Fungal polyketides are a diverse class of natural products that possess many pharmacological properties, including anticancer properties. These properties are evident in the resorcylic acid lactones, a family of polyketides, including zearalenone and radicicol, which shows potent inhibition of tumour cell growth. The key step in the biosynthesis of these lactones is macrocyclization of a linear carboxylic acid into the macrolactone. This reaction is catalyzed by a polyketide synthase (PKS) thioesterase enzyme. Bacterial PKS thioesterases (TEs) have been extensively studied and their substrate specificity has been characterized in vitro. They are highly substrate selective for the macrocyclization reaction. Since Fungal PKS TEs show little sequence homology to bacterial TEs, we have begun investigating their substrate specificity. In particular we are examining the ability of fungal TEs to macrocyclize compounds with varying ring sizes, stereogenic configuration, and nucleophiles. Herein we present the synthesis of a number of diverse TE substrates and the in vitro macrocyclization results for the TEs from zearalenone and radicicol biosynthetic pathway with these substrates.

Fungal polyketides are a diverse class of natural products that possess many pharmacological properties, including anticancer properties. These properties are evident in the resorcylic acid lactones, a family of polyketides, including zearalenone and radicicol, which shows potent inhibition of tumour cell growth. The key step in the biosynthesis of these lactones is macrocyclization of a linear carboxylic acid into the macrolactone. This reaction is catalyzed by a polyketide synthase (PKS) thioesterase enzyme. Bacterial PKS thioesterases (TEs) have been extensively studied and their substrate specificity has been characterized in vitro. They are highly substrate selective for the macrocyclization reaction. Since Fungal PKS TEs show little sequence homology to bacterial TEs, we have begun investigating their substrate specificity. In particular we are examining the ability of fungal TEs to macrocyclize compounds with varying ring sizes, stereogenic configuration, and nucleophiles. Herein we present the synthesis of a number of diverse TE substrates and the in vitro macrocyclization results for the TEs from zearalenone and radicicol biosynthetic pathway with these substrates.

In 1908, H. Emil Fisher attempted to prepare glycinal, an unprotected amino aldehyde, which he found to be inherently unstable and prone to polymerization. This instability arises from the propensity of amines to condense with aldehydes. Accordingly, amino aldehydes require protection of the amine functional group. On the contrary, aziridines do not condense with aldehydes; the aziridine ring-strain precludes the formation of an iminium ion. Predicated upon this orthogonal reactivity, a stable class of unprotected amino aldehydes has been prepared, and an in-depth investigation into their chemical reactivity has been undertaken. Reactions designed to utilize both their nucleophilic (amine) and electrophilic (aldehyde) centres have demonstrated their capacity to forge multiple bonds in a single transformation, and have been implemented in the synthesis of complex heterocycles and cyclic peptides.

In 1908, H. Emil Fisher attempted to prepare glycinal, an unprotected amino aldehyde, which he found to be inherently unstable and prone to polymerization. This instability arises from the propensity of amines to condense with aldehydes. Accordingly, amino aldehydes require protection of the amine functional group. On the contrary, aziridines do not condense with aldehydes; the aziridine ring-strain precludes the formation of an iminium ion. Predicated upon this orthogonal reactivity, a stable class of unprotected amino aldehydes has been prepared, and an in-depth investigation into their chemical reactivity has been undertaken. Reactions designed to utilize both their nucleophilic (amine) and electrophilic (aldehyde) centres have demonstrated their capacity to forge multiple bonds in a single transformation, and have been implemented in the synthesis of complex heterocycles and cyclic peptides.

Polyketide secondary metabolites constitute a structurally-diverse and clinically-important family of natural products. The wide range of biological activities represented by these substrates have contributed to therapeutic agents with annual sales exceeding $20B USD. Large multi-domain proteins called polyketide synthases (PKSs) use simple building blocks to generate highly-oxygenated and stereochemically-rich frameworks with astonishing selectivity. These substrates often feature rigidifying biases imposed by macrocyclic lactones and substituted heterocycles, which can impact their bioactive conformation. The work of this dissertation combines synthetic chemistry and biochemistry to investigate chemoenzymatic production of macrocyclic polyketide natural products. Research focused on validating a transannular oxa-conjugate addition strategy to assembly 2,6-cis-tetrahydropyran (THP) ring systems, as demonstrated by synthesis of the macrocyclic core to neopeltolide. Ultimately, we wish to apply this chemistry to de novo PKS pathways for rapid, reliable, and sustainable production of THP-bearing products like neopeltolide, and toward building SAR libraries. Additionally, a second study probed the specificity of the macrolactonizing thioesterase (TE) domain from the 6-deoxyerythronolide B (DEBS) biosynthetic pathway. This pathway is the paradigm for type-I PKS systems, and is responsible for producing the macrolide core of erythromycin. Our on-going research evaluates the limits of promiscuity within this specific catalytic domain, to characterize the structural elements required to accurately predict macrolactonization. The long-term goal of this study is to assess the potential applicability of DEBS TE as a generalized cyclization biocatalyst for combinatorial biochemistry and chemoenzymatic research.

Ce mémoire présente une nouvelle méthode pour la synthèse de 1,3-énynes par dimérisation d’alcynes terminaux. La méthode de synthèse utilisée est la métallaphotorédox, une technologie qui s’est largement développée au cours des dernières années. Celle-ci nécessite l’utilisation de la lumière visible comme source d’énergie, un photocatalyseur qui peut absorber la lumière ainsi qu’un catalyseur métallique qui peut interagir avec le photocatalyseur. La dimérisation d’alcyne peut être achevée en soumettant un alcyne terminal face à de la lumière bleue en présence du photocatalyseur 4CzIPN, du catalyseur Co(BF4)2·6H2O, du ligand DPPP et de la base DIPEA qui fait office d’anode sacrificielle, dans l’acétonitrile. Les réactions d’homo-dimérisation (un alcyne avec un autre alcyne identique) fournissent des rendements de 48 à 90 % avec un rapport E:Z de >99:1 pour l’ényne formé. Des réactions d’hétéro-dimérisation (un alcyne avec un alcyne différent) peuvent également être effectuées entre un alcyne aliphatique ou aromatique et un alcyne de silyle (TMS ou TIPS) et les rendements varient de 47 à 99 % avec un rapport E:Z de >99:1 pour l’ényne formé. Cette méthodologie a ensuite été appliquée pour des réactions de macrocyclisation. Trois macrocycles à 17, 18 et 19 chaînons ont pu être synthétisés de cette manière avec des rendements respectifs de 25, 91 et 37 %. / This thesis presents a new methodology for the synthesis of 1,3-enynes by dimerization of terminal alkynes. The synthetic method used is based on metallaphotoredox, a technology that was largely developed during the last few years. The latter uses visible light as an energy source, a photocatalyst that can absorb the light and a metal-based catalyst that interacts with the photocatalyst. The dimerization of alkyne can be achieved by submitting a terminal alkyne under blue light irradiation in the presence of the photocatalyst 4CzIPN, the catalyst Co(BF4)2·6H2O, the ligand DPPP and the base DIPEA which is used as a sacrificial anode, in acetonitrile. The reactions of homo-dimerization (one alkyne with an identical alkyne) provides yields ranging from 48 to 90 % and a E:Z ratio of >99:1 for the resulting enyne. Reactions of hetero-dimerization (one alkyne with a different alkyne) can also be achieve with an aliphatic or aromatic alkyne and a silyl alkyne (TMS or TIPS) with yields ranging from 47 to 99 % and a E:Z ratio of >99:1 for the resulting enyne. This methodology was then applied for macrocyclization reactions. Macrocycles with 17-, 18- and 19- members ring were synthesized that way, with yield of 25, 91 and 37 % respectively.

[ES] En la presente tesis doctoral se ha llevado a cabo un estudio de nuevas reacciones orgánicas de interés catalizadas por diferentes especies de paladio, tanto clústeres como complejos metálicos. En primer lugar, se ha estudiado la reacción intramolecular regioirregular de Mizoroki-Heck catalizada por clústeres de paladio en disolución, donde se han puesto en conflicto las propiedades electrónicas y estéricas de la reacción de Mizoroki-Heck, obteniendo la reacción de ciclación con una regioselectividad inversa. También se han realizado estudios mecanísticos mediante experimentos cinéticos y computacionales. También se ha desarrollado un catalizador sólido basado en una zeolita, concretamente la zeolita X, que tras intercambiar los cationes de compensación de carga e introducir el Pd, cataliza la reacción intermolecular de Mizoroki-Heck regioirregular. Se ha conseguido obtener un centro activo en la zeolita con las condiciones tanto estéricas como electrónicas óptimas para realizar la reacción intermolecular regioirregular de Mizoroki-Heck entre estirenos y iodobencenos, con buenos rendimientos y selectividades. Por otro lado, se ha estudiado una nueva ruta de síntesis de una fragancia comercial, la deshidromuscona, donde se obtiene un rendimiento global de síntesis mayor que el obtenido actualmente en la industria, todo ello en un menor número de pasos. Se ha llevado a cabo un estudio más intenso sobre el paso donde se realiza la macrociclación en alta concentración, para poder ser viable industrialmente. Mediante la reacción de Mizoroki-Heck estudiada arriba, se ha llevado a cabo un estudio de macrociclación a alta concentración donde, a partir de unos sustratos modelo, se realiza la reacción de macrociclación catalizada por clústeres de paladio en disolución, y se obtienen los macrociclos con buenos rendimientos en una concentración de hasta 1M. También se han realizado estudios mecanísticos tanto computacionales como cinéticos. Por último, se ha realizado la síntesis de dos fosfinas voluminosas basadas en una fragancia, y su posterior estudio de coordinación con el paladio y su alcance catalítico en reacciones orgánicas de interés, como son la telomerización de isopreno, y las reacciones de acoplamiento Buchwald-Hartwig y Suzuki con cloroderivados, donde se comprueba la capacidad catalítica de los complejos formados con nuestras fosfinas y el paladio. / [CA] En la present tesi doctoral s'ha dut a terme un estudi de noves reaccions orgàniques d'interés catalitzades per diferents espècies de pal·ladi, tant clústers com complexos metàl·lics. En primer lloc, s'ha estudiat la reacció intramolecular regioirregular de Mizoroki- Heck catalitzada per clústers de pal·ladi en dissolució, on s'han posat en contrast les propietats electròniques i estèriques de la reacció de Mizoroki-Heck obtenint la reacció de ciclació amb una regioselectivitat inversa. També s'han realitzat estudis mecanístics mitjançant estudis cinètics i computacionals. També s'ha desenvolupat un catalitzador sòlid basat en zeolita, concretament la zeolita X, que després de fer-li intercanvi de cations i introduir el Pd que catalitza la reacció a l'interior, s'ha aconseguit obtindre un centre actiu amb les condicions tant estèriques com electròniques òptimes per a realitzar la reacció intermolecular regioirregular de Mizoroki-Heck entre estirens i iodobencenos amb bons rendiments i selectivitats. D'altra banda, s'ha estudiat una nova ruta de síntesi d'una fragància comercial, la deshidromuscona, on s'obté un rendiment global de síntesi major que l'obtingut actualment en la indústria, tot això en un menor nombre de passos, on s'ha dut a terme un estudi més intens d'un pas on es realitza una macrociclació en alta concentració per a poder ser viable industrialment. Mitjançant la reacció de Mizoroki-Heck estudiada anteriorment, s'ha dut a terme un estudi de macrociclació en alta concentració on a partir uns substrats model es realitza la reacció de macrociclació catalitzada per clústers de pal·ladi en dissolució on s'obtenen els macrocicles amb bons rendiments en una concentració fins del 1M. També s'han realitzat estudis mecanístics tant computacionals com cinètics. Finalment, s'ha realitzat la síntesi de dues fosfines voluminoses basades en fragàncies i el seu posterior estudi de coordinació amb el pal·ladi i el seu abast catalític en reaccions orgàniques difícils, com són la telomerización, la Buchwald-Hartwig o la Suzuki, comprovant la capacitat catalítica dels complexos formats amb les nostres fosfines i el pal·ladi. / [EN] In this doctoral thesis, a study of new organic reactions of interest catalyzed by different palladium species, both clusters and metal complexes, has been carried out. Firstly, the intramolecular regioirregular Mizoroki-Heck reaction catalyzed by palladium clusters in solution has been studied, where the electronic and steric properties of the Mizoroki-Heck reaction have been contrasted, obtaining the cyclization reaction with an inverse regioselectivity. Mechanistic studies have also been carried out by kinetic and computational studies. A solid catalyst based on a zeolite has also been developed, specifically zeolite X, which after making cation exchange and introducing the Pd that catalyzes the reaction inside, it has been possible to obtain an active center with the optimum steric and electronic conditions to carry out the intermolecular regioirregular Mizoroki-Heck reaction between styrenes and iodobenzenes, with good yields and selectivities. On the other hand, a new route for the synthesis of a commercial fragrance, dehydromuscone, has been studied, where a higher overall synthesis yield than the one currently obtained in the industry is obtained. The new synthesis is carried out in less number steps, and a more intense study on the macrocyclization reactive in high concentration has been carried out, in order to evaluate if it is industrially viable. By means of the Mizoroki-Heck reaction studied previously, a study of macrocyclization at high concentration has been carried out where, from some model substrates, the macrocyclization reaction catalyzed by palladium clusters in solution is carried out, to obtain macrocycles with good yields and in a concentration up to 1M. Both computational and kinetic mechanistic studies have also been performed. Finally, the synthesis of two bulky phosphines based on a commercial fragrance and their subsequent coordination with palladium has been studied. Their catalytic scope in challenging organic reactions, such as the telomerization of isoprene, and the Buchwald-Hartwig and Suzuki cross-coupling reactions of chloroderivatives, have been carried out, verifying the catalytic capacity of the complexes formed with our phosphines and palladium. / Garnes Pórtoles, F. (2023). Nuevas reacciones de acoplamiento cruzado de alquenos terminales altamente regioselectivas y catalizadas por paladio [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/195109

Avec les réactions de macrocyclisation, il est possible d’ajouter de nouveaux groupements chimiques dans le macrocycle afin d’influencer ses propriétés biologiques. Les thioalcynes sont des groupements fonctionnels peu présents dans les macrocycles qui pourraient offrir de nouvelles possibilités de composés utilisables en chimie médicinale et qui ouvrent également la voie à plusieurs possibilités de diversification grâce à leur réactivité prévisible. Par conséquent, la mise au point d’une méthode de synthèse permettant formation de macrocycles peptidiques contenant des thioalcynes est intéressante. Ce mémoire présente ainsi le développement d’une stratégie de synthèse pour la formation de macrocycles tripeptidiques via la formation d’un connecteur thioalcyne par couplage croisé catalysé par un système [Cu(MeCN)4]PF6/dtbbpy/2,6-lutidine. Cette méthode a permis la synthèse de 4 macrocycles tripeptidiques possédant un thioalcyne avec des rendements compris entre 30% et 58%. Nous montrons aussi des possibilités de diversifications des macrocycles par l’utilisation du thioalcyne des macrocycles comme réactif. / Macrocyclization gives the opportunity to incorporate new functional groups in macrocycles to influence their biological properties. Rarely seen in macrocycles, alkynyl sulfides are functional groups that could be introduced into interesting compounds for applications in medicinal chemistry due to their ability to open new opportunities for diversification of the macrocyclic framework with its predictable reactivity. Consequently, the development of a synthetic strategy for the synthesis of macrocycles containing alkynyl sulfide in their structure is of interest. This thesis presents the development of a synthetic strategy for the formation of macrocyclic tripeptides using an alkynyl sulfide linker which is formed by cross coupling employing a [Cu(MeCN)4]PF6/dtbbpy/2,6-lutidine catalytic system. This method was used to generate 4 macrocyclic alkynyl sulfide tripeptides with yields ranging between 30% and 58%. This thesis also shows different possibilities for the diversification the macrocycle framework by using the alkynyl sulfide linker as a building block.

PEGylation can improve the pharmacokinetic properties of protein therapeutics via decreasing renal clearance and shielding the protein surface from proteases, antibody neutrailization, and aggregation. Conformational stability enhancement can provide criteria for the identification of optimal sites for PEGylation, but how PEG influence the noncovalent interactions from the surface of proteins has not been well illustrated. Macrocyclization can effectively enhance the conformational stability of small peptides and large proteins. Combination of PEG-based conformational stability enhancement and macrocyclization-based conformational constraint has not been explored. Macrocycliziation has been employed to stabilize protein tertiary structures, but there are no general guidelines for interhelical staple to stabilize coiled-coil motifs of proteins. Chapter 1 is an introduction to peptide stapling and macrocyclization of proteins. Chapter 2 describes our test of the hypothesis that PEG increases the conformational stability of proteins by desolvating nearby salt bridges. In chapter 3, we explore the combination of PEG-based conformational stability enhancement with macrocyclization on WW domain, and find that the most important criteria for PEG stapling is ensuring the side chains cross-linked by PEG are distant in primary sequence but close in tertiary structure. In chapter 4, we further apply this macrocyclization criteria to another -sheet-based protein, SH3 domain of the chicken Src protein, and to a disulfide-bonded parallel coiled-coil heterodimer derived from the yeast transcription factor GCN4. In chapter 5, we explore the determinants of PEG-staple-based stabilization by changing the distance of the staple to the terminal interhelical disulfide bond, varying the length of staple, exploring different solvent exposed positions for stapling and employing heterochiral residues for stapling. We further apply the interhelical PEG staple to a HER-2 affibody, and find that PEG-stapling increases the conformational stability and proteolytic resistance of the stapled affibody relative to its non-stapled counterpart and to the native unmodified affibody.

Dans le domaine des machines moléculaires, la recherche d'un système produisant un mouvement orienté fait l'objet d'intenses recherches. Des résultats ont déjà été obtenus en ce sens mais n'exploitant pas le mouvement brownien comme le font les systèmes trouvés dans la nature comme la myosine. Nous proposons donc la synthèse d'une roue à rochet avec pour but de mimer ce phénomène, si besoin avec une activation de faible énergie. La première partie de ce travail a consisté en la conception par modélisation de l'équivalent synthétique d'une roue à rochet, basiquement constituée d'une roue crantée et d'un cliquet en vue d'étudier si une telle molécule pourrait produire une rotation orientée. Ce système est constitué d'un motif bishomoinositol fonctionnalisé en tant que roue et d'un cycloadduit de l'anthracène comme cliquet, liés par des bras espaceurs. La synthèse d'un système modèle dans lequel un motif bicyclo[2.2.2]octane joue le rôle de roue simplifiée a été menée afin de valider les différentes stratégies de synthèse. A cette occasion, une méthode alternative de la synthèse du diacide bicyclo[2.2.2]octanoïque a été mise au point ainsi qu'une méthodologie d'estérification compatible avec cet acide ainsi que le diacide 9,10-anthracènedioïque. Nous sommes ainsi parvenus à obtenir le produit de macrolactonisation, avec de possibles application comme roue de nanovéhicules. La synthèse du modèle a été l'occasion de pointer certaines impossibilités synthétiques au niveau des espaceurs (éthers, amides, triazoles, tétrazoles, alcènes) dans le système original. Un nouveau travail de modélisation a permis d'aboutir à une cible accessible synthétiquement conservant le comportement de roue à rochet. Dans cette optique, la synthèse du bishomoinositol fonctionnalisé par des acides en tête de pont a été entreprise par une première voie incluant ces groupements au départ de la synthèse, une seconde voie introduisant ces groupements en fin de séquence. / In the field of molecular machines, systems producing an oriented motion have been intensely looked for. Results, obtained toward this goal, do not exploit Brownian motion whereas it happens in natural systems such as myosin. Hereby we propose the synthesis of a molecular ratchet aiming to mimic this phenomenon or to be able to act as a molecular motor through low energy activation. The first part of this work consisted in the design by modelisation of a synthetic equivalent of a ratchet, basically consisting of a toothed wheel and a pawl. This study aim at determining if such a molecule could produce an oriented motion by simple Brownian motion. This system is consisting of a functionalized bishomoinositol moiety as the wheel and a cycloadduct of anthracene as the pawl, linked by spacers allowing the adjustment of the energy barriers. The synthesis of a model where a bicyclo[2.2.2]octane moiety plays the role of a simplified wheel has been conducted in order to validate various synthetic strategies. This gave the opportunity to develop an alternative method of the synthesis of the bicyclo[2.2.2]octanedicarboxylic diacid as well as an esterification methodology compatible with the bulkiness or low solubility of both partners. The macrolactonization product could be obtained and opens some possible applications as nanovehicle wheels. The synthesis of the model was the occasion to highlight synthetic limitations concerning the spacers (ethers) in the original system. A new series of targets taking into account the synthetic restrictions while keeping the ratchet behavior were designed. The discovery of another synthetic difficulties (amides, triazoles, tetrazoles and alkenes) yielded a single refined target. Toward this goal, the synthesis of the bishomoinositol functionalized in the bridgehead position was engaged in a first pathway including these functions at the beginning of the synthesis, the second way introducing them on the bishomoinositol at the end of the sequence.