Coupling reagent

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A coupling reagent (also known as coupling reagent ) is used in peptide synthesis , crosslinking , immobilization and molecule labeling to carry out reactions on amino acids .

properties

Amino acids condense with one another only slowly, both in the case of a peptide bond between two amino acids and in the case of a bond of an amino acid to a modification such as a label. By using a coupling reagent, one of the amino acids involved at the C terminus is temporarily converted into a reactive intermediate, which then readily reacts with a nucleophile (the N terminus and other amino groups as well as sulfhydryl groups must be blocked with protective groups in order to avoid undesirable reactions during peptide synthesis ), or the coupling is permanent, e.g. B. with a crosslinker or a marker. A racemization can be obtained by ionization of the hydrogen atom on the α-C atom of the C -terminal amino acid, and forming a oxazolinone - or an enol -Übergangszustands arise. For this reason, additives such as hydroxybenzotriazoles or, increasingly due to the lack of explosion hazard, Oxyma are often added to reduce racemization . The target properties in the development of coupling reagents are primarily yield , low epimerization and side reactions , and secondarily the reaction rate . There is a large number of coupling reagents, since not every combination of two amino acids with one coupling reagent has the same yield. Water-soluble coupling reagents can be used to immobilize and cross-link peptides in aqueous solutions. Coupling reagents can be divided into different substance classes.

Peptide synthesis

Carbodiimides

Examples of coupling reagents for the carbodiimides are dicyclohexylcarbodiimide (DCC) and diisopropylcarbodiimide (DIC). Water-soluble carbodiimide coupling reagents are EDC (mostly as hydrochloride) and CMC . Hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt) or ethyl hydroxyiminocyanoacetate (Oxyma) are usually used to reduce racemization . For coupling to hydroxide groups, 4- (dimethylamino) pyridine (DMAP) is used in catalytic amounts (0.15 times the stoichiometric ratio). As a side reaction, dehydration of asparagine and glutamine can occur.

Phosphonium salts

Phosphonium salts are less prone to racemization and dehydration of asparagine and glutamine than carbodiimides, e.g. B. BOP reagent , PyBOP , PyBrOP and PyOxim . BOP produces the carcinogenic hexamethylphosphoramide as a by-product . PyOxim is also an Oxyma derivative.

Immonium salts

Immonium salts in peptide synthesis are e.g. B. BOMI , BDMP, to a lesser extent AOMP, FOMP, DOMP, BPMP, and SOMP are also used.

Aminium salts

Aminium salts (originally erroneously described as uronium salts ) include e.g. B. HBTU , TBTU , HATU , HCTU , TATU . Usually, HOBt is added to reduce racemization.

Uronium salts

Uronium salts in peptide synthesis are z. B. TNTU , TPTU , TOTU , TDBTU , COMU , COMBU and TOMBU . TSTU is soluble in water. COMU, COMBU and TOMBU are also Oxyma derivatives.

Imidazolium salts

As coupling reagents, for. B. CBMIT, BOI, CIP, CIB, CMBI are used.

Other

Further coupling reagents in peptide synthesis are DEPBT , CDI and TCFH .

Marking, cross-linking and immobilization

Succinimides

N -hydroxysuccinimide , hydroxysulfosuccinimide and other succinimidyl esters react with the amino group of an amino acid.

Isothiocyanates

Phenyl isothiocyanate (PITC) and other isothiocyanates are used in Edman degradation , among others . Fluorescent derivatives are FITC and TRITC , while DABITC leads to colored coupling products. A volatile coupling reagent is trifluoroethyl isothiocyanate (TFEITC). Phenyl diisothiocyanate is used to crosslink two amino groups.

Other

Furthermore bisoxiranes , cyanogen bromide , divinyl sulfone , epichlorohydrin , glutaraldehyde , periodic acid or trichloro-s-triazine used.

Individual evidence

  1. ↑ a b Martin Holtzhauer: Biochemical laboratory methods. Springer-Verlag, 2013, ISBN 978-3-642-97111-2 , p. 88.
  2. ↑ Martin Holtzhauer: Biochemical laboratory methods. Springer-Verlag, 2013, ISBN 978-3-642-97609-4 , p. 120.
  3. ↑ a b c d e So-Yeop Han, Young-Ah Kim: Recent development of peptide coupling reagents in organic synthesis. In: Tetrahedron. 60, 2004, p. 2447, doi : 10.1016 / j.tet.2004.01.020 .
  4. ^ A b E. Valeur, M. Bradley: Amide bond formation: beyond the myth of coupling reagents. In: Chemical Society reviews. Volume 38, Number 2, February 2009, pp. 606-631, doi : 10.1039 / b701677h , PMID 19169468 .
  5. ↑ Wolfgang König, Rolf Geiger: A new method for the synthesis of peptides: Activation of the carboxyl group with dicyclohexylcarbodiimide with the addition of 1-hydroxy-benzotriazoles. In: Chemical Reports . 103, 1970, p. 788, doi : 10.1002 / cber.19701030319 .
  6. ↑ J. Klose, A. El-Faham, P. Henklein, LA Carpino, M. Bienert: Addition of HOAt dramatically improves the effectiveness of pentafluorophenyl-based coupling reagents. In: Tetrahedron Letters . 40, 1999, p. 2045, doi : 10.1016 / S0040-4039 (99) 00089-1 .
  7. ^ R. Seyer, A. Aumelas, A. Caraty, P. Rivaille, B. Castro: Repetitive BOP coupling (REBOP) in solid phase peptide synthesis. Luliberin synthesis as model. In: International journal of peptide and protein research. Volume 35, Number 5, May 1990, pp. 465-472, PMID 2198236 .
  8. ↑ A. Nagy, B. Szoke, AV Schally: Selective coupling of methotrexate to peptide hormone carriers through a gamma-carboxamide linkage of its glutamic acid moiety: benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate activation in salt coupling. In: Proceedings of the National Academy of Sciences . Volume 90, Number 13, July 1993, pp. 6373-6376, PMID 8101004 . PMC 46931 (free full text).
  9. ^ J. Coste, D. Le-Nguyen, B. Castro: PyBOP: A new peptide coupling reagent devoid of toxic by-product. In: Tetrahedron Letters. 31, 1990, p. 205, doi : 10.1016 / S0040-4039 (00) 94371-5 .
  10. ↑ Jacques Coste, Eric Frérot, Patrick Jouin, Bertrand Castro: Oxybenzotriazole free peptide coupling reagents for N-methylated amino acids. In: Tetrahedron Letters. 32, 1991, p. 1967, doi : 10.1016 / 0040-4039 (91) 85015-W .
  11. ↑ Iman Abdelmoty, Fernando Albericio, Louis A. Carpino, Bruce M. Foxman, Steven A. Kates: Structural studies of reagents for peptide bond formation: Crystal and molecular structures of HBTU and HATU. In: Letters in Peptide Science. 1, 1994, p. 57, doi : 10.1007 / BF00126274 .
  12. ^ Reinhard Knorr, Arnold Trzeciak, Willi Bannwarth, Dieter Gillessen: New coupling reagents in peptide chemistry. In: Tetrahedron Letters . 30, 1989, p. 1927, doi : 10.1016 / S0040-4039 (00) 99616-3 .
  13. ↑ LP Miranda, PF Alewood: Accelerated chemical synthesis of peptides and small proteins. In: Proceedings of the National Academy of Sciences . Volume 96, Number 4, February 1999, pp. 1181-1186, PMID 9989998 . PMC 15437 (free full text).
  14. ↑ CA Chantell, MA Onaiyekan, M. Menakuru: Fast conventional Fmoc solid-phase peptide synthesis: a comparative study of different activators. In: Journal of Peptide Science . Volume 18, Number 2, February 2012, pp. 88-91, doi : 10.1002 / psc.1419 , PMID 22147296 .
  15. ↑ CU Hjørringgaard, A. Brust, PF Alewood: Evaluation of COMU as a coupling reagent for in situ neutralization Boc solid phase peptide synthesis. In: Journal of Peptide Science . Volume 18, Number 3, March 2012, ISSN  1099-1387 , pp. 199-207, doi : 10.1002 / psc.1438 , PMID 22252935 .
  16. ^ A b Y. E. Jad, SN Khattab, BG de la Torre, T. Govender, HG Kruger, A. El-Faham, F. Albericio: TOMBU and COMBU as Novel Uronium-type peptide coupling reagents derived from Oxyma-B. In: Molecules . Volume 19, number 11, 2014, pp. 18953-18965, doi : 10.3390 / molecules191118953 , PMID 25412042 .
  17. ↑ Hubert Rehm: The Experimenter: Protein Biochemistry / Proteomics. Springer-Verlag, 2016, ISBN 978-3-662-48851-5 , p. 312.