Alanine | EPFL Graph Search

L'alanine (abréviations IUPAC-IUBMB : Ala et A) est un acide dont l'énantiomère L est l'un des aminés protéinogènes, encodé sur les ARN messagers par les codons GCU, GCC, GCA et GCG. Elle est caractérisée par une chaîne latérale apolaire aliphatique. Elle est, avec la sérine, la leucine et la glycine, l'un des acides aminés les plus fréquents dans les protéines. On trouve également dans la nature l'énantiomère D-alanine, qui participe en particulier à la construction du peptidoglycane, principal constituant de la paroi bactérienne. La D-alanine est formée à partir de L-alanine par l'action d'une isomérase, l'alanine racémase. La L-alanine est créée dans les cellules musculaires à partir du pyruvate en présence de glutamate dans un processus appelé transamination. Dans le foie, l’alanine se transforme en pyruvate. De plus, l’alanine aminotransférase (ALAT) catalyse la réaction dans laquelle le groupement aminé de l’alanine est transféré au α-cétoglutarate. C'est un acide aminé alip

Determination of 4-Hydroxy-2,5-dimethyl-3(2H)-furanone and 2(or 5)-Ethyl-4-hydroxy-5(or 2)-methyl-3(2H)-furanone in Pentose Sugar-Based Maillard Model Systems by Isotope Dilution Assays

Imre Blank

The formation of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) and 2(or 5)-ethyl-4-hydroxy-5(or 2)-methyl-3(2H)-furanone (EHMF) from pentose sugars was studied in Maillard model systems. The amts. generated at 90 DegC for 1 h were detd. by isotope diln. assay (IDA). The internal stds. used for IDA, i.e., [13C2]HDMF and [2H3]EHMF, were prepd. in good overall yields in 3 steps: addn. of labeled acetaldehyde or propionaldehyde to tert-butyloxycarbonyl (BOC)-protected and lithiated 3-butyn-2-ol; oxidn. of the BOC-protected diol with permanganate to 1,2-dione; and finally cyclization to the target mols. after removal of the protective groups under acidic conditions. Quant. data confirmed previous findings that HDMF and EHMF are preferentially formed in the presence of glycine and L-alanine, resp. The yields obtained were 2.6-5.1 mg of HDMF and 6.8-10 mg of EHMF per mmol pentose. Formation of both furanones was favored in phosphate-buffered solns. at pH 7 compared to pH 5, particularly in the presence of an excess of amino acid. These data are well in agreement with the previously proposed formation mechanism of HDMF and EHMF via Strecker-assisted chain elongation of the pentose moiety. However, both furanones were also produced to a lesser extent by sugar fragmentation-condensation reactions. [on SciFinder (R)]

1997

Mechanistic studies of DNP and applications of hyperpolarized probes to study renal physiology and metabolism

Alice Radaelli

Dissolution dynamic nuclear polarization (dDNP) is a powerful technique that enhances the magnetic resonance signal of nuclear spins by several orders of magnitude. DNP relies on the principle of cross-relaxation by electron spins driven out of equilibrium to enhance nuclear polarization. When coupled to magnetic resonance spectroscopy/imaging (MRS/I), infusion of 13C-labeled tracers hyperpolarized via DNP in vivo provides real-time estimates of metabolic fluxes, physiological parameters and/or tissue perfusion. The present work focuses on fundamental aspects of DNP, in particular how sample composition affects the DNP of low-gamma nuclei, as well as on in vivo dDNP applications targeting renal metabolism and physiology. The DNP properties of the SA-BDPA radical were studied in a sample of 13C-urea. SA-BDPA, a water-soluble derivative of BDPA, is characterized by a small g-anisotropy and unresolved hyperfine coupling to protons. As a result, at 6.7 T, 1.1 K its ESR linewidth is much smaller than the 13C Larmor frequency, which enabled the observation for the first time of 13C DNP via the solid effect and pure thermal mixing, the latter defined as the process where the electron non-Zeeman reservoir alone provides the energy required for triple-spin flips. The effect of solvent deuteration on 6Li DNP and radical ESR properties was studied in 6LiCl water:glycerol solutions doped with either the nitroxide radical TEMPOL or the trityl radical OX063. Unexpectedly, the TEMPOL-doped samples polarized better than the trityl-doped ones. Across all samples, the relationship between the degree of solvent deuteration with the buildup time constant and polarization level was notably different from what has been reported for 13C. This behavior is indicative of DNP via a combination of the cross effect and thermal mixing mechanisms. The uptake and metabolism of hyperpolarized L-[1-13C]alaninamide was investigated in the rat kidney in vivo. This probe of aminopeptidase N, which can play a role in tumor growth, was previously studied in vitro. Our study showed that alanine production from alaninamide also occurs in vivo, however, with spectral overlap of substrate and product. Alaninamide, having a pKa of 7.9, proved to be sensitive to local pH. Three spectral peaks, corresponding to at least three environments with different pH values, could be observed in the kidney. The two peaks at higher pH were assigned to the blood extra- and (partially) intracellular compartments, while the third one was mainly in the inner part of the kidney. Finally, alaninamide was shown to also be sensitive to dissolved CO2, with the rapid formation of a carbamate adduct following infusion. The renal metabolism of D-[1-13C]alanine by D-amino acid oxidase (DAO) was also studied. Conversion of hyperpolarized D-alanine to pyruvate and further metabolism to lactate and bicarbonate was observed in the kidney only when DAO was not inhibited. DAO activity could also be detected in blood, where leukocytes express the enzyme, but not in the brain and liver, in line with their lower DAO activity. Overall, this thesis provides additional insight into how the experimental conditions can favor a particular DNP mechanism over another. It also significantly expands the scope of in vivo dDNP applications, showing that additional enzyme-catalyzed processes can be detected, along with the potential of amino-acid based hyperpolarized 13C sensors for physiological studies.

EPFL2021

Gas-phase probes of kinetically trapped peptides

Liudmila Voronina

Gas-phase techniques are promising for studying intrinsically disordered peptides and proteins, as they can be implemented in a conformer-selective way. One of the issues that prevent them from finding a broader use is the question of to which extent the structure of a biological molecule is preserved when transferring it from solution to the gas phase via electrospray. We focus on cis/trans isomerization of prolyl-peptide bonds, which is known to have high barrier on the potential energy surface. Upon electrospraying, solution-like structures can find themselves kinetically trapped behind these barriers, as we explicitly show for the case of bradykinin 1-5 fragment. Using nuclear magnetic resonance, we determine that it adopts mostly all-trans conformation in solution, while three distinct conformational families are observed in the ion mobility experiment in the gas phase. Cryogenic ion spectroscopy combined with first-principles simulations allows us to identify the major conformers and show that the solution structural preferences of the prolyl-peptide bonds are preserved in the kinetically trapped conformational family, while the lowest-energy gas-phase structures have one of the prolines in the cis conformation. We show how collisional cross-sections and infrared spectra are used to guide and verify the calculations, giving rise to a new type of symbiosis between theory and experiment. This is especially valuable for the kinetically trapped structures, as in this case the energy criterion cannot be decisive. As a next step, we characterize the full nonapeptide bradykinin in the +3 charge state. Using field-asymmetric ion mobility spectrometry combined with cryogenic ion spectroscopy, we demonstrate the presence of three major conformational families in the gas phase, one of which is kinetically trapped. This result is in agreement with the drift-tube ion mobility data published previously, and we propose a correspondence between the conformational families separated by field-asymmetric and drift-tube ion mobility spectrometry. We obtain conformer-specific infrared spectra of the major conformers and assign the vibrational bands using 15N isotopic labeling. Substituting carbon atoms in the phenyl ring with their 13C isotope allows us to separate two types of structures according to their fragmentation pattern upon electronic excitation. This method can serve as a complementary way to introduce conformer-selectivity when studying large molecules. Finally, we assess the result of substituting one or two prolines in the bradykinin sequence with alanine. This method has been used previously to assign the peaks in the drift-time distributions to certain conformations of the prolyl-peptide bonds. We show that the mutants do reproduce a part of the conformers of the original peptide, but also form additional structures due to the higher flexibility of the alanine backbone compared to proline. We conclude that the proline-to-non-proline substitutions are helpful to assign the structures, but have to be used in conjunction with spectroscopic techniques, which allow detailed comparison of the structures of the mutant and the native peptide. In general, this work is one of the first steps towards a database of atomic-resolution peptide structures in the gas phase and a better understanding of the capabilities and limits of spectroscopy and ion mobility in the gas phase applied to intrinsically disordered peptides.

EPFL2016