Peptide Handbook A Guide to Peptide Design and Applications in Biomedical Research First Edition www.GenScript.com GenScript USA Inc. 860 Centennial Ave. Piscataway, NJ 08854 USA Phone: 1-732-885-9188 Toll-Free: 1-877-436-7274 Fax: 1-732-885-5878 Table of Contents The Universe of Peptides Reliable Synthesis of High-Quality Peptides Molecular structure 3 by GenScript Characteristics 5 Categories and biological functions 8 Analytical methods 10 Application of Peptides Research in structural biology 12 Research in disease pathogenesis 12 Generating antibodies 13 FlexPeptideTM Peptide Synthesis Platform which takes advantage of the latest Vaccine development 14 peptide synthesis technologies generates a large capacity for the quick Drug discovery and development 15 synthesis of high-quality peptides in a variety of lengths, quantities, purities Immunotherapy 17 and modifications. Cell penetration-based applications 18 Anti-microorganisms applications 19 Total Quality Management System based on multiple rounds of MS and HPLC Tissue engineering and regenerative medicine 20 analyses during and after peptide synthesis ensures the synthesis of Cosmetics 21 high-quality peptides free of contaminants, and provides reports on peptide Food industry 21 solubility, quality and content. Synthesis of Peptides Diverse Delivery Options help customers plan their peptide-based research Chemical synthesis 23 according to their time schedule and with peace of mind. Microwave-assisted technology 24 ArgonShield™ Packing eliminates the experimental variation caused by Ligation technology 26 oxidization and deliquescence of custom peptides through an innovative Recombinant technology 28 Modifications packing and delivery technology. 28 Purification 30 Expert Support offered by Ph.D.-level scientists guides customers from Product identity and quality control 31 peptide design and synthesis to reconstitution and application. Reconstitution, storage and handling 32 Delivery formats 34 Considerations in peptide design 35 Future of Peptides 38 Resources on Peptides Peptide-related tools 40 Troubleshooting guide 41 References 44 https://www.genscript.com/peptide-services.html https://www.genscript.com/peptide-handbook.html 1 Molecular Structure The word peptide, derived from the Greek word“peptós/digested,” refers to a chain of amino acids (AAs) linked together via amide or peptide bonds. The formation of the covalent peptide bond is an example of a condensation reaction, which generates water from the combination of the α-carboxyl group of one AA and the α-amino group of another. An AA unit within a peptide chain is called a "residue”. A peptide can be as short as two residues with only one peptide bond (named “dipeptide”) or as long as several residues forming a continuous and unbranched peptide chain (named “oligo- peptide” if containing <20 AAs or polypeptide if containing >20 AAs). Proteins are comprised of many peptide chains and sometimes the terms “polypeptide” and “protein” are used interchangeably. There are several different conventions for making a distinction between the two, but in general, molecules referred to as polypeptides have molecular weights (MWs) below 10,000 Daltons or less than 50 Chapter One AAs, and molecules above these limits are considered as proteins (Lehninger et al.) Peptide Bond Formation The Universe of Peptides Amino Acid 1 Amino Acid 2 R R H O N H H O H N H H O H H O Dipeptide Water R O H H H H N H O N O H H H O R Peptide Bond 3 In a peptide chain, the AA at the end with a free α-amino group is called the “amino-ter- minal” (N-terminal) residue, whereas the one at the other end with a free carboxyl group Characteristics is called the “carboxyl-terminal” (C-terminal) residue. The backbone carbon before the carboxyl carbon in an AA is called the “alpha carbon” (Cα) or “chiral center”, where differ- ent side chains attach. Depending on the position of carbon atoms relative to Cα, the remaining carbons are named with Greek letters of β, γ, etc. Numbering Carbon Atoms in an AA Each peptide has its own unique structural and biochemical characteristics, such as an isoelectric pH (pI) and ionization behavior. These features are derived from the peptide’s AA components. Therefore, the overall characteristics of a peptide can change depending γ ε δ β α on the quantity and type of each AA within the chain. 6 5 4 3 2 1 For example, the acid-base behavior of a peptide is defined by the nature of the free CH CH CH CH CH COO- 2 2 2 2 and bound AAs in its chain. The terminal α-amino and α-carboxyl groups sitting at opposite ends of the chain as well as the side chains (R groups) of some AAs can ionize and affect the + NH + NH 3 3 titration curve. Similarly, due to the positioning of the chiral carbon and stereoisomer configurations in all AAs except Glycine, the newly formed L or D isomer of AAs can add to Due to the possibility of forming two different enantiomers (stereoisomers) around their unique characteristics. Ultimately all these characteristics influence how a given the central carbon atom, all AAs except Glycine can exist in two isomeric configurations peptide behaves in a biological system. of D (dextrorotatory; right-handed) and L (levorotatory; left-handed). Until recently, most AAs synthesized by eukaryotes were reported to be in the L-isoform, whereas Formation of Acid or Base from an Amino Acid D-isoforms were predominantly found in bacterial cell wall proteins or synthesized chemically. However, due to latest developments in sensitive analytical methods, free D-amino acids are shown to be present in the nervous system and venom of animals as O O well (Kiriyama & Nochi). R + R OH H OH NH2 NH + Amino Acid Chirality 3 Positive Ion In acidic conditions: acting like a base. O O OH OH O O NH2 NH2 R - R 2 OH OH O- H O NH2 L-alanine D-alanine NH2 Negative Ion In basic conditions: acting like an acid. 4 5 Common Amino Acids Secondary Structure of Peptides O O O H OH OH OH OH H C H2N NH2 H NH2 NH2 N C O N C C O Alanine Isoleucine Leucine Valine C A I L V H O MW: 89.09 MW: 131.17 MW: 131.17 MW: 117.15 C H IP: 6.00 IP: 5.94 IP: 5.98 IP: 5.96 N C O C N H N O H C O O H H N OH OH 2 N C NH2 OH C NH2 N C NH2 HO O NH2 O OH C O H O Phenylalanine Tryptophan Tyrosine Asparagine C H F W Y N N C MW: 165.19 MW: 204.23 MW: 181.19 MW: 132.12 O C N IP: 5.48 IP: 5.89 IP: 5.66 IP: 5.41 H H C N C O O O O O N C S C OH O HS OH H2N OH HO OH C O NH2 NH2 NH2 H NH2 C H N C C Cysteine C Glutamine Q Methionine M Serine S O N MW: 121.16 MW: 146.15 MW: 149.21 MW: 105.09 C IP: 5.02 IP: 5.65 IP: 5.74 IP: 5.68 O O OH O NH O H O N β-sheet α-helix OH H2N OH H2N N OH OH H N NH2 NH2 NH2 NH2 Threonine T Arginine R Histidine H Lysine K MW: 119.12 MW: 174.2 MW: 155.16 MW: 146.19 IP: 5.64 IP: 11.15 IP: 7.47 IP: 9.59 GenScript’s Peptide Calculator can help you determine the chemical formula and MW of your peptide of interest. O OO O HO OH OH N H2N HO OH OH O NH2 H NH2 O Aspartic Acid D Glutamic Acid E Proline P Glycine G MW: 133.1 MW: 147.13 MW: 115.13 MW: 75.07 IP: 2.77 IP: 3.22 IP: 6.30 IP: 5.97 Aliphatic amino acids with hydrophobic side chain Amino acids with positive charged side chain Aromatic amino acids with hydrophobic side Chain Amino acids with negative charged side chain Amino acids with neutral side chain Unique amino acids MW: Molecular Weight IP: Isoelectric Point 66 7 The intrinsic characteristics of natural peptides, such as instability and proteolytic degra- Categories and Biological Functions dation limit their medicinal application. To overcome this limitation peptide analogues or “peptidomimetics” have been developed to mimic the biological actions of peptides without undesired restrictions (Olson). Peptidomimetics are synthesized by modification of an existing natural peptide or through introducing novel structural changes that render resistance to proteolytic degradation, increase thermodynamic stability and provide the capability to pass through the plasma membrane. The 7,000 naturally-occurring peptides are present in all organisms and have extensive roles in the physiology of microorganisms, plants, animals, and humans. To better study and use peptides, they can be classified using different methods. The function of peptides Peptide vs Peptidomimetics as hormones, growth factors, ion channel ligands, neurotransmitters, and immune system components in biological systems is the basis of one method of peptide classification. O R' R O Another approach is based on the way natural peptides are produced. Each method of H classification may overlap with another and can be field or application specific. N OH N OH H N H N H R' R O O Major Classes of Peptides n n Peptide Peptoid Class Source Application/Function O O H N N OH Antibiotics, Hormones, R' Ribosomal Peptides mRNA translation R Signaling peptides, Bacteriocins n β-Peptoid Toxins, Cytostatics, Sidero- Non-ribosomal peptide phores, Pigments, Antibiotics, Non-Ribosomal Peptides synthetases Immunosuppressants R O R' O O R' O H H N Enzymatic digestion or N N OH Ingredient in bacteria and H N OH Peptones acid hydrolysis of natural H H fungal growth media R R products n n β3-Peptoid β2-Peptoid Enzymatic degradation of laboratory samples Adapted from Olson, 2010.