Carboxylic acids solid phase peptide synthesis

The actual process of solid phase peptide synthesis outlined m Figure 27 15 begins with the attachment of the C terminal ammo acid to the chloromethylated polymer m step 1 Nucleophilic substitution by the carboxylate anion of an N Boc protected C terminal... 

RD Dimarchi, JP Tam, SBH Kent, RB Merrifield. Weak-acid catalyzed pyrrolidone carboxylic acid from glutamine during solid phase peptide synthesis. Minimization by rapid coupling. J Pept Prot Res 19, 88, 1982. 

When solid-phase peptide synthesis was initially being developed, the question of whether or not a separate neutralization step is necessary was considered. Since it was known from the work of others that the chloride ion promotes racemization during the coupling step in classical peptide synthesis, and since we were deprotecting the Boc group with HC1, it seemed advisable to neutralize the hydrochloride by treatment with TEA and to remove chloride by filtration and washing. This short, additional step was simple and convenient and became the standard protocol. Subsequently, we became aware of three other reasons why neutralization was desirable (1) to avoid weak acid catalysis of piperazine-2,5-dione formation, 49 (2) to avoid acid-catalyzed formation of pyroglutamic acid (5-oxopyr-rolidine-2-carboxylic acid), 50 and (3) to avoid amidine formation between DCC and pro-tonated peptide-resin. The latter does not occur with the free amine. 

The linear precursor of, for example, 34 [51] was obtained by solid-phase peptide synthesis performed on an SASRIN -resin applying the Fmoc/ Bu strategy. This allowed the preparation of a protected peptide with both free amino and carboxylic acid termini, which were head-to-tail cyclized using diphenylphosphorylazide, leading to the protected cyclic peptide. Removal of side chain protecting groups afforded 34. 

The 9-fluorenylmethoxycarbonyl group is another distinguished contribution from the Carpino laboratory198199 to the solution-phase synthesis of peptides and latterly it has been adapted to solid-phase peptide synthesis too.200 The Fmoc group is exceptionally stable towards acid thus, carboxylic acids can be converted to acid chlorides with thionyl chloride201 or terf-butyl esters using sulfuric acid and isobutene.202 Furthermore, Fmoc groups are unscathed by HBr in... 

Transport peptides can be synthesized using either t-Boc or Fmoc solid phase peptide synthesis strategies with a synthesizer or manually. We routinely synthesize CPPs in a stepwise manner on solid support using an Applied Biosystems Model 431A peptide synthesizer. tert-Butyloxycarbonyl amino acids are coupled as 1-hydroxybenzotriazole (HOBt) esters to a p-methylbenzylhydryl-amine (MBHA) resin (65). C-terminally amidated CPPs are less prone to degradation and show higher internalization efficiency than carboxylic acid derivatives. 

Ethyl l-hydroxy-l//-l,2,3-triazole-4-carboxylate (20) has been investigated as an additive to carbodiinoides particularly for solid-phase peptide synthesis. No racemization was observed when Fmoc-protected anoino acids were coupled to the resin-bound peptides, except for histidine residues which under these reaction conditions racenoized extensively unless the activation time was kept to one minute and three equivalents of ethyl 1-hydroxy-1//-1,2,3-triazole-4-carboxylate (20) were added. Under the latter conditions no racemization was detected.The efficiency of this newly developed method has been well documented by the successful synthesis of deglycosylated human erythropoietin.t ... 

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. 

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.

The 9-fluorenylmethyl group, in the guise of the 9-fluorenylmethoxycarbonyl or Fmoc group, is the keystone in modern solid-phase peptide synthesis and we will examine its enormous influence in section 8.3.5. The great virtues of the 9-fluorenylmethyl group have also been adapted to the protection of carboxylic acids as 9-fluorenylmethyl esters (see section 6.5.3). Bodanszky and Bednarek realised the opportunity for extending the principle of 9-fluorenylmethyl activation, the aromaticity of the fluorenyl anion, to the protection of cysteine in peptide synthesis. 9-Fluorenylmethyl thioethers are stable towards iodine and acidic conditions, including HF, but they easily eliminate on treatment with piperidine or DBU. 

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