Peptide Macrocycles: Methods and Protocols (Methods in Molecular Biology, 2371) 1071616889, 9781071616888

Table of contents :
Preface
Contents
Contributors
Part I: Peptide Macrocycle Synthesis
Chapter 1: Three Methods for Peptide Cyclization Via Lactamization
1 Introduction
2 Materials
3 Methods
3.1 SPPS and Solution-Phase Macrolactamization (See Note 3)
3.2 SPPS and On-Resin Macrocyclization on 2-Chlorotrityl Chloride Resin
3.3 SPPS and On-Resin Macrocyclization by Native Chemical Ligation
4 Notes
References
Chapter 2: Solid-Phase Peptide Cyclization with Two Disulfide Bridges
1 Introduction
2 Materials
2.1 Reagents
2.2 Solvents and Solutions
2.3 Consumables
2.4 Instrumentation
3 Methods
3.1 Attaching First Amino Acid: Preparation of Preloaded Resin (Fmoc-Xaa-Rink Amide AM Resin)
3.2 Determination of Resin Substitution (Loading)
3.3 Elongation of Peptide Chain
3.4 On-Resin Formation of Two Disulfide Bonds
3.5 Ellman´s Test
4 Notes
References
Chapter 3: Peptide Macrocyclization Through Palladium-Catalyzed Late-Stage C-H Activation
1 Introduction
2 Materials
2.1 Protected Amino Acids (Store at 4 C)
2.2 Solvents
2.3 Coupling Reagent (Store at 4 C)
2.4 Deprotection Reagents
2.5 Catalyst and Oxidant (Stored in Thermostatic Drier)
2.6 Other Reagents
2.7 Equipment
3 Methods
3.1 Preparation of Peptidosulfonamides by Liquid Phase Peptide Synthesis
3.2 Preparation of Linear Peptide by Solid-Phase Peptide Synthesis (SPPS)
3.3 Macrocyclization of Peptidosulfonamides by Pd-Catalyzed C-H Olefination
3.4 Pd-Catalyzed, β-Carbon Arylation Method to Peptide Macrocyclization
3.5 Macrocyclization of Peptidoarylacetamides by Pd-Catalyzed C-H Olefination
4 Notes
References
Chapter 4: Three Methods for the Solution Phase Synthesis of Cyclic Peptides
1 Introduction
2 Materials
2.1 Materials for Peptide Cyclizations Via Pentafluorophenyl Ester
2.1.1 N-Boc-Protected Linear Peptide as Cyclization Precursor
2.1.2 N-Cbz-Protected Linear Peptide as Cyclization Precursor
2.2 Materials for Peptide Cyclizations of N- and C-Terminal Deprotected Linear Peptides
3 Methods
3.1 Peptide Cyclizations Via Pentafluorophenyl Ester
3.1.1 N-Boc-Protected Linear Peptide as Cyclization Precursor
3.1.2 N-Cbz-Protected Linear Peptide as Cyclization Precursor
3.2 Peptide Cyclizations of N- and C-Terminal Deprotected Linear Peptides
4 Notes
References
Chapter 5: Computational Design of Structured and Functional Peptide Macrocycles
1 Introduction
2 Materials
2.1 Hardware
2.2 Software
2.3 Installation
3 Basics of Peptide Design with Rosetta
3.1 Conformational Sampling of Constrained Peptides
3.1.1 Generating Conformations
3.1.2 Choosing Conformations
3.2 Computational Sequence Assignment
3.2.1 Rosetta Design
3.2.2 Controlling Design
3.3 Analysis
3.3.1 Analyzing Structured Peptides: Likelihood of Folding In Vitro
3.3.2 Analyzing Functional Peptides: Interface Metrics
4 Methods
4.1 Designing Structured Peptides
4.1.1 Backbone Generation
Backbone Conformational Sampling
Backbone Clustering
4.1.2 Peptide Sequence Generation
Rosetta Design
Folding Analysis
4.2 Design for Function
4.2.1 Anchor Extension
4.2.2 Docking and Design
5 Conclusion
6 Notes
References
Chapter 6: On-Resin Peptide Cyclization Using the 3-Amino-4-(Methylamino)Benzoic Acid MeDbz Linker
1 Introduction
2 Materials
2.1 Linear Peptide Synthesis and MeNbz Formation
2.2 Peptide Cyclization and One-Pot Modifications
3 Methods
3.1 Linear Peptide Synthesis and MeNbz Formation
3.1.1 MeDbz-Resin Preparation
3.1.2 SPPS of a Linear Peptide on a MeDbz-Resin
3.1.3 MeNbz Formation: Activation of the MeDbz Linker
3.2 Peptide Cyclization and One-Pot Modifications
3.2.1 Synthesis of Cyclic Thiodepsipeptides
3.2.2 Synthesis of Cysteine-Containing Homodetic Cyclic Peptides
3.2.3 Synthesis of Homodetic Cyclic Peptides with Subsequent Desulfurization
3.2.4 Synthesis of Homodetic Cyclic Peptides with Internal Disulfide Bond
3.2.5 Synthesis of Homodetic Cyclic Peptides with Subsequent Cysteine Alkylation
4 Notes
References
Chapter 7: Scalable and Efficient In Planta Biosynthesis of Sunflower Trypsin Inhibitor-1 (SFTI) Peptide Therapeutics
1 Introduction
2 Materials
2.1 Instrumentation
2.2 Assembly of Plant Expression Constructs
2.3 Plant Growth and Agrobacterium Infiltration
2.4 Plant Harvesting and Peptide Extraction
2.5 Preparing Synthetic Standards for Structural and Functional Equivalence Assays
2.5.1 Synthesis
2.5.2 HPLC-Based Peptide Purification
2.5.3 LC-MS
2.6 Peptide Oxidation
2.7 Determining Structural Equivalency of Peptides
2.7.1 MS/MS Fragment Analysis
2.7.2 NMR
2.8 Validating Functional Equivalency of Peptides
3 Methods
3.1 Assembly of Plant Expression Vectors for AEP and SFTI-1 Precursor Genes
3.2 Agrobacterium Transformation
3.3 Small-Scale Infiltration for Selection of Optimal AEP Ligase
3.4 Large-Scale Vacuum-Based Infiltration of Whole Plants
3.5 Whole Plant Peptide Extraction and Quantification
3.6 Preparing Synthetic Standards for Structural and Functional Equivalence Assays
3.6.1 Synthesis of Peptide
3.6.2 Peptide Purification and Oxidation
3.6.3 LCMS Validation of Samples
3.7 Validating the Structure and Functional Equivalence of Plant-Produced SFTI-1 Therapeutic Candidates
3.7.1 Co-Elution Analysis
3.7.2 MS/MS Fragment Analysis
3.7.3 NMR Analysis
3.8 Validating Functional Equivalence of Synthetic and in Planta Generated Peptides
4 Notes
References
Chapter 8: Ligation, Macrocyclization, and Simultaneous Functionalization of Peptides by Multicomponent Reactions (MCR)
1 Introduction
2 Materials
3 Methods
3.1 Resin Loading and Peptide Anchoring
3.2 Elongation 1
3.3 Test Cleavage
3.4 On-Resin Ugi Ligation
3.5 Elongation 2
3.6 Allyl Ester Orthogonal Deprotection (See Note 16)
3.7 On-Resin Ugi Cyclization
3.8 Final Cleavage and Purification
4 Notes
References
Chapter 9: Diels-Alder Cycloadditions for Peptide Macrocycle Formation
1 Introduction
2 Materials (See Note 2)
2.1 Reagents
2.2 Solvents and Solutions
2.3 Consumables
2.4 Instrumentation
3 Methods
3.1 DAC Peptide Synthesis Design and Preparation (See Fig. 1)
3.2 Representative DAC-RGD Peptide Synthesis and Functionalization
3.3 DAC Peptide Cleavage and Analysis
4 Notes
References
Part II: Peptide Macrocycle Combinatorial Library Synthesis and Screening
Chapter 10: Protein Catalyzed Capture (PCC) Agents for Antigen Targeting
1 Introduction
2 Materials
2.1 Library Synthesis
2.2 Preclear, Product Screen, and Target Screen
2.3 Proteins
2.4 Cyanogen Bromide (CNBr) Cleavage
2.5 MALDI-Time-of-Flight (TOF)/TOF Tandem Mass Spectrometry
3 Methods
3.1 Macrocyclic Peptide Library Synthesis
3.2 Library Preclear
3.2.1 Manual Preclear
3.2.2 Automated Preclear (Using Large Particle Flow Cytometer and Sorter Instrumentation)
3.3 Product Screen
3.4 Target Screen
3.5 CNBr Cleavage
3.6 MALDI TOF/TOF Tandem Mass Spectrometry
4 Notes
References
Chapter 11: Preparation of Bacterial Cell-Surface Displayed Semisynthetic Cyclic Peptides
1 Introduction
2 Materials
2.1 Plasmids
2.2 Solid-Phase Peptide Synthesis
2.3 Cell Surface Protein Expression
2.4 Protein Trans-Splicing and Oxime Cyclization on E. coli Cell Surface
2.5 Characterization of Cyclic Peptides by ESI-MS
2.6 Preparation of Semisynthetic Cyclic Peptide Library
3 Methods
3.1 Solid-Phase Peptide Synthesis
3.2 Bacterial Cell Surface Protein Expression
3.3 Cell Surface Protein Trans-Splicing and Peptide Cyclization
3.4 Analysis of Cyclic Peptide by LC-MS
3.5 Preparation of Semisynthetic Cyclic Peptide Library
4 Notes
References
Chapter 12: The Discovery of Peptide Macrocycle Rescuers of Pathogenic Protein Misfolding and Aggregation by Integrating SICLO...
1 Introduction
2 Materials
2.1 Growth Media, Antibiotics, Inducers of Protein Production and Common Buffers
2.2 E. coli Strains
2.3 Plasmid Vectors
2.4 Enzymes Used for DNA Cloning
2.5 Primers Used for DNA Cloning and Sequencing
2.6 SDS-PAGE/Western Blot Reagents
2.7 Reagents for Next-Generation Sequencing
2.8 Additional Reagents
2.9 Software
2.10 Equipment
3 Methods
3.1 Construction of Head-to-Tail Cyclic Peptide Libraries in E. coli
3.2 Assessment of the Quality of the Generated pSICLOPPS Library
3.2.1 Colony PCR (See Note 26)
3.2.2 Diagnostic Digestion
3.2.3 SDS-PAGE/Western Blot Analysis
3.2.4 Deep Sequencing Analysis
3.3 In Vivo Monitoring of Protein Misfolding and Aggregation
3.3.1 Expression of MisP-GFP
3.3.2 Monitoring GFP Fluorescence on Plate Reader
3.3.3 Monitoring GFP Fluorescence by Flow Cytometry
3.3.4 Analyzing the Overexpressed MisP-GFP Protein Using SDS-PAGE/Western Blot
3.4 Identification of Cyclic Peptides with Misfolding-Rescuing Activity in an Ultrahigh-Throughput Manner
3.5 Evaluation of the Efficiency of the Selection Process for the Discovery of Putative Bioactive Cyclic Peptides
3.5.1 Assessing the Rescuing Capabilities of Individual Clones from the Sorted Sub-library Population Against MisP Misfolding
3.5.2 For the Clones Exhibiting Positive Results (i.e., Increased Fluorescence Compared to the Negative Controls)
3.5.3 Assessing Whether the Above Positive Effects Are Dependent on Intein Processing by Utilizing the Ssp DnaE Splicing-Defic...
3.5.4 Generating Splicing-Deficient Variants of Selected Clones from the Sorted pSICLOPPS Sub-library
3.5.5 Assessing Whether the Splicing-Deficient Variants Rescue the Misfolding of the MisP
3.6 High-Throughput Sequencing of the Selected pSICLOPPS Sub-library
3.7 Identification of Structure-Activity Relationships and Bioactive Motifs of the Selected Peptides
4 Notes
References
Chapter 13: In Vitro Selection of Thioether-Closed Macrocyclic Peptide Ligands by Means of the RaPID System
1 Introduction
2 Materials
2.1 Preparation N-ClAc-Aminoacyl-tRNAfMetCAU
2.2 Immobilization of the Target Protein
2.3 Preparation of Pyromycin-Tagged mRNA Library
2.4 Preparation of Macrocyclic Peptide-mRNA Fusion Library and Selection of Active Species
3 Methods
3.1 Preparation N-ClAc-Aminoacyl-tRNAfMetCAU
3.2 Immobilization of the Target Protein
3.3 Preparation of Pyromycin-Tagged mRNA Library
3.4 Preparation of Macrocyclic peptide-mRNA Fusion Library and Selection of Active Species
4 Notes
References
Chapter 14: MOrPH-PhD: A Phage Display System for the Functional Selection of Genetically Encoded Macrocyclic Peptides
1 Introduction
2 Materials
2.1 Reagents
2.2 Solvents
3 Methods
3.1 Cloning of MOrPH-PhD Libraries
3.2 Production of MOrPH-PhD Library
3.3 Panning of MOrPH-PhD Library
3.4 Deconvolution of MOrPH-PhD Library Via DNA Sequencing
3.5 Recombinant Expression and Isolation of the Macrocyclic Peptide Hits
3.6 Streptavidin-Binding Assay
4 Notes
References
Chapter 15: Discovery of Antimicrobial Peptide Macrocycles Through Bacterial Display
1 Introduction
2 Materials
2.1 Primers and dsDNA
2.2 Growth Media and Reagents
2.3 Equipment and Materials
2.4 Kits and Enzymes
2.5 Software and Algorithms
3 Methods
3.1 Peptide Library Construction
3.2 Screening and Sequencing the Peptide Library
3.3 Read Trimming and Counting
3.4 Differential Abundance Analysis
4 Notes
References
Part III: Peptide Macrocycle Characterization
Chapter 16: Assessing the Cellular Uptake, Endosomal Escape, and Cytosolic Entry Efficiencies of Cyclic Peptides
1 Introduction
2 Materials
2.1 Solid Phase Peptide Synthesis and Purification
2.2 Fluorescent Labeling of Peptides
2.3 Cell Culture and Flow Cytometry
3 Methods
3.1 Solid Phase Synthesis of Cell-Penetrating Peptides
3.2 Solution-Phase Labeling of Peptides with NF and TMR
3.3 Standard Flow Cytometry and Data Analysis
3.4 Flow Cytometry in Acidic Solution
3.5 Advantages and Limitations of the Method
4 Notes
References
Chapter 17: In Silico Analysis of Peptide Macrocycle-Protein Interactions
1 Introduction
2 Materials
2.1 Computer
2.2 Software
3 Methods
3.1 Generating an Ensemble of Protein Conformations
3.2 Generating an Ensemble of Peptide Macrocycle Conformations
3.3 Generating an Ensemble of Protein-Peptide Macrocycle Complex Conformations
3.4 Calculation of MM/GBSA Binding Free Energies
3.4.1 For the Protein
3.4.2 For the Peptide
3.4.3 For the Complex
4 Notes
References
Chapter 18: Binding Characterization of Cyclic Peptide Ligands to Target Proteins and Chemical Epitopes Using ELISA and Fluore...
1 Introduction
1.1 ELISA
1.2 Fluorescence Polarization
2 Materials
2.1 Sandwich ELISA for His6-Tagged Protein
2.2 Sandwich ELISA for Glutathione (GST)-Tagged Protein
2.3 Indirect ELISA for Demonstration of Epitope Binding to Ligands
2.4 Fluorescence Polarization of One Cyclic Ligand Against Target Protein (See Note 16)
3 Methods
3.1 Sandwich ELISA of a Cyclic Peptide for Measuring Relative Binding Affinity to His6-Tagged Protein
3.2 Sandwich ELISA of a Cyclic Peptide for Measuring Relative Binding Affinity to His6-Tagged Protein
3.3 Indirect ELISA for Demonstration of a Cyclic Peptide Binding to Target Epitope
3.4 Fluorescence Polarization of Cyclic Peptide Ligand Binding to Target Protein
3.4.1 Determination of Optimal Concentration of Fluorescent Cyclic Ligand for an FP Assay
3.4.2 Saturation Fluorescence Polarization Experiment to Determine Ligand Binding to Protein
4 Notes
References
Chapter 19: Determining the Binding Kinetics of Peptide Macrocycles Using Bio-Layer Interferometry (BLI)
1 Introduction
2 Materials
2.1 Running Buffer
2.2 Sensors
2.3 Antigens and Peptides
2.4 Equipment
2.5 Consumables
3 Methods
3.1 Preparing the Sensors
3.2 Ligand Loading
3.3 Analyte Preparation
3.4 Concentration Scout
3.5 Analysis
4 Notes
References
Part IV: Unique Applications of Peptide Macrocycles
Chapter 20: Different Approaches to Cyclize a Cell-Penetrating Peptide and to Tether Bioactive Payloads
1 Introduction
2 Materials
2.1 General Synthesis Reagents and Solvents
2.2 Side Chain-to-Side Chain Cyclization by Click Reaction
2.3 Side Chain Selective and Covalent Labeling of the Peptide and Cyclization by Click Reaction
2.4 Head-to-Tail Cyclization
2.5 Head-to-Tail Cyclization and Conjugation to a Drug
3 Methods
3.1 Side Chain-to-Side Chain Cyclization by Click Reaction
3.2 Side Chain Selective and Covalent Labeling of the Peptide and Cyclization by Click Reaction
3.3 Head-to-Tail Cyclization
3.4 Head-to-Tail Cyclization and Conjugation to a Drug
4 Notes
References
Chapter 21: Helical Stabilization of Peptide Macrocycles by Stapled Architectures
1 Introduction
2 Materials
2.1 General Method for Solid-Phase Synthesis
2.2 Ring-Closing Metathesis (RCM)
2.3 Lactamisation
2.4 Cycloaddition
2.5 Reversible Reaction
2.6 Thioether Formation
2.7 Sulfonium Center Formation
3 Methods
3.1 General Method for Solid-Phase Peptide Synthesis
3.2 Ring-Closing Metathesis (RCM)
3.3 Lactamisation
3.4 `Click´ Cycloaddition
3.5 Reversible Disulfide Cyclization Reaction (See Note 19)
3.6 Thioether Formation
3.6.1 Hexafluorbenzene Linker
3.6.2 Other Linkers
3.7 Sulfonium Center Formation (Reversible) (See Note 22)
3.7.1 Cys-Met Macrocyclization
3.7.2 N-terminal: Met Macrocyclization
4 Notes
References
Chapter 22: Development and Characterization of Light-Responsive Peptide Macrocycles
1 Introduction
2 Materials
2.1 Synthesis of Light-Responsive Macrocyclic Peptides
2.1.1 Solvents
2.1.2 Reagents
2.1.3 Laboratory Equipment and Supplies
2.2 Synthesis of Light-Responsive Bicycle Peptides
2.2.1 Solvents
2.2.2 Reagents
2.2.3 Laboratory Equipment and Supplies
3 Methods
3.1 Synthesis of Light-Responsive Macrocyclic Peptides
3.1.1 Synthesis of 1b
3.1.2 Synthesis of 1c
3.1.3 Synthesis of 1d
3.1.4 Synthesis of 1e
3.1.5 Synthesis of 3,3′-Bis(sulfonato)-4,4′-bis(chloroacetamido)azobenzene (BSBCA)
3.1.6 Synthesis of the Light-Responsive Macrocyclic Peptide
3.2 Synthesis of Light-Responsive Bicycle Peptides
3.2.1 Synthesis of 3,5-Bis(hydroxymethyl) aniline (2b)
3.2.2 Synthesis of 3,5-Bis(hydroxymethyl) aniline-tert-butyl carbamate (2c)
3.2.3 Synthesis of 3,5-Bis(chloromethyl) aniline-tert-butyl carbamate (2d)
3.2.4 Synthesis of 3,5-Bis(chloromethyl) aniline (2e)
3.2.5 Synthesis of4-Methoxy-N-phenyl-benzylamine (2f)
3.2.6 Synthesis of 2g
3.2.7 Synthesis of 2h
3.2.8 Synthesis of N-(4-{(E)-[3,5-Bis(chloromethyl)phenyl]diazenyl}phenyl)acetamide (HADCAz)
3.2.9 Oxidative Conversion of N-Terminal Serine to Glyoxal
3.2.10 Synthesis of the Bicyclic Peptide
3.2.11 Kinetics and Switching Efficiency of HADCAz and Bicyclic Peptides
4 Notes
References
Chapter 23: Hydrogel Formation with Enzyme-Responsive Cyclic Peptides
1 Introduction
2 Materials
2.1 Materials for Solid-Phase Synthesis of Linear Peptide Precursors
2.2 Materials for Solution-Phase Macrocyclization of Progelators
2.3 Materials for Peptide Purification and Analysis
2.4 Materials for Progelator Sterilization and Formulation
2.5 Materials for Enzymatic Cleavage of Cyclic Peptide Progelators
3 Methods
3.1 Solid-Phase Synthesis of Linear Peptide Precursors
3.2 Solution-Phase Macrocyclization of Progelators
3.3 Peptide Purification and Analysis
3.4 Progelator Sterilization and Formulation (See Note 28)
3.5 Enzymatic Cleavage of Cyclic Peptide Progelators (See Note 31)
3.6 Sample Preparation for Instrumental Analysis
4 Notes
References
Chapter 24: Cyclization and Self-Assembly of Cyclic Peptides
1 Introduction
2 Materials
2.1 Disposable and Measuring Material
2.2 Reagents
2.3 Solvents
2.4 Equipment
3 Methods
3.1 Cyclization of Linear Peptides by Reaction Between the Carboxy- and Amino-Termini
3.2 Aqueous Self-Assembly of Cyclic Peptides
3.2.1 Temperature Annealing
3.2.2 Assembly Triggered by pH
3.2.3 Assembly Triggered by Controlling the Ionic Strength
3.3 Characterization of Cyclic Peptide Assemblies
3.3.1 (Scanning) Transmission Electron Microscopy
3.3.2 Atomic Force Microscopy
3.3.3 Fluorescence Microscopy
3.3.4 Nuclear Magnetic Resonance
4 Notes
References
Index