20 research outputs found
Predicting the functions and specificity of triterpenoid synthases: a mechanism-based multi-intermediate docking approach
pre-printTerpenoid synthases construct the carbon skeletons of tens of thousands of natural products. To predict functions and specificity of triterpenoid synthases, a mechanism-based, multi-intermediate docking approach is proposed. In addition to enzyme function prediction, other potential applications of the current approach, such as enzyme mechanistic studies and enzyme redesign by mutagenesis, are discussed
Tuscan Varieties of Sweet Cherry Are Rich Sources of Ursolic and Oleanolic Acid: Protein Modeling Coupled to Targeted Gene Expression and Metabolite Analyses
The potential of six ancient Tuscan sweet cherry (Prunus avium L.) varieties as a source of health-promotingpentacyclictriterpenesishereevaluatedbymeansofatargetedgeneexpressionand metabolite analysis. By using a sequence homology criterion, we identify five oxidosqualene cyclase genes (OSCs) and three cytochrome P450s (CYP85s) that are putatively involved in the triterpene production pathway in sweet cherries. We performed 3D structure prediction and induced-fit docking using cation intermediates and reaction products for some OSCs to predict their function. We show that the Tuscan varieties have different amounts of ursolic and oleanolic acids and that these variations are related to different gene expression profiles. This study stresses the interest of valorizing ancient fruits as alternative sources of functional molecules with nutraceutical value. It also provides information on sweet cherry triterpene biosynthetic genes, which could be the object of follow-up functional studies
Untersuchungen zur Substratspezifität von Squalen-Hopen Zyklasen (SHCs)
Cyclic terpenoids form a large group of natural products with various biological functions. About 60,000 different cyclic terpenoids have been identified by now, containing scaffolds of ten to more than 30 carbon atoms. Among this huge amount of different cyclic compounds, there are many well-known flavors and fragrances, such as menthol or limonene, compounds which are widely used for pharmaceutical purposes like the antitumor compound Taxol and the anti-malaria agent artemisinin, or common membrane constituents and hormones such as the sterols. All of these natural products are derived from cyclization reactions of few linear precursor molecules catalyzed by terpenoid cyclases.
The main focus of the present work rests on one very interesting family of the terpenoid cyclases, the squalene-hopene cyclases (SHCs). Among the over 300 annotated SHCs, most extensive studies had been carried out characterizing the SHC from the thermophilic bacterium Alicyclobacillus acidocaldarius (AacSHC), solving the crystal structure and the complex cyclization mechanism of the C30 precursor squalene into the pentacyclic products hopene and hopanol. This reaction constitutes one of the most complex reaction mechanisms found in nature, including the stereospecific formation of nine stereocenters and 13 covalent bonds.13 Besides AacSHC, several SHCs were partially characterized in previous works. Our interest was triggered by the ethanol and sugar tolerant strain Zymomonas mobilis which is known as one of the most potent hopanoid producers. Zymomonas mobilis contains two genes encoding for SHCs: ZmoSHC1 and the formerly partially characterized ZmoSHC2.
It could be shown that the SHCs are also capable of cyclizing other linear terpenoids. For example, it was found that truncated squalene analogs were accepted as substrates by AacSHC and also the alcohol homofarnesol could be converted into the corresponding cyclic ether ambroxan.16–19 These results indicate that SHCs represent a promising family for catalysis of very different and complex cyclization reactions. Thus, we decided to investigate the SHCs’ potentials regarding their substrate specificities.
In order to characterize the squalene-hopene cyclases ZmoSHC1 and ZmoSHC2 and compare them with AacSHC especially regarding their biocatalytic activities towards unnatural substrates, the SHCs were cloned and heterologously expressed in Escherichia coli. Functional expression was confirmed by conversion of the natural substrate squalene. For the direct comparison, a protocol for partial purification of the membrane-anchored SHCs was elaborated and optimized. For this partial purification as well as for the conversion of the hydrophobic substrates in aqueous milieu a suitable detergent had to be selected. ZmoSHC1 was characterized in more detail, retrieving information about pH- and temperature-dependence of the activity and the biocatalytic stability over a long period of time as well as inhibitory effects.
All of the three enzymes were tested with unnatural substrates of C10-C18 carbon chain lengths. A special focus was laid on substrates containing functional groups such as hydroxyl , carboxy- or keto-groups expected to participate in the cyclization reaction, as shown for the hydroxyl-group of homofarnesol. Several of the substrates were accepted and cyclic products were generated. Interestingly, the functional groups were integrated in the final ring closure and to products with new properties were obtained. Homofarnesol conversion yielding the cyclic ether ambroxan, which is known as a expensive and rare flavor compound, was observed as reported in the literature. Also the corresponding carboxylic acid, homofarnesoic acid, could be converted into the cyclic lactone sclareolide. The C15 tertiary alcohol nerolidol was accepted as substrate and the bicyclic ether caparrapioxide was formed. Lastly, two ketones were accepted as substrates leading to cyclic enol ether products.
Within the present work, all of these new products were characterized after preparative biotransformation and product isolation. Not only the facts that these substrates are much shorter than the natural substrate squalene and possess different functional groups which take part in the cyclization reaction and that useful products containing new properties are formed, but also the different activities of the SHCs towards these substrates are remarkable. Thus, it could be shown that ZmoSHC1 exhibits special biocatalytic properties, as the substrate activity pattern was unexpected. While squalene was converted very poorly, good activity was found towards the reaction of homofarnesol to ambroxan. All of the other substrates were converted in low but significant rates into the corresponding cyclic products. A completely different substrate activity pattern was observed using AacSHC as biocatalyst. Besides very good squalene conversion, much lower activities towards all of the other substrates were found. Using ZmoSHC2, only very low conversion rates were found for squalene and farnesylacetone and no conversion of any of the other substrates.
Based on these observations, it can be concluded that ZmoSHC1 represents a versatile biocatalyst for complex cyclization reactions, as it shows unexpected substrate activity towards other substrates than squalene. In the present work, these and further detailed results are described. Besides the examination of the SHCs’ activities towards different substrates there were also several mutants created in order to find explanations for the differences between the SHCs regarding their substrate activities. This characterization of the triterpenoid cyclase ZmoSHC1 and discussion of their special properties leads to new conclusions about the potential of SHCs to serve as potent biocatalysts for new reactions.Die zyklischen Terpenoide stellen eine große Gruppe von Naturstoffen mit verschiedensten biologischen Funktionen dar. Bis heute konnten etwa 60.000 verschiedene zyklische Terpenoide, die aus Gerüsten von zehn bis über 30 Kohlenstoffatomen aufgebaut sind, identifiziert werden. Unter dieser großen Anzahl von zyklischen Stoffen finden sich viele bekannte Duft- und Aromastoffe, wie zum Beispiel Menthol oder Limonen, Verbindungen, die als pharmazeutisch wirksame Inhaltsstoffe in Medikamenten Anwendung finden, wie etwa der gegen Tumor wirksame Stoff Taxol oder das gegen Malaria angewendete Artemisinin oder auch die als Membranbestandteile und Hormone bekannten Steroide. All diese interessanten Naturstoffe werden durch Zyklisierung von wenigen linearen Vorläufermolekülen gebildet. Diese Zyklisierungsreaktionen werden von Terpenoid Zyklasen katalysiert.
Der Schwerpunkt der vorliegenden Arbeit liegt auf einer Unterfamilie dieser Enzymgruppe der Terpenoid Zyklasen, den Squalen-Hopen Zyklasen (SHCs). Unter den über 300 annotierten SHCs wurde die SHC vom thermophilen Bakterium Alicyclobacillus acidocaldarius (AacSHC) am besten untersucht. Neben der Kristallstruktur wurde auch der komplexe Mechanismus aufgeklärt, nach dem das lineare C30 Substrat Squalen zu den pentazyklischen Produkten Hopen und Hopanol zyklisiert wird. Der Mechanismus dieser Reaktion, bei der neun Stereozentren und 13 kovalente C-C Bindungen spezifisch entstehen, gilt als einer der komplexesten, die man in der Chemie der Naturstoffe bislang entdecken konnte. Neben AacSHC wurden in vorangehenden Arbeiten auch einige andere SHCs teilweise charakterisiert. Von besonderem Interesse ist hierbei der gegen hohe Alkohol- und Zuckerkonzentrationen tolerante Stamm Zymomonas mobilis, der als einer der besten Hopanoid-produzierenden Bakterienstämme bekannt ist und zwei Gene enthält, die für SHCs codieren: ZmoSHC1 und die in vorherigen Arbeiten partiell charakterisierte ZmoSHC2.
Es war gezeigt worden, dass SHCs neben dem natürlichen Substrat Squalen auch einige andere lineare Terpenoide als Substrate akzeptieren und diese zyklisieren. Zum Beispiel konnten verkürzte Squalen-Analoga von AacSHC zyklisiert werden und auch der C16-Alkohol Homofarnesol wurde in den entsprechenden zyklischen Ether Ambroxan umgesetzt. Diese Ergebnisse ließen darauf schließen, dass die SHCs eine vielversprechende Enzymfamilie zur Katalyse von sehr verschiedenen, komplexen Zyklisierungsreaktionen darstellen könnten und deswegen entschieden wir uns dazu, die Substratbreite der SHCs näher zu untersuchen.
Um die Squalen-Hopen Zyklasen ZmoSHC1 und ZmoSHC2 zu charakterisieren und ihre biokatalytischen Aktivitäten mit der von AacSHC vergleichen zu können, wurden die für diese Enzyme codierenden Gene kloniert und heterolog in Escherichia coli exprimiert. Die Expression funktioneller Enzyme wurde durch Umsetzung des natürlichen Substrates Squalen bestätigt. Um die Enzyme direkt miteinander vergleichen zu können, wurde ein Protokoll für die partielle Aufreinigung der membrangebundenen SHCs ausgearbeitet und optimiert. Für diese Aufreinigung sowie für die Umsetzung der hydrophoben Substrate in wässrigem Milieu musste ein geeignetes Detergenz verwendet werden. ZmoSHC1 wurde des Weiteren näher charakterisiert, wobei die pH- und Temperaturabhängigkeit der katalytischen Aktivität, die biokatalytische Stabilität des Enzyms über eine längere Zeitdauer sowie Inhibierungseffekte untersucht wurden. Die drei Enzyme wurden auf Aktivität gegenüber unnatürlichen Substraten mit C-Kettenlängen von C10-C18 getestet. Ein besonderer Fokus wurde hierbei auf Substrate gelegt, die funktionelle Gruppen enthalten, wie zum Beispiel Hydroxyl-, Carboxy- oder Ketogruppen, die, wie für die Hydroxylgruppe von Homofarnesol gezeigt, an der Zyklisierungsreaktion teilnehmen könnten. Interessanterweise wurden diese funktionellen Gruppen in den finalen Ring der polyzyklischen Produkte integriert, wodurch Produkte mit neuen, attraktiven Eigenschaften entstanden. Homofarnesol konnte in den zyklischen Ether und bekannten Duftstoff Ambroxan umgesetzt werden. Die entsprechende Carbonsäure Homofarnesolsäure wurde ebenfalls als Substrat akzeptiert und es wurde das zyklische Lakton Sclareolid erhalten. Der tertiäre C15 Alkohol Nerolidol wurde zum bizyklischen Caparrapioxid umgesetzt. Des Weiteren wurden auch zwei Ketone als Substrate akzeptiert und in zyklische Enolether umgesetzt.
Im Rahmen der vorliegenden Arbeit wurden die neuen Produkte nach präparativer Biotransformation und Aufreinigung charakterisiert. Nicht nur die Tatsache, dass diese Substrate sehr viel kürzere Kohlenstoff-Ketten als das „natürliche“ Substrat Squalen besitzen und über verschiedene funktionelle Gruppen verfügen, die an den Zyklisierungsreaktionen teilhaben und zu interessanten Produkten umgesetzt werden, sondern auch die unterschiedlichen Aktivitäten der SHCs gegenüber dieser Substrate sind bemerkenswert. Es konnte gezeigt werden, dass ZmoSHC1 über besondere Eigenschaften verfügt, da unerwartete Umsetzungsraten bei der Katalyse mit diesem Enzym bestimmt wurden. Während die Zyklisierung von Squalen von ZmoSHC1 nur sehr gering katalysiert wurde, wurde eine gute Aktivität gegenüber der Reaktion von Homofarnesol zu Ambroxan ermittelt. Alle anderen beschriebenen Substrate wurden in geringen, aber signifikanten Raten umgesetzt. Ein vollkommen anderes Aktivitäts-Muster wurde bei Umsetzungen mit AacSHC erhalten. Hier wurde neben sehr guter Umsetzung von Squalen eine viel geringere Aktivität gegenüber allen anderen Substraten bestimmt. Vom Enzym ZmoSHC2 wurden nur Squalen und Farnesylaceton mit sehr geringer Aktivität als Substrate akzeptiert, alle anderen Substrate wurden nicht umgesetzt.
Anhand dieser Ergebnisse kann gefolgert werden, dass SHCs als vielseitige Biokatalysatoren für komplexe Zyklisierungsreaktionen verwendet werden können, da diese Enzyme eine unerwartete Substrataktivität mit anderen Substraten als Squalen zeigen. In der vorliegenden Arbeit werden diese und weitere Ergebnisse im Detail beschrieben. Neben der erwähnten Untersuchung der Aktivität der verschiedenen SHCs gegenüber unterschiedlichen Substraten wurden auch Mutanten hergestellt und untersucht, die zu einer Erklärung der Aktivitätsunterschiede zwischen den verschiedenen Squalen-Hopen Zyklasen verhelfen sollten. Diese Charakterisierung der Triterpen Zyklasen und die Diskussion ihrer besonderen Eigenschaften führen zu neuen Schlussfolgerungen über das Potenzial von SHCs, als fähige Biokatalysatoren für noch nie gezeigte Reaktionen eingesetzt werden zu können
Structural And Functional Studies Of Two Bacterial Terpene Cyclases: Geosmin Synthase And Epi-Isozizaene Synthase
Terpene cyclases convert acyclic isoprenoid precursors into complex cyclic terpenoid compounds. Hydrophobic active site contours in terpene cyclases direct cyclization reactions through a cascade of carbocation intermediates by serving as templates for terpenoid product structure. Both local molecular structure at the active site and global protein structure encompassing domain organization and oligomerization state have effects on catalytic function in terpene cyclases. The goal of this work is characterization of the effects of local structural changes to the active site of a terpene cyclase, and a thorough understanding of the structure-function relationship of active site structure and domain organization in two terpene cyclases, geosmin synthase (ScGS) and epi-isozizaene synthase (EIZS) from Streptomyces coelicolor.
Geosmin synthase (ScGS) is a bifunctional class I sesquiterpene cyclase that catalyzes the conversion of FPP to germacradienol, germacrene D, and geosmin in unique cyclization and cyclization-fragmentation reactions occurring in separate active sites. We determined the X-ray crystal structure of the N-terminal domain of ScGS and homology models of the C-terminal domain of ScGS, and used small-angle X-ray scattering (SAXS) to propose models of domain association in this system. Product analysis by gas chromatography-mass spectrometry (GC-MS) in this system indicates residues that are important for catalysis in the C-terminal domain cyclization-fragmentation reaction.
EIZS is a promiscuous terpene cyclase and produces epi-isozizaene as a major product, along with five other sesquiterpene products. Single mutations at the active site of EIZS can drastically change the proportions and identities of sesquiterpene products. Mutagenesis of key residues at the active site to polar side chains results in mutant EIZS enzymes with altered catalytic properties. The X-ray crystal structures of EIZS F95N and F95C were determined and demonstrate that EIZS mutants containing polar residues at the active site do not exhibit global structural changes when compared to the wild type enzyme. GC-MS was used to analyze the products of eight new EIZS mutants, demonstrating formation of thirteen new sesquiterpene products not previously observed in this system. These results demonstrate that polar mutations are structurally and catalytically tolerated at the active site of EIZS
Towards the structure-informed engineering of enzymes in the avenacin biosynthesis pathway
Glycosylated triterpenes represent numerous and diverse plant natural products, but difficult production has limited their applications in health, food and industry.
This thesis describes the structural characterisation of three enzymes from the biosynthetic pathway of avenacin, an antifungal glycosylated triterpene from oat, to enable their rational engineering.
Avena strigosa arabinosyltransferase (AsAAT1) and transglucosidase (AsTG1), involved in the glycosylation of avenacin, were expressed in Escherichia coli and purified, but did not crystallise. Several deletion constructs proved insoluble, so molecular models were used to rationalise both the specificity of three AsAAT1 mutants for various sugar donors and the switch from glucosyl hydrolase to transglucosidase activity in AsTG1, which was suggested by a multiple sequence alignment. Molecular dynamics simulations of AsTG1 confirmed its ability to discriminate between analogous substrates.
The membrane-bound A. strigosa β-amyrin synthase (AsbAS1), which forms the triterpene scaffold of avenacin, was expressed in E. coli. Attempts to purify and crystallise it only led to the high-resolution structure of a contaminant, HPII catalase. AsbAS1 mutants were designed, inspired by a soluble homologue, to simplify this process. With no solubilised protein observed in E. coli, AsbAS1 was expressed in the yeast Pichia pastoris instead, which resulted in active protein. A homologue, Euphorbia tirucalli β-amyrin synthase (EtAS), was expressed in an active form in E. coli. These expression methods could be used to produce two different β-amyrin synthases and attempt to obtain the first crystal structure of a plant oxidosqualene cyclase. A multiple sequence alignment and models of other homologues generated with AlphaFold2 enabled the design of four AsbAS1 mutants that may have altered product specificity.
This work shows structural information for three enzymes in the avenacin biosynthesis pathway, leading to the rationalisation of the effect from various amino acids. This can now be tested by expressing mutants using the methods described
Characterization of CYP264B1 and a terpene cyclase of a terpene biosynthesis gene cluster from the myxobacterium Sorangium cellulosum So ce56
In the work presented here, CYP264B1 and the terpene cyclase GeoA of Sorangium cellulosum So ce56 have been characterized. CYP264B1 is able to convert norisoprenoids (a-ionone and b-ionone) and diverse sesquiterpene compounds, including nootkatone. Three products, 3-hydroxy-a-ionone, 3-hydroxy-b-ionone and 13-hydroxy-nootkatone were characterized using HPLC and 1H and 13C NMR. CYP264B1 is the first enzyme reported to be capable to hydroxylate regioselectively both norisoprenoids at the position C-3 as well as nootkatone at the position C-13. The kinetics (Km and Vmax) of the product formation were analyzed by HPLC. The results of docking a-/b-ionone and nootkatone into a homology model of CYP264B1 revealed the structural basis of these selective hydroxylations. In addition, an E. coli whole cell system containing CYP264B1 and its redox partners was created for the biotransformation of CYP264B1 substrates. This system was applied successfully for b-ionone conversion. FPP and GGPP were found to be substrates for GeoA. The sesquiterpene and diterpene products of GeoA are similar to valencene (89%) and neocembrene A (80%), respectively. However, these products are most likely new compounds. In order to characterize them by NMR, a whole cell system based on mevalonate pathwayengineered E. coli was created to faciliate the production of sufficient amounts. The terpene production using this system was investigated, showing that it is possible to obtain the amounts required for NMR analysis if laboratory conditions are optimized.In der vorliegenden Arbeit wurden CYP264B1 und die Terpencyclase GeoA aus So ce56 charakterisiert. CYP264B1 ist in der Lage, Norisoprenoide (a-Ionon und b-Ionon) und diverse Sesquiterpene, inklusive Nootkaton, umzusetzen. Drei Produkte (3-Hydroxy-a-Ionon, 3-Hydroxy-b-Ionone und 13-Hydroxy-Nootkaton) wurden mittels HPLC und 1H und 13C NMR charakterisiert. Damit ist CYP264B1 das erste Enzym, das die Fähigkeit besitzt, regioselektiv Norisoprenoide an Position C-3, sowie Nootkaton an Position C- 13 zu hydroxylieren. Die Kinetik der Produktbildung (Vmax und Km) wurde mittels HPLC analysiert. Durch das Docking von a/b-Ionon und Nootkaton in das Homologiemodell von CYP264B1 konnte die strukturelle Grundlage dieser selektiven Hydroxylierungen aufgeklärt werden. Des Weiteren wurde ein E. coli Ganzell- Sumsatzsystem für die Umsetzung von CYP264B1 Substraten etabliert, das neben CYP264B1 auch seine Redox Partner enthält. Dieses System wurde erfolgreich für dieUmsetzung von b-Ionon eingesetzt. FPP und GGPP wurden als Substrate von GeoA identifiziert. Die jeweiligen Sesquiterpen- und Diterpen- Produkte wiesen Ähnlichkeit mit Valencen (89%), bzw. Neocembren (80%) auf. Jedoch handelt es sich bei den gebildeten Produkten höchstwahrscheinlich um neue Verbindungen. Um ausreichende Mengen dieser Verbindungen für eine NMR Analyse zur Verfügung zu stellen, wurde ein Ganzzell- System aufgebaut, das auf E. coli Zellen die heterolog zusätzliche Proteine des Mevalonat Stoffwechselweges exprimieren, basiert. Durch weitere Optimierung dieses Systems sollte es in Zukunft möglich sein, die für eine NMR Analyse erforderlichen Produktmengen produzieren
Engineering Selina-4(15),7(11)-diene synthase for the Production of Novel Products
Terpenes are secondary natural products consisting of carbon five units produced by terpene synthase. They have been found to have functional importance ranging from medical uses (taxadiene), biofuel potential and taste and smell for cinnamon and mint. One terpene precursor substrate can produce up to 400 different products through various terpene synthase with some single enzymes producing over 50 different compounds. This broad chemical diversity is a critical focus for the engineering effort to produce novel terpenes.
Terpene synthase reactions are all initiated by the removal of a pyrophosphate to create a cation that is shaped in a hydrophobic section of the active site controlling the product structure. Little is known about how this hydrophobic region controls production, so this site is the focus of engineering work.
The aim of this project was to engineer Streptomyces pristinaespiralis selina-4(15),7(11)-diene synthase to produce novel compounds. Through this engineering, we could gain mechanistic insight into Streptomyces pristinaespiralis selina-4(15),7(11)-diene synthase and terpene synthases. Streptomyces pristinaespiralis selina-4(15),7(11)-diene synthase is a specific terpene synthase with selina-4(15),7(11)-diene comprising over 90% of the product profile and the second by-product germacrene B.
Using ligand binding software, a series of site directed mutants was produced with the aim of altering product binding and product distribution. An “in culture” GC/MS screen was developed to screen these targeted mutagenesis variants. Initially alanine scanning was performed at specific points in the active sites to identify potential hotspots for further targeted mutagenesis. While no novel products were found in the current screening, variant V187F showed a switch in specificity between the products and a Y152F showed a 50:50 ratio of wild-type products. The kinetic parameters of V187F and Y152F were determined and showed an increased Km suggesting the variant reduces the substrate affinity favouring specificity of germacrene B. It was concluded that, V187 and Y152 have a key roles in the conversion of germacrene B to selina-4(15),7(11)-diene and the formation of the two rings. This mechanistic insight may be used to engineer further terpene synthase to produce novel ring terpenes
Synthesis of Six-Membered Rings and Inhibitors of Protein Kinases
The six-membered rings have a priviledged presence in both natural products and synthetic compounds such as drug molecules. Multiple methods to prepare them in the laboratory have been developed. The Diels-Alder reaction provides several pathways toward the construction of substituted six-membered rings with a high degree of regio-, diastereo- and enantioselectivity. It can be considered to be the most important and powerful carbon-carbon bond-forming reaction of all, in synthetic organic chemistry.
A practical synthetic method for the preparation of hexahydrocinnolines was developed here, as part of continuing research on polymer-supported pericyclic reactions in preparation of biologically interesting compounds. Some cinnoline derivatives from the literature were reported to show interesting biological properties, such as antimicrobial activity and inhibition of cancer cell lines. Hexahydro-1,2,4-triazolocinnoline-1,3-diones and related compounds were synthesized via aza Diels-Alder reaction on solid-phase.
Protein kinases are key regulators of cell function that constitute one of the largest and most functionally diverse gene families. By adding phosphate groups to substrate proteins, kinase driven phosphorylation plays a significant role in a wide range of cellular processes. More than 500 protein kinase genes are present in the the human genome, constituting about 2% of all human genes. They regulate many cellular processes such as growth, differentiation, and proliferation. Protein kinases are seen as potential therapeutic targets since their mutation and dysregulation is causal in multiple human diseases, including metabolic, immunological disorders, and cancer. The consistent structure of the catalytic site among protein kinases sets limits for the development of protein kinase inhibitors. Some protein kinases, however, have regulatory domains as part of their structure, such as protein kinase C (PKC), whose regulatory (C1) domain is unique and is found only in a small number of kinases. This offers a selectivity advantage, thus making the C1-domain an attractive drug target. In fact, the utilization of the the X-ray crystal structure of the PKCδ C1b domain, with molecular modeling, led to the discovery, in this work, of novel C1 domain ligands, the tricyclic γ-amino alcohols. Synthesis of these compounds was achieved by the utilization of the Diels-Alder reactions.
In the process of modifying a naturally occurring deep-blue colored hydrocarbon guaiazulene, a novel aminoguaiazulene derivative was synthesized. This novel derivative undergoes ring annulation reactions with 1,2-dicarbonyl reagents to yield tricyclic δ-lactams, types of benzo[cd]azulenes. Benzo[cd]azulenes derived from guaiazulene, are colorful synthetic carbocyclics with interesting chemical and biological properties. Some of the benzo[cd]azulenes synthesized in this study were recently characterized as selective Pim kinase inhibitors. Pim kinases have become intriguing targets for cancer therapy that possess unique structural features, among protein kinases, that offer a great potential in the design of selective Pim-inhibitors. Based on the promising Pim-kinase inhibition results from multiple cell-based assays, a further modification of the benzo[cd]azulenes was conducted, where some interesting findings in their chemical behavior were observed; new phenolic benzo[cd]azulene compounds were formed, with potent Pim-inhibitory activities. The benzo[cd]azulenes developed in this study were found to be useful research compounds, potential Pim-selective kinase inhibitors, and putative anti-cancer drug candidates. The new synthetic methods detailed in this study will be valuable tools in the further development of additional benzo[cd]azulenes and related systems in the future.Luonnossa esiintyvistä orgaanisista molekyyleistä valtaosalla on rengasrakenne. Näistä kuusirenkaisilla yhdisteillä on merkittävä asema. Luonnonaineiden lisäksi kuusirengasrakenne on yleinen monissa synteettisissä molekyyleissä kuten lääkeaineissa. Näiden valmistamiseksi laboratoriossa on kehitetty suuri joukko erilaisia menetelmiä. Yksi merkittävimmistä on Diels-Alder-reaktio, joka kuuluu laajempaan perisyklisten reaktioiden ryhmään. Yhtenäistä perisyklisille reaktioille on konjugoitu syklinen siirtymätila, jonka seurauksena reaktiotuote syntyy ilman välituotetta. Diels-Alder-reaktiolla voidaan valmistaa tehokkaasti kuusirenkaisisia yhdisteitä ja reaktio on usein stereo- ja regioselektiivinen. Reaktio ei rajoitu pelkästään hiiltä sisältävien renkaiden syntetisointiin, vaan erilaisia heteroatomeja kuten typpeä ja happea sisältäviäkin kuusirenkaita voidaan valmistaa. Diels-Alder-reaktion katsotaan olevan ehkäpä jopa tärkein ja tehokkain menetelmä uusien hiili-hiilisidosten muodostamiseksi synteettisessä orgaanisessa kemiassa. Reaktio ei rajoitu pelkästään laboratorioon, vaan myös luonnosta on löydetty entsyymejä jotka katalysoivat Diels-Alder-reaktiota.
Tutkimusryhmässämme on valmistettu biologisesti mielenkiintoisia heterosyklisiä yhdisteitä kiinteäfaasitekniikalla, jossa reaktiot tapahtuvat kiintokantajan (polymeerin) pinnalla. Kiinteä-faasitekniikan etuina on mm. polymeeriin sidotun tuotteen helppo eristys ja puhdistus. Lisäksi suuria reagenssiylimääriä on mahdollista käyttää, sillä ne voidaan suodattaa helposti erilleen tuotteesta. Kirjallisuudessa esiintyvillä kinnoliinijohdannaisilla on havaittu mm. antibakteerisia ja syöpäsolujen kasvua estäviä vaikutuksia. Tutkimuksessa kehitettiin kiinteäfaasimenetelmä heksahydrokinnoliinirakenteen syntetisoimiseksi hetero-Diels-Alder-reaktiolla, jossa reaktiivinen typpi-typpikaksoissidos osallistuu dienofiilinä sykloadditioon polymeeriin sidotun dieenin kanssa. Reaktiotuotteiden irrotus kiintokantajista tapahtui happokäsittelyllä ja tämän jälkeen ne puhdistettiin pylväskromatografisin menetelmin. Lopuksi selvitettiin kinnoliiniyhdisteiden kyky estää 77 proteiinikinaasin aktiivisuutta.
Proteiinikinaasit ovat kinaasientsyymejä, joiden substraatti on toinen proteiini. Liittämällä fosfaattiryhmän kohdeproteiiniinsa proteiinikinaaseilla on merkittävä rooli solunsisäisessä viestinnässä. Ihmisen genomi sisältää yli 500 proteiinikinaasigeeniä, joka käsittää noin 2 % koko ihmisen perimästä. Proteiinikinaasit säätelevät monia keskeisiä solun toimintaan vaikuttavia prosesseja, kuten kasvua, erilaistumista ja jakautumista. Proteiinikinaasit ovat potentiaalinen lääkevaikutuskohde, sillä näiden entsyymien mutaatiot ja säätelyhäiriöt ovat osallisena useissa ihmisen vakavissa sairauksissa kuten syövissä. Joillakin proteiinikinaaseilla, kuten proteiinikinaasi C:llä on erillinen regulatorinen C1b-domeeni osana proteiinirakennetta. Väitöstyössä käytettiin hyväksi tunnettua proteiinikinaasi C:n C1b:n kiderakennetta ja C1b-domeeniin sitoutuvan forboliesterin rakennetta, joiden avulla mallinnettiin C1b-domeeniin uudentyyppinen inhibiittoriyhdiste. Tämän kolmirengasrakenteisen, kuusi stereokeskusta sisältävän, γ-aminoalkoholin syntetisoinnissa käytettiin hyväksi peräkkäisiä Diels-Alder-reaktioita, joiden avulla yhdisteen molekyylirunko valmistettiin.
Tutkimusryhmässämme on kehitetty menetelmiä luonnossa esiintyvän guajatsuleeni-hiilivedyn muokkaamiseksi. Tämän seskviterpeeneihin kuuluvan atsuleenin kemiallisesti muokatuilla johdoksilla on havaittu Pim-1 ja Pim-3-kinaasien toimintaa estävä vaikutus. Tutkimuksessa kehitettiin menetelmä aminoryhmän liittämiseksi guajatsuleenin rakenteeseen. Uusi aminoguajatsuleeni reagoi 1,2-dikarbonyyliyhdisteiden kanssa muodostaen δ-laktaamirakenteisia, typpiatomin sisältäviä, bentso[cd]azuleeneja. Tutkimuksessa muokattiin myös kemiallisesti jo aikaisemmin ryhmässämme syntetisoituja bentso[cd]atsuleenirakenteita, tarkoituksena valmistaa potentiaalisia ja selektiivisiä Pim-kinaasi-inhibiittoreita. Samalla havaittiin, että osalla näistä yhdisteitä on biologisten ominaisuuksien lisäksi myös ainutlaatuisia kemiallisia ominaisuuksia. Tässä tutkimuksessa valmistetut ja karakterisoidut bentso[cd]atsuleeniyhdisteet ovat osoittautuneet hyödyllisiksi tutkimuskäytössä ja voivat olla myös mahdollisia syöpälääkekandidaatteja tulevaisuudessa