Comprehensive Strategy for Peptide Synthesis: From Process to Purification

The solid-phase synthesis of peptides is a delicate art, and today we will explain in detail the entire process of peptide synthesis.

Peptide sequence design
Firstly, we need a clear peptide sequence. For example: [Cys Gly ****** - Glu Gly Ala Glu Ala Lys (Ac) - Pro Trp Tyr Cys]

Process Flow Diagram

The entire synthesis process can be summarized into several key steps, as shown in Figure 

preparation process

Solid phase synthesis:

Using Wang Resin (1.0 mmol/g), S=0.3 mmol/g, and employing Fmoc process.

First connect Fmoc Cys (Trt) - OH Wang resin.

Then, according to the above peptide sequence, the amino acids are sequentially condensed from the C-terminus to the 

N-terminus (from right to left) until the resin peptide is obtained.

Substitution synthesis:

Use R-01 Fmoc Cys (Trt) - OH and R-02 Wang resin.

Conjugate the following amino acids in sequence:

A-03 Fmoc-Tyr(tBu)-OH

A-04 Fmoc-Trp(Boc)-OH

A-05 Fmoc-Pro-OH

...

A-17 Fmoc-Gly-OH

A-18 Fmoc-Cys（Trt）-OH

Peptide resin cleavage:

Preparation of lysis reagents:

Calculate the amount of lysis reagent based on a ratio of 1g peptide resin to 15ml ± 2ml in volume.

TFA:H2O:EDT:TIS = 95:1:2:2。

Add the required lysis reagent into the lysis reaction bottle and control the temperature between 0-10 ℃.

Add peptide resin, wait for the system temperature to stabilize, and stir the reaction at 20-25 ℃ for 2 hours.

Filter out the lysate and precipitate it with 5 times the volume of concentrated liquid using ice ether. Filter out the precipitate and dry it under reduced pressure at room temperature to obtain the crude product.

oxidation

Grind the crude product finely and prepare purified water.

Slowly add finely ground crude product while stirring, while adding acetonitrile aqueous solution dropwise. When the crude product is completely dissolved, add dilute ammonia solution dropwise to adjust the pH value of the solution to 7.5-8.
Take samples every half hour and send them to QC for mass spectrometry to determine if oxidation is complete.

Purification freeze-drying

Filter the completely oxidized sample through a 0.45 μ m microporous membrane.

Using Shimadzu semi preparation, 5cm, 10um, C-18 column packing was used for separation and purification at room temperature using a suitable gradient. The target product was collected, analyzed, detected, and classified.

The impurity purity requirement is ≥ 97%. Collect the unqualified target substance and separate and purify it again using a 2cm, 5um, C-18 column with a suitable gradient.
Freeze dry the qualified main peak under reduced pressure to obtain powdery impurities.

By following the above steps, we can obtain pure peptide products.