One last factor of solid phase synthesis that one must account for is the exponentially decreasing efficacy of the process. A solid synthesis can expect ~90% yield a step. By this logic, the following efficacies may be extrapolated for varying length polypeptide reactions.
This means that glycine and proline are the only viable amino acids for N to C solid phase synthesis. Glycine contains a non-sterogenic carbon for the amino acid backbone, and the iminium intermediate required for racemization of proline is too strained to actually occur. When combining larger peptides, one would have to make sure that the C terminus of the beginning peptide ended with a glycine or proline.
As mentioned above, when using the bead you can use excess amounts of reagents, helping to solve the kinetic problem of phase transition. The polypeptide chain is always kept convalently attached to the beads, and the DCC and new amino acids are then introduced in solution. DCC and amino acids are used in excess to overcome the kinetics problem of phase transition (Le Chatelier). In solution phase synthesis, excess reagents would lead to a very tedious purification process, but here, they can simply be washed away in solution by filtration.
Solid phase peptide synthesis consists of two phases: one reactant is the C-terminus of the starting amino acid covalently bonded to an insoluble resin bead, while the other reactant is in solution. The physical properties of the bead, and its applications, vary with the material from which the bead is constructed, the amount of crosslinking, as well as the linker and handle being used. Solid phase sythnesis builds polypeptide chains in the C to N direction to maintain stereochemistry.
In this synthesis, the beginning and ending steps are unique, while the middle steps are iterative, or repetitive. The unique beginning step is the attachment of the Boc-protected amino acid to the polystyrene bead. This initial reaction is driven by the"cesium effect" or "naked anion" effect. The cesium cation has a large diameter and is therefore more easily attacked by organic molecules, making it very soluble. With the counter cation otherwise occupied, the amino acid becomes a much better nucleophile. Chlorine is very good at SN2 reactions, and the amino acid easily displaces the chlorine, forming the covalent bond between the first amino acid and the polystyrene bead. The precipitation of cesium chloride pulls the reactions forward to nearly a 100% yield (Le Chatelier's principle). Without the help of the cesium, the amino acid would remain a poor nucleophile and the reaction would then have to compete with E2 reactions.
Currently, two protecting groups (t-Boc, Fmoc) are commonly used in solid-phase peptide synthesis. Their ability to act as a protecting group is caused by the carbamate group which readily releases carbon dioxide for an irreversible decoupling step.
The reactions that have been covered in class up to this point have all occurred in a solution, which is fine if you only want to make simple di- and tripeptides. More complex peptides are not able to be synthesized in this manner. All of the polypeptides are floating in solution, which means they only encounter each other in a stochastic (or random collision) fashion, resulting in the majority of the solution being byproducts that we don't want. A tedious purification must be done before each addition of a new peptide, and yield of the reaction drops drastically. Solid phase peptide synthesis is the solution to these problems.
Solid-phase peptide synthesis was invented by Merrifield. This was a revolution in polypeptide synthesis, and is now the accepted method for creating peptides in the lab.
D7-1. Provide a solid-phase synthesis of the following dipeptide starting from chloromethyl polystyrene resin. You have the following reagents available: any appropriately protected amino acids and/or their cesium salts, trifluoroacetic acid (CF3CO2H), dicyclohexylcarbodiimide (DCC), and hydrogen fluoride (HF). You may omit all solvents, wash steps, and neutralization steps from the synthesis. All protecting groups must be specified, but any side chain protecting groups can be abbreviated as “P”.
Soybean lecithin consisting of 90–95%phosphatidylcholine and mPEG-DSPE, andMal-PEG-DSPE were purchased from Avanti Lipid(Alabaster, AL, USA). Cholesterol (CHO) was purchased from ChengduKelong Chemical Company (Chengdu, China). Rhodamine-PE waspurchased from Avanti Lipid. T7 peptide with terminal cysteine(Cys-HAIYPRH) and TAT peptide with terminal cysteine(Cys-AYGRKKRRQRRR) were produced according to the standard solidphase peptide synthesis by Shanghai Jier Bio-Pharmaceutical Co.,Ltd. (Shanghai, China). Cell culture plates were purchased fromWuxi NEST Biotechnology Co., Ltd. (Wuxi, China). Other chemicalsand reagents were of analytical grade and obtainedcommercially.