This section aims to address several questions:

• • How is a peptide made in the lab?
  • What are the key steps involved in chemical synthesis?
  • How are amino acids prone to undergo side reactions?

How are peptides made in the lab?

Chemical synthesis of peptides

Peptides are made in the lab through chemical synthesis by linking amino acids in a specific sequence. This process involves protecting reactive groups on the amino acids to prevent unwanted reactions and adding them one at a time to build the desired chain. Once the sequence is complete, the protecting groups are removed, and the peptide is purified to ensure it meets the required specifications. This method allows precise control over the peptide’s structure and any desired modifications.

A key objective of chemical synthesis is to modify the biological activity of peptides, with specific goals such as:

• Enhancing desired activity: Many biologically active peptides exhibit multiple activities; chemical synthesis can amplify the desired one.
• Improving peptide stability: Ensures longer activity in the body, addressing the challenge of rapid breakdown and limited oral bioavailability.
• Reducing side effects: Minimizes unintended effects for safer applications.
• Understanding structure-activity relationships: Helps in linking peptide structure to its function.
• Creating novel peptides: Allows synthesis of peptides not found in nature.

These capabilities make chemical synthesis an essential tool in peptide research and drug development.

The advantages of chemical synthesis

Chemical synthesis offers several advantages:

• Scalable production: Peptides can be synthesized at any scale.
• Simplified process for short peptides: Chemical methods are easier than biological approaches for shorter sequences.
• BSE/TSE-free products: Ensures safety and purity.
• Customisation: Modifications to amino acid sequences of known peptides are feasible.
• Introduction of peptide modifications: Enables tailored functionality.

What are the key steps involved in chemical synthesis?

Synthesis of a dipeptide from two different amino acids

Example l-Alanyl-l-phenylalanine (H-Ala-Phe-OH): Two amino acids react with each other to form the dipeptide, with the release of water.

This does not happen spontaneously.  The amino acids must be activated for the reaction to occur. For this purpose, “coupling reagents” have been developed. These generate more reactive derivatives of the amino acids and remove the water that is formed concomitantly from the system.

However, if a coupling reagent is simply added to a solution of alanine and phenylalanine, it results in a mixture of the four possible dipeptides; tri-and longer peptides can be produced as well:

To obtain the desired dipeptide H-Ala-Phe-OH and not a mixture, two “protecting groups” are needed. These must be stable under the conditions of the coupling reaction but be readily cleaved in a separate step after coupling is complete. First, the amino group of alanine and the acid group of phenylalanine must be blocked:

H-Ala-OH → X-Ala-OH
H-Phe-OH → H-Phe-OY

Now, only the acid group of alanine and the amino group of phenylalanine are available to react:

X-Ala-OH + H-Phe-OY → only X-Ala-Phe-OY 

Finally, the protecting groups are removed under the same conditions:

X-Ala–Phe-OY → H-Ala-Phe-OH 

Now, let us choose X and Y so that X is removed under conditions in which OY is retained. This allows to synthesize longer peptides:

Thus, a peptide can be elongated to any desired length. X is called a temporary protection group because it is only used for the coupling step. Y is a “permanent” protection group. It must be stable enough to endure all coupling and X-cleavage steps. However, it must also be removable in the last step without damaging the peptide. A peptide always has two different groups at the two ends and therefore a “direction”. The amino group at one end of a peptide is known as the “N-terminus”, whereas the carboxyl group at the other end is known as the “C-terminus”.

In chemical synthesis, the peptide is constructed from the C-terminus in the direction of the N-terminus. The terminal carboxyl group must therefore be protected throughout the entire synthesis. Irrespective of the system of notation, representing a peptide sequence, one always starts at the N-terminus (see Table 5).

Notation	Explanation
H-Gly-Leu-Ala-Phe-OH	Bachem notation, here the N- and C-terminals are easily to recognize, both are "free" and not modified.
Gly-Leu-Ala-Phe	Terminal groups are only explicitly stated if they are not free
GLAF	1-letter code. Terminal groups are only explicitly stated if they are not free
Glycyl-L- leucyl-L-ala-nyl-L-phenyl- alanine	"Written out in full" especially common for shorter peptides

Table 5: Several peptide notation systems

 

Amino protecting groups

Table 6 lists the protecting groups most used by Bachem for the α-amino group. These can be cleaved using a variety of methods. Some examples of Nα-protected amino acid derivatives are as follows:

• Z-Leu-OH 
• Boc-Ala-OH 
• Fmoc-Phe-OH 

Abbr.	Chemical name	Cleavage reagent
Fmoc	9-Fluorenylmethoxycarbonyl	Piperidine
Boc	t-Butoxycarbonyl	Trifluoroacetic acid
Z	Benzyloxy carbonyl	Catalytic hydrogenation (hydrogen/palladium)

Table 6: Common temporary amino protecting groups

 

Many Boc- or Z-amino acid derivatives are available as (di)cyclohexyl ammonium (DCHA or CHA) salts. Salt formation improves the storage stability of acid-sensitive derivatives because amino acids are quite strong acids (stronger than acetic acid). Furthermore, some Boc- or Z-amino acid derivatives are oils and can only be obtained in a solid, crystalline form as salts. Solid products are much easier to handle than oils, e.g., when weighing.

Benzyloxy carbonyl (Z or Cbz) is the oldest usable N^α protecting group for amino acids [1]. Its development brought about the start of modern peptide synthesis and hence also the abbreviation Z in honor of Leonidas Zervas. The Z group is still commonly used today to protect amino moieties, in both peptide synthesis and organic synthesis in general.

[1] Bergmann M, Zervas L: Über ein allgemeines Verfahren der Peptid-Synthese, Berichte der deutschen chemischen Gesellschaft 1932. https://doi.org/10.1002/cber.19320650722

Abbr.	Chemical name	Cleavage reagent
OtBu	t-Butyl ester	Trifluoroacetic acid
OBzl	Benzyl ester	Catalytic hydrogenation (hydrogen/palladium)
OMe	Methyl ester	Bases (OMe used only for C-terminus)

Table 7: Common permanent acid protecting groups for the C-terminus and the side chains of Asp and Glu

 

Derivative	Use
Fmoc-Arg(Pbf)-OH	Standard derivative of Arg for Fmoc-SPPS, must be activated
Fmoc-Asn(Trt)-OPfp	Standard derivative of Asn for Fmoc-SPPS, already activated as the “active
	ester” and can be used directly
Z-Glu(OtBu)-OSu	Derivative of Glu for solution synthesis, already activated
Boc-Ser(tBu)-OH	Only for N-terminal serine! Nα and sidechain protecting groups are cleaved under the same conditions

Table 8: Examples of protected amino acid derivatives from Bachem.

 

Protecting groups for carboxyl moieties

In contrast to the α-amino group, the C-terminal carboxylic acid group must be protected throughout the entire synthesis, usually as an ester. The most used protecting groups are listed in Table 7.

Some examples of carboxyl-protected amino acid esters are as follows:

• H-Ala-OtBu·HCl
• H-Val-OMe·HCl
• H-Glu(OBzl)-OBzl·p-tosylate

Esters of amino acids are only stable for storage when prepared as salts with strong acids, such as hydrochloric acid (hydrochloride, HCl) or p-toluenesulfonic acid (p-tosylate). These salts are also more readily available in crystalline form than the free amino acid esters.

The choice of amino and carboxyl protecting groups also depends on the peptide synthesis method. The two most important methods — solid-phase peptide synthesis (SPPS) and liquid-phase peptide synthesis (LPPS) are described in the next subsection. In SPPS, the C-terminal protecting group is an insoluble polymer.

Amino Acids Prone to Undergo Side Reactions

Sidechain protecting groups

The almost infinite variety of amino acid side chains and their profound influence on the properties of peptides have already been mentioned. However, some sensitive side-chain groups must be permanently protected during peptide synthesis. Some examples can be seen in Table 9.

Let us now look at the 20 proteinogenic amino acids in more detail. Ala, Leu, and Phe have side chains that do not react under the conditions of peptide synthesis. This is also the case for Ile, Pro, and Val.

Other side-chain functions may be undesirably altered during peptide synthesis if they are not protected. The carboxyl groups of Asp and Glu and the amino group of Lys can participate in unintended coupling reactions, producing a mixture of peptides with incorrect links, such as branched peptides.

Other side reactions may occur with the side-chain functions of the following amino acids: Cys, Ser, Thr, Tyr, Arg  His, Asn, Gln, Trp, Met (protection only required in special cases).

The side-chain functions of Lys and Cys must be permanently protected during peptide assembly. For the rest of the amino acids carrying a third functionality, the need for permanent side-chain protection depends on the synthesis method and peptide sequence.

The most common sidechain protecting groups are listed in Table 9 and their abbreviations are explained in Table 10. During solution synthesis, the side chains of Asn, Gln, His, Thr, Trp, and Tyr do not necessarily have to be protected. In the case of Arg, salt formation with a strong acid such as HCl may suffice. Solution synthesis also places less demand on the stability of protecting groups than SPPS.

Generally, amino acid derivatives for peptide synthesis should be available in microcrystalline form (no amorphous materials or oils) and readily soluble. Fmoc derivatives should dissolve easily and rapidly in DMF (or NMP) — this is important for fully automated Fmoc-SPPS.

Amino acid	Protecting group	Main use
Arg	Pbf, Pmc	Fmoc-SPPS
Arg	Salt formation	Solution synthesis
Asp, Glu	OtBu	Fmoc-SPPS, solution synthesis
Asp, Glu	OBzl	Solution synthesis
Asn, Gln	Trt, Mtt	Fmoc-SPPS
Cys	Trt	Fmoc-SPPS, solution synthesis
Cys	Acm	SPPS, solution synthesis
His	Trt	Fmoc-SPPS
Lys	Boc	Fmoc-SPPS, solution synthesis
Lys	Z	Solution synthesis
Ser	tBu	Fmoc-SPPS, solution synthesis
Ser	Bzl	Solution synthesis
Thr, Tyr	tBu	Fmoc-SPPS
Trp	Boc	Fmoc-SPPS

Table 9: Common sidechain protecting groups

 

Abbr.	Meaning	Main use
Acm	Acetamidomethyl	Cys; Fmoc-SPPS, solution synthesis
Boc	t-Butyloxycarbonyl	Lys and Trp; Fmoc-SPPS
tBu	t-Butyl	Ser, Thr, and Tyr; Fmoc-SPPS
Bzl	Benzyl	Ser, Thr, and Tyr; solution synthesis
OtBu	t-Butyl ester	Asp and Glu; Fmoc-SPPS
OBzl	Benzyl ester	Asp and Glu; solution synthesis
Pbf	2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl	Arg; Fmoc-SPPS
Pmc	2,2,5,7,8-Pentamethylchroman-6-sulfonyl	Arg; Fmoc-SPPS
Trt	Trityl (triphenylmethyl)	Cys, His, Asn, and Gln; Fmoc-SPPS
Z	Benzyloxycarbonyl	Lys; solution synthesis

Table 10: Abbreviations for common sidechain protecting groups

 

 

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