N‐Terminal methylation of proteasome subunit Rpt1 in yeast

The 26S proteasome is a multicatalytic protease complex that degrades ubiquitinated proteins in eukaryotic cells. It consists of a proteolytic core (the 20S proteasome) as well as regulatory particles, which contain six ATPase (Rpt) subunits involved in unfolding and translocation of substrates to the catalytic chamber of the 20S proteasome. In this study, we used MS to analyze the N‐terminal modifications of the yeast Rpt1 subunit, which contains the N‐terminal recognition sequence for N‐methyltransferase. Our results revealed that following the removal of the initiation Met residue of yeast Rpt1, the N‐terminal Pro residue is either unmodified, mono‐methylated, or di‐methylated, and that this N‐methylation has not been conserved throughout evolution. In order to gain a better understanding of the possible function(s) of the Pro‐Lys (PK) sequence at positions 3 and 4 of yeast Rpt1, we generated mutant strains expressing an Rpt1 allele that lacks this sequence. The absence of the PK sequence abolished N‐methylation, decreased cell growth, and increased sensitivity to stress. Our data suggest that N‐methylation of Rpt1 and/or its PK sequence might be important in cell growth or stress tolerance in yeast.     

Efficient racemization of N‐phenylacetyl‐D‐glufosinate for L‐glufosinate production

Most amino acids contain chiral centres and exist as both D‐enantiomer and L‐enantiomer. The optically pure enantiomer is often more valuable than the racemate. Enzymatic resolution provides an effective strategy to obtain optically pure amino acids but often results in large amounts of unwanted isomer. In this study, optically pure L‐glufosinate (L‐PPT) was obtained by coupling amidase‐mediated hydrolysis of N‐phenylacetyl‐D,L‐glufosinate with racemization of N‐phenylacetyl‐D‐glufosinate (NPDG), which exclusively exhibits effective herbicidal properties compared with its D‐enantiomer. To improve the yield of L‐PPT, the racemization reaction conditions were optimized, and through single‐factor experiments, the optimal reaction temperature, reaction time, and mole ratio of phenylacetic acid to NPDG were determined to be 150°C, 30 minutes, and 1.5, respectively. The response surface methodology was applied to further optimize the racemization conditions, and the final yield of L‐PPT reached 96.13% with optimum reaction temperature of 154°C, reaction time of 23 minutes, and phenylacetic acid/NPDG mole ratio of 1.7, respectively. Moreover, adding a small amount of acetic anhydride further raised the yield of L‐PPT to 97.02%.     

Crystallographic studies for the chiral recognition of the novel chiral stationary phase derived from (+)‐(R)‐18‐crown‐6 tetracarboxylic acid

Chiral discrimination observed in high‐performance liquid chromatography (HPLC) with the novel chiral stationary phase (CSP‐18C6I) derived from (+)‐(R)‐18‐crown‐6 tetracarboxylic acid [(+)‐18C6H₄] was investigated by X‐ray crystallographic analysis of the complex composed of the R‐enantiomer of 1‐(1‐naphthyl)ethylamine (1‐NEA) and (+)‐18C6H₄. Mixtures of 1‐NEA (the R‐ or S‐enantiomer) and (+)‐18C6H₄ were dissolved in methanol‐water (1:1) solution and allowed to stand for crystallization. The R‐enantiomer crystallized with (+)‐18C6H₄ as a co‐crystal, although the S‐enantiomer did not. This result was in good agreement with the enantiomer elution order of 1‐NEA in CSP‐18C6I. The apparent binding constants (K_a) of the enantiomers to the (+)‐18C6H₄ obtained from ¹H‐NMR experiments also supported the above‐mentioned result. The X‐ray crystal structure of the 1:1 complex of the R‐enantiomer and (+)‐18C6H₄ indicated the four sets of hydrogen bond association between the naphthylethylammonium cation and oxygen of polyether ring or carbonyl group of (+)‐18C6H₄. Chirality 11:173–178, 1999. © 1999 Wiley‐Liss, Inc.     

Application of (S)‐TBMB carboxylic acid‐based on‐line HPLC‐exciton CD analysis to acyclic vicinal alcohols and amino alcohols

Applications of the on‐line HPLC‐exciton CD analysis using (S)‐2‐tert‐butyl‐2‐methyl‐1,3‐benzodioxole‐4‐carboxylic acid [(S)‐TBMBC‐OH] that can simultaneously determine the enantiomeric compositions and the absolute configuration of cyclohexane‐1,2‐diols and diamines as well as acyclic vicinal diols and amino alcohols were studied. Di‐O‐ or di‐N,O‐(S)‐TBMBC derivatives of acyclic terminal vicinal diols, 2‐hydroxy‐1‐amines, and nonterminal vicinal diols gave symmetrical exciton CD spectra between enantiomers, indicating their absolute configurations. However, Di‐N,O‐(S)‐TBMBC derivatives of 2‐amino‐1‐ols did not always give symmetrical exciton CD spectra between enantiomers, but their 2‐phthalimido‐1‐O‐(S)‐TBMBC derivatives gave symmetrical exciton CD spectra, indicating their absolute configurations. All these (S)‐TBMBC derivatives were separated by normal‐phase HPLC and unequivocally determined by the on‐line HPLC‐exciton CD analysis without recourse to reference samples. Chirality 11:149–159, 1999. © 1999 Wiley‐Liss, Inc.     

Crystal structure of the N‐terminal methyltransferase‐like domain of anamorsin

Anamorsin is a recently identified molecule that inhibits apoptosis during hematopoiesis. It contains an N‐terminal methyltransferase‐like domain and a C‐terminal Fe‐S cluster motif. Not much is known about the function of the protein. To better understand the function of anamorsin, we have solved the crystal structure of the N‐terminal domain at 1.8 Å resolution. Although the overall structure resembles a typical S‐adenosylmethionine (SAM) dependent methyltransferase fold, it lacks one α‐helix and one β‐strand. As a result, the N‐terminal domain as well as the full‐length anamorsin did not show S‐adenosyl‐l ‐methionine (AdoMet) dependent methyltransferase activity. Structural comparisons with known AdoMet dependent methyltransferases reveals subtle differences in the SAM binding pocket that preclude the N‐terminal domain from binding to AdoMet. The N‐terminal methyltransferase‐like domain of anamorsin probably functions as a structural scaffold to inhibit methyl transfers by out‐competing other AdoMet dependant methyltransferases or acts as bait for protein–protein interactions.Proteins 2014; 82:1066–1071. © 2013 Wiley Periodicals, Inc.     

Preparation of (2S,4R)‐2,4‐thiazolidinedicarboxylic acid by separation of diastereoisomeric salts with organic amines

L ‐Cysteine was condensed with glyoxylic acid monohydrate in acetic acid at 30°C to give (4R)‐2,4‐thiazolidinedicarboxylic acid [(4R)‐TDA] as a mixture of two diastereoisomers, (2R,4R)‐ and (2S,4R)‐TDA. An attempt was made to separate (2S,4R)‐TDA from the diastereoisomeric salts of (4R)‐TDA with 1‐propylamine, 2‐methyl‐2‐propylamine, benzylamine, and (R)‐ and (S)‐1‐phenylethylamines [(R)‐ and (S)‐PEA]. The salts of (2S,4R)‐TDA were preferentially crystallized as less soluble diastereoisomeric salts. When the salt with (R)‐PEA was employed, the separation was successfully achieved to afford optically pure (2S,4R)‐TDA in a yield of 41%, based on the starting amount of the diastereoisomeric mixture of (4R)‐TDA. Chirality 11:326–329, 1999. © 1999 Wiley‐Liss, Inc.     

Syntheses and β‐adrenergic binding affinities of (R)‐ and (S)‐fluoronaphthyloxypropanolamines

The β‐adrenergic receptors mediate several physiological processes including heart rate (β₁), bronchodilation (β₂), and lipolysis (β₃). Therefore, selectivity is important for a possible therapeutic agent acting via these receptors. Aryloxypropanolamines are β‐receptor agonists or antagonists, depending on the aryl group and its substituents. We therefore hypothesized that fluorine substitution on the aromatic ring in this class could lead to significant biological effects because of the unique chemical characteristics of fluorine. Because the target compound has a chiral center, we set out to synthesize the two enantiomers so that effects of stereochemistry on biological activity could be evaluated. Syntheses of the enantiomers were performed starting with commercially available fluoronaphthalene and subsequent use of the chiral synthon (2R)‐ or (2S)‐glycidyl 3‐nitrobenzenesulfonate, depending on the desired enantiomer. High‐pressure liquid chromatography (HPLC) methods were used to characterize %ee. Each enantiomer was synthesized. They exhibited nanomolar binding activities on β‐adrenergic receptors. The (S)‐enantiomer was found to be up to 310 times more potent than the (R). It was also found to be about five‐fold more selective for β₂‐ than for β₁‐receptors. The current report demonstrates the importance of stereochemistry for the fluoroaromatic β‐receptor ligands. Chirality 11:144–148, 1999. © 1999 Wiley‐Liss, Inc.     

Enantiomeric separation of N‐protected amino acids by non‐aqueous capillary electrophoresis using quinine or Tert‐butyl carbamoylated quinine as chiral additive

A capillary electrophoretic (CE) method for the enantioseparation of N‐protected chiral amino acids was developed using quinine and tert‐butyl carbamoylated quinine as chiral selectors added to nonaqueous electrolyte solutions (NACE). A series of various N‐derivatized amino acids were tested as chiral selectands, and in order to optimize the CE enantioseparation of these compounds, different parameters were investigated: the nature of the organic solvent, the combination of different solvents, the nature and the concentration of the background electrolyte, the selector concentration, the capillary temperature, and the applied voltage. The influence of these factors on the separation of the analyte enantiomers and the electroosmotic flow was studied. Generally, with tert‐butyl carbamoylated quinine as chiral selector, better enantioseparations were achieved than with unmodified quinine. Optimum experimental conditions were found with a buffer made of 12.5 mM ammonia, 100 mM octanoic acid, and 10 mM tert‐butyl carbamoylated quinine in an ethanol–methanol mixture (60:40 v/v). Under these conditions, DNB‐Leu enantiomers could be separated with a selectivity factor (α) of 1.572 and a resolution (Rs) of 64.3; a plate number (N) of 127,000 and an asymmetry factor (As) of 0.93 were obtained for the first migrating enantiomer. Chirality 11:622–630, 1999. © 1999 Wiley‐Liss, Inc.     

Synthesis,Anticonvulsant Activity and Metabolism of 4‐chlor‐3‐methylphenoxyethylamine Derivatives of Trans‐2‐aminocyclohexan‐1‐ol

In this study, we report the synthesis, spectral characterization, antiepileptic activity and biotransformation of three new, chiral, N‐aminoalkyl derivatives of trans – 2 aminocyclohexan‐1‐ol: 1 (R enantiomer), 2 (S enantiomer) and 3 (racemate). Antiepileptic activity of the titled compounds was studied using MES and scMet. Moreover, in this study, the biotransformation of 1 , 2 and 3 in microbial model (Cunninghamella), liver microsomal assay as well as in silico studies (MetaSite) was evaluated. Studies have indicated that 1 , 2 and 3 have good antiepileptic activity in vivo, comparable to valproate. Biotransformation assays showed that the most probable metabolite (indicated in every tested assays) was M1 . The microbial model as well as in silico study showed no difference in biotransformation between tested enantiomers. However, in a rat liver microsomal study compound 1 and 2 (R and S enantiomer) had different main metabolite – M2 for 1 and M1 for 2 . MS/MS fragmentation allowed us to predict the structures of obtained metabolites, which were in agreement with 1°alcohol ( M1 ) and carboxylic acid ( M2 ). Our research has shown that microbial model, microsomal assay, and computational methods can be included as useful and reliable tools in early ADME‐Tox assays in the process of developing new drug candidates. Chirality 27:163–169, 2015. © 2014 Wiley Periodicals, Inc.     

Synthesis of enantiomers of exo‐2‐norbornyl‐N‐n‐butylcarbamate and endo‐2‐norbornyl‐N‐n‐butylcarbamate for stereoselective inhibition of acetylcholinesterase

The acetylcholinesterase inhibition by enantiomers of exo‐ and endo‐2‐norbornyl‐N‐n‐butylcarbamates shows high stereoselelectivity. For the acetylcholinesterase inhibitions by (R)‐(+)‐ and (S)‐(?)‐exo‐2‐norbornyl‐N‐n‐butylcarbamates, the R‐enantiomer is more potent than the S‐enantiomer. But, for the acetylcholinesterase inhibitions by (R)‐(+)‐ and (S)‐(?)‐endo‐2‐norbornyl‐N‐n‐butylcarbamates, the S‐enantiomer is more potent than the R‐enantiomer. Optically pure (R)‐(+)‐exo‐, (S)‐(?)‐exo‐, (R)‐(+)‐endo‐, and (S)‐(?)‐endo‐2‐norbornyl‐N‐n‐butylcarbamates are synthesized from condensations of optically pure (R)‐(+)‐exo‐, (S)‐(?)‐exo‐, (R)‐(+)‐endo‐, and (S)‐(?)‐endo‐2‐norborneols with n‐butyl isocyanate, respectively. Optically pure norborneols are obtained from kinetic resolutions of their racemic esters by lipase catalysis in organic solvent. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Efficient one‐pot synthesis of N‐ethyl amino acids

Mono‐N‐ethylated α‐amino acid esters are obtained in high yields using reductive amination procedures. Formation of imine is achieved by excess of acetaldehyde, followed by removal of acetaldehyde and reduction by NaBH(OAc)₃. The elaborated one‐pot synthesis allows for the efficient synthesis of side‐chain protected amino acid derivatives. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

A first view on the unsuspected intragenus diversity of N‐glycans in Chlorella microalgae

Chlorella microalgae are increasingly used for various purposes such as fatty acid production, wastewater processing, or as health‐promoting food supplements. A mass spectrometry‐based survey of N‐glycan structures of strain collection specimens and 80 commercial Chlorella products revealed a hitherto unseen intragenus diversity of N‐glycan structures. Differing numbers of methyl groups, pentoses, deoxyhexoses, and N‐acetylglucosamine culminated in c. 100 different glycan masses. Thirteen clearly discernible glycan‐type groups were identified. Unexpected features included the occurrence of arabinose, of different and rare types of monosaccharide methylation (e.g. 4‐O‐methyl‐N‐acetylglucosamine), and substitution of the second N‐acetylglucosamine. Analysis of barcode ITS1–5.8S–ITS2 rDNA sequences established a phylogenetic tree that essentially went hand in hand with the grouping obtained by glycan patterns. This brief prelude to microalgal N‐glycans revealed a fabulous wealth of undescribed structural features that finely differentiated Chlorella‐like microalgae, which are notoriously poor in morphological attributes. In light of the almost identical N‐glycan structural features that exist within vertebrates or land plants, the herein discovered diversity is astonishing and argues for a selection pressure only explicable by a fundamental functional role of these glycans.     

Lewis acid catalysed methylation of N‐(9H‐fluoren‐9‐yl)methanesulfonyl (Fms) protected lipophilic α‐amino acid methyl esters

This work reports an efficient Lewis acid catalysed N‐methylation procedure of lipophilic α‐amino acid methyl esters in solution phase. The developed methodology involves the use of the reagent system AlCl₃/diazomethane as methylating agent and α‐amino acid methyl esters protected on the amino function with the (9H‐fluoren‐9‐yl)methanesulfonyl (Fms) group. The removal of Fms protecting group is achieved under the same conditions to those used for Fmoc removal. Thus the Fms group can be interchangeable with the Fmoc group in the synthesis of N‐methylated peptides using standard Fmoc‐based strategies. Finally, the absence of racemization during the methylation reaction and the removal of Fms group were demonstrated by synthesising a pair of diastereomeric dipeptides. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

A novel ATP‐dependent conformation in p97 N–D1 fragment revealed by crystal structures of disease‐related mutants

Mutations in p97, a major cytosolic AAA (ATPases associated with a variety of cellular activities) chaperone, cause inclusion body myopathy associated with Paget's disease of the bone and frontotemporal dementia (IBMPFD). IBMPFD mutants have single amino‐acid substitutions at the interface between the N‐terminal domain (N‐domain) and the adjacent AAA domain (D1), resulting in a reduced affinity for ADP. The structures of p97 N–D1 fragments bearing IBMPFD mutations adopt an atypical N‐domain conformation in the presence of Mg²⁺·ATPγS, which is reversible by ADP, showing for the first time the nucleotide‐dependent conformational change of the N‐domain. The transition from the ADP‐ to the ATPγS‐bound state is accompanied by a loop‐to‐helix conversion in the N–D1 linker and by an apparent re‐ordering in the N‐terminal region of p97. X‐ray scattering experiments suggest that wild‐type p97 subunits undergo a similar nucleotide‐dependent N‐domain conformational change. We propose that IBMPFD mutations alter the timing of the transition between nucleotide states by destabilizing the ADP‐bound form and consequently interfere with the interactions between the N‐domains and their substrates.     

Indole and its alkyl‐substituted derivatives protect erythrocyte and DNA against radical‐induced oxidation

The antioxidant properties of 1,2,3,4‐tetra‐hydrocarbazole, 6‐methoxy‐1,2,3,4‐tetrahydrocar‐bazole (MTC), 2,3‐dimethylindole, 5‐methoxy‐2,3‐dimethylindole, and indole were investigated in the case of hemolysis of human erythrocytes and oxidative damage of DNA induced by 2,2′‐azobis(2‐amidinopropane hydrochloride) (AAPH), respectively. The aim of this work was to explore the influence of methoxy, methyl, and cyclohexyl substituents on the antioxidant activities of indole derivatives. These indole derivatives were able to protect erythrocytes and DNA in a concentration‐dependent manner. The alkyl‐substituted indole can protect erythrocytes and DNA against AAPH‐induced oxidation. Especially, the structural features of cyclohexyl and methoxy substituents made MTC the best antioxidant among the indole derivatives used herein. Finally, the interaction between these indole derivatives and 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonate) radical cation and 2,2′‐diphenyl‐1‐picrylhydrazyl, respectively, provided direct evidence for these indole derivatives to scavenge radicals and emphasized the importance of electron‐donating groups for the free radical–scavenging activity of indole derivatives. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:273–279, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20289     

The determination of enantiomer composition of 1‐((3‐chlorophenyl)‐(phenyl)methyl) amine and 1‐((3‐chlorophenyl)(phenyl)‐methyl) urea (Galodif) by NMR spectroscopy,chiral HPLC,and polarimetry

For the first time, a method for enantiomer resolution of the anticonvulsant Galodif (1‐((3‐chlorophenyl)(phenyl)methyl) urea) by chiral HPLC was developed, whereas the enantiomeric composition of 1‐((3‐chlorophenyl)(phenyl)methyl) amine—precursor in Galodif synthesis—cannot be resolved by this method. However, starting 1‐((3‐chlorophenyl)(phenyl)methyl) amine quantitatively forms diastereomeric N‐((3‐chlorophenyl)(phenyl)methyl)‐1‐camphorsulfonamides in reaction with chiral (1R)‐(+)‐ or (1S)‐(?)‐camphor‐10‐sulfonyl chlorides. The diastereomeric ratio of obtained camphorsulfonamides can be easily determined by NMR ¹H and ¹³C spectroscopy. The DFT calculations of specific rotation of Galodif enantiomers showed good agreement with experimental data. The absolute configuration of enantiomers was proposed for the first time.     

A novel endo‐β‐N‐acetylglucosaminidase releases specific N‐glycans depending on different reaction conditions

Milk glycoproteins are involved in different functions and contribute to different cellular processes, including adhesion and signaling, and shape the development of the infant microbiome. Methods have been developed to study the complexities of milk protein glycosylation and understand the role of N‐glycans in protein functionality. Endo‐β‐N‐acetylglucosaminidase (EndoBI‐1) isolated from Bifidobacterium longum subsp. infantis ATCC 15697 is a recently isolated heat‐stable enzyme that cleaves the N‐N′‐diacetyl chitobiose moiety found in the N‐glycan core. The effects of different processing conditions (pH, temperature, reaction time, and enzyme/protein ratio) were evaluated for their ability to change EndoBI‐1 activity on bovine colostrum whey glycoproteins using advanced mass spectrometry. This study shows that EndoBI‐1 is able to cleave a high diversity of N‐glycan structures. Nano‐LC‐Chip–Q‐TOF MS data also revealed that different reaction conditions resulted in different N‐glycan compositions released, thus modifying the relative abundance of N‐glycan types. In general, more sialylated N‐glycans were released at lower temperatures and pH values. These results demonstrated that EndoBI‐1 is able to release a wide variety of N‐glycans, whose compositions can be selectively manipulated using different processing conditions. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1323–1330, 2015     

Functional Roles of N‐Linked Glycosylation of Human Matrix Metalloproteinase 9

Matrix metalloproteinase‐9 (MMP‐9) is a secreted endoproteinase with a critical role in the regulation of the extracellular matrix and proteolytic activation of signaling molecules. Human (h)MMP‐9 has two well‐defined N‐glycosylation sites at residues N38 and N120; however, their role has remained mostly unexplored partly because expression of the N‐glycosylation‐deficient N38S has been difficult due to a recently discovered single nucleotide polymorphism‐dependent miRNA‐mediated inhibitory mechanism. hMMP‐9 cDNA encoding amino acid substitutions at residues 38 (modified‐S38, mS38) or 120 (N120S) were created in the background of a miRNA‐binding site disrupted template and expressed by transient transfection. hMMP‐9 harboring a single mS38 replacement secreted well, whereas N120S, or a double mS38/N120S hMMP‐9 demonstrated much reduced secretion. Imaging indicated endoplasmic reticulum (ER) retention of the non‐secreted variants and co‐immunoprecipitation confirmed an enhanced strong interaction between the non‐secreted hMMP‐9 and the ER‐resident protein calreticulin (CALR). Removal of N‐glycosylation at residue 38 revealed an amino acid‐dependent strong interaction with CALR likely preventing unloading of the misfolded protein from the ER chaperone down the normal secretory pathway. As with other glycoproteins, N‐glycosylation strongly regulates hMMP‐9 secretion. This is mediated, however, through a novel mechanism of cloaking an N‐glycosylation‐independent strong interaction with the ER‐resident CALR.      

Phosphorylation stabilizes the N‐termini of α‐helices

The role of phosphorylation in stabilizing the N‐termini of α‐helices is examined using computer simulations of model peptides. The models comprise either a phosphorylated or unphosphorylated serine at the helix N‐terminus, followed by nine alanines. Monte Carlo/stochastic Dynamics simulations were performed on the model helices. The simulations revealed a distinct stabilization of the helical conformation at the N‐terminus after phosphorylation. The stabilization was attributable to favorable electrostatic interactions between the phosphate and the helix backbone. However, direct helix capping by the phosphorylated sidechain was not observed. The results of the calculations are consistent with experimental evidence on the stabilization of helices by phosphates and other anions. © 1999 John Wiley & Sons, Inc. Biopoly 49: 225–233, 1999     

Comparative Enantioseparation of Ketoprofen with Trimethylated α‐, β‐, and γ‐Cyclodextrins in Capillary Electrophoresis and Study of Related Selector–Selectand Interactions Using Nuclear Magnetic Resonance Spectroscopy

The enantiomers of ketoprofen were separated by capillary electrophoresis using the (2,3,6‐tri‐O‐methyl)‐derivatives of α‐, β‐, and γ‐cyclodextrin (CyD) as chiral selectors. The affinity pattern of the ketoprofen enantiomers toward these CyDs changed depending on their cavity size. Thus, with hexakis (2,3,6‐tri‐O‐methyl)‐α‐CyD and heptakis (2,3,6‐tri‐O‐methyl)‐β‐CyD, the R enantiomer of the drug migrated first, whereas the enantiomer migration order was reversed in the presence of octakis(2,3,6‐tri‐O‐methyl)‐γ‐CyD. The change in the migration order was rationalized on the basis of changes in the structure of the complexes between the ketoprofen enantiomers and the chiral selectors as derived from nuclear magnetic resonance spectroscopy experiments. Chirality, 25:79–88, 2013.© 2012 Wiley Periodicals, Inc.