The procedures used to combine two amino acid residues to form a peptide are referred to as coupling methods. Coupling involves nucleophilic attack by the amino group of one residue at the electrophilic carbonyl carbon atom of the carboxy-containing component that has been activated by the introduction of an electron-withdrawing group Y. Activation may be carried out either in the presence of the N-nucleophile or in the absence of the N-nucleophile, which may be by choice or by necessity. When a coupling is effected by the addition of a single compound to a mixture of the two reactants, the compound is referred to as a coupling reagent. The coupling reagent requires a subsequent deprotonation of one of the reactants to effect the reaction.The activating moiety Y is composed of either a halide atom or an azide group or an oxygen atom linked to a double-bonded carbon atom (O–C=), a cationic carbon (O–C+) or phosphorus (O–P+) atom, or a nitrogen atom adjacent to a double bonded atom (O–N–X=).


Peptide bond formation is a nucleophilic substitution reaction of an amino group (nucleophile) at a carboxyl group involving a tetrahedral intermediate. Furthermore, the peptide coupling reaction must be performed under mild conditions, and preferably at room temperature. Activation of the carboxyl component is achieved by the introduction of electron accepting moieties. Carboxyl components can be activated as acyl halides, acyl azides, acylimidazoles, anhydrides, esters etc. There are different ways of coupling reactive carboxyl derivatives with an amine.

In recent years, peptide-coupling reactions have significantly advanced in accord with the development of new peptide-coupling reagents and their application to both solution and solid- phase synthesis.

The established method for the production of synthetic peptides in the lab is known as solid-phase peptide synthesis,The general SPPS procedure is one of repeated cycles of alternate N-terminal deprotection and coupling reactions.

Peptide coupling reagents

An important feature that has enabled the broad application of SPPS is the generation of extremely high yields in the coupling step. Highly efficient amide bond-formation conditions are required.and adding an excess of each amino acid (between 2- and 10-fold). The minimization of amino acid racemization during coupling is also of vital importance to avoid epimerization in the final peptide product. Amide bond formation between an amine and carboxylic acid is slow, and as such usually requires ‘coupling reagents’ or ‘activators’. A wide range of coupling reagents exist, due in part to their varying effectivness for particular couplings, many of these reagents are commercially available.

Aminium/uronium and phosphonium salts

Some coupling reagents omit the carbodiimide completely and incorporate the HOAt/HOBt moiety as an aminium/uronium or phosphonium salt of a non-nucleophilic anion (tetrafluoroborate or hexafluorophosphate).[10] Examples of aminium/uronium reagents include HATU (HOAt), HBTU/TBTU (HOBt) and HCTU (6-ClHOBt). HBTU and TBTU differ only in the choice of anion. Phosphonium reagents include PyBOP (HOBt) and PyAOP (HOAt).

These reagents form the same active ester species as the carbo di imide activation conditions, but differ in the rate of the initial activation step, which is determined by nature of the carbon skeleton of the coupling reagent.[17] Furthermore, aminium/uronium reagents are capable of reacting with the peptide N-terminus to form an inactive guanidino by-product, whereas phosphonium reagents are not.

Structure of symmetric iminium / uronium salts coupling reagent.

Later, El-Faham and Albericio reported a new family of coupling reagents based on the modification of the structures of the carbocation skeleton moiety, which feature relatively high reactivity and low racemization during peptide bond formation . Very recently, El-Faham and Albericio extended their work taking an N-containing 6-membered ring structure containing O, S, and N-CH3 for synthesis of novel coupling reagents.

Structure of morphine based coupling reagent.

Recent reports confirmed the explosive properties of HOBt derivatives . Accordingly, El-Faham and Albericio reported the new additives as well as their uronium salts derivatives as replacement for HOBt and HOAt derivatives . Among these entire additives Oxyma and its uronium salt COMU showed an excellent replacement for HOBt and its analogues.

Structure of oxime to replace of HOBt.
Structure of oximino and benzotriazolo uronium salt.

5-(hydroxyimino)-1,3- dimethylpyrimidine-2,4,6 (1H,3H,5H)-trione (Oxyma-B) reported as an excellent additive for the suppression of racemization during peptide synthesis [27]. Oxyma-B, has the same structurefuture for the carbonyl moiety in which the oxime group is flanked between the two carbonyl group as in HONM. In addition, Oxyma-B performs better as a racemization suppressor than Oxyma Pure and even better than HOAt in both stepwise and segment coupling in solid- and solution-phase peptide synthesis . Lately,a new class of O-form uronium-type coupling reagents derived from Oxyma-B were introduced TOMBU and COMBU.

Structure of Oxyma-B (16), and its uronium salt TOMBU (17), and COMBU (18).

-The acyl-azide method of coupling has been available for about a century,

-all methods used for coupling N-alkoxycarbonylamino acids can be used to couple Nαprotected peptides.

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