Effects of steric bulk and stereochemistry on the rates of diketopiperazine formation from N-aminoacyl-2,2-dimethylthiazolidine-4-carboxamides (Dmt dipeptide amides)—a model for a new prodrug linker system

A peptide-like self-immolative molecular clip is required for release of active drugs from prodrugs by endopeptidases. Upon cleavage from the carrier, this clip must collapse and release the drug rapidly. A series of aminoacyl-5,5-dimethylthiaproline (Aaa-Dmt) N-(2-(4-nitrophenyl)ethyl)amides were designed. Boc-l-aminoacyl fluorides were coupled with R-DmtOH to give Boc-l-Aaa-R-DmtOH, which were converted to the Boc-l-Aaa-R-Dmt N-(2-(4-nitrophenyl)ethyl)amides. The l,S diastereomeric series was prepared by the reaction of Boc-Aaa PFP esters with S-DmtOH. The l-Aaa-Dmt N-(2-(4-nitrophenyl)ethyl)amides were allowed to cyclise to diketopiperazines (DKPs) in aqueous buffers, expelling 2-(4-nitrophenyl)ethylamine as a model for amine-containing drugs. Reaction rates were dependant on pH. In the l,R diastereomeric series, increasing steric bulk of the Aaa side-chain (Gly, Ala, Phe, Val) led to decrease in the reaction rate. However, in the l,S series, the greatest rate of reaction was observed for the most bulky amino-acid (Val), with t_½=15 min at pH 8.0. The effects of steric bulk and stereochemistry are rationalised through conformational analysis (NMR and X-ray crystallography) of the starting dipeptide amides, the product diketopiperazines and key analogues. Since the dipeptides are (almost) exclusively in the cis-amide conformation, trans-cis interconversion is not relevant. The data suggest that steric interactions in the reacting conformations of the dipeptide amides, as they form the tetrahedral intermediates, are the controlling factors. Thus, l-Aaa-S-Dmt amides are shown to be excellent candidates for incorporation into the design of novel prodrugs.     

Rapid determination of volatile organic compounds in environmentally hazardous wastewaters using solid phase microextraction

Solid phase micro-extraction (SPME) was applied to the determination of volatile organic compounds (VOC) in wastewater discharges. Environmentally significant samples, typical of those subject to regulatory control, were examined and included discharges from pharmaceutical, petrochemical and municipal sewerage treatment plants. Analysis was performed using gas chromatography – mass spectrometry (GC-MS) following sampling using headspace or immersion SPME. Fused silica fibres, coated with either poly(dimethylsiloxane) or poly(acrylate), were examined to determine VOC which included chloroform, saturated carboxylic acids, alkylbenzenes, phenol, benzonitrile and benzofuran. Detection limits varied from 10 to 170 ng/ml and satisfactory relative standard deviations (%RSD < 10) were obtained. For most samples, headspace sampling was preferred to immersion. SPME was found to be a useful technique for the rapid screening of wastewaters for VOC.     

EFFECTS OF LIGHT BEAM SIZE ON FLUENCE DISTRIBUTION AND DEPTH OF NECROSIS IN SUPERFICIALLY APPLIED PHOTODYNAMIC THERAPY OF NORMAL RAT BRAIN

The light fluence distributions of 632.8 nm light incident on the exposed surface of normal rat brain in vivo have been measured using an interstitial, stereotactically-mounted optical fiber detector with isotropic response. The dependence of the relative fluence rate on depth and the spatial distribution of fluence were compared for incident beam diameters of 3 and 5 mm. The fluence rate at depth of 1-6 mm along the optical axis within the brain tissue was approximately 70% greater for a 5 mm diameter beam than for a 3 mm beam, at the same incident fluence rate, although the plots of the relative fluence rate vs depth were parallel over the depth range 1-6 mm. The depths of necrosis resulting from photodynamic treatment of brain tissue using the photosensitizer Photofrin and irradiation by 632 nm light with 3 and 5 mm incident beams were also measured. The observed difference in necrosis depths was consistent with the measured difference in fluence. The importance of beam size in photodynamic treatment with small diameter incident light fields is discussed.     

Dissolution of plutonium(IV) hydroxide by desferrioxamine siderophores and simple organic chelators

Plutonium is thought to exist mostly as low soluble plutonium(IV) species in the environment and, therefore, has low potential of becoming mobile. Due to their prevalence and high solution stability constants for Pu(IV), microbial siderophores could significantly affect plutonium solubility and mobility. In this study, the ability of trihydroxamate desferrioxamine siderophores to solubilize Pu(IV) solids was investigated. Both desferrioxamine B and E (DFB and DFE) are far less effective at solubilizing amorphous Pu(IV) hydroxide in neutral solution than are simple chelators, such as EDTA, citrate, and tiron, despite the fact that these chelators have smaller solution stability constants for Pu(IV) than do DFE and DFB. Positively charged, linear DFB is less effective than the neutral cyclic DFE. Hydroxamate siderophores may, in fact, passivate Pu(IV) hydroxide surfaces, thereby inhibiting solubilization by other chelators. PuO(2) solubilization under these conditions is far slower than that of Pu(IV) hydroxide.     

ACTION SPECTRA FOR THE trans TO cis PHOTOISOMERISATION OF UROCANIC ACID in vitro and IN MOUSE SKIN

Urocanic acid (UCA) is a major UV chromophore in the upper layers of the skin where it is found predominantly as the trans isomer. UV irradiation induces photoisomerisation of trans-UCA to cis-UCA which has been shown to mimic some of the immunosuppressive properties of UV exposure. We examined the wavelength dependence for trans-UCA to cis-UCA photoisomerisation in vitro and in mouse skin in vivo over the spectral range270–340 nm. The resulting action spectra were very similar with maximal effectiveness at300–315 nm and equal activity at 270 nm and325–330 nm, demonstrating that UVA-II radiation (320–340nm) is efficient at UCA photoisomerisation. These action spectra differed markedly from the trans-UCA absorption spectrum in vitro and also the reported action spectrum for UV suppression of contact hypersensitivity in mice. These findings suggest that the relationship between cis-UCA formation in skin and UV-induced immunosuppression may be complex.     

Propionaldehyde: ab initio and semi-empirical calculations

The internal rotation of propionaldehyde about the 1–2 bond has been studied by means of ab initio calculations. The most stable conformer has methyl and carbonyl eclipsed. Increasing the 1–2 dihedral angle to 60°, 120°, and 180° gives energies of 1.7, 0.4, and 0.7 Kcal/mol, respectively. The agreement with force field calculations and with experiment is reasonable.     

Crystal structure of 1:1 Complexes between urea and two crown ether derivatives of phthalic acid, 2,3,5,6,8,9,11,12,14,15-decahydrobenzo[1,4,7,10,13,16]hexaoxacyclo-octadecin-18,19-dicarboxylic acid (i) and 2,3,5,6,8,9,11,12-octahydro-benzo[1,4,7,10,13]pentaoxacyclopentadecin-15,16-dicarboxylic acid (II)

The crystal structures of the titlke compounds have been determined by X-ray diffraction. Urea, I crystallizes in the triclinic PI space group with cell dimensions a = 8.336(2), b = 11.009(2), c = 13.313(2) Å, α = 105.55(3), β = 103.62(3), γ = 104.63(3)° and Z = 2 final R value 0.072 for 2105 observations. Urea, II crystallizes in the orthorhombic P2₁2₁2₁ space group with cell dimensions a = 8.750(2), b = 10.844(3) and c = 21.215(3) Å and Z = 4, final R value 0.083 for 599 observations. All the hydrogen atoms were located in the complex urea, I ; urea molecules form hydrogen bonded dimers about centers of symmetry, these dimers are sandwiched between macrocyclic rings forming one simple and one bifurcated hydrogen bond from the “endo” hydrogen atoms to the ether oxygen atoms. These units are held by hydrogen bonding between the urea molecules and carboxylic acids in two other units; these hydrogen bonds are cyclic involving eight atoms -(N-H(exo)…O(keto)-C-O-H…O(urea)-C)-. Only one carboxylic acid group per molecule takes part in these hydrogen bonds, the other forms a short, 2.490(7) Å, internal bond to the acceptor keto oxygen atom. N(H)…O bonds range from 2.930(7) to 3.206(7) Å, O(H)…O is 2.475(6) Å. In the complex urea, II each urea is hydrogen bonded to three different host molecules and vice versa; the urea “endo” hydrogen atoms bond to the ether oxygen atoms, while both “exo” hydrogen atoms take part in cyclic hydrogen bonds to carboxylic acids. There is not internal hydrogen bond. N(H)…O bonds range from 2.83 to 3.26(2) A and the O-…O bonds are 2.55 and 2.56(2) Å.     

Analysis of chemical warfare agents. II. Use of thiols and statistical experimental design for the trace level determination of vesicant compounds in air samples

Thermal desorption with gas chromatography-mass spectrometry (TD-GC-MS) remains the technique of choice for analysis of trace concentrations of analytes in air samples. This paper describes the development and application of a method for analysing the vesicant compounds sulfur mustard and Lewisites I-III. 3,4-Dimercaptotoluene and butanethiol were used to spike sorbent tubes and vesicant vapours sampled; Lewisite I and II reacted with the thiols while sulfur mustard and Lewisite III did not. Statistical experimental design was used to optimise thermal desorption parameters and the optimum method used to determine vesicant compounds in headspace samples taken from a decontamination trial. 3,4-Dimercaptotoluene reacted with Lewisites I and II to give a common derivative with a limit of detection (LOD) of 260 microg m(-3), while the butanethiol gave distinct derivatives with limits of detection around 30 microg m(-3).     

Nitrosyliron(III) porphyrinates: porphyrin core conformation and FeNO geometry. Any correlation?

The synthesis, structural, and spectroscopic characterization of (nitrosyl)iron(III) porphyrinate complexes designed to have strongly nonplanar porphyrin core conformations is reported. The species have a nitrogen-donor axial ligand trans to the nitrosyl ligand and display planar as well as highly nonplanar porphyrin core conformations. The systems were designed to test the idea, expressly discussed for the heme protein nitrophorin (Roberts, et al. Biochemistry 2001, 40, 11327), that porphyrin core distortions could lead to an unexpected, bent geometry for the FeNO group. For [Fe(OETPP)(1-MeIm)(NO)]ClO(4).C(6)H(5)Cl (H(2)OETPP = octaethyltetraphenylporphyrin), the porphyrin core is found to be severely saddled. However, this distortion has little or no effect on the geometric parameters of the coordination group: Fe-N(p) = 1.990(9) A, Fe-N(NO) = 1.650(2) A, Fe-N(L) = 1.983(2) A, and Fe-N-O = 177.0(3) degrees. For the complex [Fe(OEP)(2-MeHIm)(NO)]ClO(4).0.5CH(2)Cl(2) (H(2)OEP = octaethylporphyrin), there are two independent molecules in the asymmetric unit. The cation denoted [Fe(OEP)(2-MeHIm)(NO)](+)(pla) has a close-to-planar porphyrin core. For this cation, Fe-N(p) = 2.014(8) A, Fe-N(NO) = 1.649(2) A, Fe-N(L) = 2.053(2) A, and Fe-N-O = 175.6(2) degrees. The second cation, [Fe(OEP)(2-MeHIm)(NO)](+)(ruf), has a ruffled core: Fe-N(p) = 2.003(7) A, Fe-N(NO) = 1.648(2) A, Fe-N(L) = 2.032(2) A, and Fe-N-O = 177.4(2) degrees. Thus, there is no effect on the coordination group geometry caused by either type of nonplanar core deformation; it is unlikely that a protein engendered core deformation would cause FeNO bending either. The solid-state nitrosyl stretching frequencies of 1917 cm(-)(1) for [Fe(OEP)(2-MeHIm)(NO)]ClO(4) and 1871 cm(-)(1) for [Fe(OETPP)(1-MeIm)(NO)]ClO(4) are well within the range seen for linear Fe-N-O groups. M?ssbauer data for [Fe(OEP)(2-MeHIm)(NO)]ClO(4) confirm that the ground state is diamagnetic. In addition, the quadrupole splitting value of 1.88 mm/s and isomer shift (0.05 mm/s) at 4.2 K are similar to other (nitrosyl)iron(III) porphyrin complexes with linear Fe-N-O groups. Crystal data: [Fe(OETPP)(1-MeIm)(NO)]ClO(4).C(6)H(5)Cl, monoclinic, space group P2(1)/c, Z = 4, with a = 12.9829(6) A, b = 36.305(2) A, c = 14.0126(6) A, beta = 108.087(1) degrees; [Fe(OEP)(2-MeHIm)(NO)]ClO(4).0.5CH(2)Cl(2), triclinic, space group Ponemacr;, Z = 4, with a = 14.062(2) A, b = 16.175(3) A, c = 19.948(3) A, alpha = 69.427(3) degrees, beta = 71.504(3) degrees, gamma = 89.054(3) degrees.     

Two-step FRET as a structural tool

The power of FRET to study molecular complexes is expanded by the use of two or more donor/acceptor pairs. A general theoretical framework for distance measurements in three-chromophore systems is presented. Three energy transfer schemes applicable to many diverse situations are considered: (I) two-step FRET relay with FRET between the first and second chromophores and between the second and third, (II) FRET from a single donor to two different acceptors, and (III) two-step FRET relay with FRET also between the first and third chromophores. Equations for the efficiencies involving multiple energy transfer steps are derived for both donor quenching and sensitized emission measurements. The theory is supported by experimental data on model systems of known structure using steady-state donor quenching, lifetime quenching, and sensitized emission. The distances measured in the three-chromophore systems agree with those in two-chromophore systems and molecular models. Finally, labeling requirements for diagnosis of the energy transfer scheme and subsequent distance measurements are discussed.