Stephen A. Salisbury

A new base-stable linker for solid-phase oligonucleotide synthesis

The succinyl-sarcosyl linker (1b), which is stable to prolonged contact with 1,8-diazabicyclo[5.4.0]undec-7-ene but cleaves rapidly with aqueous ammonia, is of potential value in solid-phase oligonucleotide synthesis.

Synthesis and enzymatic incorporation of a novel, bicyclic pyrimidine nucleoside: a thymidine mimic

Abstract Nucleophilic ring-opening and rearrangement reaction of a furanopyrimidine nucleoside with anhydrous hydrazine provided a novel, 6,6-bicyclic pyrimidopyridazin-7-one nucleoside (dH, 4 ), whose structure was confirmed by X-ray crystallography. This novel nucleoside was converted to its 5′-triphosphate (dHTP) for studies with DNA polymerases and incorporated into a template by using standard phosphoramidite chemistry. In the template, dH directed the incorporation of dATP and to a lesser extent dGTP into the transcript and dHTP was efficiently incorporated at the 3′-end of a primer opposite dA using both exonuclease free Klenow fragment (KF exo-) and Taq DNA polymerases and extended with natural dNTPs.

CYCLISATION AND REARRANGEMENT OF N4-ACYLAMINODEOXYCYTIDINES

Abstract N4-Acetylamino-2′-deoxycytidine undergoes an acid promoted cyclisation to give a 1,2,4-triazolo[4,3-c]pyrimidinone which, under basic conditions, isomerises via a Dimroth-type rearrangement to the 1,2,4-triazolo[1,5-c]pyrimidinone.

Electrophilic substitution in dihydrouracils

2′-Deoxyuridine photohydrate with dimethylamine and formaldehyde forms 5-dimethylaminomethyl-2′-deoxyuridine in a very fast reaction the mechanism of which bears on thymidylate synthetase catalysis.

An electrophysiological and spectroscopic study of the properties and structure of biological calcium channels. Investigations of a model ion channel

The N- and C-terminally protected peptide N-acetyl-Asp-Phe-Ala-Asn-Arg-Val-Leu-Leu-Ser-Leu-Phe-Thr-Ile-Glu-Met-Leu -Leu-Lys-Met-Leu-NH2, closely based on the sequence of the putative S2 membrane spanning helix of domain II of the dihydropyridine receptor calcium channel of the T-system of skeletal muscle, residues 465-486 (Tanabe et al. (1987) Nature 328, 313-318) has been synthesised. Conductance measurements in planar lipid bilayers show that the peptide is capable of inducing the transmembrane passage of calcium and barium ions, in preference to monovalent cations. No anion conductance is observed. 1H-NMR spectroscopy demonstrates that in an amphilic solvent, methanol, the peptide forms highly stable structures characterised by very slow exchange with solvent of peptide N-H protons. Double-quantum filtered phase-sensitive COSY shows that, on the basis of NH-CH alpha scalar coupling constants, most peptide torsion angles are appropriate to an overall alpha-helical conformation; the presence of some alpha-helix is also supported by CD measurements. Most side-chain connectivities have been identified in a DIPSI-TOCSY experiment. This evidence has been used to construct a low-resolution model of the ion-conducting channel of the muscle T-system dihydropyridine receptor from the sequences of the four homologous putative channel-lining stretches. It is characterised by an association of acidic residues at the putative extra-membranous face of the channel, followed by a predominantly hydrophobic band. The next prominent feature of the model is an ordered array of four acidic residues (glutamates 100, 478, 846 and 1164), followed by four lysines (104, 482, 850 and 1168) which may play a gating role.

Synthesis and incorporation of pyrrole carboxamide nucleoside triphosphates by DNA polymerases.

Abstract We have synthesised and examined the enzymatic incorporation properties of the 5′-triphosphates of 2′-deoxyribosyl pyrrole 3-monocarboxamide (dMTP) and 2′-deoxyribosyl pyrrole 3,4-dicarboxamide (dDTP). These analogues we had hoped would behave as ambivalent base analogues in that they can present two alternative hydrogen-bonding faces either by rotation about the carboxamide group or about the glycosidic bond. The two pyrrole derivatives, dMTP and dDTP, exhibit a preference for incorporation with Klenow polymerase. They are preferentially incorporated as either A or C.

A DIMROTH REARRANGEMENT OF PYRIMIDINE NUCLEOSIDES

N 4-Acylamino-2′-deoxycytidine derivatives, 4a–c, undergo acid-promoted cyclisation to give [1,2,4]triazolo[4,3-c]pyrimidin-5(6H)-ones, 5a–c, in the presence of pyridinium chloride. This reaction has been demonstrated for a series of analogues. Treatment of the cyclised products in basic media gives rise to a novel Dimroth-type rearrangement leading to [1,2,4]triazolo[1,5-c]pyrimidin-5(6H)-ones, 7a–c. The crystal structure of one such product, 15, was confirmed by X-ray analysis.

Acylation monitoring in solid phase peptide synthesis by the equilibrium distribution of coloured ions

Spectrophotometric monitoring of the release of anionic dye from the support matrix during the acylation step in solid phase peptide synthesis (counterion distribution monitoring; CDM) is a sensitive indicator of the progress of this often problematic part of the process; this new method is readily incorporated into automated instrumentation where it permits feedback control of the time allowed for the coupling reaction, with consequent improvements in product quality and the speed of synthesis.

Mismatches in DNA: measurement of reduced duplex stability using 1H n.m.r. spectroscopy

The destabilisation of a double-stranded DNA fragment, which results when substitution of a single nucleotide introduces a G · T base pair, is determined from an equilibrium monitored using imino proton resonances in the n.m.r. spectrum in H2O.