Solid-phase, anhydride synthesis

EJ Heimer, C Chang, T Lambros, J Meienhofer. Stable isolated symmetrical anhydrides of (Va-9-fluorenylmethyloxycarbonylamino acids in solid-phase peptide synthesis. hit J Pept Prot Res 18, 237, 1981. 

RB Merrifield, AR Mitchell, JE Clarke. Detection and prevention of urethane acylation during solid-phase peptide synthesis by anhydride methods. J Org Chem 39, 660, 1974. 

Tertiary aliphatic alcohol linkers have only occasionally been used in solid-phase organic synthesis [73], This might be because of the vigorous conditions required for their acylation. Esterification of resin-bound linker 4 with /V-Fmoc-prolinc [72,74] could not be achieved with the symmetric anhydride in the presence of DMAP (20 h), but required the use of /V-Fmoc-prolyl chloride (10-40% pyridine in DCM, 25 °C, 10-20 h [72]). A further problem with these linkers is that they can undergo elimination, a side reaction that cannot occur with benzyl or trityl linkers. Hence, for most applications in which a nucleophile-resistant linker for carboxylic acids is needed, 2-chlorotri-tyl- or 4-acyltrityl esters will probably be a better choice than ferf-alkyl esters. 

Less reactive than acyl halides, but still suitable for difficult couplings, are symmetric or mixed anhydrides (e.g. with pivalic or 2,6-dichlorobenzoic acid) and HOAt-derived active esters. HOBt esters smoothly acylate primary or secondary aliphatic amines, including amino acid esters or amides, without concomitant esterification of alcohols or phenols [34], HOBt esters are the most commonly used type of activated esters in automated solid-phase peptide synthesis. For reasons not yet fully understood, acylations with HOBt esters or halophenyl esters can be effectively catalyzed by HOBt and HOAt [3], and mixtures of BOP (in situ formation of HOBt esters) and HOBt are among the most efficient coupling agents for solid-phase peptide synthesis [2]. In acylations with activated amino acid derivatives, the addition of HOBt or HOAt also retards racemization [4,12,35]. 

Several syntheses of the peptide have been reported by solution methods (74-76). After the introduction of solid-phase peptide synthesis, Marshall and Merrifield conducted the first study of the synthesis of the peptide by using the new technique (77). A -Boc chemistry was used, and Merrifield resin was selected as the solid support. The side chain protections were as follows His, Arg, and Asp were protected by Bn groups Arg by a NO2 group. The Phe was esterified onto the resin in ethanol with the presence of 1 equivalent of triethylamine. The symmetric anhydride method was used for the coupling of the amino acids, and DCC was the coupling reagent. The following cycle of reactions was used to introduce each new residue (Table 7) ... 

Solid-phase peptide synthesis. The combination of the base-labile N-a-fluorenylmethoxycarbonyl (Fmoc) amino acids and the acid-labile /-butyl protecting group is valuable for solid-phase peptide synthesis, particularly with polar resins. Intermediate Fmoc-peptide resins are deprotected with 20% piperidine or 5% piperazine in DMF. Six amino acid groups can be added per day without difficulty. This new strategy was used for synthesis of human /3-endorphin (31 residues), with 29 residues added as the anhydrides of Fmoc-amino acids. The last residue was the N-a-Boc derivative of 0-/-butyltyrosine. The peptide resin was cleaved with anhydrous CF3COOH. The overall yield of isolated polypeptide was 41 %. This method does not require vigorous acidic conditions. 

In peptide chemistry two methods are used frequently. The reaction of phosgene with protected amino acids leads to symmetric anhydrides (equation 4), after disproportionation and release of carbon dioxide, which can be readily used to build up a growing peptide chain. - More recently, symmetrical amino acid anhydrides have been generated by carbodiimides like DCC, water soluble caibodiimide and diisopropylcarbodiimides in situ. They are extensively applied to autmnated solid phase peptide synthesis. Attention has to be paid to the proper solvent system, as eq)rotic dipolar solvents tend to slow down reactions and increase formation of side products. ... 

One of the first dedicated applications of microwaves in solid-phase chemistry was in the synthesis of small peptide molecules, as described by Wang and coworkers [22]. As a preliminary test, the authors coupled Fmoc-Ile and Fmoc-Val, respectively, with Gly-preloaded Wang resin using the corresponding symmetric anhydrides (Scheme 7.1). 

The reagents and methods employed for coupling in solid-phase synthesis are the same as for synthesis in solution, but a few are excluded because they are unsuitable. The mixed-anhydride method (see Section 2.6) and l-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline (see Section 2.15) are not used because there is no way to eliminate aminolysis at the wrong carbonyl of the anhydride. Acyl azides (see Section 2.13) are too laborious to make and too slow to react. The preparation of acyl chlorides (see Section 2.14) is too complicated for their routine use this may be rectified, however, by the availability of triphosgene (see Section 7.13). That leaves the following choices, bearing in mind that a two to three times molar excess of protected amino acid is always employed. 

During the first decade when solid-phase synthesis was executed using Fmoc/tBu chemistry, the first Fmoc-amino acid was anchored to the support by reaction of the symmetrical anhydride with the hydroxymethylphenyl group of the linker or support. Because this is an esterification reaction that does not occur readily, 4-dimethylaminopyridine was employed as catalyst. The basic catalyst caused up to 6% enantiomerization of the activated residue (see Section 4.19). Diminution of the amount of catalyst to one-tenth of an equivalent (Figure 5.21, A) reduced the isomerization substantially but did not suppress it completely. As a consequence, the products synthesized during that decade were usually contaminated with a small amount of the epimer. In addition, the basic catalyst was responsible for a second side reaction namely, the premature removal of Fmoc protector, which led to loading of some dimer of the first residue. Nothing could be done about the situation,... 

The major side reaction associated with the use of mixed anhydrides is aminolysis at the carbonyl of the carbonate moiety (Figure 7.4, path B). The product is a urethane that resembles the desired protected peptide in properties, except that the amino-terminal substituent is not cleaved by the usual deprotecting reagents. Hence, its removal from the target product is not straightforward. The problem is serious when the residues activated are hindered (Val, lie, MeXaa), where the amounts can be as high as 10%. Other residues generate much less, but the reaction cannot be avoided completely, with the possible exception of activated proline (see Section 7.22). This is one reason why mixed anhydrides are not employed for solid-phase synthesis. 

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