The library was synthesized on 500 mg of TentaGel microbeads (130 m, ~7

The library was synthesized on 500 mg of TentaGel microbeads (130 m, ~7.8 105 beads/g, ~350 pmol peptides/bead). recognize Rabbit polyclonal to ACTN4 specific phosphoserine (pSer)/phosphothreonine (pThr)-Pro motifs in their protein substrates.4,5 Through cis-trans isomerization of specific pSer/pThr-Pro bonds, Pin1 regulates the levels, activities, as well as intracellular localization of a wide variety of phosphoproteins.6 For example, Pin1 controls the in vivo stability of cyclin D17,8 and cyclin E9 and switches c-Jun,10 c-Fos,11 and NF-B12 between their inactive unstable forms and active stable forms. Isomerization by Pin1 also regulates the catalytic activity of numerous cell-cycle signaling proteins such as phosphatase CDC25C13,14 and kinase Wee1.15 Finally, Pin1-catalyzed conformational changes in -catenin16 and NF-B12 lead to subcellular translocation. Given its essential tasks in cell-cycle rules and improved manifestation levels and activity in human being cancers,17 Pin1 has been proposed like a potential target for the development of anticancer medicines.18,19 Pin1 is also implicated in neural degenerative diseases such as Alzheimers disease.20 Therefore, there have been significant interests in developing specific inhibitors against Pin1. Small-molecule inhibitors such as Juglone,21 PiB,22 dipentamenthylene thiauram monosulfide23 and halogenated phenyl-isothiazolone (TME-001)24 generally lack sufficient potency and/or specificity.25 A number of potent peptidyl Pin1 inhibitors have been reported and are more selective than the small-molecule inhibitors.26C31 However, peptidyl inhibitors are generally impermeable to the cell membrane and therefore have limited energy as therapeutics or in vivo probes. We recently reported a cell-permeable bicyclic peptidyl inhibitor against Pin1, in which one ring (A ring) presented a Pin1-binding phosphopeptide motif [D-pThr-Pip-Nal, where Pip and Nal are (R)-piperidine-2-carboxylic acid and L-naphthylalanine, respectively] while the second ring (B ring) contained a cell-penetrating peptide, Phe-Nal-Arg-Arg-Arg-Arg (Number 1, peptide 1).32 Even though bicyclic peptidyl inhibitor is potent (KD = 72 nM) and active in cellular assays, we anticipated that its D-pThr moiety might be metabolically labile due to hydrolysis by nonspecific phosphatases. The bad costs of the phosphate group might also impede the cellular access of the inhibitor. In this work, we found out a nonphosphorylated bicyclic peptidyl inhibitor against Pin1 by testing a peptide library and hit optimization. The producing bicyclic peptidyl inhibitor is definitely potent and selective against Pin1 in vitro, cell-permeable, and metabolically stable in biological assays. Open in a separate window Number 1 Development of bicyclic peptide inhibitors against Pin1. The structural moieties derived from library screening are demonstrated in red, while the changes made during optimization are demonstrated in blue. RESULTS AND Conversation Bicyclic Peptide Library Design, Synthesis, and Screening We previously found that although removal of the phosphoryl group of peptide 1 significantly reduced its potency against Pin1, the nonphosphorylated peptide (Number 1, peptide 2) was still a relatively potent Pin1 inhibitor (KD = 0.62 M).32 We hypothesized the potency of peptide 2 might be further improved by optimizing the sequences flanking the D-Thr-Pip-Nal motif. We consequently designed a second-generation bicyclic peptide library, bicyclo[Tm-(X1X2X3-Pip-Nal-Arg-Ala-D-Ala)-Dap-(Phe-Nal-Arg-Arg-Arg-Arg-Dap)]–Ala–Ala-Pra–Ala-Hmb–Ala–Ala-Met-resin (Number 1, where Tm was trimesic acid, Dap was 2,3-diaminopropionic acid, -Ala was -alanine, Pra was L-propargylglycine, and Hmb was 4-hydroxymethyl benzoic acid), by randomizing the three N-terminal residues of peptide 2. X1 and X2 displayed any of the 27 amino acid building blocks that included 12 proteinogenic L-amino acids [Arg, Asp, Gln, Gly, His, Ile, Lys, Pro, Ser, Thr, Trp, and Tyr], 5 nonproteinogenic -L-amino acids [L-4-fluorophenylalanine (Fpa), L-norleucine (Nle), L-ornithine (Orn), L-phenylglycine (Phg), and L-Nal], 6 -D-amino acids [D-Ala, D-Asn, D-Glu, D-Leu, D-Phe, and D-Val], and 4 N-methylated L-amino acids [L-N-methylalanine (Mal), L-N-methyleucine (Mle), L-N-methylphenylalanine (Mpa), and sarcosine (Sar)], while X3 was Asp, Glu, D-Asp, D-Glu, or D-Thr. Incorporation of these nonproteinogenic amino acids was expected to increase both the structural diversity and the proteolytic stability of the library peptides. The library experienced a theoretical diversity of 5 27 27 or 3645 different bicyclic peptides, most (if not all) of which were expected to become cell-permeable. The library was synthesized on 500 mg of TentaGel microbeads (130 m, ~7.8 105 beads/g, ~350 pmol peptides/bead). Peptide cyclization was achieved by forming three amide bonds between Tm and the N-terminal amine and the sidechain amines of the two Dap residues.33 The -Ala provides a flexible linker, while Pra serves as a handle.In particular, replacement of D-Phe with D-4-fluorophenylalanine (D-Fpa)] resulted in the most potent Pin1 inhibitor of this series (KD = 0.12 M for peptide 37) (Figures 1 and ?and2a).2a). activity of numerous cell-cycle signaling proteins such as phosphatase CDC25C13,14 and kinase Wee1.15 Finally, Pin1-catalyzed conformational changes in -catenin16 and NF-B12 lead to subcellular translocation. Given its critical tasks in cell-cycle rules and increased manifestation levels and activity in human being cancers,17 Pin1 has been proposed like a potential target for the development of anticancer medicines.18,19 Pin1 is also implicated in neural degenerative diseases such as Alzheimers disease.20 Therefore, there have been significant interests in developing specific inhibitors against Pin1. Small-molecule inhibitors such as Juglone,21 PiB,22 dipentamenthylene thiauram monosulfide23 and halogenated phenyl-isothiazolone (TME-001)24 generally lack sufficient potency and/or specificity.25 A number of potent peptidyl Pin1 inhibitors have been reported and are more selective than the small-molecule inhibitors.26C31 However, peptidyl inhibitors are generally impermeable to the cell membrane and therefore have limited energy as therapeutics or in vivo probes. We recently reported a cell-permeable bicyclic peptidyl inhibitor against Pin1, in which one ring (A ring) presented a Pin1-binding phosphopeptide motif [D-pThr-Pip-Nal, where Pip and Nal are (R)-piperidine-2-carboxylic acid and L-naphthylalanine, respectively] while the second ring (B ring) contained a cell-penetrating peptide, Phe-Nal-Arg-Arg-Arg-Arg (Number 1, peptide 1).32 Even though bicyclic peptidyl inhibitor is potent (KD = 72 nM) and active in cellular assays, we anticipated that its D-pThr moiety might be metabolically labile due to hydrolysis by nonspecific phosphatases. The bad charges of the phosphate group might also impede the cellular entry of the inhibitor. With this work, we found out a nonphosphorylated bicyclic peptidyl inhibitor against Pin1 by testing a peptide library and hit optimization. The producing bicyclic peptidyl inhibitor is definitely potent and selective against Pin1 in vitro, cell-permeable, and metabolically stable in biological assays. Open up in another window Body 1 Progression of bicyclic peptide inhibitors against Pin1. The structural moieties produced from library testing are proven in red, as the adjustments produced during marketing are proven in blue. Outcomes AND Debate Bicyclic Peptide Library Style, Synthesis, and Testing We previously discovered that although removal of the phosphoryl band of peptide 1 considerably reduced its strength against Pin1, the nonphosphorylated peptide (Body 1, peptide 2) was still a comparatively powerful Pin1 inhibitor (KD = 0.62 M).32 We hypothesized the fact that strength of peptide 2 may be further improved by optimizing the sequences flanking the D-Thr-Pip-Nal theme. We as a result designed a second-generation bicyclic peptide collection, bicyclo[Tm-(X1X2X3-Pip-Nal-Arg-Ala-D-Ala)-Dap-(Phe-Nal-Arg-Arg-Arg-Arg-Dap)]–Ala–Ala-Pra–Ala-Hmb–Ala–Ala-Met-resin (Body 1, where Tm was trimesic acidity, Dap was 2,3-diaminopropionic acidity, -Ala was -alanine, Pra was L-propargylglycine, and Hmb was 4-hydroxymethyl benzoic acidity), by randomizing the three N-terminal residues of peptide 2. X1 and X2 symbolized the 27 amino acidity blocks that included 12 proteinogenic L-amino acids [Arg, Asp, Gln, Gly, His, Ile, Lys, Pro, Ser, Thr, Trp, and Tyr], 5 nonproteinogenic -L-amino acids [L-4-fluorophenylalanine (Fpa), L-norleucine (Nle), L-ornithine (Orn), L-phenylglycine (Phg), and Niraparib R-enantiomer L-Nal], 6 -D-amino acids [D-Ala, D-Asn, D-Glu, D-Leu, D-Phe, and D-Val], and 4 N-methylated L-amino acids [L-N-methylalanine (Mal), L-N-methyleucine (Mle), L-N-methylphenylalanine (Mpa), and sarcosine (Sar)], while X3 was Asp, Glu, D-Asp, D-Glu, or D-Thr. Incorporation of the nonproteinogenic proteins was likely to increase both structural diversity as well as the proteolytic balance from the collection peptides. The library acquired a theoretical variety of 5 27 27 or 3645 different bicyclic peptides, most (if not absolutely all) which were likely to end up being cell-permeable. The library was synthesized on 500 mg of TentaGel microbeads (130 m, ~7.8 105 beads/g, ~350 pmol peptides/bead). Peptide cyclization was attained by developing three amide bonds between Tm as well as the N-terminal amine as well as the sidechain amines of both Dap residues.33 The -Ala offers a flexible linker, while Pra acts as a deal with for on-bead labeling from the bicyclic peptides with fluorescent probes through click chemistry. The ester linkage of Hmb allows selective release from the bicyclic peptides in the resin for solution-phase binding evaluation. Finally, the C-terminal Met enables peptide release in the resin by CNBr cleavage ahead of MS evaluation. The library (100 mg of resin) was screened against a S16A/Y23A mutant Pin1, that includes a faulty WW area.34.Peptide 37 bound weakly to BSA (KD ~20 M), however, not the various other six protein (Body S3). D17,8 and cyclin E9 and switches c-Jun,10 c-Fos,11 and NF-B12 between their inactive unpredictable forms and energetic steady forms. Isomerization by Pin1 also regulates the catalytic activity of several cell-cycle signaling protein such as for example phosphatase CDC25C13,14 and kinase Wee1.15 Finally, Pin1-catalyzed conformational changes in -catenin16 and NF-B12 result in subcellular translocation. Provided its critical assignments in cell-cycle legislation and increased appearance amounts and activity in individual malignancies,17 Pin1 continues to be proposed being a potential focus on for the introduction of anticancer medications.18,19 Pin1 can be implicated in neural degenerative diseases such as for example Alzheimers disease.20 Therefore, there were significant passions in developing particular inhibitors against Pin1. Small-molecule inhibitors such as for example Juglone,21 PiB,22 dipentamenthylene thiauram monosulfide23 and halogenated phenyl-isothiazolone (TME-001)24 generally absence sufficient strength and/or specificity.25 Several potent peptidyl Pin1 inhibitors have already been reported and so are more selective compared to the small-molecule inhibitors.26C31 However, peptidyl inhibitors are usually impermeable towards the cell membrane and for that reason have limited tool as therapeutics or in vivo probes. We lately reported a cell-permeable bicyclic peptidyl inhibitor against Pin1, where one band (A band) highlighted a Pin1-binding phosphopeptide theme [D-pThr-Pip-Nal, where Pip and Nal are (R)-piperidine-2-carboxylic acidity and L-naphthylalanine, respectively] as the second band (B band) included a cell-penetrating peptide, Phe-Nal-Arg-Arg-Arg-Arg (Body 1, peptide 1).32 However the bicyclic peptidyl inhibitor is potent (KD = 72 nM) and dynamic in cellular assays, we anticipated that its D-pThr moiety may be metabolically labile because of hydrolysis by non-specific phosphatases. The harmful charges from the phosphate group may also impede the mobile entry from the inhibitor. Within this function, we uncovered a nonphosphorylated bicyclic peptidyl inhibitor against Pin1 by verification a peptide collection and hit marketing. The causing bicyclic peptidyl inhibitor is certainly powerful and selective against Pin1 in vitro, cell-permeable, and metabolically steady in natural assays. Open up in another window Body 1 Progression of bicyclic peptide inhibitors against Pin1. The structural moieties produced from library testing are proven in red, as the adjustments produced during marketing are proven in blue. Outcomes AND Debate Bicyclic Peptide Library Style, Synthesis, and Testing We previously discovered that although removal of the phosphoryl band of peptide 1 considerably reduced its strength against Pin1, the nonphosphorylated peptide (Body 1, peptide 2) was still a comparatively powerful Pin1 inhibitor (KD = 0.62 M).32 We hypothesized the fact that strength of peptide 2 may be further improved by optimizing the sequences flanking the D-Thr-Pip-Nal theme. We as a result designed a second-generation bicyclic peptide collection, bicyclo[Tm-(X1X2X3-Pip-Nal-Arg-Ala-D-Ala)-Dap-(Phe-Nal-Arg-Arg-Arg-Arg-Dap)]–Ala–Ala-Pra–Ala-Hmb–Ala–Ala-Met-resin (Body 1, where Tm was trimesic acidity, Dap was 2,3-diaminopropionic acidity, -Ala was -alanine, Pra was L-propargylglycine, and Hmb was 4-hydroxymethyl benzoic acidity), by randomizing the three N-terminal residues of peptide 2. X1 and X2 symbolized the 27 amino acidity blocks that included 12 proteinogenic L-amino acids [Arg, Asp, Gln, Gly, His, Ile, Lys, Pro, Ser, Thr, Trp, and Tyr], 5 nonproteinogenic -L-amino acids [L-4-fluorophenylalanine (Fpa), L-norleucine (Nle), L-ornithine (Orn), L-phenylglycine (Phg), and L-Nal], 6 -D-amino acids [D-Ala, D-Asn, D-Glu, D-Leu, D-Phe, and D-Val], and 4 N-methylated L-amino acids [L-N-methylalanine (Mal), L-N-methyleucine (Mle), L-N-methylphenylalanine (Mpa), and sarcosine (Sar)], while X3 was Asp, Glu, D-Asp, D-Glu, or D-Thr. Incorporation of the nonproteinogenic proteins was likely to increase both structural diversity as well as the proteolytic balance from the collection peptides. The library got a theoretical variety of 5 27 27 or 3645 different bicyclic peptides, most (if not absolutely all) which were likely to become cell-permeable. The library was synthesized on 500 mg of TentaGel microbeads (130 m, ~7.8 105 beads/g, ~350 pmol peptides/bead). Peptide cyclization was attained by developing three amide bonds between Tm as well as the N-terminal amine as well as the sidechain amines of both Dap residues.33 The -Ala offers a flexible linker, while Pra acts as a.Peptide 37 had zero influence on the catalytic activity of cyclophilin or FKBP12 A up to 5 M focus. Pin1 also regulates the catalytic activity of several cell-cycle signaling protein such as for example phosphatase CDC25C13,14 and kinase Wee1.15 Finally, Pin1-catalyzed conformational changes in -catenin16 and NF-B12 result in subcellular translocation. Provided its critical jobs in cell-cycle rules and increased manifestation amounts and activity in human being malignancies,17 Pin1 continues to be proposed like a potential focus on for the introduction of anticancer medicines.18,19 Pin1 can be implicated in neural degenerative diseases such as for example Alzheimers disease.20 Therefore, there were significant passions in developing particular inhibitors against Pin1. Small-molecule inhibitors such as for example Juglone,21 PiB,22 dipentamenthylene thiauram monosulfide23 and halogenated phenyl-isothiazolone (TME-001)24 generally absence sufficient strength and/or specificity.25 Several potent peptidyl Pin1 inhibitors have already been reported and so are more selective compared to the small-molecule inhibitors.26C31 However, peptidyl inhibitors are usually impermeable towards the cell membrane and for that reason have limited electricity as therapeutics or in vivo probes. We lately reported a cell-permeable bicyclic peptidyl inhibitor against Pin1, where one band (A band) presented a Pin1-binding phosphopeptide theme [D-pThr-Pip-Nal, where Pip and Nal are (R)-piperidine-2-carboxylic acidity and L-naphthylalanine, respectively] as the second band (B band) included a cell-penetrating peptide, Phe-Nal-Arg-Arg-Arg-Arg (Shape 1, peptide 1).32 Even though the bicyclic peptidyl inhibitor is potent (KD = 72 nM) and dynamic in cellular assays, we anticipated that its D-pThr moiety may be metabolically labile because of hydrolysis by non-specific phosphatases. The adverse charges from the phosphate group may also impede the mobile entry from the inhibitor. With this function, we found out a nonphosphorylated bicyclic peptidyl inhibitor against Pin1 by testing a peptide collection and hit marketing. The ensuing bicyclic peptidyl inhibitor can be powerful and selective against Pin1 in vitro, cell-permeable, and metabolically steady in natural assays. Open up in another window Shape 1 Advancement of bicyclic peptide inhibitors against Pin1. The structural moieties produced from library testing are demonstrated in red, as the adjustments produced during marketing are demonstrated in blue. Outcomes AND Dialogue Bicyclic Peptide Library Style, Synthesis, and Testing We previously discovered that although removal of the phosphoryl band of peptide 1 considerably reduced its strength against Pin1, the nonphosphorylated peptide (Shape 1, peptide 2) was still a comparatively powerful Pin1 inhibitor (KD = 0.62 M).32 We hypothesized how the strength of peptide 2 may be further improved by optimizing the sequences flanking the D-Thr-Pip-Nal theme. We consequently designed a second-generation bicyclic peptide collection, bicyclo[Tm-(X1X2X3-Pip-Nal-Arg-Ala-D-Ala)-Dap-(Phe-Nal-Arg-Arg-Arg-Arg-Dap)]–Ala–Ala-Pra–Ala-Hmb–Ala–Ala-Met-resin (Shape 1, where Tm was trimesic acidity, Dap was 2,3-diaminopropionic acidity, -Ala was -alanine, Pra was L-propargylglycine, and Hmb was 4-hydroxymethyl benzoic acidity), by randomizing the three N-terminal residues of peptide 2. X1 and X2 displayed the 27 amino acidity blocks that included 12 proteinogenic L-amino acids [Arg, Asp, Gln, Gly, His, Ile, Lys, Pro, Ser, Thr, Trp, and Tyr], 5 nonproteinogenic -L-amino acids [L-4-fluorophenylalanine (Fpa), L-norleucine (Nle), L-ornithine (Orn), L-phenylglycine (Phg), and L-Nal], 6 -D-amino acids [D-Ala, D-Asn, D-Glu, D-Leu, D-Phe, and D-Val], and 4 N-methylated L-amino acids [L-N-methylalanine (Mal), L-N-methyleucine (Mle), L-N-methylphenylalanine (Mpa), and sarcosine (Sar)], while X3 was Asp, Glu, D-Asp, D-Glu, or D-Thr. Incorporation of the nonproteinogenic proteins was likely to increase both structural diversity as well as the proteolytic balance from the collection peptides. The library got a theoretical variety of 5 27 27 or 3645 different bicyclic peptides, most (if not absolutely all) which were likely to become cell-permeable. The library was synthesized on 500 mg of TentaGel microbeads (130 m, ~7.8 105.Incorporation of the nonproteinogenic proteins was likely to increase both structural diversity as well as the proteolytic balance from the collection peptides. consists of an N-terminal WW site and a C-terminal catalytic site, both which recognize particular phosphoserine (pSer)/phosphothreonine (pThr)-Pro motifs within their proteins substrates.4,5 Through cis-trans isomerization of specific pSer/pThr-Pro bonds, Pin1 regulates the amounts, activities, aswell as intracellular localization of a multitude of phosphoproteins.6 For instance, Pin1 settings the in vivo balance of cyclin D17,8 and cyclin E9 and switches c-Jun,10 c-Fos,11 and NF-B12 between their inactive unstable forms and dynamic steady forms. Isomerization by Pin1 also regulates the catalytic activity of several cell-cycle signaling protein such as for example phosphatase CDC25C13,14 and kinase Wee1.15 Finally, Pin1-catalyzed conformational changes in -catenin16 and NF-B12 result in subcellular translocation. Provided its critical jobs in cell-cycle rules and increased manifestation amounts and activity in human being malignancies,17 Pin1 continues to be proposed as a potential target for the development of anticancer drugs.18,19 Pin1 is also implicated in neural degenerative diseases such as Alzheimers disease.20 Therefore, there have been significant interests in developing specific inhibitors against Pin1. Small-molecule inhibitors such as Juglone,21 PiB,22 dipentamenthylene thiauram monosulfide23 and halogenated phenyl-isothiazolone (TME-001)24 generally lack sufficient potency and/or specificity.25 A number of potent peptidyl Pin1 inhibitors have been reported and are more selective than the small-molecule inhibitors.26C31 However, peptidyl inhibitors are generally impermeable to the cell membrane and therefore have limited utility as therapeutics or in vivo probes. We recently reported a cell-permeable bicyclic peptidyl inhibitor against Pin1, in which one ring (A ring) featured a Pin1-binding phosphopeptide motif [D-pThr-Pip-Nal, where Pip and Nal are (R)-piperidine-2-carboxylic acid and L-naphthylalanine, respectively] while the second ring (B ring) contained a cell-penetrating peptide, Phe-Nal-Arg-Arg-Arg-Arg (Figure 1, peptide 1).32 Although the bicyclic peptidyl inhibitor is potent (KD = 72 nM) and active in cellular assays, we anticipated that its D-pThr moiety might be metabolically labile due to hydrolysis by nonspecific phosphatases. The negative charges of the phosphate group might also impede the cellular entry of the inhibitor. In this work, we discovered a nonphosphorylated bicyclic peptidyl inhibitor against Pin1 by screening a peptide library and hit optimization. The resulting bicyclic peptidyl inhibitor is potent and selective against Pin1 in vitro, cell-permeable, and metabolically stable in biological assays. Open in a separate window Niraparib R-enantiomer Figure 1 Evolution of bicyclic peptide inhibitors against Pin1. The structural moieties derived from library screening are shown in red, while the changes made during optimization are shown in blue. RESULTS AND DISCUSSION Bicyclic Peptide Library Design, Synthesis, and Screening We previously found that although removal of the phosphoryl group of peptide 1 significantly reduced its potency against Pin1, the nonphosphorylated peptide (Figure 1, peptide 2) was still a relatively potent Pin1 inhibitor (KD = 0.62 M).32 We hypothesized that the potency of peptide 2 might be further improved by optimizing the sequences flanking the D-Thr-Pip-Nal motif. We therefore designed a second-generation bicyclic peptide library, bicyclo[Tm-(X1X2X3-Pip-Nal-Arg-Ala-D-Ala)-Dap-(Phe-Nal-Arg-Arg-Arg-Arg-Dap)]–Ala–Ala-Pra–Ala-Hmb–Ala–Ala-Met-resin (Figure 1, where Tm was trimesic acid, Dap was 2,3-diaminopropionic acid, -Ala was -alanine, Pra was L-propargylglycine, and Hmb was 4-hydroxymethyl benzoic acid), by randomizing the three N-terminal residues of peptide 2. X1 and X2 represented any of the 27 amino acid building blocks Niraparib R-enantiomer that included 12 proteinogenic L-amino acids [Arg, Asp, Gln, Gly, His, Ile, Lys, Pro, Ser, Thr, Trp, and Tyr], 5 nonproteinogenic -L-amino acids [L-4-fluorophenylalanine (Fpa), L-norleucine (Nle), L-ornithine (Orn), L-phenylglycine (Phg), and Niraparib R-enantiomer L-Nal], 6 -D-amino acids [D-Ala, D-Asn, D-Glu, D-Leu, D-Phe, and D-Val], and 4 N-methylated L-amino acids [L-N-methylalanine (Mal), L-N-methyleucine (Mle), L-N-methylphenylalanine (Mpa), and sarcosine (Sar)], while X3 was Asp, Glu, D-Asp, D-Glu, or D-Thr. Incorporation of these nonproteinogenic amino acids was expected to increase both the structural diversity and the proteolytic stability of the library peptides. The library had a theoretical diversity of 5 27 27 or 3645 different bicyclic peptides, most (if not all) of which were expected to be cell-permeable. The library was synthesized on 500 mg of TentaGel microbeads (130 m, ~7.8 105 beads/g, ~350 pmol peptides/bead). Peptide cyclization was achieved by forming three amide bonds between Tm and the N-terminal amine and the sidechain amines of the two Dap residues.33 The -Ala provides a flexible linker, while Pra serves as a.

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