6-Deoxyhexoses froml-Rhamnose in the Search for Inducers of the Rhamnose Operon: Synergy of Chemistry and Biotechnology

In the search for alternative non‐metabolizable inducers in the l ‐rhamnose promoter system, the synthesis of fifteen 6‐deoxyhexoses from l ‐rhamnose demonstrates the value of synergy between biotechnology and chemistry. The readily available 2,3‐acetonide of rhamnonolactone allows inversion of configuration at C4 and/or C5 of rhamnose to give 6‐deoxy‐d ‐allose, 6‐deoxy‐d ‐gulose and 6‐deoxy‐l ‐talose. Highly crystalline 3,5‐benzylidene rhamnonolactone gives easy access to l ‐quinovose (6‐deoxy‐l ‐glucose), l ‐olivose and rhamnose analogue with C2 azido, amino and acetamido substituents. Electrophilic fluorination of rhamnal gives a mixture of 2‐deoxy‐2‐fluoro‐l ‐rhamnose and 2‐deoxy‐2‐fluoro‐l ‐quinovose. Biotechnology provides access to 6‐deoxy‐l ‐altrose and 1‐deoxy‐l ‐fructose

One-pot Enzymatic Synthesis of Deoxy-thymidine-diphosphate (TDP)-2-deoxy-∝-d-glucose Using Phosphomannomutase

Production of deoxy-thymidine-diphosphate (TDP)-sugars as substrates of glycosyltransferases, has been one of main hurdles for combinatorial antibiotic biosynthesis, which combines sugar moiety with aglycon of various antibiotics. Here, we report the one-pot enzymatic synthesis of TDP-2-deoxy-glucose employing high efficient TMP kinase (TMK; E.C. 2.7.2.12), acetate kinase (ACK; E.C. 2.7.1.21), and TDP-glucose synthase (TGS; E.C. 2.7.7.24) with phosphomannomutase (PMM; E.C. 5.4.2.8). In this study, replacing phosphoglucomutase (PGM; E.C. 5.4.2) by PMM from Escherichia coli gave four times higher specific activity on 2-deoxy-6-phosphate glucose, suggesting that the activity on 2-deoxy-glucose-6-phosphate was mainly affected by PMM activity, not PGM activity. Using an in vitro system starting from TMP and 2-deoxy-glucose-6-phosphate glucose, TDP-2-deoxy-glucose (63% yield) was successfully synthesized. Considering low productivity of NDP-sugars from cheap starting materials, this paper showed how production of NDP-sugars could be enhanced by controlling mutase activity

Greater V˙O2peak is correlated with greater skeletal muscle deoxygenation amplitude and hemoglobin concentration within individual muscles during ramp-incremental cycle exercise.

It is axiomatic that greater aerobic fitness (V˙O2peak) derives from enhanced perfusive and diffusive O2 conductances across active muscles. However, it remains unknown how these conductances might be reflected by regional differences in fractional O2 extraction (i.e., deoxy [Hb+Mb] and tissue O2 saturation [StO2]) and diffusive O2 potential (i.e., total[Hb+Mb]) among muscles spatially heterogeneous in blood flow, fiber type, and recruitment (vastus lateralis, VL; rectus femoris, RF). Using quantitative time-resolved near-infrared spectroscopy during ramp cycling in 24 young participants (V˙O2peak range: ~37.4-66.4 mL kg-1 min-1), we tested the hypotheses that (1) deoxy[Hb+Mb] and total[Hb+Mb] at V˙O2peak would be positively correlated with V˙O2peak in both VL and RF muscles; (2) the pattern of deoxygenation (the deoxy[Hb+Mb] slopes) during submaximal exercise would not differ among subjects differing in V˙O2peak Peak deoxy [Hb+Mb] and StO2 correlated with V˙O2peak for both VL (r = 0.44 and -0.51) and RF (r = 0.49 and -0.49), whereas for total[Hb+Mb] this was true only for RF (r = 0.45). Baseline deoxy[Hb+Mb] and StO2 correlated with V˙O2peak only for RF (r = -0.50 and 0.54). In addition, the deoxy[Hb+Mb] slopes were not affected by aerobic fitness. In conclusion, while the pattern of deoxygenation (the deoxy[Hb+Mb] slopes) did not differ between fitness groups the capacity to deoxygenate [Hb+Mb] (index of maximal fractional O2 extraction) correlated significantly with V˙O2peak in both RF and VL muscles. However, only in the RF did total[Hb+Mb] (index of diffusive O2 potential) relate to fitness

Lactose as an inexpensive starting material for the preparation of aldohexos-5-uloses: synthesis of L-ribo and D-lyxo derivatives

SUMMARY: Partially protected derivatives of L-ribo- and D-lyxo-aldohexos-5-ulose have been prepared starting from triacetonlactose dimethyl acetal derivatives. Key steps of the synthetic sequences are a) the synthesis of 4'-deoxy-4'-eno- and 6'-deoxy-5'-eno lactose derivatives, and b) the epoxidation-methanolysis of the above enol ethers to give 1,5-bis-glycopyranosides, masked form of the target 1,5-dicarbonyl hexoses

Substrate specificity provides insights into the sugar donor recognition mechanism of O-GlcNAc transferase (OGT).

O-Linked β-N-acetylglucosaminyl transferase (OGT) plays an important role in the glycosylation of proteins, which is involved in various cellular events. In human, three isoforms of OGT (short OGT [sOGT]; mitochondrial OGT [mOGT]; and nucleocytoplasmic OGT [ncOGT]) share the same catalytic domain, implying that they might adopt a similar catalytic mechanism, including sugar donor recognition. In this work, the sugar-nucleotide tolerance of sOGT was investigated. Among a series of uridine 5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc) analogs tested using the casein kinase II (CKII) peptide as the sugar acceptor, four compounds could be used by sOGT, including UDP-6-deoxy-GlcNAc, UDP-GlcNPr, UDP-6-deoxy-GalNAc and UDP-4-deoxy-GlcNAc. Determined values of Km showed that the substitution of the N-acyl group, deoxy modification of C6/C4-OH or epimerization of C4-OH of the GlcNAc in UDP-GlcNAc decreased its affinity to sOGT. A molecular docking study combined with site-directed mutagenesis indicated that the backbone carbonyl oxygen of Leu653 and the hydroxyl group of Thr560 in sOGT contributed to the recognition of the sugar moiety via hydrogen bonds. The close vicinity between Met501 and the N-acyl group of GlcNPr, as well as the hydrophobic environment near Met501, were responsible for the selective binding of UDP-GlcNPr. These findings illustrate the interaction of OGT and sugar nucleotide donor, providing insights into the OGT catalytic mechanism

Synthese des 18F-markierten Coenzyms Uridindiphosphatglucose als Basis für die 18F-Glykosylierung von Glykoproteinen

The chemo-enzymatic radiosynthesis of no carrier added (n.c.a.) uridine diphospho-2-deoxy- 2-[$^{18}$F]fluoro-$\alpha$-D-glucose (UDP-[$^{18}$F]FGlc) was developed. In order to overcome the problem of poor regioselectivity when using the commonly strategy to label proteins via $^{18}$F-labelled prosthetic groups, the use of enzyme systems in addition to the corresponding $^{18}$F-labelled coenzymes was shown to be a reliable, regioselective and mild labelling method. With regard to the comparison and evaluation of the stereoselectivity of the phosphorylating agents used in the chemical synthesis of cold uridine diphospho-2-deoxy-2-fluoro-$\alpha$-Dglucose, $^{31}$P-decoupled and $^{1}$H-NMR-studies were successfully realized. Uridine diphospho- 2-deoxy-2-fluoro-$\alpha$-D-glucose was obtained in a 7 step synthesis. Tetrabenzylpyrophosphate was shown to be a highly stereoselective phosphorylating agent for FDG ($\alpha /\beta$=3:1). Moreover, a multienzymatic pathway for the synthesis of uridine diphospho-2-deoxy-2-fluoro-$\alpha$- D-glucose was adopted starting from FDG and four commercially available enzymes. This strategy was adjusted to a mg-scale synthesis providing 35% chemical yield. Within the scope of this procedure, a comparison of the natural substrate $\alpha$-D-glucose-1-phosphate with 2-fluoro-2-deoxy-$\alpha$-D-glucose-1-phosphate indicated that the enzyme activity of UDP-glucose pyrophosphorylase (UDP-Glc PPase) was decreased by a factor of 30. With regard to the adaptability of the multiple enzyme system for the radiosynthesis of n.c.a. uridine diphospho-2-deoxy-2-[$^{18}$F]fluoro-$\alpha$-D-glucose a rapid hexokinase-mediated phosphorylation of [$^{18}$F]FDG utilizing ATP or UTP as phosphate donor was performed. A further enzymatic isomerization of n.c.a [$^{18}$F]FDG-6-phosphate to n.c.a. [$^{18}$F]FDG-1-phosphate was limited due to the formation of [$^{18}$F]FDG-1.6-diphosphate as main product. Experiments using a multiple enzyme system to develop a fully enzymatic synthetic route to UDP-[$^{18}$F]FGlc turned out to be less efficient due to the necessity of carrier added conditions. Thus, a chemo-enzymatic synthesis of n.c.a. UDP-[$^{18}$8F]FGlc has been developed, starting from 1.3.4.6-tetra-O-acetyl-2-[$^{18}$F]fluoro-2-deoxy-D-glucose, which occurs as an intermediate in the [$^{18}$F]FDG synthesis. The chemical phosphorylation via MacDonald reaction and subsequent deprotection led to a radiochemical yield of 55% of [$^{18}$F]FDG-1-phosphate. UDP- [$^{18}$F]FGlc was synthesized enzymatically by condensation of [$^{18}$F]FDG-1-phosphate with UTP in presence of UDP-Glc PPase. In order to overcome the problem of decreased enzyme acitivty the reaction was performed in a minimized reaction volume and optimized UTP-concentration of 0.5 mmol/l leading to an overall radiochemical yield of 20% of UDP-[$^{18}$F]FGlc within 110 min. The $^{18}$F-labelled coenzyme UDP-[$^{18}$F]FGlc was used as a tool for $^{18}$F-glycosylation of N-acetylglucosamine mediated by $\beta$-1.4-galactosyltransferase. The $^{18}$F-glycosylated product was obtained in a radiochemical yield of 56% and was easily isolated by solid phase extraction. In addition to the general availability of [$^{18}$F]FDG worldwide, this new strategy for enzymatic transfer of "activated [$^{18}$F]FDG" has demonstrated its potential as a highly selective and mild $^{18}$F-labelling method of glycosylated biopolymers to study their pharmacokinetics using positron-emission-tomography

Preparative Synthesis of dTDP-L-Rhamnose Through Combined Enzymatic Pathways

dTDP-L-rhamnose, an important precursor of O-antigen, was prepared on a large scale from dTMP by executing an one-pot reaction in which six enzymes are involved. Two enzymes, dTDP-4-keto-6-deoxy-D-glucose 3,5-epimerase and dTDP-4-keto-rhamnose reductase, responsible for the conversion of dTDP-4-keto-6-deoxy- D-glucose to dTDP-L-rhamnose, were isolated from their putative sequences in the genome of Mesorhizobium loti, functionally expressed in Escherichia coli, and their enzymatic activities were identified. The two enzymes were combined with an enzymatic process for dTDP-4- keto-6-deoxy-D-glucose involving TMP kinase, acetate kinase, dTDP-glucose synthase, and dTDP-glucose 4,6- dehydratase, which allowed us to achieve a preparative scale synthesis of dTDP-L-rhamnose using dTMP and glucose-1-phosphate as starting materials. About 82% yield of dTDP-L-rhamnose was obtained based on initial dTMP concentration at 20 mM dTMP, 1 mM ATP, 10 mM NADH, 60 mM acetyl phosphate, and 80 mM glucose-1- phosphate. From the reaction with 20 ml volume, approximately 180 mg of dTDP-L-rhamnose was obtained in an overall yield of 60% after two-step purification, that is, anion exchange chromatography and gel filtration for desalting. The purified product was identifiedbyHPLC, ESI-MS,andNMR,showingabout95%purity

The Human SLC25A33 and SLC25A36 Genes of Solute Carrier Family 25 Encode Two Mitochondrial Pyrimidine Nucleotide Transporters

The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport inorganic anions, amino acids, carboxylates, nucleotides, and coenzymes across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. Here two members of this family, SLC25A33 and SLC25A36, have been thoroughly characterized biochemically. These proteins were overexpressed in bacteria and reconstituted in phospholipid vesicles. Their transport properties and kinetic parameters demonstrate that SLC25A33 transports uracil, thymine, and cytosine (deoxy)nucleoside di- and triphosphates by an antiport mechanism and SLC25A36 cytosine and uracil (deoxy)nucleoside mono-, di-, and triphosphates by uniport and antiport. Both carriers also transported guanine but not adenine (deoxy)nucleotides. Transport catalyzed by both carriers was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. In confirmation of their identity (i) SLC25A33 and SLC25A36 were found to be targeted to mitochondria and (ii) the phenotypes of Saccharomyces cerevisiae cells lacking RIM2, the gene encoding the well characterized yeast mitochondrial pyrimidine nucleotide carrier, were overcome by expressing SLC25A33 or SLC25A36 in these cells. The main physiological role of SLC25A33 and SLC25A36 is to import/export pyrimidine nucleotides into and from mitochondria, i.e. to accomplish transport steps essential for mitochondrial DNA and RNA synthesis and breakdown

The effect of reducing ATP levels on reorientation of the secondary palate

The force for directing palate shelf reorientation appears to be associated with elements of the presumptive hard palate (Brinkley & Vickerman, 1979; Bulleit & Zimmerman, 1985). The palatal elements that mediate this process do not require palate cells to be metabolically active for expression of the force. This contention was demonstrated using an in vitro system that allows substantial reorientation of the hard palate to occur. ATP levels were reduced by treatment with metabolic inhibitors and the degree of reorientation was measured 1 h following pretreatment with inhibitors. Treatment of cultured embryonic heads under anoxic conditions with 2,4-dinitrophenol or KCN had noeffect on the degree of reorientation occurring in vitro. These agents reduced ATP levels by 71 % and 62 %, respectively. Treatment of cultured heads with 2-deoxy-D-glucose under anoxia also had no effect on reorientation. This treatment reduced ATP levels in embryonic heads by 92–94%. A similar reduction was observed if ATP levels were measured in palate tissue alone. The treatment of cultured heads with 2-deoxy-D-glucose and anoxia not only reduced levels of ATP but also reduced CTP, GTP and UTP. These results indicate that palate shelf reorientation is independent of cellular metabolic activity and supports the hypothesis that reorientation is dependent on a pre-existing infrastructure within the palate shelves