Alternative E.COSY techniques for the measurement of 3J(C i ′ −1,C i β ) and 3 J(H i N ,C i β ) coupling constants in proteins

Experiments are proposed for the measurement of the vicinal coupling constants between -carbons and either amide protons of the same or carbonyl carbons of the preceding amino acid residue in 13C/15N-labeled proteins. Both couplings depend on the backbone torsional angle . The three-dimensional pulse sequences give rise to E.COSY-like multiplet patterns in which heteronuclear one-bond couplings separate the doublet components corresponding to the two spin states of the respective passive nuclei. Thus, in contrast to previously published pulse schemes which employed the homonuclear 1J(C,C) interaction, difficulties due to overlap of spectral regions of active and passive spins are avoided. A major drawback of the novel sequences is their limited sensitivity. Nevertheless, application to Desulfovibrio vulgaris flavodoxin yielded coupling constants for more than 85% of all non-glycine and non-proline residues.     

A New Experiment for the Measurement of nJ(C,P) Coupling Constants Including 3J(C4′i,Pi) and 3J(C4′i,Pi+1) in Oligonucleotides

A new experiment for the measurement of nJ(C,P) coupling constants along the phosphodiester backbone in RNA and DNA based on a quantitative-J HCP experiment is presented. In addition to coupling constants, in which a carbon atom couples to only one phosphorus atom, both the intraresidual 3J(C4i,Pi) and the sequential 3J(C4i,Pi+1) for the C4 resonances that couple to two phosphorus atoms can be obtained. Coupling constants obtained by this new method are compared to values obtained from the P-FIDS experiment. Together with 3J(H,P) coupling constants measured using the P-FIDS experiment, the backbone angles and can be determined.     

Determination of 3J(H infi supN,C infi sup′ ) coupling constants in proteins with the C′-FIDS method

Summary We introduce the C-FIDS-¹H,¹⁵N-HSQC experiment, a new method for the determination of ³J(H _infi ^supN ,C _infi ^sup ) coupling constants in proteins, yielding information about the torsional angle . It relies on the ¹H,¹⁵N-HSQC or HNCO experiment, two of the the most sensitive heteronuclear correlation experiments for isotopically labeled proteins. A set of three ¹H,¹⁵N-HSQC or HNCO spectra are recorded: a reference experiment in which the carbonyl spins are decoupled during t₁ and t₂, a second experiment in which they are decoupled exclusively during t₁ and a third one in which they are coupled in t₁ as well as t₂. The last experiment yields an E.COSY-type pattern from which the ²J(H _infi ^supN ,C _infi-1 ^sup ) and ¹J(N_i,C _infi-1 ^sup ) coupling constants can be extracted. By comparison of the coupled multiplet (obtained from the second experiment) with the decoupled multiplet (obtained from the first experiment) convoluted with the ²J(H _infi ^supN ,C _infi-1 ^sup ) coupling, the ³J(H _infi ^supN ,C _infi ^sup ) coupling can be found in a one-parameter fitting procedure. The method is demonstrated for the protein rhodniin, containing 103 amino acids. Systematic errors due to differential relaxation are small for ⁿJ(H^N,C) couplings in biomacromolecules of the size currently under NMR spectroscopic investigation.     

Measurement of 2J(H,C)- and 3J(H,C)-coupling constants by α/β selective HC(C)H-TOCSY

A new heteronuclear NMR pulse sequence for the measurement of ⁿJ(C,H) coupling constants, the /selective HC(C)H-TOCSY, is described. It is shown that the S³E element (Meissner et al., 1997a,b) can be used to obtain spin state selective coherence transfer in molecules, in which adjacent CH moieties are labeled with ¹³C. Application of the / selective HC(C)H-TOCSY to a 10nt RNA tetraloop 5-CGCUUUUGCG-3, in which the four uridine residues are ¹³C labeled in the sugar moiety, allowed measurement of two bond and three bond J(C,H) coupling constants, which provide additional restraints to characterize the sugar ring conformation of RNA in cases of conformational averaging.     

A 4D TROSY-based pulse scheme for correlating 1HNi,15Ni,13Cαi,13C′i−1 chemical shifts in high molecular weight, 15N,13C, 2H labeled proteins

A 4D TROSY-based triple resonance experiment, 4D-HNCO_i–1CA_i, is presented which correlates intra-residue ¹HN, ¹⁵N, ¹³ C chemical shifts with the carbonyl (¹³C) shift of the preceding residue. The experiment is best used in concert with recently described 4D TROSY-HNCOCA and -HNCACO experiments [Yang, D. and Kay, L.E. (1999) J. Am. Chem. Soc., 121, 2571–2575]. In cases where degeneracy of (¹HN,¹⁵N) spin pairs precludes assignment using the HNCOCA and HNCACO, the HNCO_i–1CA_i often allows resolution of the ambiguity by linking the ¹³C and ¹³C spins surrounding the (¹HN,¹⁵N) pair. The experiment is demonstrated on a sample of ¹⁵N, ¹³C, ² H labeled maltose binding protein in complex with -cyclodextrin that tumbles with a correlation time of 46 ns.     

Measurement of the protein backbone dihedral angle φ based on quantification of remote CSA/DD interference in inter-residue 13C′(i − 1)-13Cα(i) multiple-quantum coherences

A novel triple-resonance NMR method is presented for the measurement of the protein backbone dihedral angle based on differential multiple-quantum relaxation induced by relaxation interference between ¹H(i)-¹³C(i) dipolar and ¹³C(i–1) (carbonyl) chemical shift anisotropy mechanisms. The method employs a simultaneous transfer of ¹⁵N magnetization to the inter- and intra-residue ¹³C carbons as well as the directly attached carbonyl carbon ¹³C. Results obtained on ¹³C,¹⁵N-labeled ubiquitin demonstrate the potential of the method.     

Syntheses and antitumor activities of N′1,N′3-dialkyl-N′1,N′3-di-(alkylcarbonothioyl) malonohydrazide: The discovery of elesclomol

A series of N′¹,N′³-dialkyl-N′¹,N′³-di(alkylcarbonothioyl) malonohydrazides have been designed and synthesized as anticancer agents by targeting oxidative stress and Hsp70 induction. Structure–activity relationship (SAR) studies lead to the discovery of STA-4783 (elesclomol), a novel small molecule that has been evaluated in a number of clinical trials as an anticancer agent in combination with Taxol.     

C1′ Acylated Derivatives of 2′-Deoxyuridine. Photolabile Precursors of 2′-Deoxyuridin-1′-yl

Abstract

ABSTRACT

C1′ acylated derivatives of 2′-dcoxyuiidinc (1a-c) were synthesised from 1-[3-deoxy-β-D-psieofiiraiiosylliii.acil (6). The acyl group is introduced via the C1′ aldehyde (11). Following nucleophilic addition, the ketones (1a-c) are obtained via periodinane oxidation and desilylation with NH₄F.     

Structural studies of an Inv (1, −2) kappa light chain

A Bence Jones protein with phenotype Inv (1, –2) was isolated from the urine of a patient with multiple myeloma. Inv typing of the patient's relatives established the presence of anInv ¹ allele in the kindred, and that the patient's genotype wasInv ¹/Inv ³. Hence, the absence of Inv (2) in the Bence Jones protein was shown to be genetic and not an artifact caused by the disease. The tryptic peptide-containing residues 191 through 194 were isolated and shown to be composed of Leu, Tyr, Ala, Cys, with Leu at the amino end. Hence, the residue at 191 is the same as that present in Inv (1, 2) Bence Jones proteins. More detailed study of the tryptic peptides established that residue 153 is Val rather than Ala as in all other K chains thus far studied. The primary sequence: Ala¹⁵³, Leu¹⁹¹ determines Inv (1, 2); Ala¹⁵³, Val¹⁹¹ determines Inv (3); and Val¹⁵³, Leu¹⁹¹ determines Inv (1). The Val¹⁵³, Val¹⁹¹ sequence has not been observed. It may correspond to Inv (–). These data are strikingly similar to the data for the Kern and Oz isotypes (changes at 154 and 191, respectively) in the chain. As in the case of theK chain, only three of the four possible combinations have been observed. The implications of this parallelism and of crystallographic findings on chains, reported by others, are discussed.     

Selectively 13C-enriched DNA: Dynamics of the C1′-H1′ vector in d(CGCAAATTTGCG)2

Summary In order to examine the internal dynamic processes of the dodecamer d(CGCAAATTTGCG)₂, the ¹³C-enriched oligonucleotide has been synthesized. The three central thymines were selectively ¹³C-labeled at the C1′ position and their spin-lattice relaxation parameters R(C_Z), R(C_X,Y), R(H_Z→C_Z), R(2H_ZC_Z), R(2H_ZC_X,Y) and R(H _infZ ^supC ) were measured. Density functions were computed for two models of internal motions. Comparisons of the experimental data were made with the spin-lattice relaxation rates rather than with the density functions, whose values were altered by accumulation of the uncertainties of each relaxation rate measurement. The spin-lattice relaxation rates were computed with respect to the motions of the sugar around the C1′-N1 bond. A two-state jump model between the anti- and syn-conformations with P(anti)/P(syn)=91/9 or a restricted rotation model with Δχ=28° and an internal diffusion coefficient of 30×10⁷ s^-1 gave a good fit with the experimental data. Twist, tilt or roll base motions have little effect on ¹³C1′ NMR relaxation. Simulation of spin-relaxation rates with the data obtained at several temperatures between 7 and 32 °C, where the dodecamer is double stranded, shows that the internal motion amplitude is independent of the temperature within this range, as expected for internal motion. Using the strong correlation which exists in a B-DNA structure between the χ and δ angle, we suggest that the change in the glycosidic angle value should be indicative of a sugar puckering between the C1′-exo and C2′-endo conformations.     

Untersuchungen zum C′3-Polymorphismus (β 1c-Globulin)

Zusammenfassung Serumproben von 1322 Blutspendern aus Hessen, 40 Familien mit 89 Kindern, 20 Mutter-Kind-Kombinationen und 268 Sera einer Bantupopulation aus Portugiesisch Angola wurden mit einer modifizierten Technik der Hochspannungs-Dünnschichtelektrophorese auf Agarosegel hinsichtlich des C3-Polymorphismus untersucht. Die Genfrequenzen für Weiße (C3^S=0.779, C3^F=0.215) und Neger (C3^S=0.95, C3^F=0,048) stimmen gut mit den Werten anderer Autoren überein. Insgesamt ließen sich bei Weißen 9 Phänotypen sicher abgrenzen, bei Negern 3. Familienstudien bestätigten den für die Allele C3^S und C3^F angenommenen Vererbungsmodus (autosomal codominant) ausnahmslos. Die Frage der Lagerungsstabilität des C3 wurde abschließend untersucht.

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Investigations on C3-polymorphism ( _1c-Globulin)Gene frequencies and family studies in blood donors from Hessen and a Bantu population

Summary Serum samples of 1322 unrelated individuals from Hessen (Germany), 40 families with 89 children, 20 mother-child-combinations and 268 sera of a Bantu population from Angola were examined for C3 polymorphism using a modified technique of high voltage agarose gel electrophoresis. The gene frequencies for Caucasians (C3^S=0.779, C3^F=0.215) and negroes (C3^S=0.95, C^F=0.048) are in good accordance with those obtained by other authors. In total 9 different phenotypes were observed in Caucasians, 3 phenotypes in negroes. Family studies verify the supposed way of inheritance (autosomal codominant for C3^S and C3^F) without exception. Finally the problem of C3-inactivation by storage was investigated.

     

MQ-HCN-based pulse sequences for the measurement of 13C1′-1H1′, 13C1′-15N, 1H1′-15N, 13C1′-13C2′, 1H1′-13C2′, 13C6/8-1H6/8, 13C6/8-15N, 1H6/8-15N, 13C6-13C5, 1H6-13C5 dipolar couplings in 13C, 15N-labeled DNA (and RNA)

A suite of multiple quantum (MQ) HCN-based pulse sequences has been developed for the purpose of collecting dipolar coupling data in labeled nucleic acids. All the pulse sequences are based on the robust MQ-HCN experiment which has been utilized for assignment purposes in labeled nucleic acids for a number of years and provides much-needed resolution for the dipolar coupling measurements. We have attempted to collect multiple couplings centered on the 13C1' and 13C6/8 positions. Six pulse sequences are described, one each for measurement of one-bond 13C1'-1H1' and 13C6/8-1H6/8 couplings, one for measurement of one-bond 13C1'-15N and two-bond 1H1'-15N couplings, one for measurement of one-bond 13C6/8-15N and two-bond 1H6/8-15N couplings, one for measurement of one-bond 13C1'- 13C2' and two-bond 1H1'-13C2' couplings, and one for measurement of one-bond 13C6-13C5 and two-bond 1H6-13C5 couplings in the bases of C and T. These sequences are demonstrated for a labeled 18 bp DNA duplex in a 47 kDa ternary complex of DNA, CBFbeta, and the CBFalpha Runt domain, thus clearly demonstrating the robustness of the pulse sequences even for a very large complex.     

Unsymmetrical 1,1′-disubstituted Ferrocenes: Synthesis of Co(ii), Cu(ii), Ni(ii) and Zn(ii) Chelates of Ferrocenyl -1-thiadiazolo-1′-tetrazole, -1-thiadiazolo-1′-triazole and -1-tetrazolo-1′-triazole with Antimicrobial Properties

Condensation reactions of 1,1"-diacetylferrocene with different heteroaromatic amines such as, 2-amino-1,3,4-thiadiazole, 5-aminotetrazole and 3-amino-1,2,4-triazole to form unsymmetrically 1,1′-disubstituted ferrocenes have been studied. The obtained compounds have been further investigated for their liganding and biological properties upon chelation with Co(II), Cu(II), Ni(II) and Zn(II) metal ions. The synthesized compounds have been characterized by physical, spectral and analytical data and have been screened against pathogenic bacterial strains e.g., Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, showing moderate activity as antibacterials in vitro.     

Measurement of two- and three-bond 13C−1H J couplings to the Cδ carbons of leucine residues in staphylococcal nuclease

Summary A new ¹H-detected 3D NMR experiment is described that permits quantitative measurement of two- and three-bond ¹³C–¹H couplings in proteins with selectively ¹³C-enriched methyl sites. The method is demonstrated for staphylococcal nuclease selectively [5,5 ¹³C]-labeled in all 11 leucine positions and ligated with thymidine 3,5-biphosphate and Ca²⁺. Two- and three-bond ¹³C methyl-proton couplings are reported and, together with the measured three-bond J_CC in uniformly ¹³C-enriched staphylococcal nuclease, the ²- and the stereospecific assignments of the C methyl group with respect to the prochiral -protons were determined. The same residues that were previously found to have high degrees of internal mobility on the basis of ¹³C relaxation times have measured coupling constants that are indicative of motional averaging.     

Metabolism of O,O-Dipropyl S-[2-(2′-Methyl-1′-piperidinyl)-2-oxo-ethyl] Phosphorodithioate (C 19 490) in Paddy Rice

The fate of O,O-dipropyl S-[2-(2′-methyl-1′-piperidinyl)-2-oxo-ethyl] phosphorodithioate (C 19 490, piperophos) was followed in greenhouse-grown rice after application of 1.6 kg/ha ¹⁴C-labeled C 19 490 into the paddy water. At maturity the shoots contained 2.5 ppm and the hulled grains 0.16 ppm ¹⁴C-C 19 490 equivalents. Small amounts of unchanged C 19 490 were found in the shoots but none in the grains.

The main metabolites found were:- 2-(2′-methyl-1′-piperidiny])-2-oxo-ethane sulfonic acid, 2-(2?-methyl-1?-piperidinyl)-2-oxo-ethanoic acid and a fraction of polar unknown substances. O,O-Dipropyl S-[2-(2?-methyl-1′ -piperidinyl)-2-oxo-ethyl] phosphorothioate, 2-(2?-methyl-1?-piperidinyl)- 1-methyl-sulfinyl-2-oxo-ethane, 2-(2?-methyl-1?-piperidinyl)-1-methyl-sulfonyl-2-oxo- ethane, 2-methyl-piperidine and CO₂ were found in smaller amounts.     

The oxidation of N,N′-bis(4-aminophenyl)-N,N′-dimethylethylenediamine (BED) by rat liver

We have examined the oxidation of N,N′-bis(4-aminophenyl)-N,N′-dimethylethylenediamine (BED) by tissue homogenates and fractions of liver homogenates. We find that this agent both gives osmiophilic deposits in tissue blocks and readily increases the uptake of oxygen by hepatic homogenates. The highest activity was in the mitochondrial and, next, in the microsomal fractions. Kinetic evidence indicates that the former represents two enzymatic activities while the latter is only a single site. The activity was greatest in the outer membrane of the mitochondria, in agreement with electron micrographic studies and in the rough microsomal fraction. Further, it was very sensitive to both formaldehyde and detergents. The activity was not well associated with either monamine oxidase (benzylamine substrate) or xanthine oxidase activities. Activity was observed in a large number of tissues.     

Syntheses,properties, and use of fluorescent N-(5′-phospho-4′-pyridoxyl)amines in assay of pyridoxamine (pyridoxine) 5′-phosphate oxidase

A sensitive and simple fluorometric assay has been developed for detection of pyridoxamine (pyridoxine) 5′-phosphate oxidase. This technique utilizes fluorescent N-(5′-phospho-4′-pyridoxyl)amines as substrates that, upon incubation with the oxidase, release the free fluorescent amine. The substrates were prepared by condensation of pyridoxal 5′-phosphate with fluorescent amines and subsequent hydrogenation of the Schiff bases. Since N-(1-naphthyl)ethylenediamine is 15 times less fluorescent in the intramolecularly quenched substrate than the product amine, the direct increase of fluorescence, as well as selective extraction of more fluorescent product, can be utilized for assay. The apparent K_m value for this substrate is 8 μm, which is slightly less than that of pyridoxamine 5′-phosphate; V is larger than the natural substrate value. The greater sensitivity gained by this fluorimetric method allows detection of the oxidase in smaller quantities than can be determined by the conventional colorimetric assay.     

Crystal Structure of 2′-Deoxycytidine Hemidihydrogenphosphate Reveals C+·C Base Pairs and Tight,Hydrogen-Bonded (H2PO4 −)∞ Columns (1)

Abstract

2′-Deoxycytidine hemidihydrogenphosphate has been crystallized in the hexagonal space group P6₂ with α=25.839(3), c = 12.529(1) Å. The structure has been solved using the Patterson search method. The asymmetric unit contains two protonated, base-paired 2′-deoxycytidine dimers and two H₂PO₄ ^? anions. The C⁺·C base pairs are composed of a protonated and a neutral species each and are triple H-bonded, the central N(3)…N(3) bonds being 2.850(7) and 2.884(5) Å. The conformations of the four nucleosides fall in the same category (sugar puckers 2·-endo, glycosidic links anti) but in one of them the glycosidic torsion angle is quite low with consequences in other geometrical parameters. The H₂PO₄ ^? anions are located on twofold axes and form two types of tight columns with P…P separations about 4.18 Å The neighboring units along a column are linked via two very short O…H…O hydrogen bonds (O…O about 2.49 Å) leading to effective equalization of the P-O bonds. The base pairs of the two dC⁺·dC cations are coplanar and form layers perpendicular to the phosphate columns repeating every c/3. Within the layers, the dimers form a network through 0(5′)…O(2) hydrogen bonds but their primary intermolecular interactions have the form of H-bond anchors [N(4)-H…O-P and 0(3′)-H…O-P] to the phosphate groups.     

Selective synthesis of cerebrosides: (2S, 3R, 4E)-1-O-β-d-galactopyranosyl-N-(2′R and 2′S)-2′-hydroxytetracosanoyl-sphingenine

The cerebrosides were first isolated by Thudicum in 1874 and the structures were established by Carteret al. in 1950 (for review, see [2]). In 1961 Shapiro and Flowers [3] reported the first total synthesis of a cerebroside1 (Fig. 1) which was identified with the natural sample, only through comparison of their i.r. data. In order to confirm the absolute configuration at C-2 of natural cerebroside1, we describe here an unambiguous synthesis of two stereoisomeric cerebrosides1 and2, and found that the¹H-NMR spectra of the synthetic1 (Fig. 2) was completely identical with that of the natural cerebroside reported recently by Dabrowskiet al. [4].In planning the synthetic route, the target structures1 and2 were disconnected at the dotted lines to give three key synthetic intermediates3, 4 and5 or6 (Fig. 1).Abbreviations Bu butyl - Ph phenyl - t-BuPh₂SiCl t-butyldiphenylsilyl chloride - MTPA -methoxy--trifluoromethylphenylacetic acid - THF tetrahydrofuran Part 36 in the series Synthetic Studies on Cell-surface Glycans, for part 35, see [1]     

Synthesis of Two 3-(7′-Theophyllyl)glycals, 3-Deoxy-3-(7′-theophyllyl)-d-xylo-hex-1-enopyranose and Methyl 3-Deoxy-3-(7′-theophyllyl)-d-xylo-hex-1-enopyranuronate,and their Derivatives

Two 3-(7′-theophyllyl)glycals, (IV) and (V), were synthesized by fusion of theophylline and the appropriate glycals in the presence of p-toluenesulfonic acid. The structure and stereochemistry of the glycals were determined mainly from NMR analysis of their dihydro and 1,6-anhydro derivatives.