Walter P. Niemczura

Inhibitors of Ornithine Carbamoyltransferase from Pseudomonas syringae pv. phaseolicola. st]Revised Structure of Phaseolotoxin

Abstract The structure of phaseolotoxin, a toxin produced by cultured Pseudomonas syringae pv. phaseolicola , the causal agent of halo blight disease in bean plants is revised to 3. The structure of octicidin, isolated from leaves of bean plants infected with pv. phaseolicola . has been determined to be 4, a protease degradation product of 3 which is formed in the plant.

Cooperativity between the Hydrophobic and Cross-linking Domains of Elastin

The principal protein component of the elastic fiber found in elastic tissues is elastin, an amorphous, cross-linked biopolymer that is assembled from a high molecular weight monomer. The hydrophobic and cross-linking domains of elastin have been considered separate and independent, such that changes to one region are not thought to affect the other. However, results from these solid-state 13C NMR experiments demonstrate that cooperativity in protein folding exists between the two domain types. The sequence of the EP20-24-24 polypeptide has three hydrophobic sequences from exons 20 and 24 of the soluble monomer tropoelastin, interspersed with cross-linking domains constructed from exons 21 and 23. In the middle of each cross-linking domain is a “hinge” sequence. When this pentapeptide is replaced with alanines, as in EP20-24-24[23U], its properties are changed. In addition to the expected increase in α-helical content and the resulting increase in rigidity of the cross-linking domains, changes to the organization of the hydrophobic regions are also observed. Using one-dimensional CPMAS (cross-polarization with magic angle spinning) techniques, including spectral editing and relaxation measurements, evidence for a change in dynamics to both domain types is observed. Furthermore, it is likely that the methyl groups of the leucines of the hydrophobic domains are also affected by the substitution to the hinge region of the cross-linking sequences. This cooperativity between the two domain types brings new questions to the phenomenon of coacervation in elastin polypeptides and strongly suggests that functional models for the protein must include a role for the cross-linking regions.

Sucrose-based epoxy monomers and their reactions with diethylenetriamine

Two sets of sucrose-based epoxy monomers, namely, epoxy allyl sucroses (EAS), and epoxy crotyl sucroses (ECS), were prepared by epoxidation of octa-O-allyl and octa-O-crotyl sucroses (OAS and OCS, respectively). Synthetic and structural characterization studies showed that the new epoxy monomers were mixtures of structural isomers and diastereoisomers that contained varying numbers of epoxy groups per sucrose. EAS and ECS can be tailored to contain an average of one to eight epoxy groups per sucrose. Quantitative 13C-NMR spectrometry and titrimetry were used independently to confirm the average number of epoxy groups per sucrose. Sucrose-based epoxy monomers were cured with diethylenetriamine (DETA) in a differential scanning calorimeter (DSC), and their curing characteristics were compared with those of diglycidyl ether of bisphenol A (DGEBA) and diepoxycrotyl ether of bisphenol A (DECEBA). EAS and DGEBA cured at 100 to 125°C and exhibited a heat of cure of about 108.8 kJ per mol epoxy. ECS and DECEBA cured at 150 and 171°C, respectively, and exhibited a heat of cure of about 83.7 kJ per mol epoxy. Depending upon the degree of epoxidation (average number of epoxy groups per sucrose) and the concentration of DETA, glass transition temperatures (Tgs) of cured EAS varied from −17 to 72°C. DETA-cured ECS containing an average of 7.3 epoxy groups per sucrose (ECS-7.3) showed no DSC glass transition between −140 and 220°C when the ratio of amine (NH) to epoxy group was 1:1 and 1.5:1. Maximum Tgs obtained for DETA-cured DGEBA and DECEBA polymers were 134 and 106°C, respectively. DETA-cured bisphenol A-based epoxy polymers degraded at about 340°C, as observed by thermogravimetric analysis (TGA). DETA-cured sucrose-based epoxy polymers degraded at about 320°C. Sucrose-based epoxies cured with DETA were found to bind aluminum, glass, and steel. Comparative lap shear tests (ASTM D1002–94) showed that DETA-cured epoxy allyl sucroses with an average of 3.2 epoxy groups per sucrose (EAS-3.2) generated a flexible adhesive comparable in bond strength to DGEBA. However, DETA-cured ECS-7.3 outperformed the bonding characteristics of both DGEBA and EAS-3.2. All sucrose-based epoxy polymers were crosslinked and insoluble in water, N,N-dimethylformamide, tetrahydrofuran, acetone, and dichloromethane.

The physical and NMR characterizations of allyl‐ and crotylcelluloses

Tri-O-allylcellulose (degree of polymerization, DP ∼112) was prepared in ∼91% yield, and tri-O-crotylcellulose (DP ∼138) was prepared in ∼56% yield from microcrystalline cellulose (DP ∼172, and polydispersity index, PDI ∼1.95) using modified literature methods. Number-average molecular weight (Mn = 31,600), weight-average molecular weight (Mw = 191,800), and PDI = 6.07 data suggested that tri-O-allylcellulose may be crosslinking in air to generate branched chains. The polymer was stabilized with 100 ppm butylated hydroxy toluene (BHT). The material without BHT experienced glass transition (Tg, differential-scanning calorimetry, DSC) between −2 and +3 °C, crosslinked beyond 100 °C, and degraded at 298.6 °C (by thermogravimetric analysis, TGA). Mn (45,100), Mw (118,200), PDI (2.62), and thermal data (Tg − 5 to +3 °C, melting point 185.8 °C, recrystallization 168.9 °C, and degradation 343.6 °C) on tri-O-crotylcellulose suggested that the polymer was formed with about the same polydispersity as the starting material and is heat stable. While allylcellulose generated continuous flexible yellow films by solution casting, crotylcellulose precipitated from solution as brittle white flakes. Dynamic mechanical analysis (DMA) data on allylcellulose films (Tg − 29.1 °C, Youngs modulus 5.81 × 108 Pa) suggest that the material is tough and flexible at room temperature. All 1H and 13C resonances in the NMR spectra were identified and assigned using the following methods: Double-quantum filter correlation spectroscopy (DQF COSY) was used to assign the network of seven protons in the anhydroglucose portion of the repeat unit. The proton assignments were verified and confirmed by total correlation spectroscopy (TOCSY). A combination of heteronuclear single-quantum coherence (HSQC) and 13C spectroscopies were used to identify all bonded carbon–hydrogen pairs in the anhydroglucose portion of the repeat unit, and assign the carbon nuclei chemical shift values. Heteronuclear multiple bond correlation (HMBC) spectroscopy was used to connect the resonances of methines and methylenes at positions 2, 3, and 6 to the methylene resonances of the allyl ethers. TOCSY was used again to identify the fifteen 1H resonances in the three pendant allyl groups. Finally, a combination of HSQC, HMBC, and 13C spectroscopies were used to identify each carbon in the allyl pendants at 2, 3, and 6. Because of line broadening and signal overlap, we were unable to identify the conformational arrangement about the C5 and C6 bond in tri-O-allyl- and tri-O-crotylcelluloses.

Characterization of tri-o-methylcellulose by one- and two-dimensional NMR methods†

Tri-O-methylcellulose was prepared from partially O-methylated cellulose and its chemical shifts ( 1 H and 13 C), and proton coupling constants were assigned using the following NMR methods: (1) One-dimensional 1 H and 13 C spectra of the title compound were used to assign functional groups and to compare with literature data; (2) double quantum filtered proton-proton correlation spectroscopy ( 1 H, 1 H DQF-COSY) was used to assign the chemical shifts of the network of 7 protons in the anhydroglucose portion of the repeat unit; (3) the heteronuclear single-quantum coherence (HSQC) spectrum was used to establish connectivities between the bonded protons and carbons; (4) the heteronuclear multiple-bond correlation (HMBC) spectrum was used to connect the hydrogens of the methyl ethers to their respective sugar carbons; (5) the combination of HSQC and HMBC spectra was used to assign the 13 C shifts of the methyl ethers; (6) all spectra were used in combination to verify the assigned chemical shifts; (7) first-order proton coupling constants data (J H,H in Hz) were obtained from the resolution-enhanced proton spectra. The NMR spectra of tri-O-methylcellulose and other cellulose ethers do not resemble the spectra of similarly substituted cellobioses. Although the 1 H and 13 C shifts and coupling constants of 2,3,6-tri-O-methylcellulose closely resemble those of methyl tetra-O-methyl-β-D-glucoside, there are differences with regard to the chemical shifts and the order of appearances of the resonating nuclei of the methyl ether appendages and the proton at position 4 in the pyranose ring. H4 in tri-O-methylcellulose is deshielded by the acetal system comprising the β-1→4 linkage, and it resonates downfield. H4 in the permethylated glucoside is not as deshielded by the equitorial O-methyl group at C4, and it resonates upfield. The order of appearance of the 1 H and 13 C resonances in the spectra of the tri-O-methylcellulose repeat unit (from upfield to downfield) are H2 < H3 < H5 < H6a < H3a < H2a < pro R H6B < H4 < pro S H6A « H1 and C6a < C3a < C2a < C6 < C5 < C4 < C2 < C3 « C1, respectively. Close examination of the pyranose ring coupling constants of the repeat unit in tri-O-methylcellulose supports the 4 C 1 arrangement of the glucopyranose ring. Examination of the proton coupling constants about the C5-C6 bond (J 5,6A and J 5,6B ) in the nuclear Overhauser effect difference spectra revealed that the C6 O-methyl group is predominantly in the gauche gauche conformation about the C5-C6 bond for the polymer in solution.

Structural insights into the elastin mimetic (LGGVG)6 using solid-state 13C NMR experiments and statistical analysis of the PDB.

Elastin is a crosslinked hydrophobic protein found in abundance in vertebrate tissue and is the source of elasticity in connective tissues and blood vessels. The repeating polypeptide sequences found in the hydrophobic domains of elastin have been the focus of many studies that attempt to understand the function of the native protein on a molecular scale. In this study, the central residues of the (LGGVG)(6) elastin mimetic are targeted. Using a combination of a statistical analysis based on structures in the Brookhaven Protein Data Bank (PDB), 1D cross-polarization magic-angle-spinning (CPMAS) NMR spectroscopy, and 2D off-magic-angle-spinning (OMAS) spin-diffusion experiments, it is determined that none of the residues are found in a singular regular, highly ordered structure. Instead, like the poly(VPGVG) elastin mimetics, there are multiple conformations and significant disorder. Furthermore, the conformational ensembles are not reflective of proteins generally, as in the PDB, suggesting that the structure distributions in elastin mimetics are unique to these peptides and are a salient feature of the functional model of the native protein.

Selection of side-chain carbons in a high-molecular-weight, hydrophobic peptide using solid-state spectral editing methods

Solid-state spectral editing techniques have been used by others to simplify 13C CPMAS spectra of small organic molecules, synthetic organic polymers, and coals. One approach utilizes experiments such as cross-polarization-with-polarization-inversion and cross-polarization-with-depolarization to generate subspectra. This work shows that this particular methodology is also applicable to natural-abundance 13C CPMAS NMR studies of high-molecular-weight biopolymers. The editing experiments are demonstrated first with model peptides and then with α-elastin, a high-molecular-weight peptidyl preparation obtained from the elastic fibers in mammalian tissue. The latter has a predominance of small, nonpolar residues, which is evident in the crowded aliphatic region of typical 13C CPMAS spectra. Spectral editing is particularly useful for simplifying the aliphatic region of the NMR spectrum of this elastin preparation.Solid-state spectral editing techniques have been used by others to simplify 13C CPMAS spectra of small organic molecules, synthetic organic polymers, and coals. One approach utilizes experiments such as cross-polarization-with-polarization-inversion and cross-polarization-with-depolarization to generate subspectra. This work shows that this particular methodology is also applicable to natural-abundance 13C CPMAS NMR studies of high-molecular-weight biopolymers. The editing experiments are demonstrated first with model peptides and then with α-elastin, a high-molecular-weight peptidyl preparation obtained from the elastic fibers in mammalian tissue. The latter has a predominance of small, nonpolar residues, which is evident in the crowded aliphatic region of typical 13C CPMAS spectra. Spectral editing is particularly useful for simplifying the aliphatic region of the NMR spectrum of this elastin preparation.

Resolving Nitrogen-15 and Proton Chemical Shifts for Mobile Segments of Elastin with Two-dimensional NMR Spectroscopy

Background: Little is known about the structure of elastin, the abundant elastomeric protein in vertebrate tissue. Results: Observed chemical shifts do not match predictions for random coil, helix, or sheet. Conclusion: The structural makeup of elastin is heterogeneous and possibly unique in nature. Significance: The chemical shift index needs refinement for structural studies of Gly-rich proteins. In this study, one- and two-dimensional NMR experiments are applied to uniformly 15N-enriched synthetic elastin, a recombinant human tropoelastin that has been cross-linked to form an elastic hydrogel. Hydrated elastin is characterized by large segments that undergo “liquid-like” motions that limit the efficiency of cross-polarization. The refocused insensitive nuclei enhanced by polarization transfer experiment is used to target these extensive, mobile regions of this protein. Numerous peaks are detected in the backbone amide region of the protein, and their chemical shifts indicate the completely unstructured, “random coil” model for elastin is unlikely. Instead, more evidence is gathered that supports a characteristic ensemble of conformations in this rubber-like protein.

Regioselective synthesis and characterization of naphthylethylcarbamoyl-β-cyclodextrins

Regioselective reactions of 1-(1-naphthyl)ethyl isocyanate (NEIC) with beta-cyclodextrin (beta-CD) were studied with and without NaH activation of beta-CD in N,N-dimethylformamide (DMF) and pyridine. All six possible monosubstituted CD products were separated and characterized by proton NMR. Primary substitution product predominates when the reaction was carried out under reflux condition in pyridine without NaH activation. The C-2 substitution product predominates when the reaction was carried out in DMF. Conversion of 2-O-(1-(1-naphthyl)ethylcarbamoyl)-beta-CD to 6-O-(1-(1-naphthyl)ethylcarbamoyl)-beta-CD was observed when NaH was used to activate hydroxyl groups of CD.

Backbone motion in elastin's hydrophobic domains as detected by 2H NMR spectroscopy†

The elasticity of vertebrate tissue originates from the insoluble, cross-linked protein elastin. Here, the results of variable-temperature (2) H NMR spectra are reported for hydrated elastin that has been enriched at the Hα position in its abundant glycines. Typical powder patterns reflecting averaged quadrupolar parameters are observed for the frozen protein, as opposed to the two, inequivalent deuterons that are detected in a powder sample of enriched glycine. The spectra of the hydrated elastin at warmer temperatures are dominated by a strong central peak with features close to the baseline, reflective of both isotropic and very weakly anisotropic motions.