Background
Melanotan I, also known as Afamelanotide, is a linear polypeptide composed of 13 amino acids that is poised to play an increasingly significant role in pharmaceutical research. The primary English names for Melanotan I are Melanotan-1 and Afamelanotide. As a synthetic analog of α-melanocyte-stimulating hormone α-MSH), Melanotan I can be used to treat photosensitivity disorders caused by erythropoietic protoporphyria. Currently, most Melanotan I products are labeled for research use only and are not yet intended for human application. In traditional solid-phase synthesis, the choice of resin significantly impacts the coupling efficienc and final purity of the product.
Basic Information
Chinese Name: 美拉诺坦 1
English Name: Melanotan-1
Company No.: GT-A006
CAS No.: 75921-69-6
Sequence: Ac-SYS{Nle}EH{D-Phe}RWGKPV-NH2
Molecular Formula: C78H111N21O19
Molecular Weight: 1646.88
Preparation method
I. Preparation of Fmoc-Val-Amino Resin
Step 1: Weigh 11.11g (5mmol) of Ramage Amide AM resin with a substitution degree of 0.45mmol/g and add it to a solid-phase reactor. Add DCM to swell the resin for 30 minutes, then drain. Wash three times with DMF. Add a DMF solution containing 20% piperidine (v/v) and react for 5 minutes. Add another 20% piperidine DMF solution and react for 10 minutes, with one DMF wash in between. After the reaction completes, drain and wash three times with DMF.
Step 2: Dissolve 5.09g of Fmoc-Val-OH, 2.02g of HOBT (1-hydroxybenzotriazole), and 2.32ml of DIC (N,N'-diisopropylcarbodiimide) in DMF at 0°C. After complete dissolution, add the mixture to the reaction column and react at room temperature for 1 hour. Monitor the reaction progress using the ninhydrin test, ensuring a negative result. After the reaction completes, wash three times with DMF to obtain Fmoc-Val-amino resin.
II. Preparation of Fully Protected Resin Peptide of Melanotan I.
Step 1: Add 20% piperidine in DMF solution (volume ratio) to the above Fmoc-Val-aminoresin. React for 5 minutes, then add another portion of 20% piperidine in DMF solution and react for 10 minutes. Wash once with DMF during the interval. After the reaction is complete, dry under vacuum and wash three times with DMF.
Step 2: Dissolve 5.06g Fmoc-Pro-OH, 2.02g HOBT (1-hydroxybenzotriazole), and 2.32ml DIC (N,N'-diisopropylcarbodiimide) in DMF at 0°C. After complete dissolution, add the mixture to the reaction column and react at room temperature for 1 hour. Monitor the reaction progress using the ninhydrin test (Kaiser test) until negative results are obtained. Upon completion, wash three times with DMF to obtain Fmoc-Pro-Val-aminoresin.
Step 3: Following the method described in Step 2, sequentially couple each amino acid according to the sequence of Melanotan I. After completion of the Ac-Ser(tBu)-OH coupling reaction, wash three times with DMF, three times with DCM (dichloromethane), and three times with methanol. Dry under vacuum to obtain 23.89g of fully protected Melanotan I resin-bound peptide.
III. Preparation of Melanotan I Crude Peptide
23.89 g of fully protected Melanotan I resin-bound peptide was placed in a round-bottom flask. At 0°C, 250 ml of a pre-prepared cleavage cocktail (composed of TFA:thioanisole:phenol:triisopropylsilane:water = 82.5:7.5:5:3:2 by volume) was slowly added under stirring. The mixture was reacted at low temperature for 0.5 hours, followed by reaction at room temperature for 2 hours. The cleavage solution was then filtered under vacuum. This solution was slowly added to 2 L of ice-cold anhydrous diethyl ether under stirring. The crude peptide was isolated by filtration, washed three times with ice-cold diethyl ether, and 8.55 g of Melanotan I crude peptide was obtained.
IV. Preparation of Melanotan I Purified Peptide
Step 1: Dissolve the crude Melanotan I peptide in 100 ml of purified water and filter through a 0.45 μm membrane to obtain a crude peptide solution.
Step 2: Purify the crude Melanotan I peptide using high-performance liquid chromatography (HPLC). Utilize a DAC-HB50 dynamic axial compression column with mobile phase A (0.05% trifluoroacetic acid aqueous solution) and mobile phase B (0.05% trifluoroacetic acid in acetonitrile). Perform gradient elution for separation and purification. Detect the sample using a UV detector and collect the peptide solution corresponding to the target peak in fractions.
Step 3: After HPLC purification, obtain 180 ml of Melanotan I trifluoroacetate solution with a purity exceeding 99%. Concentrate the solution to 50 ml via rotary evaporation.
Step 4: Equilibrate the chromatography column with deionized water and load 50 ml of the purified Melanotan I trifluoroacetate solution (purity >99%). Elute for 50 minutes in a 2% acetic acid aqueous solution system. Concentrate the collected target product to 80 ml via rotary evaporation. Perform pre-freeze drying and freeze-drying to ultimately obtain 5.33 g of Melanotan I purified peptide, with a yield of 32.33%.
Post time: 2026-01-05