**2. Solid-phase peptide synthesis (SSPS)**

The specific characters of peptides (high bioactivity, high specificity, and low toxicity) have made them attractive therapeutic agents. The synthesis of the peptides may provide sufficient material to enable further studies and to determine the structure-activity relationships or may provide discovery of new analogues with improved properties [1–7]. The peptides are able to synthesize in three methods: in a solution medium, on a solid support, or as a combination of the solid and the solution synthesis. Although peptide synthesis is often carried out by the solid-phase

**4**

*Peptide Synthesis*

**References**

[1] Grant GA. Synthetic Peptides: A User's Guide. New York: Oxford

[2] Varkey JT, Rajasekharan Pillai VN. Merrifield resin and newly developed 1,6-hexanediol diacrylate

[3] Johnson ECB, Kent SBH. Insights into the mechanism and catalysis of the native chemical ligation reaction. Journal of the American Chemical Society. 2006;**128**:6640-6646

[4] Hamada Y, Matsumoto H, Yamaguchi S, et al. Water-soluble prodrugs of dipeptide HIV protease inhibitors based on *O*→*N* intramolecular acyl migration: Design, synthesis and kinetic study. Bioorganic & Medicinal Chemistry.

[5] Hamada Y, Kiso Y. New directions for protease inhibitors directed drug discovery. Biopolymers.

[6] Copolovici DM, Langel K, Eriste E, Langel U. Cell-penetrating peptides: Design, synthesis, and applications. ACS Nano. 2014;**8**(3):1972-1994

[7] Epand RM, Vogel HJ. Diversity of antimicrobial peptides and their mechanisms of action. Biochimica et Biophysica Acta (BBA) - Biomembranes.

[8] Wilson SS, Wiens ME, Smith JG. Antiviral mechanisms of human defensins. Journal of Molecular Biology.

resin for solid phase peptide synthesis—A comparative study. Journal of Applied Polymer Science.

University Press; 2002

1999;**71**:1933-1939

2004;**12**:159-170

2016;**106**:563-579

1999;**1462**(1-2):11-28

2013;**425**(24):4965-4980

method, the solution method was preferred by the pharmaceutical companies in the 1970s and 1980s [8]. In the solution medium synthesis method, except for the reversible protection of the N-amino group of the first amino acid or fragment, the orthogonal protection of carboxyl groups of the second amino acid or the fragment is needed. On the solid-phase method, the synthesis is carried out on a solid support, also called a resin. The peptide is separated from the resin after each amino acid in the peptide sequence is sequentially bound. In the solid-phase technique, the peptide that bounded to the insoluble resin is separated without any significant loss during the washing or filtration of the resin. All reactions are carried out in a single reaction vessel, and possible losses are prevented during processes such as exchange and transfer of reaction vessels [9]. Another one is hybrid synthesis which is the composition of the solution and SPPS methods. Herein, the peptide to be synthesized is obtained after the condensation from a solution of two or more suitable peptide sequences, which are obtained mainly by solid-phase synthesis [10].

The principle of peptide synthesis in the solid phase is quite simple. The peptide chain is attached to the stable solid phase. The peptide sequence remains bound on this resin for the duration of the synthesis. During the synthesis that includes deprotection, activation, and coupling steps, other soluble chemicals outside the resin beads are removed by filtration and washing. In the last stage, the desired product is separated from the solid phase by cleavage procedure. Purification and characterization procedures are carried out in the free solution of the desired product [11]. In SPPS, although there are two main strategies, the Boc approach requires the use of hard acids such as HF; thus the majority of synthetic peptides are nowadays synthesized by using Fmoc chemistry. The most important advantage of the Fmoc method compared to Boc strategy is that it does not require corrosive acids such as TFA during the synthesis cycle, and thus the synthesis can be automated. In this method, the N-terminal amine groups, the acid-labile side-chain protecting groups, and the linkers forming the protecting group of the C-terminal amino acid are protected by the base-labile Fmoc group [12, 13]. Solid support materials (resins), linkers, protecting groups, and cleavage cocktail solutions are general components of the solid-phase peptide synthesis.

*The resin* is a spherical polymer ball bearing the active groups to which the first amino acid (indicates the first amino acid at the C-terminus of the sequence) of the sequence to be synthesized can be attached. The solid supports used in the solid-phase peptide synthesis must be stable against mechanical mixing, various temperature conditions, and different types of solvents, must have a narrow range of resin bead sizes, and must have high swelling properties (should be able to swell up to 5–6 times in DCM, dimethylformamide (DMF), etc.); thus the reagents can reach the active regions of the resin. The solid support material is usually used in a size of about 100–200 mesh or 200–400 mesh, and the peptide chain is extended by the addition of amino acids on each other on the resin. The most common classes of resins are the classic polystyrene (PS) resins (crosslinked with 1% of divinylbenzene (DVB)), the PS-functionalized polyethylene glycol (PEG) resins, pure cross-linked PEG resins, and polystyrene cross-linked with 1,6-hexanediol diacrylate resin (PS-HDODA) [14–20]. The solid support used in the synthesis can be of two kinds depending on whether or not the initial amino acid of the sequence to be synthesized is bound to the resin. Accordingly, there are two types of resin, either pre-loaded or unloaded, but pre-loaded resin is generally preferred. The pre-loaded resins differ depending on whether they are lowly loaded or highly loaded. When the highly loaded resin is used, aggregation of the peptide sequences growing on the resin increases, and this will lead to the deletion of an amino acid which is an undesirable situation; therefore, the use of lowly loaded resins is common [19].

**7**

*Synthesis and Applications of Synthetic Peptides DOI: http://dx.doi.org/10.5772/intechopen.85486*

on the type of the linker on the resin beads [13, 19, 21, 22].

protected by Fmoc or Boc [23–33].

gentle shaking for 1–3 h [12, 22, 34, 35].

In order to bind the first amino acid to the resins, chemical structures containing functional group(s) called *linker* are attached. The number of these active groups on the resin (substitution) is important in calculating the theoretical yield of a peptide to be synthesized and in determining the amount of chemicals (deprotection, activation, coupling) required for the synthesis of a peptide. Depending on the functional group of the C-terminal end of the target peptide sequence, peptide acids or peptide amides can be synthesized by means of an ester or an amide bond, respectively. While the peptide acids are obtained using 4-benzyloxybenzyl alcohol (Wang), 2-(4-hydroxymethyl) phenoxyacetic acid (HMPA), superacid-sensitive resin (SASRIN), and 2-chlorotrityl linkers, the peptide amides rink amide, 4-methyl benzhydryl amide (MBHA), and Sieber and primary amide (PAL) linkers are required (**Figure 1**) [13]. After the cleavage procedure, C-terminus of the peptide sequence remains ▬COOH or ▬NH2 depending

The reaction between the activated carboxylic group of one amino acid and the amino group of another amino acid is needed for a peptide linkage. In order to prevent side reactions from occurring, the remaining functional groups of each amino acid must be appropriately protected. For this purpose, many *protecting groups* are used to prevent the reaction of different types of functional groups. Because each amino acid contains different groups in its side chains, the protecting groups which prevent these groups from reacting vary according to the amino acid and used methodology (Fmoc or Boc chemistry). Some amino acids have not any functional groups to react in the side groups; only alpha-amino groups of these amino acids are

*Cleavage* is one of the most important steps in solid-phase peptide synthesis. Exposing a resin to a cleavage cocktail is not an easy reaction; it brings a series of competitive reactions. Unless appropriate reagents and reaction conditions are selected in the cleavage process, the desired peptide can be irreversibly modified or damaged. Also, since the DMF used in the solid-phase peptide synthesis can inhibit TFA acidolysis, before starting the cleavage, the peptide-bound resin should be washed with DCM to remove all DMF. The goal of cleavage is to split the synthesized peptide sequence from the resin while also removing amino acid side-chain protecting groups. For this process, strong acids such as anhydrous HF, TFMSA, or TMSOTf are used in Boc chemistry, while cocktails containing TFA are preferred in the Fmoc process [12, 22, 34]. Additional substances such as EDT, phenol, and thioanisole called *scavengers* are put into the cleavage medium to prevent the cationic products occurring during the cleavage process to attack the amino acids that are having electron-rich side chains such as Trp, Tyr, and Met. Considering the amino acid types involved in the resin-linked peptide sequence synthesized by the solid-phase peptide synthesis method, it is decided to which type and how much scavenger add to the cleavage cocktail. For example, when there is one or more Trp in the peptide sequence, using EDT in the cleavage will substantially protect Trp against oxidation. Typically, the peptide-bound resin is treated using 95% TFA with

The crude peptides synthesized by solid-phase peptide synthesis with the deprotection, activation, and coupling and cleavage steps are mostly analyzed on analytical HPLC using C18 columns at a concentration of 1 mg/mL with gradient elution method of water (0.1% TFA) and acetonitrile (0.1% TFA) as mobile phase. If purification of the synthesized peptide is required according to the application to be used, preparative HPLC is used. For the analysis, the peptide should be dissolved in a minimum volume of 0.1% TFA in water, and the acetonitrile and water gradient elution is adjusted according to the polarity of the peptide [12, 22, 35–37]. The molecular weight of the synthesized peptide is confirmed by mass spectroscopy. The most commonly used methods are LC-MS, MALDI-TOF, and LC-QTOF-MS. Systems

*Synthesis and Applications of Synthetic Peptides DOI: http://dx.doi.org/10.5772/intechopen.85486*

*Peptide Synthesis*

method, the solution method was preferred by the pharmaceutical companies in the 1970s and 1980s [8]. In the solution medium synthesis method, except for the reversible protection of the N-amino group of the first amino acid or fragment, the orthogonal protection of carboxyl groups of the second amino acid or the fragment is needed. On the solid-phase method, the synthesis is carried out on a solid support, also called a resin. The peptide is separated from the resin after each amino acid in the peptide sequence is sequentially bound. In the solid-phase technique, the peptide that bounded to the insoluble resin is separated without any significant loss during the washing or filtration of the resin. All reactions are carried out in a single reaction vessel, and possible losses are prevented during processes such as exchange and transfer of reaction vessels [9]. Another one is hybrid synthesis which is the composition of the solution and SPPS methods. Herein, the peptide to be synthesized is obtained after the condensation from a solution of two or more suitable peptide sequences, which are obtained mainly by solid-phase synthesis [10].

The principle of peptide synthesis in the solid phase is quite simple. The peptide chain is attached to the stable solid phase. The peptide sequence remains bound on this resin for the duration of the synthesis. During the synthesis that includes deprotection, activation, and coupling steps, other soluble chemicals outside the resin beads are removed by filtration and washing. In the last stage, the desired product is separated from the solid phase by cleavage procedure. Purification and characterization procedures are carried out in the free solution of the desired product [11]. In SPPS, although there are two main strategies, the Boc approach requires the use of hard acids such as HF; thus the majority of synthetic peptides are nowadays synthesized by using Fmoc chemistry. The most important advantage of the Fmoc method compared to Boc strategy is that it does not require corrosive acids such as TFA during the synthesis cycle, and thus the synthesis can be automated. In this method, the N-terminal amine groups, the acid-labile side-chain protecting groups, and the linkers forming the protecting group of the C-terminal amino acid are protected by the base-labile Fmoc group [12, 13]. Solid support materials (resins), linkers, protecting groups, and cleavage cocktail solutions are general

*The resin* is a spherical polymer ball bearing the active groups to which the first amino acid (indicates the first amino acid at the C-terminus of the sequence) of the sequence to be synthesized can be attached. The solid supports used in the solid-phase peptide synthesis must be stable against mechanical mixing, various temperature conditions, and different types of solvents, must have a narrow range of resin bead sizes, and must have high swelling properties (should be able to swell up to 5–6 times in DCM, dimethylformamide (DMF), etc.); thus the reagents can reach the active regions of the resin. The solid support material is usually used in a size of about 100–200 mesh or 200–400 mesh, and the peptide chain is extended by the addition of amino acids on each other on the resin. The most common classes of resins are the classic polystyrene (PS) resins (crosslinked with 1% of divinylbenzene (DVB)), the PS-functionalized polyethylene glycol (PEG) resins, pure cross-linked PEG resins, and polystyrene cross-linked with 1,6-hexanediol diacrylate resin (PS-HDODA) [14–20]. The solid support used in the synthesis can be of two kinds depending on whether or not the initial amino acid of the sequence to be synthesized is bound to the resin. Accordingly, there are two types of resin, either pre-loaded or unloaded, but pre-loaded resin is generally preferred. The pre-loaded resins differ depending on whether they are lowly loaded or highly loaded. When the highly loaded resin is used, aggregation of the peptide sequences growing on the resin increases, and this will lead to the deletion of an amino acid which is an undesirable situation; therefore, the use of

components of the solid-phase peptide synthesis.

lowly loaded resins is common [19].

**6**

In order to bind the first amino acid to the resins, chemical structures containing functional group(s) called *linker* are attached. The number of these active groups on the resin (substitution) is important in calculating the theoretical yield of a peptide to be synthesized and in determining the amount of chemicals (deprotection, activation, coupling) required for the synthesis of a peptide. Depending on the functional group of the C-terminal end of the target peptide sequence, peptide acids or peptide amides can be synthesized by means of an ester or an amide bond, respectively. While the peptide acids are obtained using 4-benzyloxybenzyl alcohol (Wang), 2-(4-hydroxymethyl) phenoxyacetic acid (HMPA), superacid-sensitive resin (SASRIN), and 2-chlorotrityl linkers, the peptide amides rink amide, 4-methyl benzhydryl amide (MBHA), and Sieber and primary amide (PAL) linkers are required (**Figure 1**) [13]. After the cleavage procedure, C-terminus of the peptide sequence remains ▬COOH or ▬NH2 depending on the type of the linker on the resin beads [13, 19, 21, 22].

The reaction between the activated carboxylic group of one amino acid and the amino group of another amino acid is needed for a peptide linkage. In order to prevent side reactions from occurring, the remaining functional groups of each amino acid must be appropriately protected. For this purpose, many *protecting groups* are used to prevent the reaction of different types of functional groups. Because each amino acid contains different groups in its side chains, the protecting groups which prevent these groups from reacting vary according to the amino acid and used methodology (Fmoc or Boc chemistry). Some amino acids have not any functional groups to react in the side groups; only alpha-amino groups of these amino acids are protected by Fmoc or Boc [23–33].

*Cleavage* is one of the most important steps in solid-phase peptide synthesis. Exposing a resin to a cleavage cocktail is not an easy reaction; it brings a series of competitive reactions. Unless appropriate reagents and reaction conditions are selected in the cleavage process, the desired peptide can be irreversibly modified or damaged. Also, since the DMF used in the solid-phase peptide synthesis can inhibit TFA acidolysis, before starting the cleavage, the peptide-bound resin should be washed with DCM to remove all DMF. The goal of cleavage is to split the synthesized peptide sequence from the resin while also removing amino acid side-chain protecting groups. For this process, strong acids such as anhydrous HF, TFMSA, or TMSOTf are used in Boc chemistry, while cocktails containing TFA are preferred in the Fmoc process [12, 22, 34]. Additional substances such as EDT, phenol, and thioanisole called *scavengers* are put into the cleavage medium to prevent the cationic products occurring during the cleavage process to attack the amino acids that are having electron-rich side chains such as Trp, Tyr, and Met. Considering the amino acid types involved in the resin-linked peptide sequence synthesized by the solid-phase peptide synthesis method, it is decided to which type and how much scavenger add to the cleavage cocktail. For example, when there is one or more Trp in the peptide sequence, using EDT in the cleavage will substantially protect Trp against oxidation. Typically, the peptide-bound resin is treated using 95% TFA with gentle shaking for 1–3 h [12, 22, 34, 35].

The crude peptides synthesized by solid-phase peptide synthesis with the deprotection, activation, and coupling and cleavage steps are mostly analyzed on analytical HPLC using C18 columns at a concentration of 1 mg/mL with gradient elution method of water (0.1% TFA) and acetonitrile (0.1% TFA) as mobile phase. If purification of the synthesized peptide is required according to the application to be used, preparative HPLC is used. For the analysis, the peptide should be dissolved in a minimum volume of 0.1% TFA in water, and the acetonitrile and water gradient elution is adjusted according to the polarity of the peptide [12, 22, 35–37]. The molecular weight of the synthesized peptide is confirmed by mass spectroscopy. The most commonly used methods are LC-MS, MALDI-TOF, and LC-QTOF-MS. Systems

with such a combination of chromatography and spectroscopy are useful for the complete characterization of synthetic peptides. While chromatographic analyses allow us to have knowledge about the peptides' purity, the molecular weight of the peptides is determined by MS analyses. It is also found in some systems that determine the amino acid sequence of the peptide molecule like amino acid analyzer and LC-MS-MS. Information on structural properties of peptides can also be obtained by NMR and FTIR methods. With NMR and FTIR, the structures of the peptides are further elucidated via specific binding and functional groups [12, 35]. Moreover circular dichroism (CD) spectroscopy gives information about the conformation and secondary structures of polypeptides. This technique can be used to distinguish between random coil, alpha-helix, or beta-sheet structures [38, 39].

**9**

*Synthesis and Applications of Synthetic Peptides DOI: http://dx.doi.org/10.5772/intechopen.85486*

The increase in studies on peptide vaccines in recent years shows that peptide vaccines will be an important part of new-generation vaccine systems. Vaccines are indispensable in protecting human and community health in terms of reducing infectious diseases, disability, and deaths and, most importantly, eliminating and eradicating the long-term disease. The development of the vaccine, which began with the observations of Edward Jenner at the end of the eighteenth century, has significantly reduced the number of infections and diseases until now [40–42]. Traditional vaccines (live-attenuated or inactivated vaccines) have been widely used over the last hundred years to develop effective vaccines against many diseases. However, the use of this technology is a source of concern because of the problems encountered in the preparation and use of such vaccines. The disadvantages of classical vaccines such as the increased need for safety, high cost in the production of large-scale vaccine preparations, high genetic variability of pathogens, side effects, and risk of leak of the disease agent have necessitated research to develop new technological vaccines [43, 44]. The basis for new vaccine technologies is the production of high-purity subunit vaccines that contain a small part of the pathogen necessary to generate an immune response. Subunit vaccines can be composed of polysaccharides and proteins or their peptide fragments. Synthetic peptide vaccines consist of 20–30 amino acids containing the specific epitope of a corresponding antigen against various diseases. There is no limitation from cancer diseases to allergies in diseases targeted by peptide vaccines. The advantages of peptide vaccines have enabled peptides to be preferred in vaccine technology [45–48]. Although many peptide vaccine studies continue, there is no approved peptide vaccine for human use. According to the database of clinical studies maintained on ClinicalTrials.gov, in the search on the topic of peptide vaccine until mid-December 2018, 374 clinical trials in phase I, 272 clinical trials in phase II, 14 clinical trials in phase III, and 2 clinical trials in phase IV have been found [47, 49]. Furthermore, it is reported in the literature that a veterinary peptide vaccine against canine parvovirus, which is targeted against animal diseases, is effective [50]. Advantages involved in the choice of peptide-based vaccine can be summarized as follows: Peptide-based vaccines are more reliable than classical vaccines because they are produced using chemical synthetic approaches almost exclusively. It does not produce any unwanted immune response as it does not contain biological material. Also, this situation eliminates the risk of biological contamination. The rapid, easy, inexpensive, and high-purity synthesis of peptides by microwave-assisted solid-phase peptide synthesis methods is an important advantage for the use of peptides in vaccine systems. These synthesized peptide antigens can be fully characterized by analytical methods. "Cold chain" is not required generally during transport and storage of peptide vaccines. Peptide vaccines have high stability and are typically water-soluble. Allergic and/ or reactogenic effects are avoided by the usage of peptide vaccines. Peptide vaccines can be customized using the peptide epitope of one or more antigens [46, 51, 52]. Despite all its advantages, there are some limitations of peptide-based vaccines. The peptides alone are generally weakly immunogenic, have poor release properties and are easily undergo enzymatic degradation. These disadvantages can be overcome using adjuvants and delivery systems. Adjuvants, derived from the Latin word "adjuvare," which means "to help," play a crucial role to the elicitation of a strong immune response to an antigen [49, 53–56]. The first step in the development of peptide vaccines is the identification of the structure of peptide epitopes which will provide effective and long-term immunity. After the synthesis and characterization of specific antigens to be determined, the appropriate adjuvant and delivery system selection is carried out [43, 46]. Currently, instead of classical adjuvants, the use of

**3. Peptide vaccines**

**Figure 1.** *Some of the fundamental linkers used on the Fmoc-based SPSS [22].*
