Active Site Design in a Chemzyme: Development of a Highly Asymmetric and Remarkably Temperature-Independent Catalyst for the Imino Aldol Reaction**

The asymmetric aldol reaction of an enolate or enolate equivalent with an imine is a reaction of established synthetic importance for the synthesis of chiral amines in general and bamino esters in particular. [1] The development of chiral catalysts for this reaction has proven to be a difficult task and had eluded all attempts until recently when Kobayashi and co-workers examined imines derived from o-aminophenol. [2±4] Their method involves the catalysis of the reactions of these imines and ketene acetals with a catalyst generated from zirconium(iv) tert-butoxide and two equivalents of (R)-6,6'-dibromoBINOL (BINOL 1,1'-binaphth-2-ol). Our interest in the synthesis of chiral amines led us to investigate the use of VAPOL-derived catalysts A comparison of catalysts prepared from BINOL, 6,6'-dibromoBINOL and VAPOL ligands on the asymmetric induction in the reaction of the phenyl-substituted imine 1 and acetal 2 is summarized in [2] The VAPOL catalyst could be prepared in either methylene chloride or toluene, but for solubility reasons, the BINOL catalysts were prepared in methylene chloride. The VAPOL and Br 2 BINOL catalysts were superior to the BINOL catalyst at À 45 8C. The asymmetric induction dropped for the Br 2 BINOL catalyst when the temperature was raised from À 45 8C to room temperature, but curiously, the asymmetric induction for the VAPOL catalyst was essentially unchanged over this same temperature range. Only a small drop-off is noted (85 % ee) when the temperature is raised to 41 8C and the substrate-to-catalyst ratio is raised to 200:1 (entry 5). Both the R enantiomers of BINOL and Br 2 BINOL ligands give the R enantiomer of the product 3, whereas with the VAPOL ligand, it is the S enantiomer that gives the R product. This reversal is not unexpected given the structures of the ligands where the zirconium is in the minor groove of the BINOL ligands and in the major groove of the VAPOL ligand. [2g] It is clear from the examination of space-filling CPK models that it is possible to bind two VAPOL ligands to one zirconium atom but only with a facial arrangement of the four oxygen atoms as is illustratred by structure 6 in Scheme 1. This is supported by 1 H NMR experiments on a catalyst generated from zirconium tetraisopropoxide and VAPOL in the presence of two equivalents of N-methyl imidiazole. A clean spectrum is only observed with two equivalents of VAPOL relative to zirconium and the spectrum is consistent with a single C 2 -symmetrical species were performed by using the TEXSAN [13] crystallographic software package. Crystallographic data (excluding structure factors) for the structure reported in this paper have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC-153832. Copies of the data can be obtained free of charge on application to CCDC

Identification of metabolic and biomass QTL in Arabidopsis thaliana in a parallel analysis of RIL and IL populations

Plant growth and development are tightly linked to primary metabolism and are subject to natural variation. In order to obtain an insight into the genetic factors controlling biomass and primary metabolism and to determine their relationships, two Arabidopsis thaliana populations [429 recombinant inbred lines (RIL) and 97 introgression lines (IL), derived from accessions Col-0 and C24] were analyzed with respect to biomass and metabolic composition using a mass spectrometry-based metabolic profiling approach. Six and 157 quantitative trait loci (QTL) were identified for biomass and metabolic content, respectively. Two biomass QTL coincide with significantly more metabolic QTL (mQTL) than statistically expected, supporting the notion that the metabolic profile and biomass accumulation of a plant are linked. On the same basis, three out the six biomass QTL can be simulated purely on the basis of metabolic composition. QTL based on analysis of the introgression lines were in substantial agreement with the RIL-based results: five of six biomass QTL and 55% of the mQTL found in the RIL population were also found in the IL population at a significance level of P ≤ 0.05, with >80% agreement on the allele effects. Some of the differences could be attributed to epistatic interactions. Depending on the search conditions, metabolic pathway-derived candidate genes were found for 24–67% of all tested mQTL in the database AraCyc 3.5. This dataset thus provides a comprehensive basis for the detection of functionally relevant variation in known genes with metabolic function and for identification of genes with hitherto unknown roles in the control of metabolism

Blocking the Metabolism of Starch Breakdown Products in Arabidopsis Leaves Triggers Chloroplast Degradation

In most plants, a large fraction of photo-assimilated carbon is stored in the chloroplasts during the day as starch and remobilized during the subsequent night to support metabolism. Mutations blocking either starch synthesis or starch breakdown in Arabidopsis thaliana reduce plant growth. Maltose is the major product of starch breakdown exported from the chloroplast at night. The maltose excess 1 mutant (mex1), which lacks the chloroplast envelope maltose transporter, accumulates high levels of maltose and starch in chloroplasts and develops a distinctive but previously unexplained chlorotic phenotype as leaves mature. The introduction of additional mutations that prevent starch synthesis, or that block maltose production from starch, also prevent chlorosis of mex1. In contrast, introduction of mutations in disproportionating enzyme (DPE1) results in the accumulation of maltotriose in addition to maltose, and greatly increases chlorosis. These data suggest a link between maltose accumulation and chloroplast homeostasis. Microscopic analyses show that the mesophyll cells in chlorotic mex1 leaves have fewer than half the number of chloroplasts than wild-type cells. Transmission electron microscopy reveals autophagy-like chloroplast degradation in both mex1 and the dpe1/mex1 double mutant. Microarray analyses reveal substantial reprogramming of metabolic and cellular processes, suggesting that organellar protein turnover is increased in mex1, though leaf senescence and senescence-related chlorophyll catabolism are not induced. We propose that the accumulation of maltose and malto-oligosaccharides causes chloroplast dysfunction, which may by signaled via a form of retrograde signaling and trigger chloroplast degradation

A plastid-localized glycogen synthase kinase 3 modulates stress tolerance and carbohydrate metabolism

Glycogen synthase kinase 3 (GSK-3) was originally identified as a regulator of glycogen synthesis in mammals. Like starch in plants, glycogen is a polymer of glucose, and serves as an energy and carbon store. Starch is the main carbohydrate store in plants. Regulation of starch metabolism, in particular in response to environmental cues, is of primary importance for carbon and energy flow in plants but is still obscure. Here, we provide evidence that MsK4, a novel Medicago sativa GSK-3-like kinase, connects stress signalling with carbon metabolism. MsK4 was found to be a plastid-localized protein kinase that is associated with starch granules. High-salt stress rapidly induced the in vivo kinase activity of MsK4. Metabolic profiling of MsK4 over-expressor lines revealed changes in sugar metabolism, including increased amounts of maltose, the main degradation product of starch in leaves. Plants over-expressing MsK4 showed improved tolerance to salt stress. Moreover, under high-salinity conditions, MsK4-over-expressing plants accumulated significantly more starch and showed modified carbohydrate content compared with wild-type plants. Overall, these data indicate that MsK4 is an important regulator that adjusts carbohydrate metabolism to environmental stress