Editing Peptide synthesis (section)

===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>