Composition of Peptides-Amino Acids
20 Common Amino Acids (except glycine, where R is H) are all L-type (left-handed) amino acids (with proline being an L-type imino acid).
The 20 common amino acids are all linked to the α-carbon atom via an amino group, a carboxyl group, a hydrogen atom, and an R-group side chain.
Essential Amino Acids
These are amino acids that the human body cannot synthesize or cannot synthesize at a fast enough rate to meet the body's needs. They must be obtained directly from food.
Infants cannot synthesize histidine, so they require an additional essential amino acid compared to adults.
Phenylalanine, methionine, lysine, threonine, tryptophan, leucine, isoleucine, and valine.
Non-Essential Amino Acids
These are amino acids that the human body can synthesize and do not depend on direct supply from food.
Glycine, alanine, tyrosine, glutamic acid, glutamine, cysteine, arginine, serine, aspartic acid, asparagine, proline, and histidine.
Isoelectric Point of Amino Acids
At a pH above or below the isoelectric point of an amino acid, the amino acid carries a net negative/positive charge. Due to the repulsive forces between like charges, the solubility of the amino acid is relatively high.
At the isoelectric point, on the one hand, the repulsive forces between like charges disappear. On the other hand, the molecule carries both positive and negative charges internally, leading to intermolecular electrostatic interactions that cause aggregation and precipitation of the molecules.
Optical Properties of Amino Acids
The side chains of certain amino acids, such as phenylalanine (Phe), tryptophan (Trp), and tyrosine (Tyr), contain aromatic rings that absorb near ultraviolet light in the wavelength range of 250-300 nm. Among these, tryptophan and tyrosine also exhibit fluorescence (useful for ultraviolet quantification).
20 common amino acids at Creative Peptides
Peptides and Their Properties
Peptides are composed of many amino acids linked by amide bonds, so they inherit the physicochemical properties of amino acids, but their characteristics are also influenced by the number, types, and arrangement of amino acids.
Amphoteric Dissociation and Isoelectric Point of Peptides
- Similar to amino acids, peptides exist as zwitterions (amphoteric ions) in aqueous solutions.
- The acid-base properties of peptides mainly depend on the free amino and carboxyl groups at the ends of the peptide chain, as well as the dissociable groups on the side chains of the amino acid residues. For example, at low pH, the dissociable groups in the peptide chain get protonated, causing the peptide to carry a net positive charge and exist in a cationic form, which migrates toward the negative electrode during electrophoresis. At higher pH, the dissociable groups release protons, causing the peptide to carry a net negative charge and exist in an anionic form, migrating toward the positive electrode. The pH at which the peptide has an equal number of positive and negative charges, resulting in a net charge of zero, is called the isoelectric point (pI). At this pH, the peptide's solubility is minimal, and it is prone to aggregation or precipitation (used for peptide separation).
- Generally, peptides with more acidic amino acids have a lower isoelectric point, while peptides with more basic amino acids have a higher isoelectric point.
Optical Properties of Peptides
Aromatic amino acids (tryptophan, tyrosine, and phenylalanine) exhibit light absorption and fluorescence characteristics in the near-ultraviolet range (quantitative detection). The polarity of the environment surrounding these amino acids also affects their UV absorption and fluorescence properties.
Color Reactions of Peptides
Linear peptides with free α-amino groups can undergo the Dansyl reaction, Sanger reaction, and Edman degradation (for quantification and N-terminal amino acid identification and sequence analysis).
Biuret Reaction of Peptides
Peptides contain peptide bonds that exhibit a biuret-like structure. When treated with copper sulfate, a blue complex is formed, and further reaction produces a purple-red or purple-blue complex. This is used for peptide content measurement.
Acid-Base Properties of Peptides
When a peptide contains more basic amino acid residues than acidic ones, it will be basic in nature; conversely, it will be acidic if it contains more acidic amino acid residues.
Water Solubility of Peptides
Hydrophilic groups, such as hydroxyl, amino, carboxyl, and amide groups, contribute to the peptide's hydrophilicity. Hydrophobic groups, such as alkyl chains, make peptides more hydrophobic. Peptides with hydrophilic groups but not excessively long side chains, such as serine, aspartic acid, glutamic acid, and asparagine, are more soluble in water, which also promotes the overall hydrophilicity of the peptide.
Solubility and modification of active peptides
Biologically active peptides have excellent stability and bioavailability, which enable highly effective formulations. Peptides have smaller molecular weights than proteins, simpler structures, and are easier to absorb. They offer dual functions: regulating physiological functions in the body and providing nutrition. Almost all cells are influenced by peptide regulation, including processes such as cell differentiation, neurotransmitter regulation, signal transduction, and immune response modulation.
Solubility of Peptides
- Molecular Design Based on Hydrophilic/Hydrophobic Nature of Amino Acids: Peptides are designed by considering the hydrophilic and hydrophobic properties of amino acid residues.
- Excessive Hydrophobic Amino Acids in Peptides: A high content of hydrophobic amino acids in a peptide generally has a negative impact on its water solubility.
- Designing Water-Soluble Peptides: From a molecular design perspective, a general guideline is to ensure that for every five amino acids in the peptide, at least one should carry a charge. If this cannot be achieved, amino acids that do not have a critical function for the target application can be replaced with charged amino acids (without considering the precursor of the substituent group).
- Peptides with Fewer Than Five Amino Acids: Peptides with fewer than five amino acids typically have good water solubility, unless they contain hydrophobic amino acids.
- Peptides with More Than 25% Hydrophilic Amino Acids: Peptides that have more than 25% hydrophilic amino acids and less than 25% hydrophobic amino acids generally have better water solubility.
- Peptides with Over 50% Hydrophobic Amino Acids: Peptides with more than 50% hydrophobic amino acids typically have poor water solubility.
- Peptides with More Than 75% of Residues Capable of Forming Intermolecular Hydrogen Bonds: Peptides where over 75% of the residues can form intermolecular hydrogen bonds (cross-linking) are generally insoluble and tend to form gels.
Peptide Composition
In addition to the length of the peptide, the specific amino acid composition also influences peptide synthesis, purity, solubility, and stability.
Some peptides may be derived from natural proteins, where the sequence contains amino acids that are essential for the peptide's target function, but also contains residues that do not contribute to its final performance and only serve structural purposes. For these peptides, synthetic modifications can be made to replace amino acids that do not contribute to performance but may have negative impacts on factors like side effects, purity, or synthesis difficulty.
Examples of Modifications
Cysteine and Methionine: Cysteine and methionine are prone to oxidation. During peptide synthesis and subsequent purification, they are difficult to separate from protecting groups. Cysteine is often replaced by serine, and methionine is replaced by leucine.
Cysteine and Disulfide Cross-Linking: If a peptide contains multiple cysteine residues, disulfide cross-links can form, which may affect the activity of the peptide, especially in antibody applications. To mitigate this, reducing agents like dithiothreitol (DTT) are often added, or cysteine can be replaced by tryptophan to avoid disulfide linkages.
One of the key advantages of synthetic peptides is their ability to undergo modification at the molecular level, allowing them to be tailored for specific applications and improving their functionality for the target use.
Types of Peptide Modifications
- N-terminal Modification (Amino Group Modification): Capping the N-terminus with an acyl group via acylation reactions to prevent unwanted reactivity and improve stability.
- C-terminal Modification (Carboxyl Group Modification): Capping the C-terminus with an amide group through amidation reactions, improving peptide stability and preventing peptide degradation.
- ε-amino Group (Tryptophan): The ε-amino group of lysine residues can be modified for specific functional applications, such as for conjugation or introducing chemical groups that improve peptide targeting or solubility.
- Hydroxyl Groups of Serine, Threonine, and Tyrosine: The hydroxyl group in these amino acids can be modified to introduce phosphorylation sites or other functional groups that are crucial for specific biological activities, such as signal transduction or enzyme activity regulation.
- Guanidine Group of Arginine: The guanidine group of arginine residues can be modified for improving the interaction with specific receptors or enhancing peptide stability.
- Thiol Group of Cysteine: The thiol group in cysteine residues can be modified to prevent disulfide bond formation or to introduce specific linkers for peptide conjugation or cross-linking.
- Other Modifications (Induced - Genetic Engineering): Additional modifications can be introduced using genetic engineering techniques, such as site-directed mutagenesis or the incorporation of non-natural amino acids to enhance peptide functionality, improve solubility, or enable targeted delivery.
Directed modification of peptides can enhance their stability, functionality, skin affinity, and lipophilicity, making them more effective for specific applications. Various chemical modifications can be used to achieve these improvements:
Glycosylation Reaction: The addition of carbohydrate groups (glycans) to peptides through glycosylation can improve the peptide's stability, solubility, and bioactivity. Glycosylation is commonly used to enhance the peptide's recognition by receptors or to increase its half-life in circulation.
Esterification Reaction: In esterification, an ester group is introduced into the peptide, which can enhance its lipophilicity and skin permeability. This is often used to improve the peptide's ability to penetrate cellular membranes or to enhance its stability in non-aqueous environments.
Acylation Reaction (e.g., Acetylation with CH3CO-): Acetylation is a common modification where an acetyl group (CH₃CO-) is added to the N-terminal amino group of the peptide. This can prevent unwanted degradation, improve stability, and enhance the peptide's membrane permeability and bioavailability.
Substitution Reactions: Substitution reactions involve replacing one amino acid residue with another, often a non-natural or modified amino acid, to improve the peptide's stability, activity, or specificity. For instance, replacing a hydrophobic amino acid with a hydrophilic one could improve water solubility or facilitate interactions with specific cellular receptors.
Phosphorylation Reaction: Phosphorylation adds a phosphate group to specific amino acids like serine, threonine, or tyrosine. This modification is crucial for regulating enzyme activities, signal transduction, and cellular functions, making it useful for peptides involved in signaling pathways or cell cycle regulation.
Alkylation Reaction: Alkylation involves adding an alkyl group (such as a methyl or ethyl group) to a peptide. This can increase lipophilicity, which may improve the peptide's ability to cross biological membranes, especially in drug delivery systems.
Redox Reactions (Oxidation/Reduction): Oxidation and reduction reactions can modify the redox state of certain amino acid residues, such as cysteine, which has a thiol group (-SH). These reactions are used to manipulate the structure and function of peptides, particularly in the formation of disulfide bonds or the stabilization of peptide structures.
Peptide modifications services at Creative Peptides
Active peptide efficacy
Antioxidant Properties of Peptides
Amino acids, such as cysteine, have antioxidant properties. Cysteine plays a significant role in scavenging free radicals, contributing to the antioxidant activity of peptides. Similarly, methionine also has antioxidant effects.
Cysteine and Free Radical Scavenging
Hydrogen Atom Donation: Cysteine can quench free radicals by donating a hydrogen atom. In this process, the antioxidant loses an H· (hydrogen atom) to the free radical (A·), transforming it into a stable compound (AH). This effectively terminates the free radical chain reaction, as the antioxidant itself becomes a relatively stable radical (B·), which does not easily initiate new free radical chain reactions.
Electron Donation: Cysteine can also act through electron transfer, where both electron and proton transfer steps are involved to neutralize free radicals. This results in the stabilization of the free radical and termination of the oxidative chain.
In most cases, peptides exhibit higher antioxidant activity than individual amino acids due to the short-range interactions between amino acids. These interactions can enhance the overall efficiency of free radical scavenging in the peptide.
Anti-inflammatory and Antiallergic Properties of Peptides
Biologically active peptides are commonly used in three main ways to counteract inflammation and allergies:
Regulation of Cell Inflammatory Factors
Peptides can regulate the secretion of cytokines, inhibiting the synthesis and release of pro-inflammatory factors while enhancing the expression of anti-inflammatory factors. This helps alleviate allergic reactions induced by inflammatory factors. Key cytokines involved include Interleukin-1 (IL-1) and Tumor Necrosis Factor (TNF), which work synergistically.
Inhibition of Inflammatory Mediator Synthesis and Release
Chemical factors involved in inflammatory and allergic reactions are termed inflammatory mediators or chemical mediators. These include:
- Vasoreactive amines
- Vasoreactive peptides
- Complement fragments
- Lipid mediators (chronic inflammation)
- Cytokines
- Chemokines (chronic inflammation)
- Proteases
Regulation of Inflammatory Signaling Pathways
The NF-κB pathway plays a central role in inflammation. When cells are stimulated, the NF-κB factor binds to specific sequences in DNA, initiating the transcription of genes that promote cytokine release. Cytokines, in turn, further activate NF-κB, amplifying the inflammation. Many bacteria activate NF-κB by interacting with receptors on the cell membrane. Antimicrobial peptides often exhibit anti-inflammatory effects by regulating this pathway.
Signal Peptides
During skin injury, proteases break down damaged tissue, forming peptide fragments. These peptides act as messengers to signal the skin to produce various types of tissue for repair. Signal peptides can "trick" the skin into thinking it needs extra proteins for healing. These peptides typically contain active amino acid sequences that can stimulate the formation of collagen, elastin, laminin, hyaluronic acid, and other proteins involved in skin repair and regeneration.
Inhibitory Peptides
Neurotransmitter inhibitory peptides work similarly to botulinum toxin (Botox) by inhibiting the synthesis of SNARE receptors. This suppresses the excessive release of catecholamines and acetylcholine at the skin, blocking signals that cause muscle contraction, leading to relaxation of facial muscles and reduction of fine lines. These peptides are often used in areas with concentrated facial muscles (e.g., around the eyes, face, and forehead).
Enzyme Inhibitory Peptides
Peptides can directly or indirectly inhibit enzymes involved in various skin processes, such as:
- Tyrosinase: Responsible for skin pigmentation, inhibition leads to skin whitening.
- Matrix Metalloproteinases (MMP-1, 2, and 9): These enzymes degrade skin tissue, including collagen. Inhibiting them helps maintain skin elasticity by preserving collagen levels.
- Angiotensin-Converting Enzyme (ACE): Inhibition of ACE reduces the conversion of angiotensin I to angiotensin II, improving blood circulation and reducing puffiness under the eyes and dark circles.
Carrier Peptides
Carrier peptides facilitate the transport of trace elements such as copper, magnesium, and manganese, which are vital for processes like wound healing and enzymatic reactions.
Antimicrobial peptides are often cationic and exhibit dual properties, with a positive charge below their isoelectric point or a negative charge above it. At the isoelectric point, antimicrobial peptides tend to have minimal charge, improving skin permeability. When they are cationic, they can interact with anionic polymers, which may impact stability.
The mechanisms of action of antimicrobial peptides are not fully understood, but there are two main hypotheses:
- Membrane Disruption Mechanism: Peptides interact with bacterial cell membranes, causing protein aggregation, inactivation, and formation of ion channels. This alters membrane permeability, leading to bacterial death.
- Intracellular Damage Mechanism: Peptides bind to specific receptors on bacterial membranes, triggering intracellular damage and eventual bacterial death.
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