Activated Esters Reactivity

FIGURE 7.8 Aminolysis of an activated ester produces a tetrahedral intermediate, the decomposition of which is the rate-limiting step of the reaction. 

Chloroform Dioxane Dioxane- Pyridine Ethyl Dimethyl- 

FIGURE 7.9 Half-life in minutes of Z-Phe-OC6H2Cl3 (1CHM) in the presence of benzylamine (10 2 M) in various solvents at 25°C.20 C6H2C13 = 2,4,5-trichlorophenyl. 

G Kupryszewski, M Formela. Amino acid chlorophenyl esters. HI N-Protected amino acid pentachlorophenyl esters. Rocz Chem 35, 931, 1961. 

J Pless, RA Boissonnas. On the velocity of aminolysis of a variety of new activated N-protected a-amino-acid phenyl esters, in particular 2,4,5-trichlorophenyl esters. Helv Chim Acta 46, 1609, 1963. 

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In the second major method of peptide synthesis the carboxyl group is activated by converting it to an active ester, usually a p-nitrophenyl ester. Recall from Section 20.12 that esters react with ammonia and amines to give fflnides. p-Nitrophenyl esters are much more reactive than methyl and ethyl esters in these reactions because p-nitrophenoxide is a better (less basic) leaving group than methoxide and ethoxide. Simply allowing the active ester and a C-protected amino acid to stand in a suitable solvent is sufficient to bring about peptide bond formation by nucleophilic acyl substitution. 

Polymer-supported esters are widely used in solid-phase peptide synthesis, and extensive information on this specialized protection is reported annually. Some activated esters that have been used as macrolide precursors and some that have been used in peptide synthesis are also described in this chapter the many activated esters that are used in peptide synthesis are discussed elsewhere. A useful list, with references, of many protected amino acids (e.g., -NH2, COOH, and side-chain-protected compounds) has been compiled/ Some general methods for the preparation of esters are provided at the beginning of this chapter conditions that are unique to a protective group are described with that group/ Some esters that have been used as protective groups are included in Reactivity Chart 6. 

A. Synthetic Methods.—There have been no strikingly new approaches to the general problem of phosphorylation, but several ingenious methods of preparing suitable active esters under mild conditions have been reported. Typical of these is the reactive intermediate (1) formed from reaction of a mono- or di-ester of phosphoric acid with (2), itself produced by reaction of triphenylphosphine with bis(2-pyridyl) disulphide (preferably in the presence of mercuric ion as scavenger for the 2-mercaptopyridine liberated). 

Activated esters of halogenated alcohols, such as 2-chloroethanol, 2,2,2-trifluoroethanol, and 2,2,2-trichloroethanol, have been often used as substrate for enzymatic synthesis of esters, owing to an increase in the electrophilicity (reactivity) of the acyl carbonyl and avoid significant alcoholysis of the products by decreasing the nucleophilicity of the leaving alcohols. ... 

The pairs of dyes more commonly used to generate FRET have been extensively reviewed [86-88] and their conjugation to different types of molecules has been facilitated by the incorporation of a range of reactive groups. Some examples are activated esters and iso thiocyanates for reaction with amino groups of proteins or peptides,... 

Commercially available glass chips provide reactive aldehyde-, amino-, mercapto-, isothiocyante-, active ester- or epoxy-groups for covalent binding of DNA (www.arrayit.com www.picorapid.com www.prolinx.com www.accelr8.com www4.amershambiosciences.com las.perkinelmer.com www.schleicher-schuell.com www.quantifoil.com www.xenopore.com ... 

The carbodiimide of choice used to couple cystamine to carboxylate- or phosphate-containing molecules is most often the water-soluble carbodiimide, EDC hydrochloride Chapter 3, Section 1.1). This reagent rapidly reacts with carboxylates or phosphates to form an active ester intermediate, which is highly reactive toward primary amines. The reaction is efficient from pH 4.7 to 7.5, and a variety of buffers may be used, providing they don t contain competing groups. 

Carboxylic acids may be covalently modified with adipic acid dihydrazide or carbohydrazide to yield stable imide bonds with extending terminal hydrazide groups. Hydrazide functionalities don t spontaneously react with carboxylate groups the way they do with aldehyde groups (Section 4.5, this chapter). In this case, the carboxylic acid first must be activated with another compound that makes it reactive toward nucleophiles. In organic solutions, this may be accomplished by using a water-insoluble carbodiimide (Chapter 3, Section 1.4) or by creating an intermediate active ester, such as an NHS ester (Chapter 2, Section 1.4). 

In aqueous solutions, the easiest method for forming this type of bond is to use the water-soluble carbodiimide EDC (Chapter 3, Section 1.1). For proteins and other water-soluble macromolecules, EDC reacts with their available carboxylate groups to form an intermediate, highly reactive, o-acylisourea. This active ester species may further react with nucleophiles such as a hydrazide to yield a stable imide product (Figure 1.109). 

Figure 3.3 EDC may be used in tandem with sulfo-NHS to create an amine-reactive protein derivative containing active ester groups. The activated protein can couple with amine-containing compounds to form amide bond linkages.

Three main forms of amine-reactive AMCA probes are commonly available. One of them is simply the free acid form of AMCA, which can be used to couple to amine-containing molecules using the carbodiimide reaction (Chapter 3, Section 1.1). The other two are active-ester derivatives of AMCA—the water-insoluble NHS ester and the water-soluble sulfo-NHS ester forms—both of which spontaneously react with amines to create stable amide linkages. All of them react under mild conditions with primary amines in proteins and other molecules to form highly fluorescent derivatives. 

Activated esters (see Section 2.9) with 1-hydroxybenzotriazole as a catalyst are employed — pentafluorophenyl or 4-oxo-3,4-dihydrobenzotriazin-3-yl esters in particular for continuous-flow systems and special cases such as dicarboxylic amino acids. Other activated esters are not reactive enough. An alternative is preparation of benzotriazolyl esters using a carbodiimide followed by addition of the solution to the peptide-resin. 

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