Editing Peptide synthesis (section)

=== General mechanism ===
Each amino acid to be coupled to the peptide chain N-terminus must be [[protecting group|protected]] on its N-terminus and side chain using appropriate protecting groups such as [[tert-Butyloxycarbonyl protecting group|Boc]] (acid-labile) or [[Fluorenylmethyloxycarbonyl protecting group|Fmoc]] (base-labile), depending on the side chain and the protection strategy used (see below).<ref name="Isidro-Llobet09" />

The general SPPS procedure is one of repeated cycles of alternate N-terminal deprotection and coupling reactions. The resin can be washed between each steps.<ref name="chan00" /> Reactions in SPPS are conducted as follows:<ref>{{Cite web |title=What is solid phase peptide synthesis? |url=https://www.biotage.com/blog/what-is-solid-phase-peptide-synthesis |access-date=2025-03-14 |website=www.biotage.com |language=en}}</ref>

# The amine group of the first amino acid is protected with Fmoc or Boc group
# Protected amino acid is coupled with free amino groups attached to resin beads
# Protecting group is removed (see: [[#Protecting groups schemes|Protecting groups schemes]])
# The second amino acid with a ''N''-protecting group is coupled with the first one. Coupling reagents are employed to help the formation of the peptide bond.
# The above cycle is repeated until the desired sequence has been synthesised
# Optionally, the ''N''-terminal amino group undergoes capping, thereby preventing the obtained peptide from further reaction
# The crude product is purified using either:
#* [[High-performance liquid chromatography|reverse-phase high-performance liquid chromatography]] (HPLC)<ref name="Mant07">{{cite book |title=Peptide Characterization and Application Protocols |vauthors=Mant CT, Chen Y, Yan Z, Popa TV, Kovacs JM, Mills JB, Tripet BP, Hodges RS |date=2007 |publisher=Humana Press |isbn=978-1-59745-430-8 |series=Methods in Molecular Biology |volume=386 |pages=3–55 |chapter=HPLC analysis and purification of peptides |doi=10.1007/978-1-59745-430-8_1 |pmc=7119934 |pmid=18604941 |ref=Mant07 |display-authors=6}}</ref><ref>{{Cite web |date=November 2021 |title=Custom peptide synthesis service. HPLC refers to High Performance Liquid Chromatography |url=https://www.remetide.com/services/custom-peptide-service/ |website=Remetide Biotech}}</ref>
#* [[multicolumn countercurrent solvent gradient purification]] (MCSGP) which is utilised mainly in the case of longer peptides, due to accumulation of numerous minor byproducts that have similar properties to the desired peptide product. This process is used to maximise the yield without sacrificing purity.<ref>{{Cite journal |vauthors=Lundemann-Hombourger O |date=May 2013 |title=The ideal peptide plant |url=https://www.polypeptide.com/web/upload/medias/1401701074538c42d265d03.pdf |url-status=dead |journal=Speciality Chemicals Magazine |pages=30–33 |archive-url=https://web.archive.org/web/20180404202124/https://www.polypeptide.com/web/upload/medias/1401701074538c42d265d03.pdf |archive-date=4 April 2018 |access-date=4 April 2018}}</ref>

[[File:Solid-phase peptide synthesis.png|center|thumb|816x816px|Solid-phase peptide synthesis ('''PG''' – protecting group)]]


SPPS is limited by [[chemical yield|reaction yields]] due to the exponential accumulation of by-products, and typically peptides and proteins in the range of 70 amino acids are pushing the limits of synthetic accessibility.<ref name="chan00" /> Synthetic difficulty also is sequence dependent; typically aggregation-prone sequences such as [[amyloid]]s<ref name="Tickler04">{{cite journal | vauthors = Tickler AK, Clippingdale AB, Wade JD | title = Amyloid-beta as a "difficult sequence" in solid phase peptide synthesis | journal = Protein and Peptide Letters | volume = 11 | issue = 4 | pages = 377–384 | date = August 2004 | pmid = 15327371 | doi = 10.2174/0929866043406986 | ref = Tickler04 | name-list-style = vanc }}</ref> are difficult to make. Longer lengths can be accessed by using ligation approaches such as [[native chemical ligation]], where two shorter fully deprotected synthetic peptides can be joined in solution.