Data & Resources

Hop (Humulus lupulus L. Cannabaceae) is an economically important crop. In addition to its role in beer brewing, its pharmaceutical applications have been of increasing importance in recent years. Bitter acids (prenylated polyketides), prenylflavonoids and essential oils, are the primary phytochemical components that account for hop medicinal value. An integrated approach utilizing nuclear magnetic resonance (NMR) and mass spectrometry (MS) techniques was used for the first large-scale metabolite profiling in Humulus lupulus. Resins and extracts prepared from 13 hop cultivars were analysed using NMR, liquid chromatography (LC)-MS and fourier transform ion cyclotron resonance (FTICR)-MS in parallel and subjected to principal component analysis (PCA). A one pot extraction method, compatible with both MS and NMR measurement was developed to help rule out effects due to differences in extraction protocols. Under optimised conditions, we were able to simultaneously quantify and identify 46 metabolites including 18 bitter acids, 12 flavonoids, 3 terpenes, 3 fatty acids and 2 sugars. Cultivars segregation in PCA plots generated from both LC-MS and NMR data were found comparable and mostly influenced by differences in bitter acids composition among cultivars. FTICR-MS showed inconsistent PCA loading plot results which are likely due to preferential ionisation and also point to the presence of novel isoprenylated metabolites in hop. This comparative metabolomic approach provided new insights for the complementariness and coincidence for these different technology platform applications in hop and similar plant metabolomics projects.

The synthesis of a new generation of highly cytotoxic tubulysin analogues (i.e., tubugis) is described. In the key step, the rare, unstable, and synthetically difficult to introduce tertiary amide–N,O-acetal moiety required for high potency in natural tubulysins is replaced by a dipeptoid element formed in an Ugi four-component reaction. Two of the four components required are themselves produced by other multicomponent reactions (MCRs). Thus, the tubugis represent the first examples of the synthesis of natural-product-inspired compounds using three intertwined isonitrile MCRs.

Reactions of ω-diphenylphosphinofunctionalized alkyl phenyl sulfides Ph2P(CH2)nSPh (n = 1, 1a; 2, 2a; 3, 3a), sulfoxides Ph2P(CH2)nS(O)Ph (n = 1, 1b; 2, 2b; 3, 3b) and sulfones Ph2P(CH2)nS(O)2Ph (n = 1, 1c; 2, 2c; 3, 3c) with dinuclear chlorido bridged rhodium(I) complexes [(RhL2)2(μ-Cl)2] (L2 = cycloocta-1.5-diene, cod, 4; bis(diphenylphosphino)ethane, dppe, 5) afforded mononuclear Rh(I) complexes of the type [RhCl{Ph2P(CH2)nS(O)xPh-κP}(cod)]1 (n/x = 1/0, 6a; 1/1, 6b; 1/2, 6c; 2/0, 8a; 2/1, 8b; 2/2, 8c; 3/0, 10a; 3/1, 10b; 3/2, 10c) and [RhCl{Ph2P(CH2)nS(O)xPh-κP}(dppe)] (n/x = 1/0, 7a; 1/1, 7b; 1/2, 7c; 2/0, 9a; 2/1, 9b; 2/2, 9c; 3/0, 11a; 3/1, 11b; 3/2, 11c) having the P^S(O)x ligands κP coordinated. Addition of Ag[BF4] to complexes 6–11 in CH2Cl2 led with precipitation of AgCl to cationic rhodium complexes of the type [Rh{Ph2P(CH2)nS(O)xPh-κP,κS/O}L2][BF4] having bound the P^S(O)x ligands bidentately in a κP,κS (13a–18a, 15b–18b) or a κP,κO (13b, 14b, 13c–18c) coordination mode. Unexpectedly, the addition of Ag[BF4] to 6a in THF afforded the trinuclear cationic rhodium(I) complex [Rh3(μ-Cl)(μ-Ph2PCH2SPh-κP:κS)4][BF4]2·4THF (12·4THF) with a four-membered Rh3Cl ring as basic framework. Addition of sodium bis(trimethylsilyl)amide to complexes 6–11 led to a selective deprotonation of the carbon atom neighbored to the S(O)x group (α-C) yielding three different types of organorhodium complexes: a) Organorhodium intramolecular coordination compounds of the type [Rh{CH{S(O)xPh}CH2CH2PPh2-κC,κP}L2] (22a–c, 23a–c), b) zwitterionic complexes [Rh{Ph2PCHS(O)xPh-κP,κS/O}L2] having κP,κS (21a, 21b) and κP,κO (20b/c, 21c) coordinated anionic [Ph2PCHS(O)xPh] ligands, and c) the dinuclear rhodium(I) complex [{Rh{μ-CH(SPh)PPh2-κC:κP}(cod)}2] (19). All complexes were fully characterized spectroscopically and complexes 15b, 15c, 12·4THF and 19·THF additionally by X-ray diffraction analysis. DFT calculations of zwitterionic complexes gave insight into the coordination mode of the [Ph2PCHS(O)Ph] ligand (κP,κS versus κP,κO).

A dsRNAi approach silencing a key enzyme of sinapate ester biosynthesis (UDP-glucose:sinapate glucosyltransferase, encoded by the UGT84A9 gene) in oilseed rape (Brassica napus) seeds was performed to reduce the anti-nutritive properties of the seeds by lowering the content of the major seed component sinapine (sinapoylcholine) and various minor sinapate esters. The transgenic seeds have been produced so far to the T6 generation and revealed a steady suppression of sinapate ester accumulation. HPLC analysis of the wild-type and transgenic seeds revealed, as in the previous generations, marked alterations of the sinapate ester pattern of the transformed seeds. Besides strong reduction of the amount of the known sinapate esters, HPLC analysis revealed unexpectedly the appearance of several minor hitherto unknown rapeseed constituents. These compounds were isolated and identified by mass spectrometric and NMR spectroscopic analyses. Structures of 11 components were elucidated to be 4-O-glucosides of syringate, caffeyl alcohol and its 7,8-dihydro derivative as well as of sinapate and sinapine, along with sinapoylated kaempferol glycosides, a hexoside of a cyclic spermidine alkaloid and a sinapine derivative with an ether-bridge to a C6–C3-unit. These results indicate a strong impact of the transgenic approach on the metabolic network of phenylpropanoids in B. napus seeds. Silencing of UGT84A9 gene expression disrupt the metabolic flow through sinapoylglucose and alters the amounts and nature of the phenylpropanoid endproducts.

Reactions of fac-[PtMe3(4,4′-R2bpy)(Me2CO)][BF4] (R = H, 1a; tBu, 1b) and fac-[PtMe3(OAc-κ2O,O′)(Me2CO)] (2), respectively, with thioglycosides containing thioethyl (ch-SEt) and thioimidate (ch-STaz, Taz = thiazoline-2-yl) anomeric groups led to the formation of the carbohydrate platinum(IV) complexes fac-[PtMe3(4,4′-R2bpy)(ch*)][BF4] (ch* = ch-SEt, 8–14; ch-STaz, 15–23) and fac-[PtMe3(OAc-κ2O,O′)(ch*)] (ch* = ch-SEt, 24–28; ch-STaz = 29–35), respectively. NMR (1H, 13C, 195Pt) spectroscopic investigations and a single-crystal X-ray diffraction analysis of 19 (ch-STaz = 2-thiazolinyl 2,3,4,6-tetra-O-benzoyl-1-thio-β-D-galactopyranose) revealed the S coordination of the ch-SEt glycosides and the N coordination of the ch-STaz glycosides. Furthermore, X-ray structure analyses of the two decomposition products fac-[PtMe3(bpy)(STazH-κS)][BF4] (21a) and 1,6-anhydro-2,3,4-tri-O-benzoyl-β-D-glucopyranose (23a), where a cleavage of the anomeric C–S bond had occurred in both cases, gave rise to the assumption that this decomposition was mediated due to coordination of the thioglycosides to the high electrophilic platinum(IV) atom, in non-strictly dried solutions. Reactions of fac-[PtMe3(Me2CO)3][BF4] (3) with ch-SEt as well as with ch-SPT and ch-Sbpy thioglycosides (PT = 4-(pyridine-2-yl)-thiazole-2-yl; bpy = 2,2′-bipyridine-6-yl), having N,S and N,N heteroaryl anomeric groups, respectively, led to the formation of platinum(IV) complexes of the type fac-[PtMe3(ch*)][BF4] (ch* = ch-SEt, 36–40, ch-SPT 42–44, ch-Sbpy45, 46). The thioglycosides were found to be coordinated in a tridentate κS,κ2O,O′, κS,κN,κO and κS,κ2N,N′ coordination mode, respectively. Analogous reactions with ch-STaz ligands succeeded for 2-thiazolinyl 2,3,4-tri-O-benzyl-6-O-(2,2′-bipyridine-6-yl)-1-thio-β-D-glucopyranoside (5h) resulting in fac-[PtMe3(ch-STaz)][BF4] (41, ch-STaz = 5h), having a κ3N,N′,N′′coordinated thioglycoside ligand.

Reactions of [PtMe3(bpy)(Me2CO)][BF4] (2) with the thionucleobases 2-thiouracil (s2Ura), 4-thiouracil (s4Ura) and 2,4-dithiouracil (s2s4Ura) resulted in the formation of complexes of the type [PtMe3(bpy)(L-κS)][BF4] (L = s2Ura, 3; s4Ura, 4; s2s4Ura, 5). The complexes were characterized by NMR spectroscopy (1H, 13C, 195Pt), IR spectroscopy as well as microanalyses. The coordination through the C4S groups (4, 5) was additionally confirmed by DFT calculations, where it was shown that these complexes [PtMe3(bpy)(L-κS4)]+ (L = s4Ura, s2s4Ura) are about 5.8 (4b) and 3.3 kcal/mol (5b), respectively, more stable than the respective complexes, having thiouracil ligands bound through the C2X groups (X = O, 4a; S, 5a). For [PtMe3(bpy)(s2Ura-κS2)][BF4] (3) no preferred coordination mode could be assigned solely based on DFT calculations. Analysis of NMR spectra showed the κS2 coordination. In vitro cytotoxic studies of complexes 3−5 on nine different cell lines (8505C, A253, FaDu, A431, A549, A2780, DLD-1, HCT-8, HT-29) revealed in most cases moderate activities. However, 3 and 5 showed significant activity towards A549 and A2780, respectively, possessing IC50 values comparable to those of cisplatin. Cell cycle perturbations and trypan blue exclusion test on cancer cell line A431 using [PtMe3(bpy)(s2s4Ura-κS4)][BF4] (5) showed induction of apoptotic cell death. Furthermore, the reaction of [PtMe3(OAc-κ2O,O′)(Me2CO)] (6) with 4-thiouracil yielded the dinuclear complex [(PtMe3)2(μ-s4Ura–H)2] (7), which has been characterized by microanalysis, NMR (1H, 13C, 195Pt) and IR spectroscopy as well as ESI mass spectrometry. X-ray diffraction analysis of crystals yielded in an isolated case exhibited the presence of a hexanuclear thiouracilato platinum(IV) complex, possessing each three different kinds of methyl platinum(IV) moieties and 4-thiouracilato ligands. This exhibited the ability of 4-thiouracil platinum(IV) complexes to form multinuclear complexes.

Reactions of dinuclear μ-chlorido rhodium(I) complexes [(RhL2)2(μ-Cl)2] (L2 = cycloocta-1,5-diene, cod, 3; L2 = P∧P: Ph2PCH2PPh2, dppm, 4a; Ph2P(CH2)2PPh2, dppe, 4b; Ph2P(CH2)3PPh2, dppp, 4c; Me2P(CH2)2PMe2, dmpe, 4d) with γ-phosphino-functionalized propyl phenyl sulfides PhSCH2CH2CH2PR2 (R = Ph, 1; Cy, 2) afforded mononuclear rhodium(I) complexes of the type [RhCl(R2PCH2CH2CH2SPh-κP)L2] ] (R = Ph/L2 = P∧P, 5a−c; R = Ph/L2 = cod, 6; R = Cy/L2 = P∧P, 7a−d; R = Cy/L2 = cod, 8). Single-crystal X-ray diffraction analysis of 7b·C6H6 exhibited the expected square-planar coordination of the rhodium atom having coordinated dppe-κ2P,P′, Cy2PCH2CH2CH2SPh-κP, and a chlorido ligand. Deprotonation of complexes 5b/c, 6, 7b/c, and 8 with lithium diisopropyl amide (LDA) yielded, with a selective deprotonation of the CH2 group next to the sulfur atom (α-CH2 group), complexes of the type [Rh{CH(SPh)CH2CH2PR2-κC,κP}L2] (13b/c, 14, 15b/c, 16), thus being organorhodium intramolecular coordination compounds. Unexpectedly, reactions of the dppm complexes 5a and 7a with LDA led to deprotonation of the CH2 group of the dppm ligand, resulting in formation of mononuclear rhodium complexes with a bis(diphenylphosphino)methanide-κ2P,P′ ligand and a R2P∧SPh-κP,κS ligand, as well (17, 18). Single-crystal X-ray diffraction analysis of [Rh(dppm−H-κ2P,P′)(Cy2PCH2CH2CH2SPh-κP,κS)]·THF (18·THF) shows the rhodium atom located in the center of a distorted square-planar environment having bound the P∧S-κP,κS ligand and the anionic dppm−H-κ2P,P′ ligand with a very small P2−Rh−P3 angle (68.8(2)°) reflecting the small bite of that ligand. Addition of Tl[PF6] to complexes 5−8 afforded cationic rhodium(I) complexes of the type [Rh(R2PCH2CH2CH2SPh-κP,κS)L2][PF6] (9−12) bearing bidentately coordinated neutral co-ligands (P∧P: 9, 11; cod, 10, 12) and κP,κS-coordinated γ-phosphino-functionalized propyl phenyl sulfide ligands, as well. Single-crystal X-ray diffraction analysis of 10 reveals that the rhodium atom adopts a slightly distorted square-planar conformation. Complexes 9a−c and 11a−d were found to react with carbon monoxide, yielding cationic rhodium carbonyl complexes [Rh(CO)(R2PCH2CH2CH2SPh-κP,κS)(P∧P-κ2P,P′)]+ (19, 20), being in a dynamic equilibrium between two diastereomers each at room temperature, which was additionally verified by DFT calculations.

An integrated approach using targeted metabolite profiles and modest EST libraries each containing approximately 3500 unigenes was developed in order to discover and functionally characterize novel genes involved in plant‐specialized metabolism. EST databases have been established for benzylisoquinoline alkaloid‐producing cell cultures of Eschscholzia californica , Papaver bracteatum and Thalictrum flavum , and are a rich repository of alkaloid biosynthetic genes. ESI‐FTICR‐MS and ESI‐MS/MS analyses facilitated unambiguous identification and relative quantification of the alkaloids in each system. Manual integration of known and candidate biosynthetic genes in each EST library with benzylisoquinoline alkaloid biosynthetic networks assembled from empirical metabolite profiles allowed identification and functional characterization of four N‐ methyltransferases (NMTs). One cDNA from T. flavum encoded pavine N‐ methyltransferase (TfPavNMT), which showed a unique preference for (±)‐pavine and represents the first isolated enzyme involved in the pavine alkaloid branch pathway. Correlation of the occurrence of specific alkaloids, the complement of ESTs encoding known benzylisoquinoline alkaloid biosynthetic genes and the differential substrate range of characterized NMTs demonstrated the feasibility of bilaterally predicting enzyme function and species‐dependent specialized metabolite profiles.

Aspalathin and nothofagin are typical ingredients of unfermented rooibos (Krafczyk, N.; Glomb, M. A. J. Agric. Food Chem.2008, 56, 3368). During oxidation these dihydrochalcones were degraded to higher molecular weight browning products under aerated and nonenzymatic conditions. In the early stages of browning reactions aspalathin formed two dimers. These two compounds were unequivocally established as atropisomers stemming from oxidative A to B ring coupling. Multilayer countercurrent chromatography (MLCCC) and preparative high-performance liquid chromatography (HPLC) were applied to obtain pure substances. The purity and identity of isolated dimers were confirmed by different NMR experiments, HPLC-DAD-MS, and HR-MS. In parallel to the formation of chromophores during the fermentation of black tea, the formation of aspalathin dimers implies an important mechanistic channel for the generation of color during the processing of rooibos.

The phytosterol, tocopherol, and tocotrienol profiles for mkukubuyo, Sterculia africana, manketti, Ricinodendron rautanenni, mokolwane, Hyphaene petersiana, morama, Tylosema esculentum, and moretologa‐kgomo, Ximenia caffra, seed oils from Botswana have been determined. Normal‐phase HPLC analysis of the unsaponifiable matter showed that among the selected oils, the most abundant tocopherol and tocotrienol were γ‐tocopherol (2232.99 μg/g) and γ‐tocotrienol (246.19 μg/g), detected in manketti and mkukubuyo, respectively. Mokolwane oil, however, contained the largest total tocotrienol (258.47 μg/g). Total tocol contents found in manketti, mokolwane, mkukubuyo, morama, and moretologa‐kgomo oils were 2238.60, 262.40, 246.20, 199.10, and 128.0 μg/g, respectively. GC–MS determination of the relative percentage composition of phytosterols showed 4‐desmethylsterols as the most abundant phytosterols in the oils, by occurring up to 90% in moretologa‐kgomo, mkukubuyo, and manketti seed oils, with β‐sitosterol being the most abundant. Mokolwane seed oil contained the largest percentage composition of 4,4‐dimethylsterols (45.93%). Besides 4‐desmethylsterols (75%), morama oil also contained significant amounts of 4,4‐dimethylsterols and 4‐monomethylsterols (15.72% total). GC–MS determination of the absolute amounts of 4‐desmethylsterols, after SPE fractionation of the unsaponifiable matter, confirmed that β‐sitosterol was the most abundant phytosterol in the test seed oils, with manketti seed oil being the richest source (1326.74 μg/g). The analysis showed total 4‐desmethylsterols content as 1617.41, 1291.88, 861.47, 149.15, and 109.11 μg/g for manketti, mokolwane, mkukubuyo, morama, and moretologa‐kgomo seed oils, respectively.