Editing Peptide synthesis (section)

==Solid-phase synthesis==
The established method for the production of synthetic peptides in the lab is known as solid phase peptide synthesis (SPPS).<ref name="chan00" /> Pioneered by [[Robert Bruce Merrifield]],<ref>{{cite journal | vauthors = Merrifield RB | author-link = Robert Bruce Merrifield | title = Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide | year = 1963 | journal = [[J. Am. Chem. Soc.]] | volume = 85 | issue = 14 | pages = 2149–2154 | doi = 10.1021/ja00897a025}}</ref><ref name="Mitchell08">{{cite journal | vauthors = Mitchell AR | title = Bruce Merrifield and solid-phase peptide synthesis: a historical assessment | journal = Biopolymers | volume = 90 | issue = 3 | pages = 175–184 | date = 2008 | pmid = 18213693 | doi = 10.1002/bip.20925 | ref = Mitchell08 | s2cid = 30382016 | doi-access =  }}</ref> SPPS allows the rapid assembly of a peptide chain through successive reactions of amino acid derivatives on a macroscopically insoluble solvent-swollen beaded resin support.<ref name=":4">{{Cite book |last=Morrison |first=Robert Thornton |title=Organic Chemistry |last2=Boyd |first2=Robert Neilson |publisher=Allyn and Bacon, Inc. |year=1973 |isbn=9780205032396 |edition=3rd |pages=1149}}</ref>

=== Characteristics of solid support ===
The solid support consists of small, polymeric resin beads functionalized with reactive groups (such as amine or hydroxyl groups) that link to the nascent peptide chain.<ref name="chan00" /> Since the peptide remains covalently attached to the support throughout the synthesis, excess reagents and side products can be removed by washing and filtration. This approach circumvents the comparatively time-consuming isolation of the product peptide from solution after each reaction step, which would be required when using conventional solution-phase synthesis.<ref name=":4" />

=== 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.

===Peptide coupling reagents===

An important feature that has enabled the broad application of SPPS is the generation of extremely high yields in the coupling step.<ref name="chan00"/> Highly efficient [[amide]] bond-formation conditions are required. To illustrate the impact of suboptimal coupling yields for a given synthesis, consider the case where each coupling step were to have at least 99% yield: this would result in a 77% overall crude yield for a 26-amino acid peptide (assuming 100% yield in each deprotection); if each coupling were 95% efficient, the overall yield would be 25%.<ref name="El-Faham11">{{cite journal | vauthors = El-Faham A, Albericio F | title = Peptide coupling reagents, more than a letter soup | journal = Chemical Reviews | volume = 111 | issue = 11 | pages = 6557–6602 | date = November 2011 | pmid = 21866984 | doi = 10.1021/cr100048w | ref = El-Faham11 }}</ref><ref name="Montalbetti05">{{cite journal| vauthors = Montalbetti CA, Falque V |title=Amide bond formation and peptide coupling|journal=Tetrahedron|date=2005|volume=61|issue=46|pages=10827–10852|doi=10.1016/j.tet.2005.08.031|ref=Montalbetti05}}</ref> and adding an excess of each amino acid (between 2- and 10-fold). The minimization of amino acid [[racemization]] during coupling is also of vital importance to avoid [[epimer]]ization in the final peptide product.{{cn|date=January 2024}}

Amide bond formation between an amine and carboxylic acid [[amide#amide synthesis|is slow]], and as such usually requires  'coupling reagents' or 'activators'. A wide range of coupling reagents exist, due in part to their varying effectiveness for particular couplings,<ref>{{cite journal | vauthors = Valeur E, Bradley M | title = Amide bond formation: beyond the myth of coupling reagents | journal = Chemical Society Reviews | volume = 38 | issue = 2 | pages = 606–631 | date = February 2009 | pmid = 19169468 | doi = 10.1039/B701677H }}</ref><ref>{{cite journal | vauthors = El-Faham A, Albericio F | title = Peptide coupling reagents, more than a letter soup | journal = Chemical Reviews | volume = 111 | issue = 11 | pages = 6557–6602 | date = November 2011 | pmid = 21866984 | doi = 10.1021/cr100048w }}</ref> many of these reagents are commercially available.

====Carbodiimides====
[[File:DIC HOBt coupling.svg|thumb|[[Amide]] bond formation using DIC/HOBt.<ref name="Montalbetti05" />]]
[[Carbodiimide]]s such as [[dicyclohexylcarbodiimide]] (DCC) and [[diisopropylcarbodiimide]] (DIC) are frequently used for amide bond formation.<ref name="Montalbetti05" /> The reaction proceeds via the formation of a highly reactive ''O''-acyliso[[urea]]. This reactive intermediate is attacked by the peptide N-terminal amine, forming a peptide bond. Formation of the ''O''-acyliso[[urea]] proceeds fastest in non-polar solvents such as dichloromethane.<ref>{{Cite journal|vauthors=Singh S|date=January 2018|title=CarboMAX - Enhanced Peptide Coupling at Elevated Temperatures|url=https://cem.com/media/contenttype/media/literature/ap0124v2-cem.pdf|journal=AP Note|volume=0124|pages=1–5|archive-date=6 August 2020|access-date=7 August 2018|archive-url=https://web.archive.org/web/20200806041631/https://cem.com/media/contenttype/media/literature/ap0124v2-cem.pdf|url-status=dead}}</ref>

DIC is particularly useful for SPPS since as a liquid it is easily dispensed, and the [[urea]] byproduct is easily washed away. Conversely, the related carbodiimide [[1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide]] (EDC) is often used for solution-phase peptide couplings as its urea byproduct can be removed by washing during aqueous [[Work-up (chemistry)|work-up]].<ref name="Montalbetti05" />

[[File:HOBT.png|thumb|left|100px|HOBt]]
[[File:1-hydroxy-7-aza-benzotriazole.svg|thumb|100px|left|HOAt]]
[[File:HOAt neighbouring.gif|thumb|200px|right|Neighbouring group effect of HOAt]]

Carbodiimide activation opens the possibility for [[racemization]] of the activated amino acid.<ref name="Montalbetti05" /> Racemization can be circumvented with 'racemization suppressing' additives such as the [[triazole]]s [[hydroxybenzotriazole|1-hydroxy-benzotriazole]] (HOBt), and [[1-Hydroxy-7-azabenzotriazole|1-hydroxy-7-aza-benzotriazole]] (HOAt). These reagents attack the ''O''-acylisourea intermediate to form an [[active ester]], which subsequently reacts with the peptide to form the desired peptide bond.<ref name=Joullié>{{cite journal | vauthors = Joullié MM, Lassen KM |journal=Arkivoc|year=2010|volume=viii|pages=189–250|title=Evolution of Amide Bond Formation |issue=8 |doi=10.3998/ark.5550190.0011.816 |url=http://www.arkat-usa.org/get-file/34631/|doi-access=free|hdl=2027/spo.5550190.0011.816|hdl-access=free}}</ref> [[Ethyl cyanohydroxyiminoacetate]] (Oxyma), an additive for carbodiimide coupling, acts as an alternative to HOAt.<ref name="subiros09">{{cite journal | vauthors = Subirós-Funosas R, Prohens R, Barbas R, El-Faham A, Albericio F | title = Oxyma: an efficient additive for peptide synthesis to replace the benzotriazole-based HOBt and HOAt with a lower risk of explosion | journal = Chemistry: A European Journal | volume = 15 | issue = 37 | pages = 9394–9403 | date = September 2009 | pmid = 19575348 | doi = 10.1002/chem.200900614 | ref = subiros09 }}</ref>

====Amidinium and phosphonium salts====
[[File:ABFreagents1.png|center|frameless|600x600px]]
To avoid epimerization through the O-acylisourea intermediate formed when using a carbodiimide reagent, an [[Amidine|amidinium]]- or [[phosphonium]]-reagent can be employed These reagents have two parts: an electrophilic moiety which deoxygenates the carboxylic acid ('''blue''') and masked nucleophilic moiety ('''red'''). Nucleophilic attack of the carboxylic acid on the electrophilic amidinium or phosphonium moiety leads to a short lived intermediate which is rapidly trapped by the unmasked nucleophile to form the activated ester intermediate and either a [[urea]] or [[phosphoramide]] by-product. These cationic reagents have non-coordinating counteranions such as a [[hexafluorophosphate]] or a [[tetrafluoroborate]].<ref name="El-Faham11" /> The identity of this anion is typically indicated by the first letter in the reagent’s acronym, although the nomenclature can be inconsistent. For example '''<u>H</u>'''BTU is a hexafluorophosphate salt while '''<u>T</u>'''BTU is a tetrafluoroborate salt. In addition to [[HBTU]] and [[HATU]] other common reagents include [[HCTU]] (6-ClHOBt), [[TCFH]] (chloride) and COMU (ethyl cyano(hydroxyimino)acetate). Amidinium reagents incorporating [[hydroxybenzotriazole]] moieties can exist in an N-form (guanadinium) or an O-form (uronium), but the N-form is generally more stable.<ref>{{Cite journal |last1=Carpino |first1=Louis A. |last2=Imazumi |first2=Hideko |last3=El-Faham |first3=Ayman |last4=Ferrer |first4=Fernando J. |last5=Zhang |first5=Chongwu |last6=Lee |first6=Yunsub |last7=Foxman |first7=Bruce M. |last8=Henklein |first8=Peter |last9=Hanay |first9=Christiane |last10=Mügge |first10=Clemens |last11=Wenschuh |first11=Holger |last12=Klose |first12=Jana |last13=Beyermann |first13=Michael |last14=Bienert |first14=Michael |date=2002-02-01 |title=The Uronium/Guanidinium Peptide Coupling Reagents: Finally the True Uronium Salts |url=https://onlinelibrary.wiley.com/doi/10.1002/1521-3773(20020201)41:33.0.CO;2-N |journal=Angewandte Chemie International Edition |language=en |volume=41 |issue=3 |pages=441–445 |doi=10.1002/1521-3773(20020201)41:3<441::AID-ANIE441>3.0.CO;2-N|pmid=12491372 |url-access=subscription }}</ref> Phosphonium reagents include [[BOP reagent|BOP]] (HOBt), [[PyBOP]] (HOBt) and [[PyAOP]] (HOAt).<ref>{{Cite journal |last1=Mansour |first1=Tarek |last2=Bardhan |first2=Sujata |last3=Wan |first3=Zhao-Kui |date=2010 |title=Phosphonium- and Benzotriazolyloxy-Mediated Bond-Forming Reactions and Their Synthetic Applications |url=http://www.thieme-connect.de/DOI/DOI?10.1055/s-0029-1219820 |journal=Synlett |language=en |volume= |issue=8 |pages=1143–1169 |doi=10.1055/s-0029-1219820 |issn=0936-5214|url-access=subscription }}</ref> Although these reagents can lead to the same activated ester intermediates as a carbodiimide reagent, the rate of activation is higher due to the high electrophilicty of these cationic reagents.<ref name="Albericio98">{{cite journal| vauthors = Albericio F, Bofill JM, El-Faham A, Kates SA |title=Use of Onium Salt-Based Coupling Reagents in Peptide Synthesis|journal=J. Org. Chem.|date=1998|volume=63|issue=26|pages=9678–9683|doi=10.1021/jo980807y|ref=Albericio98}}</ref> Amidinium reagents are capable of reacting with the peptide N-terminus to form an inactive [[guanidine|guanidino]] by-product, whereas phosphonium reagents are not.<ref>{{Cite journal |last1=Albericio |first1=Fernando |last2=Cases |first2=Marta |last3=Alsina |first3=Jordi |last4=Triolo |first4=Salvatore A. |last5=Carpino |first5=Louis A. |last6=Kates |first6=Steven A. |date=1997-07-07 |title=On the use of PyAOP, a phosphonium salt derived from HOAt, in solid-phase peptide synthesis |url=https://www.sciencedirect.com/science/article/pii/S0040403997010113 |journal=Tetrahedron Letters |volume=38 |issue=27 |pages=4853–4856 |doi=10.1016/S0040-4039(97)01011-3 |issn=0040-4039|url-access=subscription }}</ref>
==== Propanephosphonic acid anhydride ====
Since late 2000s, [[propanephosphonic acid anhydride]], sold commercially under various names such as "T3P", has become a useful reagent for amide bond formation in commercial applications. It converts the oxygen of the carboxylic acid into a leaving group, whose peptide-coupling byproducts are water-soluble and can be easily washed away. In a performance comparison between propanephosphonic acid anhydride and other peptide coupling reagents for the preparation of a nonapeptide drug, it was found that this reagent was superior to other reagents with regards to yield and low epimerization.<ref>{{Cite journal |last1=Hiebl |first1=J. |last2=Baumgartner |first2=H. |last3=Bernwieser |first3=I. |last4=Blanka |first4=M. |last5=Bodenteich |first5=M. |last6=Leitner |first6=K. |last7=Rio |first7=A. |last8=Rovenszky |first8=F. |last9=Alberts |first9=D.P. |last10=Bhatnagar |first10=P.K. |last11=Banyard |first11=A.F. |last12=Baresch |first12=K. |last13=Esch |first13=P.M. |last14=Kollmann |first14=H. |last15=Mayrhofer |first15=G. |date=1999 |title=Large-scale synthesis of hematoregulatory nonapeptide SK&F 107647 by fragment coupling |url=https://onlinelibrary.wiley.com/doi/10.1034/j.1399-3011.1999.00089.x |journal=The Journal of Peptide Research |language=en |volume=54 |issue=1 |pages=54–65 |doi=10.1034/j.1399-3011.1999.00089.x |pmid=10448970 |issn=1397-002X|url-access=subscription }}</ref>

===Solid supports===
[[File:Poly(styrene-co-divinylbenzene).png|thumb|right|288px|Cross-linked polystyrene is the most common solid support used in SPPS.]]
Solid supports for peptide synthesis are selected for physical stability, to permit the rapid filtration of liquids.  Suitable supports are inert to reagents and solvents used during SPPS and allow for the attachment of the first amino acid.<ref name=AlbericioPractical00>{{cite book | vauthors = Albericio F | title = Solid-Phase Synthesis: A Practical Guide | year = 2000 | location = Boca Raton | edition = 1 | publisher = CRC Press | page = 848 | isbn = 978-0-8247-0359-2 }}</ref> Swelling is of great importance because peptide synthesis takes place inside the swollen pores of the solid support.<ref>{{cite journal | vauthors = Kent SB | title = Chemical synthesis of peptides and proteins | journal = Annual Review of Biochemistry | volume = 57 | issue = 1 | pages = 957–989 | date = 1988 | pmid = 3052294 | doi = 10.1146/annurev.bi.57.070188.004521 }}</ref>

Three primary types of solid supports are: gel-type supports, surface-type supports, and composites.<ref name=AlbericioPractical00/> Improvements to solid supports used for peptide synthesis enhance their ability to withstand the repeated use of TFA during the deprotection step of SPPS.<ref>{{cite journal | vauthors = Feinberg RS, Merrifield RB | title = Zinc chloride-catalyzed chloromethylation of resins for solid phase peptide synthesis | year = 1974 | journal = Tetrahedron | volume = 30| issue = 17 | pages = 3209–3212 | doi=10.1016/S0040-4020(01)97575-1 }}</ref> Two primary resins are used, based on whether a C-terminal carboxylic acid or amide is desired. The Wang resin was, {{as of|1996|lc=y}}, the most commonly used resin for peptides with C-terminal carboxylic acids.<ref>{{cite journal | doi = 10.1016/S0040-4020(97)00279-2 | vauthors = Hermkens PH, Ottenheijm HC, Rees DC | title = Solid-phase organic reactions II: A review of the literature Nov 95 – Nov 96 | year = 1997 | journal = Tetrahedron | volume = 53 | issue = 16 | pages = 5643–5678 }}</ref>{{update inline|date=September 2018}}

===Protecting groups schemes===
{{more citations needed section|date=June 2017}}

As described above, the use of N-terminal and side chain [[protecting groups]] is essential during peptide synthesis to avoid undesirable side reactions, such as self-coupling of the activated amino acid leading to ([[polymerization]]).<ref name="Isidro-Llobet09" /> This would compete with the intended peptide coupling reaction, resulting in low yield or even complete failure to synthesize the desired peptide.{{cn|date=January 2024}}

Two principle protecting group schemes are typically used in solid phase peptide synthesis: so-called Boc/benzyl and Fmoc/''tert-''butyl approaches.<ref name="chan00" /> The Boc/Bzl strategy utilizes [[trifluoroacetic acid|TFA]]-labile N-terminal [[tert-Butyloxycarbonyl protecting group|Boc]] protection alongside side chain protection that is removed using anhydrous [[hydrogen fluoride]] during the final cleavage step (with simultaneous cleavage of the peptide from the solid support). Fmoc/tBu SPPS uses base-labile [[Fluorenylmethyloxycarbonyl protecting group|Fmoc]] N-terminal protection,<ref>{{cite journal |doi=10.1002/psc.2836 |title=Advances in Fmoc solid-phase peptide synthesis |date=2016 |last1=Behrendt |first1=Raymond |last2=White |first2=Peter |last3=Offer |first3=John |journal=Journal of Peptide Science |volume=22 |issue=1 |pages=4–27 |pmid=26785684 |pmc=4745034 }}</ref> with side chain protection and a resin linkage that are acid-labile (final acidic cleavage is carried out via TFA treatment). Both approaches, including the advantages and disadvantages of each, are outlined in more detail below.

====Boc/Bzl SPPS====
{{Main|Tert-butyloxycarbonyl protecting group}}
[[File:Boc deprotection peptide.svg|thumb|720x720px|Cleavage of the Boc group|center]]
Before the advent of SPPS, solution methods for chemical peptide synthesis relied on ''tert''-butyloxycarbonyl (abbreviated 'Boc') as a temporary N-terminal α-amino protecting group. The Boc group is removed with acid, such as [[trifluoroacetic acid]] (TFA). This forms a positively charged amino group in the presence of excess TFA (note that the amino group is not protonated in the image on the right), which is neutralized and coupled to the incoming activated amino acid.<ref name=KentBocNeutr07>{{cite journal | doi = 10.1007/s10989-006-9059-7 | vauthors = Schnolzer MA, Jones A, Alewood D, Kent SB | title = In Situ Neutralization in Boc-chemistry Solid Phase Peptide Synthesis | year = 2007 | journal = Int. J. Peptide Res. Therap. | volume = 13 | issue = 1–2 | pages = 31–44 | s2cid = 28922643 }}</ref> Neutralization can either occur prior to coupling or ''in situ'' during the basic coupling reaction.

The Boc/Bzl approach retains its usefulness in reducing peptide [[Protein aggregation|aggregation]] during synthesis.<ref>{{cite journal | doi = 10.1016/0040-4039(92)80014-B | vauthors = Beyermann M, Bienert M | title = Synthesis of difficult peptide sequences: A comparison of Fmoc-and BOC-technique | year = 1992 | journal = Tetrahedron Letters | volume = 33 | issue = 26 | pages = 3745–3748 }}</ref> In addition, Boc/benzyl SPPS may be preferred over the Fmoc/''tert-''butyl approach when synthesizing peptides containing base-sensitive moieties (such as [[depsipeptide]]s or thioester moeities), as treatment with base is required during the Fmoc deprotection step (see below).

Permanent side-chain protecting groups used during Boc/benzyl SPPS are typically benzyl or benzyl-based groups.<ref name="Isidro-Llobet09" /> Final removal of the peptide from the solid support occurs simultaneously with side chain deprotection using anhydrous [[hydrogen fluoride]] via hydrolytic cleavage. The final product is a fluoride salt which is relatively easy to solubilize. Scavengers such as [[cresol]] must be added to the HF in order to prevent reactive cations from generating undesired byproducts.

====Fmoc/''t''Bu SPPS====
{{See also|Fluorenylmethyloxycarbonyl protecting group}}
[[File:Fmoc-PG_Cleavage.png|thumb|300px|right|'''Cleavage of the Fmoc group.''' Treatment of the Fmoc-protected amine with [[piperidine]] results in proton abstraction from the [[methine group]] of the [[fluorene|fluorenyl]] ring system. This leads to release of a [[carbamate]], which decomposes into carbon dioxide ([[carbon dioxide|CO<sub>2</sub>]]) and the free amine. [[Dibenzofulvene]] is also generated. This reaction is able to occur due to the acidity of the [[fluorene|fluorenyl]] proton, resulting from stabilization of the [[aromatic]] anion formed. The [[dibenzofulvene]] by-product can react with [[nucleophiles]] such as the piperidine (which is in large excess), or potentially the released amine.<ref>{{cite book | vauthors = Jones J | date = 1992 | title = Amino Acid and Peptide Synthesis | location = Oxford, UK | publisher = Oxford University Press }}</ref>]]

The use of N-terminal [[Fluorenylmethyloxycarbonyl protecting group|Fmoc]] protection allows for a milder deprotection scheme than used for Boc/Bzl SPPS, and this protection scheme is truly orthogonal under SPPS conditions.<ref>{{cite journal | vauthors = Luna OF, Gomez J, Cárdenas C, Albericio F, Marshall SH, Guzmán F | title = Deprotection Reagents in Fmoc Solid Phase Peptide Synthesis: Moving Away from Piperidine? | journal = Molecules | volume = 21 | issue = 11 | pages = 1542 | date = November 2016 | pmid = 27854291 | pmc = 6274427 | doi = 10.3390/molecules21111542 | doi-access = free }}</ref> Fmoc deprotection utilizes a base, typically 20–50% [[piperidine]] in [[Dimethylformamide|DMF]].<ref name=AlbericioPractical00/> The exposed amine is therefore neutral, and consequently no neutralization of the peptide-resin is required, as in the case of the Boc/Bzl approach. The lack of electrostatic repulsion between the peptide chains can lead to increased risk of aggregation with Fmoc/''t''Bu SPPS however. Because the liberated fluorenyl group is a chromophore, Fmoc deprotection can be monitored by UV absorbance of the reaction mixture, a strategy which is employed in automated peptide synthesizers.

The ability of the Fmoc group to be cleaved under relatively mild basic conditions while being stable to acid allows the use of side chain protecting groups such as Boc and ''t''Bu that can be removed in milder acidic final cleavage conditions (TFA) than those used for final cleavage in Boc/Bzl SPPS (HF). Scavengers such as water and [[triisopropylsilane]] (TIPS) are most commonly added during the final cleavage in order to prevent side reactions with reactive cationic species released as a result of side chain deprotection. Nevertheless, many other scavenger compounds could be used as well.<ref>{{cite journal | vauthors = Huang H, Rabenstein DL | title = A cleavage cocktail for methionine-containing peptides | journal = The Journal of Peptide Research | volume = 53 | issue = 5 | pages = 548–553 | date = May 1999 | pmid = 10424350 | doi = 10.1034/j.1399-3011.1999.00059.x }}</ref><ref>{{Cite web |title=Cleavage Cocktails; Reagent B; Reagent H; Reagent K; Reagent L; Reagent R |url=https://www.peptide.com/resources/solid-phase-peptide-synthesis/cleavage-cocktails/ |access-date=2022-11-13 |website=AAPPTEC |language=en-US}}</ref><ref>{{cite book | vauthors = Dick F | chapter = Acid Cleavage/Deprotection in Fmoc/tBiu Solid-Phase Peptide Synthesis |date=1995 | title =Peptide Synthesis Protocols | series = Methods in Molecular Biology | volume = 35 |pages=63–72 | veditors = Pennington MW, Dunn BM |place=Totowa, NJ |publisher=Humana Press |language=en |doi=10.1385/0-89603-273-6:63 | pmid = 7894609 |isbn=978-1-59259-522-8 }}</ref> The resulting crude peptide is obtained as a TFA salt, which is potentially more difficult to solubilize than the fluoride salts generated in Boc SPPS.

Fmoc/''t''Bu SPPS is less [[Atom economy|atom-economical]], as the fluorenyl group is much larger than the Boc group. Accordingly, prices for Fmoc amino acids were high until the large-scale piloting of one of the first synthesized peptide drugs, [[enfuvirtide]], began in the 1990s, when market demand adjusted the relative prices of Fmoc- vs Boc- amino acids.

====Other protecting groups====

=====Benzyloxy-carbonyl=====
{{See also|Carboxybenzyl}}
The (Z) group is another carbamate-type amine protecting group, discovered by [[Leonidas Zervas]] in the early 1930s and usually added via reaction with [[benzyl chloroformate]].<ref name=":1">{{Cite book|url=https://org.chem.uoa.gr/istoriki_exelixi_toy_ergastirioy_organikis_chimeias_historical_development_of_organic_chemistry_laboratory/afieroma_ston_leonida_zerba_dedication_to_prof_leonidas_zervas/i_symboli_toy_l_zerba_stin_epistimi_tis_chimeias_contribution_of_l_zervas_in_the_science_of_chemistry/|title=The Chemistry of Polypeptides|publisher=Plenum Press|year=1973|isbn=978-1-4613-4571-8|veditors=Katsoyannis PG|location=New York|doi=10.1007/978-1-4613-4571-8|s2cid=35144893|archive-date=13 October 2022|access-date=1 April 2021|archive-url=https://web.archive.org/web/20221013010830/https://org.chem.uoa.gr/istoriki_exelixi_toy_ergastirioy_organikis_chimeias_historical_development_of_organic_chemistry_laboratory/afieroma_ston_leonida_zerba_dedication_to_prof_leonidas_zervas/i_symboli_toy_l_zerba_stin_epistimi_tis_chimeias_contribution_of_l_zervas_in_the_science_of_chemistry/|url-status=dead}}</ref>
[[File:Cbz to protect N-terminus.svg|center|thumb|620x620px|Introduction of the Z protecting group from reaction with benzyl chloroformate (Z-chloride)]]
It is removed under harsh conditions using [[hydrogen bromide|HBr]] in [[acetic acid]], or milder conditions of catalytic [[hydrogenation]].

This methodology was first used in the synthesis of oligopeptides by Zervas and [[Max Bergmann]] in 1932.<ref name=":0">{{cite journal| vauthors = Bergmann M, Zervas L |author-link2=Leonidas Zervas|year=1932|title=Über ein allgemeines Verfahren der Peptid-Synthese|journal=[[Berichte der deutschen chemischen Gesellschaft]]|volume=65|issue=7|pages=1192–1201|doi=10.1002/cber.19320650722|name-list-style=vanc|author-link1=Max Bergmann}}</ref> Hence, this became known as the Bergmann-Zervas synthesis, which was characterised "epoch-making" and helped establish synthetic peptide chemistry as a distinct field.<ref name=":1" /> It constituted the first useful lab method for controlled peptide synthesis, enabling the synthesis of previously unattainable peptides with reactive side-chains, while Z-protected amino acids are also prevented form undergoing [[racemization]].<ref name=":1" /><ref name=":0" />

The use of the Bergmann-Zervas method remained the standard practice in peptide chemistry for two full decades after its publication, superseded by newer methods (such as the Boc protecting group) in the early 1950s.<ref name=":1" /> Nowadays, while it has been used periodically for α-amine protection, it is much more commonly used for side chain protection.

=====Alloc and miscellaneous groups=====
The allyloxycarbonyl (alloc) protecting group is sometimes used to protect an amino group (or carboxylic acid or alcohol group) when an [[orthogonal deprotection]] scheme is required. It is also sometimes used when conducting on-resin cyclic peptide formation, where the peptide is linked to the resin by a side-chain functional group. The Alloc group can be removed using [[tetrakis(triphenylphosphine)palladium(0)]].<ref>{{Cite journal | vauthors = Thieriet N, Alsina J, Giralt E, Guibé F, Albericio F | doi = 10.1016/S0040-4039(97)01690-0| title = Use of Alloc-amino acids in solid-phase peptide synthesis. Tandem deprotection-coupling reactions using neutral conditions| journal = Tetrahedron Letters| volume = 38| issue = 41| pages = 7275–7278| year = 1997 }}</ref>

For special applications like synthetic steps involving [[protein microarray]]s, protecting groups sometimes termed "lithographic" are used, which are amenable to [[photochemistry]] at a particular wavelength of light, and so which can be removed during [[lithography|lithographic]] types of operations.<ref>{{cite journal | vauthors = Shin DS, Kim DH, Chung WJ, Lee YS | title = Combinatorial solid phase peptide synthesis and bioassays | journal = Journal of Biochemistry and Molecular Biology | volume = 38 | issue = 5 | pages = 517–525 | date = September 2005 | pmid = 16202229 | doi = 10.5483/BMBRep.2005.38.5.517 | doi-access = free }}</ref><ref>{{cite journal | vauthors = Price JV, Tangsombatvisit S, Xu G, Yu J, Levy D, Baechler EC, Gozani O, Varma M, Utz PJ, Liu CL | display-authors = 6 | title = On silico peptide microarrays for high-resolution mapping of antibody epitopes and diverse protein-protein interactions | journal = Nature Medicine | volume = 18 | issue = 9 | pages = 1434–1440 | date = September 2012 | pmid = 22902875 | pmc = 3491111 | doi = 10.1038/nm.2913 }}</ref><ref>{{Cite journal | vauthors = Hedberg-Dirk EL, Martinez UA |date=2010-08-08 |title=Large-Scale Protein Arrays Generated with Interferometric Lithography for Spatial Control of Cell-Material Interactions |journal=Journal of Nanomaterials |language=en |volume=2010 |pages=e176750 |doi=10.1155/2010/176750 |issn=1687-4110|doi-access=free }}</ref><ref>{{cite journal | vauthors = Fodor SP, Read JL, Pirrung MC, Stryer L, Lu AT, Solas D | title = Light-directed, spatially addressable parallel chemical synthesis | journal = Science | volume = 251 | issue = 4995 | pages = 767–773 | date = February 1991 | pmid = 1990438 | doi = 10.1126/science.1990438 | bibcode = 1991Sci...251..767F }}</ref>

====Regioselective disulfide bond formation====
The formation of multiple native disulfides remains challenging of native peptide synthesis by solid-phase methods. Random chain combination typically results in several products with nonnative disulfide bonds.<ref>{{cite journal | doi = 10.1021/ja00017a044 | vauthors = Tam JP, Wu CR, Liu W, Zhang JW | year = 1991 | title = Disulfide bond formation in peptides by dimethyl sulfoxide. Scope and applications | journal = J. Am. Chem. Soc. | volume = 113 | pages = 6657–6662 | issue = 17 }}</ref> Stepwise formation of disulfide bonds is typically the preferred method, and performed with thiol protecting groups.<ref>{{cite journal | vauthors = Sieber P, Kamber B, Hartmann A, Jöhl A, Riniker B, Rittel W | title = [Total synthesis of human insulin. IV. Description of the final steps (author's transl)] | journal = Helvetica Chimica Acta | volume = 60 | issue = 1 | pages = 27–37 | date = January 1977 | pmid = 838597 | doi = 10.1002/hlca.19770600105 }}</ref> Different thiol protecting groups provide multiple dimensions of orthogonal protection. These orthogonally protected cysteines are incorporated during the solid-phase synthesis of the peptide. Successive removal of these groups, to allow for selective exposure of free thiol groups, leads to disulfide formation in a stepwise manner. The order of removal of the groups must be considered so that only one group is removed at a time.

Thiol protecting groups used in peptide synthesis requiring later regioselective disulfide bond formation must possess multiple characteristics.<ref name=":2">{{cite journal | vauthors = Spears RJ, McMahon C, Chudasama V | title = Cysteine protecting groups: applications in peptide and protein science | journal = Chemical Society Reviews | volume = 50 | issue = 19 | pages = 11098–11155 | date = October 2021 | pmid = 34605832 | doi = 10.1039/D1CS00271F | s2cid = 238258277 | doi-access = free }}</ref><ref>{{cite journal | vauthors = Laps S, Atamleh F, Kamnesky G, Sun H, Brik A | title = General synthetic strategy for regioselective ultrafast formation of disulfide bonds in peptides and proteins | journal = Nature Communications | volume = 12 | issue = 1 | pages = 870 | date = February 2021 | pmid = 33558523 | pmc = 7870662 | doi = 10.1038/s41467-021-21209-0 | bibcode = 2021NatCo..12..870L }}</ref> First, they must be reversible with conditions that do not affect the unprotected side chains. Second, the protecting group must be able to withstand the conditions of solid-phase synthesis. Third, the removal of the thiol protecting group must be such that it leaves intact other thiol protecting groups, if orthogonal protection is desired. That is, the removal of PG A should not affect PG B. Some of the thiol protecting groups commonly used include the acetamidomethyl (Acm), ''tert''-butyl (But), 3-nitro-2-pyridine sulfenyl (NPYS), 2-pyridine-sulfenyl (Pyr), and [[trityl]] (Trt) groups.<ref name=":2" /> Importantly, the NPYS group can replace the Acm PG to yield an activated thiol.<ref>{{cite journal | vauthors = Ottl J, Battistuta R, Pieper M, Tschesche H, Bode W, Kühn K, Moroder L | title = Design and synthesis of heterotrimeric collagen peptides with a built-in cystine-knot. Models for collagen catabolism by matrix-metalloproteases | journal = FEBS Letters | volume = 398 | issue = 1 | pages = 31–36 | date = November 1996 | pmid = 8946948 | doi = 10.1016/S0014-5793(96)01212-4 | s2cid = 24688988 | doi-access = free }}</ref>

Using this method, Kiso and coworkers reported the first total synthesis of insulin in 1993.<ref name = Kiso>{{cite journal | doi = 10.1021/ja00077a043 | vauthors = Akaji K, Fujino K, Tatsumi T, Kiso Y | year = 1993 | title = Total synthesis of human insulin by regioselective disulfide formation using the silyl chloride-sulfoxide method | journal = Journal of the American Chemical Society | volume = 115 | issue = 24 | pages = 11384–11392 }}</ref> In this work, the A-chain of insulin was prepared with following protecting groups in place on its cysteines: CysA6(But), CysA7(Acm), and CysA11(But), leaving CysA20 unprotected.<ref name = Kiso/>