camel milk protein
Other sources for the production of bioactive peptides include hydrolyzed egg white proteins [ 88 ], milk, and whey proteins. Whey is one of the byproducts of dairy factories with large quantities and low cost, as well as nutritional properties. Additionally, it has been observed that the antioxidant activity of whey hydrolysates is due to the amino acid cysteine, which participates in the synthesis of glutathione. The composition of whey albumin also chelates heavy metals and reduces the risk of oxidation [ 89 ].
From both nutritional and medical perspectives, researchers’ view of egg proteins and peptides extracted from this source is very promising. Today, opinions on eggs are that they are not just a basic nutritional source. For example, a decade ago, the Arg-Val-Pro-Ser-Leu peptide from eggs was chemically synthesized and observed to have ACE inhibitory activity. In addition, the resistance of this peptide in the gastrointestinal tract has raised hopes for the use of these peptides orally [ 90 ]. Due to its rich source of amino acids and long shelf life, egg white protein powder (EWPP) is currently used in many sectors of the food and pharmaceutical industries [ 91 ]. Major efforts have been made to extract egg peptides through enzymatic digestion. In this way, enzymes such as pepsin, thermolysin, chymotrypsin, alcalase, and trypsin have been used [ 92 ].
Approximately 5.3% of the total weight of egg white proteins is the lysozyme [ 93 ]. Egg white lysozyme is a rich source of biologically active peptides, such as antimicrobial [ 94 ], anticancer, immune regulator, and antihypertensive. Hydrolyzed lysozyme is used in the industry as a natural preservative to prevent the growth of bacteria in meat products, such as sausages, beef, and pork. Heat-denatured lysozyme has been shown to have good antimicrobial activity [ 95 ].
Due to the indiscriminate consumption of red meat in some communities, most people today are trying to talk about the disadvantages of this useful food, unaware that this substance is a rich source of essential amino acids, folic acid, vitamin B12, and iron for the human body. Peptides derived from red meat have been shown to exhibit properties such as antioxidant, antimicrobial, and antihypertensive activities [ 11 , 96 , 97 ]. The ACE-inhibitory effect is one of the classic applications of BPs derived from meat. For the first time, Arihara et al. noticed this activity in peptides derived from porcine skeletal muscle proteins [ 98 ]. In addition to porcine, this biological activity has also been observed in beef-protein-derived peptides [ 99 ]. Additionally, the antioxidant activity of peptides derived from red meat should not be overlooked. It is found that there is 2700 mg of carnosine per kilogram of pork meat. Carnosine shows well-documented antioxidant activity, which stems from the ability of the carnosine peptide to trap transition metals, such as copper, cobalt, and zinc [ 100 ].
Subsequently, after enzymatic hydrolysis (actinase E and papain) of pig protein, Saiga et al. obtained a source of antioxidant peptides [ 101 ]. Papain enzyme was also used by Di Bernardini et al. to hydrolyze sarcoplasmic proteins from sarcoplasmic proteins. After fractionation, 10 and 3 kDa fractions showed antioxidant activities [ 102 ].
The striated muscles make up about 40% to 50% of livestock meat’s weight. These muscles are made up of fibers of muscle cells. About 55% of red meat proteins contain myofibrillar proteins. These proteins are insoluble in water but are soluble in dilute saline solutions. In the food industry, these proteins have beneficial properties. They comprise relatively large amounts of essential amino acids, which is why they have been considered to be 70% of the nutritional value of meat. These proteins also affect the capacity of the meat emulsion so that 90% of this capacity is due to the presence of these proteins. On the other hand, about 97% of the storage capacity of water is due to the presence of these proteins in the structure of red meat [ 103 ]. Sarcoplasmic proteins are given a lot of attention in the production of BPs and makeup about 22% to 25% of the total weight of muscle tissue proteins [ 104 ]. Other proteins that receive less attention (due to their lower solubility in water) include connective tissue proteins, such as collagen and elastin. In the food industry, the role of connective tissue proteins is not favorable as they reduce the quality of meat.
Scientists’ views on many things have changed today. One of these cases is venom, a composite that mainly includes peptides and other substances [ 105 ]. With new uses being discovered for peptides today, some researchers are trying to figure out more applications to access this hidden treasury by identifying the different components of venom [ 106 ]. Early efforts in this area focused on the separation of the various parts of venoms and the determination of their active component(s). After separating the active component and performing some other studies, such as sequencing and structuring the active part, it will be possible to study more applications of the discovered component. Efforts in this area have intensified to the point that researchers are trying to build a library of ‘expressed sequence tags’ related to the venom glands of various animals. Although these studies are expensive, they are worth the benefits that will be discovered in the future [ 107 ]. The studies in this field are numerous and varied, and Table 3 presents a brief classification overview of different families of venom peptides along with their applications.
Different families of venom peptides.
Classification | Example | Host | Applications | Ref. |
---|---|---|---|---|
Bradykinin potentiating peptides | TsTX-Ka and TsTX-KO | Hypotensive effects, ACE inhibitor | [ ] | |
BPPs | | ACE inhibitor | [ ] | |
Antimicrobial peptides | IsCTs | Antimicrobial Cytolytic activity | [ ] | |
Hormonelike peptides | Mini-Ins | Insulin-like activity | [ ] | |
Therapeutic peptides | Ziconotide | Pain killer | [ ] |
In addition to the aforementioned sources, some other animals can be a significant source of BPs. For example, bacterial antimicrobial peptides are called bacteriocins, which contain neutral or positively charged peptides and are secreted by a variety of Gram-negative and positive bacteria. These peptides are not a factor in defending against viral infection, but help bacteria kill other bacteria in the competition for environmental supplements [ 113 ].
Most bacteriocins target the cell membrane with hydrophilicity or hydrophobicity, while some also inhibit the biosynthesis of biopolymers or the activity of enzymes. The peptides are not synthesized by ribosomes and instead undergo complex step-by-step compression reactions derived from peptide synthetases. Large nonribosomal (NRPS) peptides are often composed of nonprotein amino acids, including D-type amino acids, hydroxy acids, or other unusual compounds, and exhibit a wide range of inhibitory and functional mechanisms [ 114 ]. Bacteriocins are resistant to heat, low pH, weak organic solvents, cold, ice, and salts and are therefore applicable to food protection systems. Isolation and purification of these compounds are necessary to determine the exact mechanism of their inhibitory activity on food spoilage bacteria and foodborne bacteria. Bacteriocins are generally sensitive to human intestinal proteases, making them a valuable resource for food preservation without potential harmful effects on human health [ 115 ]. Additionally, antimicrobial peptides can be extracted from the hemolymph of insects or outside their bodies. Insects in the face of microorganisms secrete different antimicrobial peptides because they can detect different types of invasive organisms and secrete the appropriate antimicrobial peptide. Antimicrobial peptides in primitive organisms are an alternative to primary responses [ 116 ].
More attention is also being paid to BPs derived from marine microorganisms. Some marine bioactive compounds are produced by microbes that coexist with marine species. Marine actinomycetes are a source of secondary bioactive compounds that have anticancer and antimicrobial activity. Cyclomarins A, B, and C are three ring-shaped heptapeptides isolated from marine actinomycetes, such as Streptomyces sp. Cyclomarin A is composed of three common amino acids and four unusual amino acids and has shown anti-inflammatory and anticellular activity in laboratory studies [ 117 , 118 ]. Salinamides A–E are types of peptides that have been extracted from Streptomyces sp. Salinamides A and B are two bioactive compounds with large rings that have local anti-inflammatory and antimicrobial activity against Gram-negative bacteria and are used in the treatment of tissue inflammation and some infections. Salinamides C, D, and E are small BPs with anti-inflammatory activity. The structure of type D is similar to that of A, but contains valine residues at the isoleucine site of type A. Salinamides E and C are also shown as a single-ring peptide [ 119 ].
Additionally, amphibians have a high level of defense system that includes innate and acquired immunity. In this group of organisms, the skin is protected by innate immunity mediated by macrophages, neutrophils, complement-mediated lysis, natural killer cells, and secreted antimicrobial peptides. Peptides of this group are synthesized in the granular glands of the skin. Due to environmental stimuli or damage to the sympathetic nerves, the peptides are activated and the contents of the glands are secreted to the surface of the skin [ 120 ]. Antimicrobial peptides in this group mostly have an alpha-helix structure. It has been shown that Rana frogs secrete the ranalexin antimicrobial peptide, which has a cyclic structure with disulfide bridges [ 121 ].
Traditionally, more attention has been paid to peptides derived from animals than plants. Nevertheless, it should be noted that plant proteins are rich sources of proteins without saturated fatty acids that can carry useful ingredients. Recently, certain activities have been discovered in plant-derived peptides that can perform important functions in humans. Antidiabetic, immunomodulatory, antimicrobial, hypocholesterolemic, opioid, antihypertensive, and antioxidant activities are of these benefits.
BPs are derived from plant sources such as plant proteins and/or directly extracted from them. Plant sources of BPs, due to their cost-effectiveness and lower immunogenic effects, have recently received more attention from experts in this field. Most plant proteins are incomplete due to a lack of essential amino acids, but wheat germ protein contains essential amino acids and is therefore categorized as valuable as animal proteins, such as meat or chicken eggs [ 122 ]. From an immunological point of view, the skin and its components are the first level of defense against invading microorganisms. The same rule is more or less true for plants. Antimicrobial peptides in plants also help plants in the early stages of the fight against invading microorganisms. Antimicrobial peptides with beta plates have been identified in plants, with two groups of these well-studied peptides including thionin and defensins. Thionins are the first identified peptides from plants that play a significant role in protecting plants against invading bacteria. This group of peptides is toxic to a variety of Gram-negative and Gram-positive bacteria, and even mammalian bacteria [ 123 ]. Although plant-derived peptides work to inhibit the growth of various types of microorganisms, there are more detailed classifications, such as antivirals, antifungals, and antiparasitics. It is believed that the antibacterial activity of peptides is due to the presence of motifs rich in positive amino acids and the amphiphilic nature of the peptide sequence [ 124 , 125 ]. Ultimately, these properties of antimicrobial peptides help them penetrate the bacterial membrane to create pores and eliminate the disturbing bacteria by altering homeostasis. To help readers’ knowledge of this article, the table below ( Table 4 ) lists the types of antimicrobial peptides found in plants. Additionally, in the following sections, several images are used to explain the mechanism of antimicrobial activity. Although many plant antifungals or antivirals have more than 50 amino acid sequences, such as 2S albumin-like from Malva parviflora [ 126 , 127 ], lipid transfer proteins (LTPs) [ 126 , 128 ], and puroindolines [ 129 , 130 ], they are omitted here because they do not belong to the peptide category.
Plant-derived antimicrobial peptides.
Plant | Peptide | Peptide Size | Biological Activity | Ref. |
---|---|---|---|---|
Heveins | 43 residues, 4.7 kDa | Antibacterial and antifungal | [ ] | |
ND | 2.2 and 6 kDa | Antibacterial and antifungal | [ , ] | |
Peptides | ND | Antiviral | [ ] | |
Shepherins | 28 residues | Antibacterial and antifungal | [ ] | |
Thionins | 45–47 residues | Antibacterial | [ , , ] | |
Cyclotides | 28–37 residues | Antibacterial, Antifungal, Insecticide, Nematicide | [ , ] | |
PAFP-S | 36–37 residues | Antibacterial | [ ] | |
Alpha-1-purothionin | 45 residues | Antibacterial | [ ] | |
Defensins | 5 kDa | Antibacterial and antifungal | [ ] | |
Hispidulin | 5.7 kDa | Antibacterial and antifungal | [ ] |
ND: not determined.
4.1. antioxidant activity of bp and its mechanism of action.
In recent years, the trend of technological developments in human societies has caused fundamental changes in human lifestyle. By reducing the physical activity of people in the community, which can be considered a special type of stress, the incidence of some diseases, including cardiovascular complications and different types of cancers, has increased in people in developed and developing societies. Looking through the literal definition of stress, it is the general and nonspecific response of the organism to maintain homeostasis against any factor that threatens or impairs the body’s compensatory abilities [ 140 ]. In its general definition, stress is a factor that interferes with a person’s physical and mental balance, causes psychosomatic problems, and reduces a person’s efficiency in various aspects of life [ 141 ].
Oxidative stress is a change in the balance between pro-oxidants and antioxidants. The production of some free radicals such as superoxide can be physiologically beneficial, but oxidative stress occurs when the balance between the production of reactive oxygen/nitrogen species (ROS/NOS) and the antioxidant defense system is upset. Oxidative stress, therefore, changes the balance between pro-oxidants/antioxidants in favor of pro-oxidants, potentially leading to biological damages. Diseases associated with ROS production include cancer, Parkinson’s, and Alzheimer’s [ 142 ] diseases. Oxidative stress can cause serious damage to important cellular macromolecules, including lipids, nucleic acids, and proteins. In biological systems, the production of free radicals of ROS is inevitable, and the body partially neutralizes their harmful effects by designing antioxidant defense mechanisms. The most important components of the enzymatic antioxidant defense system include the enzymes superoxide dismutase, glutathione peroxidase, and catalase. Antioxidant enzymes, which are responsible for detoxifying free radicals or repairing antioxidant molecules, are an indicator of stress levels in cells or tissues. In addition to the primary defense barrier created by antioxidant enzymes, the second defense barrier is created by small molecules (antioxidants) that react with free radicals to produce less dangerous radical compounds [ 143 ].
BPs have a strong antioxidant activity against free radicals and other reactive species. These antioxidant peptides contain 5–16 amino acids [ 144 ]. The mechanism by which peptides exert their antioxidant effects has not been fully elucidated, although various studies have shown that hydrolyzed peptides and proteins prevent enzymatic and nonenzymatic oxidation by removing free radicals and chelating metal ions. Several peptides have been found in protein constituents that have antioxidant capacity, and their biological activities have been extensively studied. Although the energy of free radicals (such as hydroxyl) is high, in general, all 20 amino acids found in proteins can have internal interactions with free radicals. Food-derived antioxidant peptides are safe and healthy compounds with low molecular weight, low cost, high activity, and easy absorption. The antioxidant properties of peptides are mostly related to their composition, structure, and hydrophobicity [ 144 , 145 ].
The presence of some amino acids and their position in the peptide sequence has an important effect on their antioxidant activity [ 146 ]. Aromatic amino acids, such as tyrosine, histidine, tryptophan, and phenylalanine, and hydrophobic amino acids, such as valine, leucine, methionine, glycine, and alanine, are essential for the antioxidant role of the peptide. The higher oxidation of peptides compared with free amino acids is attributed to their unique chemical and physical properties by the amino acid sequence itself. In a study, the His-Gly-Pro-Lue-Gly-Pro-Lue antioxidant peptide, the presence of two replicate sequences, Gly-Pro, and the placement of Lue in the carboxylic position and His at the amine end increased the free radical scavenging property [ 147 ]. The presence of hydrophilic amino acids such as proline, alanine, valine, and leucine in the N position and the amino acids tyrosine, valine, methionine, leucine, isoleucine, glutamine, and tryptophan in the C-terminal position was associated with the antioxidant properties of peptides [ 148 ]. Additionally, fat-soluble free radicals (peroxyl radicals) produced during the oxidation process of unsaturated fatty acids are neutralized by hydrophobic amino acids such as leucine, valine, alanine, and proline [ 149 ]. Amino acids such as histidine, tyrosine, methionine, and cysteine inactivate free radicals by giving them protons. Aromatic amino acids (phenylalanine, tryptophan, and tyrosine) convert free radicals into stable molecules by giving them electrons [ 150 ].
In addition to a peptide sequence, the molecular weight of peptides can affect their antioxidant activity [ 147 , 151 ]. Research has shown that the antioxidant activity of corn gluten hydrolyzed protein is related to its concentration and molecular weight. The antioxidant activity of peptides with a molecular weight of between 500–1500 Daltons is stronger than that of peptides with a molecular weight of higher than 1500 Daltons or lower than 500 Daltons [ 152 ]. In some cases, the higher antioxidant power of smaller peptides compared with large chain peptides was attributed to their easier access to free radicals and a more effective removal of these radicals [ 153 ]. However, it has been repeatedly shown that the higher the degree of hydrolysis, the lower the antioxidant activity of the peptides. This is due to the further breakdown of peptides into free amino acids that have little or no antioxidant activity [ 146 ].
Most food-derived antioxidant peptides include hydrophobic amino acids such as valine or leucine at the N-terminal and proline, histidine, tyrosine, tryptophan, methionine, and cysteine in their sequence. Hydrophobic amino acids such as valine or leucine can increase the affinity of peptides in the fat phase, thus facilitating access to free radicals produced in the fat phase [ 153 , 154 ].
In the last two decades, many peptides with antibacterial, antiviral, and antifungal activities have been identified in both vertebrates and invertebrates, which form an important part of the host’s innate immune system. In most cases, the mechanism of action of antimicrobial peptides appears to be different from that of conventional antibiotics (see Figure 3 ). For this reason, these peptides are very interesting as new drugs to fight infectious agents [ 155 ]. Thus, antimicrobial peptides have opened a new chapter in the sciences, which has attracted the attention of many scientists and researchers, especially since they are much simpler in structure than proteins, which simplifies the study of the function–structure relationship and makes it possible to construct them by nonbiological ways (such as chemical synthesis).
( A ) Types of mechanisms of action of antimicrobial peptides on bacterial cells. ( B ) How antimicrobial peptides penetrate the cell membrane.
The factors of the effectiveness of these biologically active peptides as antimicrobial agents depend on structural properties (e.g., peptide size, amino acid composition, or charge) [ 156 ]. However, antimicrobial peptides have some common features. Most antimicrobial peptides are composed of fewer than 50 amino acids, 50% of which are hydrophobic [ 157 ]. Among these peptides, those with essential amino acids (lysine and arginine) have the highest antimicrobial properties [ 158 , 159 ]. Additionally, cationic and amphipathicity amino acids are important structural features for the antimicrobial activity of these BPs [ 160 , 161 ]. These peptides are also referred to as cell-penetrating peptides, protein transport domains, membrane sequences, or trojan peptides. Today, these peptides are used to transport many membrane materials. Recent studies have shown that these peptides can transport a wide range of drugs, proteins, liposomes, and nanoparticles into animal cells [ 162 ]. In 1988, a membrane-permeable peptide was discovered from the Tat protein of the HIV-I virus with tree sequences. This peptide was found to be able to cross the membrane of cultured cells and accumulate in the nucleus [ 163 , 164 ].
It has been concluded that in the case of some antimicrobial peptides, although the peptides reduce the growth of harmful microbes, they do not directly interact with the target microbes or microorganisms, but do so with the help of the host immune system [ 165 ]. For example, milk protein hydrolyzate has been shown to stimulate the host immune system. These activities include stimulating the proliferation of the natural killer cell, stimulating macrophage phagocytosis, and encouraging the expression of many antibodies, cytokines, and chemokines [ 166 ].
The anti-inflammatory function of peptides is usually related to their antimicrobial activity [ 167 ]. Inflammation is the response of the immune system to harmful stimuli (which can be invasive agents or damaged tissues) that are necessary to protect living organisms. In other words, inflammation is a complex biological response of host cells, vascular tissue, proteins, and other mediators to eliminate the primary causes of cell damage, tissue hemorrhage, and necrotic cells, which ultimately leads to the elimination of infection and treatment. During an inflammatory response, by increasing blood flow and vascular permeability, immune system components can escape from the blood vessels to the affected area, resulting in five symptoms that may indicate inflammation: redness, heat, swelling, pain, and loss of function.
Inflammation is normally controlled and limited on its own. Inflammatory mediators are activated only in response to harmful stimuli and have a short lifespan, and when the harmful agents are removed, they are destroyed or inactivated. Additionally, at this time, the acute inflammatory response is over, infection is removed, and damaged tissue is repaired. In addition, various anti-inflammatory mechanisms are activated. If the causative agent cannot be eliminated quickly, it may lead to chronic inflammation that can have serious pathological consequences. At the end of the inflammation, several different regulatory mechanisms are activated: (1) inflammatory mediators that are short-lived are destroyed or inactivated; (2) leukocyte migration stops; (3) the permeability of the vessels decreases and returns to normal; (4) the expression of proinflammatory molecules decreases, and conversely, the expression of anti-inflammatory molecules increases, which causes the transfer of monocytes instead of neutrophils. Monocytes clean dead and damaged tissues, and tissue repair begins [ 168 , 169 ].
The production of natural antimicrobial agents by phagocytes has long been known. These antimicrobial peptides provide the first line of defense against pathogens in eukaryotic organisms and are generally effective against bacteria, fungi, and viruses. In addition to the direct killing of microbes, these compounds also participate in processes related to inflammation and innate and acquired immunity. Antimicrobial peptides, which are innate immune mediators, increase phagocytosis and trigger the release of prostaglandins. They also neutralize the shock effects of liposaccharides caused by bacteria. These peptides transport and accumulate immune cells at the site of inflammation, induce angiogenesis, and heal wounds. The production of cytokines is also affected by these peptides [ 113 ]. Antimicrobial peptides also have a chemotactic role [ 170 ]. All of these actions eliminate the cells of bacteria. The results showed that these compounds are bactericidal at high concentrations of mg/mL and have a safety regulatory role at lower concentrations [ 171 ].
Given all of the above, antimicrobial peptides are probably involved in all stages of host defense. In addition to enhancing the immune response, these compounds prevent uncontrolled inflammation by suppressing proinflammatory responses. Despite the specific overlap, the antimicrobial peptides interact with each other, complementing each other to guide effective cells to the site of inflammation and modulate the local immune response [ 172 ]. Phagocytes, neutrophils, and monocytes are absorbed via alpha-defensins, human neutrophil peptides 1HNP1-3, and beta-defensins such as human β-defensins 2hBD3 and 3hBD4, while mast cells are adsorbed via HNP1-3, LL-37, and 4B. In addition, hBD1 and hBD3 are chemotactic for immature dendritic cells and memory T cells.
The combination of these peptides and cytokines at the site of injury will help these immature dendritic cells to mature and enable them to process antigens and migrate to nearby lymph nodes, where antigens are present. Antimicrobial peptides indirectly play a chemotactic role by inducing or increasing chemokine secretion. For example, LL-37 induces the release of interleukin-8 by lung epithelial cells, and human defensin HNP1-3 induces the activation and degranulation of mast cells. In addition, these human peptides increase neutrophil invasion, stimulate further transcription, and produce interleukin-8 by bronchial epithelial cells [ 173 ].
Antimicrobial peptides have a dual property: on the one hand, they protect the host against harmful pathogens through antimicrobial activity, and on the other hand, they protect the host from the harmful effects of excessive inflammatory responses. In other words, these peptides stimulate the production of proinflammatory cytokines, increase the accumulation of dendritic cells and monocytes at the site of injury, and increase phagocytosis and maturation of dendritic cells, while simultaneously protecting the organism from the harmful effects of an inflammatory response. As a result, these peptides have both proinflammatory and anti-inflammatory roles.
Cathelicidin is an important family of cationic peptides. In humans, the cathelicidin gene encodes an inactive precursor protein that finally matured to active 37-amino acid peptide (IL-37). It causes a balance between proinflammatory and anti-inflammatory signals. Such peptides can inhibit the host’s harmful proinflammatory responses without losing the beneficial innate defense [ 174 ]. Another example, α-melanocyte stimulating hormone (α-MSH), is a neuropeptide that belongs to the melanocortin family with anti-inflammatory effects and shares several properties with antimicrobial peptides. Recent studies indicate the direct antimicrobial activity of this peptide against fungi and pathogenic bacteria. It reduces the concentration of proinflammatory mediators and thus protects the brain and peripheral organs from inflammatory disorders. Therefore, α-MSH is an anti-inflammatory peptide with antimicrobial properties [ 175 ].
In 2000, there were 972 million cases of hypertension in the world, and this number is expected to reach 1.56 billion by 2025 [ 176 ]. The angiotensin-converting enzyme plays an important role in regulating and increasing blood pressure. This enzyme catalyzes the transformation of inactive angiotensin I (decapeptide) to activate angiotensin II (octapeptide), which is a strong vasoconstrictor. Angiotensin II also has a regulatory effect on the enzyme cellular lipoxygenase, which accelerates the oxidation of low-density lipoprotein (LDL) and is associated with atherogenesis. It is also an inhibitor of bradykinin, a potent vasodilator.
Clinical studies have shown that ACE inhibitors significantly reduce mortality in patients with myocardial infarction or heart failure [ 177 ]. Captopril and enalapril inhibitors are used for hypertension, but they have many different side effects, including coughing, changes in taste, pimples, and edema, so there is a great deal of interest in using natural antihypertensive peptides [ 178 ]. In addition to milk proteins [ 179 ], other sources of antihypertensive peptides have been investigated. These sources include egg protein, mainly ovalbumin [ 180 ]; meat protein [ 98 ]; beef hemoglobin [ 181 ]; gelatin [ 182 ]; fish skin protein [ 183 ]; and several plant proteins, such as soy [ 184 ], sesame [ 185 ], broccoli [ 186 ], buckwheat [ 187 ], and transgenic rice protein [ 188 ]. In most cases, for the body to use the antihypertensive peptides, these molecules must be absorbed intact through the intestines and enter the bloodstream.
Although pain is an important sign that there is a problem somewhere in the body, it is an unpleasant feeling that is often accompanied by severe and destructive stimuli. Chronic pain is associated with high levels of depression and anxiety. Additionally, in some physical conditions, reduced physical activity due to the generation of pain causes other diseases, such as obesity and heart harm. Chronic pain originates in the brain and/or spinal cord and is often difficult to manage [ 189 ]. Opioid drugs are currently used to relieve such pain, despite being associated with undeniable side effects. Pain is also one of the most important challenges in the management and/or treatment of cancer, and it has been seen that the psychological effects of pain in cancer patients have a negative effect on their recovery [ 190 , 191 ]. The probability of pain in advanced stages of cancer is close to 70% to 80% [ 192 ]. Pain is also seen in 90% of patients who have experienced cancer metastasis [ 193 ].
For these reasons, understanding the principles of pain and its management is critical for this group of patients. Pain is divided into two categories in terms of location: peripheral pain and nerve pain. Peripheral pain is pain that originates outside the central nervous system, including superficial pain, deep pain, or visceral pain. Nerve pain may be due to a pathophysiological condition of the central nervous system, such as deep disturbance or secretion of microbial–chemical substances and irritation due to heat or cold. Central pain, neuritis, neuralgia, and causalgia are types of nerve pain [ 194 ].
So far, various therapeutic measures have been used to control pain. Common therapeutic measures include the administration of non-narcotic and narcotic analgesics (opioids). The types of drugs used are [ 195 ]:
Most drugs reduce blood pressure and respiratory depression, bradycardia, and confusion [ 196 , 197 ]. Scientific methods of acute postoperative pain relief introduce the use of both narcotic and non-narcotic drugs along with the use of nerve blocks as the method of choice for complete postoperative pain relief. Oral and injectable methods (intramuscular, intravenous, and subcutaneous) for dermal or mucosal absorption or central or peripheral nerve blocks or without catheter placement are a variety of ways to administer the drug [ 198 ].
As mentioned, one of the ways to control pain is to use narcotics. These drugs are generally peptides between 5 and 80 amino acids, which generally have two sources, endogenous and exogenous [ 199 ]. Drug peptides bind to their receptors on the surface of nerve cells, triggering a signal that ultimately reduces pain. According to studies, most drug peptides act as agonists. In addition to pain, these peptides have been shown to reduce stress levels. The internal sources of opioid peptides are usually in the form of either hormones (secreted by the glands) or a neurotransmitter that is secreted by nerve cells and acts on the terminals of other cells [ 200 ].
Enkephalin was the first known endogenous peptide. Many endogenous peptides have been shown to have a conserved Tyr-Gly-Gly-Phe sequence at the end of their N-terminal [ 201 , 202 ]. Exorphins or exogenous opioid peptides with morphine-like activity enter the body from food sources, or in emergencies through drugs and supplements. Among the available food sources, dairy products are the best source for exorphins due to the similarity of the sequence of peptides derived from them to endogenous opioid peptides. Some observations suggest that the product of enzymatic digestion of dairy proteins, especially milk, can bind to opioid receptors on the cell surface. For example, the Arg-Tyr-Leu-Gly-Tyr-Leu-Glu peptide derived from bovine milk casein alpha protein has been shown to have narcotic activity [ 203 ]. It is important to note that peptides resulting from the digestion of digestive enzymes can be easily administered orally to humans [ 204 ]. Interestingly, opioid peptides of animal origin generally bind to µ receptors, and peptides of plant origin bind to ẟ receptors [ 205 ].
Proteins interact with ions through their amino acid side chain. For example, alpha-casein and beta-casein interact with divalent and trivalent cations, such as calcium. In addition to proteins, peptides also have the ability to bind minerals. For example, casein-derived phosphopeptides, also known as casein phosphopeptides, have this activity [ 206 ]. These phosphopeptides are involved in maintaining calcium, phosphorus, and other mineral elements in solution in intestinal pH. This activity is due to the presence of the amino acid phosphorylated serine, which can make salts with minerals, such as calcium. Enzymatic digestion of milk produces a diverse group of these peptides [ 207 ]. The type of amino acid composition present in the phosphorylated region plays an essential role in the amount of calcium-binding activity in this group of peptides [ 208 ]. These peptides are also effective in preventing tooth decay, osteoporosis, insomnia, and hypertension. Animal studies have shown a positive effect of these peptides on calcium absorption. A group of researchers has shown that fermentation of whey protein with Lactobacillus holoticus is effective in the proliferation of osteoblasts in vitro. There have also been other reports of the increased bioavailability of iron in rat models [ 209 ].
Hyperlipidemia, especially high cholesterol, is one of the most important risk factors for cardiovascular disease. Many studies show that soy-derived peptides can lower blood cholesterol levels in animal models of hepatotoxicity as well as in humans. Soy-rich diets have become one of the most effective dietary treatments for high cholesterol, although the mechanism has not yet been fully elucidated. It is believed that soy peptides derived from protease actively cut cholesterol traveling in the gut and thus reduce cholesterol uptake [ 210 ]. Other research has shown that hydrophobic peptides derived from soy proteins are able to interact with bile acids, thereby increasing the excretion of fatty acids in the feces [ 97 , 209 ].
It has been indicated that LPYPR and IAVPGEVA peptides derived from soy glycinin protein, which have structural similarity with endostatin and VPDPR, showed a cholesterol-lowering effect. These peptides inhibit 3-hydroxy-3-methylglutaryl-coenzyme, a reductase, which is a key enzyme during the biosynthesis of cholesterol [ 97 ]. Milk is another important source of BPs with a cholesterol-lowering effect. In 1999, Nagaoko et al. discovered a cholesterol-lowering peptide from digested beta-lactoglobulin hydrolase. Hydrolyzed plant protein with cholesterol-lowering activity such as soy and hydrolyzed Brassica carinata proteins have also been reported [ 211 ]. Such effects from aquatic animals, such as sardine [ 212 ] and zebrafish [ 213 ], have been shown on blood lipids. It has been shown that peptides that are lower in proportion to the amino acids methionine, glycine, lysine, and arginine are better able to cause hyperlipidemia. However, bovine casein protein, which has a higher proportion of these amino acids, especially methionine and glycine, raises cholesterol levels [ 214 ].
Peptides can affect the absorption of nutrients in the intestines, especially the small intestine, thereby reducing appetite. Many studies have shown that peptides derived from dietary proteins can send satiety signals to the brain and thus prevent the consumption of more foods [ 214 ]. Casein-derived peptides have been shown to regulate eating in the body by activating the cholecystokinin A (CCK-A) receptor [ 215 ].
A wide range of plant-derived peptides can help diabetics through a variety of pathways. The pathways that have been studied so far include inhibitory properties on alpha-amylase, dipeptidyl peptidase IV, glucose transporter system, and mimicking insulin activity [ 216 ].
During the aging process, the production of extracellular matrix proteins, such as collagen, fibronectin, elastin, and laminin, decreases, and their breakdown increases. In addition to protecting the cell structure, the extracellular matrix is effective on cellular behaviors, such as proliferation and differentiation. Such functions are controlled by small peptides derived from the breakdown of extracellular matrix proteins called matrikine [ 217 , 218 ]. Following the destruction of the extracellular matrix, the elasticity of the skin gradually decreases, and the first lines of aging and wrinkles appear [ 219 , 220 ].
The aging process of the skin is controlled by internal and external factors. External factors include exposure to ultraviolet radiation, environmental air pollution, and smoking. Exposure to ultraviolet light increases reactive oxygen species (ROS), disrupts collagen synthesis, and induces collagenase production and enzymes that break down proteins in the extracellular matrix, thereby causing cell DNA damage, and ultimately destructs the skin integrity. Interior factors include the formation of large amounts of reactive oxygen species during cellular metabolism and genetic factors that cause the destruction of extracellular matrix proteins and reduced blood flow, and the function of skin cells is reduced [ 221 ].
Gradually, with the discovery of more functions of BPs, it was seen that these molecules are able to rejuvenate the skin and increase its repair power. Today, peptides are used to stimulate collagen, heal wounds, and smooth out skin wrinkles and for antioxidant, antibacterial, and skin whitening effects [ 222 , 223 , 224 ]. Cosmetic peptides can affect skin freshness through several mechanisms. These pathways can be used to signal peptides that modulate collagen levels, such as Lys-Thr-Thr-Lys-Ser [ 225 ]; reduce IL-6 secretion and stimulate collagen and elastin synthesis, such as palmitoyl tetrapeptide-7 [ 226 ]; stimulate human skin fibroblasts and increase skin angiogenesis, such as Val-Gly-Val-Ala-Pro-Gly peptide; and inhibit tyrosine enzyme activity, such as Nonapeptide-1 [ 227 ].
Some peptides act as inhibitors of neurotransmitters. The mechanism of action of neurotransmitter-inhibitory peptides similar to botulinum neurotoxin type A under the brand names Botox ® [ 228 ] and/or Dysport ® [ 229 ] is the most widely used skin beauty product, which weakens muscle contractions and thus reduces wrinkles by inhibiting the signal path of neurotransmitters in nerve–muscle connections. In the use of these peptides, the most important challenge being studied today is their transfer from the skin layers to the lower and living parts of the skin.
Some studies have suggested that linear peptides have not been able to meet expectations in some applications. For example, the most important drawback of linear peptides is their low stability, which can cast a shadow on their therapeutic applications. In recent decades, a new gate of research has been opened to design cyclic peptides to overcome some of the challenges of linear peptides. Cyclic peptides have been shown to be less susceptible to proteolysis [ 230 ] and have a higher receptor binding capacity than their linear counterparts [ 231 ]. It is believed that the cyclization of peptides ultimately reduces their spatial vibrations, provides a large surface area for interacting with the target, and consequently strengthens their binding to the target molecule and also increases metabolic stability [ 232 ]. Although in nature, similar to the examples given in the section on marine peptides, there are many sources of cyclic peptides, solid-phase synthesis of them has now become a common method in many laboratories around the world [ 233 ]. Nearly three decades ago, the production cost of cyclic peptides was very high, almost prohibitive for mass production, but much effort in the chemical synthesis of peptides, development of new methods for the purification of the peptides, and significant reduction in the price of amino acids compared with the past have led to more attention to cyclic peptides [ 233 ]. The table below shows a number of cyclic peptides with their function and source ( Table 5 ).
Different well-known cyclic peptides along with their application and source.
Name | Source | Application | Ref. |
---|---|---|---|
Gramicidin S | Antibiotic activity towards Gram-negative and Gram-positive and even several pathogenic fungi. | [ , ] | |
Tyrocidine | By antibiotic action, it can disrupt the cell membrane function. | [ ] | |
Plitidepsin | Antitumor, antiviral, and immunosuppressive activities. | [ ] | |
Cyclosporin A | As a calcineurin inhibitor, it can decrease the function of lymphocytes. | [ ] | |
Alisporivir | Chemically synthesized from ciclosporin | Inhibits cyclophilin A, and it is believed that it may have a potential effect on Alzheimer’s disease and hepatitis C. | [ , ] |
Romidepsin | By apoptotic activity, it has an anticancer activity on many types of malignant cell lines. | [ , ] | |
Ziconotide | Acts as an analgesic agent; strong pain killer. | [ ] | |
Vancomycin | A board range antibacterial compound that is used in many bacterial infections. | [ ] |
Computer-based methods, such as proteomic and peptidomic studies, are very helpful in researching peptides. Using computer-based techniques, it is possible to predict the production of peptides from specific dietary proteins. With this method, the selection of enzymes, proteins, and hydrolysis products, as well as the study of the secondary structure and physical and chemical properties of the produced peptides, would be possible. The classic method for identifying and processing BPs involves in vitro digestion and chromatographic purification of the hydrolysis product. After the bioactivity test, the peptide sequence will be usually identified. Most of the time, this process continues with the confirmation of the biological activity of the chemically synthesized peptide sequence.
The main problem in the classical method is the low yield and limitation in the number of peptide samples that are studied at one time. On the other hand, proteomics-based methods are based on high-efficiency protein digestion and techniques for predicting peptide activity using computer-based systems (in silico) that provide biological and chemo-metric information about the desired peptide sequence. The key steps in this method are as follows: first, the protein databases are examined to select the desired proteins with known amino acid sequences. The proteins are then digested in silico using the appropriate proteolytic enzymes for the selected protein. Peptides produced in silico are then examined for structural properties and potential biological activities, including toxicity and allergenicity [ 245 ]. A convenient and useful list of the different databases for structural and physical properties of peptides was recently created [ 246 ].
BPs can be identified as specific amino acid sequences that have beneficial physiological effects. Some of these peptides are inactively buried in the structure of proteins and are activated by extraction from parental proteins. Technology for the production of BPs including protein hydrolysis by microbial enzymes, plant or animal enzymes, and fermentation using different amino acids to produce peptides with separate or multiple biological functions provided a promising way to reach a better quality of life. Today, BPs are known as products of protein hydrolysis of various foods. These peptides play a variety of biological roles, one of the most important of which is antioxidant activity. The inverse relationship between antioxidant activity and the occurrence of diseases has been proven in several studies. The results showed that the antioxidant power of hydrolyzed protein was lower than standard antioxidant solutions, such as ascorbic acid and ethylenediaminetetraacetic (EDTA), but due to the fact that natural antioxidants are usually used as alternatives in larger quantities due to their lower potency than synthetic antioxidants, in this case too, higher doses can be recommended for greater effectiveness.
Marine BPs also showed a variety of physiological functions, such as immune stimulation, hypotension, antidiabetic, antioxidant stress, antiobesity, skin protection, and wound healing. These effects have been investigated in animal and human models, and the results indicate the promising effect of peptides as beneficial compounds in the production of food–drug compounds and other drug supplements. Regarding BPs originating from the oceans, it is worth mentioning a few suggestions: (I) Investigation of different extracts of different marine species such as sponges and algae will identify compounds with medicinal properties in order to produce new drugs. (II) Bioactive compounds with marine sources are a good alternative to land-based drugs that have shown drug resistance in humans. (III) Marine environments account for the largest area of the planet relative to land and contain unknown and undiscovered compositions that confirm the need for further research.
Past and current research on antimicrobial peptides has shown that these compounds have great potential for use in the food and medical industries. Continuous discoveries of new antimicrobial peptides and understanding of the process, biological systems involved in the synthesis, safety, and regulation of antimicrobial peptides have paved the way for advances in this field with an emphasis on practical applications in the industry. Genetic engineering or chemical modification of bacteriocins to improve their functional properties has been considered in recent years, which has led to significant development in bacteriocin technology. While most classical research has focused on finding antimicrobial peptides on prokaryotic sources, there is ample evidence that most life forms produce small peptides with antimicrobial properties. These include not only bacterial but also hydrophilic proteinlike compounds in eukaryotes, such as mammalian defensin and cathelicidins, frog magainin, insect thanatin, and plant thionin.
In addition, a large number of antimicrobial peptides are known that have not yet been properly used, and their applications have not satisfactorily been discovered. Therefore, more work needs to be done to investigate the applications of these compounds, especially in the new sectors of the food and medical industries. For example, there is increasing knowledge about the role of antimicrobial peptides in reducing the prevalence of some cancers, especially colon cancer. The exact mechanism of action is not known, but it is likely due to the control of mutagenic compounds in the intestine by direct binding to carcinogens or inhibition of the microbes that produce these agents. Therefore, there is a high potential for the use of antimicrobial peptides, and more research in this field can lead to promising results that have significant effects in the food and medical industries.
Conceptualization, M.A. and V.N.U.; methodology, M.A., A.K., S.E. and V.N.U.; validation, M.A. and V.N.U.; formal analysis, M.A. and V.N.U.; investigation, M.A., A.K., S.E. and V.N.U.; writing-original draft preparation, M.A.; writing-review and editing, M.A. and V.N.U.; visualization, M.A.; project administration, M.A. and V.N.U. All authors have read and agreed to the published version of the manuscript.
This research received no external funding.
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The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
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A historical overview of peptide chemistry from T. Curtius to E. Fischer to M. Bergmann and L. Zervas is first presented. Next, the fundamentals of peptide synthesis with a focus on solid phase peptide synthesis by R. B. Merrifield are described. Immobilization strategies to attach the first amino acid to the resin, coupling strategies in stepwise peptide chain elongation, and approaches to synthesize difficult peptide sequences are also shown. A brief comparison between tert-butyloxycarbonyl (Boc)/benzyl (Bzl) strategy and 9-fluorenylmethoxycarbonyl (Fmoc)/tert-butyl (t -Bu) strategy utilized in solid phase peptide synthesis is given with an emphasis on the latter. Finally, the review focuses on the discovery and development of peptide ligation and the latest advances in this field including native amide bond formation strategies, these include the native chemical ligation, α-ketoacid-hydroxylamine ligation, and serine/threonine ligation which are the most commonly used chemoselective ligation methods that provide amide bond at the ligation site. This review provides an overview of the literature concerning the most important advances in the chemical synthesis of proteins and peptides covering the period from 1882 to 2017.
Keywords: Chemical protein synthesis; Chemoselective ligation; Coupling reagents; Difficult peptide sequences; Resins; SPPS.
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Lay summary, introduction, materials and methods, data availability, supplementary data, acknowledgements, rna-directed peptide synthesis across a nicked loop.
The first two authors should be regarded as Joint First Authors.
Meng Su, Samuel J Roberts, John D Sutherland, RNA-directed peptide synthesis across a nicked loop, Nucleic Acids Research , 2024;, gkae702, https://doi.org/10.1093/nar/gkae702
Ribosomal translation at the origin of life requires controlled aminoacylation to produce mono-aminoacyl esters of tRNAs. Herein, we show that transient annealing of short RNA oligo:amino acid mixed anhydrides to an acceptor strand enables the sequential transfer of aminoacyl residues to the diol of an overhang, first forming aminoacyl esters then peptidyl esters. Using N -protected aminoacyl esters prevents unwanted peptidyl ester formation in this manner. However, N -acyl-aminoacyl transfer is not stereoselective.
The Central Dogma dominates our understanding of modern biology. However, the mechanisms of how RNA directed peptide synthesis (translation) developed at the dawn of life remain a puzzle. In this study, we demonstrate that short peptides can spontaneously form at the end of a duplex RNA with an overhang. Contemporary proteins are composed exclusively of L-amino acids and our research reveals that L-amino acids with free amino groups are also more likely to participate in this prebiotic peptide synthesis mechanism. Conversely, when one of the amino acids is protected as it is in modern bacteria, this preference disappears. We demonstrate this chemistry can afford a variety of peptide sequences in a sequence-dependent manner.
Modern RNA-coded peptide synthesis is complicated, including the ribosome and various translation factors to enable peptidyl transfer at the ( N -acyl-)aminoacylated 3′-CCA termini of two juxtaposed tRNAs ( 1 ). Although the details of this mechanism have been elucidated in recent years ( 2 , 3 ), it is not known how translation could have first emerged.
According to the RNA world hypothesis ( 4 ), first posited in 1986, self-replicating RNA molecules proliferated before the evolution of DNA and proteins. In support of this proposal, RNA has been found to be capable of both storing genetic information and catalysing essential biochemical reactions. Given RNA’s key role in the synthesis of proteins there has been a long-standing interest in understanding how translation developed on early Earth. Several cases of RNA-catalysed or RNA-mediated peptide synthesis have been reported. A 196-nt ribozyme was evolved to perform a peptidyl transfer reaction to yield Met-Phe dipeptide ( 5 , 6 ). A triphenylalanyl-RNA ester was formed in a nicked RNA/DNA duplex without additional ribozymes ( 7 ). Oligophenylalanine up to a pentapeptide was detected using a five-nucleotide ribozyme and phenylalanyl adenylate ( 8 ). However, whether phenylalanine can be deemed as a prebiotically plausible amino acid is still in doubt ( 9–11 ). Crucially, no relationship between RNA sequence and peptide sequence (or coding) has been demonstrated with these systems. Two examples of RNA-templated peptide synthesis, in a nicked duplex or across a duplex terminus, were published recently ( 12 , 13 ). A condensation buffer containing an excess of a conventional chemical activating agent, rather than a prebiotically plausible one, was required in either case, as the coupling reactions do not appreciably occur without them. Furthermore, an adequate hypothesis for how either of these mechanisms could have transitioned to the extant ribosomal mechanism over time is yet to be proposed. As such, a question remains over their relevance to the origin and early evolution of life.
We have previously reported an aminoacyl-transfer reaction within a stem-overhang RNA structure (Figure 1A ) ( 14 ). Starting from a chemically synthesised aminoacyl-phosphate mixed anhydride at the 5′-terminus of an RNA strand, in combination with a longer overhanging complementary RNA, the aminoacyl-residue is spontaneously transferred to the 3′-terminus forming a 2′/3′-aminoacyl-ester. The 5-mer UUCCA overhang was found to be the most efficient CCA-ending sequence for the transfer reaction. This mechanism has clear parallels to that of extant biology which utilises an aminoacyl adenylate mixed anhydride and aminoacyl tRNA synthetases to synthesize 2′/3′-aminoacyl-ester tRNAs. We have also reported recently how the transfer varies with RNA sequence and different amino acids and the possible consequences for the origin of the genetic code ( 15 ). Furthermore, we have demonstrated that the transfer is stereoselective for L-amino acyl residues when using D-RNA. Biology also uses these same enantiomers of bio-molecules and establishing this stereochemical relationship would be vital at the origins of translation. We were interested to see if further equivalents of mixed anhydride would allow subsequent aminoacyl transfer to the aminoacyl-ester amino group to give a 2′/3′-dipeptidyl-ester.
Peptide synthesis via alanyl mixed anhydride transfer. ( A ) Schematic representation of aminoacyl transfer; ( B ) HPLC trace of alanyl mixed anhydride transfer over time. The broken box shows a new peak other than the 10-mer acceptor strand and its 2′/3′-Ala-esters, (indicated by the dashed arrow); ( C ) time courses of the 2′/3′-Ala-esters (thin line) and the 2′/3′-dipeptidyl-esters (putatively) corresponding to the new peaks over time (corrected for the mixed anhydride yield); ( D ) MALDI-TOF results for the aminoacyl-transfer reaction, 2′/3′-Ala-Ala-esters, calculated 3260.6, found 3260.7; 2′/3′-trialanyl-esters, calculated 3331.7, found 3331.8. A and G in bold and coloured circles indicate amino acids alanine and glycine. Conditions: acceptor strand 100 μM, Ala-mixed anhydride donor strand (3 equivalents relative to the acceptor strand), HEPES 50 mM, NaCl 100 mM, MgCl 2 5 mM, pH 6.8, 16°C.
Reagents and solvents were obtained from Acros Organics , Santa Cruz Biotechnology , Sigma-Aldrich , and were used without further purification. For solid-phase RNA synthesis, primer support 5G with a loading of 300 μmol/g was purchased from Cytiva . Phosphoramidites for RNA synthesis were purchased from Link Technologies . RNA oligomers used in this study were synthesized using an ÄKTA Oligopilot plus 10 ( Cytiva ) on a 20–50 μmol scale. Mettler Toledo SevenEasy pH Meter S20 combined with a ThermoFisher Scientific Orion 8103BN Ross semi-micro pH electrode was used to measure and adjust the pH to the desired value. Mass spectra were acquired on an Agilent 1200 LC–MS system equipped with an electrospray ionization (ESI) source and a 6130 quadrupole spectrometer (LC solvents: A, 0.2% formic acid in H 2 O; B, 0.2% formic acid in acetonitrile), or Bruker Ultraflex III MALDI-TOF/TOF. High-performance liquid Chromatography (HPLC) was run on Dionex Ultimate 3000 ( Thermo Scientific ) using an A tlantis T3, 3 μm, 4.6 × 150 mm column (unless stated otherwise). Oligonucleotide concentrations were determined by UV absorbance at 260 nm using a NanoDrop ND-1000 spectrophotometer. 1 H- and 13 C-nuclear magnetic resonance (NMR) spectra were acquired using a Bruker Ultrashield 400 Plus operating at 400.13 and 100.61 MHz, respectively. Data for 1 H- experiments are reported as chemical shifts (relative integral, splitting, coupling constant, assignment) whilst for 13 C- are reported as chemical shifts (assignment). Chemical shifts ( δ ) are shown in ppm. Coupling constants ( J ) are given in Hertz; s, singlet; d, doublet; q, quartet; qd, quartet of doublets; m, multiplet; br, broad signal.
After automated synthesis, RNAs were first cleaved from the solid support by treating with 3 ml of a 1:1 mixture of NH 3 aqueous solution (28% wt) and CH 3 NH 2 ethanol solution (33% wt) at 55°C for 80 min in a tube with a sealed cap. The solid was removed by filtration and washed with 50% EtOH/H 2 O. The solutions were combined and evaporated to dryness under reduced pressure. Silyl protecting groups were removed by treating the residues with 2 ml of 1:1 mixture of triethylamine trihydrofluoride and DMSO at 65°C for 150 min in a tube with a sealed cap. After brief cooling at –32°C, Cold NaClO 4 in acetone (50 mM, 40 ml) was added to the solution to precipitate the oligoribonucleotides. The resulting mixture was centrifuged and the RNA pellets were dissolved in water and purified by preparative HPLC then lyophilized. The purified RNA was redissolved in 2 ml of water and passed through a Waters Sep-Pak C18 Cartridge with 10 g sorbent. The cartridge was pre-washed with 50 ml of acetonitrile then 50 ml of water before sample loading, washed with 100 ml of H 2 O and 50 ml of 20% acetonitrile aq. Eluates containing RNA were checked on a Nanodrop, combined and lyophilized. The resulting white powder was stored at -32°C for future use.
Synthesis of the protected amino acids and oligoribonucleotide mixed anhydrides, transfer reaction, yield calculation and kinetic regression analysis were adapted from previous methods ( 14 ). Hydrolysis was executed for at least one half-life. MALDI-TOF characterization was conducted after desalting the reaction mixture with a Sep-Pak C18 column. See Table S1 for oligonucleotides used in this study.
N -Formyl-alanine (23.4 mg, 0.20 mmol, 1 eq.) was dissolved in dichloromethane (3 ml) and cooled to 0°C. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 42.0 mg, 0.22 mmol, 1.1 eq.) was added and the reaction was stirred overnight. The solution was dried in vacuo and used directly with no further purification.
1 H NMR (400 MHz, CDCl 3 ) δ = 7.50 (1H, d, J = 2.5 Hz, H2), 4.09 (1H, qd, J = 7.6, 2.5 Hz, α), 1.40 (3H, d, J = 7.6Hz, β).
13 C NMR (100 MHz, CDCl 3 ) δ = 178.2 (C1), 152.9 (C2), 58.3 (α), 16.3 (β).
Alanyl alanine (240 mg, 1.5 mmol) was dissolved in aqueous sodium hydroxide solution (1 M, 2.9 ml) and tetrahydrofuran (2.0 ml) and cooled on ice. Ditertbutyl dicarbonate (393 mg, 1.8 mmol) was dissolved in tetrahydrofuran (THF, 1.6 ml) and added dropwise over 5 min. The reaction was stirred on ice for 30 min before being stirred overnight at room temperature. The solution was cooled to 0°C and acidified to pH = 4 with aqueous HCl (2 M). The aqueous layer was extracted with diethyl ether (20 ml × 3). The combined organic fractions were dried with magnesium sulphate and concentrated to dryness in vacuo . The solid was recrystallised from diethyl ether affording 100 mg (26% yield) of colourless needle-like crystals.
1 H NMR (400 MHz, CDCl 3 ) δ = 6.97 (1H, d, J = 7.2 Hz, NH), 5.27 (1H, s, NH), 4.56 (1H, q, J = 7.2 Hz, α1), 4.24 (1H, br, α2), 1.45 (12H, m, β1 + Boc), 1.36 (3H, d, J = 7.1 Hz, β2).
13 C NMR (100 MHz, CDCl 3 ) δ = 210.9 (CO-Boc), 175.6 (CONH), 173.1 (CO 2 H), 80.3 (Boc (C(CH 3 ))), 50.1 (α2), 48.3 (α1), 28.4 (Boc (CH 3 )) 18.3 (β2), 18.1 (β1).
Adenosine (1.3 mg, 4.9 μmol, 1.0 eq.) was dissolved in a mixture of H 2 O (375 μl), D 2 O (50 μl) and DMSO (25 μl) with the aid of a heat gun. Carbonyl diimidazole (CDI, 4.4 mg, 27.4 μmol, 5.6 eq.) and the desired N -protected amino acid or Boc-Ala-Ala (27.4 μmol, 5.6 eq.) were dissolved in acetonitrile (250 μl) and vortexed for 3 min at room temperature. An aliquot of the amino acid solution (50 μl) was added to the adenosine solution. The mixture was shaken (300 rpm) for 10 min at room temperature. If the amino acid was Boc protected, the solution was acidified to pH 4 with aqueous HCl (1 M), then concentrated in vacuo . Trifluoroacetic acid (200 μl) was added and the mixture was left to stand at room temperature for 10 min before being concentrated in vacuo and redissolved in a mixture of H 2 O (450 μl), D 2 O (50 μl). If the amino acid was formyl protected, solutions were taken on directly. All samples were analysed both by HPLC with 260 nm UV detection. Solvent A: 25 mM TEAA (triethylamine buffered to pH 4.5 with acetic acid); solvent B: acetonitrile; flow rate 1 ml/min; column compartment 25°C; gradient 0 min, 0%; 18 min, 25%; 19 min, 85%; 22 min 85%; 23 min, 0%; 26 min, 0%. For formyl-glycylalanyl ester adenosine, a dichloromethane solution (5 ml) of N , N ′-dicyclohexyl-carbodiimide (DCC, 107 mg, 0.52 mmol, 2 eq.) and formic acid (20 μl, 0.52 mmol, 2 eq.) were stirred at room temperature for 5 min before glycyl alanine ( Santa Cruz , sc-479092, 38 mg, 0.26 mmol, 1 eq.) was added. The mixture was further stirred at room temperature for 8 h. The precipitate was filtered, and the dichloromethane solvent was removed. The residue was redissolved in acetonitrile (500 μl) and stirred with CDI (16.9 mg, 0.10 mmol, 0.4 eq.) for 3 min before an aqueous suspension (2 ml) of adenosine (13.9 mg, 0.05 mmol, 0.2 eq.) was added. The mixture was further stirred for 10 min while monitoring the pH to 7.0, before being quenched with 10% formic acid. The mixture was purified using HPLC. Solvents as above, flow rate 5 ml/min. See Supplementary Figures S23 – S30 for 1 H NMR spectrum for the above adenylates.
When necessary, samples of a desired retention time were collected from analytical HPLC and then lyophilized. Sodium acetate solution (pH 4.0, 0.5 M, 1 μl) was added to either an aliquot of crude reaction (9 μl) or redissolved lyophilizate (9 μl, H 2 O). RNase A (1 μl, 10 mg/ml, Thermo Fischer , EN0531) was added and the solution was shaken for 30 min (20°C, 300 rpm). Methanol (11 μl) was added and the mixture was centrifuged for 30 min (4°C, 21 000 × g). The supernatant was recovered and analysed by HPLC with Atlantis T3, 5 μm, 4.6 × 250 mm column. Solvents and gradients as above.
In the HPLC trace of transfer reaction of alanyl (Ala) residues (3 eq. 5′-L-Ala-pCUG mixed anhydride as donor strand mixed with 1 eq. 5′-CGCAG UUCCA as acceptor stand, Figure 1A , see supplementary data for schematic representations) we observed a new peak with a longer retention time than that of the 2′/3′-Ala-ester acceptors (Figure 1B ). The peak reached its maximum yield of 8% after 140 min, compared to 70 min for the known 2′/3′-Ala-esters (Figure 1C , Supplementary Table S2 , Supplementary Figure S1 ). This new peak decreased in intensity slowly with a half-life of 14 h at 16°C, more than double that of the 2′/3′-Ala-esters. After isolation of species responsible for the new peak, MALDI-TOF characterization was consistent with a 10-mer strand plus two Ala residues (Figure 1D ). Although it is possible in theory to produce a bis-2′,3′-Ala,Ala-diester (with Ala residues both at terminal or internal 2′ and terminal 3′ positions), we would expect such species to have comparable lability to the 2′/3′-Ala-esters. However, we expected the 2′/3′-dialanyl-esters (Ala-Ala) to be more stable than the monoalanyl esters and therefore tentatively assigned the new peak to result from the formation of the 2′/3′-Ala-Ala-ester acceptor strands.
We had previously used RNase A digestion to analyse N -acetyl-aminoacylation of RNA ( 14 ). RNase A cleaves the terminal adenosine nucleoside of the acceptor stem whilst maintaining any aminoacyl ester bonds. By comparison against synthetic standards, the 2′/3′-( N -acetyl)-Ala-ester adenosines could be identified in the digestion products of N -acetyl-aminoacylated-RNA when analysed by HPLC, and thus the presence of terminal 2′/3′- N -acetyl-aminoacyl-esters in the undigested RNA could be deduced. To provide further verification that the new peak we had observed in the current work indicated the formation of 2′/3′-Ala-Ala-esters, we HPLC purified the species responsible for the peak, then lyophilised and digested it with RNase. The chromatogram of the digested material contained peaks consistent with both 2′- and 3′-Ala-Ala-ester adenosines ( Supplementary Figure S2 , Supplementary Figure S3 ) when compared to synthetic standards. The region of the chromatogram corresponding to the bis-2′,3′-Ala,Ala-diester adenosine standard was quite congested in the digested sample, but very low in intensity. We conclude the new peak to result from the formation of 2′/3′-Ala-Ala-esters. Furthermore, we were able to identify a mass peak corresponding to the 10-mer strand plus three alanine residues by MALDI (Figure 1D ), which is logically even more likely to include at least one peptide bond.
In prokaryotes, peptide synthesis starts with N -formyl-methionine (fMet) encoded by the start codon AUG. The formyl group allows the corresponding aminoacyl-tRNA to be accommodated at the P site of the ribosome. N -formyl-amino acids have been demonstrated to be prebiotically plausible species on early Earth ( 16 ). Therefore, we studied N -formyl-alanyl (fAla) and N -formyl-glycyl transfer (fGly) (Figure 2A , B , Supplementary Table S3 , Supplementary Figure S4 , Supplementary Figure S5 ). First, we demonstrated that both fAla and fGly themselves directly transferred to the 3′-termini of a 10-mer acceptor bearing no other aminoacyl residues (maximum observed yields 16% and 21% at pH 8.0, Figure 2A , B ctrl lines). Next, we tested whether a sequential addition could form peptidyl-RNA. Starting with the same sequence 10-mer acceptor strand, an Ala residue was first transferred to the 10-mer 3′-terminus using an alanyl mixed anhydride-tetramer donor (5′-L-Ala-pCUGC). When the yields of 2′/3′-Ala-esters reached their peaks, 2 eq. fAla- or fGly-mixed anhydride-pentamer donor (5′-fAla-/fGly-pCUGCG) was added to the system.
Peptide synthesis via transfer of an alanyl residue followed by N -formyl-aminoacyl transfer. HPLC traces of ( A ) fAla and ( B ) fGly transfer to 2′/3′-Ala-ester acceptors. The dotted lines show two new peaks corresponding to 2′/3′- N -formyl-dipeptidyl-ester acceptors over time. fAla and fGly ctrl indicate the reaction of fAla-/fGly-mixed anhydride donor transferred directly to the 2′/3′-diol of a 10-mer acceptor to give 2′/3′-fAla/fGly-esters. * indicates 2′/3′-fAla/fGly-esters. ( C ) The respective MALDI spectra and data. A and G in bold and coloured circles indicate amino acids alanine and glycine. Conditions: acceptor strand 100 μM, Ala-mixed anhydride donor strand (1 eq.) and N -formyl-aminoacyl mixed anhydride donor strand (2 eq.) to the acceptor strand, HEPES 50 mM, NaCl 100 mM, MgCl 2 5 mM, pH 6.8, 10°C. fAla/fGly ctrl was performed at pH 8.0. Stacked HPLC traces are staggered by 15 s each for clarity.
After the addition of N -formyl-aminoacyl mixed anhydride, the peak corresponding to the 2′/3′-Ala-esters was observed to decrease much faster (half-life = 1.9 h for fGly and 3.3 h for fAla) than without the N -formyl-aminoacyl mixed anhydride (half-life = 6.6 h). New peaks with similar kinetic profiles to each other were detected ( Supplementary Figure S4 , Supplementary Figure S5 ) as well as peaks identifiable as 2′/3′- N -formyl-aminoacyl-esters by comparison with a control reaction (Figure 2A , B ctrl lines). After isolation, the species responsible for the new peaks produced MALDI peaks consistent with RNA plus both Ala- and fGly/fAla- residues (Figure 2C ). Despite our previous assumption that no bis-2′,3′-products were formed, we entertained the possibility these new peaks may correspond to the 2′,3′-Ala-,fAla-/Ala-,fGly-diesters. We would expect that both 2′,3′-Ala-,fAla- and 2′,3′-Ala-,fGly-diesters would first hydrolyse to 2′/3′-fAla- or 2′/3′-fGly-esters. However, the corresponding peaks for these species did not increase as the new peaks degraded, supporting the formation of 2′/3′-dipeptidyl esters instead.
As there was always a substantial proportion of unreacted 10-mer acceptor strand after the first step, we also observed the direct formation of 2′/3′-fAla-/fGly-esters from said acceptor. Due to the increased nucleophilicity of a 2′,3′-diol compared to a single hydroxyl, we would expect more 2′/3′-fAla-/fGly-ester to form than any bis-species. However, in the case of fAla transfer (Figure 2A , Supplementary Table S3 ), we found the rate constants of formation of 2′/3′-fAla-ester and the new species to be nearly identical (0.0066 and 0.0067 min –1 respectively). In the case of fGly transfer (Figure 2B , Supplementary Table S3 ), the integration of the new peaks was 2-fold higher than the integration of the 2′/3′-fGly-ester peak. All these observations suggest that the new peaks did not correspond to bis-species, but instead indicated the formation of 2′/3′-formyl-dipeptidyl-esters.
We further studied the transfer by RNase A digestion of the products of fGly transferred onto preformed 2′/3′-Ala-esters. By comparison of the HPLC chromatograms of the digestion products with synthetic standards, we identified peaks corresponding to the 2′/3′-fGly-Ala-esters of adenosine, and none of the bis-2′,3′-Ala-,fGly-diesters, demonstrating that the transfer reaction does not produce bis-2′,3′-diesters. ( Supplementary Figure S3 , Supplementary Figure S6 )
In the case of the fAla transfer onto preformed 2′/3′-Ala-esters, the question remained, why were there two new peaks with significant differences in retention time? When a fAla-residue was transferred from a pentamer donor to the 3′-terminus of the 10-mer acceptor as a control experiment, two overlapping peaks were observed (Figure 2A ). This could be consistent with racemization of fAla during the chemical synthesis of the mixed anhydride followed by transfer of both D- and L-fAla-residues. Previous experiments had demonstrated that D-alanyl mixed anhydrides would only transfer inefficiently whilst D- N -acetyl-alanyl (D-AcAla) mixed anhydrides transferred equally well as L- across a nicked loop ( 14 , 15 ). Alternatively, the two new peaks could be due to a mixture of 2′/3′-aminoacyl-esters resolving differently on the column. In the case of fGly transfer to a 2′,3′-diol, only one peak was identified, which could suggest either the transfer was regioselective for one alcohol or that both 2′/3′-aminoacyl-esters have equal retention times.
The yield of peptide bond formation differed between these formylaminoacyl residues (2′/3′-fAla-Ala-esters 23%, 2′/3′-fGly-Ala-esters 57%). Unsurprisingly, due to the difference in charge state, 2′/3′-fAla-Ala-esters were less labile to cleavage ( t |$\frac{1}{2}$| = 4.1 days at 10°C, pH 6.8) than 2′/3′-Ala-Ala-esters ( t |$\frac{1}{2}$| = 1 day at 10°C, pH 6.8) which (as well as direct hydrolysis by hydroxide) can additionally cleave to form diketopiperazines (see ESI for schematic representations). The half-life of the 2′/3′-fGly-Ala-esters under the same conditions was t |$\frac{1}{2}$| = 3.4 days, a bit shorter than the 2′/3′-fAla-Ala-esters.
Next, we broadened our aminoacyl mixed anhydride scope in the first step to include leucyl- and prolyl-residues (Leu and Pro respectively). For the second transfer, fAla- or fGly-mixed anhydride pentamer donor strand was used as with previous experiments (Figure 3 , Supplementary Table S3 , Supplementary Figure S7 - S10 ). Transfers of fAla onto Leu- or Pro-acceptors produced four new HPLC peaks with identical kinetics, whilst transfers of fGly onto Leu or Pro produced two new ones. This is consistent with both the racemization of fAla and a resolution of the mixture of 2′/3′-esters in the products. We did not further differentiate these peaks. HPLC and MALDI analysis confirmed that all four 2′/3′-formyl-dipeptidyl-esters were correctly synthesized, with distinct yields and synthesis/hydrolysis kinetics. In the first step, Ala transferred better than Leu and Pro as reported previously ( 15 ). In the second step, fGly generally transferred better or as well as fAla, depending on the identity of the first amino acid transferred.
Time courses and MALDI characterization of 2′/3′- N -formyl-dipeptidyl RNA. Timepoint 0 was set at the peak yield of 2′/3′-aminoacyl-esters at which point the formylaminoacyl-mixed anhydride donor was added. The solid line in the time course represents the percentage yield of 2′/3′-fGly-/fAla-dipeptidyl-esters relative to the 2′/3′-aminoacyl-esters. The broken line represents the consumption due to hydrolysis and formylaminoacylation of the 2′/3′-aminoacyl-esters. A, G, L, P in bold and coloured circles indicate amino acids alanine, glycine, leucine, and proline. Conditions: acceptor strand 100 μM, aminoacyl-mixed anhydride tetramer donor strand (1 eq.) and N -formyl-aminoacyl mixed anhydride pentamer donor strand (2 eq.) to the acceptor strand, HEPES 50 mM, NaCl 100 mM, MgCl 2 5 mM, pH 6.8, 10°C.
Both the constituent amino acids in the formyl-dipeptidyl-ester affected the ester lability, despite their inability to form diketopiperazines. It has previously been shown that a Leu-tRNA ester was more stable than an Ala-tRNA ester or a Pro-tRNA ester ( 17 ). We observed the same trend. Furthermore, the 2′/3′-formyl-dipeptidyl-esters containing fAla are always found to be more stable than those containing fGly.
The duplex-overhang 2′/3′-formyl-dipeptidyl-esters are equivalent to the acceptor stem of a peptidyl-tRNA. In extant translation, releasing the mature peptides is assisted by the Gly-Gly-Gln motif in the Class I release factors ( 18 ). But hydrolysis of 2′/3′-peptidyl-esters in a prebiotic context has not been documented to the best of our knowledge. In this work, with the help of aminoacyl transfer chemistry, we have built a small but diverse series of 2′/3′-( N -formyl)-dipeptidyl-esters and characterized their hydrolytic stability (Table S3). The longest half-life among the six 2′/3′-formyl-dipeptidyl-esters is 2′/3′-fAla-Leu-ester ( t |$\frac{1}{2}$| = 6.6 d at 10°C, pH 6.8). This suggests that under these conditions peptidyl RNA conjugates can form, and become enriched without auxiliary macromolecules.
In all three examples where fGly-mixed anhydride was used in the second transfer, we identified two peaks with longer retention time and greater stability toward hydrolysis (peaks marked with * in Supplementary Figure S5 , Supplementary Figure S8 , Supplementary Figure S10 ). These peaks may correspond to 2′/3′-tripeptidyl-esters terminating in fGly. This would require fGly-mixed anhydride to react with the 2′/3′-dipeptidyl-esters we identified at the beginning of this communication (Figure 1B , Supplementary Figure S1 ). Due to the lack of material, we were unable to characterize these peaks further.
We investigated the mechanism of the multi-step N -formyl-aminoacyl transfer. We prepared the alanyl mixed anhydride donor in three different lengths, trimer (5′-L-Ala-pCUG), tetramer (5′-L-Ala-pCUGC) and pentamer (5′-L-Ala-pCUGCG). As the first transfer reached its maximum, two equivalents of a fGly-mixed anhydride pentamer (5′-fGly-pCUGCG) were added. With the increasing length of the original alanyl-mixed anhydride donor, it becomes harder for the fGly-mixed anhydride pentamer donor to replace it, thus reducing the yield and rate of dipeptidyl ester formation. The 5′-L-Ala-pCUG followed by 5′-fGly-pCUGCG transfer gave the highest dipeptide yield (65%) in the shortest time (325 min) (Figure 4 , Supplementary Table S4 ). The 5′-L-Ala-pCUGC followed by 5′-fGly-pCUGCG transfer yield was 57% (at 410 min), whilst the 5′-L-Ala-pCUGCG followed by 5′-fGly-pCUGCG transfer was the slowest, reaching to maximum yield of 40% after 1000 min. These data indicate the necessity of strand exchange before N -formyl-dipeptidyl ester formation.
Peptidyl-RNA synthesis is achieved by donor strand replacement. Time courses for the formation of 2′/3′-fGly-Ala-esters using different length Ala-mixed anhydride donor strands. A and G in bold and coloured circles indicate amino acids alanine and glycine. Conditions: acceptor strand 100 μM, Ala-mixed anhydride initial donor strand (1 eq.), N -formyl-glycyl-mixed anhydride donor strand (2 eq.) added at the peak formation of 2′/3′-Ala-esters, HEPES 50 mM, NaCl 100 mM, MgCl 2 5 mM, pH 6.8, 10°C.
When N -acyl amino acids are activated at their C-termini they form oxazolones (see Supplementary Figure S11 - S15 and Supplementary Figure S22 for relevant structures) which are reactive electrophiles ( 19 ). It is possible the formyl-aminoacyl-mixed anhydrides could undergo reversible cleavage from 5-( 4H )-oxazolones, which then subsequently react intermolecularly with the nucleophilic amine of the 2′/3′-aminoacyl-ester. To test this hypothesis we incubated the 10-mer acceptor strand and a pentamer (pCUGCG) containing a 5′-phosphate then added the water-soluble carbodiimide EDC and fGly/fAla to provide in situ formation of oxazolone ( Supplementary Figures S11 and S12 ). This failed to produce any acyl transfer products, mixed anhydride or other RNA esters. Repeating the reaction instead using preformed Ala 5-( 4H )-oxazolone up to 2000 eq. also failed to produce any peaks where we would expect transfer products nor mixed anhydride, in contrast to previous studies with nucleosides ( 19 ) ( Supplementary Figures S13 and S14 ). Similar addition of Ala 5-( 4H )-oxazolone to preformed 2′/3′-Ala-esters did not produce new peaks with the correct retention time or stability ( Supplementary Figure S15 ). As no transfer to the 10-mer strand is observed at such high concentrations of oxazolone and given the relative stability of the formyl-aminoacyl-mixed anhydrides under these conditions, we think it unlikely that 5-( 4H )-oxazolone is implicated in the formation of N -formyl-dipeptidyl-2′/3′-esters or N -formyl-aminoacyl-2′/3′-esters presented above.
As diastereoselectivity of transfer had only been studied for the transfer of AcAla residues, we studied the transfer of the more prebiotically plausible fAla residues used in this work. A 5-mer mixed anhydride synthesised from L-fAla as a donor strand was mixed with a 10-mer acceptor strand (1 eq. 5′-fAla-pAGCGA mixed anhydride with 1 eq. 5′-UCGCU UUCCA forming a nicked loop) or with a 10-mer template strand and 8-mer acceptor strand (1 eq. 5′-fAla-pAGCGA mixed anhydride with 1 eq. 5′-UCGCU UUCCA template and 1 eq. 5′-UAA UGGAA acceptor making a nicked duplex). fAla transfer to their acceptors was identified across a nicked loop ( Supplementary Figure S16 , 16%) or nicked duplex ( Supplementary Figure S17 , 61%) by the presence of new peaks with increased retention time consistent with our previous experiments. The crude mixtures were digested by RNase A as before and analysed by HPLC ( Supplementary Figures S18 and S19 ). Comparison of these chromatograms with those of synthetic standards confirmed the presence of nearly equal amounts of both L- and D-2′/3′-fAla-ester adenosines. These results were consistent with racemization of the formylalanyl residue during synthesis of the fAla mixed anhydride 5-mer, followed by the transfer of both L- and D-fAla residues. Repeating these experiments, but starting with D-fAla, afforded similar transfer yields (13% nicked loop and 50% nicked duplex, Supplementary Figures S16 and S17 ) and resulted in nearly identical chromatograms ( Supplementary Figures S18 and S19 ).
At this point, it seemed odd that only one out of the four possible combinations of N -acetyl-, and N -formyl-aminoacyl transfers across either a nicked-loop or nicked-duplex was stereoselective. We synthesised 5-mer mixed anhydrides using either L- or D-AcAla as a donor strand, then added them to a nicked duplex scenario containing the same 10-mer template strand and 8-mer acceptor strand as our earlier experiments (1 eq. 5′-AcAla-pAGCGA mixed anhydride with 1 eq. 5′-UCGCU UUCCA template and 1 eq. 5′-UAA UGGAA acceptor). We identified the transfer of AcAla residues across a nicked duplex by formation of a new peak as before (starting from Ac-L-Ala 25%, starting from Ac-D-Ala 22%; Supplementary Figure S20 ). The species responsible for this peak were collected, lyophilised and then digested with RNase. Subsequent HPLC analysis and comparison to synthesised standards demonstrated the presence of equal amounts of L- and D- 2′/3′-AcAla-ester adenosines ( Supplementary Figure S21 ).
Oxazolones are known to racemize ( 20 ). The synthetic chemistry used to make formylaminoacyl mixed anhydrides in this work would likely produce 5-( 4H )-oxazolones as reactive intermediates, so we would expect both diastereoisomers of the formyl-aminoacyl mixed anhydrides to be formed. Stereoselectivity of transfer of N -acyl-aminoacyl residues would have been important to nascent translation to ensure diasteromerically pure proteins. We and others have previously demonstrated that the transfer of aminoacyl residues from a mixed anhydride in a nicked loop ( 14 ) or nicked duplex ( 21 ) configuration is stereoselective with some exceptions for specific sequences ( 15 ). However, nicked loop transfer of AcAla residues is not stereoselective ( 14 ). This last observation is interesting in comparison to a previous report that AcAla residues do in fact transfer stereoselectively in a nicked duplex ( 22 ).
In contrast to the previous report, we conclude that N -acyl-aminoacyl residues do not transfer stereoselectively in either nicked-loop or nicked-duplex configurations. Due to the lack of experimental detail reported by Tamura et al. ( 22 ), it is difficult to see where our conflicting results originate. It is possible that the different sequences used demonstrate different levels of selectivity, something we have seen in some exceptional cases with the transfer of aminoacyl residues in a nicked loop ( 15 ). Alternatively, simple experimental errors in the previous work such as differing loading onto gels (no loading control was shown) or significantly hydrolysed mixed anhydride starting material synthesised from D-AcAla, are potential causes.
Our results here demonstrate the possibility of assembling prebiotic peptidyl-RNA via strand replacement and transfer from N -formyl-aminoacyl mixed anhydrides. We note dipeptides have been shown to have sequence specific activities relevant to the origins of life, such as vesicle growth ( 23 ). In our scenarios, stable duplex and strand replacement are the prerequisites for peptidyl assembly. Here, the peptide directly forms at the CCA-ending overhang, as in extant tRNA. The hydrolytic stability of formyl-dipeptidyl-RNA varies with amino acid sequence. Our investigations into the mechanism of N -formyl amino acid transfer suggest that 5-( 4H )-oxazolones are not implicated in the mechanism of transfer. The transfer of N -acyl amino acids onto 3′-termini in a nicked-duplex or a nicked-loop, and onto aminoacyl esters in nicked loops show no stereoselectivity. Due to the similarity in chemistry, it is conceivable that a transition from trimer-mixed anhydride donors to adenylate-mixed anhydrides which we see in modern biology is simpler than those template chemistries presented previously ( 12 , 13 ). Our results also highlight the difficulty of avoiding peptidyl-RNA formation in systems capable of transferring aminoacyl residues from amino acid:phosphate mixed anhydrides to the 3′-termini of RNA.
The data underlying this article are available in the article and in its online supplementary material.
Supplementary Data are available at NAR Online.
We thank Dr Holger Kramer for his assistance in mass experiments and other J.D.S. group members for fruitful discussions.
Medical Research Council (MC_UP_A024_1009); Simons Foundation (290362 to J.D.S.).
Conflict of interest statement . None declared.
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The greening of peptide synthesis †.
* Corresponding authors
a Green Chemistry Centre of Excellence, Department of Chemistry, University of York, York, UK E-mail: [email protected]
The synthesis of peptides by amide bond formation between suitably protected amino acids is a fundamental part of the drug discovery process. However, the required coupling and deprotection reactions are routinely carried out in dichloromethane and DMF, both of which have serious toxicity concerns and generate waste solvent which constitutes the vast majority of the waste generated during peptide synthesis. In this work, propylene carbonate has been shown to be a green polar aprotic solvent which can be used to replace dichloromethane and DMF in both solution- and solid-phase peptide synthesis. Solution-phase chemistry was carried out with Boc/benzyl protecting groups to the tetrapeptide stage, no epimerisation occurred during these syntheses and chemical yields for both coupling and deprotection reactions in propylene carbonate were at least comparable to those obtained in conventional solvents. Solid-phase peptide synthesis was carried out using Fmoc protected amino acids on a ChemMatrix resin and was used to prepare the biologically relevant nonapeptide bradykinin with comparable purity to a sample prepared in DMF.
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S. B. Lawrenson, R. Arav and M. North, Green Chem. , 2017, 19 , 1685 DOI: 10.1039/C7GC00247E
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August 19th, 2024
Bachem AG (Switzerland) and SpheriTech Ltd (UK) collaborate to develop water-based SPPS.
Peptides, crucial for modern medicine, are synthesized using organic solvents, which pose environmental challenges. Bachem is collaborating with UK based SpheriTech to develop water-based solid-phase peptide synthesis (SPPS) to reduce ecological impact.
Peptides are essential for modern medicine as active ingredients for an increasing number of drugs. Today’s manufacturing of peptides currently relies on large quantities of organic solvents. Organic solvents play a crucial role in peptide synthesis, facilitating chemical reactions and ensuring efficient coupling of amino acids. Unfortunately, their use comes at a cost. These solvents can be toxic, flammable, and contribute to greenhouse gas emissions. As the demand for peptide-based drugs grows, finding sustainable alternatives becomes imperative in an industry that must secure the long-term supply of medical ingredients for millions of patients worldwide. A growing pipeline of clinical projects, including a new generation of medicines against diabetes and obesity, all based on peptides, and combined with tightening environmental regulatory trends, are expected to challenge the current large scale use of solvents approach in the decade to come.
Bachem has a long track record as a trailblazing innovator, developing and applying new ways to improve peptide manufacturing technology. That is why Bachem has joined forces with UK-based SpheriTech to investigate a new water-based approach to solid-phase peptide synthesis (SPPS). Solid-phase peptide synthesis involves assembling amino acids step by step on a solid support. Traditionally, this process occurs in organic solvents. Water-based SPPS, however, replaces these solvents with water as the primary medium.
The collaboration aims to reduce the reliance on organic solvents while maintaining high-quality peptide production. By combining SpheriTech’s cutting-edge innovation with Bachem’s extensive large-scale peptide manufacturing experience, both companies are working together to bring water-based SPPS to a stage where it can be implemented in a commercially viable form. After the successful conclusion of a research phase, SpheriTech will apply the water-based technology to small-scale peptide synthesis, while Bachem will begin to develop the technology for larger scale synthesis.
For more information about SpheriTech visit: https://spheritech.com/
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The concept of solid-phase peptide synthesis (SPPS) is to retain chemistry that has been proven in solution but to add a covalent attachment step that links the nascent peptide chain to an insoluble polymeric support (resin). Subsequently, the anchored peptide is extended by a series of addition cycles ( Fig. 18.1.1 ).
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A linker plays a dual role during peptide synthesis. It offers protection to overcome aggregation during the elongation of the peptidic sequence while it provides a reversible linkage between the peptide chain and the solid support [15].Linkers are easily categorized in low and high acid-labile linkers based on the conditions that are used for cleavage, usually TFA solution.
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Authoritative and practical, Peptide Synthesis: Methods and Protocols serves as an essential guide to the many crucial processes that will allow researchers to efficiently prepare, purify, characterize, and use peptides for chemical, biochemical, and biological studies.
Abstract. Peptide drug development has made significant progress over the last century. The discovery of solid-phase peptide synthesis has enabled chemists to synthesize various peptides with divergent sequence patterns. However, due to the increased demand for various peptide sequences in the modern pharmaceutical industry, there is always ...
Solid-Phase Peptide Synthesis (SPPS) is a mature technique widely used in research and in production. There are different approaches that fulfill the diverse requirements, regarding the number, quantity and quality of peptides. We have implemented three laboratory protocols of synthesis that cover these needs. These protocols have been tested ...
Isolation of peptides from animal sources continued till the evolution of peptide synthesis methods [3]. With the inception of solution phase peptide synthesis during the early 1950s, chemical synthesis of oxytocin was performed in 1953 [4], followed by vasopressin in 1958 by Vincent du Vigneaud [5]. However, the search for more peptide-based ...
During these days, we are celebrating the 60th anniversary of the first public announcement of the Solid-Phase Peptide Synthesis (SPPS) technique at the Federation Meeting held in Atlantic City in 1962 by the Nobel Laureate R. Bruce Merrifield. The first publication appeared a year after this event [ 5 ].
Chemical synthesis of peptides by soluble-phase methods was first used in 1953 to produce pharmaceutical insulin peptides . The basis of this method is the reaction of amino acids in a soluble medium. ... Use of Computer-Based Techniques in Peptide Research. Computer-based methods, such as proteomic and peptidomic studies, are very helpful in ...
Solid-phase peptide synthesis (SPPS) is the method of choice for the synthesis of peptides for research and production purposes. Despite having several positive features, it remains a challenge to reduce the amount of solvent waste generated during the synthesis. We proposed a 3-step protocol (in-situ Fmoc removal) where washing after coupling ...
Next, the fundamentals of peptide synthesis with a focus on solid phase peptide synthesis by R. B. Merrifield are described. Immobilization strategies to attach the first amino acid to the resin, coupling strategies in stepwise peptide chain elongation, and approaches to synthesize difficult peptide sequences are also shown.
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Peptide therapeutics have experienced a rapid resurgence over the past three decades. While a few peptide drugs are biologically produced, most are manufactured via chemical synthesis. The cycle of prior protection of the amino group of an α-amino acid, activation of its carboxyl group, aminolysis with the free amino group of a growing peptide chain, and deprotection of the N-terminus ...
The greening of peptide synthesis S. B. Lawrenson, R. Arav and M. North, Green Chem., 2017, 19, 1685 DOI: 10.1039/C7GC00247E This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given.
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The coupling reaction, i.e. the formation of an amide bond between amino acids and/or peptides, is the crucial step in peptide synthesis. The chemistry behind and the most important coupling reagents will be presented in this blog. The reaction consists of two consecutive steps: 1. Activation of the carboxy moiety 2. Acylation of the amino group
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Peptides are essential for modern medicine as active ingredients for an increasing number of drugs. Today's manufacturing of peptides currently relies on large quantities of organic solvents. Organic solvents play a crucial role in peptide synthesis, facilitating chemical reactions and ensuring efficient coupling of amino acids.
In the absence of the peptide catalyst, an equimolar mixture of pyr, PM, Ala, and PL reacts reversibly to give a number of intermediate species . Aldimine species A is formed between Ala and PL, which appears to conserve the stereochemistry of the amino acid; when L-Ala is employed, Ala ee = 98% after 6 h in the absence of peptide catalyst. A ...
Improved Synthesis of a Macrocyclic Peptide-Like C5aR Antagonist for Intravenous Applications. Organic Process Research & Development 2023 , 27 (11) , 2010-2019.
The HSV polymerase is a heterodimer of Pol and a processivity subunit (UL42) that promotes long-chain DNA synthesis. 3 HSV Pol has characteristic thumb, palm, and fingers domains, a 3′-5′ exonuclease domain (Exo), and an NH 2-terminal domain (NTD).Pol also has a herpesvirus-specific pre-NH 2 domain of unknown function. 4 UL42 resembles a monomer of the eukaryotic sliding clamp protein ...
Loading and release of MK2i from the surface of the balloons were quantified (Figure 2 D).Initial coating with 6 layers using solutions of 50 μM MK2i and 5 μM PPAA (6L-50M-5P) yielded 1.5-2 μg/cm 2 of MK2i loading. Increasing the number of layers (12L-50M-5P) and increasing the concentration of the loading solutions (6L-100M-10P) both increased the peptide loading, with 6L-100M-10P balloons ...