High level production of flavonoid rhamnosides by metagenome-derived Glycosyltransferase C in Escherichia coli utilizing dextrins of starch as a single carbon source.

Flavonoids exert a wide variety of biological functions that are highly attractive for the pharmaceutical and healthcare industries. However, their application is often limited by low water solubility and poor bioavailability, which can generally be relieved through glycosylation. Glycosyltransferase C (GtfC), a metagenome-derived, bacterial glycosyltransferase, was used to produce novel and rare rhamnosides of various flavonoids, including chrysin, diosmetin, biochanin A, and hesperetin. Some of them are to our knowledge firstly described within this work. In our study we deployed a new metabolic engineering approach to increase the rhamnosylation rate in Escherichia coli whole cell biotransformations. The coupling of maltodextrin metabolism to glycosylation was developed in E. coli MG1655 with the model substrate hesperetin. The process proved to be highly dependent on the availability of maltodextrins. Maximal production was achieved by the deletion of the phosphoglucomutase (pgm) and UTP-glucose-1-phosphate uridyltransferase (galU) genes and simultaneous overexpression of the dTDP-rhamnose synthesis genes (rmlABCD) as well as glucan 1,4-alpha-maltohexaosidase for increased maltodextrin degradation next to GtfC in E. coli UHH_CR5-A. These modifications resulted in a 3.2-fold increase of hesperetin rhamnosides compared to E. coli MG1655 expressing GtfC in 24 h batch fermentations. Furthermore, E. coli UHH-CR_5-A was able to produce a final product titer of 2.4 g/L of hesperetin-3'-O-rhamnoside after 48 h. To show the versatility of the engineered E. coli strain, biotransformations of quercetin and kaempferol were performed, leading to production of 4.3 g/L quercitrin and 1.9 g/L afzelin in a 48 h time period, respectively. So far, these are the highest published yields of flavonoid rhamnosylation using a biotransformation approach. These results clearly demonstrate the high potential of the engineered E. coli production host as a platform for the high level biotransformation of flavonoid rhamnosides.

Densely Packed Hydrophobic Clustering: Encapsulated Valerates Form a High-Temperature-Stable {Mo132 } Capsule System.

Porous molecular nanocontainers of {Mo132 }-type Keplerates offer unique opportunities to study a wide variety of relevant phenomena. An impressive example is provided by the highly reactive {Mo132 -CO3 } capsule, the reaction of which with valeric acid results in the very easy release of carbon dioxide and the uptake of 24 valerate ions/ligands that are integrated as a densely packed aggregate, thus indicating the unique possibility of hydrophobic clustering inside the cavity. Two-dimensional NMR techniques were used to demonstrate the presence of the 24 valerates and the stability of the capsule up to ca. 100 °C. Increasing the number of hydrophobic parts enhances the stability of the whole system. This situation also occurs in biological systems, such as globular proteins or protein pockets.

A unique fluoride nanocontainer: porous molecular capsules can accommodate an unusually high number of "rather labile" fluoride anions.

The present work refers to the challenging issue of fluoride anion recognition/binding in water by taking advantage of the unique possibilities offered by the porous molecular nanocontainers of the {Mo132} Keplerate type allowing the study of a variety of new phenomena. Reaction of the highly reactive carbonate-type capsule with aqueous HF results in the release of carbon dioxide and integration of an unprecedentedly large number of fluoride anions--partly as coordinated ligands at both the pentagonal units and the linkers, partly as a disordered water/fluoride assembly inside the cavity. The internal assembly and some of the fluoride ligands are easily released, which provides interesting options for future studies regarding coordination chemistry and catalysis under confined conditions.

Chemical adaptability: the integration of different kinds of matter into giant molecular metal oxides.

Unique properties of the two giant wheel-shaped molybdenum-oxides of the type {Mo(154)}≡[{Mo(2)}{Mo(8)}{Mo(1)}](14) (1) and {Mo(176)}≡[{Mo(2)}{Mo(8)}{Mo(1)}](16) (2) that have the same building blocks either 14 or 16 times, respectively, are considered and show a "chemical adaptability" as a new phenomenon regarding the integration of a large number of appropriate cations and anions, for example, in form of the large "salt-like" {M(SO(4))}(16) rings (M = K(+), NH(4)(+)), while the two resulting {Mo(146)(K(SO(4)))(16)} (3) and {Mo(146)(NH(4)(SO(4)))(16)} (4) type hybrid compounds have the same shape as the parent ring structures. The chemical adaptability, which also allows the integration of anions and cations even at the same positions in the {Mo(4)O(6)}-type units of 1 and 2, is caused by easy changes in constitution by reorganisation and simultaneous release of (some) building blocks (one example: two opposite orientations of the same functional groups, that is, of H(2)O{Mo=O} (I) and O={Mo(H(2)O)} (II) are possible). Whereas Cu(2+) in [(H(4)Cu(II)(5))Mo(V)(28)Mo(VI)(114)O(432)(H(2)O)(58)](26-) (5 a) is simply coordinated to two parent O(2-) ions of {Mo(4)O(6)} and to two fragments of type II, the SO(4)(2-) integration in 3 and 4 occurs through the substitution of two oxo ligands of {Mo(4)O(6)} as well as two H(2)O ligands of fragment I. Complexes 3 and now 4 were characterised by different physical methods, for example, solutions of 4 in DMSO with sophisticated NMR spectroscopy (EXSY, DOSY and HSQC). The NH(4)(+) ions integrated in the cluster anion of 4 "communicate" with those in solution in the sense that the related H(+) ion exchange is in equilibrium. The important message: the reported "chemical adaptability" has its formal counterpart in solutions of "molybdates", which can form unique dynamic libraries containing constituents/building blocks that may form and break reversibly and can lead to the isolation of a variety of giant clusters with unusual properties.

Picking up 30 CO2 molecules by a porous metal oxide capsule based on the same number of receptors.

30 receptors in waiting position: In the porous (pentagon)(12)(linker)(30)-type molybdenum oxide capsule (see picture), the 30 positively charged linkers (five unsaturated shown for illustration in green, the others contain CO(3)(2-) ligands) can act as receptors for neutral and negatively charged ligands. Bubbling CO(2) into the solution containing the acetate-type capsules leads to the upload of CO(2) based on 30 coordinated CO(3)(2-) ligands.

Hydrophobic interactions and clustering in a porous capsule: option to remove hydrophobic materials from water.

The investigation of hydrophobic interactions under confined conditions is of tremendous interdisciplinary interest. It is shown that based on porous capsules of the type {(pentagon)}(12){(linker)}(30) ≡ {(Mo)Mo(5)(12){Mo(2)(ligand)}(30), which exhibit different hydrophobic interiors-achieved by coordinating related ligands to the internal sites of the 30 {Mo(2)} type linkers-there is the option to study systematically interactions with different uptaken/encapsulated hydrophobic molecules like long-chain alcohols as well as to prove the important correlation between the sizes of the related hydrophobic cavities and the option of water encapsulations. The measurements of 1D- and 2D-NMR spectra (e.g. ROESY, NOESY and HSQC) allowed the study of the interactions especially between encapsulated n-hexanol molecules and the hydrophobic interior formed by propionate ligands present in a new synthesized capsule. Future detailed studies will focus on interactions of a variety of hydrophobic species with different deliberately constructed hydrophobic capsule interiors.

Cation behavior at an artificial cell interface: binding distinguished by ion hydration energetics and size.

The unique molybdenum oxide-based nucleophilic porous capsule/artificial cell [{(MoVI)MoVI5O21(H2O)6}12{MoV2O4(SO4)30}]72-, according to an X-ray crystallographic study, traps [Al(H2O)6]3+ complexes above the pores while interacting with the latter via hydrogen bonds; this is supported by 27Al NMR studies of the interaction of the capsule with hydrated Al3+ cations in aqueous solution.

Cellular cation transport studied by 6/7Li and 23Na NMR in a porous Mo132 Keplerate type nano-capsule as model system.

Li+ ions can interplay with other cations intrinsically present in the intra- and extra-cellular space (i.e. Na+, K+, Mg2+ and Ca2+) have therapeutic effects (e.g. in the treatment of bipolar disorder) or toxic effects (at higher doses), likely because Li+ interferes with the intra-/extra-cellular concentration gradients of the mentioned physiologically relevant cations. The cellular transmembrane transport can be modelled by molybdenum-oxide-based Keplerates, i.e. nano-sized porous capsules containing 132 Mo centres, monitored through 6/7Li as well as 23Na NMR spectroscopy. The effects on the transport of Li+ cations through the 'ion channels' of these model cells, caused by variations in water amount, temperature, and by the addition of organic cationic 'plugs' and the shift reagent [Dy(PPP)2](7-) are reported. In the investigated solvent systems, water acts as a transport mediator for Li+. Likewise, the counter-transport (Li+/Na+, Li+/K+, Li+/Cs+ and Li+/Ca2+) has been investigated by 7Li NMR and, in the case of Li+/Na+ exchange, by 23Na NMR, and it has been shown that most (in the case of Na+ and K+, all (Ca2+) or almost none (Cs+) of the Li cations is extruded from the internal sites of the artificial cell to the extra-cellular medium, while Na+, K+ and Ca2+ are partially incorporated.

Reactions inside a porous nanocapsule/artificial cell: encapsulates' structuring directed by internal surface deprotonations.

In the cavities of unprecedentedly functionalised, spherical, porous capsules of the type {Pentagon}12{Linker}30 identical with [{(Mo)Mo5O21(H2O)6}12{Mo2O4(ligand)}30]n- reactions with the ligands -i.e. at the internal shell surfaces - can be performed, in the present case deliberate aquation/hydration and deprotonation reactions at the linker fragments {(Mo2O4)C2O4H}+ similar to that reported in the literature for [(NH3)5CoC2O4H]2+ in solution.

Porous inorganic capsules in action: modelling transmembrane cation-transport parameter-dependence based on water as vehicle.

Insight into basic principles of cation transport through "molecular channels", and especially details of the related fundamental H2O vehicle function, could be obtained via7Li NMR studies of the Li+ uptake/release processes by the unique porous nanocapsule [{(MoVI)MoVI5O21(H2O)6}12{MoV2O4(SO4)}30]72- which behaves as a semi-permeable inorganic membrane open for H2O and small cations; channel traffic as well as internal cavity distribution processes show a strong dependence on "environmental" effects such as exerted by solvent properties, the amount of water present, and competing complexing ligands, and end up in a complex equilibrium situation as in biological leak channels.

Structural characterisation of humic acid-bound PAH residues in soil by 13C-CPMAS-NMR-spectroscopy: evidence of covalent bonds.

The fate of 13C-labelled phenanthrene and fluoranthene in different soil systems during biodegradation was studied. The soil humic acid fraction was isolated followed by structural characterisation using 13C-cross polarisation magic angle spinning nuclear magnetic resonance spectroscopy (13C-CPMAS-NMR). It could be demonstrated that especially the ratio between the concentrations of polycyclic aromatic hydrocarbons (PAHs) and soil humus matrix limits the usefulness of this analytical tool. Based on these results a ratio of 13C-activity(PAH)/13C-activity(soil) approximately 1.5/1.0 in the test material was suggested. The chemical transformation of a PAH and its bound residue formation in a soil system detected by changes of chemical shifts in the 13C-NMR spectrum was proven for the first time. Structural information obtained by NMR spectra were verified by alkaline hydrolysis of PAH/humus-associations and following identification of cleavage products. Ester-bound phenanthrene metabolites such as 1-hydroxy-2-naphthoic acid, ortho-phthalic acid and 3,4-dihydroxybenzoic acid were detected. Additional structural assignments indicated the presence of ether-bound phenanthrene derivatives as well. Using isotopic labelling techniques a quantitative evaluation of bound residue distribution was undertaken. Fifty to seventy percent of phenanthrene metabolites which could be related to the added 13C(1)-phenanthrene were ester bound via their carboxyl groups.

Mimicking biological cation-transport based on sphere-surface supramolecular chemistry: simultaneous interaction of porous capsules with molecular plugs and passing cations.

Spherical capsules of the type [{(Mo)Mo(5)}(12){Mo(2)(ligand)}(30)](n-) exhibiting 20 {Mo(9)O(9)} pores with crown ether functions allowed us to perform a sophisticated study of the title phenomenon based on synthetic work as well as NMR spectroscopy. The pores of the host system interact in solution specifically with guests that can be noncovalently bonded, such as formamidinium and acetamidinium cations, while having different affinities to the pores. The exchange between the guest species present in solution and in the pores was investigated, including, besides the extreme scenarios of complete pore closing and complete opening, that of stepwise pore plugging. Because of this option it was possible to model for the first time passive transmembrane cation transport based on gated pores/channels. These have the appropriate dimensions and can even adopt different structure flexibilities in response to different cations. The present investigation is based on related syntheses as well as on numerous detailed (7)Li NMR studies of Li(+) transport/exchange equilibria in dependence of the pore environment/guest situations. One compound containing capsules with sulfate ligands (2) could be obtained in which all the pores are plugged with formamidinium cations and another corresponding one was obtained with additionally encapsulated Ca(2+) ions (3); these were taken up after temporary release of some of the formamidinium plugs/guests upon short heating of the related solution.

Countercation transport modeled by porous spherical molybdenum oxide based nanocapsules.

Porous nanosized polyoxomolybdate capsule anions of composition [{Mo(VI)(Mo(VI)5O21)(H2O)6}12(linker)30]n-, where (linker)30 is {Mo(V)2O4(SO4)}30 (n = 72) (1 a) or {Mo(V)2O4(SO4)}24{Mo(V)2O4(CH3COO)}6 (n = 64) (2 a), model the (competitive) cellular transmembrane transport of Li+, Na+, K+, and Ca2+ ions along ion channels. According to X-ray crystallography and 7Li and 23Na NMR spectroscopy, Li+ and Na+, the counterions for 1 a and 2 a, respectively, occupy internal sites of the capsule. This study of the counterion transport phenomenon shows that, while Li+ ions can be replaced to a large extent by Na+ and K+ ions and completely by Ca2+ ions added to a solution of 1 a, external Li+ ions do not replace the incorporated Na+ ions of 2 a in an analogous experiment. In this context, related properties of the capsules and especially of their flexible channels, in connection with the complex pathways of cation uptake, are discussed briefly. The relevance of these investigations for lithium-based therapies is also addressed.