Permeability of membranes to amino acids and modified amino acids: Mechanisms involved in translocation

Summary The amino acid permeability of membranes is of interest because they are one of the key solutes involved in cell function. Membrane permeability coefficients (P) for amino acid classes, including neutral, polar, hydrophobic, and charged species, have been measured and compared using a variety of techniques. Decreasing lipid chain length increased permeability slightly (5-fold), while variations in pH had only minor effects on the permeability coefficients of the amino acids tested in liposomes. Increasing the membrane surface charge increased the permeability of amino acids of the opposite charge, while increasing the cholesterol content decreased membrane permeability. The permeability coefficients for most amino acids tested were surprisingly similar to those previously measured for monovalent cations such as sodium and potassium (approximately 10^–12–10^–13 cm · s^–1). This observation suggests that the permeation rates for the neutral, polar and charged amino acids are controlled by bilayer fluctuations and transient defects, rather than partition coefficients and Born energy barriers. Hydrophobic amino acids were 10² more permeable than the hydrophilic forms, reflecting their increased partition coefficient values.External pH had dramatic effects on the permeation rates for the modified amino acid lysine methyl ester in response to transmembrane pH gradients. It was established that lysine methyl ester and other modified short peptides permeate rapidly (P = 10^–2 cm · s^–1) as neutral (deprotonated) molecules. It was also shown that charge distributions dramatically alter permeation rates for modified di-peptides. These results may relate to the movement of peptides through membranes during protein translocation and to the origin of cellular membrane transport on the early Earth.Abbreviations DCP dicetylphosphate - DMPC dimyristoyl phosphatidylcholine - EPC egg phosphatidylcholine - LUV large unilamellar vesicle - MLV multilamellar vesicle - PLM planar lipid membrane - SUV small unilamellar vesicle - pH transmembrane pH gradient     

Lipophilicity of amino acids

Summary The lipophilicity (or hydrophobicity) of amino acids is an important property relevant for protein folding and therefore of great interest in protein engineering. For peptides or peptidomimetics of potential therapeutic interest, lipophilicity is related to absorption and distribution, and thus indirectly relates to their bioactivity. A rationalization of peptide lipophilicity requires basic knowledge of the lipophilicity of the constituting amino acids. In the present contribution we will review methods to measure or calculate the lipophilicities of amino acids, including unusual amino acids, and we will make a comparison between various lipophilicity scales.     

Cobalt hydroxide nanoparticles modified glassy carbon electrode as a biosensor for electrooxidation and determination of some amino acids

The electrochemical behavior of some amino acids was investigated on cobalt hydroxide nanoparticles modified glassy carbon (CHM-GC) electrode in alkaline solution. The process of oxidation and its kinetics were established by using cyclic voltammetry, chronoamperometry techniques, and steady-state polarization measurements. The results revealed that cobalt hydroxide promotes the rate of oxidation by increasing the peak current, so these bimolecular reactions are oxidized at lower potentials. Cyclic voltammograms and chronoamperometry indicate a catalytic EC′ mechanism to be operative with electrogeneration of Co(IV) as the electrochemical process. Also, the process is diffusion controlled and the current-time responses follow Cottrellian behavior. This result was confirmed by steady-state measurements. The rate constants of the catalytic oxidation of amino acids and the electron transfer coefficients are reported.     

Synthesis of novel acetylene-containing amino acids

Summary.  Novel synthetic procedures for the modification of non-proteinogenic acetylene-containing amino acids have been developed. The functionalization either proceeds via zinc/copper-mediated introduction of alkyl substituents, or via tungsten-catalyzed ring-closing alkyne metathesis reactions. Received March 28, 2002 Accepted October 3, 2002 Published online December 18, 2002 Acknowledgements These investigations are supported (in part) by the Netherlands Research Council for Chemical Sciences (CW) with financial aid from the Netherlands Technology Foundation (STW). Authors' address: Floris P. J. T. Rutjes, Prof. Dr., Department of Organic Chemistry, University of Nijmegen, Toernooiveld 1, NL-6525 ED Nijmegen, The Netherlands, E-mail: rutjes@sci.kun.nl  2, selected data: ¹H NMR (300 MHz, CDCl₃) δ 5.32 (d, J = 7.7 Hz, 1H), 4.44–4.40 (m, 1H), 3.76 (s, 3H), 2.75–2.73 (d, J = 5.0 Hz, 2H), 1.44 (s, 9H); ¹³C NMR (75 MHz, CDCl₃) δ 171.0, 155.0, 80.3, 74.6, 52.6, 51.9, 41.7, 28.3, 24.0; mp = 55°C.  Typical procedure for 5: zinc dust (116 mg, 1.408 mmol) was weighed into a 20 mL flask, which was repeatedly evacuated (with heating using a heat gun) and flushed with argon. Dry DMF (0.5 mL, distilled from CaH₂) and 1,2-dibromoethane (9.2 μL, 0.106 mmol) were added and the flask was heated at 80°C for 40 min. The reaction mixture was allowed to cool to room temperature, trimethylsilyl chloride (4 μL, 0.035 mmol) was added and the resulting mixture was stirred vigorously for a further 30 min under argon. Iodocyclohexane (69 μl, 0.528 mmol) was added and stirred at room temperature for 3 h more after which stirring was ceased to settle the zinc. CuCN (41 mg, 0.458 mmol) and LiCl (40 mg, 0.915 mmol) were heated to 150°C for 2 h and cooled to room temperature. Addition of DMF (1 mL) formed a soluble CuCN·2LiCl complex within 5 min. After cooling the Cu-complex to −15°C, the organozinc reagent was added dropwise followed by the bromoacetylene 2 (116 mg, 0.352 mmol). The mixture was allowed to stir overnight at room temperature. Water was added and the suspension was extracted using heptane, washed with brine, dried (MgSO₄) and concentrated. Purification using flash column chromatography (10% EtOAc in heptane) yielded 5 (100 mg, 81%) as a colorless oil. 5: IR ν 3355, 2929, 2852, 2359, 2337, 1749, 1717, 1498, 1447, 1365, 1251, 1181, 1060; ¹H NMR (300 MHz, CDCl₃) δ 5.28 (d, J = 7.7 Hz, 1H), 4.43–4.38 (m, 1H), 3.73 (s, 3H), 2.69–2.63 (m, 2H), 2.13 (m, 1H), 1.73–1.22 (m, 10H), 1.43 (s, 9H); ¹³C NMR (75 MHz, CDCl₃) δ 171.4, 155.0, 88.1, 79.9, 73.8, 52.3, 32.7, 32.7, 28.8, 28.2, 25.8, 24.6, 23.1; HRMS (EI): calculated for C₁₇H₂₇NO₄ 309.1940, found 309.1937.  A solution of the tungsten catalyst (7 mg, 10 mol%) in C₆H₅Cl (2 mL) was treated with a solution of 14 (49.0 mg, 0.120 mmol) in C₆H₅Cl (5.0 mL) under an argon atmosphere and the resulting mixture was heated at 80°C for 3 h. Evaporation followed by flash column chromatography (80% EtOAc in heptane) afforded 15 (21.0 mg, 50%; 64% after correction for starting material) and 14 (16 mg, 33%) as colorless oils. 15: [α]_D =–14.6 (c = 1, CH₂Cl₂); IR ν 3313, 2931, 2865, 2249, 1744, 1667, 1520, 1366, 1170; ¹H NMR (400 MHz, CDCl₃) δ 7.14 (d, J = 8.7 Hz, 1H), 6.08 (d, J = 8.3 Hz, 1H), 4.78 (q, J = 6.8 Hz, 1H), 4.27 (q, J = 7.9 Hz, 1H), 3.73 (s, 3H), 2.17–2.15 (m, 4H), 2.07–1.96 (m, 2H), 1.79–1.52 (m, 4H), 1.45 (s, 9H), 0.89–0.83 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 173.2, 171.8, 155.8, 80.4, 80.2, 79.3, 53.8, 52.5, 51.2, 32.8 (2×), 28.1, 24.6, 24.2, 18.3 (2×); HRMS (EI): calculated for C₁₈H₂₈N₂O₅     

ABRF ESRG 2005 study: identification of seven modified amino acids by Edman sequencing.

Identification of modified amino acids can be a challenging part for Edman degradation sequence analysis, largely because they are not included among the commonly used phenylthiohydantion amino acid standards. Yet many can have unique retention times and can be assigned by an experienced researcher or through the use of a guide showing their typical chromatography characteristics. The Edman Sequencing Research Group (ESRG) 2005 study is a continuation of the 2004 study, in which the participating laboratories were provided a synthetic peptide and asked to identify the modified amino acids present in the sequence. The study sample provided an opportunity to sequence a peptide containing a variety of modified amino acids and note their retention times relative to the common amino acids. It also allowed the ESRG to compile the chromatographic properties and intensities from multiple instruments and tabulate an average elution position for these modified amino acids on commonly used instruments. Participating laboratories were given 2000 pmoles of a synthetic peptide, 18 amino acids long, containing the following modified amino acids: dimethyl- and trimethyl-lysine, 3-methyl-histidine, N-carbamyl-lysine, cystine, N-methyl-alanine, and isoaspartic acid. The modified amino acids were interspersed with standard amino acids to help in the assessment of initial and repetitive yields. In addition to filling in an assignment sheet, which included retention times and peak areas, participants were asked to provide specific details about the parameters used for the sequencing run. References for some of the modified amino acid elution characteristics were provided and the participants had the option of viewing a list of the modified amino acids present in the peptide at the ESRG Web site. The ABRF ESRG 2005 sample is the seventeenth in a series of studies designed to aid laboratories in evaluating their abilities to obtain and interpret amino acid sequence data.     

Plasma amino acids in four models of experimental liver injury in rats

Summary We studied the plasma amino acid profiles in four models of hepatic injury in rats. In partially hepatectomized rats (65% of liver was removed) we observed significant increase of aromatic amino acids (AAA; i.e. tyrosine and phenylalanine), taurine, aspartate, threonine, serine, asparagine, methionine, ornithine and histidine. Branched-chain amino acids (BCAA; i.e. valine, leucine and isoleucine) concentrations were unchanged. In ischemic and carbon tetrachloride acute liver damage we observed extreme elevation of most of amino acids (BCAA included) and very low concentration of arginine. In carbon tetrachloride induced liver cirrhosis we observed increased levels of AAA, aspartate, asparagine, methionine, ornithine and histidine and decrease of BCAA, threonine and cystine. BCAA/AAA ratio decreased significantly in partially hepatectomized and cirrhotic rats and was unchanged in ischemic and acute carbon tetrachloride liver damage. We conclude that a high increase of most of amino acids is characteristic of fulminant hepatic necrosis; decreased BCAA/AAA ratio is characteristic of liver cirrhosis; and decrease of BCAA/AAA ratio may not be used as an indicator of the severity of hepatic parenchymal damage.Abbreviations BCAA branched-chain amino acids (i.e. valine, leucine and isoleucine) - AAA aromatic amino acids (i.e. tyrosine and phenylalanine)     

D-L amino acid analysis using automated precolumn derivatization with 1-fluoro-2,4-dinitrophenyl-5-alanine amide

Summary An automated procedure for the precolumn derivatization of enantiomeric amino acid mixtures with 1-fluoro-2,4-dinitrophenyl-5-alanine amide and a liquid chromatographic method for the separation of the derivatives with UV detection are reported. The system described allows to perform routine analyses using microbore columns with a sensitivity at the picomol level. Improvements for the use of this reagent in the protocol of a subtractive Edman degradation procedure of peptides to determine the sequence position of amino acid residues with concomitant identification of their chirality are also described.     

Study of the resolution of amino acids and aminoalcohols in organic solvents

Summary The enzymatic resolution of racemic phenylglycine, phenylglycinol and phenylalaninol has been studied in organic solvents under a variety of experimental conditions. Subtilisin in 3-methyl-3-pentanol was effective for the resolution of phenylglycine esters, via N-acylation with trifluoroethyl butyrate. Porcine pancreatic lipase in ethyl acetate gave satisfactory results in the resolution of phenylglycinol and phenylalaninol; the or position of the phenyl group was found to influence both the rate and the chemioselectivity of the reaction.     

Changes of plasma insulin, urea, amino acids and rumen metabolites in somatotropin treated dairy cows

Summary An experiment was performed to evaluate the effects of somatotropin on plasma free amino acid, urea and insulin concentrations and rumen fermentation pattern and to assess their relationships. Four Italian Friesian dairy cows fitted with rumen cannulae were used in a switch-back design. Slow releasing recombinant bovine somatotropin (640 mg/cow) was injected every 28 days for two consecutive periods. Rumen fluid and blood samples were collected before and after feeding at 0, 7 and 21 days after rbST injection. Exogenous rbST increased plasma insulin concentration and the insulin response to feeding, and decreased plasma urea and free essential and branched chain amino acid concentrations. rbST did not affect rumen fermentation pattern. No correlation was found between rumen and plasma parameters measured after feeding. Our results are consistent with the notion that the main effect of somatotropin is post-absorptive.     

Free amino acids in some sponges

Most invertebrates, particularly those of marine origin, have relatively high concentrations of free amino acids which are considered an important constituent of their osmoregulatory mechanisms [1]. Very little information is available on the free amino acid distribution in Porifera [2,3]. Common amino acids in some sponges were recognised by paper chromatography by Inskip and Cassidy [4] and Ackermann et al. [5,6] included a few sponges in their survey of the occurence of nitrogen compounds in marine invertebrates. More recently Bergquist and Hartman [7] surveyed semiquantitatively the distribution of free amino acids in several sponges. In the present paper we report on the amino acid composition of 12 species of sponges belonging to the class Demospongiae as a part of a study on the metabolites of Porifera [8]. Fresh sponges were extracted with aqueous ethanol. The organic solvent was removed and the aqueous solution, after removal of the ether soluble compounds, was separated into cationic, anionic and neutral fractions by ion-exchange chromatography. The cation fraction was analysed for amino acids using an automatic amino acid analyser. The results, which are presented in Table 1, show that all species of sponges examined have a similar composition in common amino acids. Glycine almost always appears as the dominant protein amino acid, followed by high concentrations of alanine and glutamic acid, whereas relatively lower concentrations of basic amino acids are present. In Axinella cannabina, Chondrosia reniformis, Chondrilla nucula, Cliona viridis and Hymeniacidon sanguinea, glycine represents more than 77% of the total amino acids. The high percentage of free glycine (90.4%) in Chondrosia reniformis is noteworthy. The anionic and the neutral fractions were examined for sulfur-containing amino acids using PC. Taurine (Table 2) was detected in all the Porifera examined; this is in agreement with previous observations [5–7]. N-Methyltaurine was identified in some of the species examined, whereas neither N,N-dimethyltaurine nor N,N,N-trimethyltaurine were found.     

Plasma amino acids are highly dependent on acid-base balance in uremic rats

Summary Acidosis is a common finding in uremia. We analysed the effect of changes in acid-base balance in 42 Sprague-Dawley rats having longstanding uremia (> 3 mon). The rats were treated with CaCO₃ or CaCl₂ in a random cross-over trial after being divided into two dietary groups (8% or 18% protein). At the end of each observation period parameters were assessed. The main finding was that plasma amino and keto acid concentrations varied significantly with the acid-base balance and the protein intake. Furthermore a considerable degree of catabolism and anorexia was observed. In addition other confounding variables were observed, e.g. increased corticosterone excretion, electrolyte losses, and proteinuria. Our data underline that plasma amino acid concentrations in uremic rats are highly dependent on acid-base balance and overall protein intake.     

A modified spray reagent for the detection of amino acids on thin layer chromatography plates

Summary. Identification of amino acids is extremely important for the evaluation of protein structure. Thin layer chromatography is an important tool for detecting amino acids by variety of spray reagents. Among these ninhydrin is the most popular due to its high sensitivity. However, ninhydrin produces the same purple/violet color with most amino acids. A spray reagent with high sensitivity for easy and rapid identification of amino acids on thin-layer plates has been introduced. Received March 14, 2000 Accepted August 31, 2000     

Application of tritium high resolution NMR spectroscopy to analysis of tritium-labelled amino acids and peptides

Summary A method has been developed for the qualitative and quantitative analysis of complex isotopic mixtures of tritium-labelled amino acids and peptides by using high resolution³H NMR spectroscopy at 266.8 MHz. Determined were tritium distribution in alanine, glycine, tryptophan and 4-hydroxyproline amino acids, as well as in glycine and valine residues of peptides. Approaches have been worked out for the determination of spin coupling constants and isotope chemical shifts for the strongly coupled nonequivalent atoms of the methylene groups.     

Neuroexcitatory amino acids: 4-methylene glutamic acid derivatives

Summary A short synthesis of 4-methylene glutamic acid was achieved. Under thermal conditions the corresponding anhydride reacted with 2,3 dimethylbutadiene to afford the corresponding DIELS-ALDER adduct in good yield. L-4-methylene glutamic acid essentially acts on glutamate metabotropic receptors and is as potent as L-Glu in producing IPs.     

Determination of amino acids in human serum by capillary gas chromatography

A simple and rapid method for the determination of serum amino acids by gas chromatography (GC) has been developed. Following deproteinization of serum with perchloric acid, free amino acids in the supernatant were converted into their N(O,S)-isobutoxycarbonyl methyl ester derivatives and measured by GC with flame ionization detection using a DB-17 capillary column. All the derivatives of the 22 protein amino acids were completely resolved as single peaks within 9 min by GC. The calibration curves were linear in the range 0.2–50 μg of each amino acid, and the correlation coefficients were above 0.998. By using this method, serum amino acids could be directly analysed without prior clean-up procedure such as ion-exchange column chromatography except for deproteinization of the samples, and without any interference from coexisting substances. Overall recoveries of amino acids added to serum samples were 88–108%. Analytical results for serum amino acids from normal subjects are presented.     

Influence of high dietary threonine on growth and amino acids in blood and tissues of rats

Summary Diets containing 8 or 15% protein from casein plus limiting amino acids, 25% fat and adequate levels of other nutrients for rat growth were supplemented with 0, 0.5, 1.0, 2.0 or 4% of excess L-threonine. Addition of up to 1% excess threonine had little effect on weight gains or food intakes of weanling rats, but addition of 2 and 4% threonine caused a drastic reduction in weight gains or food intakes (up to 41%); the adverse effect being more severe in rats fed lower protein diets. Addition of graded levels of excess threonine resulted in (5 to 47-fold and 4 to 20-fold) increase in concentration of free threonine in rat plasma and brain, respectively. Addition of excess threonine also caused up to 5-fold increase in plasma level of 3-methylhistidine, suggesting increased muscle protein breakdown.     

Nonprotein amino acids in edible lentil and garden pea seedlings

Summary. Commercial edible seedlings of garden pea (Pisum sativum L.) and lentil (Lens culinaris L.) contain high concentration of nonprotein amino acids and trigonelline. Both seedlings grown in the laboratory or purchased in a supermarket were studied by HPLC. Samples from both origins contained trigonelline, α-aminoadipic acid, homoserine, β-(isoxazolin-5-on-2-yl)-alanine (BIA), and γ-glutamyl-BIA. Garden pea seedlings also contained a uracil-alanine derivative (isowillardiine) in substantial amount. Some of these compounds such as BIA and α-aminoadipic acid have neurotoxic activity. Received December 17, 1999 Accepted February 15, 2000     

Unusual amino acids VIII. Asymmetric hydrogenation of some heteroaryl-N-CBZ and N-BOC aminocinnamic acid derivatives

Summary (Z)--[(Benzyloxy)- or (tert.-butyloxy)carbonylamino]- (thienyl)-or (furyl)-acrylic acids and their esters were prepared by known methods and hydrogenated to the corresponding optically active alanine derivatives with optical yields in the range of 58–93% ee using the cationic rhodium complex of PROPRAPHOS.     

Changes with aging in the levels of amino acids in rat CNS structural elements II. Taurine and small neutral amino acids

Taurine (Tau) and the small neutral amino acids glycine (Gly), serine (Ser), threonine (Thr), and alanine (Ala) were measured in 53 brain areas of 3- and 29-month-old male Fisher 344 rats. The ratio of highest to lowest level was 34 for Tau, 9.1 for Thr, 7.6 for Gly and Ser, and 6.5 for Ala. The heterogeneity was found in numerous areas; for example, Tau levels were more than 90 nmol/mg protein in 6 areas, and less than 20 nmol/mg protein in 10 areas. Similar heterogeneity was found with the other amino acids. The relative distribution of the small neutral amino acids showed several similarities; Tau distribution was different. With age, four amino acids decreased in 10–18 areas, and increased in only 1–3, while Thr increased in more areas than it decreased. The five amino acids of this paper, and the four of the previous paper, are among the amino acids at highest level in the brain; the sequence in their levels shows considerable regional heterogeneity.     

Sugar amino acids in designing new molecules

Emulating the basic principles followed by Nature to build its vast repertoire of biomolecules, organic chemists are developing many novel multifunctional building blocks and using them to create ‘nature-like’ and yet unnatural organic molecules. Sugar amino acids constitute an important class of such polyfunctional scaffolds where the carboxyl, amino and hydroxyl termini provide an excellent opportunity to organic chemists to create structural diversities akin to Nature’s molecular arsenal. This article describes some of our works on various sugar amino acids and many other related building blocks, like furan amino acids, pyrrole amino acids etc. used in wide-ranging peptidomimetic studies. Published in 2005.Based on the invited lecture presented at the XVII International Symposium on Glycoconjugates held in January 12–16, 2003 at Bangalore, India.