Pernille Harris

Cis−Trans Amide Bond Rotamers in β‑Peptoids and Peptoids: Evaluation of Stereoelectronic Effects in Backbone and Side Chains

Non-natural peptide analogs have significant potential for the development of new materials and pharmacologically active ligands. One such architecture, the β-peptoids (N-alkyl-β-alanines), has found use in a variety of biologically active compounds but has been sparsely studied with respect to folding propensity. Thus, we here report an investigation of the effect of structural variations on the cis-trans amide bond rotamer equilibria in a selection of monomer model systems. In addition to various side chain effects, which correlated well with previous studies of α-peptoids, we present the synthesis and investigation of cis-trans isomerism in the first examples of peptoids and β-peptoids containing thioamide bonds as well as trifluoroacetylated peptoids and β-peptoids. These systems revealed an increase in the preference for cis-amides as compared to their parent compounds and thus provide novel strategies for affecting the folding of peptoid constructs. By using NMR spectroscopy, X-ray crystallographic analysis, and density functional theory calculations, we present evidence for the presence of thioamide-aromatic interactions through C(sp(2))-H···S(amide) hydrogen bonding, which stabilize certain peptoid conformations.

Rhamnogalacturonan lyase reveals a unique three-domain modular structure for polysaccharide lyase family 4.

Rhamnogalacturonan lyase (RG‐lyase) specifically recognizes and cleaves α‐1,4 glycosidic bonds between l‐rhamnose and d‐galacturonic acids in the backbone of rhamnogalacturonan‐I, a major component of the plant cell wall polysaccharide, pectin. The three‐dimensional structure of RG‐lyase from Aspergillus aculeatus has been determined to 1.5 Å resolution representing the first known structure from polysaccharide lyase family 4 and of an enzyme with this catalytic specificity. The 508‐amino acid polypeptide displays a unique arrangement of three distinct modular domains. Each domain shows structural homology to non‐catalytic domains from other carbohydrate active enzymes.

Synthesis and Characterization of Mixed Chalcogen Triangular Complexes with New Mo3(μ3-S)(μ2-Se2)34+ and M3(μ3-S)(μ2-Se)34+ (M = Mo, W) Cluster Cores

In our pursuit of mixed chalcogen-bridged cluster complexes, solids of the compositions Mo(3)SSe(6)Br(4) and W(3)SSe(6)Br(4) were prepared using high-temperature synthesis from the elements. Treatment of Mo(3)SSe(6)Br(4) with Bu(4)NBr in a vibration mill yielded (Bu(4)N)(3){[Mo(3)(mu(3)-S)(mu(2)-Se(2))(3)Br(6)]Br} (I). Its all-selenide analogue (Bu(4)N)(3){[Mo(3)(mu(3)-Se)(mu(2)-Se(2))(3)Br(6)]Br} (II) was prepared from Mo(3)Se(7)Br(4) in a similar way. Both compounds were characterized by IR, Raman, and (77)Se NMR spectroscopy. The structure of II was determined by X-ray single-crystal analysis. Compound I is isostructural with II and contains the new Mo(3)(mu(3)-S)Se(6)(4+) cluster core. By treatment of a 4 M Hpts solution of I with PPh(3) followed by cation-exchange chromatography, the new mixed chalcogenido-molybdenum aqua ion, [Mo(3)(mu(3)-S)(mu(2)-Se)(3)(H(2)O)(9)](4+), was isolated and characterized using UV-vis spectroscopy and, after derivatization into [Mo(3)(mu(3)-S)(mu(2)-Se)(3)(acac)(3)(py)(3)](+), electrospray ionization mass spectrometry. From HCl solutions of the aqua ion, a supramolecular adduct with cucurbit[6]uril (CB[6]), {[Mo(3)(mu(3)-S)(mu(2)-Se)(3)(H(2)O)(6)Cl(3)](2)CB[6]}Cl(2) x 11 H(2)O (III), was isolated and its structure determined using X-ray crystallography. W(3)SSe(6)Br(4) upon reaction with H(3)PO(2) gave a mixture of all of the [W(3)S(x)Se(4-x)(H(2)O)(9)](4+) species. After repeated chromatography, crystals of {[W(3)(mu(3)-S)(mu(2)-Se)(3)(H(2)O)(7)Cl(2)](2)CB[6]}Cl(4) x 12 H(2)O (IV) were crystallized from the fraction rich in [W(3)(mu(3)-S)Se(3)(H(2)O)(9)](4+) and structurally characterized.

Mechanism of dTTP inhibition of the bifunctional dCTP deaminase:dUTPase encoded by Mycobacterium tuberculosis.

Recombinant deoxycytidine triphosphate (dCTP) deaminase from Mycobacterium tuberculosis was produced in Escherichia coli and purified. The enzyme proved to be a bifunctional dCTP deaminase:deoxyuridine triphosphatase. As such, the M. tuberculosis enzyme is the second bifunctional enzyme to be characterised and provides evidence for bifunctionality of dCTP deaminase occurring outside the Archaea kingdom. A steady-state kinetic analysis revealed that the affinity for dCTP and deoxyuridine triphosphate as substrates for the synthesis of deoxyuridine monophosphate were very similar, a result that contrasts that obtained previously for the archaean Methanocaldococcus jannaschii enzyme, which showed approximately 10-fold lower affinity for deoxyuridine triphosphate than for dCTP. The crystal structures of the enzyme in complex with the inhibitor, thymidine triphosphate, and the apo form have been solved. Comparison of the two shows that upon binding of thymidine triphosphate, the disordered C-terminal arranges as a lid covering the active site, and the enzyme adapts an inactive conformation as a result of structural changes in the active site. In the inactive conformation dephosphorylation cannot take place due to the absence of a water molecule otherwise hydrogen-bonded to O2 of the alpha-phosphate.

Crystal Structure of Tryptophan Hydroxylase with Bound Amino Acid Substrate

Tryptophan hydroxylase (TPH) is a mononuclear non-heme iron enzyme, which catalyzes the reaction between tryptophan, O 2, and tetrahydrobiopterin (BH 4) to produce 5-hydroxytryptophan and 4a-hydroxytetrahydrobiopterin. This is the first and rate-limiting step in the biosynthesis of the neurotransmitter and hormone serotonin (5-hydroxytryptamine). We have determined the 1.9 A resolution crystal structure of the catalytic domain (Delta1-100/Delta415-445) of chicken TPH isoform 1 (TPH1) in complex with the tryptophan substrate and an iron-bound imidazole. This is the first structure of any aromatic amino acid hydroxylase with bound natural amino acid substrate. The iron coordination can be described as distorted trigonal bipyramidal coordination with His273, His278, and Glu318 (partially bidentate) and one imidazole as ligands. The tryptophan stacks against Pro269 with a distance of 3.9 A between the iron and the tryptophan Czeta3 atom that is hydroxylated. The binding of tryptophan and maybe the imidazole has caused the structural changes in the catalytic domain compared to the structure of the human TPH1 without tryptophan. The structure of chicken TPH1 is more compact, and the loops of residues Leu124-Asp139 and Ile367-Thr369 close around the active site. Similar structural changes are seen in the catalytic domain of phenylalanine hydroxylase (PAH) upon binding of substrate analogues norleucine and thienylalanine to the PAH.BH 4 complex. In fact, the chicken TPH1.Trp.imidazole structure resembles the PAH.BH 4.thienylalanine structure more (root-mean-square deviation for Calpha atoms of 0.90 A) than the human TPH1 structure (root-mean-square deviation of 1.47 A).

Novofumigatonin, a new orthoester meroterpenoid from Aspergillus novofumigatus.

Novofumigatonin (1), a new metabolite, has been isolated from Aspergillus novofumigatus. The structure and relative stereochemistry were determined from HR ESI MS, one- and two-dimensional NMR, and single-crystal X-ray analysis. The absolute configuration was assigned using vibrational circular dichroism in combination with density functional calculations.

Experimental and Theoretical Mechanistic Investigation of the Iridium-Catalyzed Dehydrogenative Decarbonylation of Primary Alcohols

The mechanism for the iridium-BINAP catalyzed dehydrogenative decarbonylation of primary alcohols with the liberation of molecular hydrogen and carbon monoxide was studied experimentally and computationally. The reaction takes place by tandem catalysis through two catalytic cycles involving dehydrogenation of the alcohol and decarbonylation of the resulting aldehyde. The square planar complex IrCl(CO)(rac-BINAP) was isolated from the reaction between [Ir(cod)Cl]2, rac-BINAP, and benzyl alcohol. The complex was catalytically active and applied in the study of the individual steps in the catalytic cycles. One carbon monoxide ligand was shown to remain coordinated to iridium throughout the reaction, and release of carbon monoxide was suggested to occur from a dicarbonyl complex. IrH2Cl(CO)(rac-BINAP) was also synthesized and detected in the dehydrogenation of benzyl alcohol. In the same experiment, IrHCl2(CO)(rac-BINAP) was detected from the release of HCl in the dehydrogenation and subsequent reaction with IrCl(CO)(rac-BINAP). This indicated a substitution of chloride with the alcohol to form a square planar iridium alkoxo complex that could undergo a β-hydride elimination. A KIE of 1.0 was determined for the decarbonylation and 1.42 for the overall reaction. Electron rich benzyl alcohols were converted faster than electron poor alcohols, but no electronic effect was found when comparing aldehydes of different electronic character. The lack of electronic and kinetic isotope effects implies a rate-determining phosphine dissociation for the decarbonylation of aldehydes.

Total Synthesis and Full Histone Deacetylase Inhibitory Profiling of Azumamides A–E as Well as β2- epi-Azumamide E and β3-epi-Azumamide E

Cyclic tetrapeptide and depsipeptide natural products have proven useful as biological probes and drug candidates due to their potent activities as histone deacetylase (HDAC) inhibitors. Here, we present the syntheses of a class of cyclic tetrapeptide HDAC inhibitors, the azumamides, by a concise route in which the key step in preparation of the noncanonical disubstituted β-amino acid building block was an Ellman-type Mannich reaction. By tweaking the reaction conditions during this transformation, we gained access to the natural products as well as two epimeric homologues. Thus, the first total syntheses of azumamides B-D corroborated the originally assigned structures, and the synthetic efforts enabled the first full profiling of HDAC inhibitory properties of the entire selection of azumamides A-E. This revealed unexpected differences in the relative potencies within the class and showed that azumamides C and E are both potent inhibitors of HDAC10 and HDAC11.

Crystal Structure, Vibrational Spectroscopy and ab Initio Density Functional Theory Calculations on the Ionic Liquid forming 1,1,3,3-Tetramethylguanidinium bis{(trifluoromethyl)sulfonyl}amide

The salt 1,1,3,3-tetramethylguanidinium bis{(trifluoromethyl)sulfonyl}amide, [((CH(3))(2)N)(2)C=NH(2)](+)[N(SO(2)CF(3))(2)](-) or [tmgH][NTf(2)], easily forms an ionic liquid with high SO(2) absorbing capacity. The crystal structure of the salt was determined at 120(2) K by X-ray diffraction. The structure was found to be monoclinic, space group P2(1)/n with a = 11.349(2), b = 11.631(2), c = 11.887(2) A, and beta = 90.44(3) degrees . Raman and IR spectra are presented and interpreted. The results are interpreted using ab initio quantum mechanics calculations that also predicted vibrational spectra. The relationship between the transoid (C(2) symmetry) structure of the [NTf(2)](-) ion and the conformationally sensitive bands is discussed.

The crystal structure of human dopamine β-hydroxylase at 2.9 Å resolution

This first structure of the enzyme converting dopamine to norepinephrine provides new perspectives on numerous disorders. The norepinephrine pathway is believed to modulate behavioral and physiological processes, such as mood, overall arousal, and attention. Furthermore, abnormalities in the pathway have been linked to numerous diseases, for example hypertension, depression, anxiety, Parkinson’s disease, schizophrenia, Alzheimer’s disease, attention deficit hyperactivity disorder, and cocaine dependence. We report the crystal structure of human dopamine β-hydroxylase, which is the enzyme converting dopamine to norepinephrine. The structure of the DOMON (dopamine β-monooxygenase N-terminal) domain, also found in >1600 other proteins, reveals a possible metal-binding site and a ligand-binding pocket. The catalytic core structure shows two different conformations: an open active site, as also seen in another member of this enzyme family [the peptidylglycine α-hydroxylating (and α-amidating) monooxygenase], and a closed active site structure, in which the two copper-binding sites are only 4 to 5 Å apart, in what might be a coupled binuclear copper site. The dimerization domain adopts a conformation that bears no resemblance to any other known protein structure. The structure provides new molecular insights into the numerous devastating disorders of both physiological and neurological origins associated with the dopamine system.