Using remote substituents to control solution structure and anion binding in lanthanide complexes.

A study of the anion-binding properties of three structurally related lanthanide complexes, which all contain chemically identical anion-binding motifs, has revealed dramatic differences in their anion affinity. These arise as a consequence of changes in the substitution pattern on the periphery of the molecule, at a substantial distance from the binding pocket. Herein, we explore these remote substituent effects and explain the observed behaviour through discussion of the way in which remote substituents can influence and control the global structure of a molecule through their demands upon conformational space. Peripheral modifications to a binuclear lanthanide motif derived from α,α'-bis(DO3 Ayl)-m-xylene are shown to result in dramatic changes to the binding constant for isophthalate. In this system, the parent compound displays considerable conformational flexibility, yet can be assumed to bind to isophthalate through a well-defined conformer. Addition of steric bulk remote from the binding site restricts conformational mobility, giving rise to an increase in binding constant on entropic grounds as long as the ideal binding conformation is not excluded from the available range of conformers.

Photorelease of tyrosine from α-carboxy-6-nitroveratryl (αCNV) derivatives.

The synthesis of photolabile tyrosine derivatives protected on the phenolic oxygen by the α-carboxy-6-nitroveratryl (αCNV) protecting group is described. The compounds undergo rapid photolysis at wavelengths longer than 300 nm to liberate the corresponding phenol in excellent yield (quantum yield for the deprotection of tyrosine = 0.19). Further protection of caged tyrosine is possible, yielding N-Fmoc protected derivatives suitable for direct incorporation of caged tyrosine in solid-phase peptide synthesis.

Cycloaddition of homochiral dihydroimidazoles: a 1,3-dipolar cycloaddition route to optically active pyrrolo[1,2-a]imidazoles.

N-Alkylation of optically active 1-benzyl-4-phenyl-4,5-dihydroimidazole with active alkyl halides and treatment of the so-formed 4,5-dihydroimidazolium ions with DBU in the presence of a range of electron-deficient alkene dipolarophiles, constitutes a 'one-pot' cascade terminating in a 1,3-dipolar cycloaddition reaction that affords optically active pyrrolo[1,2-a]imidazoles. Three bonds of the so-formed pyrrolidine moiety are constructed in this cascade. The cycloaddition follows an endo approach of dipole and dipolarophile with anti geometry of the dipole and facial selectivity derived from the phenyl substituent. Inter- and intramolecular cycloaddition modes are observed.

Stereoselective synthesis of 2,4,5-trisubstituted piperidines via radical cyclization.

A novel approach to 2,4,5-trisubstituted piperidines is reported, involving the 6-exo cyclization of stabilized radicals onto α,ß-unsaturated esters. Only two of the four possible diastereoisomers are observed, with diastereomeric ratios ranging from 3:2 to 40:1 when the radical stabilizing group is vinyl or phenyl. Cyclization of a (triethylsilyl)vinyl-stabilized radical gives the corresponding piperidine radical as a single diastereoisomer that may either be trapped by tributyltin hydride to afford the 2,4,5-trisubstituted piperidine or undergo a second 5-endo cyclization onto the (triethylsilyl)vinyl substituent to produce the 3,5,7-trisubstituted octahydro[2]pyrindene as a single diastereoisomer.

Alpha-carboxy-6-nitroveratryl: a photolabile protecting group for carboxylic acids.

The synthesis of a new photolabile protecting group for carboxylic acids, alpha-carboxy-6-nitroveratryl (alphaCNV), is described. Bromide 3, prepared in four steps from 3,4-dimethoxyphenylacetic acid, was used to alkylate carboxylic acids under mild conditions in good yield. Palladium-catalyzed deallylation afforded the acids 4a-h, which underwent rapid and quantitative photolysis at wavelengths longer than 300 nm to liberate the carboxylic acid in good to quantitative yield. The rate of photolysis and quantum yield were determined to be 325 s(-1) and 0.17.

Changing the local coordination environment in mono- and bi- nuclear lanthanide complexes through "click" chemistry.

Alkyne appended lanthanide complexes derived from DO3A undergo copper catalysed cycloaddition reactions with azides to form triazole appended complexes: coordination of one of the triazole nitrogen atoms to the metal centre changes the local coordination environment and the spectroscopic properties of the complex.

Using the Ugi multicomponent condensation reaction to prepare families of chromophore appended azamacrocycles and their complexes.

The Ugi reaction offers an effective method for preparing chromophore-appended DOTA-monoamide ligands, which can readily be elaborated to their lanthanide complexes.

Stereoselective synthesis of 2,4,5-trisubstituted piperidines by carbonyl ene and Prins cyclisations.

An approach to 2,4,5-trisubstituted piperidines is reported, in which the key step is the Prins or carbonyl ene cyclisation of aldehydes of the type 1. Prins cyclisation catalysed by concentrated hydrochloric acid in CH(2)Cl(2) at -78 degrees C afforded good yields of two of the four possible diastereomeric piperidines, with the 4,5-cis product 7 predominating in a diastereomeric ratio of up to 94:6. The diastereoselectivity of the cyclisation decreased as the 2-substituent increased in size, becoming unselective for very bulky 2-substituents. In contrast, cyclisation catalysed by MeAlCl(2) in CH(2)Cl(2) or CHCl(3) at temperatures of between 20-60 degrees C, favoured the 4,5-trans diastereomer 8, in a diastereomeric ratio of up to 99:1. The low-temperature cyclisations catalysed by HCl proceed under kinetic control via a mechanism involving the development of significant carbocationic character, in which the 4,5-cis cation is more stable than the 4,5-trans cation as a result of overlap with the neighbouring oxygen. The cyclisations catalysed by MeAlCl(2) proceed under thermodynamic control, affording the product in which both the 4- and 5-substituents are equatorial.

Synthesis and ex vivo profiling of chemically modified cytomegalovirus CMVpp65 epitopes.

The effect of substituting unnatural hydrophobic amino acids into the critical MHC binding residues of an HLA-A*0201-restricted cytomegalovirus CMVpp65 epitope, NLVPMVATV, has been investigated. A new set of peptides containing the amino acids tert-butyl glycine (Tgl), cyclohexyl glycine (Chg), neo-pentyl glycine (Npg), cyclohexyl alanine (Cha) and cyclo leucine (Cyl), at either position 2, to mimic Leu, or position 9, to mimic Val, have been synthesised. Immunological profiling using class I MHC stabilisation assays to assess MHC binding affinity, and enzyme-linked immunospot (ELISPOT) assays to assess the ability of the modified peptides to re-stimulate a specific cytotoxic T-lymphocyte (CTL) response, compared to the native epitope, have been performed. It was found that the majority of the unnatural substitutions resulted in a decrease in either HLA-A*0201 binding affinity or cytotoxic T-cell activity. However, the HLA-A*0201 binding affinity was unrelated to the ability to re-stimulate a T-cell response. Minimisation and molecular dynamics studies proved helpful in dissecting the ELISPOT responses. Two principal peptide binding modes were found by minimisation, designated kinked and straight. Peptides that bound in a kinked conformation were poor at re-stimulating a T-cell response. Of the peptides that bound in a straight conformation, molecular dynamics (MD) simulations revealed that those capable of re-stimulating the strongest responses had the greatest degree of flexibility (as determined by RMSD values across the MD simulation) around the P6 residue, one of the residues important for T-cell receptor recognition.

Synthesis and biological evaluation of a C5-biphenyl thiolactomycin library.

Fifteen novel C5 analogues of thiolactomycin (13 biphenyl analogues and two biphenyl mimics) have been synthesised and assessed for their in vitro mtFabH and whole cell Mycobacterium bovis BCG activity, respectively. Analysis of the 15 compounds revealed that six possessed enhanced in vitro activity in a direct mtFabH assay. Encouragingly analogues 11, 12 and 13 gave a significant enhancement in in vitro activity against mtFabH. Analogue 13 (5-(4-methoxycarbonyl-biphenyl-4-ylmethyl)-4-hydroxy-3,5-dimethyl-5H-thiophen-2-one) gave an IC(50) value of 3 microM compared to the parent drug thiolactomycin (75 microM) against mtFabH. The biological analysis of this library reaffirms the requirement for a linear pi-rich system containing hydrogen bond accepting substituents attached to the para-position of the C5 biphenyl analogue to generate compounds with enhanced activity.

Stereoselective synthesis of 3,4-disubstituted and 3,4,5-trisubstituted piperidines by Lewis acid-catalysed ene cyclisation of 4-aza-1,7-dienes.

The thermal or Lewis acid-catalysed ene cyclisation of a variety of 4-aza-1,7-dienes afforded 3,4-disubstituted or 3,4,5-trisubstituted piperidines. Activation of the enophile with a single ester facilitated a thermal ene cyclisation, although the reaction was not amenable to Lewis acid catalysis. With other activating groups on the enophile it was found that Lewis acid catalysis was facile, although there was a fine balance between the desired ene cyclisation and the competing hetero-Diels-Alder reaction, with the product distribution being influenced by the activating group on the enophile, the nature of the ene component, and the Lewis acid used. Activation of the enophile with an oxazolidinone function facilitated Lewis acid-catalysed cyclisation to afford mixtures of ene and hetero-Diels-Alder products. Activating the enophile with two ester groups gave a substrate that underwent a very facile ene cyclisation catalysed by MeAlCl(2) to give the corresponding trans 3,4-disubstituted piperidines with diastereomeric ratios of >200 : 1.

Structures of the dI2dIII1 complex of proton-translocating transhydrogenase with bound, inactive analogues of NADH and NADPH reveal active site geometries.

Transhydrogenase couples the redox reaction between NADH and NADP+ to proton translocation across a membrane. The enzyme comprises three components; dI binds NAD(H), dIII binds NADP(H), and dII spans the membrane. The 1,4,5,6-tetrahydro analogue of NADH (designated H2NADH) bound to isolated dI from Rhodospirillum rubrum transhydrogenase with similar affinity to the physiological nucleotide. Binding of either NADH or H2NADH led to closure of the dI mobile loop. The 1,4,5,6-tetrahydro analogue of NADPH (H2NADPH) bound very tightly to isolated R. rubrum dIII, but the rate constant for dissociation was greater than that for NADPH. The replacement of NADP+ on dIII either with H2NADPH or with NADPH caused a similar set of chemical shift alterations, signifying an equivalent conformational change. Despite similar binding properties to the natural nucleotides, neither H2NADH nor H2NADPH could serve as a hydride donor in transhydrogenation reactions. Mixtures of dI and dIII form dI2dIII1 complexes. The nucleotide charge distribution of complexes loaded either with H2NADH and NADP+ or with NAD+ and H2NADPH should more closely mimic the ground states for forward and reverse hydride transfer, respectively, than previously studied dead-end species. Crystal structures of such complexes at 2.6 and 2.3 A resolution are described. A transition state for hydride transfer between dihydronicotinamide and nicotinamide derivatives determined in ab initio quantum mechanical calculations resembles the organization of nucleotides in the transhydrogenase active site in the crystal structure. Molecular dynamics simulations of the enzyme indicate that the (dihydro)nicotinamide rings remain close to a ground state for hydride transfer throughout a 1.4 ns trajectory.

Synthesis and biological evaluation of two chemically modified peptide epitopes for the class I MHC protein HLA-B*2705.

The T-cell receptor of a CD8(+) T-cell recognises peptide epitopes bound by class I major histocompatibility complex (MHC) glycoproteins presented in a groove on their upper surface. Within the groove of the MHC molecule are 6 pockets, two of which mostly display a high degree of specificity for binding amino acids capable of making conserved and energetically favourable contacts with the MHC. One type of MHC molecule, HLA-B*2705, preferentially binds peptides containing an arginine at position 2. In an effort to increase the affinity of peptides for HLA-B*2705, potentially leading to better immune responses to such a peptide, we synthesised two modified epitopes where the amino acid at position 2 involved in anchoring the peptide to the class I molecule was replaced with the alpha-methylated beta,gamma-unsaturated arginine analogue 2-(S)-amino-5-guanidino-2-methyl-pent-3-enoic acid. The latter was prepared via a multi-step synthetic sequence, starting from alpha-methyl serine, and incorporated into dipeptides which were fragment-coupled to resin-bound heptameric peptides yielding the target nonameric sequences. Biological characterisation indicated that the modified peptides were poorer than the native peptides at stabilising empty class I MHC complexes, and cells sensitised with these peptides were not recognised as well by cognate CD8(+) T-cells, where available, compared to those sensitised with the native peptide. We suggest that the modifications made to the peptide have decreased its ability to bind to the peptide binding groove of HLA-B*2705 molecules which may explain the decrease in recognition by cytotoxic T-cells when compared to the native peptide.

Synthesis of 2,4-disubstituted piperidines via radical cyclization: unexpected enhancement in diastereoselectivity with tris(trimethylsilyl)silane.

A novel approach to 2,4-disubstituted piperidines is reported, involving the radical cyclization of 7-substituted-6-aza-8-bromooct-2-enoates. Cyclization with tributyltin hydride affords the trans piperidines with trans/cis diastereomeric ratios ranging typically from 3:1 to 6:1. Cyclization with tris(trimethylsilyl)silane affords the same products with diastereomeric ratios of up to 99:1 in certain cases. The enhancement in diastereoselectivity results from the selective rearrangement of the minor stereoisomer through a cascade process involving radical cyclization to the piperidine radical, 1,5-radical translocation, and attack of the translocated radical onto the sulfonamide with extrusion of SO2 in a Smiles-type rearrangement. Slower trapping of the piperidine radical by tris(trimethylsilyl)silane compared to tributyltin hydride accounts for the occurrence of the rearrangement cascade in the former case.

Synthesis of 3,4-disubstituted piperidines by carbonyl ene and prins cyclizations: switching between kinetic and thermodynamic control with Brønsted and Lewis acid catalysts.

A novel approach to cis and trans 3,4-disubstituted piperidines is described. Carbonyl ene cyclization of aldehydes 4a-e catalyzed by MeAlCl(2) in refluxing chloroform afforded the trans piperidines 7a-e with diastereomeric ratios of up to 93:7, while aldehyde 4f afforded solely the cis product 6f, which was resistant to isomerization to the trans isomer. It was demonstrated for 4a that the cyclization catalyzed by a variety of Lewis acids at low temperature proceeded under kinetic control to afford predominantly the cis piperidine 6a, and this isomerized to the thermodynamically more stable trans piperidine 7a on warming. In contrast, Prins cyclization of 4a-e catalyzed by concentrated hydrochloric acid in CH2Cl2 at low temperature afforded cis piperidines 6a-e with diastereomeric ratios of up to >98:2. The yield and diastereoselectivity of these cyclizations could be improved by using HCl-saturated CH2Cl2 to form the corresponding chloride, followed by elimination of HCl effected by ammonia. Aldehydes 4f and 4galso cyclized in good yield under the latter conditions. Mechanistic studies supported by DFT calculations (B3LYP/6-31G(d)) suggest that the cyclizations proceed via a mechanism with significant carbocationic character, with the cis carbocation being more stable than the trans carbocation. DFT calculations (B3LYP/6-31G(d)) of the transition state energies for concerted cyclization show that the cis piperidine is also the favored product from cyclization through a more concerted mechanism.

A luminescent probe containing a tuftsin targeting vector coupled to a terbium complex.

Orthogonal protection strategies have been used to prepare a series of luminescent and MRI active lanthanide complexes containing a tuftsin targeting vector that are internalised by macrophage cells.

Stereoselective synthesis of 2,4,5-trisubstituted piperidines by carbonyl ene and Prins cyclisations.

Cyclisation of aldehydes 3-e catalysed by concentrated hydrochloric acid affords predominantly the all cis 2,4,5-trisubstituted piperidines 4a-ewhen the 2-substituent is small, while catalysis by MeAlCl2 in refluxing chloroform gives the trans piperidines 5a-e with diastereomeric ratios of up to 99:1.

Al-isopropoxydiisobutylalane: a study of the effect of solvent on the rate and stereoselectivity of cyclic ketone reduction.

The effect of solvent on the rate and stereoselectivity of cyclic ketone reduction by Al-isopropoxydiisobutylalane (DIBA(i)OPr) has been investigated. In dichloromethane, DIBA(i)OPr behaves as a bulky reducing agent, approaching the carbonyl group along an equatorial trajectory to produce the axial alcohol with >10:1 stereoselectivity. In sharp contrast, reduction in toluene gives the complementary outcome, affording the thermodynamically more stable isomer with >99:1 stereoselectivity.

Tris(trimethylsilyl)silane: an unprecedented enhancement in the diastereoselectivity of radical cyclisations to give 2,4-disubstituted piperidines.

Cyclisation of bromides 4a-f mediated by tributyltin hydride affords predominantly the trans piperidines 5a-f with modest diastereomeric ratios, while cyclisation with tris(trimethylsilyl)silane affords the same products with diastereomeric ratios of up to 99 : 1.

Synthesis of 3-substituted 4-piperidinones via a one-pot tandem oxidation-cyclization-oxidation process: stereodivergent reduction to 3,4-disubstituted piperidines.

A novel approach to 3-substituted 4-piperidinones is described. The one-pot tandem oxidation-cyclization-oxidation of unsaturated alcohols 1a-e by PCC or PCC and trifluoromethanesulfonic acid affords piperidinones 2a-e in good yield. Reduction of 2a-e by L-Selectride gives the corresponding cis 3,4-disubstituted piperidines with diastereomeric ratios of >99:1. By contrast, reduction of 2a-e by Al-isopropoxydiisobutylalane gives the trans products with diastereomeric ratios of up to 99:1.