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Amino Acids, Peptides and Proteins Volume 37
By Etelka Farkas, Maxim Ryadnov
The Royal Society of ChemistryCopyright © 2012 The Royal Society of Chemistry
All rights reserved.
Amino acid and peptide bioconjugates
Nikolett Mihala and Ferenc Hudecz
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.
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 only found limited application in the synthesis of peptide-bioconjugates.
1.2.2 Chemoselective ligation. In 2006 J.W. Bode and co-workers reported on a mechanistically unique, highly chemoselective amide forming reaction. The ligation of unprotected fragments is accomplished by an unusual decarboxylative condensation of α-ketoacids with N-alkylhydroxylamines as depicted in Scheme 2. No reagents are required for the ligation and no by-products, other than carbon dioxide and water are produced and more importantly there is no need for a special amino acid at the ligation site. Lately, α-ketoacid–hydroxylamine amide ligation (KAHA) was applied for the synthesis of human glucagon-like peptide, GLP-1 (7–36). The 30-amino-acid residue peptide was prepared in good yields and purity.
Recently X. Li et al. has published their preliminary investigations on a new chemoselective ligation method. The reaction which involves a peptide having an O-salicylaldehyde ester at the C-terminus and Ser/Thr peptide possessing a protecting group at the C-terminus proceeds rapidly in Py/AcOH (1:1). The forming N,O-benzylidene acetal intermediate in a separate step is treated with a mixture of TFA/H2O/i-Pr3SiH to give a native peptide bond (Xx- Ser/Thr) at the ligation site (Scheme 3).
Another study on a novel chemical ligation resulting in Xxx-Thr bond was reported by F. B. Dyer and co-workers. The reaction between an unprotected peptide thioacid and an aziridine-2-carbonyl containing peptide proceeds in the presence of stoichiometric amount of Cu(II) ion (Scheme 4). To suppress epimerization 1-hydroxybenzotriazole (HOBt) was added to the mixture of DMF-buffer. The forming aziridine - without isolation - was converted using water as nucleophile into the ligation product containing a Thr residue at the ligation site. The amino, carboxyl and aliphatic/aromatic hydroxyl groups were found compatible with this ligation method.
2 Reactions yielding a non-amide bond
2.1 Imine ligation
The condensation reaction of a nitrogen base with aldehydes or ketones is accomplished in aqueous solutions at neutral pH forming a carbon-nitrogen bond is a highly chemoselective reaction and as such has found wide application in the conjugation of biomolecules even though it shows sluggish reaction kinetics.
When the nitrogen base is a hydrazine the forming products are hydra- zones (C=N–N) while oximes (C=N–O) are generated when the nitrogen base is an alkoxyamine (Scheme 5). Both hydrazones and oximes are significantly more stable than those of simple imines (C=N), the products of condensation reaction of amines with aldehydes or ketones. Furthermore hydrazone can be reduced with sodium borohydride to produce the more stable hydrazide.
Recently, anilines were suggested to be effective catalysts of these reactions. This could greatly broadened their utility by allowing the use of a significantly lower concentration of reagents and reaction conditions at a pH value closer to neutral.
2.1.1 Hydrazone ligation. Hydrazone ligation chemistry has became central to the synthesis of a wide range of bioinspired compounds such as hydrazide reactive peptide tags for protein labelling. Further utility of the aniline catalyzed hydrazone ligation has been shown for attaching protein capture reagents, aldehyde-modified antibodies to model silicon dioxide surface demonstrating that the method can be exploited for biosensor applications in the biomedical field. Hydrazone ligation resulted an efficient and versatile method also for the covalent modification of quantum dots (QD).
J. B. Blanco-Canosa and co-workers reported on a straightforward aniline catalyzed arylhydrazone ligation strategy with chemoselective covalent modification of CdSe ZnS QDs. The reaction between the benzaldehyde functionalized QDs and 2-hydrazinonicotinoyl (HYNIC) protease substrate oligopeptide with a fluorescent label was found compatible with neutral aqueous buffer. The reaction proceeded quantitatively at micromolar concentrations without the use of an excess of the peptide target. Average peptide/QD ratios from 2:1 to 11:1 (mol/mol) were achieved with excellent control over the desired valency.
G. Iyer and co-workers developed a very similar method for the synthesis of modified QDs. It is based on bis-aryl hydrazone bond formation mediated by aromatic aldehyde and hydrazinonicotinate acetone hydrazone (HYNIC) activated peptide coated quantum dots. The versatility of the approach was demonstrated by controlled preparation of antibody–QD bioconjugates (Scheme 6).
In an elegant study, F.M. Brunel and co-workers described a sequential hydrazone ligation strategy to assemble multifunctional viral nanoparticles. The Cowpea mosaic virus was functionalized by benzaldehyde moiety and than conjugated to a tumour targeting peptide bearing aryl-hydrazone group. The kinetics of covalent modification can be monitored spectroscopically at λ ~350 nm due to the strong absorbance of the forming bisarylhydrazone product. In a second step, the conjugate was incubated with an arylhydrazido group modified and fluorescent PEGylated peptide. This sequential arylhydrazone ligation strategy enables the introduction of different labels in a ratio that is controlled by the reaction conditions. It is important to note that the modular nature of the method can be tailored for specific applications.
A two-step modular linkage strategy was described by D.E. Prasuhn and co-workers for the multivalent display of oligopeptides and DNA on CdSe/ZnS core/shell QDs. The approach exploits the chemoselective, ani-line-catalyzed hydrazone coupling chemistry to append hexahistidine sequences onto oligopeptides and DNA for subsequent self-assembly to QDs. This specifically facilitates the ratiometrical self-assembly to hydro- philic QDs. The versatility of these QD-biomolecular conjugates were demonstrated in a variety of targeted biosensing, hybridization, and cellular uptake assays.
A.R. Blanden et al. studied the reaction between 3-formyltyrosine and hydrazine-containing fluorophores using 4-aminophenylalanine as catalyst. The authors found that 4-aminophenylalanine preserved ~70% of the catalytic efficacy of aniline with the advantage of being much more compatible with biomolecules.
2.1.2 Oxime ligaton. Oxime ligation is a simple, yet powerful tool suited for application in many areas of bioconjugate chemistry due its high chemoselectivity and the stability of forming oxime bond under physiological conditions. Of late, a number of synthetic strategies for the introduction of functionalities allowing oxime ligation was described.
M. R. Carrasco et al. showed a highly efficient synthetic strategy for Boc- protected aminooxy and N-alkylaminooxy amines starting from two- and three-carbon Cbz-protected amino alcohols. The amino functionality enables the incorporation of the aminooxy moiety by standard amide formation protocol into biomolecules and after removal of the protecting group the forming aminooxy and N-alkylaminooxy groups enables conjugation with desired target molecules establishing various distances between the reaction partners.
R. S. Goody and co-workers established a simple and general method to incorporate oxyamino functionality into proteins. The homofunctional 1,2-bis(oxyamino)ethane linker could be introduced into protein thioester. The oxyamino - modified proteins react rapidly with keto fluorescent dyes under mild condition (Scheme 7).
M. B. Francis reported screening studies that have identified the most reactive N-terminal residues in the previously described pyridoxal 5'-phosphate (PLP)-mediated reaction. This site-specifically oxidizes the N-terminal amine of a proteins to afford a ketone functionality. A generalizable combinatorial peptide library screening platform was developed, which enables the identification of highly reactive motifs toward a desired bioconjugation reaction.
E. H. M. Lempens et al. developed a surface immobilization technique based on aniline-catalyzed oxime ligation chemistry. A set of aldehyde- or glyoxyl-functionalized peptide and protein was conjugated to biosensor chip modified with alkoxyamines.
In another study of the same research group, the controlled conjugation of tumour homing oligopeptides to an AB5-type dendritic scaffold via aniline-catalyzed oxime chemistry was reported. A short linear and a cyclic tumour homing peptide modified on solid phase by levulinic acid were incubated using 6:1 peptide/dendron ratio (mol/mol) to the aminooxy-functionalized scaffold obtaining complete substitution. The resulting pentavalent compounds were investigated for tumour homing and found that the peptide properties and overall size greatly influenced their biological behaviour (Scheme 8).
O. Renaudet et al. presented an iterative oxime ligation strategy for the assembly of multivalent bioconjugates. The method was illustrated by the assembly of structurally diverse tetravalent and hexadecavalent glycoclusters on a cyclodecapeptide template.
2.2 Staudinger ligation
The roots of this ligation method go back to the investigations of Hermann Staudinger, who in 1919 described the reaction of azides with triarylphosphines through an aza-ylide intermediate, which in aqueous medium rapidly hydrolyzes to the corresponding amine (Scheme 9).
The Staudinger ligation developed by C. Bertozzi and co-workers is a variant of this classical Staudinger reduction in which the triaryl phosphine substituted with electophilic group (i.e. methylester) captures the nucleo- philic aza-ylide intermediate by an intermolecular cyclization and creates a stable amide (Scheme 10).
Of particular note is the high compatibility of the Staudinger ligation with biological systems thus this method can offer a very attractive alternative or complement to the more traditional ligation techniques. However, it can not be applied to visualize low-abundance entities or to follow rapid biological processes due to its slow kinetics.
It is not surprising that - even considering its serious drawbacks, like the aforementioned sluggish kinetics or the sensitivity of phosphines to air oxidation - the Staudinger ligation, as a conjugation strategy, has been widely applied for the synthesis of oligopeptides and proteins through coupling chemical probes to biomolecules as well as to cell surface engineering in vivo.
Excerpted from Amino Acids, Peptides and Proteins Volume 37 by Etelka Farkas, Maxim Ryadnov. Copyright © 2012 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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