Peptide Characterization. Peptide Synthesis and Modifications

Keywords: peptide; peptide hydrolysis; peptide bonds; peptide modifications; peptide synthesis; …

Keywords: peptide; peptide hydrolysis; peptide bonds; peptide modifications; peptide synthesis; peptide bond; peptide hormones; peptide analogues; salting out; amidation; acetylation

Peptides

Peptides are chemical compounds built similarly to proteins, from amino acids. They are formed by joining two or more amino acids through a peptide bond as a result of a condensation process, in which, alongside the peptide, a water molecule is also produced. (Fig. 1) They are a subject of broad interest, fulfilling important biological functions. Many hormones and neurotransmitters are in fact peptides. In the case of endogenous peptides, they act antimicrobially, functioning as the body's defense system. Naturally occurring peptides and their synthetic analogues are considered attractive compounds of therapeutic significance due to their high degree of activity, low toxicity and lack of interaction with drugs. In medical practice, only a few peptides find application due to biological instability and rapid degradation, however peptide synthesis allows for obtaining stable forms. The same applies, for example, in the case of synthesis of peptides from natural sources. Peptides occur in an unbranched form and possess only two specific ends. One of them is called the amino terminus, where an amino acid with a free α-amino group is present. The other is called the carboxyl terminus or C-terminus, where an amino acid with a free α-carboxyl group is present.

Peptide nomenclature

Peptide nomenclature begins with the name of the N-terminal amino acid residue, followed by the names of successive amino acid residues, and ends with the name of the C-terminal amino acid. The sequence of amino acids is recorded using three-letter or one-letter symbols.

Peptide bond

Carbon, as a result of the reaction of the α-carboxyl group, binds with the nitrogen of the α-amino group through a single bond – a peptide bond. It is presumed that this bond forms in the shape of two structures that remain in a specific mutual equilibrium. The C-N bond transitions into C=N and vice versa. Rotation around the C=N axis is not possible, which means the peptide bond is rigid enough to possess the characteristics of a double bond. In the case of a peptide bond involving the imino group of proline or hydroxyproline with the carboxyl group of another amino acid, a different, distinct structure forms. In this case, nitrogen is incorporated into the structure of the pyrrolidine ring; there is no hydrogen substituent, which means there is no possibility of rotation relative to bonds formed in the presence of nitrogen. Amino acids that participate in the formation of a peptide bond lose fragments of their molecules – specifically the -OH from the carboxyl group and -H from the amino group. This is why amino acids found in peptides and proteins are called amino acid residues. The resulting peptide bonds are stable and their breakdown can only occur under the action of strong bases and acids at simultaneously high temperatures.

Breaking the peptide bond

Breaking of the peptide bond occurs as a result of a peptide hydrolysis reaction, which is based on the rupture of formed peptide bonds and the restoration of individual amino acids. Water participates in this reaction, with its molecules breaking down into hydroxyl groups (-OH) and hydrogen atoms (H), which then combine with the released bonds of the substance.

Classification of peptides

The classification of peptides is established based on the number of amino acids from which they are built. In the general classification of peptides, we distinguish:

• Dipeptides – products formed from the reaction of two amino acids while retaining the free amino group of one amino acid and the free carboxyl group of the second amino acid;

• Oligopeptides – peptides composed of several to over a dozen amino acids;

• Polypeptides – longer peptides containing several dozen amino acid residues;

• Proteins – accepted when a molecule consists of more than one hundred amino acid residues.

Spectrum of peptide activity

Peptides show a broad spectrum of biological activity and are used in the treatment of bacterial infections, viral diseases, cardiovascular diseases, skeletal system diseases, nervous system diseases, diabetes and osteoporosis.

Advantages of peptides

• High activity and selectivity
• Wide range of molecular targets
• Potentially lower toxicity compared to low-molecular-weight compounds
• Low tissue accumulation
• High chemical and biological diversity
• Discoverable at the gene level
• Easy synthesis of analogues

Peptide synthesis

Depending on the peptide we wish to obtain, we need an appropriate method of synthesis. In a brief explanation, we will attempt to present peptide synthesis in relation to its size. To obtain a dipeptide, a reagent must be used that will activate the carboxyl group of the arylating amino acid, or the acylating amino acid must be converted into an anhydride. A more labor-intensive and difficult process is the synthesis of larger peptides, which are obtained from a dipeptide, involving removal of the protecting group of the N-terminal amino acid and its acylation with the next N-protected amino acid. This process is particularly time-consuming, as the aforementioned steps are repeated until a peptide of the planned sequence is obtained. In the case of obtaining large peptides, the Merrifield method is the best-performing and easiest approach. This method is carried out on a solid phase. The C-terminal amino acid is attached to a polymer and then successive amino acids are attached until the desired chain length is achieved.

Biologically active peptides

Peptide hormones and protein hormones are widely present in the environment surrounding us. Previously known mostly as relatively unstable forms. Under the influence of synthesis, peptide therapy can be increasingly confidently chosen to be durable and effective depending on the needs of the body. This is precisely why it is worth skillfully and safely engaging with hormonal stimulation. Taking into account some biologically active peptides, we can give as an example glutathione, which as a tripeptide with a specific structure is composed of glutamate, cysteine and glycine. Glutamate occurs as the N-terminal amino acid. The connection of glutamate with cysteine is, however, atypical for peptides and proteins, because here it is not the α-carboxyl group of glutamate but the γ-carboxyl group that is present. Glutathione therefore occurs in a reduced and an oxidized form, being γ-glutamylcysteinylglycine. In the reduced form it possesses a free sulfhydryl group, while in the oxidized form a pair of hydrogen atoms detaches from the -SH groups. The sulfur atoms remain deprived of hydrogen, resulting in the formation of a disulfide bridge. The ability of glutathione to be modified into an oxidized or reduced state is important in oxidation-reduction processes.

Further examples are oxytocin and vasopressin, which as nona-peptides produced by hypothalamic neurons and released by the posterior pituitary gland, differ by only two amino acids. Cysteine occurs in two positions, thereby leading to the formation of a disulfide bridge. Oxytocin occurs as a hormone stimulating the contractile activity of the uterus. Vasopressin, on the other hand, stimulates water reabsorption in the renal tubules. Vasopressin also plays an important role in regulating the secretion of adrenocorticotropic hormone (ACTH) in stressful situations.

Peptide hormones

Adrenocorticotropic hormone (ACTH)

Adrenocorticotropic hormone, as a 39-amino acid peptide, is produced as a result of degradation of a much larger precursor molecule – proopiomelanocortin (POMC). Proopiomelanocortin also serves as the source of other active peptides. Two peptides are contained within the structure of ACTH itself. These include α-melanotropic hormone (α-MSH), which is structurally identical to the first 13 amino acids of ACTH, and corticotropin-like intermediate lobe peptide – fragment 18-39 of ACTH. The basic function of ACTH is considered to be the stimulation of the adrenal cortex in such a way that it is capable of secreting steroid hormones. Adrenocorticotropic hormone is responsible for regulating activity at the level of the zona fasciculata and zona reticularis. The first 18 amino acids are responsible for the biological activity of ACTH. Regulation of ACTH occurs through corticotropin-releasing hormone (CRH), a hormone present in the hypothalamus, which releases corticotropin through cortisol via negative feedback. This means that a cortisol deficiency stimulates CRH and ACTH, while its excess inhibits secretion. By releasing cortisol, many important vital functions are thus regulated, including mobilization of the body to stress conditions, elevation of blood pressure and anti-inflammatory capabilities. ACTH secreted pulsatilely in a circadian rhythm means that its highest concentration is observed in the morning hours when this is most desirable, then decreasing as the day progresses. An increase in ACTH secretion is observed in such disease states as adrenocortical insufficiency, Cushing's disease or Nelson's syndrome.

Insulin and C-peptide

Insulin and C-peptide are secreted in the pancreas by the human body continuously. During insulin production, in the process of its biosynthesis, C-peptide is produced. Pancreatic cells produce preproinsulin in the first stage, which undergoes further modification through the detachment of amino acids, leading to the formation of proinsulin composed of two chains A and B connected by C-peptide, followed by the detachment of proinsulin from C-peptide, resulting in the final form. When glucose appears in the body, the pancreas receives a signal to release granules containing the stored insulin and C-peptide molecules. C-peptide is retained in the liver significantly longer than insulin, due to the fact that it is not degraded there. Its breakdown occurs mainly in the kidneys. In the case of both insulin and C-peptide, elevated or excessively low concentrations lead to the development of type I or II diabetes as well as Cushing's disease. In the case of C-peptide, concentration fluctuations may also indicate chronic renal insufficiency or the presence of metastases or local tumor recurrence, which is why maintaining proper concentration norms is so important.

Motilin

Motilin is a hormone associated with the smooth muscles of the stomach and intestines, controlled by vagus nerve fibers. Synthesized in endocrine cells. As a peptide hormone composed of 22 amino acids located in a specific sequence, it is produced by cells of the small intestine. Produced by endocrine M (Mo) cells of the digestive system, it participates in the regulation of gastrointestinal motility. Motilin is an important hormone participating in the formation of phase III of the migrating motor complex (MMC), in which the stomach and small intestine have the task of emptying the stomach of unnecessary food remnants and desquamated epithelial cells through stimulation of peristaltic movements. The hormone additionally influences gallbladder emptying during the interdigestive period at the highest motilin concentration.

Glucagon

Glucagon is one of the hormones involved in regulating glucose concentration; this peptide is secreted by endocrine cells of the pancreas. It is a polypeptide composed of 29 amino acids, produced from a precursor with a structure of 180 amino acids. Changes in glucose concentration allow for glucagon secretion. Production of the hormone glucagon occurs in the pancreatic islets, in which glucagon and glicentin-related pancreatic polypeptide (GRPP) are produced from proglucagon. The main task of glucagon is to maintain proper glucose concentration in the serum during its decline between meals or during physical exertion. Its reserves in such situations are released from the liver to provide the body with appropriate protection. Additionally, it can participate in regulation during food intake, which means that the feeling of satiety may appear earlier. Glucagon can potentially inhibit ghrelin release and also inhibit intestinal peristalsis.

Peptide analogues

Peptide analogues are defined as appropriate chemical compounds in which one atom is replaced by another relative to the parent compound, while the general structure of the peptide remains unchanged. Peptide analogues include helix-structure analogues and β-turn and β-sheet analogues. In the first type, helices are one of the key structural elements of bioactive peptides. Stabilizing short oligomer fragments in a helical conformation results in increased activity. In β-turn and β-sheet analogues, D-amino acid residues or β,γ,δ-amino acid residues are inserted. Peptide analogues allow us to obtain new peptide compounds that will be more stable, find application in a broader symptomatic spectrum and allow for innovative solving of problems associated with the action of existing pre-analogue forms.

Salting out of peptides

The salting-out process involves changing protein charges. Protein charges are neutralized by the anions and cations of salt. Protein molecules do not attract each other and do not form aggregates, and the protein itself is precipitated as a result of losing its hydration shell. The salting-out process is reversible. In the reverse process, salt is removed by dialysis or its concentration is reduced by adding water. Based on our earlier articles, it can be confidently stated that salting out, which led to the formation of a stable form of BPC-157 peptide, is an innovative method for ensuring peptide stability and consequently expanding the biological action of peptides.

Acetylation of peptides

Acetylation involves the attachment of acetyl radicals to substrates, which are compounds with NH2, OH or SH groups, with the participation of the enzyme N-acetyltransferase. The source of the acetyl radical is acetyl-CoA. The main function of N-acetyltransferases is to facilitate the connection of the acetyl group with the amino group of aromatic amines and hydrazines (N-acetylation reaction), i.e. the detoxification of potentially toxic exogenous compounds.

Amidation of peptides

When peptide bonds are broken and consequently fragmentation of the polypeptide chain occurs, carbonyl groups form. Oxidation of a protein molecule by a hydroxyl radical begins with the detachment of a hydrogen atom at the α-carbon of an amino acid. The resulting alkyl radical reacts with oxygen to form an alkylperoxyl radical transitioning into an alkyl hydroperoxide. The alkoxyl radical formed from it can transform into a hydroxylated α-carbon amino acid residue or can lead to fragmentation of the polypeptide chain. The presence of an alkoxyl radical favors fragmentation of the polypeptide chain. Cleavage of the peptide bond can occur via α-amidation or diamidation pathways. The N-terminal peptide formed during α-amide fragmentation has an amide group at its C-terminus, while the second peptide contains an N-α-ketoacyl derivative at its N-terminus. Fragmentation via the diamide pathway is characterized by the formation of an N-terminal peptide containing a diamide structure and a peptide derived from the C-terminus of the protein molecule containing an isocyanate structure at its N-terminus.

Bibliography

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3. Kołodziejczak A. Amino acids and peptides. 2006