Ashit K. Ganguly

Electrospray ionization mass spectrometry for the study of non-covalent complexes : an emerging technology

The detection of non-covalent complexes in the mass range 19,000-34,000 Da, using electrospray ionization mass spectrometry (ESI-MS), is reviewed. The examples discussed include (1) a protein-ligand interaction (ras-GDP), (2) an inhibitor-protein-ligand interaction (SCH 54292/SCH 54341-ras-GDP), (3) a protein-protein interaction (gamma-IFN homodimer) and (4) a protein-metal complex [HCV (1-181)-Zn]. In each case, the ESI-MS method is capable of releasing the intact non-covalent complex from its native solution state into the gas phase in the form of multiply-charge ions. The molecular masses of these complexes were determined with a mass accuracy of better than 0.01%, which is far superior to the traditional methods of sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel permeation chromatography. The method provides the researcher with a quick, reliable and reproducible method for probing difficult biological problems. The key to success in the study of non-covalent complexes depends on careful understanding and manipulation of ESI source parameters and sample solution conditions; special care must be taken with the source orifice potential and the solution pH and organic co-solvents must be avoided. This paper also illustrates the usefulness of ESI-MS for addressing biological problems leading to the discovery of new therapeutics; the approach involves the rapid screening of potential drug candidates, such as weakly bound inhibitors.

Ras oncoprotein inhibitors: The discovery of potent, ras nucleotide exchange inhibitors and the structural determination of a drug-protein complex

The nucleotide exchange process is one of the key activation steps regulating the ras protein. This report describes the development of potent, non-nucleotide, small organic inhibitors of the ras nucleotide exchange process. These inhibitors bind to the ras protein in a previously unidentified binding pocket, without displacing bound nucleotide. This report also describes the development and use of mass spectrometry, NMR spectroscopy and molecular modeling techniques to elucidate the structure of a drug-protein complex, and aid in designing new ras inhibitor targets.

One pot solid phase synthesis of isoxazolines

Abstract 1,3 Dipolar cycloadditions of nitrile oxides generated in situ on solid phase in the presence of a variety of dipolaraphiles provided a library of isoxazolines and isoxazoles.

Application of electrospray mass spectrometry in probing protein-protein and protein-ligand noncovalent interactions.

A novel mass spectrometry-based methodology using electrospray ionization (ESI) is described for the detection of protein-protein [interferon (IFN)-γ dimer] and protein-ligand [ras-guanosine diphosphate (GDP)] noncovalent interactions. The method utilizes ESI from aqueous solution at appropriate pH. The presence of the noncovalent complex of the IFN-γ dimer was confirmed by the observed average molecular weight of 33,819 Da. The key to the detection of the IFN-γ dimer is the use of an alkaline solution (pH ≈ 9) for sample preparation and for mass spectrornetry analysis. The effect of the declustering energy in the region of the ion sampling orifice and focusing quadrupole on the preservation of the gas-phase noncovalent complex (IFN-γ dimer) was also studied. The effect of the declustering energy on complex dissociation was further extended to probe the noncovalent protein-ligand association of ras-GDP. It was found that little energy is required to dissociate the IFN-γ dimer, whereas a substantial amount of energy is required to dissociate the gas-phase ras-GDP complex.

Concise asymmetric routes to 2,2,4-trisubstituted tetrahydrofurans via chiral titanium imide enolates: Key intermediates towards synthesis of highly active azole antifungals SCH 51048 and SCH 56592

Two complimentary approaches to the key (−)-(2R)-cis-tosylate 1 and its (+)-(2S)-enantiomer 15 via generation of chiral imide enolates having a 2,2-disubstituted olefin functionality in the β-position, are described. In a “protecting group free” sequence, reaction of the titanium enolate generated from (4R)-benzyl-2-oxazolidinone derived imide 5b with s-trioxane provided a convenient intermediate 19 which could be directly subjected to 2,4-diastereoselective iodocyclization.

Controlling benzylic functionality and stereochemistry: 2. Synthesis of the pseudopterosin aglycone

Homologation, cyclisation, and reduction converted the tetralin (2) to the hexahydrophenalenol (8), which was methylated to afford (19), via alkoxide-directed metalation. The degree of stereoselectivity resulting from reactions of (19) and congeners with allylsilane - Lewis acid combinations was markedly dependent upon substitution patterns, whereas Et2AlCN-SnCl4 produced pseudoaxial nitriles. The trimethyl nitrile (24) was elaborated to the pseudopterosin aglycone (4).

Discovery of novel nonpeptide tricyclic inhibitors of ras farnesyl protein transferase

A comprehensive structure-activity relationship (SAR) study of novel tricyclic amides has been undertaken. The discovery of compounds that are potent FPT inhibitors in the nanomolar range has been achieved. These compounds are nonpeptidic and do not contain sulfhydryl groups. They selectively inhibit farnesyl protein transferase (FPT) and not geranylgeranyl protein transferase-1 (GGPT-1). They also inhibit H-Ras processing in Cos monkey kidney cells.

Synthesis of an optically pure 3-unsubstituted β-lactam using an asymmetric Reformatsky reaction and its conversion to cholesterol absorption inhibitors

Abstract Asymmetric induction by several chiral alcohols in the reaction of their bromoaceates with imines in the presence of activated Zn (Reformatsky reaction) was studied. Trans -2-phenylcyclohexanol and phenyl menthol gave β-lactam 9 , obtained by cyclizing the diastereoisomeric β-aminoesters 8 , in > 99%ee. The resulting chiral 3-unsubstituted azetidin-2-one 9 was converted to 3-substituted products 11, 12 , and 13 which exhibit cholesterol absorption inhibitory activity.

Antitumor 8-chlorobenzocycloheptapyridines: a new class of selective, nonpeptidic, nonsulfhydryl inhibitors of ras farnesylation.

Ras farnesylation by farnesyl protein transferase (FPT) is an intracellular event that facilitates the membrane association of the ras protein and is involved in the signal transduction process. FPT inhibition could be a novel, noncytotoxic method of treating ras dependent tumor growth. We report here three structural classes of 8-chlorobenzocycloheptapyridines as novel, nonpeptidic, nonsulfhydryl FPT inhibitors having antitumor activity in mice when dosed orally. We discuss structural and conformational aspects of these compounds in relation to biological activities as well as a comparison to the conformation of a bound tetrapeptide FPT inhibitor.

Total synthesis and absolute stereochemistry of the antifungal dipeptide Sch 37137 and its 2S,3S - isomer

Abstract The absolute stereochemistry of the epoxide moiety in Sch 37137 has been established as 2R, 3R by its synthesis from L-tartaric acid. .