An Approach To Prepare Melanotan I

Melanotan I, also known as Afamelanotide, is a linear polypeptide composed of 13 amino acids that is bound to play a greater role in drug research. The main English names of melanotan I are Melanotan-1 and Afamelanotide, as an artificial synthetic analogue of α-melanotropin (α-MSH), melanotan I can be used to treat photosensitive diseases caused by erythropoietic protoporphyria, and most melanotan I products are only labeled for scientific research and are not used in the human body for the time being. In traditional solid-phase synthesis, the choice of resin will have a great impact on the coupling rate and final purity of the product.

Basic information
Chinese name: melanotan I
English name: Melanotan-1
CAS Number: 75921-69-6
Sequence: Ac-SYS{Nle}EH{D-Phe}RWGKPV-NH2
Formula: C78H111N21O19
Molecular weight: 1646.88
Preparation Approach

1. Preparation of Fmoc-Val-amino resin
   Step 1: Weigh 11.11g (5mmol) of Ramage Amide AM resin resin with a substitution of 0.45mmol/g and add it to the solid-phase reactor, add DCM swelling resin for 30 minutes, drain it, wash it three times with DMF, add a volume ratio of 20% hexahydropyridine DMF solution, react for 5 minutes, add 20% hexahydropyridine DMF solution to react for 10 minutes, wash the DMF in the middle once, and drain it at the end of the reaction. DMF wash 3 times.
   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, and add it to the reaction column for room temperature reaction for 1 hour after complete dissolution.
   
2. Preparation of melanotan I fully protective resin peptideS
   tep 1: Add 20% hexahydropyridine DMF solution to the above Fmoc-Val-amino resin, react for 5 minutes, add 20% hexahydropyridine DMF solution again for 10 minutes, wash the middle DMF once, drain it at the end of the reaction, and wash the DMF 3 times.
   Step 2: 5.06g of Fmoc-Pro-OH, 2.02g of HOBT (1-hydroxybenzotriazole), and 2.32ml of DIC (N,N'-diisopropylcarbodiaimide) will be dissolved in DMF at 0°C, and after complete dissolution, add to the reaction column for room temperature reaction for 1 hour.
   Step 3: According to the method of step 2, according to the sequence of melanotane I, conjugate each amino acid in turn, react to Ac-Ser(tBu)-OH After completion, DMF washes three times, DCM washes three times, methanol washes three times, and drains 23.89g of melanotane I full protection resin peptide.
3. Preparation of melanotan I crude peptide
   Put 23.89g of melanotan I fully protective resin peptide into a round-bottom flask, slowly add 250ml of the configured lysate at 0°C (the volume ratio of the reagent formula used in the lysate is TFA: benzosulfide: phenol: triisopropylsilane: water = 82.5:7.5:5:3:2) and stir slowly, Reaction at low temperature for 0.5 hours, reaction at room temperature for 2 hours, filter to obtain lysate, slowly add the lysate to 2L of anhydrous ice ether and stir, filter and separate crude peptides, wash the ice ether 3 times, and obtain 8.55g of melanotan I crude peptides.
   
4. Preparation of melanotan I high-quality peptidesS
   tep 1: Dissolve the crude peptide of melanotan I in 100ml of purified water and filter it with a 0.45μm filter membrane to obtain a crude peptide solution.
   Step2. Purification of melanotan I crude peptide using high performance liquid chromatography instrument: Through the DAC-HB50 dynamic axial compression column, mobile phase A is an aqueous solution of 0.05% trifluoroacetic acid and B is a solution of 0.05% acetonitrile trifluoroacetate with a mass concentration of 0.05%, and the peptide solution of the target peak is collected by gradient elution.
   Step 3: After high-performance liquid phase purification, 180ml of melanotane I trifluoroacetic acid liquid with a purity greater than 99% is obtained, and 50ml of liquid is obtained after spin steaming and concentration.
   Step 4: The column was equilibrated with deionized water and loaded, the sample volume was 50ml, the purity was greater than 99% melanotane I trifluoroacetic acid liquid, eluted in 2% acetic acid aqueous solution system for 50min, the collected target product was rotated and concentrated to 80ml, pre-lyophilized and lyophilized, and finally 5.33g of melanotan I fine peptide was obtained, with a yield of 32.33%.