Morten Grøtli

Structural and Mechanistic Basis of Pre- and Posttransfer Editing by Leucyl-tRNA Synthetase

The aminoacyl-tRNA synthetases link tRNAs with their cognate amino acid. In some cases, their fidelity relies on hydrolytic editing that destroys incorrectly activated amino acids or mischarged tRNAs. We present structures of leucyl-tRNA synthetase complexed with analogs of the distinct pre- and posttransfer editing substrates. The editing active site binds the two different substrates using a single amino acid discriminatory pocket while preserving the same mode of adenine recognition. This suggests a similar mechanism of hydrolysis for both editing substrates that depends on a key, completely conserved aspartic acid, which interacts with the alpha-amino group of the noncognate amino acid and positions both substrates for hydrolysis. Our results demonstrate the economy by which a single active site accommodates two distinct substrates in a proofreading process critical to the fidelity of protein synthesis.

The crystal structure of asparaginyl‐tRNA synthetase from Thermus thermophilus and its complexes with ATP and asparaginyl‐adenylate: the mechanism of discrimination between asparagine and aspartic acid

The crystal structure of Thermus thermophilus asparaginyl‐tRNA synthetase has been solved by multiple isomorphous replacement and refined at 2.6 Å resolution. This is the last of the three class IIb aminoacyl‐tRNA synthetase structures to be determined. As expected from primary sequence comparisons, there are remarkable similarities between the tertiary structures of asparaginyl‐tRNA synthetase and aspartyl‐tRNA synthetase, and most of the active site residues are identical except for three key differences. The structure at 2.65 Å of asparaginyl‐tRNA synthetase complexed with a non‐hydrolysable analogue of asparaginyl‐adenylate permits a detailed explanation of how these three differences allow each enzyme to discriminate between their respective and very similar amino acid substrates, asparagine and aspartic acid. In addition, a structure of the complex of asparaginyl‐tRNA synthetase with ATP shows exactly the same configuration of three divalent cations as previously observed in the seryl‐tRNA synthetase–ATP complex, showing that this a general feature of class II synthetases. The structural similarity of asparaginyl‐ and aspartyl‐tRNA synthetases as well as that of both enzymes to the ammonia‐dependent asparagine synthetase suggests that these three enzymes have evolved relatively recently from a common ancestor.

Inhibitors and promoters of tubulin polymerization: Synthesis and biological evaluation of chalcones and related dienones as potential anticancer agents

A series of dihalogenated chalcones and structurally related dienones were synthesized and evaluated for their antiproliferative activity in 10 different cancer cell lines and for their effect on microtubule assembly. All compounds showed cytotoxic activity, with IC(50) values in the 5-280 μM range depending on the chalcone structure and the cell line. Five of the compounds were found to be tubulin polymerization inhibitors. In contrast, one of the compounds was found to stabilize tubulin to the same extent as the anticancer drug docetaxel. Molecular modeling suggested that the tubulin inhibitors bind to the colchicine binding site of β-tubulin while the novel tubulin stabilization agent seems to interact with the paclitaxel binding site.

2′-O-Propargyl oligoribonucleotides: Synthesis and hybridisation

Abstract Fully modified oligonucleotide sequences containing 2′- O -propargylribonucleotides were synthesised on automated DNA-synthesisers using the phosphoramidite approach. A highly selective alkylation procedure was used to introduce the propargyl functionality, thereby enabling the synthesis of protected 2′- O -propargyl-3′- O -phosphoramidites, building blocks for the assembly of 2′- O -propargyl oligoribonucleotides. The suitability of phosphoramidite chemistry for the introduction of this modified nucleoside was proven using MALDI or ES mass spectrometry of the final oligomer. The 2′- O -propargyl oligoribonucleotides showed an increase in the Tm of duplexes with complementary RNA relative to the corresponding RNA homoduplex. These analogues should prove useful for a variety of antisense applications.

Characterization of photophysical and base-mimicking properties of a novel fluorescent adenine analogue in DNA

To increase the diversity of fluorescent base analogues with improved properties, we here present the straightforward click-chemistry-based synthesis of a novel fluorescent adenine-analogue triazole adenine (AT) and its photophysical characterization inside DNA. AT shows promising properties compared to the widely used adenine analogue 2-aminopurine. Quantum yields reach >20% and >5% in single- and double-stranded DNA, respectively, and show dependence on neighbouring bases. Moreover, AT shows only a minor destabilization of DNA duplexes, comparable to 2-aminopurine, and circular dichroism investigations suggest that AT only causes minimal structural perturbations to normal B-DNA. Furthermore, we find that AT shows favourable base-pairing properties with thymine and more surprisingly also with normal adenine. In conclusion, AT shows strong potential as a new fluorescent adenine analogue for monitoring changes within its microenvironment in DNA.

Solid-Phase Peptide Synthesis in Water Using Microwave-Assisted Heating

An approach using water as a solvent (coupling and deprotection) was developed for the solid-phase synthesis of peptides using the most common Boc-amino acid derivatives. Key aspects of this methodology are the use of a PEG-based resin, EDC-HONB as a coupling method, and microwave irradiation as an energy source.

Synthesis of 3-(1,2,3-triazol-1-yl)- and 3-(1,2,3-triazol-4-yl)-substituted pyrazolo[3,4-d]pyrimidin-4-amines via click chemistry: potential inhibitors of the Plasmodium falciparum PfPK7 protein kinase

Efficient routes to 3-(1,2,3-triazol-1-yl)- and 3-(1,2,3-triazol-4-yl)pyrazolo[3,4-d]pyrimidin-4-amines using a one-pot two-step reaction are presented. The two routes give easy access to two different isomers of 1,4-disubstituted triazoles and the target compounds are obtained from a variety of readily available aromatic and aliphatic halides without isolation of potentially unstable organic azide intermediates. Two compounds show activity towards the PfPK7 kinase (IC(50) 10-20 microM) of P. falciparum, the organism responsible for the most virulent form of malaria, and can be regarded as hits useful for further development into lead compounds.

Preparation of 3-Substituted-1-Isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amines as RET Kinase Inhibitors

A series of 3-substituted-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amines have been designed, synthesized, and evaluated as RET protein kinase inhibitors. On the basis of docking results, a small library of pyrazolopyrimidine compounds with an extended hydrophobic side arm was synthesized. The most promising of the compounds (7a) displayed efficient inhibition in vitro and good selectivity when tested on a panel of kinases. Furthermore, 7a inhibited GDNF-induced RET phosphorylation of ERK1/2 in MCF-7 breast cancer cells at concentrations as low as 100 nM.

Synthesis of 2-Alkyl-Substituted Chromone Derivatives Using Microwave Irradiation

A base-promoted condensation between 2-hydroxyacetophenones and aliphatic aldehydes has been studied. The reaction has been optimized to afford 2-alkyl-substituted 4-chromanones in an efficient manner using microwave heating. Performing the reaction using diisopropylamine in EtOH at 170 degrees C for 1 h gave moderate to high yields (43-88%). The 4-chromanones could be further converted into highly functionalized 2,3,6,8-tetrasubstituted chromones in which a 3-substituent (acetate, amine, or bromine) was introduced via straightforward chemical transformations.

Enhanced microwave-assisted method for on-bead disulfide bond formation: Synthesis of α-conotoxin MII

A novel enhanced microwave‐assisted disulfide bridge formation method has been developed. To optimize the synthesis of the biologically important bicyclic peptide α‐conotoxin MII (α‐CtxMII), several cyclization methods have been tested and are discussed herein. By using m.w.‐assisted heating, we achieved high yields for the first loop cyclization of α‐CtxMII on‐bead. This method has the advantage of avoiding intermolecular by‐products during the cyclization step. Furthermore, the method gives higher yields compared with the common on‐bead cyclization methods. The second disulfide bridge of α‐CtxMII was formed using a simple oxidation method after the cleavage of the intermediate monocyclic peptide from the resin. This method has the potential to be efficient for the synthesis of other disulfide rich biologically important peptides.