**3. Solid-phase peptide synthesis (SPPS)**

A historical overview of peptide chemistry from T. Curtius (who achieved the first synthesis of peptide in 1882) and Fischer (who synthesized the first dipeptide in 1901) to M. Bergmann and L. Zervas is first in presenting the Solid-Phase peptide synthesis. Next, the fundamentals of peptide synthesis with a focus on SPPS by R. B. Merrifeld are described. Although the peptides can be synthesized in three methods: in a solution medium, on a solid support, or as a combination of the solid and the solution synthesis, this chapter emphasizes an overview of peptide synthesis giving importance on SPPS. Currently, most of the peptides for research,

vaccination or therapeutic drugs for cancer and brain diseases are synthesized by SPPS methods. Successful peptide synthesis depends on the appropriate selection of suitable resins, linkers, amino acid derivatives and coupling reagents, as well as the side chain (de) protection and cleavage conditions, and the correct synthesis of the assay. In the SPPS method, the solid support is attached at the end of the first amino acid-COOH at the carboxyl end a polymeric support insoluble in the newly formed peptide chain is referred to as resin. A covalent binding step that binds the resin is important for the reaction [24]. The peptides may be gradually joined between the C and N terminus using N-protected amino acids. The Nα protecting group (Boc) is unstable in the presence of intermediate acid (trifluoroacetic acid; TFA), the side chain protecting benzyl (Bzl) based groups and the peptide/resin linkage are stable in the presence of intermediate acid and are variable in the presence of strong acid (HF). Fmoc group is important for solid-phase applications. Fmoc-based strategies are also available, and hydroxymethylphenoxy-based binders are used to add peptide to the resin with t-butyl (tBu) based side chain protection [25]. The solid phase peptide synthesis method consists of three basic steps. According to this, deprotection of the carboxyl group activation and peptide bond formation (Coupling). Following this procedure, the final deprotection of the last added amino acid is removed and the N- terminal is released. Cleavage and deprotection of the resinbound peptide from the solid support [26].

The stepwise representation of solid phase peptide synthesis is illustrated in **Figure 2**. The starting amino acid masked by a non-persistent protecting group at the N-α terminus is loaded from the C-terminus to the resin. A semi-permanent protection group can also be used to mask the side chain if necessary (**Figure 3**, Step 1). The synthesis of the peptide, repeated deprotection of the N-α-transient protecting group, and binding of the next protected amino acid (**Figure 3**, Step 3).

#### **Figure 2.**

*The antigens and immunomodulators that can be used for inclusion in liposomes; it is shown in different strategies depending on the target and structure of the molecule [28].*

**29**

**Figure 3.**

**Table 1.**

After the last amino acid is loaded (**Figure 3**, Step 4), the peptide is separated from

*Synthetic cycle and important reagents with time and conditions in microwave-assisted SPPS [27].*

**Synthetic cycle Reagents Time & conditions**

1–5 min (70°C Fmoc and Boc)

50–70°C

(Fmoc)

Couplings Amino acids, HBTU/HATU/HOBt/HOAt/DIC, DIPEA 5–15 min

The development of microwave-assisted solid-phase peptide synthesis has been developed by the synthesis of linear and complex peptide sequences and long

the resin support and the Fmoc or Boc groups are removed [25].

Deprotections Trifluoroacetic acid (Boc) and 20% piperidine in DMF

*Stepwise representation of solid phase peptide synthesis [25].*

*New Generation Peptide-Based Vaccine Prototype DOI: http://dx.doi.org/10.5772/intechopen.89115*

*New Generation Peptide-Based Vaccine Prototype DOI: http://dx.doi.org/10.5772/intechopen.89115*

*Current and Future Aspects of Nanomedicine*

bound peptide from the solid support [26].

vaccination or therapeutic drugs for cancer and brain diseases are synthesized by SPPS methods. Successful peptide synthesis depends on the appropriate selection of suitable resins, linkers, amino acid derivatives and coupling reagents, as well as the side chain (de) protection and cleavage conditions, and the correct synthesis of the assay. In the SPPS method, the solid support is attached at the end of the first amino acid-COOH at the carboxyl end a polymeric support insoluble in the newly formed peptide chain is referred to as resin. A covalent binding step that binds the resin is important for the reaction [24]. The peptides may be gradually joined between the C and N terminus using N-protected amino acids. The Nα protecting group (Boc) is unstable in the presence of intermediate acid (trifluoroacetic acid; TFA), the side chain protecting benzyl (Bzl) based groups and the peptide/resin linkage are stable in the presence of intermediate acid and are variable in the presence of strong acid (HF). Fmoc group is important for solid-phase applications. Fmoc-based strategies are also available, and hydroxymethylphenoxy-based binders are used to add peptide to the resin with t-butyl (tBu) based side chain protection [25]. The solid phase peptide synthesis method consists of three basic steps. According to this, deprotection of the carboxyl group activation and peptide bond formation (Coupling). Following this procedure, the final deprotection of the last added amino acid is removed and the N- terminal is released. Cleavage and deprotection of the resin-

The stepwise representation of solid phase peptide synthesis is illustrated in **Figure 2**. The starting amino acid masked by a non-persistent protecting group at the N-α terminus is loaded from the C-terminus to the resin. A semi-permanent protection group can also be used to mask the side chain if necessary (**Figure 3**, Step 1). The synthesis of the peptide, repeated deprotection of the N-α-transient protecting group, and binding of the next protected amino acid (**Figure 3**, Step 3).

*The antigens and immunomodulators that can be used for inclusion in liposomes; it is shown in different* 

*strategies depending on the target and structure of the molecule [28].*

**28**

**Figure 2.**

#### **Figure 3.**

*Stepwise representation of solid phase peptide synthesis [25].*


#### **Table 1.**

*Synthetic cycle and important reagents with time and conditions in microwave-assisted SPPS [27].*

After the last amino acid is loaded (**Figure 3**, Step 4), the peptide is separated from the resin support and the Fmoc or Boc groups are removed [25].

The development of microwave-assisted solid-phase peptide synthesis has been developed by the synthesis of linear and complex peptide sequences and long peptide sequences in a shorter time and high throughput. Time and temperature conditions, reagents and synthesis cycle for microwave assisted SPPS with using Boc or Fmoc are represented below in **Table 1**. The disadvantage of this technique could be the cost of resin (the binding procedure of the first binding amino acid to the resin in peptide synthesis can requires different and complex processes) and equipment [27, 29].
