Benzotriazoles peptide synthesis

S Chen, J Xu. A new coupling reagent for peptide synthesis. Benzotriazol-yl-bis(pyr-rolidino)-carbonium hexafluorophosphate (BCC). Tetrahedron Lett 33, 647, 1992. 

GE Reid, RJ Simpson. Automated solid-phase peptide synthesis use of 2-(lH-benzotriazol-l-yl)-l,l,3,3-tetramethyluronium tetrafluoroborate for coupling of tert-butyloxycarbonyl amino acids. Anal Biochem 200, 301, 1992. 

The utility of carbodiimide reagents was too important to consider abandoning them. Instead, it rapidly became obvious that carbodiimide activation could be used to prepare in situ active esters. Numerous nucleophilic additives were discovered and prepared for use in these reactions. The most important of these additives was 1,2,3-benzotriazol-l-ol (HOBt, 1), first reported for use in peptide synthesis by Konig and Geiger in 1970.[2(l As an example of a typical result, 1.2 equivalents of HOBt was added to the DCC coupling of Boc-Leu-Phe-OH to H-Val-OtBu in DMF as a solvent. Less than 1% of the l-d-l epimerized tripeptide was formed. When the reaction was carried out in the absence of HOBt, the amount of l-d-l product formed was 14.3%. Addition of HOBt to DCC reactions converts the intermediate G-acylisourea (2) (and any symmetrical anhydride) into the HOBt active ester 3 (Scheme 8). 

In the first step amine 11 is coupled with carboxylic acid 15 to form an amide The method employed here for coupling an a-chiral carboxylic acid with an amine was developed in the context of peptide synthesis. Its success is based on DCC-mediated formation (see Chapter 5) of the reactive 1-hydroxy benzotriazole ester 55, which reacts with an amine to give the corresponding amide. In most cases reaction takes place without raccmi/ation, and often in the absence of side reactions that cause other procedures to The alcohol is converted into a silyl ether in the second step. 

Peptide synthesis reagents such as diisopropylcarbodiimide (DIC), benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP), bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP), 2-(lH-benzotriazole-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (FlOBt), piperidine, A-methylmorpholine (NMM), trifluoroacetic acid (TEA), triisopropylsilane (TIS), A -diisopropylethylamine (DIPEA, DIEA), l,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,2-ethanedithiol (EDT), and 4-dimethylaminopyridine (DMAP) may be obtained from Sigma-Aldrich, Chemlmpex, and Novabiochem. 

This reagent, benzotriazol-l-yl-oxytripyrrolidinophosphonium hexafluorophosphate (14, PyBOP)P l (Scheme 4), was designed in order to avoid the formation of toxic HMPA during acylation. As with BOP, it is assumed that the first step is the carboxylic acid activation which involves formation of an acyloxyphosphonium salt.P This initial salt is then attacked by the benzotriazolyloxy anion to form the benzotriazolyl active ester which then reacts with the amino component. PyBOP can easily replace the BOP reagent and is especially suitable for solid-phase peptide synthesis. It is soluble in a wide range of solvents such as DMF, di-chloromethane, THF, and NMP. PyBOP is more useful in peptide synthesis on solid support than in solution. The byproduct, tris(pyrrolidino)phosphine oxide is partially water-soluble and is easily removed by washing. PyBOP is used under the same experimental conditions as BOP. Note that PyBOP is a white, crystalline and non-hygroscopic solid. It can be kept as a solid, but solutions of PyBOP cannot be stored for more than 24 hours. 

Beck-Sickinger AG, Dtirr H, Jung G (1991) Semiautomated T-bag peptide synthesis using 9-fluorcn-yl-methoxycarbonyl-strategy and benzotriazol-l-yl-tetramethyl uronium tetrafluoroborate activation, Peptide Res 4 88-94. 

Peptide synthesis. CoupUng reactions in peptide synthesis using active esters are greatly accelerated in the solution phase or the solid phase by addition of 1 -hydroxy benzotriazole. 

The preparation and implementation in peptide synthesis of analogues of phosphonium salts such as HBTU (27) and HATU (28) bearing a positive carbon atom in place of the phosphonium residue have also been reported (Fig. 7). Although they were initially assigned a uronium-type structure [29,31,70,71], presumably by analogy to the corresponding phosphonium salts, it has been determined by X-ray analysis that A -[(l/f-benzotriazol-l-yl)(dimethylamino)methylene]-iV-methylmethanaminium hexafluorophos-phate iV-oxide (HBTU), iV-[(dimethylamino)-lH-l,2,3-triazolo[4,5-fojpyridino-1 -ylmethylene] -N-methylmethanaminium hexafluorophosphate... 

Figure 2.2 Modern solid phase peptide synthesis. Process begins with a-N terminal Fmoc deprotection of resin bound C-terminal amino acid residue with piperidine (mechanism illustrated). Peptide link formation follows (typical solvent Al-methylpyrrolidone [NMP]) by carboxyl group activation with dicyclohexylcarbodiimide (DCC) (mechanism illustrated) in presence of hydroxybenzotriazole (HOBt). HOBt probably replaces DCC as an activated leaving group helping to reduce a-racemization during peptide link formation. Other effective coupling agents used in place of DCC/HOBt are HBTU 2-(lH-benzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate Py-BOP benzotriazole-l-yl-oxy-tns-pyrrolidino-phosphonium hexafluorophosphate. The Process of a-N deprotection, and peptide link formation, continues for as many times as required (n-times), prior to global deprotection and resin removal.

Microwave heating can be nsed to facilitate peptide synthesis using other non-traditional coupling methodologies. Eor example, A/-Fmoc-protected(a-aminoacyl) benzotriazoles have been nsed in the preparation of tri-, tetra-, penta-, hexa-, and heptapeptides on solid-snpport with an average crnde yield of 71% (Scheme 9.2). ° Acylbenzotriazoles are easily prepared, chirally stable analogs of acid halides that are relatively water stable. Coupling reactions of A/-protected(a-aminoacyl)... 

We have found (Laursen et al., 1980) that the addition of 1-hydroxy-benzotriazole, a catalyst frequently used in peptide synthesis (Fletcher et... 

Benzotriazole is especially usefijl as a carboxylic activating group in solution phase peptide synthesis (2013AA43). Thus the carboxylic acid groups of protected amino adds are activated by benzotriazole in the presence of thionyl chloride or DCC to afford stable, activated amino acids or peptides (e.g., 82 83) with full retention of chirality... 

---