Peptide - an overview

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PEPTIDES AND PROTEINS

The term ‘peptide’ includes a wide range of compounds varying from low to very high molecular weights and showing marked differences in physical, chemical and pharmacological properties. The lowest members are derived from only two molecules of amino acid, but higher members have many amino-acid units and form either peptides, simple proteins (albumins, globulins, prolamines, glutalins, etc.) or more complex proteins, conjugated proteins, in which other groupings form part of the molecule—for example, carbohydrate in mucoproteins, the very complex chlorophyll molecule in the protein of chloroplasts, phosphorus-containing proteins such as casein, nucleoproteins, in which proteins are combined with nucleic acid, and the lipoproteins of the cytoplasm, in which protein is combined with lipids. Among such substances with relatively low molecular weight are some antibiotics which have a cyclic polypeptide structure (e.g. gramicidin, bacitracin and polymyxin); peptide hormones such as oxytocin and vasopressin from the posterior pituitary gland; and glutathione, which is found in nearly all living cells.

All these more or less complex compounds have two or more molecules of amino acid united by a peptide linkage which results from the elimination of water, an OH coming from one amino acid and an H from the other.

Thus, a dipeptide is formed:

A dipeptide of the Sapindaceous plant Blighia sapida has hypoglycaemic properties and although more complex, penicillin also has a dipeptide structure. Tripeptides have three amino-acid components and polypeptides from ten upwards. Peptides are usually defined as protein-like substances having molecular weights below 10 000. In typical proteins the molecular weight is higher, ranging from about 30 000 to 50 000 in the relatively simple prolamines and glutelins and reaching very high values, sometimes several million, in the complex proteins such as those in sheep's wool.

Protein synthesis takes place in association with the ribosomes, which are small bodies found in the cytoplasm and particularly in the endoplasmic reticulum area (see Fig. 18.1). The amino acids are brought to the ribosomes associated with a transfer-RNA molecule and by the action of the ribosomes, using a sequence dictated by a particular messenger-RNA molecule, are linked to form the peptide chains of the particular protein. Although not directly relevant to most pharmacognostical studies, the story of the nucleic acids and their vital role in the control of cell metabolism is a fascinating one which it is suggested students study from a standard work on biochemistry.

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The peptide secretome

Peptides are short sequences of amino acids (typically ranging from 2 to 50) linked by peptide bonds. Peptides are often hormones, neurotransmitters, ion channel ligands, and growth factors [28] (Figure 2). They can be encoded, but are often synthesized from larger precursors through the action of enzymes like signal peptidases, endopeptidases, and carboxypeptidases [29,30]. Following their synthesis, they undergo post-translational modifications including amidation, acetylation, phosphorylation, sulfation, and glycosylation that can impact their stability and cellular bioactivity. In the past, the identification of peptides involved dissecting biological sources such as tissue samples, extracting, and isolating the peptides, and performing various biochemical and functional tests. This is exemplified by the discovery of glucagon-like peptide (GLP)-1 and pancreatic polypeptide hormone (PYY). GLP-1 is a peptide hormone of 30 amino acids, that originates from the proglucagon precursor protein and synthesized in the L cells of the small intestine. GLP-1 regulates both blood glucose and insulin levels [31,32]. The discovery of GLP-1 was facilitated by recombinant DNA techniques in the early 1970s [33]. The proglucagon gene was deduced through sequencing recombinant cDNA from messenger RNAs of various organisms, such as anglerfish, rats, hamsters, bovines, and humans. To elucidate the structure, the proglucagon cDNA was transfected into cells, followed by chromatography and radioimmunoassays to determine the peptides derived from this gene. While the larger proglucagon gene is present in several organs, GLP-1 and GLP-2 peptides are found in the human pancreas as a single large peptide, whereas in the small intestine, they exist as distinct smaller molecules due to tissue-specific processing [33] (Box 2). PYY belongs to the neuropeptide Y family of biologically active peptides that also includes pancreatic polypeptide (PP). PYY was first detected in porcine intestinal extracts due to the presence of an unusual COOH-terminal tyrosine amide structure. The samples were degraded with trypsin or thermolysin and measured using COOH-terminal amide determination [34]. Thin layer chromatography (TLC) was used to determine the released tyrosine amide and the peptide fragments were separated by HPLC. The PYY fraction was then subjected to trypsin and chymotrypsin degradation and the amino acid sequence was determined by Edman degradation [34]. Human PYY was identified by reverse phase chromatography, HPLC, and ion exchange FPLC followed by microsequencing, amino acid, and MS analysis of the fragments from human colon samples [35]. PYY regulates body weight by suppressing appetite while increasing energy expenditure. These two examples illustrate the rich diversity in processed peptides, with many more peptides likely to be discovered (see Box 3).

Box 2

Metabolic lessons from glucagon-derived peptides

Glucagon-derived peptides, such as GLP-1, GLP-2, gastrointestinal peptide (GIP), glicentin-related pancreatic peptide (GRPP), major proglucagon fragment (MPGF), and glucagon, are fascinating molecules that have dramatically expanded our understanding of metabolic regulation [31,73]. They are generated from the same precursor protein, proglucagon, but exhibit diverse functions through interactions with different receptors. This complexity unveils several key lessons that offer insights into metabolic regulation and endocrinology.

First, the tissue-specific expression of the enzymes proprotein convertase subtilisin/kexin (PCSK) that cleave a prohormone into different peptides is instrumental in determining their final form [74]. Different organs express specific PCSK enzymes, leading to selective production of peptides. PCSK1, mainly found in the brain and peripheral tissues, cleaves proglucagon into GRPP, GLP-1, and glucagon. PCSK1 variants are linked to human obesity [75]. PCSK2 is predominantly expressed in the brain and primarily cleaves proglucagon into GLP-2. In various tissues, PCSK3/Furin is involved in processing proglucagon to MPGF. The intestine-specific PCSK5 and PCSK7 enzymes cleave proglucagon into GLP-1, GLP-2, and glicentin. The tissue-specific nature of these enzymatic processes ensures that different peptides exert distinct biological effects based on their localized expression [73]. Second, the different glucagon-derived peptides exhibit their functions by binding to different G-protein coupled receptors (GPCRs) in targeted tissues. GLP-1 binds to GLP-1R, GIP engages the GIP receptor, GLP-2 interacts with GLP-2R, and glucagon interacts with glucagon receptor (GCGR) [32,73,76,77]. By investigating the diverse functions and interactions of these glucagon-derived peptides, and understanding their enzymatic processing by specific enzymes, we are uncovering new insights into how small peptides are generated from larger precursors. This knowledge opens the door to identifying other peptides that are processed by the same or similar enzymes, potentially revealing novel regulatory molecules similar to glucagon-derived peptides. Such discoveries may lead to the development of targeted therapies that can address various metabolic pathways, thereby enhancing our ability to treat different metabolic disorders.

Box 3

Challenges of omics approaches

Analyzing three untargeted metabolomics studies in the context of nonalcoholic fatty liver disease (NAFLD) reveals disparities in findings from similar omics approaches across research investigations. In one study involving Sprague–Dawley rats with NAFLD, untargeted metabolomics analysis of liver samples identified 4-hydroxy-6-eicosanone, 3-phosphoglyceric acid, 13-hydroxy-9-methoxy-10-oxo-11-octadecenoic acid, and taurochenodeoxycholate-3-sulfate, as metabolites positively correlated with NAFLD. Conversely, metabolites such as lysophosphatidylcholine [16:1 (9Z)], S-(9-hydroxy-PGA1)-glutathione, lysophosphatidylcholine [20:5 (5Z,8Z,11Z,14Z,17Z)], sphingomyelin (d18:1/14:0), nicotinamide adenine dinucleotide phosphate (NADP+), 5,10-methylene-tetrahydrofolate, folinic acid, N-lactoyl-glycine, and 6-hydroxy-5-methoxyindole glucuronide were found to be reduced in these animals [78].

In a separate untargeted metabolomics investigation involving plasma from nonobese individuals with NAFLD, distinct metabolites such as d-pantothenic acid, hypoxanthine, citric acid, citramalic acid, l-phenylalanine, glutamine, tramadol, 1,4-butynediol, dl-pyroglutamic acid, dehydroisoandrosterone sulfate (DHEA-S), 5-androsten-3-β,17-β-diol-3-sulfate, glyceric acid, d-ribose, and 5-α pregnan-3-α-,17-diol-20-one 3-sulfate were significantly elevated, while metabolites like β-hydroxymyristic acid, histamine-trifluoromethyl-toluidide, dl-lactic acid, and 3-methyl-2-oxopentanoic acid were notably lower in individuals with NAFLD [79].

In another untargeted metabolomics study of NAFLD, an increase in taurocholate, glutamyl dipeptides, carnitine, butyrylcarnitine, mannose, lactate, erythronate, glutamate, lysine, tyrosine, and isoleucine, was observed in plasma samples. Conversely, there was a decrease in metabolites such as glutathione, cysteine-glutathione, caprate (C10:0), 10-undecenoate (C11:1n1), and 1-oleoylglycerophosphocholine [80].

While the three aforementioned studies share common objectives, they arrive at varying conclusions, illustrating reproducibility challenges in the field of omics. These differences could be due to species differences, the dynamic nature of the metabolic secretome, variations in the samples and platforms used for analyses and differences in annotation tools used. Additionally, disparities may also arise from variations in analytical parameters employed, such as selection of cutoff values for quality assessment, methods for handling missing data, or criteria for distinguishing background noise from anticipated signals [81,82].

Improving the quality of omics studies can be achieved by adhering to established best practices for generating and analyzing data. These guidelines have been outlined by initiatives like the proteomics, lipidomics, and metabolomics standards initiatives [83–85]. Another aspect in elevating scientific rigor is the transparent documentation of methodologies and materials used. It is noteworthy that data sharing is a more common practice in genomics and proteomics research but is less prevalent in studies within the areas of lipidomics, peptidomics, and metabolomics. A positive stride in this direction could involve mandating data deposition by journals. As of now, while many journals encourage data sharing, not all of them require it [86]. Thus, collaborative efforts within the scientific community can facilitate the attainment of robust and reproducible findings in the omics era.

Peptidomics involves utilizing a variety of techniques to comprehensively analyze peptides within a biological sample. It encompasses two primary approaches: targeted and untargeted. Targeted peptidomics focuses on specific sets of peptides with known sequences and properties, and requires the availability of synthesized peptide standards that possess well-defined mass-to-charge (m/z) ratios. This targeted approach is particularly effective for well-characterized peptide groups. In contrast, untargeted peptidomics involves analyzing the entire peptide landscape within a sample without prior knowledge of the peptides present. This approach lacks synthesized peptide standards and instead aims to detect and analyze all m/z ratios present. Untargeted peptidomics is exploratory in nature, uncovering unannotated peptides and their potential roles in complex biological processes. Both processes encompass gathering and preparing samples, utilizing MS, acquiring, and processing data, identifying peptides, conducting quantitative analysis, and interpreting data. While like proteomics in many respects, one distinction lies in the fact that proteomic studies employ digestive enzymes to break down proteins into fragments for analysis, whereas peptidomics often analyzes peptides in their natural, undegraded state. A challenge encountered in peptidomics is the natural enzymatic breakdown of peptides, compounded by the presence of protein fragments generated by protease activity within the sample, and protease inhibitors might need to be added during sample collection. Another constraint stems from peptidomics relying on databases for identifying peptides. Unlike DNA and protein databases, those for peptides are still in their infancy, allowing to identify only a fraction of the total peptides present in a sample [36]. N-terminomics is an advancing branch of proteomics that detects all N-terminal peptides within a sample. It has applications in understanding the proteolytic machinery as well as the protease cleavage patterns for specific proteins. It is based on the bottom-up proteomics approach but requires an additional step for enrichment of N-terminal peptides [37]. In the future, the development of in vivo or genetic models for N-terminomics [38,39] would facilitate the discovery of peptides regulated by physiological and pathophysiological stimuli.

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1 Introduction

Proteins are linear- or branched-polymers made up of amino acids linked by peptide bonds and produced as the end-product of decoding process initiated from information in genetic material [113]. The biological functions of proteins are attributed to tertiary structures determined by the linear chain of the amino acids [130]. Secondary structures such as stretching, coiling, and spacing appear repeatedly and are formed by the interaction of backbone constituents. Large peptides may also adopt a three-dimensional tertiary conformational structure. Peptides of shorter length are produced by protein metabolism or by synthesis by recombinant methods and synthetic chemistry. The cut-off number of amino acids for the designation of a peptide is arbitrary, but it is typically set at below 50 residues [162], with insulin (55 amino acids) assigned as an exception. Endogenous peptides perform key biological functions, such as those performed by calcitonin, insulin, Glucagon-like Peptide 1, and amylin. Exogenous proteins and peptides are important as a source of essential nutrition and therapeutics. Peptides are regarded as excellent therapeutics due to their specificity for receptor targets and low off-target toxicity compared to small molecules [91]. Over 95% of marketed peptides are parenteral injections, with most having low stability in plasma and fast clearance [77], features that are unsuitable for non-parenteral routes of delivery.

Innovation in food technology, synthetic peptide chemistry, biotechnology, and formulation sciences has encouraged the use of food-sourced proteins and peptides in a range of products. The sources of proteins and peptides for the food and pharmaceutical sectors are shown in Table 1. Food-sourced proteins are a major source of hydrolysates, which contain bioactive peptides. The terms “food-derived bioactive peptides” and “protein hydrolysates” are often used interchangeably [30,31] and this terminology can often confuse. Bioactive peptides from food are defined as “food-derived peptides that exert, beyond their nutritional value, a physiological effect in humans” [116]. In this review, protein hydrolysates are defined as a source of releasable mixtures of bioactive peptides with potential health benefits. Protein hydrolysates are a complex mixture of oligopeptides, peptides, and free amino acids produced by enzymatic, chemical, or microbial hydrolysis of the original whole proteins [107,155]. Protein hydrolysates are the substrates for the subsequent production of bioactive peptides. The peptides released from hydrolysates can elicit pharmacological responses outside the GI tract upon oral ingestion only if they are absorbed sufficiently. Hydrolysates are cleaved during digestion by stomach pepsin and pancreatic serine proteases to produce bio-accessible low molecular weight (MW) peptides. These can reach the systemic circulation only if they are stable enough in the gastrointestinal GI tract and have sufficient epithelial permeability [35]. Bio-accessibility is defined as the quantity of a molecule released from its parent matrix in the intestine that is available for absorption [135]. It is a familiar term used in food sciences, whereas the pharmaceutical scientists are better acquainted with the parallel terms of dispersion and dissolution of oral solid dosage forms.

Table 1. Comparisons between therapeutic and dietary proteins.

Variables	Therapeutic use	Dietary use
Macromolecule	Proteins, polypeptides, peptides	Dietary proteins, protein hydrolysates, derived bioactive peptides
Sources	Rational, synthetic chemistry, recombinant production	Food ingredients
Mode of administration for systemic delivery	95% are parenteral (with a few FDA-approved examples for nasal, pulmonary and oral administration)	In food, nutraceuticals, and supplement formulations.
Evidence for oral BA is weak for most candidates.
Major industrial stakeholder	Biotech, Pharma	Food industry, nutraceutical companies, food supplement industry

Enzymatic hydrolysis can be considered the preferred industrial method to produce hydrolysates from food-sourced protein as it takes place under mild conditions with minimal side reactions [68]. However, there can still be some variability in the composition of the hydrolysate with enzymatic hydrolysis, which therefore requires systematic analysis of the constituents [43,94]. GI enzymes cause further digestion of the hydrolysate. The peptide structures produced by the actions of GI enzymes define the analytical range of breakdown products to be covered [120]. The endopeptidase, trypsin, catalyzes the breakdown of specific peptide chains and can be used for in vitro digestion assays of proteins to predict some of peptides arising from pancreatic secretions. Pancreatin is a digestive enzyme assay mixture which includes trypsin along with a set of exo- and endopeptidases, amylases, and lipases. The highest yield of peptides produced from a hempseed hydrolysate from the actions of pancreatin, trypsin and pepsin was in the order: pancreatic hydrolysate > tryptic hydrolysate > co-digested hydrolysate > peptic hydrolysate [5]. The composition of peptides produced in the GI tract from a hydrolysate guides the decision on the reference biomarker to be used for detection in the blood by MS. For instance, X-Hyp-Gly-type tripeptides from collagen/gelatin hydrolysates are resistant to peptidases and can reach plasma intact due to the presence of Hyp [150]. Therefore, an analytical method for identification of Hyp-containing tripeptides from plasma is essential. Another example of diversity of peptides generated by metabolism in the GI tract was an analysis of collagen hydrolysates with differing MW, which highlighted that their composition before and after digestion differs greatly and that the analysis of the components is complex [81].

To exert their biological activity, peptides need to pass through the intestinal epithelium, where brush border- and cytosolic enzymes further digest them. Hydrolysates must be assessed for digestibility and the subsequent release of bioactive peptides in relevant ex vivo intestinal models and in the GI lumen in vivo [174]. Subsequent intestinal permeability as individual peptides or combinations of peptides can be assessed in a range of in vitro and in vivo bioassays [128]. Food-derived protein hydrolysates also provide an alternative to enteral nutrition from elemental formulas containing individual amino acids, glucose polymers, and low fat [34]. Peptides within hydrolysates may improve cardiovascular, digestive, immune and nervous system function [107]. Hypoallergenic, antioxidant, antihypertensive, antidiabetic, anti-inflammatory, and attenuating muscle atrophy are useful associated functions [1,25,75,76,131,142,160].

Altered properties of food proteins result from industrial hydrolysis [154]. The properties of the hydrolyzed products to be assessed include solubility, gelation, foaming, and exposure of ionizable and hydrophobic functional groups. The industrial processes are application-oriented and are selected for either individual bioactive peptides or hydrolysates end-products. Hydrolysates go through subsequent fractionation and purification stages to produce a fraction for bioactivity testing [119]. If the intended end-product is a bioactive peptide, chromatographic techniques are applied to further fractionate those hydrolysate fractions with potential for displaying the highest bioactivity. The peptides in the fraction after chromatographic separation are then identified by mass spectrometry and synthesized.

Artificial intelligence (AI) can be used to identify novel bioactive peptides within natural proteomes from food sources. An AI approach using machine learning has successfully predicted bioactive peptides with a range of in vitro and in vivo activities such as anti-inflammatory, improvements in muscle physiology, and glucose regulation [25,27,32,36,75,76]. Prediction of specific bioactivities from structured and unstructured data involves searching scientific literature, patents, and public databases for bioactive peptide sequences [36]. The data collected is manually curated to ensure high quality standards, compiled into a training set of known bioactive peptides, which are used to build predictive architectures for bioactivity. Predicted peptides are further refined using a “predict-test-refine” feedback loop [27]. As iterations of the feedback loop continue, accuracy of successful prediction improves over several rounds. Specific predictors can be used alone or in combination to produce final predictions and to identify the most efficacious peptides within appropriate proteomes, as well as the enzymatic process required to create the hydrolysate. Protein BLAST (BLASTP) is then used to filter out known bioactive peptide homologs, resulting in a list of potential, novel bioactive peptides to be interrogated in bioassays [149].

The hydrolytic processes are early-stage steps in production and are either digestive or fermentative in nature (Fig. 1). Digestive enzymes including pepsin, chymotrypsin, and trypsin are often used to simulate GI digestion. The functional potency of hydrolysates can be compared to that of the peptides produced from them. For example, the IC50 of the initial hydrolysis product produced from Atlantic salmon skin gelatin to inhibit dipeptidyl peptidase IV in vitro was 1.35 mg/mL [95]. Fractionation of that hydrolysate led to isolation of the peptides, Gly-Pro-Ala-Glu and Gly-Pro-Gly-Ala, with much lower IC50 values of 49.6 and 41.9 μM respectively. This illustrates that potency can be improved by identifying bioactive peptides from the hydrolysate. Ultimately, the nutritional and potential health value of a hydrolysate is related to both the production methods that promote the release of individual peptides or mixtures as well as the oral bioavailability (BA). Oral BA is the fraction of the oral dose of the bioactive reaching the systemic circulation intact in its active form. It is the product remaining in plasma after permeating the intestinal epithelium while escaping intestinal metabolism and the liver first-pass effect [10].

Fig. 1. Industrial production of hydrolysates, GI metabolism to bioactive peptides, and intestinal epithelial permeation to reach the hepatic portal vein. Di- and tripeptides are substrates for the PepT1 carrier on small intestinal epithelia and transport is transcellular. Other low MW peptides with low numbers of residues can permeate tight junctions if they are sufficiently polar. Some larger proteins can also be absorbed to some extent by epithelial transcytotic pathways or through the targeting of receptor-mediated transcytotic pathways. Created using templates from BioRender.com.

The market for protein hydrolysates is expected to reach over $1 billion by 2026, with infant nutrition dominating demand. In the United States, protein hydrolysate-based baby formula milk currently accounts for 29% of sales [128]. The challenges associated with formulating oral hydrolysates include bitter tastes, hygroscopicity, limited shelf lives, and low oral BA. While wide-ranging health benefits are claimed for many hydrolysates, but evidence of the necessary oral BA of constituent bioactive peptides that would support such benefits is scarce in human studies [161]. Assessment of the possible health benefits of hydrolysates needs to be related to the oral BA values for the nominated constituent peptides [66].

What are the approaches to assessing systemic delivery of bioactive peptides found in protein hydrolysates by the oral route? In vitro assays of digestion, intestinal permeability, and intestinal/hepatic metabolism can help predict to an extent oral BA in humans. Determination of oral BA of hydrolysates focus on the effects of hydrolysis and the source of hydrolysates, as well as the need of using advanced peptidomics for plasma analysis of the permeated peptides.

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Peptides

Peptides represent another important class of antibiotics, with over 1000 known compounds (Felnagle et al., 2008; Glaser, 2013). Fifty-five peptides have uses, such as antibiotics, for chronic constipation, irritable bowel syndrome with constipation, as a pulmonary surfactant, for anemia due to chronic kidney disease in adults on dialysis, as a proteasome inhibitor for multiple myeloma, for short bowel syndrome, type 2 diabetes and for Cushing’s disease (Goldstein et al., 2010). The annual market is $15 billion and many are made by chemical synthesis.

The antimicrobial peptide group includes vancomycin, teicoplanin, streptogramins, the bacteriocins and other compounds. The glycopeptide vancomycin, the so-called “drug of last resort” for years, has been used to treat infections caused by antibiotic-resistant bacteria. However, with the emergence of VRE, other antibiotics, such as the lipoglycopeptide teicoplanin, daptomycin, linezolid, synercid, and televancin were developed. Telavancin, which was FDA-approved in 2009, is a vancomycin analog and contains a deacylaminoethyl moiety. It is more active versus C. difficile than vancomycin (Eisenstein et al., 2010). Teicoplanin is approved for vancomycin-resistant C. difficile. The advantages of telavancin over vancomycin and teicoplanin include better in vivo efficacy, better pharmacokinetics, greater safety, easier administration, and better activity against resistant staphylococci, streptococci, and enterococci. The cyclic lipopeptides include daptomycin and ramoplanin. Daptomycin is produced by Streptomyces roseosporus, and acts against Gram-positive bacteria, including VRE, MRSA, and penicillin-resistant S. pneumoniae. It is used for complicated skin and soft tissue infections, S. aureus bacteremia and endocarditis. It kills by binding irreversibly to the cell membrane, and disrupting plasma membrane function without penetrating into the cytoplasm. When it inserts its tail into the membrane, it causes efflux of K and membrane depolarization, thus destroying the ion concentration gradient. Thus, the target bacterium cannot make ATP or take in nutrients. Daptomycin was discovered at the Eli Lilly and Company laboratories in the 1970s but was thought to be toxic. It was then licensed to Cubist Pharmaceuticals Inc. (now part of Merck & Co.) and FDA-approved for Gram-positive skin infections in 2003 and for S. aureus bacteremia and endocarditis in 2006 (Mandal et al., 2014). The use of recombinant probiotics with antimicrobial peptides as a dual strategy to improve the immune response in immune-compromised patients has been reviewed by S.M. Mandal et al. (2014).

Acyl depsipeptides (ADEPs) can kill persisters which are rare, slow-growing bacteria, generally identical to the rest of the population (Conlon et al., 2013). They occur at 1 in 10,000 to a million cells of a rapidly growing population. ADEPs work well especially when combined with rifampicin, linezolid, or ciprofloxacin. ADEP action against Gram-positive bacteria involves activation of protein clpP, which is a subunit of the protease Clp. Clp is found in all bacteria and functions in protein-quality control and in the regulated degradation of specific proteins.

Additional glycopeptides include dalbavancin, used for the treatment of MRSA, as well as oritavancin. Via transcriptional engineering, production of teicoplanin by Actinoplanes teichomyceticus has been improved (Wang et al., 2014). Other improvements in teicoplanin production have been reported by L. Horbal et al. (2014). Teicoplanin is two to four times as active as the as vancomycin against methicillin-resistant and -sensitive S. aureus and is much less toxic.

Romidepsin is an interesting depsipeptide, which inhibits histone deacetylase and is produced by the Gram-negative bacterium Chromobacterium violaceum (VanderMolen et al., 2011). The organism was isolated from soil by Ueda et al. (1994) of the Fujisawa Corp. (now Astellas). It was further investigated by the US National Cancer Institute (NCI). The Fujisawa Co. carried out clinical trials in 2002 after the NCI confirmed the anticancer activity. It was licensed to Gloucester Pharmaceuticals in the United States of America in 2004, which was later acquired by the Celgene Corp. Romidepsin is active against cutaneous T-cell lymphoma (CTCL) and was approved by the FDA in 2009. In the United States of America, there are 1500 new cases and 500 deaths per year from CTCL.

The bacteriocins are produced by a number of bacteria (Maffioli et al., 2012). Comprising part of the bacteriocins are the lantibiotics, that is, peptides produced ribosomally and modified posttranslationally and the best known is nisin. Lantibiotics are gene-encoded, small (19–39 amino acid residues), and contain one or more lanthionine or methyl-lanthionine residues. They act against Gram-positive bacteria including MRSA, enterococci, and C. difficile but are inactive against Gram-negative bacteria. The latter is probably due to the outer membrane of Gram-negatives preventing the lantibiotic from entering the cell and reaching the outer side of the cytoplasmic membrane. In addition to nisins A and Z, the bacteriocins consist of subtilin, nukacin ISK-1, several lacticins, mersacidin, actagardine, cinnamycin, SapB, sublancin, gallidermin, plantaricin W, mutacin, streptins, bovicin, nukacin, and epidermins. A few are in clinical trials. Over 50 lantibiotics are produced by Gram-positive bacteria, such as Staphylococcus and Streptomyces. Despite the fact that Type A lantibiotics are effective in vivo with a low toxicity risk and potent, broad-spectrum activity (Asaduzzaman and Sonomoto, 2009), they have not been used frequently for treatment (Smith and Hillman, 2008). Their limited use is likely attributed to an expensive and time-consuming manufacturing process, along with high cost.

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Natriuretic Peptides

Peptides are biomolecules that consist of amino acids monomers and peptide (acid) bonds. The amino acid composition of these biomolecules is variable, and is considered an important factor that determines unique chemical and physical properties. The number of bound amino acids dictates the length of the peptides: dipeptides are the shortest peptides (2 amino acids and 1 single peptide bond), whereas polypeptides are long, continuous peptide chains. In contrast to biologically complex proteins, peptides have a rather simple biological composition and generally consist of ≤50 amino acids.

The family of NPs is a group of polypeptides that plays a pivotal role in maintaining the fluid homeostasis of the body by regulating intravascular volume, vascular homeostasis, and arterial pressure (Figure 1) (10). Recently, a role for the NPs in metabolic homeostasis has also been advanced (11–13). The NP system is highly preserved across species, and currently, the following 6 different NPs have been identified: ANP, BNP, C-type (CNP), D-type (DNP), ventricular NP (VNP), and the renal peptide, URO (14). NPs function as ligands for a set of transmembrane NP receptors (NPRs): CNP, evolutionarily the oldest of the NPs, mainly binds to the extracellular domain of the particulate guanylyl cyclase B receptor (pGC-B, NPR-B), whereas all other NPs bind to the transmembrane pGC-A (NPR-A) receptor (15). pGC-A receptors are expressed in various tissues, including heart, kidney, brain, adrenals, adipocytes, and vasculature (both arteries and veins) (16). pGC-B receptors are expressed in kidney, brain and veins, but less so in arteries (17).

Figure 1. Overview of Available Native and Designer NPs

ANP = A-type natriuretic peptide; BNP = B-type natriuretic peptide; CNP = C-type natriuretic peptide; DNP = D-type natriuretic peptide; NP = natriuretic peptide; pGC = particular guanylyl cyclase.

A third NPR, called NPR-C, or the clearance receptor (18), actively eliminates endogenous NPs from the circulation using hydrolysis (ranked from the greatest to the lowest degradation rate: VNP = ANP ≥ CNP > BNP = DNP). Studies also suggest a signaling role for NPR-C via modulation of cyclic adenosine monophosphate (19–21). Clearance of NPs is furthermore regulated by the enzyme NEP, which is widely expressed in endothelium and lung with the highest abundance in the kidney. CNP is the least resistant to NEP-mediated hydrolysis (ranked from greatest to lowest degradation rate: CNP > ANP > BNP > DNP) (3,10,15,16). The differences in local NPR expression, degradation and clearance rates, and NP-binding affinity cause all 6 NPs to have unique and NP-specific properties (15).

Importantly, binding of a NP to a NPR activates the membrane-bound pGC-A and pGC-B receptors, and induces a variety of autocrine, paracrine, and endocrine effects. Activated pGC receptors produce the second messenger cyclic guanosine monophosphate (cGMP) that in turn activates protein kinase G. cGMP can also be produced in a nitric oxide–dependent manner; its production is then regulated via activation of the soluble guanylyl cyclase pathway (22).

ANP and BNP are believed to be the most important in controlling body fluid and blood pressure homeostasis (23,24). ANP has renin-inhibiting properties, is a potent aldosterone inhibitor, and is an antagonist to the mineralocorticoid receptor. In addition, via alternative processing of the ANP precursor (pro-ANP) it also contributes to renal sodium and water handling via generation of URO (25–27). BNP has been identified as an NP highly relevant to HF, which is due to its natriuretic, renin-angiotensin-aldosterone system (RAAS) inhibitory, vasodilating, and lusitropic properties (28–30), as well as its robust performance as a HF diagnostic and prognostic biomarker (31–33). CNP is an autocrine and paracrine factor that currently has limited use as a therapeutic for HF, particularly because of its rapid enzymatic degradation and paucity of renal protective actions (29), although its potent antifibrotic actions provide a therapeutic opportunity (34). Moyes et al. (21) elegantly demonstrated a role for CNP in vascular homeostasis that may involve binding and activation of NPR-C (21). DNP is a unique NP that has only been isolated from the venom gland of the Green Mamba snake (35). Its function has not been entirely clarified. Currently, VNP expression has only been confirmed in the hearts of primitive ray-finned bony fish, in which it is responsible for the maintenance of fluid and salt homeostasis (17).

Overall, NPs possess a wide variety of properties that are of value in diagnosis, prognosis, and treatment of CV disease, especially HF and HTN. Despite current optimal therapies, the prognosis for HF remains poor, and 50% of patients die within 5 years after first hospitalization (36). The report of a relative deficiency or low bioavailability of NPs in HTN has also provided a therapeutic opportunity for use of designer NPs, especially in special populations, such as those with resistant HTN, in whom there remains a huge unmet therapeutic need (37). Development of new treatment regimens, with novel modes of action is therefore a high priority, and in this light, the concept of targeting (native) NPs for HF and HTN treatment strategies has attracted increased attention (15,38).

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3.3.1 Peptide/protein hormones

Peptide and protein hormones are composed of short and long amino acid chains, respectively. Synthesis occurs in the nucleus and cytoplasm of secretory cells via gene transcription, translation into the peptide chain, and finally post-translational modifications. After hormones are synthesized and packaged into secretory granules, neural and hormonal signals cause their secretion into extracellular space (Malandrino and Smith, 2018). Some peptide and protein hormones are secreted in pulsatile patterns that may have rhythmic changes that contribute to feedback mechanisms, such as the hypothalamic-pituitary-gonadotropin (HPG) axis, to control other hormone production in the body. Most peptide/protein hormones are soluble in aqueous solvents and do not need carrier proteins for transport throughout the blood stream. Therefore, they are susceptible to rapid protease degradation, leading to a short half-life and duration of action (Malandrino and Smith, 2018). TSH, a glycoprotein hormone that stimulates the production of T4, is susceptible to EDC influence. Perinatal exposure in rats to a glyphosate-based herbicide resulted in decreased TSH levels, decreased gene expression of deiodinases and transporters, and altered other genes regulated by thyroid hormones or involved in thyroid hormone metabolism in male offspring (de Souza et al., 2017). Epidemiological studies of agricultural workers have shown associations between pesticide exposure and levels of FSH and LH (Recio et al., 2005; Cremonese et al., 2017). Extensive evidence in animal studies also indicates that chlorpyrifos treatment alters levels of FSH and LH, in addition to steroid hormones (Li et al., 2019).

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4.08.7 Two Types of Peptide Synthesis

Many groups of a special class of metabolites, peptide antibiotics, are produced by an abundance of species of pathogenic as well as nonpathogenic prokaryotes such as unicellular bacteria and actinomycetes and many lower eukaryotes such as filamentous fungi.88,95,96 The unicellular bacteria and filamentous fungi, including a number of nonpathogens that inhabit soil and aquatic environments, have remarkably complex biosynthetic pathways,97 and are thought to be synthesized independently of genetic translations, through metabolic pathways.98

In contrast to this synthetic pathway, many organisms utilize an alternative pathway, in which the amino acid sequence in the peptide product is not determined directly at the gene level through nucleic acid interactions, but rather by the protein structure of the respective enzyme system.97 Thus, amino acids are incorporated into peptides during the process of biosynthesis of peptides, and the catalysis of peptide bond formation takes place by either one of the two peptide-forming strategies,99 that is, the ribosomal system or on a nonribosomal protein template.10 In both cases, the common feature is the activation of amino acids by the energy derived from hydrolysis of an ATP α–β linkage.100

4.08.7.1 Ribosomal Antibiotic Synthesis

In biological systems, the majority of cellular peptides and all proteins are of ribosomal origin.101 In the ribosomal system, amino acids are activated by aminoacyl-tRNA synthetases as tRNA esters and the peptide bond formation is initiated on the ribosome.100 The total number of 20 proteinogenic amino acids (21, including selenocysteine)102,103 is limited by the number of tRNA ligases which specifically recognize the amino acid and esterify the constituent amino acids to their cognate tRNAs.

For example, the bioactive lantibiotics represent highly stable multicyclic structures resulting from the processing of gene-encoded peptide precursors that have undergone extensive post-translational changes.65 The gene-coded antibiotic subtilin, which is synthesized as a precursor through transcription and translation, is a well-known example of ribosomal peptide antibiotic synthesis.5 Colicins and microcins are also plasmid-encoded and are of ribosomal origin.76 The type A lantibiotics, such as nisin of L. lactis, subtilisin of B. subtilis, epidermin of Staphylococcus epidermidis, gallidermin of S. gallinarum and type B lantibiotics, such as duramycins (including cinnamycin and ancovenin) of Acinomycetes are all ribosomally synthesized.

4.08.7.2 Nonribosomal Antibiotic Synthesis

In nonribosomal peptide antibiotic synthesis, amino acids are activated employing a multienzyme. Peptide bond formation takes place without ribosomal involvement.100 The synthesis of gramicidin S, tyrocidine, surfactin, and bacitracin are some of the best characterized examples of a nonribosomal biosynthetic system.

It is now accepted that nonribosomal peptide analysis, as shown in Figure 1, is an alternative means of manufacturing polypeptides, playing an important role in the production of small molecules of quite specialized function.10 A nonribosomally produced peptide can be composed of linear, cyclic and/or branched peptide chains, often containing amino acids not found in ribosomally synthesized products. Although structurally diverse, most of the nonribosomal peptides share a common mode of synthesis, the multienzyme thiotemplate mechanism facilitated by peptide synthetases. This alternative machinery for peptide synthesis, which depends on a protein template, seems to be necessary for introducing the required variability into the peptides synthesized.

Figure 1. Substrate activation in multienzymatic peptide synthesis system. Peptide synthetases are composed of highly conserved modules. They activate and covalently bind an amino acid specifically by the two-step-reaction shown in the box. Similarly to the ribosomal machinery, the multienzymatic system activates the cognate amino acids under ATP hydrolysis as amino acyl-adenylates. These relatively unstable intermediates are stabilized by thioesterification on the prosthetic 4′-phosphopantetheine groups of the synthetases. According to the multicarrier model, the growing peptide chain is transferred from one domain-linked cofactor to the next.

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2 Peptide Synthesis

2.1 Historical notes and future progress

Initial studies on synthetic peptides were based on the assumption that the amino acids constituting the protein molecule are linked together via amide bonds. Experimental research involved the condensation of aspartic acid and asparagine to a complex mixture of polymeric products, difficult to characterize, and with only a superficial resemblance to the protein molecules (Grimaux, 1882; Schaal, 1871).

The true beginning of peptide chemistry is considered to be the synthesis of benzoylglycylglycine as demonstrated by Curtius (1882) and that of glycylglycine by Fischer and Fourneau (1901).

The intensive work of these German chemists led, in the early 1900s, to the development of the first practical methods for peptide synthesis (Curtius, 1902; Fischer, 1903). The azide-coupling method was successfully employed by Curtius in the preparation of benzoylglycine oligopeptides, while the development of the acylchloride method enabled Fischer to synthesize several short peptides and complete in 1907 the synthesis of the octadecapeptide LGGGLGGGLGGGGGGGGG (Fischer, 1907).

Both approaches suffered from serious side reactions, such as racemization, the rearrangement of acyl azides and the practical impossibility to use amino acids with side-chain functionality. In addition, difficulties in obtaining enantiomerically pure amino acids and the lack of an easily removable group for the temporary protection of the amino group hampered, at that time and for years, any serious attempts to synthesize more complex peptides.

The introduction in 1932 of the carbobenzoxy group (Z) by Bergmann and Zervas (the abbreviated designation Z was chosen to honor Zervas in his lifetime) marks the origin of modern peptide synthesis (Bergman and Zervas, 1932).

The 30-year search for a satisfactory amino blocking group finally produced a protecting moiety readily removable without inducing breakdown of the labile peptide bond and with the important additional property to preserve the integrity of the amino acid chiral carbon upon “activation” (i.e., the attachment of a leaving group to the acyl carbon) of the carboxyl group.

Since then, peptide bond formation has been carried out by condensation of a Z-protected and activated amino acid with a second amino acid carrying a masked carboxyl group. The ready removal of Z allowed peptide chemists to assemble complex peptide sequences by addition of one residue at a time (“stepwise synthesis”) proceeding from the esterified or amidated C-terminal amino acid to the N-terminal amino acid or by the conjunction of protected sequences of amino acids (“fragment condensation”). The desired free peptides were obtained at the end of the synthesis by removal of Z and all other protecting groups (Fig. 5.2).

Figure 5.2. General scheme of the Boc-benzyl and Fmoc–t-butyl methods of stepwise solid-phase synthesis of linear peptides. Deprotection of the α-amino protecting group of the resin-linked growing peptide chain and coupling of the next protected amino acid constitute the repetitive cycle of the synthesis. X indicates the coupling reagent for amide bond formation. Both the α-amino group and the ε-amino group on lysine side chains can be modified for various purposes. The use of an insoluble support (resin) in a single reaction vessel allows for automation of the process. The crude peptide is obtained by resin-peptide acidolysis (liquid HF for Boc-benzyl and TFA for Fmoc–t-butyl methods) in the presence of scavengers. X: for example, diisopropylcarbodiimide–N-hydroxybenzotriazole; preformed symmetrical anhydrides; active esters; aminium salts such as HBTU [2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate], TBTU [2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumtetrafluoroborate], BOP [benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophosphate], PyBOP (benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate), or HCTU [2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate] in the presence of a tertiary base.

After about 20 years from the introduction of Z in peptide synthesis, du Vigneaud accomplished the formidable task of synthesizing the active hormone oxytocin, a nonapeptide amide with a disulfide bridge that forms a 20-atom ring containing six of the nine residues present in the molecule, with an overall yield well under 1% (du Vigneaud et al., 1954). For his landmark work, du Vigneaud was awarded the 1955 Nobel Prize in chemistry.

Between 1932 and 1963, Z as well as new amino protecting groups, preeminent among them the acid-labile tert-butyloxycarbonyl group (Boc) introduced in 1957 (Carpino, 1957; Mckay and Albertson, 1957) and more efficient coupling methods such as dicyclohexylcarbodiimide (DCCI) and active esters (Bodanszky, 1955; Schwyzer, 1953; Sheehan and Hess, 1955) were used for the synthesis of many small- and medium-size biologically active peptides in solution, the most spectacular example being the synthesis of the 39-residue porcine β-corticotropin adrenocorticotrophic hormone (ACTH) by Schwyzer and Sieber (1963).

Chinese scientists wishing to accomplish a task on a grandiose scale started in 1958 the synthesis of bovine insulin. The total synthesis of the complex molecule (two linear A and B chains with 21 and 30 aa residues, respectively, and three disulfide bridges between A6A11, A7B7, and A19B20) was fully accomplished in 1965 (Du et al., 1961; Kung et al., 1965). Crystalline bovine insulin was obtained by co-oxidation of separately prepared A and B chains and represents the first protein ever synthesized in vitro. In a recent personal reminiscence of the synthesis of insulin Zhang (2010), one of the many contributors, wrote: “when the final paper was published in 1966, the world scientific community was astonished, unable to imagine how such an important achievement was accomplished in China, a developing country not very advanced in science.”

The general strategy of peptide synthesis in solution includes: (1) selection of main-chain and side-chain protective groups, (2) choice of the activation method, (3) careful selection of segments to avoid or minimize racemization during segment condensation, and (4) solubility considerations. Solution-based methods offer the chemist the attractive opportunity to isolate and characterize the intermediates after each reaction. Perhaps the most frustrating problem faced during actual synthesis is the unpredictable solubility of the growing peptide chain. Furthermore, peptide synthesis in solution is a very labor intensive and time-consuming procedure requiring the skills of experienced peptide chemists.

In an effort to overcome many of the problems encountered in solution synthesis, Merrifield introduced the concept of stepwise peptide synthesis on an insoluble matrix by describing the first successful synthesis of the tetrapeptide Leu-Ala-Gly-Val at the Federation of American Societies for Experimental Biology (FASEB) in 1962 (Merrifield, 1962) with a full paper appearing in 1963 (Merrifield, 1963).

The historic 1963 paper represents a second major breakthrough in peptide chemistry after the introduction of the Bergman and Zervas carbobenzoxy protecting group.

In this approach, the C-terminal amino acid is bound covalently through its carboxyl group to an insoluble support and synthesis is then carried out by the successive addition of amino acids in the desired sequence. In contrast to solution synthesis, all of the intermediate steps of purification are reduced to simple resin filtration and washings. Being a rapid, cyclic process (deprotection, washing, coupling, washing, deprotection, etc.) occurring in a single vessel, the method has been automated early on in its implementation (Merrifield et al., 1966). At the end of the assembly, the peptide is released from the solid support and freed from the amino acid side-chain protecting groups (Fig. 5.2).

The original method of solid phase peptide synthesis (SPPS) was rapidly improved by Merrifield by replacement of NαZ with NαBoc-protecting groups and use of 2% cross-linked polystyrene resin in place of the nitropolystyrene support. A benzyl ester linkage to the cross-linked polystyrene remaining stable throughout the chain assembly, benzyl-based side-chain protection, in situ activation with DCCI and cleavage of the assembled peptides from the resin with HF secured a rapid stepwise synthesis of a series of biologically active peptides culminating with the total synthesis of a 124-residue enzyme with ribonuclease A activity in early 1969 (Gutte and Merrifield, 1969).

The difference in effort required between the SPPS and solution phase methods for the synthesis of ribonuclease A, attracted global attention in the scientific community. The synthesis based on the solid phase procedure was fully described in a single communication and involved only Merrifield and his first postdoctoral fellow Gutte; the synthesis in solution of ribonuclease S (which consists of residues 21–124 of ribonuclease A) was reported in detail by chemists working at the Merck laboratories in a series of communications, and involved over twenty people (Denkewalter et al., 1969).

Nevertheless, the concept of a multistep synthesis without the isolation and purification of intermediates was an anathema to most synthetic chemists. Wünsch et al. (1968) described the first synthesis in solution, by classical fragment condensation, of fully active and crystalline mammalian glucagon, a 29-aa peptide hormone secreted by the pancreas. The preparation of the hormone required the efforts of a large, skilled team over a period of several years. In 1971, he reviewed the synthesis of peptides and concluded that SPPS was “unsuitable for the satisfactory synthesis of higher natural peptides (with more than 15 aa residues)” (Wünsch, 1971).

This firm belief was demolished within a few years by Mojsov (a graduate student in the Merrifield laboratory) who prepared glucagon by SPPS in a few months and, after improving the synthetic protocol, obtained crystalline glucagon in an overall yield higher than that reported by Wünsch (Mojsov and Merrifield, 1984). By 2006, over 200 analogues (agonists and antagonists) of glucagon have been synthesized by SPPS (Unson, 2007).

As a consequence of these differences in effort and after reexamination and improvement of virtually every aspect of SPPS in Merrifield's laboratory (Merrifield, 1993), the solid phase methodology has become the more widely adopted. In 1984, Merrifield was awarded the Nobel Prize in chemistry for his invention.

The advantages of SPPS had become apparent to all but a few determined skeptics. The synthesis in solution of pure, crystalline ribonuclease A with full enzymatic activity achieved in 1981 by Yajima and Fujii marks the end of an era and represents the culmination of classical peptide synthesis in solution (Yajima and Fujii, 1981).

Sheppard and his group in Cambridge (UK) have taken the Merrifield basic concept and remodeled it very successfully with alternative chemistry (Atherton and Sheppard, 1989). The Sheppard method of SPPS has rapidly gained in popularity for the milder conditions of the synthesis protocol which uses the Fmoc group (9-fluorenylmethoxycarbonyl) for α-amino protection and ether, ester and urethane derivatives based on t-butanol for side-chain (ω) protection. The α/ω-protection is absolutely orthogonal as opposed to nearly orthogonal of the standard Merrifield approach. Fmoc is usually removed with piperidine in N,N-dimethylformamide. The final resin cleavage with simultaneous removal of mild, acid-labile side-chains protecting groups can be carried out under much gentler conditions with trifluoroacetic acid (TFA) in the presence of suitable scavengers (Fig. 5.2).

Improved chemistries, solid supports and protocols, incessantly developed over the years for both the Boc-benzyl and Fmoc–t-butyl methods, have been utilized in the stepwise assembly of many long peptides and small proteins containing 50–90 and even more than 100 aa residues (Nilsson et al., 2005). For example, the Boc method has been used by Kent and coworkers for the stepwise assembly of both L and D enantiomers of the 99-residue HIV-1 protease (1–99). The natural 198-residue HIV-1 protease obtained by the association of the monomers into an active, noncovalently bound homodimer is considered a landmark achievement in SPPS (Schneider and Kent, 1988). The stepwise synthesis (on an automatic peptide synthesizer) of the 99-residue monomer has been systematically optimized to reach a rate of synthesis of 75 residues per day and the assembly of the protected peptide in 33 h! All but two couplings proceeded in > 99.8%. As discussed by Kent, an extraordinary synthetic efficiency of each coupling step (99.9%) is necessary for preparing a 100-residue peptide in 90% overall yield; a 97.0% efficiency provides only a 5% overall yield (Kent, 1980).

Peptides control many of the processes involved in the immune system response. Since the late-1970, synthetic peptides obtained by stepwise SPPS have become increasingly important in almost all areas of immunological research. Designing new peptide vaccine candidates is one of the hottest challenges of the twenty-first century science. Vaccines based on chemically synthesized peptides offer implicit safety due to the absence of such contaminants as DNA and other viral or cellular debris. Since small peptides can hardly reproduce the secondary and tertiary structures of native protein molecules, today the best approach consists in chemically preparing by SPPS long, linear peptides (from about 50 to 100 aa residues or more), which are safe and immunogenic when formulated with various adjuvants and suitable for eliciting an antibody response in humans (Corradin et al., 2004, 2010; Olugbile et al., 2010). These long peptides can mimic native structural epitopes when isolated from the context of the whole protein and can be identified in the proteomes by using bioinformatics approaches (Corradin et al., 2007, 2010). Recently, this structure-based bioinformatics/peptide synthesis approach has been successfully applied to the identification of new malaria vaccine candidates (Olugbile et al., 2009, 2011; Villard et al., 2007).

Despite the significant gain made in advancing the technology of SPPS, most proteins cannot be synthesized by the stepwise assembly of amino acids. One successful approach used to access a protein with 238 residues (Aequorea green fluorescent protein) consisted in the assembly in solution of 26 protected peptide segments prepared by SPPS (Nishiuchi et al., 1998).

Currently, the most common approach for the total chemical synthesis of proteins is native chemical ligation (NCL) (Schnolzer and Kent, 1992). In this approach, two fragments are initially assembled stepwise on the solid phase, one with an N-terminal Cys residue and the other with a C-terminal thioacid (Fig. 5.3). After resin cleavage, side chain deblocking and purification, the thioacid is converted to a thioester, allowing the two fragments to react in aqueous solution (transthioesterification) to form a thioester bond between them. Spontaneous rearrangement of this bond results in a native amide bond between the fragments with regeneration of the free sulfhydryl on the Cys residue (Fig. 5.3). The process can then be repeated to link additional peptide fragments. The cumulative effects of stepwise synthetic errors are minimized due to the coupling of highly purified fragments in water. Examples of proteins prepared via NCL are the 116-residues anticoagulant microprotein S (Hackeng et al., 2000) and a 166-residues polyethylene glycol-modified erythropoiesis protein (Kochendoerfer et al., 2003).

Figure 5.3. Chemical ligation. The method involves a thioester-mediated condensation of two unprotected peptide segments in aqueous solution at neutral pH resulting in the ligation of the terminal cysteine of one peptide segment to the other via a native amide bond.

Long peptides and proteins can be also synthesized by solid phase NCL as described in 1999 by Kent and Dawson (Brik et al., 2000; Canne et al., 1999). This protocol allows to assemble the polypeptide chains either in the N → C or in the C → N direction. Human group V secretory phospholipase A2 (118 aa and six disulfide bonds) has been successfully prepared by three solid phase NCL reactions using purified, water-soluble peptide segments ranging from 25 to 33 aa residues (Brik et al., 2000).

An additional method for the synthesis of proteins, the expressed protein ligation (EPL) introduced in 1998 by Muir and Evans, is an amalgamation of SPPS (chemistry) and ribosomal peptide synthesis (Evans et al., 1998; Muir et al., 1998). Recombinant DNA technology is used to produce large protein fragments that after purification and characterization are ligated by NCL to peptide segments prepared by SPPS. The size of proteins that are accessible by this semi synthetic technology is thus greatly increased. EPL has been used extensively for protein structure–function analyses and to produce a functional single-chain antibody with a C-terminal thioester (Sydor et al., 2002).

Chemical peptide synthesis has enormously progressed from the time of Curtius and Fischer. As Fischer envisaged in his Nobel Prize Lecture on December 12, 1902, a century later enzymes and other proteins with full biological activity are generally considered accessible targets for synthetic chemistry (Kent, 2009).

2.2 Why to choose synthetic peptides as vaccines

The main reasons to use SPPS about 20 years ago for the discovery and development of malaria vaccines were the versatility of peptide synthesis, the speed of production and purity of the final product devoid of DNA or unrelated proteins, and the difficulty to express malarial recombinant proteins, especially the CSP from P. falciparum. These considerations not only are still valid today but increased availability of special amino acid reagents, resins, catalysts, and state of the art instrumentations has rendered peptide synthesis even more attractive and cheaper (see Section 2.1). Since the peptide length we felt comfortable to synthesize was around 100 aa residues, we were quite careful to choose domains whose structure as it appears in the native protein could be predicted or was already determined. Using our chain assembly and folding protocols biologically active, commercially available chemokines were produced (Thierry et al., 2001; Verdini et al., 2008);

Mimicry of the correct antigen structure is of extreme importance for antigens from the sporozoite or the erythrocytic stage against which specific antibodies need to exert the protective function. On the other hand, for liver stage antigens against which elicitation of a specific T cell response is needed, linear peptides of limited length (8–15 aa residues) are required. These short peptide segments can be used as such or inserted colinearly into a long peptide chain with interposed nonimmunogenic spacers NH(CH2CH2O)nCO to prevent the formation of neoepitopes (Olugbile et al., 2011).

One can argue that domains or multi subunit polyepitopes of 100 or more aa may not be sufficient to confer protection against a complicated disease such as malaria, and only larger proteins may activate the immune response to obtain a protective response. Our approach is that properly targeted epitopes or domains can indeed be effective in preventing malaria. A well-documented example is the spectacular success of monoclonal antibodies against a variety of diseases including cancer. In addition, using well-defined domains it is possible to avoid the presence of polymorphic regions, which are present in most of the antigens developed in the last 25 years and, possibly, the consequence of their failure. Furthermore, once nonpolymorphic domains are identified, recombinant DNA technology can be used to produce multivalent constructs. In addition, large constructs can also be produced by peptide synthesis; polypeptides of 150 and more aa have been prepared in our laboratory (Perlaza et al., 2001, unpublished data).

We have always adopted the Fmoc–t-butyl solid phase stepwise method to synthesize long peptides. This procedure does not require a highly skilled operator and is easy to learn. Even a high school student, well trained and supervised, is able to accurately follow the synthetic protocol. Purification of the crude material is, generally, first performed through a size exclusion chromatography, usually a Sephadex G50 resin, which very effectively eliminates smaller truncated peptides which occur most of the time in the first 10–40 aa. This size exclusion chromatography usually yields a good product (≥ 70% purity) to be used for T-cell proliferation and ELISA. Better quality products (> 90%) for animal model immunization can be obtained by reverse phase high performance liquid chromatography (RP-HPLC) (Regnier, 1983).

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24.8 Self-Assessment Questions

1.

A peptide chain (referred to as PC) has the following sequence: Lys-Gly-Ser-Arg-Ser-Phe-Gly

a.

Which amino acid is present at the N terminus?

b.

Which amino acid is present at the C terminus?

2.

Given the sequence of PC above, indicate where the serine proteases, chymotrypsin, and trypsin are expected to cleave.

3.

What would be expected in the presence of PC if there was a mutation at His57 of trypsin? Why?

4.

Cytochrome P450 enzymes typically catalyze the hydroxylation of several substrates; however, to achieve this it must break the strong double bond of the oxygen molecule.

a.

How is this achieved?

b.

What catalytic strategy do the CYP enzymes utilize?

5.

Currently, the pharmaceutical industry is dominated by small molecular drugs with singular targets. (a) What are the advantages and disadvantages of this drug design? (b) How can protein-based drugs overcome the disadvantages of small molecular drugs? (c) What are the disadvantages of protein-based drugs?

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Detection

Peptides are detected in amounts ranging from 100 to 1000 ng by absorbance at between 200 and 230 nm. Detection of peptides containing aromatic amino acids (phenylalanine, tyrosine, and tryptophan) may be performed at 254 nm; tyrosine- or tryptophan-containing peptides can also be detected at 280 nm. Rapid-scanning ultraviolet/visible detectors based on diode array technology have proved an extremely useful aid in identifying peptides that contain aromatic residues. Wavelengths of the spectrum maxima, convexity interval, and wavelength of the second derivative spectrum maxima, all over in the 190–340 nm range, allow identification of the aromatic amino acids that form the peptides, as well as identification of HPLC coeluted compounds such as the cinnamic derivatives detected in the peptide fractions of wines.

Since numerous solvents and even other sample components may absorb light in the range 200–230 nm, formation of derivatives detectable at higher wavelengths that are more specific for given chromophores is commonly employed. To this end, phenyl isothiocyanate and dansyl chloride are frequently used in peptide analysis as derivatizing agents. Peptides may also be detected using a fluorescence detector to detect either the natural fluorescence of certain amino acids in the peptide sequence or the fluorescence of derivatives formed artificially using such reagents as fluorescamine and o-phthalaldehyde. Other reagents used lately for derivatization of peptides are naphthalene-2,3-dicarboxaldehyde, 3-(4-carboxy-benzoyl) 2-quinolinecarboxaldehyde, and 6-aminoquinoyl-N-hydroxysuccimidylcarbamate.

Following the significant advances made in liquid chromatography/mass spectroscopy coupling in recent years, mass spectrometry has turned into a detection system that holds great promise for peptide analysis, since it is capable of furnishing structural information and quantitative data that are otherwise difficult to obtain.

The greatest advance in the analysis of peptides has been the coupling with spectrometric techniques, both for identification and for characterization. Their high accuracy, sensitivity, and, in some instances, tolerance to solvents make mass spectrometers ideal detectors for analyses of HPLC-separated peptides. Presently, electrospray ionization and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry – soft ionization methods – provide the suitable means to determine accurate mass on peptides and proteins with high sensitivity (subpicomol range). Furthermore, both methods are suitable for the creation of peptide ions for analysis using tandem mass spectrometry and collision-induced dissociation methods. Mass spectrometry in conjunction with database searching plays an increasingly important role in the characterization of food peptides.

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