The main reaction in the synthesis of peptides is the coupling between two amino acids.

Amino acids are coupled with each other by losing a water molecule to form the desired dipeptide. This process does not happen by itself, they have to be activated for the reaction to occur. For this purpose, coupling reagents are made. They convert amino acids into more reactive derivatives and remove water that is simultaneously formed in the system.

The following example is an example of a dipeptide, consisting of two different amino acids:

L-Alanyl-L-phenylalanine (H-Ala-Phe-OH)

However, if a coupling reagent is added to a mixture of alanine and phenylalanine, it leads to the synthesis of four different dipeptides. Peptides of three amino acids and longer may also be produced. Two protecting groups are required to obtain the desired dipeptide H-Ala-Phe-OH and not a mixture of peptides. They should not be separated from the amino acid under the conditions of the coupling reaction, but should be easily separated in a separate step after the coupling.

The amino group of alanine must be protected:

H-Ala-OH → X-Ala-OH

And also the acidic group of phenylalanine must be protected:

H-Phe-OH → H-Phe-OY

Now only the acidic group of alanine can react with the amino group of phenylalanine:

X-Ala-OH + H-Phe-OY → X-Ala-Phe-OY

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

X-Ala-Phe-OY → H-Ala-Phe-OH

In order to synthesize longer peptides, X and Y must be chosen so that X is eliminated under conditions where OY is preserved. Consider the following example:

X is called a temporary protecting group because it is only used for the coupling step. Y is a permanent protection group. Y must be stable enough to withstand all coupling and deprotection steps of X. However, it should be removable in the final step without damaging the peptide. A peptide always has two different groups at each end and thus has a “direction”. The amino group at the end of a peptide is known as N-terminal and the carboxyl group at the other end is known as C-terminal. In chemical syntheses, the peptide is built from the C-terminal to the N-terminal direction, and therefore the C-terminal group must be protected throughout the synthesis.

Protecting groups are necessary to ensure the sequence orientation of amino acids.

The main protective groups used to protect α-amine (Nα) amino acids include Fmoc (9-Fluorenylmethoxycarbonyl), Boc (t-Butoxycarbonyl) and Z (Benzyloxycarbonyl). Each of them defines the overall strategy of peptide synthesis and therefore, the protection and deprotection conditions will change accordingly. Fmoc is removed under basic conditions by elution with piperidine, while Boc requires acidic conditions such as trifluoroacetic acid elution and Z is removed by zinc/palladium catalytic hydrogenation.

Benzyloxycarbonyl (Z) or (Cbz) is the oldest group used to protect Nα amino acids. The development of Z initiated the synthesis of modern peptides, and the abbreviation Z was assigned to this protecting group in honor of Leonidas Zervas. Today, the Z group is still used to protect the amino group in organic reactions. Unlike the α-amino group, the terminal acid group must be protected throughout the synthesis. The most common protecting groups and their deprotection conditions are shown in the following table:

In addition to the alpha amine and the acidic group of amino acids, the side chains must also be protected to avoid any side reactions at this position during the coupling reaction. Indeed, if a reactive group is present in the side chain, for example, the amino acid lysine, it can react in the coupling step, leading to the formation of branches and unwanted side products.

The choice of amine and carboxyl protecting groups depends on the synthesis method and strategy. Two important methods are described in the next section.

Solid phase or liquid phase? Two different but complementary methods!

There are two standard methods for peptide synthesis: solid phase peptide synthesis (SPPS) and solution synthesis (LPPS). The table below summarizes the similarities and differences between these two methods.

Solution synthesis is still chosen for the synthesis of some very short peptides (such as dipeptides) and C-terminally modified peptides. The main advantages of solid phase synthesis compared to solution synthesis are its speed and ease. However, it is often said that the larger the scale of synthesis, the slower SPPS proceeds, as “manual work” increases. But on a small and medium scale, peptides can be produced in a fully automatic machine as shown in the image below.

Very large quantities (several kilograms of peptides) are produced in semi-automatic machines (only the washing program is run automatically) or manually. This process always involves four iterative steps:

There is a standard protocol for SPPS that works for the synthesis of most peptides, while solution synthesis requires more careful planning and no such protocol exists for solution-phase synthesis. Synthesis in the solution phase has many differences with SPPS, not only in the choice of protecting groups, but also in coupling methods, used solvents, and preparation methods.

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