Different active ester methods

Table II. Comparison of hapten density by different active ester methods...

Two different activated ester methods were used to conjugate methoprene to a protein. Both the NHS-ester and the HNSA-ester methods produced immunogens of suitable hapten density, however the HNSA-ester method can be carried out in aqueous solution and allows for the spectrophotometric determination of hapten density. 

A well-defined monodisperse penta(L-alanine)- -butylamide H-[Ala]5-NHBu was synthesized by an activated ester method " and other natural abundant polypeptides, [Ala]n-5, [Leu]n-1 and [Leu]n-2, were synthesized by the N-carboxy a-amino-acid anhydride (NCA) method.Fully N-labelled homopolypeptides, [Ala ]n (99 at.% of N purity MASSTRACE, Inc.) and [Leu ]n (99 at.% of N purity MASSTRACE, Inc.), which show characteristic differences in conformation such as the a-helix and /3-sheet forms, were prepared by the heterogeneous polymerization of the corresponding NCAs in acetonitrile with -butylamine as an initiator. Conformational characterization of these samples was made on the basis of the conformation-dependent C and chemical shifts determined from the CP-MAS NMR method and from the characteristic bands in the IR and far-IR spectra. Figs. 38 and 39 show the 75.5 MHz C and 30.4 MHz N CP-MAS NMR spectra respectively of these fully N-labelled (99 at.% purity of N) homopolypeptides adopting the a-helical and /3-sheet forms (A) [Ala ]n-2 (a-helix), (B) [Ala ]n-1 (/3-sheet), (C) [Leu ]n-2 (a-helix), (D) [Leu ]n-1 (/3-sheet) in the solid state. Synthetic conditions and conformational characteristics of these samples are summarized... 

Stereoinversion Stereoinversion can be achieved either using a chemoenzymatic approach or a purely biocatalytic method. As an example of the former case, deracemization of secondary alcohols via enzymatic hydrolysis of their acetates may be mentioned. Thus, after the first step, kinetic resolution of a racemate, the enantiomeric alcohol resulting from hydrolysis of the fast reacting enantiomer of the substrate is chemically transformed into an activated ester, for example, by mesylation. The mixture of both esters is then subjected to basic hydrolysis. Each hydrolysis proceeds with different stereochemistry - the acetate is hydrolyzed with retention of configuration due to the attack of the hydroxy anion on the carbonyl carbon, and the mesylate - with inversion as a result of the attack of the hydroxy anion on the stereogenic carbon atom. As a result, a single enantiomer of the secondary alcohol is obtained (Scheme 5.12) [8, 50a]. 

A new and efficient method for the synthesis of optically active esters and lactones having a tertiary or a quaternary stereogenic centre at the y -position has been developed.28 Treatment of optically active 1-chlorovinyl p-tolyl sulfoxides having two different substituents at the 2-position with the lithium enolate of t -butyl acetate gave optically active adducts in 99% chiral induction from the sulfur stereogenic centre. 

One method involves the use of preactivated Fmoc-protected amino acids (e.g., pentafluorophenyl ester). This method has the advantage that only one reagent is necessary since preparation of the amino acid solutions is very simple and the likelihood of mistakes is low. One disadvantage is the higher price of the amino acid derivatives, but due to the small amount of activated amino acids used the absolute difference falls in the range of a few dollars for synthesis of an entire peptide membrane array. Another disadvantage lies in the fact that activated esters are only commercially available for the standard amino acids. 

The library was designed not to contain amino acids that could be self-acylated, thus Ser, Thr, Lys, and Cys were not part of the library. His was incorporated as a known weak acylation catalyst and was expected to be selected. After reacting the library with the dye-marked Pfp ester, and thorough washing, several beads remained red, indicating covalent attachment of the dye to the resin beads. Analysis of the peptides on these red beads showed that each active bead carried at least one His. There is, therefore, no cross catalysis between different beads. The method can be used to search for catalysts of any bimolecular reaction in which one reaction partner can be attached to a solid support and the other labeled with a dye, a fluorophore, or radioactivity. 

Thus, in this method the stepwise synthesis proceeds on the soluble polymer, as in the liquid phase method the only difference is in the coupling step where the carboxyl component is an insoluble polymeric active ester. The use of the solubilizing C-terminal macromolecular protecting group overcomes the limitations encountered in the original application of the polymer reagent method of peptide synthesis. However, due to the steric limitations characteristic of the polymer-polymer interactions, the attainment of quantitative coupling in each step of the synthesis can be difficult in this method. 

To achieve comparable results (unification of the activity, i.e. one scale), all the activities were recalculated to neat/ liter. Recalculation of the activities was performed simply using U/liter= 16.6667 ncat/hter. However, in the case of different substrates (esters of choline and thiocholine), eorreetion was made according to correlation analysis (y = 2.0188x + 2.2458 and r = 0.9998). Moreover, this correlation was demonstrated previously including not only Ellman s and Winter s methods (Elhnan et al, 1961 Winter, 1960) but also the potentiometric method. 

For an excellent review of this field up to 1966, see Hamilton-Miller [93], The different amide derivatives (68) of penicillin were investigated in the very early period. Their syntheses started from penicillanic acid anhydride, but later different mixed anhydrides were applied for this purpose [94-98]. Moreover, other procedures described in peptide chemistry are also useful, e.g. the DCC method [99], or synthesis through active esters [100],... 

The second task, resolution of synthetic D,L-amino acids has been solved by conversion of the neutral amino acids into real carboxyUc acids by acylation of the amino group, either by the benzoyl or the formyl residue. The D,L-acyl amino acids then formed diastereomeric salts with optically active bases, mostly alkaloids, which differed in their solubihty in various solvents, and so could be separated by recrystallization. This method is still in use, although enzymatic procedures, specific oxidation of the D-antipode in the presence of D-amino acid oxidase or enzymatic, stereospecific removal of iV-acyl residues from d,l-AT-acetyl-amino acids by acylase, are more convenient. Certainly, L-amino acids became accessible from nature by the ester method, but without synthetic material, extended experiments of peptide couphng would have been impossible. 

The active ester AT-succinimidyl 3-(2-p3 ridyldithio)propionate (SPDP) is the classical reagent to modify proteins with pyridyldithiopropyl (PDP) groups (20). If the protein to be modified contains many free Cys-residues this creates the risk of carrier-carrier conjugation. In this case it is possible to block the free thiols with iodo- or bromoacetic acid (or amide) before reaction with SPDP (or to use a different conjugation method, as described in Section 3.2). 

A technique of peptide synthesis different from Merrifield s method has been introduced (Fridkin et aL, 1965a, 1965b, 1966 Wieland and Birr, 1966a, 1966b). This method is based on the use of polymer-supported amino acid active esters. Wunsch (1971) refers to this technique as polymeric reagent synthesis. The method is claimed to be free from certain limitations of the classical solid-phase peptide synthesis e.g., it offers the advantage of isolation and purification of the intermediate peptides, and the synthesis does not go unchecked. However, the method has certain limitations. These will be discussed later. 

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