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Incorporation of pyrrolysine derivatives into proteins and development of bioorthogonal protein modification methods
Incorporation of pyrrolysine derivatives into proteins and development of bioorthogonal protein modification methods
Site specific incorporation of non-canonical amino acids (NCAAs) into proteins is a powerful tool for e.g. studying the effects of post translational modifications, revealing enzymatic mechanisms, visualizing proteins in living cells or adding new functionalities to therapeutic and diagnostic proteins. The technology is based on orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pairs, which do not interfere with the biosynthetic machinery of the host cell (e.g. E. coli). The tRNA/aaRS pair, which was used in this thesis originates from the archaeal strain Methanosarcina mazei. It is responsible for the ribosomal incorporation of pPyrrolysine (Pyl), also known as the 22nd proteinogenic amino acid, in response to UAG ("amber-") codons. In the classic genetic code, these amber codons are stop codons and lead to translation termination. When transferring the tRNAPyl/PylRS-pair to e.g. E. coli, the incorporation of a NCAA in response to an amber codon competes with translation termination and is therefore called "amber suppression". In this thesis, the pPyrrolysine system was exploited to incorporate three newly described posttranslational lysine modifications site specifically into histone H3, where they have been identified. The amino acids ε-N-propionyl-, ε-N-butyryl-, and ε-N-crotonyl-lysine were directly incorporated at the desired position to simulate the respective naturally occuring propionylation, butyrylation or crotonylation respectively. Comprehensive bioanalytical examinations of the modified histone, which included peptide mapping-MS and western blot analyses unambiguously proved the site specific and complete incorporation of these NCAAs. Although numerous NCAAs have already been incorporated into proteins by amber suppression, the structural diversity of these is of course limited. However, the introduction of NCAAs carrying bioorthogonal modification handles opens an infinite field of possibilities since it allows post translational, specific modifications of the protein of interest. One of the most promising handles are norbornenes, which could be introduced site specifically into proteins by the Carell group.1 The modification of norbornens with tetrazines in inverse electron demand Diels-Alder reactions is one of the fastest bioorthogonal modification reactions described. Mechanistic studies in collaboration with the de Vivie-Riedle group revealed and explained that, due to a more stable transition state, exo-substituted hydroxymethyl norbornenes react around three-times faster than their endo substituted isomers. To analyze, whether this behavior could also be observed on the protein level, the endo substituted norbornyl lysine and two novel exo substituted norbornene amino acids were incorporated into model proteins. The acceptance of these new norbornene amino acids by the Pyl system including a PylRS triple mutant was supported with solid analytical data. The correct incorporation and complete labeling with fluorescent and PEGylated tetrazines was demonstrated by SDS-PAGE, intact-MS and peptide mapping analyses of the unmodified and modified protein species. A faster labeling reaction of the exo-substituted norbornyl amino acids was not observed in these in vitro protein labeling studies. In collaboration with the Schneider group, crystal structures of the used PylRS triple mutant bearing the Pyl analogs demonstrated the versatility of the triple mutant developed in the Carell group.1 In order to expand the set of bioorthogonal modification reactions, a novel click reaction, the aziridation of norbornenes using electron-deficient sulfonyl azides was developed. The reaction was characterized on small molecule level, on the peptide level and finally on the protein level using norbornene containing model proteins. The second order rate constant was determined to be k2 = 1.7 x 10-3 M-1 s-1. This value is comparable to the rate constants of the strain-promoted azide–alkyne cycloaddition of the first generation which is commonly used for biomolecule labeling applications.2,3 Chemoselective protein conjugation with fluorescent, and immuno tags on the norbornene residue was proven by SDS-PAGE, Western blot as well as by peptide mapping and intact mass spectrometric studies. The reaction proceeds efficiently under mild conditions, does not require any catalysis and is orthogonal to functional groups of native proteins. Furthermore, sulfonyl azides are easily accessible compounds, which makes the newly developed labeling reaction an attractive alternative to the existing set of bioconjugation techniques.� A general challenge of the incorporation of NCAAs into proteins is that the amino acids have to be chemically synthesized in large quantities to reach the required millimolar concentrations during protein expression. Our idea was to circumvent these laborious syntheses and hand over the work to the biosynthetic machinery of the host cell. In this thesis, a Pyl-biosynthesis "hijacking" system was developed, that allowed the biosynthesis and incorporation of unnatural 3S-ethynylpyrrolysine (ePyl), a bifunctional reaction handle, by feeding E. coli cells with 3R-methylethynyl-D-ornithine. The correct production and incorporation of the novel NCAA in a purified model protein was proved by extended analytics including mass spectrometric studies on intact and peptide level. It was shown, that site specifically incorporated ePyl can be modified by two different orthogonal click reactions at only one amino acid site providing access to highly modified proteins. Finally, to prove the feasibility of NCAA carrying proteins as useful tools in biomedical applications, site specifically modified human carbonic anhydrase-folic acid-conjugates (HCA-FA) were applied as targeting, pH responsive capping proteins for drug delivery in mesoporous silica-based nanocarriers (MSNs). The studies were carried out in collaboration with the groups of Prof. Bein and Prof. Bräuchle. Three key elements of the newly developed capping system arise from the site-specifically modified HCA-FA. First, the protein is large enough to block the gates of the MSN to prevent the cargo from escaping. Second, HCA binds phenyl-sulfonamide (phSA) and therefore sulfonamide-functionalized MSNs (MSN-phSA) in a pH depended manner. At pH values below 7, typically present during endocytosis of MSNs, the enzyme releases MSN-phSA, which ensures the release of the cargo at the exact right moment. Third, the site-specific functionalization of HCA can direct the MSNs to a specific cell type. Site specific incorporation of non-canonical amino acids (NCAAs) into proteins is a powerful tool for e.g. studying the effects of post translational modifications, revealing enzymatic mechanisms, visualizing proteins in living cells or adding new functionalities to therapeutic and diagnostic proteins. The technology is based on orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pairs, which do not interfere with the biosynthetic machinery of the host cell (e.g. E. coli). The tRNA/aaRS pair, which was used in this thesis originates from the archaeal strain Methanosarcina mazei. It is responsible for the ribosomal incorporation of pPyrrolysine (Pyl), also known as the 22nd proteinogenic amino acid, in response to UAG ("amber-") codons. In the classic genetic code, these amber codons are stop codons and lead to translation termination. When transferring the tRNAPyl/PylRS-pair to e.g. E. coli, the incorporation of a NCAA in response to an amber codon competes with translation termination and is therefore called "amber suppression". In this thesis, the pPyrrolysine system was exploited to incorporate three newly described posttranslational lysine modifications site specifically into histone H3, where they have been identified. The amino acids ε-N-propionyl-, ε-N-butyryl-, and ε-N-crotonyl-lysine were directly incorporated at the desired position to simulate the respective naturally occuring propionylation, butyrylation or crotonylation respectively. Comprehensive bioanalytical examinations of the modified histone, which included peptide mapping-MS and western blot analyses unambiguously proved the site specific and complete incorporation of these NCAAs. Although numerous NCAAs have already been incorporated into proteins by amber suppression, the structural diversity of these is of course limited. However, the introduction of NCAAs carrying bioorthogonal modification handles opens an infinite field of possibilities since it allows post translational, specific modifications of the protein of interest. One of the most promising handles are norbornenes, which could be introduced site specifically into proteins by the Carell group.1 The modification of norbornens with tetrazines in inverse electron demand Diels-Alder reactions is one of the fastest bioorthogonal modification reactions described. Mechanistic studies in collaboration with the de Vivie-Riedle group revealed and explained that, due to a more stable transition state, exo-substituted hydroxymethyl norbornenes react around three-times faster than their endo substituted isomers. To analyze, whether this behavior could also be observed on the protein level, the endo substituted norbornyl lysine and two novel exo substituted norbornene amino acids were incorporated into model proteins. The acceptance of these new norbornene amino acids by the Pyl system including a PylRS triple mutant was supported with solid analytical data. The correct incorporation and complete labeling with fluorescent and PEGylated tetrazines was demonstrated by SDS-PAGE, intact-MS and peptide mapping analyses of the unmodified and modified protein species. A faster labeling reaction of the exo-substituted norbornyl amino acids was not observed in these in vitro protein labeling studies. In collaboration with the Schneider group, crystal structures of the used PylRS triple mutant bearing the Pyl analogs demonstrated the versatility of the triple mutant developed in the Carell group.1 In order to expand the set of bioorthogonal modification reactions, a novel click reaction, the aziridation of norbornenes using electron-deficient sulfonyl azides was developed. The reaction was characterized on small molecule level, on the peptide level and finally on the protein level using norbornene containing model proteins. The second order rate constant was determined to be k2 = 1.7 x 10-3 M-1 s-1. This value is comparable to the rate constants of the strain-promoted azide–alkyne cycloaddition of the first generation which is commonly used for biomolecule labeling applications.2,3 Chemoselective protein conjugation with fluorescent, and immuno tags on the norbornene residue was proven by SDS-PAGE, Western blot as well as by peptide mapping and intact mass spectrometric studies. The reaction proceeds efficiently under mild conditions, does not require any catalysis and is orthogonal to functional groups of native proteins. Furthermore, sulfonyl azides are easily accessible compounds, which makes the newly developed labeling reaction an attractive alternative to the existing set of bioconjugation techniques.� A general challenge of the incorporation of NCAAs into proteins is that the amino acids have to be chemically synthesized in large quantities to reach the required millimolar concentrations during protein expression. Our idea was to circumvent these laborious syntheses and hand over the work to the biosynthetic machinery of the host cell. In this thesis, a Pyl-biosynthesis "hijacking" system was developed, that allowed the biosynthesis and incorporation of unnatural 3S-ethynylpyrrolysine (ePyl), a bifunctional reaction handle, by feeding E. coli cells with 3R-methylethynyl-D-ornithine. The correct production and incorporation of the novel NCAA in a purified model protein was proved by extended analytics including mass spectrometric studies on intact and peptide level. It was shown, that site specifically incorporated ePyl can be modified by two different orthogonal click reactions at only one amino acid site providing access to highly modified proteins. Finally, to prove the feasibility of NCAA carrying proteins as useful tools in biomedical applications, site specifically modified human carbonic anhydrase-folic acid-conjugates (HCA-FA) were applied as targeting, pH responsive capping proteins for drug delivery in mesoporous silica-based nanocarriers (MSNs). The studies were carried out in collaboration with the groups of Prof. Bein and Prof. Bräuchle. Three key elements of the newly developed capping system arise from the site-specifically modified HCA-FA. First, the protein is large enough to block the gates of the MSN to prevent the cargo from escaping. Second, HCA binds phenyl-sulfonamide (phSA) and therefore sulfonamide-functionalized MSNs (MSN-phSA) in a pH depended manner. At pH values below 7, typically present during endocytosis of MSNs, the enzyme releases MSN-phSA, which ensures the release of the cargo at the exact right moment. Third, the site-specific functionalization of HCA can direct the MSNs to a specific cell type., Der ortsspezifische Einbau von nicht-kanonischen Aminosäuren (engl. abgekürzt NCAAs) in Proteine ist ein leistungsfähiges Werkzeug in der chemischen Biologie. Es kann unter anderem für die Untersuchung der Auswirkungen von posttranslationalen Modifikationen, für die Aufklärung enzymatischer Mechanismen, für die Visualisierung von Proteinen in lebenden Zellen oder zum Funktionalisieren therapeutischer und diagnostischer Proteine verwendet werden. Die Technologie basiert auf orthogonalen tRNA/Aminoacyl-tRNA-Synthetase (aaRS)-Paaren, die nicht in die Biosynthesemaschinerie der Wirtszelle (z.B. E. coli) eingreifen. Das in dieser Arbeit verwendete tRNA/aaRS-Paar stammt aus dem Archaea-Stamm Methanosarcina mazei. Es ist verantwortlich für den ribosomalen Einbau von Pyrrolysin (Pyl), das auch als 22. proteinogene Aminosäure bekannt ist. Pyl wird vom sogenannten "Amber Codon" codiert. Im klassischen genetischen Code ist dieses Codon ein Stopp-Signal und führt zur Termination der Translation. Wenn das tRNAPyl/PylRS-Paar z.B. in E. coli exprimiert wird, konkurriert der Einbau einer NCAA als Antwort auf ein Amber-Codon mit der Translationsterminierung und wird daher als amber suppression bezeichnet. In dieser Arbeit wurde das Pyrrolysin-System genutzt, um drei neu beschriebene posttranslationale Lysin-Modifikationen spezifisch an Positionen in Histon H3 zu integrieren, an denen sie identifiziert wurden. Die Aminosäuren ε-N-Propionyl-, ε-N-Butyryl- und ε-N-Crotonyllysin wurden direkt an der gewünschten Position eingebaut, um die jeweilige natürlich vorkommende Propionylierung, Butyrylierung bzw. Crotonylierung zu simulieren. Umfassende bioanalytische Untersuchungen des modifizierten Histons mit peptide mapping-MS und Western-Blot-Analysen belegten den ortsspezifischen und vollständigen Einbau dieser NCAAs eindeutig. Obwohl zahlreiche NCAAs bereits durch amber suppression in Proteine eingebaut wurden, ist die strukturelle Vielfalt dieser Aminosäuren natürlich begrenzt. Die Einführung von NCAAs, die bioorthogonal modifizierbare funktionale Gruppen tragen, eröffnet jedoch nahezu unerschöpfliche Möglichkeiten, da sie spezifische Modifikationen von Proteinen nach deren Expression ermöglicht. Eine der Vielversprechendsten dieser funktionalen Einheiten sind Norbornene, die von der Carell-Gruppe spezifisch in Proteine eingebaut werden können.1 Die Reaktion von Norbornenen mit Tetrazinen in invers Elektronen-anfordernden Diels-Alder-Cycloadditionen ist eine der schnellsten beschriebenen bioorthogonalen Modifizierungsreaktionen. In mechanistischen Studien in Zusammenarbeit mit der Gruppe von Prof. de Vivie-Riedle konnte gezeigt und erklärt werden, dass exo-substituierte Hydroxymethyl-Norbornene aufgrund eines stabileren Übergangszustands etwa dreimal schneller mit Tetrazinen reagieren als ihre endosubstituierten Isomere. Um zu analysieren, ob dieses Verhalten auch auf Proteinebene beobachtet werden kann, wurden das endo-substituierte Norbornyllysin und zwei neue exo-substituierte Norbornen-Aminosäuren in Modellproteine eingebaut. Die Akzeptanz dieser neuen Norbornen-Aminosäuren durch das Pyl-System, das eine zuvor entwickelte PylRS-Dreifachmutante enthielt, wurde mit soliden analytischen Daten bewiesen. Der korrekte Einbau und die vollständige Markierung mit fluoreszierenden und PEGylierten Tetrazinen wurde durch SDS-PAGE-, Intakt-MS- und peptide mapping-Analysen der unmodifizierten und modifizierten Proteinspezies gezeigt. Eine schnellere Markierungsreaktion der exo-substituierten Norbornyl-Aminosäuren wurde bei diesen in vitro-Proteinmarkierungsstudien allerdings nicht beobachtet. In Zusammenarbeit mit der Arbeitsgruppe von Dr. Sabine Schneider konnte die Vielseitigkeit der in der Carell-Gruppe entwickelten PylRS Dreifachmutante in Kristallstrukturen mit gebundenen Pyl-Analoga gezeigt werden. 1 Um die Auswahl an bioorthogonalen Modifikationsreaktionen zu erweitern, wurde die Aziridierung von Norbornenen mit elektronenarmen Sulfonylaziden, eine neuartige Klickreaktion, entwickelt. Die Reaktion wurde auf niedermolekularer, auf Peptid- und schließlich auf Proteinebene mit Norbornenen, Norbornen-Peptiden und Norbornen-haltigen Modellproteinen charakterisiert. Die Geschwindigkeitskonstante zweiter Ordnung wurde mit k2 = 1,7×10 -3 M-1s-1 bestimmt. Dieser Wert ist vergleichbar mit den Geschwindigkeitskonstanten der Ringspannungs-unterstützten Azid-Alkin-Cycloaddition der ersten Generation, die häufig für Biomolekül-Markierungen verwendet wird. 2,3 Die chemoselektive Proteinkonjugation mit Fluoreszenz- und Immuno-reagenzien am Norbornen-Rest wurde durch SDS-PAGE, Western Blot sowie durch peptide mapping und intakte massenspektrometrische Untersuchungen nachgewiesen. Die Reaktion verläuft effizient unter milden Bedingungen, erfordert keine Katalyse und ist orthogonal zu funktionellen Gruppen von nativen Proteinen. Darüber hinaus sind Sulfonylazide leicht zugängliche Verbindungen, was die neu entwickelte Markierungsreaktion zu einer attraktiven Alternative zu den bestehenden Biokonjugationstechniken macht. Eine allgemeine Herausforderung beim Einbau von NCAAs in Proteine besteht darin, dass die Aminosäuren in großen Mengen chemisch synthetisiert werden müssen, um die erforderlichen millimolaren Konzentrationen während der Proteinexpression zu erreichen. Unsere Idee war es, diese aufwendigen Synthesen zu umgehen und die Arbeit an die biosynthetische Maschinerie der Wirtszelle zu übergeben. Dafür wurde in diesem Projekt ein Pyl-Biosynthese-"Entführungs"-System entwickelt, das die Biosynthese und den Einbau von unnatürlichem 3S-Ethiynylpyrrolysin (ePyl), einer zweifach modifizierbaren Einheit, ermöglicht, indem E. coli-Zellen mit 3R-MethylEthinyl-D-Ornithin gefüttert werden. Die korrekte Synthese und die richtige Position des Einbaus der neuen NCAA in einem gereinigten Modellprotein wurde durch umfassende Analysen einschließlich massenspektrometrischer Untersuchungen auf intakter und Peptidebene nachgewiesen. Es konnte gezeigt werden, dass ortsspezifisch eingebautes ePyl durch zwei unterschiedliche orthogonale Klickreaktionen modifiziert werden kann, was den Zugang zu hochmodifizierten Proteinen ermöglicht. Um schließlich die Anwendbarkeit von NCAA-tragenden Proteinen als nützliche Werkzeuge in biomedizinischen Anwendungen zu beweisen, wurden ortsspezifisch modifizierte humane Carboanhydrase-Folsäure-Konjugate (HCA-FA) als zielgerichtete, pH-schaltbare Capping-Proteine für die Wirkstoffabgabe in mesoporösem Siliciumdioxid Nanoträgern (engl. abgekürzt MSNs) eingesetzt. Die Studien wurden in Zusammenarbeit mit den Gruppen von Prof. Bein und Prof. Bräuchle durchgeführt. Drei Schlüsselelemente des neu entwickelten Verschlusssystems ergeben sich aus dem ortsspezifisch modifizierten HCA-FA. Erstens ist das Protein groß genug, um die Poren der MSNs zu blockieren, um zu verhindern, dass die Ladung austritt. Zweitens bindet HCA Phenylsulfonamid (phSA) und daher Sulfonamid-funktionalisierte MSNs (MSN-phSA) pH-abhäng. Bei pH-Werten unter 7, die typischerweise während der Endozytose von MSNs vorliegen, lässt das Enzym MSN-phSA los und gewährleistet damit die Freisetzung der Fracht zum genau richtigen Zeitpunkt. Drittens steuert die ortsspezifische Funktionalisierung von HCA die Anbindung der MSNs an spezifische Zelltypen.
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Gattner, Michael
2019
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Gattner, Michael (2019): Incorporation of pyrrolysine derivatives into proteins and development of bioorthogonal protein modification methods. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

Site specific incorporation of non-canonical amino acids (NCAAs) into proteins is a powerful tool for e.g. studying the effects of post translational modifications, revealing enzymatic mechanisms, visualizing proteins in living cells or adding new functionalities to therapeutic and diagnostic proteins. The technology is based on orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pairs, which do not interfere with the biosynthetic machinery of the host cell (e.g. E. coli). The tRNA/aaRS pair, which was used in this thesis originates from the archaeal strain Methanosarcina mazei. It is responsible for the ribosomal incorporation of pPyrrolysine (Pyl), also known as the 22nd proteinogenic amino acid, in response to UAG ("amber-") codons. In the classic genetic code, these amber codons are stop codons and lead to translation termination. When transferring the tRNAPyl/PylRS-pair to e.g. E. coli, the incorporation of a NCAA in response to an amber codon competes with translation termination and is therefore called "amber suppression". In this thesis, the pPyrrolysine system was exploited to incorporate three newly described posttranslational lysine modifications site specifically into histone H3, where they have been identified. The amino acids ε-N-propionyl-, ε-N-butyryl-, and ε-N-crotonyl-lysine were directly incorporated at the desired position to simulate the respective naturally occuring propionylation, butyrylation or crotonylation respectively. Comprehensive bioanalytical examinations of the modified histone, which included peptide mapping-MS and western blot analyses unambiguously proved the site specific and complete incorporation of these NCAAs. Although numerous NCAAs have already been incorporated into proteins by amber suppression, the structural diversity of these is of course limited. However, the introduction of NCAAs carrying bioorthogonal modification handles opens an infinite field of possibilities since it allows post translational, specific modifications of the protein of interest. One of the most promising handles are norbornenes, which could be introduced site specifically into proteins by the Carell group.1 The modification of norbornens with tetrazines in inverse electron demand Diels-Alder reactions is one of the fastest bioorthogonal modification reactions described. Mechanistic studies in collaboration with the de Vivie-Riedle group revealed and explained that, due to a more stable transition state, exo-substituted hydroxymethyl norbornenes react around three-times faster than their endo substituted isomers. To analyze, whether this behavior could also be observed on the protein level, the endo substituted norbornyl lysine and two novel exo substituted norbornene amino acids were incorporated into model proteins. The acceptance of these new norbornene amino acids by the Pyl system including a PylRS triple mutant was supported with solid analytical data. The correct incorporation and complete labeling with fluorescent and PEGylated tetrazines was demonstrated by SDS-PAGE, intact-MS and peptide mapping analyses of the unmodified and modified protein species. A faster labeling reaction of the exo-substituted norbornyl amino acids was not observed in these in vitro protein labeling studies. In collaboration with the Schneider group, crystal structures of the used PylRS triple mutant bearing the Pyl analogs demonstrated the versatility of the triple mutant developed in the Carell group.1 In order to expand the set of bioorthogonal modification reactions, a novel click reaction, the aziridation of norbornenes using electron-deficient sulfonyl azides was developed. The reaction was characterized on small molecule level, on the peptide level and finally on the protein level using norbornene containing model proteins. The second order rate constant was determined to be k2 = 1.7 x 10-3 M-1 s-1. This value is comparable to the rate constants of the strain-promoted azide–alkyne cycloaddition of the first generation which is commonly used for biomolecule labeling applications.2,3 Chemoselective protein conjugation with fluorescent, and immuno tags on the norbornene residue was proven by SDS-PAGE, Western blot as well as by peptide mapping and intact mass spectrometric studies. The reaction proceeds efficiently under mild conditions, does not require any catalysis and is orthogonal to functional groups of native proteins. Furthermore, sulfonyl azides are easily accessible compounds, which makes the newly developed labeling reaction an attractive alternative to the existing set of bioconjugation techniques.� A general challenge of the incorporation of NCAAs into proteins is that the amino acids have to be chemically synthesized in large quantities to reach the required millimolar concentrations during protein expression. Our idea was to circumvent these laborious syntheses and hand over the work to the biosynthetic machinery of the host cell. In this thesis, a Pyl-biosynthesis "hijacking" system was developed, that allowed the biosynthesis and incorporation of unnatural 3S-ethynylpyrrolysine (ePyl), a bifunctional reaction handle, by feeding E. coli cells with 3R-methylethynyl-D-ornithine. The correct production and incorporation of the novel NCAA in a purified model protein was proved by extended analytics including mass spectrometric studies on intact and peptide level. It was shown, that site specifically incorporated ePyl can be modified by two different orthogonal click reactions at only one amino acid site providing access to highly modified proteins. Finally, to prove the feasibility of NCAA carrying proteins as useful tools in biomedical applications, site specifically modified human carbonic anhydrase-folic acid-conjugates (HCA-FA) were applied as targeting, pH responsive capping proteins for drug delivery in mesoporous silica-based nanocarriers (MSNs). The studies were carried out in collaboration with the groups of Prof. Bein and Prof. Bräuchle. Three key elements of the newly developed capping system arise from the site-specifically modified HCA-FA. First, the protein is large enough to block the gates of the MSN to prevent the cargo from escaping. Second, HCA binds phenyl-sulfonamide (phSA) and therefore sulfonamide-functionalized MSNs (MSN-phSA) in a pH depended manner. At pH values below 7, typically present during endocytosis of MSNs, the enzyme releases MSN-phSA, which ensures the release of the cargo at the exact right moment. Third, the site-specific functionalization of HCA can direct the MSNs to a specific cell type. Site specific incorporation of non-canonical amino acids (NCAAs) into proteins is a powerful tool for e.g. studying the effects of post translational modifications, revealing enzymatic mechanisms, visualizing proteins in living cells or adding new functionalities to therapeutic and diagnostic proteins. The technology is based on orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pairs, which do not interfere with the biosynthetic machinery of the host cell (e.g. E. coli). The tRNA/aaRS pair, which was used in this thesis originates from the archaeal strain Methanosarcina mazei. It is responsible for the ribosomal incorporation of pPyrrolysine (Pyl), also known as the 22nd proteinogenic amino acid, in response to UAG ("amber-") codons. In the classic genetic code, these amber codons are stop codons and lead to translation termination. When transferring the tRNAPyl/PylRS-pair to e.g. E. coli, the incorporation of a NCAA in response to an amber codon competes with translation termination and is therefore called "amber suppression". In this thesis, the pPyrrolysine system was exploited to incorporate three newly described posttranslational lysine modifications site specifically into histone H3, where they have been identified. The amino acids ε-N-propionyl-, ε-N-butyryl-, and ε-N-crotonyl-lysine were directly incorporated at the desired position to simulate the respective naturally occuring propionylation, butyrylation or crotonylation respectively. Comprehensive bioanalytical examinations of the modified histone, which included peptide mapping-MS and western blot analyses unambiguously proved the site specific and complete incorporation of these NCAAs. Although numerous NCAAs have already been incorporated into proteins by amber suppression, the structural diversity of these is of course limited. However, the introduction of NCAAs carrying bioorthogonal modification handles opens an infinite field of possibilities since it allows post translational, specific modifications of the protein of interest. One of the most promising handles are norbornenes, which could be introduced site specifically into proteins by the Carell group.1 The modification of norbornens with tetrazines in inverse electron demand Diels-Alder reactions is one of the fastest bioorthogonal modification reactions described. Mechanistic studies in collaboration with the de Vivie-Riedle group revealed and explained that, due to a more stable transition state, exo-substituted hydroxymethyl norbornenes react around three-times faster than their endo substituted isomers. To analyze, whether this behavior could also be observed on the protein level, the endo substituted norbornyl lysine and two novel exo substituted norbornene amino acids were incorporated into model proteins. The acceptance of these new norbornene amino acids by the Pyl system including a PylRS triple mutant was supported with solid analytical data. The correct incorporation and complete labeling with fluorescent and PEGylated tetrazines was demonstrated by SDS-PAGE, intact-MS and peptide mapping analyses of the unmodified and modified protein species. A faster labeling reaction of the exo-substituted norbornyl amino acids was not observed in these in vitro protein labeling studies. In collaboration with the Schneider group, crystal structures of the used PylRS triple mutant bearing the Pyl analogs demonstrated the versatility of the triple mutant developed in the Carell group.1 In order to expand the set of bioorthogonal modification reactions, a novel click reaction, the aziridation of norbornenes using electron-deficient sulfonyl azides was developed. The reaction was characterized on small molecule level, on the peptide level and finally on the protein level using norbornene containing model proteins. The second order rate constant was determined to be k2 = 1.7 x 10-3 M-1 s-1. This value is comparable to the rate constants of the strain-promoted azide–alkyne cycloaddition of the first generation which is commonly used for biomolecule labeling applications.2,3 Chemoselective protein conjugation with fluorescent, and immuno tags on the norbornene residue was proven by SDS-PAGE, Western blot as well as by peptide mapping and intact mass spectrometric studies. The reaction proceeds efficiently under mild conditions, does not require any catalysis and is orthogonal to functional groups of native proteins. Furthermore, sulfonyl azides are easily accessible compounds, which makes the newly developed labeling reaction an attractive alternative to the existing set of bioconjugation techniques.� A general challenge of the incorporation of NCAAs into proteins is that the amino acids have to be chemically synthesized in large quantities to reach the required millimolar concentrations during protein expression. Our idea was to circumvent these laborious syntheses and hand over the work to the biosynthetic machinery of the host cell. In this thesis, a Pyl-biosynthesis "hijacking" system was developed, that allowed the biosynthesis and incorporation of unnatural 3S-ethynylpyrrolysine (ePyl), a bifunctional reaction handle, by feeding E. coli cells with 3R-methylethynyl-D-ornithine. The correct production and incorporation of the novel NCAA in a purified model protein was proved by extended analytics including mass spectrometric studies on intact and peptide level. It was shown, that site specifically incorporated ePyl can be modified by two different orthogonal click reactions at only one amino acid site providing access to highly modified proteins. Finally, to prove the feasibility of NCAA carrying proteins as useful tools in biomedical applications, site specifically modified human carbonic anhydrase-folic acid-conjugates (HCA-FA) were applied as targeting, pH responsive capping proteins for drug delivery in mesoporous silica-based nanocarriers (MSNs). The studies were carried out in collaboration with the groups of Prof. Bein and Prof. Bräuchle. Three key elements of the newly developed capping system arise from the site-specifically modified HCA-FA. First, the protein is large enough to block the gates of the MSN to prevent the cargo from escaping. Second, HCA binds phenyl-sulfonamide (phSA) and therefore sulfonamide-functionalized MSNs (MSN-phSA) in a pH depended manner. At pH values below 7, typically present during endocytosis of MSNs, the enzyme releases MSN-phSA, which ensures the release of the cargo at the exact right moment. Third, the site-specific functionalization of HCA can direct the MSNs to a specific cell type.

Abstract

Der ortsspezifische Einbau von nicht-kanonischen Aminosäuren (engl. abgekürzt NCAAs) in Proteine ist ein leistungsfähiges Werkzeug in der chemischen Biologie. Es kann unter anderem für die Untersuchung der Auswirkungen von posttranslationalen Modifikationen, für die Aufklärung enzymatischer Mechanismen, für die Visualisierung von Proteinen in lebenden Zellen oder zum Funktionalisieren therapeutischer und diagnostischer Proteine verwendet werden. Die Technologie basiert auf orthogonalen tRNA/Aminoacyl-tRNA-Synthetase (aaRS)-Paaren, die nicht in die Biosynthesemaschinerie der Wirtszelle (z.B. E. coli) eingreifen. Das in dieser Arbeit verwendete tRNA/aaRS-Paar stammt aus dem Archaea-Stamm Methanosarcina mazei. Es ist verantwortlich für den ribosomalen Einbau von Pyrrolysin (Pyl), das auch als 22. proteinogene Aminosäure bekannt ist. Pyl wird vom sogenannten "Amber Codon" codiert. Im klassischen genetischen Code ist dieses Codon ein Stopp-Signal und führt zur Termination der Translation. Wenn das tRNAPyl/PylRS-Paar z.B. in E. coli exprimiert wird, konkurriert der Einbau einer NCAA als Antwort auf ein Amber-Codon mit der Translationsterminierung und wird daher als amber suppression bezeichnet. In dieser Arbeit wurde das Pyrrolysin-System genutzt, um drei neu beschriebene posttranslationale Lysin-Modifikationen spezifisch an Positionen in Histon H3 zu integrieren, an denen sie identifiziert wurden. Die Aminosäuren ε-N-Propionyl-, ε-N-Butyryl- und ε-N-Crotonyllysin wurden direkt an der gewünschten Position eingebaut, um die jeweilige natürlich vorkommende Propionylierung, Butyrylierung bzw. Crotonylierung zu simulieren. Umfassende bioanalytische Untersuchungen des modifizierten Histons mit peptide mapping-MS und Western-Blot-Analysen belegten den ortsspezifischen und vollständigen Einbau dieser NCAAs eindeutig. Obwohl zahlreiche NCAAs bereits durch amber suppression in Proteine eingebaut wurden, ist die strukturelle Vielfalt dieser Aminosäuren natürlich begrenzt. Die Einführung von NCAAs, die bioorthogonal modifizierbare funktionale Gruppen tragen, eröffnet jedoch nahezu unerschöpfliche Möglichkeiten, da sie spezifische Modifikationen von Proteinen nach deren Expression ermöglicht. Eine der Vielversprechendsten dieser funktionalen Einheiten sind Norbornene, die von der Carell-Gruppe spezifisch in Proteine eingebaut werden können.1 Die Reaktion von Norbornenen mit Tetrazinen in invers Elektronen-anfordernden Diels-Alder-Cycloadditionen ist eine der schnellsten beschriebenen bioorthogonalen Modifizierungsreaktionen. In mechanistischen Studien in Zusammenarbeit mit der Gruppe von Prof. de Vivie-Riedle konnte gezeigt und erklärt werden, dass exo-substituierte Hydroxymethyl-Norbornene aufgrund eines stabileren Übergangszustands etwa dreimal schneller mit Tetrazinen reagieren als ihre endosubstituierten Isomere. Um zu analysieren, ob dieses Verhalten auch auf Proteinebene beobachtet werden kann, wurden das endo-substituierte Norbornyllysin und zwei neue exo-substituierte Norbornen-Aminosäuren in Modellproteine eingebaut. Die Akzeptanz dieser neuen Norbornen-Aminosäuren durch das Pyl-System, das eine zuvor entwickelte PylRS-Dreifachmutante enthielt, wurde mit soliden analytischen Daten bewiesen. Der korrekte Einbau und die vollständige Markierung mit fluoreszierenden und PEGylierten Tetrazinen wurde durch SDS-PAGE-, Intakt-MS- und peptide mapping-Analysen der unmodifizierten und modifizierten Proteinspezies gezeigt. Eine schnellere Markierungsreaktion der exo-substituierten Norbornyl-Aminosäuren wurde bei diesen in vitro-Proteinmarkierungsstudien allerdings nicht beobachtet. In Zusammenarbeit mit der Arbeitsgruppe von Dr. Sabine Schneider konnte die Vielseitigkeit der in der Carell-Gruppe entwickelten PylRS Dreifachmutante in Kristallstrukturen mit gebundenen Pyl-Analoga gezeigt werden. 1 Um die Auswahl an bioorthogonalen Modifikationsreaktionen zu erweitern, wurde die Aziridierung von Norbornenen mit elektronenarmen Sulfonylaziden, eine neuartige Klickreaktion, entwickelt. Die Reaktion wurde auf niedermolekularer, auf Peptid- und schließlich auf Proteinebene mit Norbornenen, Norbornen-Peptiden und Norbornen-haltigen Modellproteinen charakterisiert. Die Geschwindigkeitskonstante zweiter Ordnung wurde mit k2 = 1,7×10 -3 M-1s-1 bestimmt. Dieser Wert ist vergleichbar mit den Geschwindigkeitskonstanten der Ringspannungs-unterstützten Azid-Alkin-Cycloaddition der ersten Generation, die häufig für Biomolekül-Markierungen verwendet wird. 2,3 Die chemoselektive Proteinkonjugation mit Fluoreszenz- und Immuno-reagenzien am Norbornen-Rest wurde durch SDS-PAGE, Western Blot sowie durch peptide mapping und intakte massenspektrometrische Untersuchungen nachgewiesen. Die Reaktion verläuft effizient unter milden Bedingungen, erfordert keine Katalyse und ist orthogonal zu funktionellen Gruppen von nativen Proteinen. Darüber hinaus sind Sulfonylazide leicht zugängliche Verbindungen, was die neu entwickelte Markierungsreaktion zu einer attraktiven Alternative zu den bestehenden Biokonjugationstechniken macht. Eine allgemeine Herausforderung beim Einbau von NCAAs in Proteine besteht darin, dass die Aminosäuren in großen Mengen chemisch synthetisiert werden müssen, um die erforderlichen millimolaren Konzentrationen während der Proteinexpression zu erreichen. Unsere Idee war es, diese aufwendigen Synthesen zu umgehen und die Arbeit an die biosynthetische Maschinerie der Wirtszelle zu übergeben. Dafür wurde in diesem Projekt ein Pyl-Biosynthese-"Entführungs"-System entwickelt, das die Biosynthese und den Einbau von unnatürlichem 3S-Ethiynylpyrrolysin (ePyl), einer zweifach modifizierbaren Einheit, ermöglicht, indem E. coli-Zellen mit 3R-MethylEthinyl-D-Ornithin gefüttert werden. Die korrekte Synthese und die richtige Position des Einbaus der neuen NCAA in einem gereinigten Modellprotein wurde durch umfassende Analysen einschließlich massenspektrometrischer Untersuchungen auf intakter und Peptidebene nachgewiesen. Es konnte gezeigt werden, dass ortsspezifisch eingebautes ePyl durch zwei unterschiedliche orthogonale Klickreaktionen modifiziert werden kann, was den Zugang zu hochmodifizierten Proteinen ermöglicht. Um schließlich die Anwendbarkeit von NCAA-tragenden Proteinen als nützliche Werkzeuge in biomedizinischen Anwendungen zu beweisen, wurden ortsspezifisch modifizierte humane Carboanhydrase-Folsäure-Konjugate (HCA-FA) als zielgerichtete, pH-schaltbare Capping-Proteine für die Wirkstoffabgabe in mesoporösem Siliciumdioxid Nanoträgern (engl. abgekürzt MSNs) eingesetzt. Die Studien wurden in Zusammenarbeit mit den Gruppen von Prof. Bein und Prof. Bräuchle durchgeführt. Drei Schlüsselelemente des neu entwickelten Verschlusssystems ergeben sich aus dem ortsspezifisch modifizierten HCA-FA. Erstens ist das Protein groß genug, um die Poren der MSNs zu blockieren, um zu verhindern, dass die Ladung austritt. Zweitens bindet HCA Phenylsulfonamid (phSA) und daher Sulfonamid-funktionalisierte MSNs (MSN-phSA) pH-abhäng. Bei pH-Werten unter 7, die typischerweise während der Endozytose von MSNs vorliegen, lässt das Enzym MSN-phSA los und gewährleistet damit die Freisetzung der Fracht zum genau richtigen Zeitpunkt. Drittens steuert die ortsspezifische Funktionalisierung von HCA die Anbindung der MSNs an spezifische Zelltypen.