The stabilizing effect of a dG:dC base‐pair can also be imparted to a DNA duplex by a non‐hydrogen‐bonding, non‐shape‐complementary nucleoside analogue when interstrand stacking interactions come into play. This is the case, for example, with dBP, which has a bipyridyl (BP) residue as a nucleobase surrogate (the picture shows a dBP:dBP pair).

Pyrrolidino pseudo-C-nucleosides are isosteres of natural deoxynucleosides which are protonated at the pyrrolidino ring nitrogen under physiological conditions. As constituents of a triplex forming oligodeoxynucleotide (TFO), the positive charge is expected to stabilise DNA triple helices via electrostatic interactions with the phosphodiester backbone of the target DNA. We describe the synthesis of the pyrrolidino isocytidine pseudonucleoside and the corresponding phosphoramidite building block and its incorporation into TFOs. Such TFOs show substantially increased DNA affinity compared to unmodified oligodeoxynucleotides. The increase in affinity is shown to be due to the positive charge at the pyrrolidino subunit.

[figure: see text] A novel method for the synthesis of pyrrolidine C-nucleosides has been developed. The key step of the synthesis is the palladium(0)-mediated coupling of a disubstituted N-protected 2-pyrroline and 5-iodouracil. C-Nucleoside 14 and its N-methyl derivative 15 can easily be converted to the corresponding phosphoramidite building blocks for DNA synthesis.

Wasserstoffbrücken und Stapelwechselwirkungen zwischen Nucleobasen sind die wichtigsten nichtkovalenten Kräfte, die die DNAund die RNA-Doppelhelix zusammenhalten. [1,2] Deren relative Beiträge zur Stabilität werden jedoch seit der Entdeckung der Struktur der Doppelhelix kontrovers diskutiert. Neuere Berichte über die Bildung stabiler Duplexe und die selektive, DNA-Polymeraseinduzierte Replikation von Oligonucleotiden mit hydrophoben, nichtwasserstoffverbrückenden Basenpaaren [3±14] haben die Diskussion wieder entfacht und zu einer Neubewertung der Rolle von aromatischen Stapelwechselwirkungen und Formkomplementarität bei der Basenpaarbildung geführt. [15] Im Rahmen eines Projektes zur Einführung von spezifischen Metallbindungsstellen in DNA [16±18] interessieren wir uns für das Nucleosid-Analogon 1 (dBP; siehe Schema 1), welches eine 2,2'-Bipyridyl(BP)-Einheit als Basenersatz trägt, und dessen Einbau in Oligonucleotide. Hier berichten wir über die selektive und hochaffine Selbsterkennung von dBP innerhalb eines DNA-Duplexes in Abwesenheit von Übergangsmetallionen. [19] Das C-Nucleosid 1 wurde ausgehend von 2,3,5-Tri-Obenzyl-d-ribonolacton 2 [20] und 4-Iod-2,2'-bipyridin 3 [21,22] nach bekannten Methoden der C-Glycosidchemie synthetisiert (Schema 1): [23,24] Die Lithiierung von 3 und die nachfolgende Addition an das Lacton 2 lieferten die intermediären Hemiacetale, welche nach In-situ-Reduktion mit Et 3 SiH selektiv in das b-C-Glycosid 4 ( 1 H-NMR-NOE) überführt wurden. Mit einer typischen Reaktionsfolge zur Entfernung der 2'-Hydroxygruppe wurde die Synthese von 1 abgeschlossen, welches dann in das Phosphoramidit 7 umgewandelt wurde. Dieser Baustein wurde nach Standardvorschriften der Oligonucleotidsynthese [25] in den Duplex 8 (siehe Tabelle 1) eingebaut. Die thermische Stabilität der Duplexe 8 (X, Y dBP, dA, dG, dC, dT sowie das abasische Nucleosidanalogon f) wurde durch UV-spektroskopische Bestimmung der Schmelztemperatur T m ermittelt (10 mm NaH 2 PO 4 , 150 mm NaCl, pH 7.0; Abbildung 1 a; Tabelle 1). Eine genauere Be-

Nitric oxide mediates a wide array of cellular functions in many tissues. It is generated by three known isoforms of nitric oxide synthases (NOS). Recently, the endothelial isoform, NOSIII, was shown to be abundantly expressed in the rat thyroid gland and its expression increased in goitrous glands. In this study, we analyzed whether NOSIII is expressed in human thyroid tissue and whether levels of expression vary in different states of thyroid gland function. Semiquantitative RT-PCR was used to assess variations in NOSIII gene expression in seven patients with Graves' disease, one with a TSH-receptor germline mutation and six hypothyroid patients (Hashimoto's thyroiditis). Protein expression and subcellular localization were determined by immunohistochemistry (two normal thyroids, five multinodular goiters, ten hyperthyroid patients and two hypothyroid patients). NOSIII mRNA was detected in all samples: the levels were significantly higher in tissues from hyperthyroid patients compared with euthyroid and hypothyroid patients. NOSIII immunoreactivity was detected in vascular endothelial cells, but was also found in thyroid follicular cells. In patients with Graves' disease, the immunostaining was diffusely enhanced in all follicular cells. A more intense signal was observed in toxic adenomas and in samples obtained from a patient with severe hyperthyroidism due to an activating mutation in the TSH receptor. In multinodular goiters, large follicles displayed a weak signal whereas small proliferative follicles showed intense immunoreactivity near the apical plasma membrane. In hypothyroid patients, NOSIII immunoreactivity was barely detectable. In summary, NOSIII is expressed both in endothelial cells and thyroid follicular cells. The endothelial localization of NOSIII is consistent with a role for nitric oxide in the vascular control of the thyroid. NOSIII expression in thyroid follicular cells and the variations in its immunoreactivity suggest a possible role for nitric oxide in thyrocyte function and/or growth.