Preface
Contents
Contributors
Chapter 1: Peptide-Polymer Conjugation Via Copper-Catalyzed Alkyne-Azide 1,3-Dipolar Cycloaddition
1 Introduction
2 Materials
2.1 Synthesis of Peptide-Conjugate 3
2.2 Self-Assembly of Peptide-Conjugate 3
2.3 Purification of Peptide-Conjugate 3
3 Methods
3.1 Synthesis of Peptide-Conjugate 3
3.2 Self-Assembly of Peptide-Conjugate 3
3.3 Purification of Peptide-Conjugate 3
4 Notes
References
Chapter 2: Conjugation of Peptides to Gold Nanoparticles
1 Introduction
2 Materials
2.1 Chemicals
2.2 Labware and Equipment
3 Methods
3.1 Preparation of AuNPs
3.2 Conjugation of p53 Peptides on AuNPs
3.3 Characterization of AuNPs
3.4 Semiquantitative Measurement of the Amount of Peptides Conjugated on the AuNPs
3.5 Stability of Conjugated p53 Peptides on AuNPs
4 Notes
References
Chapter 3: Peptide-Protein Conjugation and Characterization to Develop Vaccines for Group A Streptococcus
1 Introduction
2 Materials
2.1 Activation of Carrier Protein
2.2 Determination of the Extent of Maleimidation Using EllmanΒ΄s Test
2.3 Conjugation of Peptide to Activated Carrier Protein
3 Methods
3.1 Activation of Carrier Protein
3.2 Determination of Extent of Maleimidation Using EllmanΒ΄s Test (Optional)
3.3 Conjugation of Peptide to Activated Protein
4 Notes
References
Chapter 4: Oxime/Hydrazone Conjugation at Histidine: Late-Stage Functionalization Approach of Unprotected Peptides
1 Introduction
2 Materials
2.1 Preparation of Peptide Substrate Using Fmoc-Solid-Phase Peptide Synthesis (Fmoc-SPPS)
2.2 LSF of Unprotected Peptide at Histidine
2.3 Conversion of the Difluoroethyl Group into a Conjugation Handle
2.4 Hydrazone Ligation with a Fluorophore
2.5 Equipment
3 Methods
3.1 Preparation of Peptide Substrate Using Fmoc-SPPS
3.2 LSF of Unprotected Peptide at Histidine
3.3 Conversion of the Difluoroethyl Group into a Conjugation Handle
3.4 Hydrazone Ligation with a Fluorophore
4 Notes
References
Chapter 5: Modification of Nanoparticles with Transferrin for Targeting Brain Tissues
1 Introduction
2 Materials
2.1 Synthesis of DOPE-PEG3,400-p-Nitrophenol (DOPE-PEG3,400-pNP)
2.2 Preparation of pNP-Activated Micelles
2.3 Preparation of Transferrin-Activated Micelles
3 Methods
3.1 Synthesis of DOPE-PEG3,400-p-Nitrophenol (DOPE-PEG3,400-pNP)
3.2 Preparation of pNP-Activated Micelles
3.3 Preparation of Transferrin-Activated Micelles
4 Notes
References
Chapter 6: Peptide-Pegylated Lipid Conjugation Via Copper-Catalyzed Alkyne-Azide 1,3-Dipolar Cycloaddition
1 Introduction
2 Materials
2.1 Synthesis of DOPE-PEG3400-Alkyne (4)
2.2 Synthesis of DOPE-PEG3400-Bombesin (6)
2.3 Self-Assembly of DOPE-PEG3400-Bombesin (6)
2.4 Purification of DOPE-PEG3400-Bombesin (6)
3 Methods
3.1 Synthesis of DOPE-PEG3400-Alkyne (4)
3.2 Synthesis of DOPE-PEG3400-Bombesin (6)
3.3 Self-Assembly of DOPE-PEG3400-Bombesin (6)
3.4 Purification of DOPE-PEG3400-Bombesin (6)
4 Notes
References
Chapter 7: Vitamin B12 - Peptide Nucleic Acid Conjugates
1 Introduction
2 Materials
2.1 Vitamin B12 Building Blocks for the Conjugation Via Copper(I)-Catalyzed Azide-Alkyne Cycloaddition
2.1.1 Vitamin B12 Azide
Equipment
Instruments
Solutions and Eluents
2.2 Vitamin B12 Carbamates with Terminal Azide Group
2.2.1 Chemicals
2.2.2 Equipment
2.2.3 Instruments
2.2.4 Solutions and Eluents
2.3 Vitamin B12 Building Block for the Conjugation Via Disulfide Bond Formation
2.3.1 Equipment
2.3.2 Instruments
2.3.3 Solutions and Eluents
2.4 PNA Oligomers
2.4.1 Chemicals
2.4.2 Equipment
2.4.3 Instruments
2.4.4 Solutions and Eluents
2.5 Vitamin B12-PNA Conjugates
2.5.1 Chemicals
2.5.2 Equipment
2.5.3 Instruments
2.5.4 Solutions and Eluents
3 Methods
3.1 Synthesis of Vitamin B12 Building Blocks for the Conjugation Via Copper(I)-Catalyzed Azide-Alkyne Cycloaddition
3.1.1 Synthesis of Vitamin B12 Azide
3.1.2 Synthesis of Vitamin B12 Carbamates with Terminal Azide Group
3.2 Synthesis of Vitamin B12 Building Block for the Conjugation Via Disulfide Bond Formation
3.3 Synthesis of PNA Oligomers
3.3.1 Synthesis of a PNA Oligomer with a Terminal Alkyne Moiety (Fig. 5b)
3.3.2 Synthesis of a PNA Oligomer with a Terminal Sulfhydryl Group (Fig. 5b)
3.4 Synthesis of Vitamin B12-Peptide Nucleic Acid Conjugates
3.4.1 Conjugation of Vitamin B12 with PNA Via Copper(I)-Catalyzed Azide-Alkyne Cycloaddition
3.4.2 Conjugation of Vitamin B12 with the PNA Via Disulfide Bond Formation
4 Notes
References
Chapter 8: Enzymatic Ligation of Disulfide-Rich Animal Venom Peptides: Using Sortase A to Form Double-Knotted Peptides
1 Introduction
2 Materials
2.1 Solid-Phase Peptide Synthesis
2.2 Oxidative Folding
2.3 Ligation
2.4 Peptide Purification and Analysis
2.5 Additional Equipment Required
3 Methods
3.1 Peptide Synthesis
3.2 Peptide Oxidation
3.3 Ligation
3.4 Peptide Purification and Analysis
4 Notes
References
Chapter 9: Solid-State, Thermal Synthesis of Peptide/Protein-Boron Cluster Conjugates
1 Introduction
2 Materials
2.1 Reaction Solutions (See Note 1)
2.2 HPLC Solvents (See Note 3)
2.3 Digestion Solutions
3 Methods
3.1 Conjugates Synthesis (Fig. 2)
3.2 HPLC Purification of TΞ²4-Boron Cluster Conjugates (See Note 3)
3.3 HPLC Purification of Lysozyme-Boron Cluster Conjugates
3.4 LC-MS Analysis
3.5 Determination of Concentrations of the Conjugates (See Note 11)
3.6 Digestion of Peptide-Boron Cluster Conjugates
3.7 Identification of the Sites of the Modification Using Tandem Mass Spectrometry (MS/MS) Analysis
4 Notes
References
Chapter 10: Bioconjugation of Peptides to Hybrid Gold Nanoparticles
1 Introduction
2 Materials
2.1 Aqua Regia
2.2 PEG-Stabilized AuNPs Synthesis
2.3 N-Terminus Peptide Conjugation to PEG-Stabilized AuNPs
2.4 Citrate-Stabilized AuNPs Synthesis
2.5 Cys-Terminal Peptide Conjugation to Citrate-Stabilized AuNPs
2.6 Structural Characterization Equipment
3 Methods
3.1 Aqua Regia
3.2 PEG-Stabilized Gold Nanoparticles Synthesis
3.3 N-Terminus Peptide Conjugation to PEG-Stabilized AuNPs
3.4 Citrate-Stabilized AuNPs Synthesis
3.5 Cys-Terminal Peptide Conjugation to Citrate-Stabilized AuNPs
3.6 Characterization of the Hybrid AuNPs
4 Notes
References
Chapter 11: Design and Synthesis of a Peptide-Based Glioma-Targeted Drug Delivery Vector gHope2
1 Introduction
2 Materials
2.1 Synthesis of the Construct
2.2 The Cleavage Mix
2.3 Conjugations
2.4 Purification and Mass-Spectrometry Analysis
2.5 Cell Culture Materials and Preparation of Solutions
2.6 24-Well Plate Preparation for Experiments with 0.1% Gelatin
3 Methods
3.1 On-Resin FAM Labeling of Peptide
3.2 Peptide Cleavage from the Resin
3.3 Solution-Based Doxorubicin Coupling
3.4 Peptide Purification and Analysis
3.5 Estimation of Cellular Uptake
3.6 Flow Cytometry
3.7 FACS Analysis
3.8 Confocal Microscopy
3.9 Cellular Uptake Experiments with Confocal Microscopy
3.10 Cell Membrane Binding Experiments
4 Notes
References
Chapter 12: Electrochemically Enabled C-Terminal Peptide Modifications
1 Introduction
2 Materials
2.1 Electrochemical Decarboxylation
2.2 Friedel-Crafts-Type Arylation
2.3 Sulfonylation
2.4 Equipment
3 Methods
3.1 Electrochemical Decarboxylation
3.2 Friedel-Crafts-Type Arylation
3.3 Sulfonylation
4 Notes
References
Chapter 13: Double Conjugation Using Mercapto-Acryloyl and Alkyne-Azide Reactions for the Synthesis of Branched Multiantigenic...
1 Introduction
2 Materials
2.1 Synthesis of N-Terminus 8Qmin Mercapto-Azide (N3CH2CO-CQAEPDRAHYNIVTF) (1)
2.2 Synthesis of N-Terminal Acryloyl E643-57 (CH2 = CHCO-QLLRREVYDFAFRDL) (2)
2.3 Synthesis of Multiantigenic Peptide Azide (3) Through Mercapto-Acryloyl Conjugation
2.4 Synthesis of Vaccine Candidate Lipopeptide 5
3 Methods
3.1 Synthesis of N-Terminus 8Qmin Mercapto-Azide (N3CH2CO-CQAEPDRAHYNIVTF) (1)
3.2 Synthesis of N-Terminal Acryloyl E643-57 (CH2 = CHCO-QLLRREVYDFAFRDL) (2)
3.3 Synthesis of Multiantigenic Peptide Azide (3) Through Mercapto-Acryloyl Conjugation
3.4 Synthesis of Vaccine Candidate Lipopeptide 5
4 Notes
References
Chapter 14: Chemical Protein Synthesis by Chemoselective Ξ±-Ketoacid-Hydroxylamine (KAHA) Ligations with 5-Oxaproline
1 Introduction
2 Materials
3 Methods
3.1 Preparation of C-Terminal Ξ±-Ketoacid Resin
3.2 Preparation of C-Terminal Carboxylic Acid Resin
3.3 Preparation of Ξ±-Ketoacid Segment (1)
3.4 Preparation of Carboxylic Acid Segment (2) with Opr
3.5 KAHA Ligation and O-to-N Acyl Shift (Figs. 3 and 4)
4 Notes
References
Chapter 15: Site-Specific Modification of Single-Chain Affinity Ligands for Fluorescence Labeling, Radiolabeling, and Bioconju...
1 Introduction
2 Materials
3 Methods
3.1 Reaction Setup
3.2 Reaction Mixture Analysis
3.3 Reaction Mixture Purification
4 Notes
References
Chapter 16: Preparation and Characterization of Quantum Dot-Peptide Conjugates Based on Polyhistidine Tags
1 Introduction
1.1 Quantum Dots
1.2 Applications of QD-Peptide Conjugates
1.3 Surface Chemistry and Bioconjugation of QDs
1.4 Bioconjugation of QDs via Polyhistidine Tags
2 Materials
2.1 Reagents
2.2 Equipment
3 Methods
3.1 Peptide Assembly
3.2 Agarose Gel Electrophoresis
3.2.1 Confirmation of Peptide Binding
3.2.2 Estimation of Peptide Loading Capacity
3.3 Capillary Polyacrylamide Gel Electrophoresis
3.3.1 Preparing the Polyacrylamide Gel-Filled Capillaries
3.3.2 Confirmation of Peptide Binding
3.4 FRET-Based Characterization
3.4.1 Confirmation of Peptide Binding
3.4.2 Assessment of Assembly Kinetics
3.4.3 Estimation of Peptide Loading Capacity via Simultaneous Addition
3.4.4 Estimation of Peptide Loading Capacity via Sequential Addition
4 Notes
References
Chapter 17: The Construction of a Genetically Encoded, Phage-Displayed Cyclic-Peptide Library
1 Introduction
2 Materials
2.1 Growth Media, Solutions, Buffers, Antibiotics, Plates
2.2 Cloning, Expression, and Selection of the Phage Library
2.3 Synthesis of the Selected Peptide CycH8a
2.4 Characterization of the Selected Peptide CycH8a
2.5 Instruments
3 Methods
3.1 Preparation of the Cyclic-Peptide Library
3.2 Expression of the Cyclic-Peptide Library
3.3 Panning Against HDAC8 Using Streptavidin Magnetic Beads
3.4 Synthesis of 5-FAM Conjugated Cyclic Octapeptide CycH8a (Fig. 2)
3.5 Fluorescence Polarization Measurement
3.6 IC50 Value Measurement
4 Notes
References
Chapter 18: The Chemical Synthesis of Site-Specifically Modified Proteins Via Diselenide-Selenoester Ligation
1 Introduction
2 Materials
2.1 Fmoc-Solid-Phase Peptide Synthesis (Fmoc-SPPS)
2.2 Diselenide-Selenoester Ligation/Native Chemical Ligation
3 Methods
3.1 Fmoc-SPPS Standard Protocol
3.1.1 Loading Resin
3.1.2 Peptide Extension Via Automated Microwave-Assisted SPPS
3.1.3 Preparative Acidolytic Cleavage/Deprotection and Purification
3.2 Preparation of Selenoester Peptide 1 for Two-Component DSL and Three-Component DSL-NCL
3.3 Preparation of Diselenide Peptide 2a for Two-Component DSL
3.4 Preparation of Diselenide-Thioester Peptide 2b for Three-Component DSL-NCL
3.5 Preparation of N-Terminal Cys Peptide 3 for Three-Component DSL-NCL
3.6 One-Pot, Two-Component DSL
3.7 Deselenization: Conversion of Selenocystine to Alanine
3.8 Oxidative Deselenization: Conversion of Selenocystine to Serine
3.9 One-Pot, Three-Component DSL-NCL
4 Notes
References
Chapter 19: Synthesis of Lipopeptides by CLipPA Chemistry
1 Introduction
2 Materials
3 Methods
3.1 Peptide Design and Synthesis
3.2 Thiol-Ene Reaction
3.3 Workup and Purification
4 Notes
References
Chapter 20: Constraining TAT Peptide by Ξ³PNA Hairpin for Enhanced Cellular Delivery of Biomolecules
1 Introduction
2 Materials
2.1 Peptide-Ξ³PNA Hybrid Synthesis and Characterization
2.2 RP-HPLC (Reversed Phase High-Performance Liquid Chromatography)
2.3 Cell Culture and Incubation
2.4 Polyacrylamide Gel
3 Methods
3.1 Peptide-Ξ³PNA Synthesis
3.2 Peptide-Ξ³PNA Purification and Characterization
3.3 Cyclized peptide Formation and Characterization
3.4 Cell Culture
3.5 Cellular delivery and Cell Imaging
3.6 TRAP (Telomere Repeat Amplification Protocol) Assay
4 Notes
References
Chapter 21: Preparation of mRNA Polyplexes with Post-conjugated Endosome-Disruptive Peptides
1 Introduction
2 Materials
2.1 Reagents
2.2 Solvent
2.3 Buffer Solutions (See Note 3)
3 Methods
3.1 BCN-PEGylation of GALA Peptide (Fig. 2)
3.1.1 Synthesis of BCN-PEG-NHS
3.1.2 Synthesis of Side Chain Protected GALA-Amine Peptide
3.1.3 Coupling BCN-PEG-NHS with Side Chain Protected GALA-Amine
3.1.4 Deprotection of BCN-PEG-GALA
3.2 mRNA polyplex Preparation
3.3 Gel Retardation Assay to Find Optimal (N/P) Ratio (See Note 10)
3.4 Preparation of GALA Modified mRNA polyplexes (See Note 12)
4 Notes
References
Chapter 22: Targeted Subcellular Protein Delivery Using Cleavable Cyclic Cell-Penetrating Peptide-Conjugates
1 Introduction
2 Material
2.1 Synthesis of Cyclic Cell-Penetrating Peptide
2.2 Genetic Fusion of Targeting Sequences to Protein Cargo
2.3 Expression and Purification of Protein Substrate
2.4 Conjugation of Cell-Penetrating Peptide to Protein
2.5 Cellular Uptake and Microscopy
3 Method
3.1 Synthesis of Cyclic Cell-Penetrating Peptide
3.1.1 Nuclear Localization Sequence (NLS)
3.1.2 Actin Binding Sequence
3.2 Expression and Purification of Protein Substrate
3.2.1 Nuclear Localization Sequence
3.2.2 Actin Binding Sequence
3.3 Conjugation of Cell-Penetrating Peptide to Protein
3.4 Cellular Uptake and Microscopy
4 Notes
References
Chapter 23: Facile Chemoselective Modification of Thioethers Generates Chiral Center-Induced Helical Peptides
1 Introduction
2 Materials
2.1 Reagents
2.2 Buffer
2.3 Equipment
3 Methods
3.1 Chiral Carbon Center-Induced Helical (CIH) Peptides and Basic Characterization
3.2 Synthesis of Unnatural Amino Acids
3.2.1 Synthesis of S5(2-Ph)
3.2.2 Synthesis of S5(2-Me)
3.2.3 Synthesis of Model Pentapeptides
Protocols for Solid-Phase Peptide Synthesis
Acetylation
FITC Labeling
3.2.4 The Ring-Closing by Thiol-ene Reaction
3.2.5 Cleavage, Purification, and Storage
Cleavage
Purification
Storage
3.2.6 Basic Characterization and Identification of CIH Peptides
LC-MS Assays
Circular Dichroism Assays
NMR Assays for Identification of CIH Peptides
Crystal Growth and Diffraction Assays
3.3 Dual-Chiral Peptide by Sulfoxide Modification
3.4 Dual-Chiral Peptide by Sulfonium Modification
4 Notes
References
Index