Lecture 1: Key Concepts in Stereoselective Synthesis
Topic: Ligation and BioconjugationIntroductionMacromolecules, especially peptides up to 30-40 amino acids can reliably synthetized by solid phase peptide synthesis. Longer sequences can be accessed with fragment coupling methods. The first possibility is to couple partially protected peptide fragments followed by the removal of protecting groups. This strategy is limited by the low solubility of the fragments and by the possible epimerization of the activated C terminus.To overcome these limitations new approaches were reported. These are called chemoselective ligations of unprotected peptides or macromolecules.“The term chemoselective ligation refers to the coupling of two mutually and uniquely reactive functional groups in aqueous environment or in the presence of biological material. Thus, even among a multitude of potentially reactive functional groups, two chemoselective partners will react only with each other.”Bertozzi Trends Biotech. 1998, 16, 506The ligation reactions can be used for the chemical synthesis macromolecules or for the modification/labeling of biomolecules. On the scheme above A and B functional groups are chemoselective reaction partners.The criteria to perform chemoselective ligation/bioconjugation in biological systemsLigation and bioconjugation are essentially used to bind high molecular mass molecules together in aqueous media or biological systems. To fulfill these challenges, ligation/bioconjugation reactions have to respect certain criteria:The functionalities of the ligation reaction must react selectively with each other under mild conditionsThe reaction must yield covalent bonds and no or harmless side products like water or carbon dioxide The reactants must be stable before the ligation reaction and not toxicThe reaction must proceed with a reasonable rateBode ACS Chem. Biol. 2015, 10, 102631032458699500The graph on the right illustrates the limitations of the current ligation reactions. The low solubility of macromolecules requires working in diluted conditions (μM concentrations). To achieve good yields under reasonable time at these conditions fast reactions are required.Types of ligation reactionsLigation at cysteine junctionsThe first type of protein ligation reactions are based on the unique reactivity of the thiol function on the side chain of cysteine. This allows the chemoselective ligation of unprotected protein fragments. Unfortunately cysteine is the second rarest amino acid in natural proteins. Lau Justus Liebigs Ann. Chem. 1953, 583, 129 Native Chemical Ligation (NCL)In 1994 Kent and co-workers reported the reaction of unprotected peptides with thioesters on the N peptide and cysteine on the C peptide.Kent Science 1994, 266, 776 The method can be applied to unprotected peptide fragments, and importantly, additional cysteine residues do not interfere with the overall reaction, since the irreversibile S -> N shift occurs uniquely on the N terminal of the cysteine residue.NCL is a highly chemoselective reaction and forms native amide bonds. The reactions proceed at low (mM) concentration and in neutral buffered aqueous solutions. NCL even tolerates denaturing reagents during the hydrophobic peptide synthesis. Compared with solution phase peptide synthesis (<10 amino acids) and solid phase peptide synthesis (< 40 amino acids), NCL can generate peptides/small proteins up to around 200 amino acids.Kent JACS?1997,?119, 4325Example for applied NCL:Total Synthesis of HIV-1 protease (203 amino acids) Kent ACIE 2007, 46, 1667The major limitation of NCL is the scarcity of cysteine in natural peptide sequences (< 2%). Substituting other amino acids with cysteine at ligation sites can cause problems and is not regarded as a non-influential replacement.Chemical modifications of NCL productsMethods have been developed to transform Cys to Ala.Dawson JACS 2001, 123, 526Danishefsky ACIE 2007, 46, 9248Dawson reported catalytic methods to convert cysteine to alanine. Danishefsky and co-workers elaborated an independent approach using radical reactions for the conversion of cysteine to alanine. (TCEP = tris(2-carboxyethyl)phosphine and AIBN = 2,2’-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride).In 2013 Danishefsky and co-workers reported the chemical synthesis of Erythropoietin. Erythropoietin is a signaling glycoprotein (166 amino acids).Danishefsky Science 2013, 342, 135735604459779000Auxiliary methodesThe usage of thiol-auxiliaries overcomes the problem of the necessity for cysteine in NCL. Auxiliary groups are facilitating the ligation and can be removed without the modification of any amino acid residue of the peptide product.Macmillan ACIE 2006, 45, 7668 Ligation at Selenocysteines site (Sec ligation)Selenols have similar reactivity to thiols, but selenols are more acidic and more nucleophilic than corresponding thiols. The ligation proceeds fast and in high yield. The selenol-incorporated products have important applications in spectroscopic structural investigation, and mechanistic studies. 77Se NMR measurement is feasible with high molecular weight proteins.Hilvert Helv. Chim. Acta. 2001, 84, 1197 Hondal Protein Pept. Lett. 2005, 12, 757 Homocysteine ligation (Met ligation)Tam Biopolymers 1998, 46, 319 Homocystein is the homolog of cysteine and its thiomethylether is the natural amino acid methionine. This approach allows to perform ligation at methionine sites since the reactivity of the homocysteine and cysteine are similar.Ketoacid Hydroxylamine Ligation (KAHA Ligation)In 2006, Bode et al. reported the decarboxylative condensation of N-hydroxylamines with ketoacids to yield amide bonds chemoselectively.Bode ACIE 2006, 45,1248This ligation allows the chemoselective coupling of large unprotected peptides.Depending on the hydroxylamine, the KAHA ligation reactions are divided into two mechanistically distinct categories.Type I KAHA ligationIn Type I ligations O-unsubstitued hydroxilamines are used and the methode is suittable for the synthesis of small peptides but dose not work well in aqueous media.The mechanism of type I KAHA ligation was extensively studied and the pathway below was proposed.Bode ACIE 2012, 51, 513Despite the limitations of the Type I KAHA ligation, the method was successfully applied to the synthesis of the human GLP-1 (7-21) protein.Bode Chem. Sci. 2011, 2, 1976Type II KAHA ligationIn Type II ligations O-substitued hydroxylamines are used succesfully in the aqueous media to form peptides with more than 100 residues. Typically 5-oxaproline, a cyclic hydroxylamine, is employed in these types of ligation reactions. The use of oxaproline results in a homoserine residue at the ligation site.Bode ACIE 2012, 51, 5114The reaction pathway of type II KAHA ligation supported by oxaproline proceeds through an ester intermediate which can be converted to the corresponding amide product.Bode ACIE 2014, 53, 12244The formation of the ester as major product can be explained by the intramolecular addition of the alcohol side chain of the homoserine residue.KAHA ligation with 5-oxaproline made possible the total chemical synthesis of small proteins from Mycobacterium, the prokaryotic-ubiquitin-like protein (Pup, 63 residues).Bode ACIE 2012, 51, 5114Potassium Acyltrifluoroborate (KAT) LigationRecently the Bode group identified the potassium acyl trifluoroborates (KATs) as a novel type of reaction partners of hydroxylamines to afford amides in chemoselective ligation reactions.Bode ACIE 2012, 51, 5683; Bode ACIE 2014, 53, 7604 The KATs react selectively with O-substitued hydroxylamines and form amide bonds. The KAT ligations are suitable to conjugate unprotected peptides with large molecules (other peptides or polymers) in aqueous media.Bode JACS 2014, 136, 5611Serine and threonine ligationRecently, Prof Li has developed a new method for peptide synthesis generating a native bond at a serine or threonine residue. In this case the elctrophile - thioester is replaced with a salicylaldehyde (SAL) derived ester. The reaction proceeds by the formation of a N,O-benzylidine acetal that is subsequently cleaved in acidic media to give the native amide bond and the conserved stereocenter.Li PNAS 2013, 110, 6657 Isopeptide chemical ligation ICLThe concept of the NCL can also be used to form isopeptide bonds. An isopeptide bond is an amide bond between the carboxyl/amine moiety of an amino acid side chain and the amine/carboxyl moiety of another amino acid located on a side chain of another peptide. A classic isopeptide bond is the one between the carboxyl terminus of a protein and the amino group of a lysine residue of another protein.The Isopetide Chemical Ligation (ICL) follows the principle of the NCL: a thioester is required as well as a “cysteine like” moiety.The synthesis of polyubiquitinated protein is a good example of this ICL methodology, where the “cysteine like” moiety is a ?-mercaptolysine. After the ligation is done, a simple desulfurization affords the native isopeptide bond.Brik PNAS 2013, 110, 17726 Ligations and modifications of peptides and proteins with chemoselective reactionsThe techniques listed below are more often (but not always) used for labeling experiments. While still of great importance, one of the reagents is quite often used in excess in such reactions. Such reactions still need to be highly chemoselective but as one of the component is in excess the rate of these reactions are not as essential as for the ligation reactions, which are used to synthesize peptides and proteins. Amide Bond Formation via Azido Groups(Non-traceless) Staudinger Ligation The non-traceless azide-phosphine ligation was first introduced in 2000 by Bertozzi group. The reaction proceeds via a cyclisation between the generated aza-ylide and an ester placed in ortho position. Considering, that phosphine and azide are chemically orthogonal to most functional groups found in nature, this method was a long sought solution to investigate cellular processes. The advantages of Staudinger ligation over copper-catalyzed Click reactions are that one can avoid the use of toxic copper salts however the reaction is much slower and often does not proceed to full conversion. (Review: Br?se Chem. Soc. Rev., 2011, 40, 4840).,Bertozzi Science 2000, 287, 2007It is possible to label enzymes using this ligation in a living cell. Attaching biotin to a peptide is a common method to prepare derivatives because biotin readily binds to streptavidin, which is a 60 kDa bacterium protein, and this non-covalent interaction is one of the strongest known noncovalent forces. Attached streptavidin is usually modified with fluorophores (imaging), antibodies (Western blot analysis) and polystyrene (capturing proteins). Ploegh ACS Chem. Biol. 2006, 1, 713 Traceless Staudinger LigationAlthough the Staudinger ligations work fine even in a cell, a modification forming amide bond without the unnatural phosphine oxide is appreciated. In this version of the Staudinger ligation the forming phosphine oxide is excised from the target moleculeRaines Org. Lett. 2000, 2, 1939Bertozzi Org. Lett. 2000, 2, 2141Staudinger-phosphite ReactionWhen phosphite reacts with an azide component, after the formation of trialkyl phosphorimidate it loses an O-bound moiety (instead of hydrolysis) and forms a pentavalent phosphorus compound. The phosphite starting material is easy to access, the reaction proceds under mild aqueous conditions and the purification of products is easy. This reaction can be used for protein-labelings.Hackenberger and coworkers demonstrated that the Staudinger-phosphite reaction is not only suitable to attach PEG side chains to proteins, which could reduce their immunogenicity and increase their half-life, bioavailability and water solubility but also they liberated the protein using light. This kind of system could be utilized for targeted release of therapeutic peptide.Hackenberger Chem. Sci. 2010, 1, 596 Oxime ligationKetones and aldehydes react with hydroxylamines to form oximes or with hydrazides to form hydrazones. The equilibrium favros the imine due to the ?-effect of the adjacent heteroatom. The reaction kinetics is largely dependent on pH of the reaction mixture, and the high reaction rate is observed under the acidic conditions (pH 4-5). Anilines are often used as catalysts to speed up the reaction. This reaction can be used for site-specific modification of peptides. In the example below, ubiquitin-peptide conjugates were prepared by the oxime ligation. The conjugates were used as nonhydrolyzable probes to investigate the affinities of deubiquitinating enzymes for K48 and K63 topoisomer mimics.Dawson ACIE 2006, 45, 7581Ovaa JACS, 2010, 132, 8834 Linkages Made by Cycloaddition1,3-Dipolar Cycloaddition ([3 + 2] Cycloaddition)1,3-Dipolar cycloadditions between an azide and an alkyne have been extensively studied by Rolf Huisgen since 1950s and therefore sometimes this reaction is termed as Huisgen cycloaddition.Inert to almost all biological functionalities and possessing a small size, azides and alkynes hardly disturb protein conformations and functions of the biological system. The triazoles have similarities to the peptide bond in the atom planarity and electronic properties. Also, the great stability of triazoles and resistance to oxidation and reduction makes Huisgen cycloaddition very attractive.Maarseveen OL 2006, 8, 919Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC)The concerted Huisgen cycloaddition has a high activation barrier (~26 kcal/mol for methyl azide and propyne) and requires high temperature and pressure for the reaction to proceed. Therefore, it had largely remained as an academic teaching tool until Sharpless and Meldal independently discovered the Cu-catalyzed azide-alkyne cycloaddition. Cu(I) catalyst dramatically accelerates the rate of the reaction and gives 1,4-substituted triazole as the only product. The ligands are often used for the reaction in order to protect Cu(I) from oxidation and/or to accelerate the reaction.Initial reports: Fokin & Sharpless ACIE 2002, 41, 2596; Meldal JOC 2002, 67, 3057Mechanism: Fokin Science 2013, 340, 457Protein transferrin was attached to the polyvalent cowpa mosaic virus scaffoldFinn Bioconjugate Chem. 2005, 16, 1572One of the drawbacks of the CuAAC is the cell toxicity of the copper reagents. The copper-chealating azide substrates showed higher reactivity and revealed the reduced cell toxicity. In the exapmle below, the azide substrates were designed to form strong, active copper complexes, and the reaction could be regarded as a bimolecular system.Taran ACIE 2014, 53, 5872Strain-promoted azide-alkyne cycloaddition (SPAAC)The great kinetic properties, chemoselectivity, and high yields make CuAAC one of the most useful reactions. However, the toxicity of metals to disturb metabolic balance in living organisms greatly limits their applications in biological systems.To increase the reaction rates of Huisgen cycloaddition without metal catalysts, the Bertozzi group applied strained cyclooctynes to promote the [3 + 2] cycloaddition. The ring strain for cyclooctyne is around 18 kcal/mol. Smaller cyclic alkynes are too unstable for practical use. The sp-hybridized bond angle in cyclooctynes is around 160, which is close to the distorted bond angles in the transition states, resulting in the great rate acceleration. Bertozzi Chem. Soc. Rev. 2010, 39, 1272In vivo imaging in developing zebrafishBertozzi Science 2008, 320, 664At the early stage of the reaction development, the reaction rate was rather slow (10–3 to 10–2 M–1 s–1). The modification of the substrate structure led to the excellent improvement of the reaction rate (ca. 1 M–1 s–1)GGG“#?[]|{}≠“?#?[2, t improvement of the reaction rate.ction rate was rather slow (10 –3 to 10 –2 M–1 s–1). .Bertozzi JACS 2012, 134, 9199Alternative 1,3-dipolar cycloaddition reactionsNitrones and cyclooctynesOne-pot three step site-specific modification of the protein, IL-8 (in situ generation of nitrones)Boons and Delft ACIE 2010, 49, 3065Nitrile oxides and norbornenes Nitrile oxides are good electrophiles and the angular strain of norbornene ring is about 6 kcal/mol.DNA modificationCarell OL 2009, 11, 2405Azides and oxanorbornadienesAlkenes have also been used in [3 + 2] cycloadditions with azides. While instead of giving triazoles, they give triazolines, which are relatively unstable. (The cycloadditions of alkynes and azides forming aromatic triazole have G~ - 60 kcal/mol.)The Rutjes group circumvented the problem of unstable triazolines by using the oxanorbornadiene with an azide. After cycloaddition, a furan was extruded through a retro Diels-Alder and gave a triazole instead.Rutjes ChemBioChem. 2008, 9, 1805Nitrile imines and alkenesLin group developed a photochemical 1,3-dipolar cycloaddition between nitrile imines and alkenes. The ligation product, pyraoline, is fluorescent and enables monitoring of the reaction in vivo. The reaction can occur at higher wavelength (365 nm) to minimize the damage to living systems caused by he irradiation. Simple unactivated alkenes can be used without problems.Mechanims:Photoinduced cycloaddition in bacterial cellsLin JACS 2008, 130, 9654Recently, Lin group has reported the synthesis of the spirocyclic alkenes as extremely reactive yet stable substrates for the photoclick reaction. The specific modification of superfolder GFP (sfGFP) was achieved with the excellent reaction rate (k > 10,000 M–1 s–1).Lin JACS 2014, 136, 4153Inverse electron-demand Diels-Alder reaction The normal Diels-Alder reaction usually requires EWG-activated dienophiles (e.g. maleimides). Those dienophiles are not suitable because of their Michael acceptor properties to various nucleophiles commonly found in biological systems.trans-Cyclooctenes and tetrazinesBioorthogonal reactions between tetrazines and trans-cyclooctenes proceed very fast in water (k = 102-104 M–1s–1) without any catalyst and produce N2 as a sole byproduct. The fast kinetics result from the ring strain of trans-cyclooctenes (16 kcal/mol). However, there is also a concern of the isomerization of the double bond from trans to cis over time. Also cyclooctenes and tetrazines are large in size and may complicate the metabolic or enzymatic incorporation into biomolecules. Fox JACS 2008, 130, 13518Norbornenes and tetrazinesThis cycloaddition is based on the inverse electron-demand Diels-Alder reaction of tetrazines to norbornenes, followed by retro-Diels-Alder elimination of molecular nitrogen to afford the expected dihydropyridazines and their regioisomers. Although the reaction kinetics is slower (k = 10–1-10 M–1 s–1) compared to the reaction with trans-cyclooctenes, norbornenes are bench stable. Hilderbrand Bioconjugate Chem. 2008, 19, 2297Cyclopropenes and tetrazinesFunctionalized cyclopropenes were recently found to be good substrates for IED-DA reactions with tetrazines. Cyclopropenes are stable yet reactive and compatible with various metabolic pathways sue to their small size. The reaction rate is, however, slower than that of the reaction with trans-cyclooctenes.Prescher JACS 2012, 134, 18638Metal-mediated bioconjugation reactionsRu-mediated cross-metathesisCross-metathesis appears as an attractive method to install modifications onto biomolecules through the formation of a stable C=C bond. Ruthenium-catalyzed metathesis is a remarkable reaction for its high selectivity and tolerance toward different functional groups. However, the reactions are usually run with organic co-solvents. In the case below, the method was applied to the serine protease subtilisin Bacillus lentus (SBL). Davis JACS 2008, 130, 9642Pd-mediated Suzuki-Miyaura cross-couplingThe Suzuki-Miyaura cross-coupling reaction is an emerging tool for bioconjugations. The reaction can be utilized such as for modification of the protein surface and DNA. This bioconjugation method does not require organic solvents.Davis JACS 2009, 131, 16346Mutually orthogonal bioconjugationA new challenge in bioconjugation areas is identification of the mutually orthogonal bioconjugation reactions. Those reactions allow the simultaneous monitoring of the multiple biomolecules in a single biological system.- Some cyclooctynes are reactive toward tetrazines, and they cannot be used for the multilabeling purpose. However, the judicious choice of the substrate structure allowed double [3 + 2] cycloaddition simultaneously in the cell imaging by taking advantage of the difference in reaction rates. The substituents on the tetrazine was critical. Hilderbrand demonstrated the simultaneous labeling of two different cancer cell types.Hilderbrand ACIE 2012, 51, 920- Houk and Prescher have recently reported the unique reactivity of the substituted cyclopropenes. 1,3-disubstituted cyclopropenes showed high reactivity toward tetrazines to undergo the IED-DA reaction while 3,3-disubstituted cyclopropenes did not react with tetratines. Instead, 3,3-disubstituted cyclopropenes showed the facile reaction with nitrile imines (generated by photolysis) to undergo the 1,3-dipolar cycloaddition. They tested the mutual orthogonality by labeling the model protein, BSA (Scheme below). Houk & Prescher JACS 2013, 135, 13680 ................
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