Activated ester method

A general step ahead in polycondensation was achieved by the application of the active ester method by DeTar et al.19) and Kovacs et al.291 Very soon, the nitrophenyl ester, the pentachlorophenyl ester, or the hydroxysucdnimido ester were used exclusively. The esters of the protected tripeptides could be purified by crystallization, then the N-protecting group was split off and the free peptide esters were purified again. Addition of base starts the polycondensation, resulting quickly in the formation of a viscous solution at low temperature. 

Figure 16 Conjugation of an amine and a carboxylic acid via the (V-hydroxysuccinimide (NHS)-activated ester method. NHS esters may be isolated and characterized and are stable to long term storage as the powder. Alternatively, the NHS esters may be used immediately upon formation without isolation. Details of the reaction are given in Table 4...

Polyamides containing thymine photodimer units in the main chain (17a,b) were prepared by polycondensation of thymine photodimer derivatives (15a,b), which were obtained by the photochemical reaction of the monomeric compound, and various diamines by the activated ester method (Figures 4 and 5) (17, 19). 

Carboxyethyl derivatives of the nucleic acid bases were grafted onto poly-L-lysine by using the activated ester method ( ). 

Several synthetic methods are available for the preparation of Fc peptide conjugates, of which the active ester method is most compatible with biological environments.41 This method works under mild conditions with an isolable FcCO-active ester, in which reactive heterocyclic isolable esters are formed. These can be isolated or reacted in situ with suitable peptides to give the desired Fc-peptide conjugate. These active esters can be used as stoichiometric Fc delivery reagents, which make them suitable for automated solid-phase synthesis of Fc-peptide conjugates. 

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 ... 

Solid-phase techniques can be applied to the N-activated esters.16 In the activated ester method, 4-nitrophenyl, 12 21-27 2,4-dinitrophenyl, 614 28 2,4,5-trichlorophenyl, 29 and penta-fluorophenyl 8 esters can be used, the amide bonds being formed with aminolytic release of the corresponding phenol (Scheme 4). For example, the use of pentafluorophenyl esters in the synthesis of pure azapeptides (named azatides for the first time) proved to be advantageous. 8 ... 

This coupling method seems to be equally suited to the synthesis of normal azaamino acid compared to the activated ester method. 

In general, the active ester method gives the better yield because the coupling reaction can be run in a single-phase solvent system such as DMF/H20. In such a solvent system, the active ester and salt of 1-aminoalkylphosphonic acids (sodium or triethylamine) are soluble. 

Dialkyl 1-aminoalkylphosphonates are excellent substrates for the synthesis of peptides with a C-terminal phosphonate moiety. The DCC (Scheme 10) and active ester methods (Scheme 11) provide good to excellent yields of the product protected at the N-terminal and P-terminal. Several selective methods to remove the protecting groups are available. Total deprotection is achieved by the action of HBr in AcOH. 

The literature data on the preparation of phosphono-dipeptides from 1-aminoalkanephosphonic acids showed 7-7 that the yields of condensation reactions are usually small or moderate. Moreover,the use of bulky N-blocked amino acids drastically decreased the reaction yield. Thus following Martell s method— we wre unsuccesful in the preparation of dipeptides containing N-terminal valine or leucine,while peptides of phenylalanine were obtained in 5—10% yield.Also the active ester method appeared to give small yields of the desired products. Our studies using p-nitrophenyl- and cyanomethyl esters of N-phtaloyl amino acids confirmed these observations. 

The Active Esters Method. Selected amino-protecting groups and conditions required for their deprotection in organic solvents are shown in Table III. Active N-hydroxysuccinimide esters of f-butyloxycarbonyl-L-... 

Figure 2. General scheme for covalent attachment of amino acids to proteins by the active-esters method. Reaction conditions are described in Ref. 22 TFA = trifluoroacetic acid.

Then another N-protected amino acid is coupled to the free amino group of the polymer-bound substrate using the dicyclohexylcarbodiimide activation or the active ester method. The N-deblocking and coupling steps are repeated until the desired sequence is formed. Finally the resin-peptide bond is split by a suitable acid cleavage reaction with HBr—AcOH, trifluoroacetic acid or HF. This results in a simultaneous N-deblocking and deprotection of most of the side-chain functionalities. 

The polymeric active ester method has been used successfully for the preparation of several small- to medium-sized peptides in very pure form the potentiality of the method has also been illustrated by the synthesis of a number of biologically active protein sequences like bradykinin 106), thyrotropin-releasing hormone 107), ACTH sequences 105), and LH—RH 108> in good overall yields. 

This technique is characterized by an activation of probe functionalities that easily react with the modified surface. The methods for activation are derived from protein chemistry and occur in the activation of the carboxyl group (carbodiimide method, active ester method, reactive anhydrides [13,14]). Due to the similarity to the carboxyl group the activation methods are applied to phosphate and sulfonate groups as well (Fig. 11). 

Activated ester method The reactivity of carboxylates and phosphates can be increased by electron drawn and polarizing compounds, l-chloro-4-nitrobenzene or chloroacetonitril are added to form a reactive ester that polarizes the 5 -phosphate group to a partially positive charge. As noted above, the positivated phosphor atom is susceptible to a nucleophilic attack forming a covalent bond. 

The reaction of 42 with 43 was attempted with dicyciohexylcarbodiimide (DCC) or water-soluble carbodiimide in solvent systems. All these reactions seemed to be unsuccessful because of the low solubility and low reactivity of 43. Among the methods of activation of a carboxy group, only the activated ester method of p-nitrophenyl ester was successful. In the acid chloride method a side reaction with the amino group of adenine was reported56. ... 

The poly-L-lysine derivatives containing pendant nucleic add bases can be prepared alternatively by using a polymer modification reaction69 (Scheme 19). Carboxyethyl derivatives of the bases were grafted onto poly-L-lysine by using the activated ester method . Poly-L-lysine was allowed to react in this case as trifluoroacetate71. ... 

In fact, many methods discussed above, such as that of Corey and Mukaiyama, in the final analysis, fall into the activated ester class. Several other real activated ester methods are given below. 

Many syntheses of 3-endorphin have been achieved in the past, either in solution or on solid-phase. For example, an A -Fmoc synthesis on solid-phase was reported by Atherton et al. (134). / -Alkoxybenzyl ester resin was selected as the solid support coupling was done by using the symmetric anhydride and HOBt active ester method, except that Asn and Ghi were coupled as / -nitrophenyl esters. The A"-Fmoc groups were deprotected by 20% piperidine in DMF. After completing the... 

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