Synthesis of N3- and 2-NH2-substituted 6,7-diphenylpterins and their use as intermediates for the preparation of oligonucleotide conjugates designed to target photooxidative damage on single-stranded DNA representing the bcr-abl chimeric gene

Two 17-mer oligodeoxynucleotide-5'-linked-(6,7-diphenylpterin) conjugates, 2 and 3, were prepared as photosensitisers for targeting photooxidative damage to a 34-mer DNA oligodeoxynucleotide (ODN) fragment 1 representing the chimeric bcr-abl gene that is implicated in the pathogenesis of chronic myeloid leukaemia (CML). The base sequence in the 17-mer was 3'G G T A G T T A T T C C T T C T T5'. In the first of these ODN conjugates (2) the pterin was attached at its N3 atom, via a -(CH2)3OPO(OH)- linker, to the 5'-OH group of the ODN. Conjugate 2 was prepared from 2-amino-3-(3-hydroxypropyl)-6,7-diphenyl-4(3H)-pteridinone 10, using phosphoramidite methodology. Starting material 10 was prepared from 5-amino-7-methylthiofurazano[3,4-d]pyrimidine 4 via an unusual highly resonance stabilised cation 8, incorporating the rare 2H,6H-pyrimido[6,1-b][1,3]oxazine ring system. In the characterisation of 10 two pteridine phosphazenes, 15 and 29, were obtained, as well as new products containing two uncommon tricyclic ring systems, namely pyrimido[2,1-b]pteridine (20 and 24) and pyrimido[1,2-c]pteridine (27). In the second ODN conjugate the linker was -(CH2)5CONH(CH2)6OPO(OH)- and was attached to the 2-amino group of the pterin. In the preparation of 3, the N-hydroxysuccinimide ester 37 of 2-(5-carboxypentylamino)-6,7-diphenyl-4(3H)-pteridinone was condensed with the hexylamino-modified 17-mer. Excitation of 36 with near UV light in the presence of the single-stranded target 34-mer, 5'T G A C C A T C A A T A A G14 G A A G18 A A G21 C C C T T C A G C G G C C3' 1 caused oxidative damage at guanine bases, leading to alkali-labile sites which were monitored by polyacrylamide gel electrophoresis. Cleavage was observed at all guanine sites with a marked preference for cleavage at G14. In contrast, excitation of ODN-pteridine conjugate 2 in the presence of 1 caused oxidation of the latter predominantly at G18, with a smaller extent of cleavage at G15 and G14 (in the double-stranded portion) and G21. These results contrast with our previous observation of specific cleavage at G21 with ruthenium polypyridyl sensitisers, and suggest that a different mechanism, probably one involving Type 1 photochemical electron transfer, is operative. Much lower yields were found with the ODN-pteridine conjugate 3, perhaps as a consequence of the longer linker between the ODN and the pteridine in this case.

Changes in functional behavior of 93 % Pb(Mg1/3Nb2/3)O-3-7 % PbTiO3 thin films induced by ac electric fields

The dielectric properties of Au/[93%Pb(Mg1/3Nb2/3)O-3-7%PbTiO3] (PMN-PT)/(La0.5Sr0.5)CoO3/MgO thin-film capacitor heterostructures, made using pulsed laser deposition, have been investigated, with particular emphasis on the changes in response associated with increasing the magnitude of the ac measuring field. It was found that increasing the ac field caused a change in the frequency spectrum of relaxators, increasing the speed of response of "slow" relaxators, with an associated decrease in the freezing temperature (T-f) of the relaxor system; in addition, other characteristic parameters relating to polar relaxation (activation energy E-a and attempt frequency 1/tau(0)), described by fitting of the dielectric response to a Vogel-Fulcher expression, were found to change continuously as ac field levels were increased.

SOCS3 Targets Siglec 7 for Proteasomal Degradation and Blocks Siglec 7-mediated Responses

CD33-related Siglecs (sialic acid-binding immunoglobulin-like lectins) 5–11 are inhibitory receptors that contain a membrane proximal ITIM (immunoreceptor tyrosine-based inhibitory motif) (I/V/L/)XYXX(L/V), which can recruit SHP-1/2. However, little is known about the regulation of these receptors. SOCS3 (suppressor of cytokine signaling 3) is up-regulated during inflammation and competes with SHP-1/2 for binding to ITIM-like motifs on various cytokine receptors resulting in inhibition of signaling. We show that SOCS3 binds the phosphorylated ITIM of Siglec 7 and targets it for proteasomal-mediated degradation, suggesting that Siglec 7 is a novel SOCS target. Following ligation, the ECS E3 ligase is recruited by SOCS3 to target Siglec 7 for proteasomal degradation, and SOCS3 expression is decreased concomitantly. In addition, we found that SOCS3 expression blocks Siglec 7-mediated inhibition of cytokine-induced proliferation. This is the first time that a SOCS target has been reported to degrade simultaneously with the SOCS protein and that inhibitory receptors have been shown to be degraded in this way. This may be a mechanism by which the inflammatory response is potentiated during infection.

Role of conformational constraints of position 7 of the disulphide bridge of h-alpha-CGRP derivatives in their agonist versus antagonist properties

Previous structure-activity studies have shown that the disulphide bridge of calcitonin gene-related peptide (CGRP) is important for the highly potent, CGRP receptor-mediated effects of this peptide. In this study penicillamine (Pen) was substituted for one or both of the cysteinyl residues to determine conformational and topographical properties of the disulphide bridge favourable for binding to CGRP receptors and/or receptor activation. Pen constrains the conformational flexibility of disulphide bridges in other peptides. Binding affinities were measured using a radioligand binding assay with membranes prepared from pig coronary arteries and I-125-h-alpha-CGRP. Functional effects were characterized using a previously reported pig coronary artery relaxation bioassay. The binding affinity of [Pen(2)]h-alpha-CGRP was not significantly different from that of h-alpha-CGRP. All other analogues showed reduced affinity for CGRP receptors. [Pen(2)]h-alpha-CGRP also caused relaxation of coronary arteries. The remaining analogues either caused relaxation with significantly reduced potency or failed to relax the arteries at concentrations up to 1 x 10(-5) M. All analogues that did not relax coronary arteries contained a D-Pen in position 7 and inhibited CGRP-induced relaxation. [D-Pen(2,7)]h-alpha- CGRP was the most potent antagonist with a K-B value of 630 nM. This affinity is similar to that of the classical CGRP receptor antagonist, h-alpha-CGRP(8-37), on these arteries (K-B, 212 nM). These studies show that modifying the topography of the disulphide bridge can cause large and variable effects on ligand binding and activation of CGRP receptors. The contribution of position 7 to the conformation and topography of the disulphide bridge of h-alpha-CGRP is crucial to the future design of agonists of CGRP receptors. Furthermore, position 7 is important for the development of new CGRP receptor antagonists with structures based on the whole sequence of h-alpha-CGRP.