CONFORMATIONAL CHANGES OF BOVINE LENS CRYSTALLINS IN A PHOTODYNAMIC SYSTEM

Abstract— Conformational changes of bovine lens crystallins in a photodynamic system generating singlet oxygen, have been investigated. The formation of intersubunit crosslinks was observed in all three classes (α-, β and γ-) of crystallins by irradiation in the presence of the photosensitizer methylene blue. Near-UV circular dichroism (CD) spectra of the crystallins were significantly altered by irradiation under these conditions, indicating changes in tertiary structure but the far-UV CD remained unchanged suggesting that the secondary structure ((β-sheet conformation) remains unchanged. Significant changes in the absorption and fluorescence spectra were also observed. Measurement of total sulfhydryl content showed a decrease of 27%, 50% and 37% for α-, β- and γ-crystallins respectively, after irradiation. Fluorescence lifetime measurements of N-iodoacetyl-N'-(5-sulfo-l-naphthyl)ethylenediamine-labeled crystallins showed a significant decrease of the lifetime of the major decay components of the label bound to sulfhydryl groups of α- and γ-crystallins, but showed no change in the microenvironment of the sulfhydryl groups of β-crystallin. The results are consistent with the microenvironments of the tryptophan and sulfhydryl groups predicted from sequence studies.     

Rapid and sensitive identification of epitope-containing peptides by direct matrix-assisted laser desorption/ionization tandem mass spectrometry of peptides affinity-bound to antibody beads

A method has been developed for rapid and sensitive identification of epitope-containing peptides, based on direct MALDI-MS/MS analysis of epitope-containing peptides affinity bound to affinity beads. This technique provides sequence information of the epitope that allows unambiguous identification of the epitope either by database searching or de novo sequencing. With MALDI-MS, affinity beads with bound peptides can be placed directly on the MALDI target and analyzed. Coupling a MALDI source to an orthogonal injection quadrupole time-of-flight (QqTOF) mass spectrometer allows direct sequencing of the bound peptides. In contrast to ESI-MS/MS, elution of the affinity-bound peptides followed by additional concentration and purification steps is not required, thus reducing the potential for sample loss. Direct mass spectrometric sequencing of affinity-bound peptides eliminates the need for chemical or enzymatic sequencing. Other advantages of this direct MALDI-MS/MS analysis of epitope-containing peptides bound to the affinity beads include its sensitivity (femtomole levels) and speed. In addition, direct analysis of peptides on affinity beads does not adversely affect the high mass accuracy of a QqTOF, and database searching can be performed on the MS/MS spectra obtained. In proof-of-principle experiments, this method has been demonstrated on beads containing immobilized antibodies against phosphotyrosine, the c-myc epitope tag, as well as immobilized avidin. Furthermore, de novo sequencing of epitope-containing peptides is demonstrated. The first application of this method was with anti-FLAG-tag affinity beads, where direct MALDI MS/MS was used to determine an unexpected enzymatic cleavage site on a growth factor protein.     

Three 4‐amino­quinolines of anti­malarial interest

The structures of three compounds with potential anti­malarial activity are reported. In N,N‐diethyl‐N′‐(7‐iodo­quinolin‐4‐yl)ethane‐1,2‐diamine, C₁₅H₂₀IN₃, (I), the mol­ecules are linked into ribbons by N—H⋯N and C—H⋯N hydrogen bonds. In N‐(7‐bromo­quinolin‐4‐yl)‐N′,N′‐diethyl­ethane‐1,2‐diamine dihydrate, C₁₅H₂₀BrN₃·2H₂O, (II), two amino­quino­line mol­ecules and four water mol­ecules form an R₅⁴(13) hydrogen‐bonded ring which links to its neighbours to form a T5(2) one‐dimensional infinite tape with pendant hydrogen bonds to the amino­quinolines. The phosphate salt 7‐chloro‐4‐[2‐(diethyl­ammonio)ethyl­amino]quinolinium bis­(dihydrogen­phosphate) phospho­ric acid, C₁₅H₂₂ClN₃²⁺·2H₂PO₄⁻·H₃PO₄, (III), was prepared in order to establish the protonation sites of these compounds. The phosphate ions form a two‐dimensional hydrogen‐bonded sheet, while the amino­quino­line cations are linked to the phosphates by N—H⋯O hydrogen bonds from each of their three N atoms. While the conformation of the quinoline region hardly varies between (I), (II) and (III), the amino side chain is much more flexible and adopts a significantly different conformation in each case. Aromatic π–π stacking inter­actions are the only supramolecular inter­actions seen in all three structures.     

Comparison of total folate concentrations in foods determined by microbiological assay at several experienced U.S. commercial laboratories

Analysis of total folate concentration measured by microbiological assay in a variety of foods submitted in a routine manner to experienced laboratories that regularly perform folate analysis on fee-for-service basis was evaluated. Homogenates of fresh strawberries, frozen spinach, orange juice, frozen meat and vegetable pizza, dry macaroni, and dried pinto beans were prepared and stored under conditions previously determined to maintain stability of folate content. An aliquot of each composite and of 3 certified reference materials were sent on each of 4 occasions to 4 laboratories. Results for macaroni and pizza, the only folic acid-fortified foods, had considerably lower between-laboratory variation (CV(B)) with CV(B) of 9-11% versus >45% for other foods. Mean total folate ranged from 14 to 279 microg/100 g for a mixed vegetable reference material, from 5 to 70 microg/100 g for strawberries, and from 28 to 81 microg/100 g for wholemeal flour. Only 1 laboratory reported using a tri-enzyme extraction, and all laboratories used folic acid fortified foods as internal control materials. Users of commercial total folate analysis should understand the uncertainty in values determined by microbiological assay, particularly for foods containing primarily naturally occurring folate, which may not be apparent when replicate samples are not submitted for analysis.     

Kinetic and equilibria studies of the aquation of the trinuclear platinum phase II anticancer agent [(trans-PtCl(NH(3))(2))(2)(mu-trans-Pt(NH(3))(2)(NH(2)(CH(2))(6)NH(2))(2))](4+) (BBR3464)

The hydrolysis profile of the bifunctional trinuclear phase II clinical agent [(trans-PtCl(NH(3))(2))(2)(mu-trans-Pt(NH(3))(2)(NH(2)(CH(2))(6)NH(2))(2))](4+) (BBR3464, 1) has been examined using [(1)H,(15)N] heteronuclear single quantum coherence (HSQC) 2D NMR spectroscopy. Reported are estimates of the rate and equilibrium constants for the first and second aquation steps, together with the acid dissociation constant (pK(a1) approximately equal to pK(a2) approximately equal to pK(a3)). The equilibrium constants for the aquation determined by NMR at 298 and 310 K (I = 0.1 M, pH 5.3) are similar, pK(1) = pK(2) = 3.35 +/- 0.04 and 3.42 +/- 0.04, respectively. At lower ionic strength (I = 0.015 M, pH 5.3) the values at 288, 293, and 298 K are pK(1) = pK(2) = 3.63 +/- 0.05. This indicates that the equilibrium is not strongly ionic strength or temperature dependent. The aquation and anation rate constants for the two-step aquation model at 298 K in 0.1 M NaClO(4) (pH 5.3) are k(1) = (7.1 +/- 0.2) x 10(-5) s(-1), k(-1) = 0.158 +/- 0.013 M(-1) s(-1), k(2) = (7.1 +/- 1.5) x 10(-5) s(-1), and k(-2) = 0.16 +/- 0.05 M(-1) s(-1). The rate constants in both directions increase 2-fold with an increase in temperature of 5 K, and rate constants increase with a decrease in solution ionic strength. A pK(a) value of 5.62 plus minus 0.04 was determined for the diaqua species [(trans-Pt(NH(3))(2)(OH(2)))(2)(mu-trans-Pt(NH(3))(2)(NH(2)(CH(2))(6)-NH(2))(2))](6+) (3). The speciation profile of 1 under physiological conditions is explored and suggests that the dichloro form predominates. The aquation of 1 in 15 mM phosphate was also examined. No slowing of the initial aquation was observed, but reversible reaction between aquated species and phosphate does occur.     

Tetramethylcyclopentadienylselenium derivatives

Bis(1,2,3,4-tetramethylcyclopentadienyl)selane (1) has been prepared by the reaction of tetramethylcyclopentadienyllithium (Cp(t)Li) with selenium bis(diethyldithiocarbamate). Treatment of Cp(t)Li with elemental selenium, followed by air oxidation, led to loss of the allylic hydrogen atom, and formation of the novel tricyclic compound 1,4,5,6,7,10,11,12-octamethyltricyclo[7.3.0.0]-2,8-diselenadodeca-3,5,9,11-tetraene (2). The sulfur analogue of 2 has been obtained by a similar procedure. The X-ray crystal structures of compounds 1 and 2 have been determined, and the molecular geometry observed for has been probed using DFT calculations.     

Influence of catalyst depletion or deactivation on polymerization kinetics. III. Solution polymerization of acrylonitrile in N,N-dimethylformamide at −30°C.

The experimental results on homogeneous polymerization of acrylonitrile initiated with the sodium triethylthioisopropoxyaluminate, NaAlEt₃S(i-Pr), catalyst in DMF at ?30°C. are compared with the prediction of equations based on a postulated mechanism. The agreement between the calculated and observed number-average molecular weight combined with the kinetic data and the relationship between the conversion and the initial catalyst concentration provides a rigorous test concerning the validity of the equations and the mechanism of the polymerization. A plausible mechanism is postulated as follows: The initiation must be relatively fast in accordance with the rate equations and the growing polymer undergoes propagation, transfer (to monomer), and deactivation simultaneously. The infrared spectrum of the low molecular weight polymer prepared at a high catalyst concentration showed strong absorption at 2337, 2205, and 1620 cm.^?1 but no absorption at 900 cm.^?1, indicating that there are two nitriles in the polymer, one of which is conjugated. The possibility of having ? CH?CH₂ groups in the polymer is ruled out by the absence of the band at 900 cm.^?1. In view of these facts, it is concluded that the polymer has a ? CH?CHCN endgroup resulting from the transfer reaction.     

Amidrazones. VII. Formation of s-triazines by thermolysis of N1-benzyl-substituted amidrazone ylides

The preparation of ylides of the general structure is described. Thermolysis of 14a (R = CH₃, R' = H, Ar = C₆H₅) gave dimethylamine and 2,4-dimethyl-6-phenyl-s-triazine. Thermolysis of ylides 14b (R = C₆H₅; R' = CH₃, Ar = C₆H₅) and 14c (R = C₆H₅, R' = CH₃, Ar = p-tolyl) gave dimethylamine, ArCH = NCH₃ and 1-methyl-2-Ar-4,6-diphenyl-1,2-dihydro-s-triazines ( 19a,b ). Triazines 19a and 19b were also prepared by condensation of N-methylbenzamidine with benzaldehyde and p-tolualdehyde, respectively. Thermolysis of 14d (R = C₆H₅, R¹ = CH₂C₆H₅,Ar = C₆H₅) gave 1-benzyl-2,4,6-triphenyl-1,2-dihydro-s-triazine ( 19c ) and N-benzylidenebenzylamine. Mechanistic aspects of these reactions are discussed.     

Complexation of Cu2+ by Binucleating Macrocycles Containing the N6 Donor Set

The hexadentate ligands 1,4,7,12,15,18-hexaazacyclododecosane ([2]aneN₆) and 1,4,7,14,17,20-hexaazacyclohexacosane ([26]aneN₆) both form eight complexes with Cu²⁺, three of them being binuclear. The corresponding stability constants have been determined potentiometrically, and the electronic absorption spectra have been obtained from spectrophotometric data. Possible interactions of the Cu²⁺ pairs in the three binuclear complexes have, in addition, been investigated by ESR and and linear -sweep voltammetry (LSV). The binuclear complex of [22]aneN₆ with one addditional OH group is exceptionally stable, ESR-silent, and the results of the LSV-experiments are characteristically different from those of the other binuclear complexes with both ligands. This indicates that [22]aneN₆ forms a very stable hydroxo-bridged binuclear Cu²⁺ complex Cu₂L(OH)³⁺, whereas in the case of [26]andN₆ no bridged Cu²⁺ pair exists.     

Synthesis and anti-cancer activity of dinuclear platinum(II) complexes containing bis(thioalkyl)dicarba-closo-dodecaborane(12) ligands

A series of novel, dinuclear (2,2':6',2'-terpyridine)platinum(ii) complexes containing bis(thioalkyl)-dicarba-closo-dodecaborane(12)(carborane) ligands were prepared and characterised, and their preliminary anti-cancer characteristics have been determined in vitro; the complexes are the first examples of bis-intercalator complexes containing a boron-rich carborane cage.