NMR of conotoxins: structural features and an analysis of chemical shifts of post-translationally modified amino acids

Conotoxins are small conformationally constrained peptides found in the venom of marine snails of the genus Conus. They are usually cysteine rich and frequently contain a high degree of post-translational modifications such as C-terminal amidation, hydroxylation, carboxylation, bromination, epimerisation and glycosylation. Here we review the role of NMR in determining the three-dimensional structures of conotoxins and also provide a compilation and analysis of H-1 and C-13 chemical shifts of post-translationally modified amino acids and compare them with data from common amino acids. This analysis provides a reference source for chemical shifts of post-translationally modified amino acids. Copyright (C) 2006 John Wiley & Sons, Ltd.

Amino acids and diabetes: implications for endocrine, metabolic and immune function

Aberrant alterations in glucose and lipid concentrations and their pathways of metabolism are a hallmark of diabetes. However, much less is known about alterations in concentrations of amino acids and their pathways of metabolism in diabetes. In this review we have attempted to highlight, integrate and discuss common alterations in amino acid metabolism in a wide variety of cells and tissues and relate these changes to alterations in endocrine, physiologic and immune function in diabetes.

Inhibition of L-type amino acid transport with non-physiological amino acids in the Pahenu2 mouse model of phenylketonuria

Phenylketonuria, an autosomal recessive Mendelian disorder, is one of the most common inborn errors of metabolism. Although currently treated by diet, many suboptimal outcomes occur for patients. Neuropathological outcomes include cognitive loss, white matter abnormalities, and hypo- or demyelination, resulting from high concentrations and/or fluctuating levels of phenylalanine. High phenylalanine can also result in competitive exclusion of other large neutral amino acids from the brain, including tyrosine and tryptophan (essential precursors of dopamine and serotonin). This competition occurs at the blood brain barrier, where the L-type amino acid transporter, LAT1, selectively facilitates entry of large neutral amino acids. The hypothesis of these studies is that certain non-physiological amino acids (NPAA; DL-norleucine (NL), 2-aminonorbornane (NB; 2-aminobicyclo-(2,1,1)-heptane-2-carboxylic acid), α-aminoisobutyrate (AIB), and α-methyl-aminoisobutyrate (MAIB)) would competitively inhibit LAT1 transport of phenylalanine (Phe) at the blood-brain barrier interface. To test this hypothesis, Pah-/- mice (n=5, mixed gender; Pah+/-(n=5) as controls) were fed either 5% NL, 0.5% NB, 5% AIB or 3% MAIB (w/w 18% protein mouse chow) for 3 weeks. Outcome measurements included food intake, body weight, brain LNAAs, and brain monoamines measured via LCMS/MS or HPLC. Brain Phe values at sacrifice were significantly reduced for NL, NB, and MAIB, verifying the hypothesis that these NPAAs could inhibit Phe trafficking into the brain. However, concomitant reductions in tyrosine and methionine occurred at the concentrations employed. Blood Phe levels were not altered indicating no effect of NPAA competitors in the gut. Brain NL and NB levels, measured with HPLC, verified both uptake and transport of NPAAs. Although believed predominantly unmetabolized, NL feeding significantly increased blood urea nitrogen. Pah-/-disturbances of monoamine metabolism were exacerbated by NPAA intervention, primarily with NB (the prototypical LAT inhibitor). To achieve the overarching goal of using NPAAs to stabilize Phe transport levels into the brain, a specific Phe-reducing combination and concentration of NPAAs must be found. Our studies represent the first in vivo use of NL, NB and MAIB in Pah-/- mice, and provide proof-of-principle for further characterization of these LAT inhibitors. Our data is the first to document an effect of MAIB, a specific system A transport inhibitor, on large neutral amino acid transport.

Selective adsorption of l- and d-amino acids on calcite: Implications for biochemical homochirality

The emergence of biochemical homochirality was a key step in the origin of life, yet prebiotic mechanisms for chiral separation are not well constrained. Here we demonstrate a geochemically plausible scenario for chiral separation of amino acids by adsorption on mineral surfaces. Crystals of the common rock-forming mineral calcite (CaCO3), when immersed in a racemic aspartic acid solution, display significant adsorption and chiral selectivity of d- and l-enantiomers on pairs of mirror-related crystal-growth surfaces. This selective adsorption is greater on crystals with terraced surface textures, which indicates that d- and l-aspartic acid concentrate along step-like linear growth features. Thus, selective adsorption of linear arrays of d- and l-amino acids on calcite, with subsequent condensation polymerization, represents a plausible geochemical mechanism for the production of homochiral polypeptides on the prebiotic Earth.

Contribution of transmembrane domain V amino acids to β1l-adrenoceptor activity and affinity

Introduction. There are two binding sites on the β1-adrenoceptor (AR), β1H and β1L corresponding to high and low affinity binding sites respectively, which can be activated to cause cardiostimulation. Some β-blockers that block β1AR and β2ARs can activate β1LARs at higher concentrations than those required to cause blockade. The β2AR does not form a corresponding low affinity binding site and therefore we postulated that heterologous amino acids are responsible for the formation of β1LAR. Aim. To investigate whether heterologous amino acids of transmembrane domain V (TMDV) of β1AR and β2ARs contribute to β1LAR. Methods. β1ARs, β2ARs and mutant β1ARs containing all (β1(β2TMDV)AR) or single amino acids of TMDV of the β2AR were prepared and stably expressed in Chinese Hamster Ovary cells. Concentration-effect curves for cyclicAMP accumulation were carried out for (-)-CGP12177 in the absence or presence of (-)-bupranolol. Results. The potencies (pEC50) of (-)-CGP12177 were β2AR (9.24 ± 0.14, n = 5), β1(V230I)AR (9.07 ± 0.07, n = 10), β1(β2TMDV)AR (8.86 ± 0.10, n = 15), β1(R222Q)AR (8.09 ± 0.29, n = 6), β1AR (8.00 ± 0.11, n = 11). The affinities (pKB) of (-)-bupranolol were β2AR (9.82 ± 0.52, n = 5), β1(V230I)AR (7.64 ± 0.12, n = 8), β1(β2TMV)AR (8.06 ± 0.17, n = 8), β1(R222Q)AR (7.33 ± 0.23, n = 5), β1AR (7.23 ± 0.23, n = 5). Discussion. The potency of (-)-CGP12177 was higher at β2AR than at β1AR consistent with activation through a low affinity site at the β1AR (β1LAR). The presence of V230 in β1AR accounted for the lower potency of (-)-CGP 12177. The affinity of (-)-bupranolol was lower at β1AR compared to β2AR. The presence of V230 in β1AR accounted in part for the lower affinity. In conclusion TMDV of the β1AR contributes in part to the low affinity binding site of β1AR.

Contribution of transmembrane domain V amino acids to β1l-adrenoceptor activity and affinity

There are two binding sites on the β1-adrenoceptor (AR), β1H and β1L corresponding to high and low affinity binding sites respectively, which can be activated to cause cardiostimulation (reviewed Kaumann and Molenaar, 2008). Some β-blockers that block β1AR and β2ARs can activate β1LARs at higher concentrations than those required to cause blockade. The β2AR does not form a corresponding low affinity binding site (Baker et al 2002) and therefore we postulated that heterologous amino acids are responsible for the formation of β1LAR. Our aim was to investigate whether heterologous amino acids of transmembrane domain V (TMDV) of β1AR and β2ARs contribute to β1LAR. β1ARs, β2ARs and mutant β1ARs containing all (β1(β2TMDV)AR) or single amino acids of TMDV of the β2AR were prepared and stably expressed in Chinese Hamster Ovary cells. Concentration-effect curves for cyclicAMP accumulation were carried out for (-)-CGP12177 or (-)-isoprenaline in the absence or presence of (-)-bupranolol. _______________________________________________________________________ (-)-CGP 12177 (-)-Bupranolol affinity (pKB) pEC50 vs (-)-CGP 12177 vs (-)-isoprenaline _______________________________________________________________________ β1AR 8.00 ± 0.11 (11) 7.23 ± 0.23 (5) 9.52 ± 0.28 (5) β2AR (high density) 9.24 ± 0.14 (5) 9.82 ± 0.52 (8) xPaulxxxxxxx β2AR (low density) no effect β1(β2TMV)AR 8.86 ± 0.10 (15) 8.06 ± 0.17 (8) 9.08 ± 0.22 (6) β1(V230I)AR 9.07 ± 0.07 (10) 7.64 ± 0.12 (8) 9.36 ± 0.28 (9) β1(R222Q)AR 8.09 ± 0.29 (6) 7.33 ± 0.23 (5) 9.36 ± 0.08 (6) β1(V230A)AR 7.59 ± 0.09 (6) 7.32 ± 0.24 (4) 8.62 ± 0.18 (5) _______________________________________________________________________ The potency of (-)-CGP12177 was higher at β2AR than at β1AR consistent with activation through a low affinity site at the β1AR (β1LAR) but not β2AR. The presence of V230 in β1AR accounted for the lower potency of (-)-CGP 12177. The affinity of (-)-bupranolol at β1AR and mutants was higher when determined with (-)-isoprenaline than with (-)-CGP 12177. The affinity of (-)-bupranolol determined against (-)-CGP 12177 was lower at β1AR compared to β2AR. The presence of V230 in β1AR accounted in part for the lower affinity. In conclusion V230 of the β1AR contributes in part to the low affinity binding site of β1AR. Baker JG, Hall IP, Hill SJ (2002). Pharmacological characterization of CGP12177 at the human β2-adrenoceptor. Br J Pharmacol 137, 400−408 Kaumann AJ, Molenaar P (2008) The low-affinity site of the β1-adrenoceptor and its relevance to cardiovascular pharmacology. Pharmacol Ther 118, 303-336

Preparative deracemization of unnatural amino acids

Unnatural amino acids are a growing class of intermediates required for pharmaceuticals, agrochemicals and other industrial products. However, no single method has proven sufficiently versatile to prepare these compounds broadly at scale. To address this need, we have developed a general chemoenzymatic process to prepare enantiomerically pure L- and D-amino acids in high yield by deracemization of racemic starting materials. This method involves the concerted action of an enantioselective oxidase biocatalyst and a non-selective chemical reducing agent to effect the stereoinversion of one enantiomer and can result in an enantiomeric excess of >99% from the starting racemate, and product yields of over 90%. This approach compares very favourably with resolution processes, which have a maximum single-pass yield of 50%. We have developed efficient methods to adapt the process towards new target compounds and to optimize key factors that influence process efficiency and offer competitive economics at scale.

Chemo-enzymatic synthesis of unnatural amino acids

A general chemo-enzymatic process has been developed to prepare enantiomerically pure L- and D-amino acids in high yield by deracemisation of racemic starting materials. The method has been developed from initial academic studies to be a robust, scalable industrial process. Unnatural amino acids, in high optical purity, are a rapidly growing class of intermediates required for pharmaceuticals, agrochemicals and other fine chemical applications. However, no single method has proven sufficiently adaptable to prepare these compounds generally at large scale. Our approach uses an enantioselective oxidase biocatalyst and a non-selective chemical reducing agent to effect the stereoinversion of one enantiomer and can result in an enantiomeric excess of > 99 % from a starting racemate, and product yields over 90 %. The current approach compares very favourably to resolution methods which have a maximum single pass yield of 50 %. Efficient methods have been developed to adapt the biocatalyst used in this process towards new target compounds and to optimise key factors which improve the process efficiency and offer competitive economics at scale.

X-ray studies on crystalline complexes involving amino acids and peptides. XXXIII. Crystal structures of L- and DL-arginine complexed with oxalic acid and a comparative study of amino acid oxalic acid complexes

The DL- and L-arginine complexes of oxalic acid are made up of zwitterionic positively charged amino acid molecules and semi-oxalate ions. The dissimilar molecules aggregate into separate alternating layers in the former. The basic unit in the arginine layer is a centrosymmetric dimer, while the semi-oxalate ions form hydrogen-bonded strings in their layer. In the L-arginine complex each semi-oxalate ion is surrounded by arginine molecules and the complex can be described as an inclusion compound. The oxalic acid complexes of basic amino acids exhibit a variety of ionization states and stoichiometry. They illustrate the effect of aggregation and chirality on ionization state and stoichiometry, and that of molecular properties on aggregation. The semi-oxalate/oxalate ions tend to be planar, but large departures from planarity are possible. The amino acid aggregation in the different oxalic acid complexes do not resemble one another significantly, but the aggregation of a particular amino acid in its oxalic acid complex tends to have similarities with its aggregation in other structures. Also, semi-oxalate ions aggregate into similar strings in four of the six oxalic acid complexes. Thus, the intrinsic aggregation propensities of individual molecules tend to be retained in the complexes.

x-ray studies on crystalline complexes involving amino-acids and peptides .10. head-to-tail sequences in the crystal-structure of l-lysine acetate

l-Lysine acetate crystallises in the monoclinic space group P21 with a = 5.411 (1), b = 7.562(1), c= l2.635(2) Å and β = 91.7(1). The crystal structure was solved by direct methods and refined to an R value of 0.049 using the full matrix least squares method. The conformation and the aggregation of lysine molecules in the structure are similar to those found in the crystal structure of l-lysine l-aspartate. A conspicuous similarity between the crystal structures of l-arginine acetate and l-lysine acetate is that in both cases the strongly basic side chain, although having the largest pK value, interacts with the weakly acidic acetate group leaving the α-amino and the α-carboxylate groups to take part in head-to-tail sequences. These structures thus indicate that electrostatic effects are strongly modulated by other factors so as to give rise to head-to-tail sequences which have earlier been shown to be an almost universal feature of amino acid aggregation in the solid state.

Click Chemistry Inspired Synthesis of Novel Ferrocenyl-Substituted Amino Acids or Peptides

This work reports on the synthesis of a wide range of ferrocenyl-substituted amino acids and peptides in excellent yield. Conjugation is established via copper-catalyzed 1,3-dipolar cycloaddition. Two complementary strategies were employed for conjugation, one involving cycloaddition of amino acid derived azides with ethynyl ferrocene 1 and the other involves cycloaddition between amino acid derived alkynes with ferrocene-derived azides 2 and 3. Labeling of amino acids at multiple sites with ferrocene is discussed. A new route to 1,1'-unsymmetrically substituted ferrocene conjugates is reported. A novel ferrocenophane 19 is accessed via bimolecular condensation of amino acid derived bis-alkyne 9b with the azide 2. The electrochemical behavior of some selected ferrocene conjugates has been studied by cyclic voltammetry.