3.2.2 Permanent protecting groups

But Side chain protecting groups are known as permanent protecting groups as they remain at all the multiple cycles of chemical treatment during the synthesis phase and they can be removed only on treatment with strong acids after synthesis is complete (Figure 4).

Peptide synthesis involves coupling of carboxyl group and the incoming amino acid to the N-terminus of the growing peptide chain. It is a step wise method and also a cyclic process. It involves the following steps.

1.Blocking of amino group of amino acid by N-terminal protecting groups

Prior to protein synthesis, individual amino acids are reacted with these protecting groups. Two common N-terminal protecting groups are tertbutoxycarbonyl (Boc) and 9-fluorenylmethoxycarbonyl (Fmoc), and each group has distinct characteristics that determine their use (Table 1; Figure 5).

2.Blocking of carbonyl group of amino acid by C-terminal protecting groups

Similarly the carbonyl group of amino acid is protected by C-terminal protecting group. The C-terminal protecting group depends on the type of peptide

> synthesis used. For liquid-phase peptide synthesis C-terminus of the first amino acid is protected by chemical reagent while in solid-phase peptide synthesis solid support (resin) acts as the protecting group for the C-terminal amino acid

Once peptide synthesis is completed, all the protecting groups are removed from

carbodiimides like dicyclohexylcarbodiimide (DCC) or diisopropylcarbodiimide (DIC). These coupling reagents respond with the carboxyl gathering to shape an exceptionally receptive O-acylisourea moderate that is immediately dislodged by nucleophilic assault from the deprotected essential amino gathering on the

3.Coupling of the protected amino acids to form a peptide

Fmoc strategy (Wang resin) and Boc strategy (Merrifield resin).

S. No Fmoc Boc

and derivatives

3 Acid-sensitive peptides

DOI: http://dx.doi.org/10.5772/intechopen.90184

1 Routine synthesis Requires special equipment 2 Relatively safe Potentially dangerous

Determination of Substrate Specificity of the Purified Novel Plant Cysteine Protease Solanain…

4 Frequent aggregation Moderate aggregation 5 TFA final deprotection HF final deprotection

Base-labile peptides

the nascent peptides. First C-terminal carboxylic acid is activated using

(Figure 6).

Figure 6.

69

Amino acid functional group protection.

Figure 5.

Table 1.

Difference between Fmoc and Boc.

#### Figure 4.

Solid-phase synthesis of a dipeptide victimization associate degree (amine-functionalized) organic compound. The N-terminal protective cluster (PG) will be Fmoc or Boc, reckoning on the protective cluster theme used (see below). The amino alkanoic acid facet chains (R1, R2, etc.) are orthogonally protected (not shown).

Determination of Substrate Specificity of the Purified Novel Plant Cysteine Protease Solanain… DOI: http://dx.doi.org/10.5772/intechopen.90184


#### Table 1.

• Temporary protecting groups

• Permanent protecting groups

N-terminal ends of amino acids are protected by groups called 'temporary' protecting groups as they are easily removed to allow peptide bond formation.

But Side chain protecting groups are known as permanent protecting groups as they remain at all the multiple cycles of chemical treatment during the synthesis phase and they can be removed only on treatment with strong acids after synthesis

Peptide synthesis involves coupling of carboxyl group and the incoming amino acid to the N-terminus of the growing peptide chain. It is a step wise method and

1.Blocking of amino group of amino acid by N-terminal protecting groups Prior to protein synthesis, individual amino acids are reacted with these protecting groups. Two common N-terminal protecting groups are tert-

butoxycarbonyl (Boc) and 9-fluorenylmethoxycarbonyl (Fmoc), and each group

2.Blocking of carbonyl group of amino acid by C-terminal protecting groups

Solid-phase synthesis of a dipeptide victimization associate degree (amine-functionalized) organic compound. The N-terminal protective cluster (PG) will be Fmoc or Boc, reckoning on the protective cluster theme used (see below). The amino alkanoic acid facet chains (R1, R2, etc.) are orthogonally protected (not shown).

Similarly the carbonyl group of amino acid is protected by C-terminal protecting group. The C-terminal protecting group depends on the type of peptide

has distinct characteristics that determine their use (Table 1; Figure 5).

3.2.1 Temporary protecting groups

Peptide Synthesis

3.2.2 Permanent protecting groups

also a cyclic process. It involves the following steps.

is complete (Figure 4).

Figure 4.

68

Difference between Fmoc and Boc.

#### Figure 5. Fmoc strategy (Wang resin) and Boc strategy (Merrifield resin).

synthesis used. For liquid-phase peptide synthesis C-terminus of the first amino acid is protected by chemical reagent while in solid-phase peptide synthesis solid support (resin) acts as the protecting group for the C-terminal amino acid (Figure 6).

#### 3.Coupling of the protected amino acids to form a peptide

Once peptide synthesis is completed, all the protecting groups are removed from the nascent peptides. First C-terminal carboxylic acid is activated using carbodiimides like dicyclohexylcarbodiimide (DCC) or diisopropylcarbodiimide (DIC). These coupling reagents respond with the carboxyl gathering to shape an exceptionally receptive O-acylisourea moderate that is immediately dislodged by nucleophilic assault from the deprotected essential amino gathering on the

Figure 6. Amino acid functional group protection.

these free reactive species. This cycle of deprotection and coupling is repeated. The remaining protecting groups are cleaved by acidolysis, using; strong acids such as hydrogen fluoride (HF), hydrogen bromide (HBr) or trifluoromethane sulphonic

Determination of Substrate Specificity of the Purified Novel Plant Cysteine Protease Solanain…

Peptides are purified by the following techniques namely Ion exchange chroma-

tography (IEC), Partition chromatography, Size-exclusion chromatography, Reverse-phase chromatography (RPC) and High-performance liquid chromatogra-

The Peptide synthesis (Figure 9) holds varied applications including

• Synthetic peptides are used to study enzyme-substrate interactions within important enzyme classes such as kinases and proteases, which play a crucial

• The development of epitope-specific antibodies against pathogenic proteins.

• The study of protein functions and the identification and characterization of

For about a century, the enzymes that play the central role in the degradation of proteins by hydrolysing peptide bonds have been known as 'proteases' and the term protease is therefore equivalent to 'peptide hydrolase'. They hold first place in the

acid (TFMSA) (Figure 8).

DOI: http://dx.doi.org/10.5772/intechopen.90184

5. Peptide purification

6. Applications of synthetic peptides

role in cell signalling.

proteins.

7. Proteases

Polypeptide formation.

Figure 9.

71

phy (HPLC).

#### Figure 7.

Diagram of peptide synthesis. Peptide bond formation between the deprotected N-terminus of the first amino acid and the activated C-terminus of the incoming amino acid. This cycle of deprotection and coupling is repeated until the full-length peptide is formed.

N-end of the developing peptide chain to frame the beginning peptide security. To affirm total coupling a test is performed called Kaiser Test (Figure 7).

4.Deblocking the amino group of amino acid

Removal of specific protecting groups from the newly added amino acid immediately after coupling to allow the next incoming amino acid to bind to the growing peptide chain is called deprotection. Boc is removed using moderately strong acid such as trifluoroacetic acid (TFA) while Fmoc is a base-labile protecting group that is removed with a mild base such as piperidine.
