For the analysis of the spectra of complex biofluids, preprocessing methods play a crucial role in rendering the subsequent data analyses more robust and accurate. Normalization is a preprocessing method, which accounts for different dilutions of samples by scaling the spectra to the same virtual overall concentration. In the field of 1H NMR metabonomics integral normalization, which scales spectra to the same total integral, is the de facto standard. In this work, it is shown that integral normalization is a suboptimal method for normalizing spectra from metabonomic studies. Especially strong metabonomic changes, evident as massive amounts of single metabolites in samples, significantly hamper the integral normalization resulting in incorrectly scaled spectra. The probabilistic quotient normalization is introduced in this work. This method is based on the calculation of a most probable dilution factor by looking at the distribution of the quotients of the amplitudes of a test spectrum by those of a reference spectrum. Simulated spectra, spectra of urine samples from a metabonomic study with cyclosporin-A as the active compound, and spectra of more than 4000 samples of control animals demonstrate that the probabilistic quotient normalization is by far more robust and more accurate than the widespread integral normalization and vector length normalization.

No abstract

The human recombinant Interferon-alpha(2a) (IFNalpha(2a)) is a potent drug (Roferon-A) to treat various types of cancer and viral diseases including Hepatitis B/C infections. To improve the pharmacological properties of the drug, a new pegylated form of IFNalpha(2a) was developed (PEGASYS). This 40 kDa PEG-conjugated IFNalpha(2a) ((40)PEG-IFNalpha(2a)) is obtained by the covalent binding of one 40 kDa branched PEG-polymer to a lysine side chain of IFNalpha(2a). (40)PEG-IFNalpha(2a) is a mixture of mainly six monopegylated positional isomers modified at K31, K134, K131, K121, K164, and K70, respectively. Here we report the detailed structural and biophysical characterization of (40)PEG-IFNalpha(2a) and its positional isomers, in comparison with IFNalpha(2a), using NMR spectroscopy, analytical ultracentrifugation, circular dichroism, fluorescence spectroscopy, and differential scanning calorimetry. Our results show that the three-dimensional structure of IFNalpha(2a) is not modified by the presence of the polymer in all positional isomers constituting (40)PEG-IFNalpha(2a). Regardless of where the PEG-polymer is attached, it adopts a very mobile and flexible random coil conformation, producing a shield for the protein without a permanent coverage of the protein surface. Hydrodynamic data indicate that the protein-attached PEG has a slightly more compact random-coil structure than the free PEG-polymer. Our results also provide evidence of significant structural and physicochemical advantages conferred by the pegylation: increase of the effective hydrodynamic volume and modification of the molecular shape, higher temperature stability, and reduced tendency for aggregation. These results are of tremendous pharmacological interest and benefit as was clinically shown with PEGASYS. This study constitutes a new standard for the characterization of pegylated proteins and enables an important step toward the understanding on a molecular level of the binding of (40)PEG-IFNalpha(2a) and its positional isomers to its cellular receptors.

Biocatalysis employing imine reductases is ap romising approach for the one-step generation of chiral amines from ketones. The enzymes reported for this process suffer from low activity and moderate stereoselectivity.W ei dentified as et of enzymest hat facilitate this reaction with high to quantitative conversionsf rom al ibrary of 28 imine reductases. This enabled the conversion of ketones with ammonia, methylamine, or butylamine into the corresponding amines. Most importantly,w e performed preparative (> 100 mg) scale syntheses of amines such as (1S,3R)-N,3-dimethylcyclohexylamine and (R)-N-methyl-2-aminohexane with excellent stereochemical purities (98 % de, 96 % ee)i ng ood yields.Chiral amines are keys tructuralm otifs that are frequently employedf or the preparation of pharmacologically active compounds.[1] From av ariety of asymmetric synthesis methods, transition-metal-catalyzed reductionso fi mine or enamine precursors [2] dominate the spectrum of approaches that have been reported.[3] However,t hese reactions equences usually comprise severals teps, including protection and deprotection of the amine and the necessity to introduce nitrogen activating groups for the reduction (Scheme 1, routes Aand B). [2] Biocatalysis offers an attractive alternative for synthetic chemists, with ac ontinuouslyi ncreasing toolboxo fe nzymes for the preparation of chiral amines. [1b, 4] Amine transaminases [1b, 5] and amine dehydrogenases, [6] for example, have proven to be applicable in the one-step preparation of primary amines,a lso on industrial scale. [5c, 7] Thus, these enzymes can providea na ttractive synthetic shortcut towards chiral amines. Additionally,C odexis lately filed ap atento ne ngineered opine dehydrogenases that catalyze reductive aminations.[8] NADPHdependenti mine reductases (IREDs) also catalyze the equivalent single-step reduction (Scheme 1, route C) but are, unlike amine transaminases anda mine dehydrogenases, not limited to the preparation of primary amines. IREDs are amenable to access primary,secondary,and tertiary amines starting from ketones or the respective imine or iminium ion. Whereas imine reductionsh ave been knownf or some time to occur in several metabolic pathways such as dihydrofolate, opine, opioid, and antibiotic synthesis, [9] it was only over the last five years that NADPH-dependentI REDs were reported in the context of synthetic applications.[10] Immediately after the isolation of the first amino acid sequences, [10] several research groups thoroughly studied IREDs for the reduction of variousc yclic imines and iminium ion substrates, also including artificial metal-dependentI REDs.[11] Very recently,t he group of Müllerr eported the first IRED-catalyzed reductivea mination of ak etone with methylamine in ap roofo fp rinciple study.[11e] With this work, he paved the way for an ew synthetic application of IREDs. Nestl and co-workersd emonstrated that IRED-catalyzedr eductive amination is, in principle, applicable for different amines, which thuso pened the gate for the sy...

Recombinant 15N- and 13C-labeled human granulocyte colony-stimulating factor (rh-metG-CSF) has been studied by 2D and 3D NMR using uniformly labeled protein, as well as residue-specific 15N-labeled samples. Assignment of 90% of the backbone resonances and 85% of side-chain resonances has enabled the determination of both the secondary and tertiary structures of the protein. The fold is similar to those of the human growth hormone and other growth factors. Four stretches of helices were identified between residues 11 and 41 (helix A), 71 and 95 (helix B), 102 and 125 (helix C), and 145 and 170 (helix D), which form a left-handed four-helix bundle with helices A and B aligned parallel to one another (up-up) and antiparallel to helices C and D (down-down). An additional short fifth helix (E) is part of the AB loop connecting helices A and B. Examination of the protein's relaxation behavior, based on the model-free approach of Lipari and Szabo, shows that the G-CSF backbone has a well-defined structure of limited conformational flexibility in helices. In contrast, the long loop connecting helices C and D exhibits substantial fast internal motion compared to the overall rotational correlation time of the whole molecule, which is on the order of 13 ns.