Introduction to peptide synthesis — working principle
Peptide synthesis commonly-seen in a chemical synthesis lab. How are peptide synthesized? The synthesis of peptide uses solid-phase synthesis as the reaction principle. In a sealed explosion-proof glass reactor, amino acids are continuously added, reacted, synthesized in a known sequence, and finally through operation, we obtain the polypeptide carrier. The solid-phase synthesis method greatly reduces the difficulty of product purification at each step.
To prevent side reactions, two measures are carried out. One, the side chains of the amino acids participating in the reaction are protected. Two, the carboxyl end is free and must be activated before the reaction.
There are two solid-phase synthesis methods, namely Fmoc and tBoc. Because Fmoc has many advantages over tBoc, in most cases, we adopt the Fmoc for synthesis. But for some short peptides, tBoc is still used by many companies because of the high yield.
Introduction to peptide synthesis — synthesis process
The specific peptide synthesis process involves three cycles.
Peptide synthesis process — step 1
Deprotection: The amino protective group existing in Fmoc protected columns and monomers must be removed with an alkaline solvent (piperidine).
Peptide synthesis process — step 2
Activation and cross-linking: The carboxyl group of the next amino acid is activated by an activator. A peptide bond is formed through the reaction between the activated monomer and the free amino group. In this step, a large amount of super-concentration reagent is usually used to accelerate the reaction process. Circulation: The two-step reaction repeatedly circulates until the synthesis is completed.
Peptide synthesis process — step 3
Elution and deprotection: The peptide is eluted from the column, and its protecting group is eluted and deprotected by a deprotection agent (TFA).
Introduction to peptide synthesis — synthesis methods
How are peptide synthesized? Besides its working principle, you should also know the methods of peptide synthesis. Here are the details.
Methods of peptide synthesis — acid anhydride method
In peptide synthesis, the initial consideration of the use of acid anhydrides dates back to the early research on the synthesis of benzoylglycine by Theodor Curtius in 1881. From the reaction of silver aminoacetate and benzoyl chloride, in addition to benzoylaminoacetic acid, BZ-Glyn-OH (n=2-6) was also obtained. In the early days, it was believed that when treated with benzoyl chloride, N-benzoyl amino acids or N-benzoyl peptides and benzoic acid formed the active intermediate asymmetric anhydride. About 70 years later, Theodor Wieland used these findings to apply the mixed acid anhydride method to modern peptide synthesis.N-carboxyanhydride
At present, in addition to this method, symmetrical anhydrides and N-carboxyanhydride (NCA, Leuchs anhydrides) formed by the carboxyl and carbamate of amino acids are also condensed with peptides. Finally, it should be mentioned that asymmetric anhydrides often participate in acylation reactions in biochemical reactions.
Methods of peptide synthesis — mixed acid anhydride method
Both organic and inorganic acids can be used for the formation of mixed anhydrides. However, only a few have found widespread practical applications, and in most cases, alkyl chloroformate is used. Ethyl chloroformate, which was frequently used in the past, is currently mainly replaced by isobutyl chloroformate.
The regioselectivity of the ammonolysis reaction of the mixed anhydride formed from the carboxyl component and the chloroformate depends on the electrophilicity and/or steric hindrance of the two competing carbonyl groups. When N-protected amino acid carboxylate (carboxyl component) and alkyl chloroformate (activating component, for example, derived from alkyl chloroformate) form a mixed anhydride, the nucleophile amine mainly attacks the carboxyl group of the amino acid component to form the expected peptide derivative and release the active ingredient in free acid form.
Methods of peptide synthesis — Acyl azide method
The acyl azide method was introduced into peptide chemistry as early as 1902, so it is one of the oldest condensation methods. In alkaline aqueous solutions, in addition to free amino acids and peptides condensed with acyl azides, amino acid esters can be used in organic solvents. Unlike many other condensation methods, it does not require the addition of an auxiliary base or another equivalent amino component to capture hydrazone acid.
For a long time, it has been considered that the azide method is the only condensation method without racemization. With the introduction of selectively cleavable amino acid protecting groups, this method has experienced a large-scale revival. The starting materials of this method are crystalline amino acid hydrazide or peptide hydrazide 64, which can be easily obtained by hydrazinolysis of the corresponding ester.
Methods of peptide synthesis — symmetric anhydride method
Symmetric anhydrides of Nα-acyl amino acids are highly active intermediates for peptide bond formation. Contrary to the mixed acid anhydride method of peptide synthesis, its reaction with amine nucleophiles has no ambiguous regioselectivity. But the peptide condensation yield is the highest, 50% (based on the carboxyl component).
Although the free Na-acyl amino acids formed by the aminolysis of the symmetrical anhydride can be recovered together with the target peptide by extraction with saturated sodium bicarbonate solution, the practical value of this method is extremely low at the beginning. Symmetric anhydrides can be prepared by the reaction of Na-protected amino acids with phosgene or convenient carbodiimide. The reaction of two -normal Na-protected amino acid with one-normal carbodiimide is beneficial to the formation of symmetrical anhydrides. The symmetrical anhydrides can be separated or used directly in the subsequent condensation reaction without purification. Symmetric anhydrides based on Na-alkoxycarbonyl amino acids are stable to hydrolysis and can be purified by a method similar to the above-mentioned purification of mixed acid anhydrides.