Elisha Berman

(7) Glucosylation and (1-6) rhamnosylation of exogenous flavanones by undifferentiated Citrus cell cultures

Abstract The ability of citrus cultures to biotransform flavanones was tested. Only one undifferentiated sour orange ( Citrus aurantium L.) ovule derived cell culture glucosylated exogenous naringenin at position 7 and further rhamnosylated the product at position 6 of the glucose, synthesizing narirutin. The products were verified by 1 H-NMR. Other ovule-derived cell lines, one from sour orange ( Citrus aurantium L.) and one from a Poorman × Poncirus trifoliata (L.) Raf. hybrid were only able to glucosylate exogenous naringenin and hesperetin at position 7, as previously shown in C. paradisi Macf. cv. Duncan cell cultures. TLC analyses indicated that cultures of ‘Ponderosa’ lemon ( C. limon (L.) Burm. f. cv. Ponderosa) and grapefruit ( C. paradisi Macf. cv. White Marsh) also glucosylated exogenous hesperetin at position 7. None of the cell-lines tested accumulated flavanone-glycosides without exogenously supplying aglycones.

An early transient increase of intracellular Na+ may be one of the first components of the mitogenic signal. Direct detection by 23Na-NMR spectroscopy in quiescent 3T3 mouse fibroblasts stimulated by growth factors

23Na-NMR spectroscopy was designed to allow for continuous recording of intracellular Na+ in 3T3 fibroblasts stimulated by serum growth-factors in the presence of ion transport inhibitors. The metabolic state of cells at rest and following stimulation was monitored by 31P-NMR spectra of ATP and related high-energy phosphates. The study demonstrates that early activation of ion transporters by addition of serum is marked by the appearance of transient increase of the intracellular Na+, beginning 3 min after addition of serum to quiescent culture and lasting approx. 20 min. The initial rise in cellular Na+ results from an increased activity of the bumetanide-sensitive Na+/K+/Cl- cotransport and of the amiloride-sensitive Na+/H+ antiport. It is suppressed by any one of these inhibitors. Subsequent activation of the ouabain-sensitive Na+/K(+)-ATPase results in an increased Na+ efflux, leading to a return of intracellular Na+ to its initial baseline. Previous work had shown that the early activation of bumetanide-sensitive and amiloride sensitive ion-transporters by growth-factors was essential for induction of cell division, at least in some cell types. Preventing ion activation by adding ion-transport inhibitors lead to the inhibition of DNA synthesis 18 h later. This process was reversible upon elimination of these inhibitors. Even though alternative non-specific effects of these inhibitors cannot be ruled out, the observed transient peak in intracellular Na+ may be one of the earliest components of the mitogenic signal. On the basis of previous works, its effect seems to be related to the activation of Ca(2+)-dependent and cyclic AMP second messenger pathways. The different mechanisms whereby the activated Na+/K+/Cl- cotransport and the Na+/H+ antiport contribute to this signal need to be further investigated.

Reinvestigation of the carbohydrate chains of calf fetuin using 13C-n.m.r. spectroscopy☆

The triantennary structure of the N-linked oligosaccharide side-chain of fetuin, elucidated by a combination of 13C-n.m.r. spectroscopy and enzymic degradations, accords with that reported earlier with respect to the branching pattern, but the ratio of the N-acetylneuraminic acid linkages to the galactose residues [alpha-(2----3) vs. alpha-(2----6)] was found to be 1:1, indicating structural heterogeneity of the side chains. Also, one out of nine galactosyl residues is linked to 2-acetamido-2-deoxy-beta-D-glucose by a (1----3) rather than a (1----4) linkage. The chemical shifts reported are in excellent agreement with those for the intact glycoprotein. Unusual chemical shift effects lead to the conclusion that the alpha-NeuAc-(2----6) residues interact with other parts of the oligosaccharide side-chain. The action of beta-D-galactosidase from Aspergillus niger on desialylated fetuin removed approximately 85% of the beta-Gal residues (1----4)-linked to GlcNAc and 65% of the beta-Gal residues (1----3)-linked to GalNAc, but none of the beta-Gal residues (1----3)-linked to GlcNAc.

One- and two-dimensional 90.5-MHz 13C-NMR spectroscopy of the N-linked triantennary oligosaccharide units of calf fetuin

Complete assignments of all anomeric resonances in the proton and carbon spectra of the N‐linked oligosaccharide units of fetuin were made using one‐ and two‐dimensional NMR spectroscopy. We are able to confirm the presence of microheterogeneity in the N‐acetylneuraminic acid linkages to the galactose residues and the presence of a unique triantennary structure which carries a side chain: NeuAcα(1–3)Galβ(1–3) GlcNacβ(1–4)‐. Anomeric carbon chemical shifts changes resulting from long‐range conformational effects were observed.

One- and two-dimensional NMR studies of the N-terminal portion of glycophorin A at 11.7 Tesla

One- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy (at 11.7 Tesla) was used to gain some structural and spectral information about glycophorin AM, glycophorin AM tryptic glycopeptide, a related pentapeptide, and two related monoglycosylated pentapeptides. The protein spectral information suggests that the highly glycosylated N-terminus of glycophorin does not seem to possess a unique tertiary structure. Furthermore, the spectral information provided by the carbohydrate residues also indicates that there is no strong carbohydrate-protein interaction resulting in a unique tertiary structure. This result does not preclude any unique protein-carbohydrate interactions. For the small monoglycosylated pentapeptide containing α-d-GalNAc attached to Thr, a unique NOESY cross-peak was observed between the anomeric proton and the β-proton of Thr. A cross-peak between the β-proton of Ser and the anomeric proton was not observed for a related monoglycosylated pentapetide containing α-d-GalNAc O-linked to Ser.

Conformation equilibria in vitamins D. The synthesis of 1α-hydroxy-3-epivitamin D3(1α-hydroxy-3α-cholecalciferol)

The ration of the two conformers of 1α-hydroxy-3-epivitamin D3, which has been synthesized from 1α,3β-dihydroxycholest-Δ6-ene, has been established.

Urodiolenone from grapefruit juice, a urinary metabolite found in hypertensive subjects

Abstract Urodiolenone, a compound isolated from the urine of hypertensive subjects, is a 1:1 epimeric mixture of two dihydroxy derivatives of nookatone, a constituent of grapefruit. The structure of urodiolenone was suggested by NMR spectroscopy and confirmed by partial synthesis from nootkatone, a sesquiterpenoid ketone with a valencene skeleton. In addition, we show that urodiolenone itself is also present in the grapefruit in free form and possibly also as a glucuronide. The question whether urodiolenone is produced exogenously or endogenously is discussed.

Photoexcited nitrobenzene for benzylic hydroxylation: the synthesis of 17β-acetoxy-9α-hydroxy-3-methoxy-estra-1,3,5(10)-triene

Abstract A novel method for benzylic hydroxylation is introduced based on the use of photo-excited nitrobenzene. Applying this method 17β-acetoxy-3-methoxy-estra-1,3,5(10)-triene (I) is efficiently converted to 17β-acetoxy-9α-hydroxy-3-methoxy-estra-1,3,5(10)-triene (IIIa).

13C-NMR study of the binding of [1-13C]galactose-labelled N-acetyllactosamine and [1-13C]galactose-enriched hen ovalbumin to soybean agglutinin

The binding of the tide compounds to soybean agglutinin was investigated using 13C-NMR spectroscopy. The equilibrium constant for the binding of N-acetyllactosamine was found to be smaller than that obtained for the binding of ovalbumin (1.1 X 10(3) vs. 7.4 X 10(3) M-1). Only two binding sites per lectin tetramer were determined for the binding of ovalbumin, which is half the number of binding sites reported for the binding of small ligands to the lectin. Steric interference between the bulky ovalbumin molecules is believed to be the reason for the observed decrease in the apparent number of binding sites on the lectin.

Tin derivatives for synthesis: preparation of chiral macrocycles

The efficient preparation of macrocyclic amino acid derivatives via the use of tin as covalent template is described.