Mass spectrometry characterization of Escherichia coli K4 oligosaccharides from 2-mers to more than 20-mers

The separation and characterization of oligosaccharides obtained by hyaluronidase [E.C. 3.2.1.35] digestion of Escherichia coli K4 polysaccharide using online high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) are presented. Complete identification and structural information for oligosaccharides containing 2-24 monomers (from 2- to 24-mers) were obtained. In particular, smaller K4 species, from 2-mers to 4-mers, exhibited mainly [M-H](-1) anions, whereas the 6- to 8-mers existed predominantly at the charge state of -2. The K4 oligomers from 10-mers to 14-mers were mainly represented by [M-3H](-3) anions while species from 16- to 20-mers were characterized by a charge state of -4. K4 oligosaccharides from 22- to 24-mers existed as [M-4H](-4) and [M-5H](-5) anions and, for this latter species, ions having a charge state of -6 appeared. For smaller K4 species, in particular from 6-mers to 10-mers, ESI-MS revealed anions related to the loss of one monosaccharide unit from the oligomers due to apparent collisional activation and ion source fragmentation. However, no odd-numbered anions were produced for K4 2/4-mer species or for oligosaccharides greater than 12-mers, while for K4 species 8/10-mer, ESI-MS revealed odd-numbered anions generally in low relative abundance making the interpretation of the spectra easier. The ESI-MS spectra of oligosaccharides separated by online HPLC were applied to the evaluation of the K4 polymerization process, confirming that the addition of fructose units is not critical for chain elongation as variously fructosylated oligomer species were detected directly on the K4 carbohydrate backbone.     

Quantitative parameters of cooperative interactions of oligonucleotides within tandem complexes

Quantitative parameters of cooperative interactions of deoxyribooligonucleotides within perfect complementary complexes with a nick in one strand and imperfect complexes containing one mismatched base pair in the nick were obtained. One complementary strand was represented by 22-mer oligonucleotides, while the other, by two short 8-mer oligonucleotides forming a tandem complex with the central part of the 22-mer. In the tandem complexes, the 8-mers form contacts of the following types: 5"-Py*pPy-3", 5"-Pu*pPy-3", and 5"-Pu*pPu-3", where p is phosphate, Py and Pu are pyrimidine and purine nucleosides, respectively, and * stands for a nick. In each incompletely complementary complex, the mismatched base pair in the nick is formed by the 3"-end nucleoside of the 8-mer oligonucleotide and by the nucleoside located in the middle of the 22-mer oligonucleotide. The alkylating 4-[N-(2-chloroethyl)-N-methylamino]phenyl}methylamino group (RCl) is linked through the 5"-end phosphate of the 8-mers (reagents) close to 3"-ends of the 22-mers. The dependences of the limit extents of alkylation of 22-mers (targets) at zero and saturating concentrations of the neighbor oligonucleotides (effectors) on the initial concentration of RCl-derivatives of oligonucleotides (reagents) were used to calculate the association constants K _X of the reagent with the target. The ratio of these constants was used to determine the parameters of contact cooperativity , which characterize the interactions at the junction of two oligonucleotides within the tandem complexes.     

Affinity determination of ricinus communis agglutinin ligands identified from combinatorial O- and S-,N-glycopeptide libraries

Two combinatorial glycopeptide libraries were synthesized on solid support via the "split-and-mix" method combined with the ladder synthesis strategy. The O-glycopeptide library contained Gal(beta1-O)Thr, whereas the S-,N-glycopeptide library contained both Gal(beta1-S)Cys and Gal(beta1-N)Asn. In this model study, the two libraries were screened against the fluorescently labeled lectin Ricinus communis agglutinin (RCA120). The screening results showed that both O- and S- or S-,N-glycopeptides were recognized by the lectin with similar amino acid recognition patterns. Surface plasmon resonance interaction studies demonstrated that both the selected S- or S-,N-glycopeptide hits and the O-glycopeptides bound to the lectin with a similar affinity. Structure 19, containing two glycosylated cysteine residues, bound to the receptor with the highest affinity (KA = 3.81 x 10(4) M(-1)), which is comparable to N-acetyllactosamine. Competition assays, in which some selected glycopeptides and methyl beta-d-galactopyranoside competed for the binding site of immobilized RCA120, showed that the glycopeptide-lectin interaction was carbohydrate-specific. Incubation of the O- and S-,N-glycopeptides with beta-galactosidase demonstrated the complete stability of S-,N-glycopeptides toward enzymatic degradation, whereas O-glycopeptides were not completely stable.     

Colorimetric detection of Ricinus communis Agglutinin 120 using optimally presented carbohydrate-stabilised gold nanoparticles

Ricin is a toxic lectin which presents a potential security threat. Its rapid detection is highly desirable. Here we present a colorimetric bioassay based on the aggregation of carbohydrate-stabilised gold nanoparticles which has been used to detect Ricinus communis Agglutinin 120 (RCA(120)) - a ricin surrogate. To achieve a stable and robust sensing system the anchor chain length and the density of the assembled carbohydrates on the gold particle surface has been examined to determine the optimal coverage for maximal aggregation with both RCA(120) and Concanavalin A (Con A) lectins. Gold nanoparticles were stabilised with either a thiolated galactose derivative (9-mercapto-3,6-diaoxaoctyl-beta-d-galactoside) or a thiolated mannose derivative (9-merapto-3,6-dioxaoctyl-alpha-d-mannoside), for RCA(120) and Con A respectively, diluted in each instance with varying ratios of a thiolated triethylene glycol derivative. Aggregation was induced with the respective cognate lectin with the reaction monitored by UV-visible spectrophotometry. The results obtained show that a particle surface with at least 7.5% galactose is required for aggregation with RCA(120) and 6% mannose coverage is required for aggregation with Con A. For each lectin the sensitivity of the assay could be controlled by adjustment of the carbohydrate density on the gold nanoparticles, but with differing results. Maximal aggregation with Con A was achieved with a monolayer consisting of 100% mannose, whereas for RCA(120) maximal aggregation occurred with 70% coverage of galactose. The limit of detection for RCA(120) using the optimally presented galactose-stabilised nanoparticles was 9 nM.     

Quantitative and reversible lectin-induced association of gold nanoparticles modified with alpha-lactosyl-omega-mercapto-poly(ethylene glycol)

Gold nanoparticles (1-10 nm size range) were prepared with an appreciably narrow size distribution by in situ reduction of HAuCl(4) in the presence of heterobifunctional poly(ethylene glycol) (PEG) derivatives containing both mercapto and acetal groups (alpha-acetal-omega-mercapto-PEG). The alpha-acetal-PEG layers formed on gold nanoparticles impart appreciable stability to the nanoparticles in aqueous solutions with elevated ionic strength and also in serum-containing medium. The PEG acetal terminal group was converted to aldehyde by gentle acid treatment, followed by the reaction with p-aminophenyl-beta-D- lactopyranoside (Lac) in the presence of (CH(3))(2)NHBH(3). Lac-conjugated gold nanoparticles exhibited selective aggregation when exposed to Recinus communis agglutinin (RCA(120)), a bivalent lectin specifically recognizing the beta-D-galactose residue, inducing significant changes in the absorption spectrum with concomitant visible color change from pinkish-red to purple. Aggregation of the Lac-functionalized gold nanoparticles by the RCA(120) lectin was reversible, recovering the original dispersed phase and color by addition of excess galactose. Further, the degree of aggregation was proportional to lectin concentration, allowing the system to be utilized to quantitate lectin concentration with nearly the same sensitivity as ELISA. This simple, yet highly effective, derivatization of gold nanoparticles with heterobifunctional PEG provides a convenient method to construct various colloidal sensor systems currently applied in bioassays and biorecognition.     

Preparation of a novel aggregate like sugar-ball micelle composed of poly(methylglutamate) and poly(ethyleneglycol) modified by lactose and its molecular recognition by lectin

We report the preparation and characteristics of a novel micellar aggregate of an amphiphilic diblock copolymer, poly(methylglutamate) (PMG)-poly(ethyleneglycol) (PEG), whose terminus was modified by lactose lactone (LA). Due to the terminal LA moiety, this aggregate could be specifically recognized by RCA120 lectin. PMG-PEG-LA was synthesized by polymerizing the N-carboxy anhydride of L-glutamic acid gamma-methyl ester with H2N-PEG-LA as a polymerization initiator. By applying a fluorescence method using pyrene as a probe molecule, we found that PMG-PEG-LA could form the aggregate in aqueous solution. Fluorescence measurements showed that the critical aggregation concentration (C.A.C.) was 1.1 x 10(-5) M. The average diameter of the aggregate was 220 nm at 25 degrees C, as determined by the dynamic light scattering method. Circular dichroism measurements for the aggregate solution showed that the PMG residue took an alpha-helical structure, and that they associated to constitute the hydrophobic core of the aggregate. By adding RCA120 lectin to the aggregate solution, the turbidity of the solution increased rapidly, due to association of the aggregates. This implies that the aggregate could be recognized by lectin, and also suggests that sugar residues locate at the surface of the aggregates. From these findings, we concluded that the PMG-PEG-LA molecules form an aggregate like a "sugar ball" micelle, whose surface is covered by the sugar moieties. Application of the present aggregate system as a drug carrier is briefly discussed.     

Monitoring oligomer formation from self-aggregating amylin peptides using ESI-IMS-MS

Amyloid diseases are a serious cause for concern world-wide. To understand the mechanism of formation of the fibrillar structures associated with such disorders, it is necessary to study the progression from soluble protein or peptide monomer through an array of oligomers to the final, insoluble, fibrils. The protein IAPP is found in vivo in the form of insoluble amyloid deposits in the pancreatic islets of diabetes type II sufferers. Here, we have studied the in vitro self-aggregation of three fibril-forming peptides from the amyloidogenic core of IAPP. Using electrospray ionization—mass spectrometry coupled with ion mobility spectrometry, the mass and cross-sectional area of each oligomer present in the heterogeneous assembly mixtures can be determined individually in a single, rapid experiment over time. For the three peptides studied, oligomers ≤20-mer were characterized. Conversely, no oligomers higher than a dimer were detected for a non-assembling peptide control. The rate in which the cross-sectional area of the oligomers increases with increasing number of peptide sub-units indicates that assembly for the amyloid-forming peptides proceeds in a linear fashion until an oligomer of a certain size is attained. After this, a step increase in cross-sectional area occurs for the next higher-order oligomer. This behaviour can be explained by molecular modelling of singly, doubly, triply and quadruply stacked β-stranded structures. Using one peptide as an example, the cross-sectional areas of the lower order oligomers (dimer to pentamer) were found to be consistent with a single β-sheet model, whereas the higher order oligomers were consistent with double-stranded (hexamer to decamer oligomers), triply-stranded (11-mers to 15-mers) and quadruply-stranded (16-mers to 20-mers) β-sheet models.     

Binding of Ricinus communis agglutinin to a galactose-carrying polymer brush on a colloidal gold monolayer

A polymer with many pendent galactose residues was prepared by atom-transfer radical polymerization (ATRP) of galactose-carrying vinyl monomer, 2-lactobionamidoethyl methacrylate (LAMA), with a disulfide-carrying ATRP initiator, 2-(2'-bromoisobutyroyl)ethyl disulfide (DT-Br). The galactose-carrying polymer obtained (DT-PLAMA) was accumulated as a polymer brush via Au-S bond on a colloidal gold monolayer deposited on a cover glass. For comparison, a disulfide which carried one galactose residue at both ends (2-lactobionamidoethyl disulfide, Cys-Lac) was accumulated as a self-assembled monolayer (SAM) on the colloidal gold monolayer, too. The association and dissociation processes of galactose residues on the colloidal gold with a lectin, Ricinus communis agglutinin (RCA(120)), were observed by the increase and decrease in absorbance at 550nm corresponding to localized surface plasmon resonance (LSPR) phenomena. The Cys-Lac SAM-carrying glass chip showed a strong non-specific adsorption of the lectin, whereas the DT-PLAMA brush-carrying one reversibly associated with the lectin, indicating reusability of the latter device. The apparent association constant of the lectin with the galactose residues in the DT-PLAMA brush was much larger than the association constant for free galactose, and the detection limit of RCA(120) by the glycopolymer brush-modified device was satisfactorily low. Furthermore, a microscopic observation clearly indicated that the DT-PLAMA brush could reversibly associate with a HepG2 cell having galactose receptors, though these processes could not be observed spectrophotometrically due to a gigantic size of the cell.     

Synthesis and hybridization properties of oligonucleotides containing polyamines at the C-2 position of purines: a pre-synthetic approach for the incorporation of spermine into oligodeoxynucleotides containing 2-(4,9,13-triazatridecyl)-2'-deoxyguanosine

We have developed a synthesis of spermine-containing oligonucleotides (ODN-sper) which allows incorporation of multiple polyamine residues. This approach was based on the pertrifluoroacetylated 5'DMT-dGsper phosphoramidite synthon. Its coupling yield with resin-bound ODN decreased dramatically when close to the 3'-end. Optimization of the coupling conditions allowed 22-mer ODNs containing up to six spermine residues to be synthesized. Several ODNs of different sequences with 1-4 pendent spermines could be purified and their hybridization properties were evaluated. Duplex melting temperatures increased linearly with the number of polyamine residues (deltaTm/sper = 3.0 +/- 0.2 degrees C in 100mM NaCl). This compares very favorably with values reported for duplexes of similar initial stability containing other cation-substituted bases. Moreover, the stability increase was neither sequence nor position-dependent, and even contiguous spermine residues did not cross-talk. Extrapolation based on these findings leads to the conclusion that a duplex formed with a 22-mer oligonucleotide containing seven spermine residues would be as stable as genomic DNA, which highlights its potential for DNA strand invasion.     

Carbohydrate-protein interactions at interfaces: comparison of the binding of Ricinus communis lectin to two series of synthetic glycolipids using surface plasmon resonance studies

Two C-lactosyl lipids and the related C-galactosyl lipids have been synthesised and their binding to RCA120 plant lectin was compared with a second series of thiolactosylethoxyalkanes. The interactions were measured quantitatively in real time by surface plasmon resonance (BIAcore) at a range of concentrations and temperatures from 5 to 30 degrees C. The C-galactosyl lipid (1,3-dimethyl-5-[beta-D-galactopyranosyl]-5-(4-octadecyloxybenzyl)pyrimidine-2,4,6-trione) bound much more weakly with a K(A) = 8.86 x 10(5) than the corresponding C-lactosyl lipid (1,3-dimethyl-5-[beta-D-galactopyranosyl-(1 --> 4)-beta-D-glucopyranosyl]-5-(4-octadecyloxybenzyl)pyrimidine-2,4,6-trione) (K(A) = 2.31 x 10(7)). The influence of the linker region of the two different series of lactosyl lipids was clearly demonstrated by the differences in the binding to RCA120 lectin. The changes in kinetic values and in the enthalpic and entropic contribution to the free energy of binding reflected the importance of the linker and the hydrocarbon anchor holding the synthetic glycolipids in the neomembrane.     

Dynamic light scattering as an efficient tool to study glyconanoparticle-lectin interactions

Glyconanomaterials, an emerging class of bio-functional nanomaterials, have shown promise in detecting, imaging and targeting proteins, bacteria, and cells. In this article, we report that dynamic light scattering (DLS) can be used as an efficient tool to study glyconanoparticle (GNP)--lectin interactions. Silica and Au nanoparticles (NPs) conjugated with D-mannose (Man) and D-galactose (Gal) were treated with the lectins Concanavalin A (Con A) and Ricinus communis agglutinin (RCA(120)), and the hydrodynamic volumes of the resulting aggregates were measured by DLS. The results showed that the particle size grew with increasing lectin concentration. The limit of detection (LOD) was determined to be 2.9 nM for Con A with Man-conjugated and 6.6 nM for RCA(120) with Gal-conjugated silica NPs (35 nm), respectively. The binding affinity was also determined by DLS and the results showed 3-4 orders of magnitude higher affinity of GNPs than the free ligands with lectins. The assay sensitivity and affinity were particle size dependent and decreased with increasing particle diameter. Because the method relies on the particle size growth, it is therefore general and can be applied to nanomaterials of different compositions.     

Carbohydrate-protein interactions at interfaces: synthesis of thiolactosyl glycolipids and design of a working model for surface plasmon resonance

Thiolactosyl lipids designed for carbohydrate-protein binding studies have been synthesised. One representative was selected for binding studies with a plant lectin RCA120, the agglutinin from Ricinus communis. The interactions were measured quantitatively in real time using a BIAcore surface plasmon resonance instrument. Removal of much of the galactose from the thiolactosyl lipid in situ with beta-galactosidase showed that the lectin binding was highly specific. A dissociation constant KD = 8.77 x 10(-8) M was measured for 1-[2-[2-(2-[beta-D-galactopyranosyl-(1-->4)-1-thio-beta-D -glucopyranosyl]ethoxy)ethoxy]ethoxy]octadecane 30 which is four orders of magnitude greater than that determined for binding to lactose in solution. A concentration of lactose of > 80 mM was required to block the lectin binding to thiolactosyl lipid in a neomembrane.     

Microanalysis of oligodeoxynucleotides by cathodic stripping voltammetry at amalgam-alloy surfaces in the presence of copper ions

The application of gold amalgam-alloy electrode (AuAE) for a sensitive voltammetric detection of different oligodeoxynucleotides (ODNs) containing the purine units within the ODN-chains in the presence of copper is described. The detection of ODNs is based on the following procedure: (i) the first step includes an acidic hydrolysis of the ODN (ahODN) samples performing the release of the purine bases from ODN-chain; (ii) the second step includes an electrochemical accumulation of the complex of the purine base residues released from ODN-chain with copper ions Cu(I) (ahODN-Cu(I) complex) at the potential of reduction of copper ions Cu(II) on the amalgam-alloy electrode surfaces; (iii) finally followed the cathodic stripping of the electrochemically accumulated ahODN-Cu(I) complex from the electrode surface. The proposed electrochemical method was used for: (a) detection of different ODN lengths containing only adenine units (the number of adenine units within the ODN-chains was changed from 10 to 80), and (b) determination of the number of purine units within the 30-mer ODNs containing a random sequence segments involving both the purine and pyrimidine units. The intensity of the cathodic stripping current density peak (j_CSP) of the electrochemically accumulated ahODN-Cu(I) complex increased linearly with the increasing number of purine units within the ODN-chains. We observed a good correlation between the percentage content of purine units to the whole length of different 30-mer ODNs and the percentage content of the intensity of the j_CSP of the electrochemically accumulated 30-mer ahODN-Cu(I) complexes. The detection of acid hydrolysed 80-mer (A₈₀) in the bulk solution and in a 20-μl volume is possible down to 200 pM and 2 nM at the AuAE, respectively. For the shortest 10-mer (A₁₀) a detectable value of 5 nM in the bulk solution on the AuAE was observed. The sensitive detection of different ODNs containing the purine units in their chains in the presence of copper can be also performed at the platinum amalgam-alloy (PtAE) and copper amalgam-alloy (CuAE) contrary to a lower sensitivity at the silver amalgam-alloy (AgAE) electrode.     

Molecularly defined caprolactone oligomers and polymers: synthesis and characterization

The synthesis of molecularly defined epsilon-caprolactone oligomers and polymers up to the 64-mer, via an exponential growth strategy, is described. By careful selection of orthogonal protecting groups, t-butyldimethylsilyl (TBDMS) ether for the hydroxyl group and benzyl (Bn) ester for the carboxylic acid group, a highly efficient synthetic strategy was developed with yields for both deprotection steps being essentially quantitative and for the coupling reactions using 1,3-dicyclohexylcarbodiimide (DCC), yields of 80-95% were obtained even at high molecular weights. This allows monodisperse dimers, tetramers, octamers, 16-mers, 32-mers and 64-mers to be prepared in gram quantities and fully characterized using mass spectroscopy, size exclusion chromatography (SEC), and IR and NMR spectroscopy. Thermal and physical properties were measured using thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), atomic force microscopy (AFM), and small-angle X-ray scattering (SAXS). These results conclusively show a distinct structure/property relationship with a close correlation between the number of repeat units and physical properties. In addition, a number of marked differences were observed on comparison with the parent poly(caprolactone) polymer.     

Specific and nonspecific metal ion-nucleotide interactions at aqueous/solid interfaces functionalized with adenine, thymine, guanine, and cytosine oligomers

This article reports nonlinear optical measurements that quantify, for the first time directly and without labels, how many Mg(2+) cations are bound to DNA 21-mers covalently linked to fused silica/water interfaces maintained at pH 7 and 10 mM NaCl, and what the thermodynamics are of these interactions. The overall interaction of Mg(2+) with adenine, thymine, guanine, and cytosine is found to involve -10.0 ± 0.3, -11.2 ± 0.3, -14.0 ± 0.4, and -14.9 ± 0.4 kJ/mol, and nonspecific interactions with the phosphate and sugar backbone are found to contribute -21.0 ± 0.6 kJ/mol for each Mg(2+) ion bound. The specific and nonspecific contributions to the interaction energy of Mg(2+) with oligonucleotide single strands is found to be additive, which suggests that within the uncertainty of these surface-specific experiments, the Mg(2+) ions are evenly distributed over the oligomers and not isolated to the most strongly binding nucleobase. The nucleobases adenine and thymine are found to bind only three Mg(2+) ions per 21-mer oligonucleotide, while the bases cytosine and guanine are found to bind eleven Mg(2+) ions per 21-mer oligonucleotide.     

Group epitope mapping by saturation transfer difference NMR to identify segments of a ligand in direct contact with a protein receptor.

A protocol based on saturation transfer difference (STD) NMR spectra was developed to characterize the binding interactions at an atom level, termed group epitope mapping (GEM). As an example we chose the well-studied system of galactose binding to the 120-kDa lectin Ricinus communis agglutinin I (RCA(120)). As ligands we used methyl beta-D-galactoside and a biantennary decasaccharide. Analysis of the saturation transfer effects of methyl beta-D-galactoside showed that the H2, H3, and H4 protons are saturated to the highest degree, giving evidence of their close proximity to protons of the RCA(120) lectin. The direct interaction of the lectin with this region of the galactose is in excellent agreement with results obtained from the analysis of the binding specificities of many chemically modified galactose derivatives (Bhattacharyya, L.; Brewer, C. F. Eur. J. Biochem. 1988, 176, 207-212). This new NMR technique can identify the binding epitope of even complex ligands very quickly, which is a great improvement over time-consuming chemical modifications. Efficient GEM benefits from a relatively high off rate of the ligand and a large excess of the ligand over the receptor. Even for a ligand like the biantennary decasaccharide with micromolar binding affinity, the binding epitopes could easily be mapped to the terminal beta-D-Gal-(1-4)-beta-D-GlcNAc (beta-D-GlcNAc = N-acetyl-D-glucosamine) residues located at the nonreducing end of the two carbohydrate chains. The binding contribution of the terminal galactose residue is stronger than those of the penultimate GlcNAc residues. We could show that the GlcNAc residues bind "edge-on" with the region from H2 to H4, making contact with the protein. Analysis of STD NMR experiments performed under competitive conditions proved that the two saccharides studied bind at the same receptor site, thereby ruling out unspecific binding.     

Symmetry-adapted-cluster/symmetry-adapted-cluster configuration interaction methodology extended to giant molecular systems: ring molecular crystals

The symmetry adapted cluster (SAC)/symmetry adapted cluster configuration interaction (SAC-CI) methodology for the ground, excited, ionized, and electron-attached states of molecules was extended to giant molecular systems. The size extensivity of energy and the size intensivity of excitation energy are very important for doing quantitative chemical studies of giant molecular systems and are designed to be satisfied in the present giant SAC/SAC-CI method. The first extension was made to giant molecular crystals composed of the same molecular species. The reference wave function was defined by introducing monomer-localized canonical molecular orbitals (ml-CMO's), which were obtained from the Hartree-Fock orbitals of a tetramer or a larger oligomer within the electrostatic field of the other part of the crystal. In the SAC/SAC-CI calculations, all the necessary integrals were obtained after the integral transformation with the ml-CMO's of the neighboring dimer. Only singles and doubles excitations within each neighboring dimer were considered as linked operators, and perturbation selection was done to choose only important operators. Almost all the important unlinked terms generated from the selected linked operators were included: the unlinked terms are important for keeping size extensivity and size intensivity. Some test calculations were carried out for the ring crystals of up to 10 000-mer, confirming the size extensivity and size intensivity of the calculated results and the efficiency of the giant method in comparison with the standard method available in GAUSSIAN 03. Then, the method was applied to the ring crystals of ethylene and water 50-mers, and formaldehyde 50-, 100-, and 500-mers. The potential energy curves of the ground state and the polarization and electron-transfer-type excited states were calculated for the intermonomer distances of 2.8-100 A. Several interesting behaviors were reported, showing the potentiality of the present giant SAC/SAC-CI method for molecular engineering.     

Cyclic and hairpin peptide complexes of heme

We have synthesized and characterized a new class of heme-peptide complexes using disulfide-linked hairpin-turn and cyclic peptides and compared these to their linear analogues. The binding affinities, helicities, and mechanism of binding of linear, hairpin, and cyclic peptides to [FeIII(coproporphyrin-I)]+ have been determined. In a minimalist approach, we utilize amphiphilic peptide sequences (15-mers), where a central histidine provides heme ligation, and the hydrophobic effect is used to optimize heme-peptide complex stability. We have incorporated disulfide bridges between amphiphilic peptides to make hairpin and even cyclic peptides that bind heme extremely well, roughly 5 x 106 times more strongly than histidine itself. CD studies show that the cyclic peptide heme complexes are completely alpha-helical. NMR spectra of paramagnetic complexes of the peptides show that the 15-mer peptides bind sequentially, with an observable monopeptide, high-spin intermediate. In contrast, the cyclic peptide complexes ligate both imidazoles cooperatively to the heme, producing only a low-spin complex. Electrochemical measurements of the E1/2 of the FeIII(coproporphyrin-I)+ complexes of these peptides are all at fairly low potentials, ranging from -215 to -252 mV versus NHE at pH 7.     

Dipyrido[3,2-a:2′,3′-c]phenazine-Tethered Oligo-DNA: Synthesis and Thermal Stability of Their DNA⋅DNA and DNA⋅RNA Duplexes and DNA⋅DNA⋅DNA Triplexes

Dipyrido[3,2-a:2′,3′-c]phenazine (dppz) derivatives were conjugated to 9-mer and 18-mer DNA (ODN) at a site without nucleobase, either at the 5′- or 3′-end or at a internucleotide position, via linkers of 7, 12, or 18 atoms lengths. These dppz-linked ODNs were synthesized using novel backbone glycerol phosphoramidites: Glycerol, serving as artificial nucleoside without nucleobase, was modified to amines 10 , 23 , and 24 , which were suitable for the subsequent key reaction with dppz-carboxylic acid 3 (Schemes 2 and 3). The products of these reactions (see 5 – 7 ) were then transformed to the standard phosphoramidite derivatives (see 27 , 29 , and 30 ) or used for loading on a CPG support (see 28 , 31 , and 32 ). The dppz-modified ODNs were subsequently assembled in the usual manner using automated solid-phase DNA synthesis. The 9-mer ODN-dppz conjugates 35 – 43 were tested for their ability to form stable duplexes with target DNA or RNA strands (D11 ( 60 ) or R11 ( 61 )), while the 18-mer ODN-dppz conjugates 48 – 56 were tested for their ability to form stable triplexes with a DNA target duplex D24⋅D24 ( 62 ) (see Tables 1 and 2). The presence of the conjugated dppz derivative increases the stability of DNA⋅DNA and DNA⋅RNA duplexes, typically by a ΔT_m of 7.3 – 10.9° and 4.5 – 7.4°, respectively, when the dppz is tethered at the 5′- or 3′-terminal (Table 2). The dppz derivatives also stabilize triplexes when attached to the 5′- or 3′-end, with a ΔT_m varying from 3.8 – 11.1° (Table 3). The insertion of a dppz building block at the center of a 9-mer results in a considerably poorer stability of the corresponding DNA⋅DNA duplexes (ΔT_m=0.5 to 4.2°) and DNA⋅RNA duplexes (ΔT_m=−1.5 to 0.9°), while the replacement of one interior nucleotide by a dppz building unit in the corresponding 8-mer ODN does not reveal the formation of any duplex at all. Different types of modifications in the middle of the 18-mer ODN, in general, do not lead to any triplex formation, except when the dppz derivative is tethered to the ODN through a 12-atom-long linker (Entry 9 in Table 3).