HOBt active ester formation

In 2003, Devocelle and colleagues reported a convenient two-step procedure for the parallel synthesis of hydroxamic acids (or O-protected hydroxamic acids 207) from carboxylic acids and hydroxylamine. It involves the formation of a polymer-bound HOBt active ester 206 from 204 and the acid 205 and subsequent reaction with O-protected or free hydroxylamine (Scheme 89). The use of free hydroxylamine leads to increased yields while maintaining high purities. Recycling of the exhausted resin 204 to prodnce the same or a different hydroxamic acid has been achieved by a three-step protocol, which is easily amenable to automation and cost-economical. 

There are examples of the use of supported carbodiimide 2 (R = Cy) for the formation of N-hydroxybenzotriazole (HOBt)-derived active esters from N-Boc-protected amino acids (see below), which have been subsequently employed in coupling reactions for the synthesis of dipeptide p-nitroanilides and dipeptide diphenyl phosphonates [12, 13]. Such HOBt-active esters have also been generated from pyrazinone-derived acids using 2 (R = Cy) [14], as well as from difluorophenyl acetic acids [15] for further amidation reactions in the parallel synthesis of tissue factor Vila inhibitors. 

On-resin lactam (amide) formation or cyclization can be achieved via HOBt active esters using a number of reagents. Carbodiimide and phosphonium activation have all been used for ring closure with minimal loss of chiral integrity of the activated amino acid residue. The choice of reagent and auxiliary nucleophile (i.e. HOBt, HOAt) is at the discretion of the operator (see Chapters 2 and 3). However, it is worth noting that successful peptide cyclization is, to an extent, sequence dependent. Nominal ring closure is not uncommon and is due in part to the spatial orientation of the peptide backbone and steric hindrance. 

The Z-protected derivative, again prepared by standard methods using benzyl chloroformate,t208 may serve in the case of racemic pipecolic acid for resolution into the pure enantiomers by fractional crystallization with L-tyrosine hydrazide/208 Acylation with N-protected pipecolic acid or of pipecolyl peptides is performed by standard procedures via the active ester methods, e.g. A-hydroxysuccinimide ester/121 by the mixed anhydride method, e.g. with isobutyl chloro-formate 95-114 or pivalic acid chloride/121 as well as by DCC/HOBt/118 In the synthesis on solid support, longer coupling times are required when compared to N-protected proline.1[235 ... 

In this procedure the acylating molecule is the active ester formed between the amino acyl moiety and the hydroxy group of HOAt. 40 Compared to the earlier methods utilizing HOSu 41 and HOBt, 42 HOAt maintains the known ability to suppress racemization, but is much more effective in catalyzing peptide-bond formation. This efficiency of HOAt may be attributed to the possible assistance of both N2 and N7 to the amine nucleophilic attack (Scheme 2). If the preferred active ester conformation found in the crystalline state (the heterocyclic At plane lies approximately perpendicular to that of the carboxylic ester moiety)143 is preserved in solution, then assistance to nucleophilic attack on both faces of the ester is likely. 

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]. 

In the presence of weakly acidic additives, such as Ai-hydroxysuccinimide (HOSu),P l 1,2,3-benzotriazol-l-ol (HOBt),f l 3-hydroxy-l,2,3-benzotriazin-4(3//)-one (HODhbt),b l or 7-aza-l,2,3-benzotriazol-l-ol (HOAt),f l the O N acyl migration as well as racemization/ epimerization (vide infra) are largely prevented.f d Concurrently, conversion of the highly reactive O-acylisourea derivatives and symmetrical anhydrides into the related less reactive active esters 13 derived from these additives takes place which leads to still sufficiently activated species to allow rapid in situ amide formation. 

Mixed anhydrides are also useful reagents for the generation of A-hydroxysuccinimide esters,as well as HOBt and HOAt esters. After formation of the mixed anhydride, as described in the experimental procedures below, typically, 2 equivalents of the A-hydroxy component are added followed by 2 equivalents of NMM. The active ester is rapidly formed and in about 10 min the amine component to be coupled is added. The procedure is useful for the condensation of hindered amines where wrong-way opening would otherwise be a problem. Prolylprolyl dipeptides have been obtained rapidly and in high yield with less than 2% wrong opening. 

To prevent racemization of sensitive amino acid derivatives, the activation time should be kept as short as possible (5 min) and the activation should be run at 4 °C rather than room temperature. Optimized conditions were developed for intermediate formation of active esters (DIC/HOBt or DIC/HOAt in DMF/CH2CI2, 1 1, for 5 min) and symmetrical anhydrides (activation in DMF/CH2CI2, 9 1, and coupling in DMF/CH2CI2, 1 1). 

Scheme 7.33 Synthesis of P-HOBt (20) and formation of supported active esters from N-protected amino acids.

A convergent synthesis of the potent selective inhibitor of the enzyme phosphodiesterase sildenafil (Viagra) has been based on polymer supported reagents [134], In this synthesis, the HOBt-supported resin 126 has been used for the isolation and preparation of the resin-bound active ester 128, performed by coupling polymer 126 with the benzoic acid sulfonamide derivative 127 by means of PyBrop (Scheme 7.40). Subsequent reaction of active ester 128 with aminopyrazole 129 gave rise to the clean synthesis of amide 130, which has been transformed into sildenafil (131) after a base-promoted pyrimidinone formation. 

Additives such as HOBt or DMAP can be used while attached to a polymer. Thus, the polymeric //-benzyl-1-hydroxybenzotriazole-6-sulfona-mide (19) [47] and the polymeric 1-hydroxybenzotriazole (20) [48] have been shown to be highly efficient for the solution synthesis of amides. The efficiency of 19 could be attributed to its high acidity, conferred by the sulfonyl moiety. The procedure for amide construction involves the formation of an activated ester on the derivatized polymer followed, in a second step, by treatment with an amine to generate the amide in solution. This HOBt-supported polymer has also been applied for the preparation of N-hydroxysuccinimide esters, useful for the modification of proteins [49]. Polymeric DMAP is a less basic compound and generally gives very low racemization [50]. 

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