Peptide synthesis
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In organic chemistry, peptide synthesis is the creation of peptides, which are organic compounds in which multiple amino acids bind via peptide bonds.
Chemistry
Peptides are synthesized by combining the carboxyl group of one amino acid with the amino group of another. Much like peptides created biologically, synthetic peptides are built from the C-terminus (Carboxyl) to the N-terminus (Amine).Liquid-phase synthesis
Liquid-phase peptide synthesis is a classical approach to peptide synthesis. It has been replaced in most labs by solid-phase synthesis (see below). However, it retains usefulness in large-scale production of peptides for industrial purposes.Solid-phase synthesis
Solid phase peptide synthesis was pioneered by Merrifield, resulting in a paradigm shift within the peptide synthesis community. In solid-phase synthesis, small beads are treated with linkers on which peptide chains can be built. The synthesis beads will retain strong bondage to the peptides until cleaved by a reagent such as trifluoroacetic acid. The beads create a synthesis environment in which the peptide chains being created will not pass through a filter material while the reagents used to create them will.Protective groups
Due to amino acid excesses used to ensure complete coupling during each synthesis step, polymerization of amino acids is common in reactions where each amino acid is not protected. In order to prevent this polymerization, protective groups are used. This adds additional deprotection phases to the synthesis reaction, creating a repeating design flow as follows:- Protective group is removed from trailing amino acids in a deprotection reaction
- Deprotection reagents washed away to provide clean coupling environment
- Protected amino acids dissolved in a solvent such as dimethylformamide (DMF) are combined with coupling reagents are pumped through the synthesis column
- Coupling reagents washed away to provide clean deprotection environment
Fmoc protective group
The Fmoc (9-fluorenylmethyl carbamate) is currently a widely used protective group that is generally removed from the N terminus of a peptide in the iterative synthesis of a peptide from amino acid units. The advantage of Fmoc is that it is cleaved under very mild basic conditions (e.g. piperidine), but stable under acidic conditions. This allows mild acid labile protecting groups that are stable under basic conditions, such as Boc and benzyl groups, to be used on the side-chains of amino acid residues of the target peptide. This orthogonal protecting group strategy is common in the art of organic synthesis.
Boc protective group
Before the Fmoc group became popular, the Boc group was commonly used for protecting the terminal amine of the peptide, requiring the use of more acid stable groups for side chain protection in orthoganol strategies. It retains usefulness in reducing aggregation of peptides during synthesis. Boc groups can be added to peptide with boc anhydride and a suitable base.Synthesizing long peptides
Stepwise elongation, in which the amino acids are connected step-by-step in turn, is ideal for small peptides containing between 2 and 100 amino acid residues. Another method is fragment condensation, in which peptide fragments are coupled. Although the former can elongate the peptide chain without racemization, the yield drops if only it is used in the creation of long or highly polar peptides. Fragment condensation is better than stepwise elongation for synthesizing sophisticated long peptides, but its use must be restricted in order to protect against racemization. Fragment condensation is also undesirable since the coupled fragment must be in gross excess, which may be a limitation depending on the length of the fragment. One way around this limitation is native chemical ligation.An example of solid phase peptide synthesis
The following is an outline of the synthetic steps for peptide synthesis on polyamide or polystyrene resin, using the base labile 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group. Using the techniques outlined below, one will obtain a peptide which is capped on the N-terminus with and acetyl group, and on the C-terminus with a primary amide (CONH2).Setting up glassware for manual peptide synthesis
Manual peptide synthesis can be accomplished in a fritted-filter reaction vessel with a three-way valve fitted onto a 1 L side arm vacuum flask by way of a 1-hole stopper. One valve is used to bubble nitrogen, which is first passed through a small column of Drierite, and then into the reaction mixture to agitate the solution and mix reagents. The other valve is used to evacuate excess reaction solutions and wash solvent using a vacuum flask. All glass pieces to be used in Solid-phase synthesis should be treated with a silanizing agent (such as 1-5% dimethyldichlorosilane in DCM) prior to use, to avoid accumulation of static charge, which makes the resin very difficult to handle.
Preparation of polyamide-Rink resin
Polyamide (PL-DMA) resin (1g) is treated with ethylene diamine (40 ml) in a 50 ml Falcon tube overnight on a rocker, then filtered, washed with 5x10 ml of 1:1 dimethylformamide (DMF):dichloromethane (DCM) solution, 5x10 ml of 1:1 DCM, and loaded with Fmoc-Rink using Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (3 eq), 1-Hydroxybenzotriazole Hydrate (HOBt) (3 eq), and Diisopropylethylamine (DIPEA) (6 eq) in 1:1 DCM:DMF. It can then be dried under vacuum and stored at -15oC until needed.
Handling the resin before, and during synthesis
The resin is first swelled for 15 minutes in 10 ml of 1:1 DCM:DMF and drained. The resin is also washed with 5x10 ml of 1:1 DCM and DMF after each completed amino acid coupling.
Fmoc-deprotection
Fmoc deprotection after each amino acid coupling is accomplished using 2x10 ml of 20% piperidine in DMF, with N2 agitation for 10 minutes each treatment. The resin is then washed with 5x10 ml DMF, followed by 5x10 ml of 1:1 DCM:DMF.
Adding amino acids
To begin peptide synthesis on Fmoc-polyamide-Rink resin the Fmoc group is by removed by treating the resin with piperidine. The first amino acid is then coupled to the Fmoc-deprotected N-terminal amine of the rein, or previously coupled amino acid, using PyBOP (3 eq), HOBt (3 eq), and DIPEA (6 eq) in 1:1 DCM:DMF until the resin is negative to ninhydrin.
Monitoring the progress of amino acid couplings
The progress of amino acid couplings can be followed using ninhydrin, or p-chloranil. The ninhydrin solution turns dark blue (positive result) in the presence of a free primary amine but is otherwise colorless (negative result). The p-chloranil solution will turn the resin beads dark black or blue in the presence of a primary amine if acetaldehyde is used as the solvent or in the presence of a secondary amine, if acetone is used instead; the beads remain colorless or pale yellow otherwise. (The tests are outlined below)
Testing by Ninhydrin (1)
Add 2 drops of 40% phenol in ethanol, 2 drops of 0.014 M KCN in pyridine, and 4 drops of 5% ninhydrin in ethanol to an Eppendorf tube along with a spatula tip size sample of resin, then vortex the mixture and heat for 5 minutes at 100 oC.
Testing by Chloranil (2)
Add 5 drops of acetone or acetaldehyde, 5 drops of a saturated solution of p-chloranil in toluene, plus a small spatula-tip-size sample of resin to an Eppendorf tube, then vortex the mixture and allow to stand at room temperature for 5 minutes. Acetone is used for the detection of secondary amines, where acetaldehyde is used for primary amines.
Continuing peptide extension
Once the coupling of the amino acid is complete, the resin is washed, the Fmoc group deprotected with piperidine, and the resin washed again to prepare it for the next coupling. This process is repeated until all necessary amino acids have been added.
Acetylating the N-terminus
After the peptide sequence is completed, the N-terminal amine can be acetylated with 2 ml of 1:1 acetic anhydride and triethylamine in 10 ml of 1:1 DCM:DMF for 1 hour or until negative to ninhydrin, and the resin then washed with 5x10 ml of 1:1 DCM:DMF, before the peptide is cleaved from the resin. The N-terminal can also be left as the free amine is required.
Cleaving the peptide from the resin
The resin is treated with 3x10 ml of 96:2:2 trifluoroacetic acid (TFA), triisopropylsilane (TIPS), and water (H2O) for 10 min each treatment, the resin then filtered away, and the combined filtrates allowed to stand for 1 hour to ensure removal of the acid labile protecting groups.
Workup of peptides after cleavage from the resin
The TFA is evaporated to dryness (or a heavy oil or glass if it does not solidify) on the rotary evaporator, followed by the addition of 5 ml of diethyl ether to the flask to precipitate the peptide, and remove the bulk of the by-products. The suspended mixture of peptide and ether is added to a 50 ml Falcon tube and spun at 3000 rpm for 10–20 min (IEC centrifuge) until the ether can be decanted off without losing any peptide. More ether is added, and the peptide resuspeded by vortexing. The mixture is centrifuged again, the ether decanted, and the washing process repeated twice more. Finally the product can be air dried overnight in the Falcon tube in a descicator.
Typically preparative HPLC is used to purify the final product. Mass spectrometry data is obtained to ensure the target peptide was obtained.
References
1. Atherton, E., Sheppard, R.C. Solid Phase peptide synthesis: a practical approach. IRL Press, Oxford, England, 1989.
2. Stewart J.M., Young, J.D. Solid phase peptide synthesis, 2nd edition, Pierce Chemical Company, Rockford, 1984, pp 91.
Peptide Synthesis Companies
Peptide synthesis companies include Bachem, Anaspec, and American Peptide Company.
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