Amino acid and peptide bioconjugates

Nikolett Mihala and Ferenc Hudecz

DOI: 10.1039/9781849734677-00001

Introduction

Bioconjugate research is a dynamic and trans-disciplinary field with fast development. During the last two decades the main focus of bioconjugate chemistry has been the chemical synthesis and functional characterization of two- or three, sometimes even multi-component systems in which the partner molecules are attached by covalent bond and preserve relevant functional properties (like biological activity or "reporter properties") after conjugation.

To achieve this, from the viewpoint of organic chemistry there are two main "restrictions": No. 1. Only that part of the partner molecule could be chosen (or structurally modified/derivatized, if needed) for conjugation, which has no or negligible effect on the desired functional property of the product. No. 2. There is a need to develop conjugation strategy (e.g. development a new site of reaction, inclusion a spacer entity), which will lead to a conjugate compound possessing the selected characteristics of both parent molecules (e.g. after coupling a fluorophore to a peptide hormone, the conjugate must be able to act as a hormone as well as a fluorophore).

Considering these "limitations" several new and exciting organic synthetic approaches and strategies, like "click chemistry", bioorthogonal chemistry and also novel analytical methodologies were developed. The intellectual challenge and the practical importance of this research field could be demonstrated by the emerging appreciation of the journal of Bioconjugate Chemistry, established by the American Chemical Society in 1990 as documented by the IF > 5.0 value.

Several comprehensive books and reviews published in the last years have summarized and discussed the state-of-the-art in the area of synthesis and application of bioconjugate techniques. However, many aspects of the synthesis of peptide-bioconjugate that can not be encompassed by this chapter. These include, for instance the synthesis of glycopeptide/glycoprotein, peptide-oligonucleotide bioconjugates or the application of peptide bioconjugates in nanoscience.

We have made an attempt to provide an adequate coverage on the more widely used and available methods. The references and suggestions for further reading will hopefully provide good starting points for the inquisitive reader.

Current trends

Synthesis

The state-of-art in the repertoire of organic synthetic transformations enables the access of highly complex molecular structures. Obviously the chemical toolbox for the conjugation of amino acids/peptides is much more restricted because should proceed in mild conditions. i.e. in aqueous solution and at near-ambient temperature. Moreover, such modifications must be highly chemoselective in the sense that only a single target moeity is to be modified in the presence of a myriad of other functionalities.

Bioconjugation methods of peptides rely heavily on chemoselective modification of functionalities of the side chains of amino acids. Lysine (tipically by acylation or alkylation) and cysteine (by alkylation, acylation and redox reactions) side chains are the most commonly functionalized amino acids. Alternatively, the carboxylic functionality of aspartate and glutamate residues can be activated by formation of active ester.

Recently, significant advancements have opened new avenues in bioconjugation chemistry. Several strategies have emerged that allow specific tailoring of oligo- and polypeptides through either endogenous residues or introduced functionality.

At the same time existing techniques have been expanded to enable the synthesis of a wider range of bioconjugates. Classical organic reactions have been explored in the context of bioconjugation as well. Furthermore a number of potentially useful bioconjugation reactions have been described, but have not yet been used in diverse conjugation applications.

Few methods – if any – are designed to cope with all reaction conditions, but all known factors and practical consideration should be taken into account when selecting a strategy.

1 Amide bond forming reactions

The peptide/amide bond formation is one of the most fundamental and widespread chemical tool in nature, underlying the properties of a huge array of organic biomolecules, synthetic polymers and materials, including peptides and proteins. The outstanding stability of amides is attractive especially in the synthesis of peptide-bioconjugates.

For the experimental (bio)chemist the number of methods available for the synthesis of amide linkage are nearly beyond counting. However this very high number of the chemical reactions emphasizes the need for improved strategies for synthesis of amide functionality. V. R. Pattabiraman and J. W. Bode has recently given a critical overview of examples of ground breaking, alternative amide bond forming strategies.

1.1 Coupling reagents

Traditional approaches for formation of amide bond rely on coupling reagents. These agents convert the unreactive carboxylic function into an activated form for the reaction with a suitable amine to produce the desired amide. The most recently developed coupling reagents have been thoroughly reviewed and their potentials and limitations assessed by E. Valeur and M. Bradley and by A. El-Faham and F. Albericio.

1.2 Ligation methods

Chemoselective ligation methods have been lately summarized and thoroughly reviewed by T.K. Tiefenbrunn and P.E. Dawson and also by C.P.R. Hackenberger and D. Schwarzer.

1.2.1 Native chemical ligation. The native chemical ligation (NCL) method described by S.B. Kent and co-workers in 1994 exploits a chemo-selective reaction between two unprotected fragments, a C-terminal thioester and an N-terminal cysteine, in aqueous solution at neutral pH thus forming a "native" amide bond at the ligation site (Scheme 1). First an equilibration between the nucleophilic thiol of the N-terminal cysteine and the electophilic thioester takes place then the newly generated thioester undergoes spontaneously an irreversible intramolecular S- to N- acyl transfer yielding a native bond between the two fragments. The requirement for cysteine at the ligation juncture is an intrinsic restriction of the NCL strategy.

To overcome this limitation a number of different techniques (for reviews see) has been developed. Although NCL and related technologies has became a powerful tool for peptide hence protein synthesis surprisingly, so far have...