the production of macrocyclic peptide within living bacterial cells. 
     For these studies, the constructs corresponding to Entries 13 through 15 of Table 1 were utilized. The corresponding precursor polypeptides were expressed in BL21(DE3)  E. coli  cells in the presence of the Mj-pOgY2-RS/MjtRNA CUA   Tyr  to achieve the site-selective incorporation of the unnatural amino acid p-2beF into these proteins via amber stop codon suppression. These constructs were designed to contain an Asp residue in the position preceding the GyrA intein moiety in order to favor premature N-terminal splicing of this intein during expression ( FIG. 8 ). We previously established that certain amino acid substitutions at the level of the I-1 site, and in particular Asp and Lys, can strongly promote premature splicing of GyrA intein during recombinant expression (Frost, Vitali et al. 2013). This effect is likely due to the ability of these residues to favor hydrolysis of the intein-catalyzed thioester linkage through their nucleophilic side-chain groups. This reactivity is leveraged here for mediating the spontaneous release of the macrocyclic peptide from the precursor protein after ribosomal expression as outlined in  FIG. 8  (path B). Thus, according to our strategy ( FIGS. 1A and 2A ), these precursor polypeptides were expected to result in the formation of macrocyclic peptides inside the living cell expression host ( E. coli ) via the intramolecular, thioether bond-forming reaction between the cysteine and p-2beF residue, followed by release of the cyclic peptide via spontaneous N-terminal splicing of the intein moiety. These constructs were also designed to contain a streptavidin-binding motif (HPQ) within the sequence of the resulting macrocyclic peptides (Table 1) in order to allow for the isolation of these peptides via streptavidin-affinity capturing directly from bacterial lysates. Accordingly,  E. coli  cells expressing these precursor polypeptides were grown overnight and lysed by sonication. The cell lysates were then passed over streptavidin-coated beads, from which streptavidin-bound material was eluted. LC-MS analysis of the eluates revealed the occurrence of the expected peptide macrocycle in each case, as illustrated by the LCMS chromatograms and MS/MS spectra in  FIGS. 25-27 . Since the uncyclized peptide could also be captured through this procedure, these analyses also showed that the desired macrocyclic product was formed with high efficiency in each case (i.e. &gt;95% for Strep1-Z5C(p-2beF); 70% for Strep2-Z7C(p-2beF); 85% Strep3-Z11C(p-2beF)). Furthermore, the precursor polypeptides were found to have undergone complete splicing in vivo ( FIGS. 33 a - d   ). Since p-2beF-mediated alkylation of the intein catalytic cysteine would prevent protein splicing, the latter results further highlighted the high degree of chemo- and regioselectivity of the macrocyclization reaction. Furthermore, the cyclization yield observed with these sequences correlated very well with the reactivity trend measured across the other p-2beF-containing contructs ( FIG. 9A ), suggesting that this parameter is rather predictable on the sole basis of the Cys/p-2beF distance and in spite of the difference in the composition of the target peptide sequence. 
     Altogether, these results further demonstrate the versatility of the methods described herein for enabling the ribosomal synthesis of macrocyclic peptides of varying length and compositions. In addition, they demonstrate the possibility to apply these methods to enable the production of macrocyclic peptides in vivo, i.e. inside a living cell. Finally, they demonstrate that these in vivo produced macrocyclic peptides can be functional, that is capable of specifically bind to a target biomolecule (i.e., streptavidin). 
     Experimental Details. 
     Isolation and Analysis of HPQ-Containing Macrocyclic Peptides. 
     Protein expression was performed as described above (Example 5). After centrifugation, cells were resuspended in 50 mM Tris, 300 mM NaCl and 20 mM imidazole (pH 7.5) and lysed by sonication. Cell lysates were incubated with streptavidin-coated beads for 1 hour under gentle shaking on ice. Beads were washed two times with the same buffer followed by incubation with acetonitrile:H 2 O (70:30 v/v) for one minute to release any streptavidin-bound peptides. Eluates were lyophilized and the identity of the peptides evaluated using MALDI-TOF MS and LC-MS as described above (Example 5). 
     6.6. Example 6: Preparation and Isolation of Macrocyclic Peptides Generated Via Cysteine Cross-Linking with Different Electrophilic Amino Acids 
     This example further demonstrates the formation and isolation of macrocyclic peptides produced via the cyclization of ribosomally derived precursor polypeptides of general formula (I). In particular, this example demonstrates how different cysteine-reactive unnatural amino acids of general structure (III) can be used for the purpose of generating macrocyclic peptides starting from ribosomally produced polypeptide precursors according to the methods described herein. 
     As described in Example 3, orthogonal AARS/tRNA pairs could be readily identified to achieve the specific incorporation of the unnatural amino acids 2becK, 2cecK, p-1beF, or bdnK into a precursor polypeptide of choice. Each of these amino acids contains an electrophilic side-chain functionality (i.e., alkylbromide group in 2becK and p-1beF; alkylchloride group in 2cecK; allenamide group in bdnK), which was expected to react chemoselectively with a neighboring cysteine residue within the precursor polypeptide sequence according to the general methods provided herein. To test the ability of 2becK and 2cecK to mediate peptide macrocyclization, the constructs corresponding to Entries 1 through 9 of Table 1 were expressed in  E. coli  as described above (Example 5) using the appropriate AARS/tRNA pairs (Example 3) for the incorporation of either 2becK or 2cecK as the cysteine-reactive residue (Z residue, Table 1). To establish the occurrence of the desired macrocyclization reaction, these proteins were purified by Ni-affinity chromatography and then reacted with benzyl mercaptan to splice the GyrA intein and release the macrocyclic peptide. Detection and quantification of the cyclic product was carried by LC-MS and MS/MS analysis as described in Example 4. These analyses revealed the occurrence of the desired macrocyclic peptide product in each case, as shown by the representative LC-MS extracted-ion chromatograms and MS/MS spectra in  FIGS. 18-22 . As summarized in  FIG. 9B , 2becK- and 2cecK-mediated peptide macrocyclization was found to occur very efficiently (&gt;80%) when the cysteine residue is located within a six-residue distance from the electrophilic amino acid (i.e., with constructs 12mer-Z1C through 12mer-Z1C). Beyond this spacing distance, the % cyclization decreases significantly (&lt;20%). Interestingly, the reactivity of 2becK- and 2cecK as cysteine cross-linking residues nicely complement that of p-2beF, as evidenced from comparison of % cyclization data in  FIGS. 9A and 9B . For example, whereas the 12mer-Z1C construct undergoes efficient cyclization in the presence of 2becK (or 2cecK) but not p-2beF as the cysteine-reactive residue, the opposite holds true in the context of the large macrocycles formed from the constructs 12mer-Z10C and 12mer-Z12C. Thus, these results show how different cysteine-reactive amino acids can be appropriately chosen and applied to obtain macrocyclic peptides of varying ring size according to the methods provided herein. 
     To further investigate the generality of the methods presented herein, two additional amino acids, p-1beF and bdnK, were synthesized (Example 1) and tested here for their ability to induce peptide macrocyclization upon reaction with a proximal cysteine in the precursor polypeptide. p-1beF contains a benzylic, secondary alkyl bromide group, thus enabling the formation of more compact peptide ring structures as compared to those generated using p-2beF-mediated cysteine alkylation. On the other hand, bdnK was designed to contain an allenamide group, which is known to react chemoselectively with cysteine via a Michael addition reaction (Abbas, Xing et al. 2014). Using the appropriate AARS/tRNA pair as determined in Example 3, p-1beF was incorporated into the construct 12mer-Z4C (Entry 4, Table 1) to give 12mer-Z4C(p-1beF), whereas bdnK was incorporated into the construct 12mer-Z6C (Entry 6, Table 1) to give 12mer-Z6C(bdnK). After expression in  E. coli  and purification via Ni-affinity chromatography, these proteins were made react with benzyl mercaptan to splice the GyrA intein and release the macrocyclic peptide. The desired macrocyclic peptide product could be observed in each case ( FIGS. 23 and 24 ). Altogether, the results included in this example illustrate how a variety of structurally diverse cysteine-reactive amino acids can be designed and applied in the context of the general peptide cyclization methods described in this application. 
     6.7. Example 7: Preparation and Isolation of Macrocyclic Peptides Precursor Polypeptides of General Formula (II) 
     This example demonstrates the formation and isolation of macrocyclic peptides produced via the cyclization of ribosomally derived precursor polypeptides of general formula (II). As such, this example demonstrates certain embodiments as schematically described in  FIGS. 1B and 2B . 
     For these studies, the constructs corresponding to Entries 10 through 12 of Table 1 were used. Three different cysteine-reactive amino acids, p-2beF, 2becK, and 2cecK, were tested as the Z residue in these constructs. The corresponding p-2beF-, 2becK-, or 2cecK-containing precursor polypeptides were expressed in BL21(DE3)  E. coli  cells using the appropriate AARS/tRNA pair as determined in Example 3 (Mj-pOgY2-RS/MjtRNA CUA   Tyr  pair for the p-2beF-containing proteins and Mb-CrtK-RS/Mm/MtRNA CUA   Pyl  for the 2becK and 2cecK-containing proteins). In these constructs, the reactive Cys is located upstream of the unnatural amino acid, and specifically at position Z-4, Z-6 and Z-8. Analysis of the p-2beF-containing proteins according to the procedure described above (Example 4) revealed the occurrence of the desired cyclic peptide as the largely predominant product (95-99%) for all of the constructs tested ( FIG. 9A ,  FIGS. 34-35 ). For the 2becK- and 2cecK-containing proteins, efficient inter-side-chain cyclization (80-95%) was observed when the cysteine and unnatural amino acid are three (Z-4) and five residue apart, while a lower % of cyclization was noted at the larger spacing distance (Z-8) ( FIG. 9B ). These data clearly demonstrated that the thioether bond-forming reactivity of the cysteine-reactive amino acids is preserved when the order of Cys and Z residue is reversed, thus enabling structural variation of the resulting macrocyclic peptide products. Furthermore, quantitative thiol-induced splicing of the GyrA intein from the aforementioned proteins indicated that no reaction had occurred between the side-chain of the unnatural amino acid and the catalytic I+1 cysteine residue of the intein ( FIGS. 17 a - d   ). 
     6.8 Example 8: In Vivo Production and Isolation of Bicyclic Peptides 
     This example demonstrates certain embodiments as schematically described in  FIG. 4A . In particular, this example demonstrates how bicyclic peptides can be generated from precursor polypeptides of general formula (I) via the combination of a split intein-mediated trans-splicing reaction and inter-side-chain cyclization reaction mediated by a cysteine and a cysteine-reactive unnatural amino acid according to the methods described herein. While split intein-mediated trans-splicing has proven useful for the generation and isolation of head-to-tail cyclic peptides in a variety of context (Scott, Abel-Santos et al. 1999; Tavassoli and Benkovic 2005; Tavassoli and Benkovic 2007; Tavassoli, Lu et al. 2008; Young, Young et al. 2011) (see also U.S. Pat. Nos. 7,354,756, 7,252,952, and 7,105,341), there are reports of the application of this technique (called SICLOPPS) to obtain bicyclic peptides of the general structure described in  FIGS. 4A-B . This example demonstrates the possibility to apply the general methods disclosed herein, and specifically in its embodiments as outlined in  FIGS. 4A-B , to enable the efficient production of bicyclic peptides inside a living cell. In addition, the advantage conferred by the bicyclic structure and thus by the inter-side-chain thioether linkage toward improving the functional (i.e., protein-binding) properties of the macrocyclic peptide is demonstrated. 
     For these studies, the constructs corresponding to Entries 16 through 20 of Table 1 were utilized. The corresponding precursor polypeptides were expressed in BL21(DE3)  E. coli  cells in the presence of the Mj-pOgY2-RS/MjtRNA CUA   Tyr  for incorporation of the unnatural amino acid p-2beY into these proteins via amber stop codon suppression, as described above (Example 5). These constructs were designed to comprise the C-domain and N-domain of split intein DnaE within the N-terminal tail and the C-terminal tail, respectively, of the precursor polypeptide. According to our strategy ( FIG. 4A ), these precursor polypeptides were expected to result in the formation of bicyclic peptides in  E. coli  by means of an intramolecular, thioether bond-forming reaction between the cysteine and p-2beF residues and a DnaE-catalyzed trans-splicing reaction leading to ring closure (i.e. N-to-C-end cyclization) of the peptide sequence comprised between the C- and N-domain of the split intein. To facilitate the identification and isolation of these bicyclic peptides, a streptavidin-binding motif (HPQ) was included within the sequence targeted for macrocyclization (Table 1). Accordingly, using an analogous procedure as that described in Example 5, lysates of  E. coli  cells expressing the aforementioned precursor polypeptides were passed over streptavidin-coated beads, from which streptavidin-bound material was eluted. 
     Notably, the desired bicyclic peptide was isolated as the largely predominant product in each case (70-95%), as determined by LC-MS ( FIGS. 28-32 ). The bicyclic structure of these compounds was further evidenced by the corresponding MS/MS fragmentation spectra ( FIGS. 28-32 ). Treatment of the bicyclic peptide obtained with the thiol-alkylating iodoacetamide resulted in a 57 Da increase in molecular mass and shift of the peptide retention time for the bicyclic product of the cStrep3(C)-Z3C(p-2beF) precursor protein but not for that of cStrep3(S)-Z3C(p-2beF), which is consistent with the presence of a free thiol in the former (from Intc+1 cysteine) but not in the latter. To allow measurement of the extent of post-translational self-processing of these precursor polypeptides in vivo, a chitin-binding domain was included at the C-terminus of the IntN domain in each construct (Table 1). LC-MS analysis of the protein fraction eluted from chitin beads showed that the split intein-mediated cyclization has occurred nearly quantitatively or nearly quantitatively (&gt;85%) for all the constructs tested (see representative MS spectra in  FIGS. 33 a - d   ). Overall, the successful generation of bicyclic structures across target sequences of varying length and composition supports the functionality and broad scope of the present methodology for the ribosomal synthesis of bicyclic peptides through the integration of split intein-mediated peptide circularization with inter-side-chain thioether bridge formation. 
     The increased conformational rigidity imposed by the intra-side-chain thioether bridge is expected to improve the functional and/or stability properties of these bicyclic peptides as compared to the head-to-tail cyclized peptide counterpart. To investigate this aspect, the streptavidin-binding affinity of the bicyclic peptides obtained via cyclization of the cStrep3(S)-Z3C(p-2beF) and cStrep3(C)-Z8C(p-2beF) constructs was measured through an in-solution inhibition assay and compared with that of a ‘monocyclic’ counterpart (cyclo[S(OpgY)TNCHPQFANA] (SEQ ID NO: 189) where OpgY is O-propargyl-tyrosine). In this assay ( FIG. 39A ), a streptavidin-binding surface is first created by immobilizing the bicyclic peptide obtained from the cStrep3(C)-Z8C(p-2beF) construct on maleimide-coated microtiter plates. Then, a fixed amount of streptavidin-horseradish peroxidase conjugate is added to the plate in the presence of varying amount of the bicyclic or cyclic peptide. After washing, the amount of bound streptavidin is determined based on the residual peroxidase activity using a standard (ABTS) colorimetric assay. Using this assay, the IC 50  value for the head-to-tail monocyclic peptide cyclo[S(OpgY)TNCHPQFANA (SEQ ID NO: 189) was determined to be 1.9 μM, while the thioether-constrained bicyclic peptides from the cStrep3(S)-Z3C(p-2beF) and cStrep3(C)-Z8C(p-2beF) constructs exhibited an IC 50  of 3.7 and 0.77 μM, respectively ( FIG. 39B ). The &gt;2-fold increase in streptavidin binding affinity exhibited by the latter as compared to the monocyclic counterpart exemplifies the inherent advantage provided by presence of the additional intramolecular thioether linkage. 
     Experimental Details. 
     Preparation and Isolation of Bicyclic Macrocycles. 
     Protein expression of constructs 16-20 was performed as described in the previous Examples with the difference that cells were incubated for additional 3 hours at 37° C. after overnight growth. Cells were harvested, lysed and the cell lysate treated as described above to isolate and analyze the streptavidin-bound peptides by LC-MS. To analyze the amount of protein splicing occurred in vivo, the same cell lysate samples were incubated with chitin beads for 1 h on ice. Beads were washed two times with buffer followed by incubation with acetonitrile:H 2 O (70:30 v/v) for one minute to release any chitin-bound protein. Eluates were analyzed by LC-MS. 
     6.9 Example 9: Polycyclic Peptides 
     This example demonstrates the feasibility of generating polycyclic peptides using the methods provided herein. In particular, it demonstrates the formation and isolation of polycyclic peptides obtained via the post-translational cyclization of precursor polypeptides containing multiple Z/Cys pairs. It also demonstrates the formation and isolation of polycyclic peptides produced via the cyclization of ribosomally derived precursor polypeptides of general formula (V). In particular, this example demonstrates certain embodiments as schematically described in  FIGS. 37A-B . 
     For these studies, the constructs corresponding to Entries 21 and 22 of Table 1 were utilized. In Strep6_Z4C7C4Z, a Z/Cys pair encompassing a four-amino acid target peptide sequence (HPQF (SEQ ID NO: 185)) is followed by a second Cys/Z pair encompassing a different target peptide sequence (NTSK) after a spacer sequence (ENLYFQS). To demonstrate the possibility to obtain polycyclic peptides in this manner, the corresponding precursor polypeptide was expressed in BL21(DE3)  E. coli  cells in the presence of the Mj-pOgY2-RS/MjtRNA CUA   Tyr  to achieve the site-selective incorporation of the unnatural amino acid p-2beF in correspondence of the two Z residues. Although two possible bicyclic products could be generated via p-2beF-mediated cysteine alkylation, the structure-reactivity studies described in  FIG. 9A  would predict that each p-2beF would react preferentially or exclusively with its most proximal cysteine residue (i.e., p-2beF3 with Cys8 and p-2beF21 with Cys16, Table 1). Indeed, LC-MS analysis of the small molecular weight products obtained after thiol-induced splicing of purified Strep6_Z4C7C4Z(p-2beF) revealed the occurrence of the expected 2beF3-Cys8/p-2beF21-Cys16 linked product ( FIG. 36 ) as the only bicyclic product. A small amount of the monocyclic 2beF3-Cys8-linked peptide was also observed. Overall, these studies demonstrate the possibility to generate precursor polypeptides with multiple Z/Cys pairs in order to obtain macrocyclic peptides featuring a polycyclic structure. Whereas this example illustrates the specific case in which two copies of the same cysteine-reactive amino acid are incorporated into the precursor polypeptide, a person skilled in the art would immediately recognize that this approach can be readily extended to the use of two different cysteine-reactive amino acids, such as those described in  FIGS. 5 and 6 . The ribosomal incorporation of two different cysteine-reactive unnatural amino acids into the precursor polypeptide can be achieved using methods known in the art, i.e. via suppression of two different stop codons (Wan, Huang et al. 2010) or via suppression of a stop codon and a four-based codon (Chatterjee, Sun et al. 2013; Sachdeva, Wang et al. 2014). As shown above, results from structure-reactivity studies such as those described in  FIGS. 9A-B  can guide the design of appropriate precursor polypeptides for the formation of a polycyclic peptide with the desired pattern of thioether linkages (i.e., through the judicious choice of spacing distances between the different Z and Cys residues). 
     The successful formation of cyclic peptides via the ribosomal incorporation of cysteine-reactive amino acids into precursor polypeptides as illustrated by the previous Examples suggested that macrocyclic peptide with a polycyclic architecture could also be obtained through the use of amino acids containing more than one cysteine-reactive functional group in their side chain, i.e. using amino acids with the general formula (VI) or (VII). To illustrate this aspect, one such amino acid, ObdpY, was designed and synthesized according to Scheme 6 of  FIG. 6 . A suitable, orthogonal AARS/tRNA pair for the ribosomal incorporation of ObdpY in response to an amber stop codon was then identified as described in Example 3. Using ObdpY and the Mj-pOgY2-RS/MjtRNA CUA   Tyr  pair, the precursor polypeptide corresponding to Entry 22 of Table 1 was expressed in  E. coli  and purified by Ni-affinity chromatography. In this protein (called Strep7_C 5 Z4C(ObdpY)), two cysteine residues flank the ObdpY residue encompassing two different target peptide sequences (i.e., AYDSG (SEQ ID NO: 188) and HPQF (SEQ ID NO: 185)). Analysis of the small molecular weight product obtained after thiol-induced splicing of the GyrA intein revealed the occurrence of the desired bicyclic peptide product ( FIG. 38 ). A small amount of the monocyclic peptide resulting from reaction of ObdpY side chain with only one of the cysteine residue was also observed. Altogether, these studies demonstrate the feasibility of certain embodiments as schematically illustrate in  FIGS. 37A-B . As noted above, structure-activity studies such as those presented in  FIGS. 9A-B  can guide the judicious choice of suitable Z2 residues of general formula (VI) or (VII) and of target sequence lengths in order to the obtain a polycyclic peptide carrying a desired pattern of thioether linkages. 
     6.10 Example 10: MOrPH-PhD: An Integrated Phage Display Platform for the Discovery of Functional Genetically-Encoded Peptide Macrocycles 
     This example demonstrates the display of macrocyclic peptides according to the methods disclosed herein. In particular, it illustrates the phage display of macrocyclic peptides derived from precursor polypeptides of general formula (I) and (II) using the cysteine-reactive unnatural amino acid O 2 beY. More broadly, this example demonstrates certain embodiments of the present disclosure as schematically described in  FIGS. 42 and 43 . 
     The envisioned strategy for the generation and functional screening of genetically encoded thioether-bridged macrocyclic peptides displayed on bacteriophage is schematically described in  FIG. 43 . To implement this method, a M13 pIII display system based on a pSEX phagemid and M13K07ΔpIII helper phage (Rondot S et al., 2001, Nat. Biotechnol., 19:75-78) was chosen, as this system enables low valency display and it was reported to facilitate the phage display of complex polypeptides (Broders O et al., 2003, Methods Mol. Biol., 205:295-302; Rulker T et al., 2012, PLoS One, 7:e37242). As shown in  FIG. 43 , the present macrocyclic peptide phage display (MOrPH-PhD) system was designed to feature a cyclic peptide genetically fused to the N-terminus of the M13 phage coat protein pIII, which is present in only few (3-5) copies on the tip of the phage particle. To obtain this, a macrocycle precursor sequence (MPS) was inserted between a pelB leader sequence and the pIII coat protein in a phagemid (pSEX) vector which contains an intergenic region (IGR) for packaging into the phage particle but lacks the remainder of the M13 phage genes. The pelB signal sequence directs the cargo polypeptide to the periplasmic space of  E. coli , where it is proteolytically cleaved by a signal peptidase. The macrocycle precursor sequence consists of a peptide sequence containing a cysteine residue and the non-canonical amino acid O 2 beY, which is genetically incorporated via amber stop codon (TAG) suppression (Liu C C et al., 2010, Annu. Rev. Biochem., 79:413-444; Wang L et al., 2006, Annu. Rev. Bioph. Biom., 35:225-249) using an engineered aminoacyl-tRNA synthetase/tRNACUA pair derived from  Methanococcus jannaschii  tyrosyl-tRNA synthetase and its cognate tRNA (Bionda N et al., 2014, ACS Chem. Biol., 9:2008-2013). Incorporation of O 2 beY in close proximity to a downstream or upstream cysteine (e.g., 5-10 residues apart) is sufficient for these residues to undergo a nucleophilic substitution reaction to yield a thioether-bridged macrocyclic peptide (Bionda N et al., 2014, ACS Chem. Biol., 9:2008-2013). Maturation of the phage particles incorporating the macrocycle-pIII fusions is made possible through infection of the host  E. coli  cell (TOP10F′) with the helper phage, whose genome lacks the pIII gene and the IGR. Since the phagemid is the only source of pIII protein and the latter is only expressed upon suppression of the amber stop codon with O 2 beY, this system ensures that (a) only the macrocycle-pIII fusion protein is incorporated into the mature phage particles and (b) the phagemid vector containing the gene that encodes for the macrocycle precursor sequence is integrated into the phage, thus establishing the required link between phenotype and genotype for library screening and deconvolution. The phage library is then panned against a target of choice and higher affinity binders are enriched through multiple rounds of affinity-based selection and amplification, followed by hit deconvolution via DNA sequencing ( FIG. 43 ). 
     Display of O 2 beY-Containing Sequences on M13 Phage Particles. 
     To assess the feasibility of the strategy outlined above, studies were performed to first establish the successful incorporation of O 2 beY in mature M13 phage particles by means of the engineered aminoacyl-tRNA synthetase O 2 beY-RS. To this end, a phagemid (pSEX81) construct was generated that encodes for an arbitrary linear peptide sequence containing an amber stop codon (TAG) and no cysteines (NB9=(amber stop)TGSKLAEYG; SEQ ID NO:205), fused to the N-terminal end of M13 phage coat protein pIII. This construct was then transformed into  E. coli  TOP10F′ cells containing a pEVOL-based plasmid (Young TS eta al., 2010, J. Mol. Biol., 395:361-374) encoding for the O 2 beY-RS synthetase and the cognate amber suppressor tRNA (Bionda N et al., 2014, ACS Chem. Biol., 9:2008-2013), followed by infection with M13K07ΔpIII helper phage. Since O 2 beY-RS was previously shown to selectively incorporate a non-canonical amino acid structurally similar to O 2 beY, i.e., O-propargyl-tyrosine (OpgY) (Bionda N et al., 2014, ACS Chem. Biol., 9:2008-2013), this ncAA was also used to assess the amber stop codon suppression efficiency of O 2 beY-RS in the phage-producing  E. coli  cells. Production of the phage in the absence of the non-canonical amino acid was expected to result in a reduced phage titer upon amplification in  E. coli  due to reduced expression of the minor coat protein pill. Following optimization of the expression conditions, a &gt;1,000-fold higher M13 phage titer was eventually obtained in the presence of either ncAA (O 2 beY or OpgY) compared to identical expression conditions in the absence of it ( FIG. 44A - FIG. 44C ). These results indicated that the amber stop codon had been successfully suppressed with the non-canonical amino acid, leading to the production of full-length, functional pill. 
     To assess whether O 2 beY maintains an integer side-chain alkyl-bromide group during phage assembly, as required for mediating peptide cyclization, the O 2 beY-containing phages were incubated with an excess of biotin-conjugated cysteine, followed by pull-down using streptavidin-coated beads. As negative control, phages displaying the peptide sequence containing OpgY, which is unable to react with thiol nucleophiles, were subjected to the same treatment. As shown in  FIG. 44C , the phages produced in the presence of O 2 beY could be recovered from the streptavidin-coated beads at significantly higher levels than the OpgY-containing phages, thus demonstrating the successful incorporation and display of functional O 2 beY on the phage particles. 
     Affinity Selection of Streptavidin-Binding Macrocyclic Peptides. 
     Studies were performed to assess the functionality of the phage display-based strategy for functional selection of thioether-linked macrocyclic peptides ( FIG. 43 ) that can bind to a target protein. To this end, streptavidin was used as the target protein and two libraries of macrocyclic peptides displayed on phage were prepared using target sequences in which a fixed HPQ motif is flanked by four fully randomized positions (NNK codon, where N=A, G, C, or T and K=G or C) and the O 2 beY/Cys pair in two different orientations ( FIG. 45A ). After production in  E. coli  (2×10 7  colony forming units (c.f.u.); total size of DNA library: 2.2×10 6 ), the phage displayed macrocycle libraries were panned against streptavidin immobilized on resin beads and subjected to four rounds of enrichment and amplification with increasing stringency and competitive elution with biotin ( FIG. 45C ). Upon deep sequencing of the enriched library after the final round of affinity selection, a clear consensus was observed revealing two major families of peptide sequences with a Cys/O 2 beY (i/i-8) connectivity ( FIG. 45A ). Based on these results, selected hits were chosen for further validation using a streptavidin binding assay. To this end, three peptide sequences from the ‘-GD’ family and two from the ‘-FD’ family, respectively, were subcloned into a pET22 vector for expression of the corresponding macrocyclic peptides fused to a N-terminal FLAG tag and a C-terminal chitin binding domain (CBD) and poly-histidine tag to facilitate purification and quantification. These constructs were recombinantly produced in  E. coli  using the O 2 beY-specific amber stop codon suppression system. After purification, each construct was found to have undergone quantitative cyclization (&gt;99%) as determined by MALDI-TOF mass spectrometry ( FIG. 48A ). Upon evaluating the FLAG-tagged macrocycles for streptavidin binding activity in a plate-based assay ( FIG. 45B ), the macrocyclic peptides were found to bind streptavidin with high affinity, exhibiting a binding dissociation constant (K D ) ranging from 20 to -2,000 nM ( FIG. 45A ). Furthermore, the best streptavidin-binding peptide, Strep-m3, was found to bind streptavidin with a K D  of 20 nM, whereas a linear counterpart obtained by replacing O 2 beY with OpgY, showed a 2-fold weaker binding affinity, highlighting a beneficial effect of the cyclic backbone for interaction with the target. The cyclic peptide Strep-m3 could be proteolytically cleaved from the CBD tag using a preinstalled Factor Xa cleavage site, followed by HPLC purification and MS characterization ( FIG. 48A ). The macrocyclic peptide (FLAG-Strep-m3) showed a binding affinity for streptavidin comparable to the CBD-fused peptide, confirming the specificity of the interaction between the targeted protein and cyclic peptide. Altogether, these experiments provided an initial validation of the functionality of the MOrPH-PhD system for the isolation of macrocyclic peptide binders to a target protein. 
     Discovery of Macrocyclic Peptides Inhibitors of the Keap1/Nrf2 Interaction. 
     The macrocyclic peptide phage display system of  FIG. 43  was further applied to discover macrocyclic peptides capable of targeting a biomedically relevant protein-protein interaction such as the interaction between the Kelch-like ECH-associated protein 1 (Keap1) and transcriptional regulator Nrf2. Disruption of the Nrf2/Keap1 interaction is a promising target for upregulating the expression of cytoprotective oxidative stress response enzymes for anti-inflammatory therapy (Steel et al. ACS Med Chem Lett 2012, 3, 407) and in neurodegenerative diseases (Sandberg et al., Neuropharmacology 2014, 79, 298). Two sets of macrocyclic peptide phage display libraries were designed for the purpose of developing macrocyclic inhibitors of the Keap1/Nrf2 interaction, the libraries featuring a i/i+7 cyclized peptide with the O 2 beY/Cys pair in two different orientation ( FIG. 46A ). The resulting MOrPH-PhD libraries, which comprise 5×10 4  unique peptide sequences, were subjected to four rounds of affinity selection against the Keap1 Kelch domain (KKD) immobilized on plate, followed by deep sequencing of the enriched clones. These analyses showed a significant enrichment only for members of the i/i+7-linked macrocycle library, indicating that this connectivity results in significantly better binders to Keap1 as compared to the i/i-7-linked macrocycles. Furthermore, a strong consensus sequence was observed across the identified hits, which could be clustered into two major sequence families corresponding to (O 2 beY)D(S/T)ETGEC and (O 2 beY)D(Φ)E(T/S)GEC ( FIG. 46A ). The most highly enriched macrocycles from each of the two families of consensus sequences (KKD-m1 and KKD-m3), along with a representative member of a less abundant family (KKD-m8), were then produced recombinantly in  E. coli  fused to a N-terminal FLAG tag and a C-terminal CBD tag for ease of detection and purification. After isolation, efficient cyclization of these target sequences was confirmed via MALDI-TOF MS analysis ( FIG. 48B ). The purified FLAG-tagged cyclic peptides were then tested for their ability to bind the Keap1 Kelch domain (KKD) using an in vitro assay, in which peptide binding to plate-immobilized KKD is detected using a HRP-conjugated anti-FLAG antibody ( FIG. 46B ). These experiments showed that all of the tested macrocycles interact with Keap1 with high affinity, with the best binder (KKD-m1) interacting with Keap1 with a K D  of 110 nM ( FIG. 46A ). A nearly identical binding affinity (K D : 120±10 nM) was measured for this macrocyclic peptide after proteolytic cleavage of the CBD tag ( FIG. 48B ), confirming the specificity of the macrocycle/Keap1 interaction. In addition, a linear version of the same peptide, prepared by substituting O 2 beY with OpgY, was found to bind Keap1 with a five-fold lower affinity (K D =555±17 nM), highlighting the importance of the cyclized structure for optimal interaction with the target protein. The macrocyclic peptide KKD-m1 was then further investigated in a competition assay, in which immobilized Keap1 was incubated with the FLAG-tagged macrocycle and increasing amounts of a Nrf2-derived peptide encompassing the Keap1-binding region (Nrf277-83). These experiments showed that the Nrf2-derived peptide is able to inhibit KKD-m1 binding to Keap1 (IC 50 =2.8 nM;  FIG. 46C ), indicating that the macrocyclic peptide is able to disrupt Keap1/Nrf2 interaction. 
     Generation and Screening of Naïve Macrocyclic Peptide Phage Display Library. 
     A larger and naïve library of phage-displayed macrocyclic peptides was then generating using a fully randomized (NNK) hexamer sequence in the format O 2 beY-(Xaa) 6 -Cys, which encompasses a billion-member gene library and -108 unique peptide sequences ( FIG. 47A ). The corresponding MOrPH-PhD library was panned against Keap1 resulting in the successful selection and identification of several hits ( FIG. 47A ). After production and isolation in CBD-fused form ( FIG. 48C ), five representative macrocyclic peptide hits were tested in the in vitro binding assay ( FIG. 47B ) and found to be able to bind Keap1 with high affinity, showing low micromolar to low nanomolar KD. For example, the best Keap1 binders isolated from this naïve library, KKD(6X)-m1 and KKD(6X)-m2, bind Keap1 with a KD of 40 nM ( FIG. 47A ). Other hits, which are sequence-unrelated to KKD(6X)-m1 and KKD(6X)-m2, bind Keap1 in the low micromolar range. 
     As demonstrated above, this work describes the successful implementation and application of a M13 bacteriophage-based platform for the display and selection of thioether bridged macrocyclic peptides. This represents a first example of a phage display system for the exploration of combinatorial libraries of genetically encoded cyclic peptides of arbitrary sequence and constrained by a stable (i.e., non-reducible) linkage. 
     Another noteworthy result from the present studies concerns the importance of the orientation of the thioether linkage, i.e., via O 2 beY/Cys vs. Cys/O 2 beY arrangement, for modulating the structure and thus the function of the macrocyclic peptide. This aspect becomes evident from the affinity selection experiments, in which libraries of macrocyclic peptides featuring identical randomized sequences but inverted thioether linkages were evaluated side-by-side. Indeed, macrocycles constrained by a Cys/O 2 beY linkage proved to be most effective for some targets, while an inverted orientation of the thioether linkage (i.e., O 2 beY/Cys) was found to be more beneficial for others. In prior studies, it was determined that the orientation of the linkage does not inherently affect the efficiency of O 2 beY-mediated macrocyclization for target sequences of identical length (Bionda N et al., 2014, ACS Chem. Biol., 9:2008-2013). The capability to modulate and fine-tune the molecular recognition properties of these cyclic peptides through variation of the ncAA/Cys linkage orientation represents another unique feature of the present system compared to currently available methods useful for the screening of cyclic peptide libraries (Ladner R C et al., 2004, Drug Discov. Today, 9:525-529; Angelini A et al., 2011, Curr. Opin. Chem. Biol., 15:355-361; Passioura T et al., 2014, Ann. Rev. Biochem., 83:727-752). In addition, while remaining accessible by solid-phase peptide synthesis (Owens A E et al., 2017, J. Am. Chem. Soc., 139:12559-12568), the macrocyclic peptide hits identified using the present system can be readily produced recombinantly and isolated in purified form for rapid validation and evaluation in downstream functional assays. 
     In conclusion, this work introduces an efficient and potentially very general platform for the creation and functional exploration of combinatorial libraries of genetically encoded cyclic peptides. This system is expected to constitute a valuable, new tool for the discovery and evolution of bioactive peptide macrocycles capable of targeting proteins and disrupting protein-mediated interactions with high potency and selectivity. The reliance of this system on the use of a readily accessible reagent (O 2 beY) and spontaneous O 2 beY-mediated cyclization, along with the capability to readily modulate the cyclic structure and molecular recognition properties of these cyclic peptide through variation of the position and orientation of the O 2 beY/Cys linkage add to the technical simplicity and versatility of this approach. As demonstrated further below, the present system is compatible with the use of many other cysteine-reactive ncAAs and ncAA-mediated peptide cyclization chemistries for the exploration of structurally diverse libraries of peptide macrocycles. 
     Experimental Details for Example 10 
     Cloning of Phage Constructs and Libraries: 
     The PhD libraries were constructed via PCR using pSEX81 (Progen) as the template and the appropriate mutagenizing primers (NNK codon randomization; forward primers #1-12, reverse primer #14; Table 2). The PCR product was cloned into the Nco I/Nhe I cassette of pSEX81, and the recombinant plasmid libraries were transformed in  E. coli  TOP10F′ electrocompetent cells and selected on 20 cm×20 cm 2XYT agar plates containing ampicillin (100 μg/mL) and tetracycline (5 mg/L). A colony forming unity (c.f.u.) count exceeding by at least 3-fold the size of the respective DNA library was utilized for all the libraries. Colonies were then collected from the plates and the plasmid library was isolated using a plasmid midi-prep kit (Qiagen). Control constructs such as pSEX-NB9T and HL2-cyc were cloned into the Nco I/Nhe I cassette of pSEX81(Progen) using a similar procedure as described above. The recombinant plasmid was transformed into  E. coli  TOP10F′, selected on 2XYT agar plates containing ampicillin (100 μg/mL) and tetracycline (5 mg/L), and confirmed by DNA sequencing. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Oligonucleotide sequences. 
               
            
           
           
               
               
               
               
            
               
                 Primer 
                   
                   
                 SEQ ID 
               
               
                 Number 
                 Primer Name 
                 Primer Sequence 5′→3′ 
                 NO: 
               
               
                   
               
               
                  1 
                 HPQ-NNK A(F) 
                 TCGCCATGGCGGGCAGCTAGNNKNNKC 
                 190 
               
               
                   
                   
                 ATCCGCAGNNKNNKTGCGGCAGCGCGG 
                   
               
               
                   
                   
                 CCGCTGGATCCAAAG 
                   
               
               
                   
               
               
                  2 
                 HPQ-NNK B(F) 
                 TCGCCATGGCGGGCAGCTGCNNKNNKC 
                 191 
               
               
                   
                   
                 ATCCGCAGNNKNNKTAGGGCAGCGCGG 
                   
               
               
                   
                   
                 CCGCTGGATCCAAAG 
                   
               
               
                   
               
               
                  3 
                 SHH-4mer(F) 
                 TCGCCATGGGCACCNNKNNKGATNNKG 
                 192 
               
               
                   
                   
                 AAGAATAGGATGGCTGCNNKGATGCGG 
                   
               
               
                   
                   
                 CCGCTGGATCCAAAG 
                   
               
               
                   
               
               
                  4 
                 Nrf2-NNK-A.1(F) 
                 TCGCCATGGCGGGCAGCTAGNNKNNKG 
                 193 
               
               
                   
                   
                 AANNKGGCGAATGCGGCAGCGCGGCCG 
                   
               
               
                   
                   
                 CTGGATCCAAAG 
                   
               
               
                   
               
               
                  5 
                 Nrf2-NNK-A.2(F) 
                 TCGCCATGGCGGGCAGCTAGGATNNKG 
                 194 
               
               
                   
                   
                 AANNKNNKGAATGCGGCAGCGCGGCCG 
                   
               
               
                   
                   
                 CTGGATCCAAAG 
                   
               
               
                   
               
               
                  6 
                 Nrf2-NNK-A.3(F) 
                 TCGCCATGGCGGGCAGCTAGNNKGAAG 
                 195 
               
               
                   
                   
                 AANNKNNKGAATGCGGCAGCGCGGCCG 
                   
               
               
                   
                   
                 CTGGATCCAAAG 
                   
               
               
                   
               
               
                  7 
                 Nrf2-NNK-B.1(F) 
                 TCGCCATGGCGGGCAGCTGCNNKNNKG 
                 196 
               
               
                   
                   
                 AANNKGGCGAATAGGGCAGCGCGGCCG 
                   
               
               
                   
                   
                 CTGGATCCAAAG 
                   
               
               
                   
               
               
                  8 
                 Nrf2-NNK-B.2(F) 
                 TCGCCATGGCGGGCAGCTGCGATNNKG 
                 197 
               
               
                   
                   
                 AANNKNNKGAATAGGGCAGCGCGGCCG 
                   
               
               
                   
                   
                 CTGGATCCAAAG 
                   
               
               
                   
               
               
                  9 
                 Nrf2-NNK-B.3(F) 
                 TCGCCATGGCGGGCAGCTGCNNKGAAG 
                 198 
               
               
                   
                   
                 AANNKNNKGAATAGGGCAGCGCGGCCG 
                   
               
               
                   
                   
                 CTGGATCCAAAG 
                   
               
               
                   
               
               
                 10 
                 SHH-NNK-N- 
                 TCGCCATGGGCNNKNNKNNKNNKTAGG 
                 199 
               
               
                   
                 Term(F) 
                 AAGCGATGGACATGTGCACCGATACCG 
                   
               
               
                   
                   
                 GAGCGGCCGCTGGATCCAAAG 
                   
               
               
                   
               
               
                 11 
                 SHH-NNK- 
                 TCGCCATGGGCACCCTGTCCTGGTAGN 
                 200 
               
               
                   
                 Loop(F) 
                 NKNNKNNKNNKTGCACCGATACCGGAG 
                   
               
               
                   
                   
                 CGGCCGCTGGATCCAAAG 
                   
               
               
                   
               
               
                 12 
                 6X-lib(F) 
                 TCGCCATGGCGGGCTAGNNKNNKNNKN 
                 201 
               
               
                   
                   
                 NKNNKNNKTGCGGCGCGGCCGCTGGAT 
                   
               
               
                   
                   
                 CCAAAGATATCAGAG 
                   
               
               
                   
               
               
                 13 
                 NB9(F) 
                 TCGCCATGGGCTAGACCGGCAGCAAAC 
                 202 
               
               
                   
                   
                 TGGCGGAATATGGCGCGGCCGCTGGAT 
                   
               
               
                   
                   
                 CCAAAG 
                   
               
               
                   
               
               
                 14 
                 pIII Reverse 
                 CCTCAAGCTAGCTGATCATTAGCACAGG 
                 203 
               
               
                   
               
            
           
         
       
     
     Phage Expression and Purification: 
     The pSEX81-based plasmid library (or single plasmid construct) was transformed in TOP10F′  E. coli  cells containing the plasmid pEVOL-O 2 beY-RS (BiondaN et al., 2014, ACS Chem. Biol., 9:2008-2013) by electroporation, cells were recovered with SOC media (2% w/v tryptone, 0.5% w/v yeast extract, 10 mM NaCl, 2.5 mM KCl, and 20 mM glucose), and incubated with shaking at 37° C. for 1 hour. Cells were then transferred to a 200 mL Erlenmeyer flask containing 20 mL 2XYT media (1.6% w/v tryptone, 1.0% w/v Yeast extract, 8.6 mM NaCl) supplemented with ampicillin (50 mg/L), chloramphenicol (34 mg/L), and tetracycline (5 mg/L). Cell cultures were grown overnight (12-16 hours) at 37° C., and then cells were recovered by centrifugation (4,000×g). The cell pellet was diluted to an OD 600  of 0.05 in fresh 2XYT media supplemented with ampicillin (50 mg/L), chloramphenicol (34 mg/L), tetracycline (5 mg/L) and allowed to reach an OD 600  of 0.6. A volume equal to 10% of the final phage expression culture volume was infected with Hyperphage (Progen) at a MOI of 20. Hyperphage was allowed to infect the cells for 1 hour at 37° C. with shaking (200 rpm), then the culture was pelleted by centrifugation (4,000×g). The pellet was resuspended in 2XYT supplemented with ampicillin (50 mg/L), chloramphenicol (34 mg/L), tetracycline (5 mg/L), kanamycin (30 mg/L), arabinose (0.06%), and non-canonical amino acid (2 mM). Cultures were grown for 18 hours at 30° C. with shaking (200 rpm) to express the desired library or phage clone. After expression, cell cultures were pelleted by centrifugation (4,000×g). The resulting supernatant was incubated at pH 8.5 for 6 hours to facilitate complete cyclization of macrocyclic peptides and then concentrated using an Amicon 30kDal spin filter to a convenient volume (250-300 μL). The concentrated supernatant was then mixed with 1:4 (v/v) 20% polyethylene glycol buffer (20% polyethylene glycol, 2.5 M NaCl) at 4° C. and incubated overnight. The precipitated phage was pelleted by centrifugation (14,000×g) for 30 minutes, and resuspended in 200 μL PBS (10 mM Na 2 HPO 4 , 1.8 mM, KH 2 PO 4 , 137 mM NaCl, 2.7 mM KCl, pH 8.5). The fully resuspended phage solution was centrifuged (14,000×g) for an additional 5 minutes to remove any insoluble cellular debris. The clarified phage solution was purified a second time and then passed through a 0.22 m filter and stored in PBS pH 7.5 buffer at 4° C. 
     Determination of Phage Titer: 
     10 μL aliquots of purified phage solutions were serially diluted in 10-fold dilutions with 2×YT media. 10 μL of each dilution is added to 90 μL of exponentially growing  E. coli  TOP10F′ cells (OD 600 =0.4-0.6) in Eppendorf tubes. The phage was allowed to infect  E. coli  cells for 1 hour at 37° C. with shaking on a desktop thermoblock. 100 μL of phage infected  E. coli  was then spread on 2XYT agar plates containing ampicillin (50 mg/L) and tetracycline (5 mg/L) and grown overnight at 37° C. The phage titer was determined by counting colony forming units. 
     Phage Biotinylation Experiment: 
     A pSEX81 plasmid encoding for the NB9 sequence N-terminally fused to pIII was expressed in the presence of O 2 beY (or OpgY) and purified as described above. Each phage preparation was diluted to a titer of 10 11  p.f.u. in 100 L of reaction buffer (50 mM potassium phosphate, 0.5 mM TCEP pH 8.5). Biotin-conjugated cysteine was then added to a final concentration of 2 mM and the reaction was allowed to proceed for 24 hours at room temperature. Phage were buffer exchanged against 50 mM potassium phosphate extensively (5 times) using a 30-KDa cutoff centrifugal concentrator (Amicon) to remove unreacted cystine functionalized biotin from the phage solution. Resulting phage was then diluted to a titer of 10 6  p.f.u. in PBS and incubated with magnetic streptavidin beads for 30 minutes at room temperature. Beads were separated from the supernatant with magnetic separation and the fraction of recovered phage was calculated from the phage titer (% recovered phages=((phage input-phage output)/phage input)*100). 
     Selection of Streptavidin Binding Macrocycles. 
     10 μl of streptavidin-coated magnetic beads (NEB) were washed 3 times with PBS to remove storage buffer and then incubated in 100 μl of PBS with 0.5% BSA for 2 hours at room temperature. The beads were removed from the solution by magnetic separation, washed once with PBS, and then incubated with 100 μL of phage in PBS (typical titer: 10 9 -10 11  p.f.u.). The phage/bead mixture was allowed to incubate with gentile shaking for 1 hour at room temperature. The beads were then removed from the solution by magnetic separation and washed 3×-5× times with PBS-Tween 20 buffer (0.05% Tween-20). The beads were then suspended in 100 μL 0.1 mM biotin for 30 minutes at room temperature. After competitive elution, the beads were removed from the solution by magnetic separation. 10 μl of the eluted phage solution was used to determine the titer of recovered phage. The remaining eluted phage was used to infect 2.5 ml mid-log TOP10F′ cells (OD 600  0.4-0.6) in 2XYT for 1 hour at 37° C. This culture was then pelleted by centrifugation and resuspended in 5 mL fresh 2XYT (AMP/TET) and allowed to grow to saturation overnight at 37° C. The plasmid was extracted from the overnight culture and the enriched plasmid pool was used to propagate new phage as described above. After four rounds of affinity selection and amplification, the enriched library was analyzed by deep sequencing. 
     Selection of Keap1 Binding Macrocycles. 
     For the selection experiments, Keap1 Kelch domain fused to a N-terminal poly-His tag via a TEV protease cleavable linker (‘Shh’) were immobilized on microtiter plates by incubating 100 μL of protein solution at 4 μM in PBS buffer overnight at 4° C., followed by blocking with 0.5% bovine serum albumin in PBS for 1.5 hour at room temperature. Plates were washed (3×150 μL of PBS with 0.5% Tween-20) prior to panning. Before each panning round, a negative selection was performed by pre-incubation of the library in BSA-blocked plates (for Keap1 binders selections) for 30 minutes at room temperature. Remining phage was then transferred to positive selection wells with immobilized Keap1 and incubated at room temperature for 1 hour. Then, wells were washed 3 to 5 times with 150 uL PBS with 0.5% Tween-20, and incubated with 50 μL elution buffer (0.2 M Glycine-HCl, pH 2.2, 1 mg/ml BSA) for 30 minutes at room temperature. The elution solution was neutralized with 10 μL Neutralization buffer (1 M Tris-HCl, pH 9.1) and the fraction of recovered phages was determined using the phage titer protocol described above. The remaining phage was used to infect TOP10F′  E. coli  cells for amplification of the library. For each target, four rounds of affinity selection and amplification were carried out. After isolation of the phagemid, the enriched libraries were analyzed by deep sequencing. 
     Screening of Naïve Library. 
     Keap1 was immobilized and the phage library was expressed and purified as described above. Prior to selection, purified phage containing the naïve macrocyclic peptide library were incubated with 50 μL of immobilized TCEP (tris(2-carboxyethyl) phosphine) for 1 hour at room temperature with gentile shaking, followed by incubation at pH 8.5 for 2 hours. The phage libraries were precipitated with 1:4 (v/v) 20% polyethylene glycol buffer (20% polyethylene glycol, 2.5 M NaCl) at 4° C. for 2 h. The precipitated phage was pelleted by centrifugation (14,000×g) for 15 minutes, and resuspended in 100 μL PBS (10 mM Na 2 HPO 4 , 1.8 mM, KH 2 PO 4 , 137 mM NaCl, 2.7 mM KCl, pH 7.4). Phage libraries (10 11  pf.u.) were then incubated with immobilized Shh (or Keap1) at room temperature for 1 hour with gentile shaking. Then, the wells were washed 3 to 5 times with 150 μL PBS with 0.5% Tween-20, and incubated with 50 μL elution buffer (0.2 M Glycine-HCl, pH 2.2, 1 mg/ml BSA) for 30 minutes at room temperature. The elution solution was neutralized with 10 μL Neutralization buffer (1 M Tris-HCl, pH 9.1) and the fraction of recovered phages was determined using the phage titer protocol described above. The remaining phage was used to infect TOP10F′  E. coli  cells for amplification of the library. Three rounds of affinity selection and amplification were performed, after which the enriched library was analyzed by DNA sequencing. 
     Recombinant Expression and Isolation of Macrocyclic Peptides: The macrocyclic peptides were produced recombinantly as fusion constructs with an N-terminal FLAG tag (MDYKDDDDKGSGS-; SEQ ID NO:204) and a C-terminal chitin-binding domain (CBD) or a GyrA intein protein containing a C-terminal polyhistidine tag, according to previously reported procedures.63 Briefly, genes encoding the desired constructs were amplified by PCR and cloned into the BamH I/Xho I cassette of a pET22 vector containing a N-terminal FLAG-tag and C-terminal His tag. For the CBD-fusion construct, a Factor Xa cleavage site was introduced between the macrocycle precursor sequence and the CBD tag. The recombinant DNA constructs were transformed in  E. coli  DH5α and selected on LB agar plates supplemented with ampicillin (100 mg/mL), followed by DNA sequencing. For expression, the desired plasmid vector was transformed in  E. coli  BL21(DE3) containing pEVOL_O2beYRS and grown overnight in 2×YT media with ampicillin (100 mg/mL) and chloramphenicol (34 mg/mL). The overnight cultures were used to inoculate new cultures at an OD 600  of 0.05 and grown at 37° C. until mid-log growth (OD 600 =0.5), at which point they were transferred to 27° C. Cultures were then supplemented with O 2 beY (or OpgY) at 2 mM and induced with arabinose (0.06% w/v); after an hour, IPTG was added at 0.5 mM). Cells were grown for 16-18 hours at 27° C. and then harvested by centrifugation (4,000×g). Pellets were lysed via sonication and clarified by centrifugation (14,000×g). The peptide constructs were purified from the lysate using Ni-NTA affinity chromatography as per the manufacturer&#39;s instructions and stored in PBS pH 8.5. The macrocyclic peptides were characterized by MALDI-TOF MS. 
     Preparation of Tag-Free Macrocycles: 
     Tag-free macrocycles were produced by proteolytic cleavage of the CBD tag with Factor Xa or thiol-induced cleavage of the GyrA tag. For cleavage of the CBD tag, the purified CBD-fused macrocycles (250 PM) were digested with 5 μg/mL of Factor Xa protease (NEB) in Factor Xa Buffer (20 mM Tris, 100 mM NaCl, 2 mM CaCl 2 , pH=8.0) overnight at room temperature. The reaction was acidified with TFA (0.1%) and purified by solid-phase extraction with a step gradient of acetonitrile in water (+0.1% TFA). Fractions containing peptide were lyophilized and peptide identity was confirmed by MALDI-TOF MS. For cleavage of the GyrA intein tag, the purified GyrA-fused macrocycles (˜200 μM) were incubated in potassium phosphate buffer (10 mM potassium phosphate, 150 mM NaCl, pH 8.5) containing 20 mM TCEP (tris(2-carboxyethyl) phosphine) and 10 mM thiophenol, for 16 hours at room temperature with gentle shaking. The solutions were then dialyzed against water to remove excess thiophenol, and then acidified with 0.1% TFA. The cleaved peptide was purified via solid-phase extraction with a step gradient of acetonitrile in water (+0.1% TFA). After lyophilization, the peptide identity was reconfirmed by MALDI-TOF MS. 
     Binding Assays. 
     For the streptavidin binding assays, streptavidin-coated plates (Sigma-Aldrich) were used. For the Keap1 binding assay, Keap1 was immobilized on microtiter plates by incubating 100 μL of a 4 μM protein solution in PBS buffer overnight at 4° C., followed by washing (3×150 μL of PBS with 0.5% Tween-20) and blocking with 0.5% bovine serum albumin in PBS for 1.5 h at room temperature. After washing, each well was incubated with 100 μL of purified FLAG-tagged peptide at varying concentrations for 1 hour at room temperature. After washing three times with wash buffer, each well was incubated with 100 μL of 1:2500 dilution of HRP-conjugate mouse anti-FLAG polyclonal antibody (Sigma-Aldrich) for 1 hour at room temperature. After three washing step with wash buffer, 100 μL of 2.2 mM o-phenylenediamine dihydrochloride, 4.2 mM urea hydrogen peroxide, 100 mM dibasic sodium phosphate, and 50 mM sodium citrate, pH 5.0, were added to each well, followed by measurement of the absorbance at 450 nm after 10-20 min using a Tecan Infinite 1000 plate reader. Equilibrium dissociation constants (K D ) were determined by fitting the dose-response curves to a 1:1 binding isotherm equation via nonlinear regression using SigmaPlot. Mean values and standard deviations were calculated from experiments performed in triplicate. 
     Keap1 Competition Assay. 
     Keap1 was immobilized on microtiter plates as described above. After washing, each well was incubated for 1 hour at room with 100 μL of serial dilutions of a peptide corresponding to Nrf277-83 in PBS buffer containing 800 nM FLAG-tagged KKD-m1. After washing, the bound peptide was quantified by means of HRP-conjugate mouse anti-FLAG polyclonal antibody and colorimetric assay as described above. The inhibitory constant (IC 50 ) was determined by fitting the dose-response curves to a 4-parameter equation via nonlinear regression using SigmaPlot. Mean values and standard deviations were calculated from experiments performed in triplicate. 
     Example 11: Multiplexed Macrocyclic Peptide Phage Display System 
     This example demonstrates the creation and application of multiplexed macrocyclic peptide phage display libraries generated through the use of multiple cysteine-reactive non-canonical amino acids (ncAAs) according to the methods disclosed herein. This example further demonstrates the use of different unnatural amino acids of general structure (III) for generating genetically encoded macrocyclic peptides. In addition, it illustrates the preparation and use of barcoding for streamlining the screening and analysis of multiplexed macrocyclic peptide phage display libraries according to the methods disclosed herein. 
     As described in Example 1, a diverse set of cysteine-reactive ncAAs, namely p-vinylsulfonamido-phenylalanine (pVsaF), p-acrylamido-phenylalanine (pAaF), p-(2-chloro-acetamido)-phenylalanine (pCaaF), and O-(4-bromobutyl)-tyrosine (O 4 bbY), was designed according to the methods disclosed herein and synthesized via the synthetic route provided  FIGS. 40 and 41 . To identify suitable orthogonal AARS/tRNA pairs for genetic incorporation of these ncAAs into the precursor polypeptides, a diverse panel of AARS/tRNA pairs were screened in the presence of each of these ncAA using the YFP(TAG)-based fluorescence assay described in Example 3 ( FIG. 49A-B ). From these experiments, it was established that the engineered aminoacyl-tRNA synthetase Mj-TyrRS42 (SEQ ID NO:209) was well suited for selective incorporation of either pCaaF or pAaF, Mj-VsF-RS (SEQ ID NO:210) was well suited for selective incorporation of pVsaF, and O 2 beY-RS (also called OpgY2-RS; SEQ ID NO:85) was well suited for selective incorporation of O 4 bbY into the reporter protein in response to an amber stop codon. To determine the efficiency of these ncAAs in mediating peptide cyclization of precursor polypeptides, each of these ncAAs was incorporated into a series of precursor polypeptide constructs featuring a varying inter-residue distance (i.e., from i/i+1 to i/i+20) and orientation of the Cys/Z pair ( FIG. 50A-B ). Using a procedure analogous to that described in Examples 4 and 6, these constructs were expressed as fusion to a C-terminal histagged-CBD domain from pET22b vectors in  E. coli  BL21(DE3) cells containing the appropriate AARS, as identified from the experiments of  FIG. 49 , along with the cognate amber stop codon suppressor tRNA. After purification via Ni-affinity chromatography, the macrocyclic peptide precursor sequence was proteolytically cleaved from the CBD tag using Factor IX. Detection and quantification of the cyclic product was carried by LC-MS and MS/MS analysis as described in Example 4. These experiments confirmed the ability of each of these ncAAs to promote the efficient cyclization of the varying precursor sequences, i.e., across target sequences of varying length and featuring different Z/Cys orientations ( FIG. 50A-B ). Accordingly, these ncAAs, along with the corresponding AARS/tRNA pairs, were chosen for implementation of the multiplexed macrocyclic peptide phage display system as described below. 
     To this end, a set of three barcoded macrocyclic peptide phage display libraries, comprising a fully randomized (NNK) pentamer target sequence in the format -Z-(Xaa) 5 -Cys-, were generated and cloned into pSEX vectors. Each library was barcoded using a non-silent barcode encoding for a different amino acid within the linker region between the pelB leader sequence (SEQ ID NO: 216) and the randomized sequence (Ala=O 4 bbY; Gly=pAaF; Thr=pCaaF), thus enabling multiplexed deconvolution of the libraries. Each barcoded DNA library was then expressed in TOP10F′ cells in the presence of the corresponding ncAAs, suppressor AARS/tRNA, and helper phage, resulting in the production of a set of barcoded phage-displayed macrocyclic peptide libraries cyclized by means of O 4 bbY, pAaF, or pCaaF. The phage libraries were then subjected to two rounds of infection/amplification in  E. coli  TOP10F′ cells in the absence (R1, R2) and in the presence (TCEP R1, TCEP R2) of phage treatment with TCEP prior to next step of infection/amplication in  E. coli . In each case, the phage display library could be efficiently propagated exhibiting no significant loss in phage titer over the enrichment rounds and maintaining a significantly higher phage titer (&gt;10e4 fold) than the no-ncAA control phage, which is indicative of the successful incorporation of the ncAA in the phage-displayed peptide, as illustrated in  FIG. 44B . Furthermore, deep-sequencing of the isolated phages after the second round of infection/amplification showed the expected presence of the amber stop codon preceding the randomized sequence ( FIG. 52B ), further confirming the successful incorporation of the ncAAs during phage production and assembly. Since it is known that panning of phages displaying randomized peptide sequence containing an odd number of invariant cysteine residues favor the enrichment of sequences containing an even number of cysteines (Chen et al. J. Am. Chem. Soc. 2013; 135(17):6562), this parameter was used as a proxy for assessing the efficiency of ncAA-mediated cyclization in the macrocyclic peptide phage display libraries investigated here. As shown in  FIG. 52A , none of the libraries showed an enrichment in cysteine-containing sequences during the rounds of infection/amplification compared to the pre-panning library (16%). Furthermore, similar results were obtained for the TCEP-treated libraries compared to the untreated libraries. Altogether, and consistent with the results of  FIG. 50 , these results demonstrated the ability of each of the ncAAs to promote efficient cyclization of the phage-displayed peptide sequences and thus the creation of multiplexed macrocyclic peptide phage display libraries Multiplexed deconvolution of these libraries was facilitated by the use of the barcode, which enabled simultaneous analysis of the pooled libraries and barcode-guided sorting of the sequences corresponding to the different ncAA-based libraries ( FIG. 52B ). 
     Further illustrating this aspect of the disclosure, a set of barcoded pSEX-based phagemid vectors was generated using a dual barcode system, in which one silent barcode was introduced within the linker region between the PelB signal sequence and the macrocyclic peptide sequence, and a second barcode was introduced within the non-coding region downstream of the pII gene. As shown in  FIG. 51 , macrocyclic peptide-pIII fusion gene in each of the barcoded phagemids could be selectively amplified using a corresponding set of barcode-specific primers. By using each of these dual-barcoded phagemids to produce a phage libraries containing a different ncAA, it becomes possible to amplify, propagate, isolate, and/or analyse each ncAA-specific subset of a multiplexed macrocyclic peptide phage display library over one or multiple rounds of selection and enrichment. Compared to a single-barcode system, the present dual-barcode system provides the key and distinct advantage that such sublibraries can be segregated and amplified in the presence of the corresponding ncAA after each round of selection, thereby preserving the association between the ncAA and the selected macrocyclic peptides generated using that ncAA over multiple rounds of selection. Another enabling aspect of this system is to enable the use of an identical codon (e.g. amber stop codon) or expression system (e.g., AARS/tRNA) pair for genetic incorporation of the Z residue, thereby further facilitating the generation, screening, and selection of multiplexed macrocyclic peptide display libraries. 
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     It will be appreciated that variants of the above-disclosed and other features and functions, or alternatives thereof, may be combined into many other different systems or applications. Various presently unforeseen or unanticipated alternatives, modifications, variations, or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims. 
     While embodiments of the present disclosure have been particularly shown and described with reference to certain examples and features, it will be understood by one skilled in the art that various changes in detail may be effected therein without departing from the spirit and scope of the present disclosure as defined by claims that can be supported by the written description and drawings. Further, where exemplary embodiments are described with reference to a certain number of elements it will be understood that the exemplary embodiments can be practiced utilizing either less than or more than the certain number of elements. 
     All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. 
     The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.