Conformation and sweet tastes of L-aspartyl dipeptide methyl esters

In order to investigate the conformational preferences to elicit tastes, conformational free energy calculations using an empirical potential (ECEPP/2) and the hydration shell model were carried out on the L -aspartyl dipeptide methyl esters, L -⁺HAsp^?-L -Xaa-OMe, in the hydrated state, where Xaa includes sweet (Phe, Tyr, Met, and Gly), bitter (Ala, Trp, Val, Leu, and Ile), and tasteless (Ser, Thr, and Abu) residues. The refined preferred conformation of the Phe dipeptide (aspartame) with side chain χ conformation g^? is g^?Fg^? in the hydrated state, which is consistent with the structure deduced from ¹H-nmr experiments. Irrespective of the Xaa and taste, all the dipeptides have the same conformation for the Asp residue, which is attributable to the hydrogen bond between protonated amino hydrogen and carboxylate oxygen and the favored hydration of the carboxylate group. This implies that the L -aspartyl residue is a necessary factor for the dipeptides to be sweet not a sufficient factor. The computed conformational preferences for sweet, bitter, and tasteless dipeptides in the hydrated state indicate to us that the conformation about the N? C^α bond of the Xaa residue, i.e., the orientation of the hydrophobic moiety with respect to the AH/B functionalities in the aspartyl moiety, seems to be crucial to elicit the tastes. In addition, the hydrophobicity and the size of the Xaa residue are found to play a major role in determining the tastes. These well accord with the related works reported previously. © 1994 John Wiley & Sons, Inc.     

Simulated dipeptide recognition by vancomycin

The antimicrobial activity of vancomycin and related glycopeptide antibiotics is due to stereospecific recognition of polypeptide components in bacterial cell walls. To better understand how these antibiotics recognize polypeptide determinants, we have developed dynamic models of the complexes formed by the vancomycin aglycon and two different dipeptide ligands, Ac-D-ala-D-ala and Ac-D-ala-gly. Molecular dynamics simulations of the two complexes, initially conditioned with distance constraints derived from two-dimensional nuclear magnetic resonance (NMR) studies, are conformationally stable and propagate in a manner consistent with the NMR-derived constraints after the constraints are removed. Free energy calculations accurately predict the relative binding affinity of these two complexes and help validate the simulation models for detailed structural analysis. Although the two ligands adopt similar conformations when bound to the antibiotic, there are clear differences in the configuration of intermolecular hydrogen bonds, the overall shape of the antibiotic, and other structural features of the two complexes. This analysis illustrates how complex structural and dynamic factors interrelate and contribute to differences in binding affinity. © 1997 John Wiley & Sons, Ltd.     

Quantitative structure-activity relationships (QSAR) in sweet aspartyl dipeptide methyl esters

In drug design the pharmacochemists frequently use physicochemicalconstants to correlate the structure with the observed potency.Curiously this approach has hardly been followed by psychophysiciststo indicate the increase of taste potency in a series of structurallyrelated compounds of the same stimulus. In the present experiments we correlated the relative sweetness(S) of 40 aspartyl dipeptide methyl esters [general formulaCH₂(COO^–). CH(NH₃⁺ ).CO.NH.CH(R).COOCH₃] with 8 physicochemicalparameters. Among the compounds we had 7 non-sweet stimuli whilethe potency of the remaining 33 peptide esters varied from 1to 27,000 (1 = sucrose). We calculated for the side chain Rthe values of the parachor parameter P, the hydrophobic fragmentalconstant f and 5 STERIMOL parameters (L, B₁ up to B₅). A multipleregression analysis programme selected by stages the most relevantparameters and tested their significance. We observed that the criterion whether a dipeptide ester issweet or not, is among others defined by the L and B₅ parametersof the side chain R. Compounds are sweet provided L is confinedto certain limits (0.50 nm<L<0.62 nm), or otherwise whenL exceeds these limits, the B₅ parameter has to be greater than0.45 nm (when L<0.50 nm) or smaller than 0.72 nm (when L>0.62nm). The sweet potency defined as log S correlated very significantlywith the parameters P and B₄ (n=33, r=0.812, s=0.60, F=29.06).When two compounds, which were shown to be situated at the borderlineof the length and volume parameters, were omitted in the analysis,the correlation improved (n=31, r=0.909, s=0.40, F=42.60). Inthe latter situation we found the following equation when theintercept was set at zero: log S=0.194f + 1.472.10^–2P—3.357B₅ A previously proposed conformation of aspartame (R=CH₂-Øat the receptor site was computed in detail. We calculated thedistances of the AH-B moieties to the third binding site (thecentre of Ø) and indicated the width of the receptoraccess for this series of sweet, structurally related, dipeptidemethyl esters.     

Nanoparticles of beta-cyclodextrin esters obtained by self-assembling of biotransesterified beta-cyclodextrins

The synthesis of decanoate beta-cyclodextrin esters (beta-CDd) and hexanoate beta-cyclodextrin esters (beta-CDh) was biocatalyzed by thermolysin from native beta-cyclodextrin (beta-CD) and vinyl hexanoate or vinyl decanoate used as acyl donors. The products were chemically characterized by infrared, NMR, and mass spectrometry. Both beta-CDd and beta-CDh esters were identified as a mixture of beta-CD preferentially substituted on the C2 position by the corresponding acyl chain. The degree of substitution varied from 2 to 7 for beta-CDd and from 4 to 8 for beta-CDh. The ability of beta-CD esters to self-organize into nanoparticles was tested using a nanoprecipitation technique in various solvents. The mean size diameter and polydispersity measured by quasi-elastic light scattering were dramatically affected by the nature of solvent (acetone, ethanol, or tetrahydrofuran) used in the nanoprecipitation technique. When directly observed using cryo-transmission electron microscopy, beta-CDh appeared as uniformly dense nanospheres, whereas beta-CDd exhibited a multilamellar onion-like organization. A structural model was rationalized for the beta-CDd nanoparticles.     

Chiral recognition of ethers by NMR spectroscopy

Enantiomers of chiral ethers and acetals are notoriously difficult to differentiate because their reactivity is low and they are poor donors to any Lewis acid or metal ion. As an exception, epoxides are somewhat better donors. This review describes the properties of ethers, explains NMR methods for their chiral recognition and describes successful examples of ether differentiation. The majority of literature reports deals with chiral lanthanide shift reagents and dirhodium tetracarboxylate complexes, which were used as enantiopure auxiliaries to create diastereomeric adducts with dispersed (1)H and (13)C NMR signals. The various methods are compared as to which is best suited for which purpose.     

Interference of rheumatoid factor activity by aspartame,a dipeptide methyl ester

Circulating autoimmune complexes of IgM rheumatoid factors (RF) bound to the Fc portions of normal, polyclonal IgG antibodies are frequently present in humans with rheumatoid arthritis (RA). The sweet tasting methyl ester of L ‐Asp‐L ‐Phe (aspartame or APM) was found to relieve pain and improve joint mobility in subjects with osteo‐ and mixed osteo/rheumatoid arthritis [Edmundson, A. B. and Manion, C. V. ( 1998 ). Clin. Pharmac. Ther. 63 , 580–593]. These clinical observations prompted the testing of the inhibition by APM of the binding interactions of human IgM RFs with IgG Fc regions. The propensity of APM to inhibit IgM RF binding was assessed by competitive enzyme immunoassays with solid‐phase human IgG. Ten RA serum samples and three purified monoclonal cryoglobulins, all of which had RF activity, were tested in this system. We found that the presence of APM significantly reduced the binding of IgM RFs. The inhibitory propensity of APM with monoclonal RF cryoglobulins was increased by the addition of CaCl₂ to the binding buffer. Similar inhibition of the binding of RA derived RFs to IgG was observed for Asp–Phe and its amidated derivative, indicating that the methyl ester is not required for APM's interaction with IgM antibodies. A human (Mez) IgM known to bind octameric peptides derived from the Fc portion of a human IgG₁ antibody was tested for binding of dipeptides by the Pepscan method of combinatorial chemistry. The relative binding constants of Asp–Phe and Phe–Asp were ranked among the highest values for 400 possible combinations of the 20 most common amino acids. Possible blocking interactions of APM were explored by computer‐assisted docking studies with the model of a complex of an RF Fab with the Fc of a human IgG₄ antibody. Modeling of ternary immune complexes revealed a few key residues, which could act as molecular recognition sites for APM. A structural hypothesis is presented to explain the observed interference with RF reactivity by APM. Extrapolations of the current results suggest that APM may inhibit the binding of IgG in a substantial proportion of IgM RFs. Interference of RF reactivity, especially in RA patients, may alleviate the pain and immobility resulting from chronic inflammation of the joints. Copyright © 1999 John Wiley & Sons, Ltd.     

Interference of rheumatoid factor activity by aspartame, a dipeptide methyl ester

Circulating autoimmune complexes of IgM rheumatoid factors (RF) bound to the Fc portions of normal, polyclonal IgG antibodies are frequently present in humans with rheumatoid arthritis (RA). The sweet tasting methyl ester of L-Asp-L-Phe (aspartame or APM) was found to relieve pain and improve joint mobility in subjects with osteo- and mixed osteo/rheumatoid arthritis [Edmundson, A. B. and Manion, C. V. (1998). Clin. Pharmac. Ther. 63, 580-593]. These clinical observations prompted the testing of the inhibition by APM of the binding interactions of human IgM RFs with IgG Fc regions. The propensity of APM to inhibit IgM RF binding was assessed by competitive enzyme immunoassays with solid-phase human IgG. Ten RA serum samples and three purified monoclonal cryoglobulins, all of which had RF activity, were tested in this system. We found that the presence of APM significantly reduced the binding of IgM RFs. The inhibitory propensity of APM with monoclonal RF cryoglobulins was increased by the addition of CaCl(2) to the binding buffer. Similar inhibition of the binding of RA derived RFs to IgG was observed for Asp-Phe and its amidated derivative, indicating that the methyl ester is not required for APM's interaction with IgM antibodies. A human (Mez) IgM known to bind octameric peptides derived from the Fc portion of a human IgG(1) antibody was tested for binding of dipeptides by the Pepscan method of combinatorial chemistry. The relative binding constants of Asp-Phe and Phe-Asp were ranked among the highest values for 400 possible combinations of the 20 most common amino acids. Possible blocking interactions of APM were explored by computer-assisted docking studies with the model of a complex of an RF Fab with the Fc of a human IgG(4) antibody. Modeling of ternary immune complexes revealed a few key residues, which could act as molecular recognition sites for APM. A structural hypothesis is presented to explain the observed interference with RF reactivity by APM. Extrapolations of the current results suggest that APM may inhibit the binding of IgG in a substantial proportion of IgM RFs. Interference of RF reactivity, especially in RA patients, may alleviate the pain and immobility resulting from chronic inflammation of the joints.     

Pattern recognition by TREM-2: binding of anionic ligands

We recently described the cloning of murine triggering receptor expressed by myeloid cells (TREM) 2, a single Ig domain DNAX adaptor protein 12-associated receptor expressed by cells of the myeloid lineage. In this study, we describe the identification of ligands for TREM-2 on both bacteria and mammalian cells. First, by using a TREM-2A/IgG1-Fc fusion protein, we demonstrate specific binding to a number of Gram-negative and Gram-positive bacteria and to yeast. Furthermore, we show that fluorescently labeled Escherichia coli and Staphylococcus aureus bind specifically to TREM-2-transfected cells. The binding of TREM-2A/Ig fusion protein to E. coli can be inhibited by the bacterial products LPS, lipoteichoic acid, and peptidoglycan. Additionally, binding can be inhibited by a number of other anionic carbohydrate molecules, including dextran sulfate, suggesting that ligand recognition is based partly on charge. Using a sensitive reporter assay, we demonstrate activation of a TREM-2A/CD3zeta chimeric receptor by both bacteria and dextran sulfate. Finally, we demonstrate binding of TREM-2A/Ig fusion to a series of human astrocytoma lines but not to a variety of other cell lines. The binding to astrocytomas, like binding to bacteria, is inhibited by anionic bacterial products, suggesting either a similar charge-based ligand recognition method or overlapping binding sites for recognition of self- and pathogen-expressed ligands.     

Improved sensitivity of iodinated histamine-prostaglandin radioimmunoassay by prostaglandin methyl esters

Use of (¹²⁵I)-labeled histamine-prostaglandin tracer increases the sensitivity of the radioimmunoassays of prostaglandin derivatives. Six different antisera were produced for prostaglandins and their derivatives (prostaglandins E₁, E₂, F_1α, F_2α, 13,14-dihydro-15-ketoprostaglandin E₂, and 13,14-dihydro-15-ketoprostaglandin F_2α) and were investigated with the corresponding tritiated and lodinated tracers. Displacement of iodinated tracers by the methyl esters of the prostaglandin compounds resulted, in most cases, in a three- to fivefold increase in sensitivity compared to unesterified inhibitors. Esterification also caused some alteration in the specificities observed. Our results suggest that conformational changes in the esterified prostaglandins (tracer and inhibitor) could explain these charges.     

Uptake of long-chain fatty acid methyl esters by mammalian cells

Albumin-bound long-chain fatty acid methyl esters (ME) were taken up and utilized by Ehrlich ascites tumor cells and slices of rat heart, liver, and kidney. Much more ME than albumin was taken up by the tumor cells, indicating that ME dissociated from the carrier protein during their uptake. 70-80% of the radioactivity associated with the cells after 1 min of incubation at 37 degrees C remained as ME. The results of studies with metabolic inhibitors and glucose suggest that uptake of ME is an energy-independent process. Changes in incubation medium pH between 7.8 and 6.5 did not markedly alter uptake of ME. Cells incubated with FFA and methanol did not synthesize ME. These findings indicate that ME are taken up intact, and they suggest that the presence of an anionic carboxyl group is not essential for the binding of a long-chain aliphatic hydrocarbon to a mammalian cell. When incubation with labeled ME was continued for 1 hr, increasing amounts of radioactivity were recovered in FFA, phospholipids, neutral lipid esters, and CO(2). ME radioactivity associated with the cells after a brief initial incubation was released in the form of ME and FFA when the cells were incubated subsequently in a medium containing albumin. If the second incubation medium contained no albumin, most of the ME radioactivity initially associated with the cells was incorporated into phospholipids, neutral lipid esters, and CO(2). These results suggest that much of the ME which is taken up, is hydrolyzed to FFA, and that the fatty acids derived from ME are available for further metabolism.     

Purification of fatty acid methyl esters by high-performance liquid chromatography

The determination by gas chromatography (GC) of fatty acid methyl esters (FAMEs) prepared from complex biological samples is subject to interference from cholesterol. During sample injection on the GC system of FAMEs prepared from tissues that contain cholesterol, we observed a major contaminant that co-eluted with docosahexaenoic acid (DHA, 22:6n-3). To address this problem, FAMEs were purified on an amino-phase high-performance liquid chromatography (HPLC) column using a hexane–isopropanol gradient. The HPLC retention times for both the FAME fraction and cholesterol were stable and reproducible when the amino column was used for sample purification. The purified extracts were analyzed by GC without artifacts or impurity peaks after 50 analytical runs. The method described here will be useful for measurement of 22:6n-3 and other fatty acids important for studies of nutrition or pathology.     

Borate esters of the methyl D-glucopyranosides

Methyl alpha- and beta-D-glucopyranoside act as moderate chelators of a boron centre through their O-4/6 binding site whereas the trans/trans arrangement of the O-2/3/4 hydroxy functions is not suited to form a chelate with the small boron central atom. In this work we report the crystal structure of the bisdiolatoborate Na2[B(Me alpha-D-Glcp4,6H(-2))(Me alpha-D-Glcp3,4,6H(-3))].NaOH.8H2O (1) along with a combined 11B and 13C NMR spectroscopic studies of borate-pyranoside solutions at different concentrations and pH. It is shown that crystallisation of a bisdiolatoborate needs both a high pH and a high total concentration.     

Preparation, X-ray crystal structure, and 195Pt NMR spectra of platinum(II) with dipeptides and dipeptide methyl esters

Three dipeptide complexes of the form K[M(dipeptide)Cl] (H2dipeptide=glycylbeta-alanine, beta-alanylglycine, beta-alanylbeta-alanine) and four dipeptide methyl ester complexes of the form K[M(dipeptideOMe)Cl2] (HdipeptideOMe=glycylalpha-alanine methyl ester, alpha-alanylglycine methyl ester, dialpha-alanine methyl ester) were newly prepared. The K[Pt(glybeta-ala)Cl] complex crystallizes in the monoclinic space group C2/c with unit cell dimensions of a=25.77(1) A, b=4.09(2) A, c= 16.432(9) A, beta=103.74(4) degrees, and Z=8. The K[Pt(glyalpha-alaOMe)Cl2] complex crystallizes in the monoclinic space group P1 with unit cell dimensions of a=7.195(2) A, b=7.977(5) A, c=10.326(3) A, alpha=72.49(3) degrees, beta=103.74(4) degrees, gamma=88.27(4) degrees and Z=2. The 195Pt NMR peaks of the complexes containing the beta-alanine moiety appeared significantly more upfield than those of the complexes containing diglycine. The ratios of the species of the platinum complexes containing the dipeptide ester in neutral solution were significantly different from those in alkaline solution at 40 degrees C for a short time.     

Chiral recognition of diastereomeric 6-cedrols by vibrational circular dichroism

The use of vibrational circular dichroism spectroscopy for the chiral recognition of the two epimers of 6-cedrol, tricyclic sesquiterpenes, which contains oxygen as the heaviest atom, is shown. Bands in the 1500-850 cm(-1) region of the spectra were analyzed to calculate the anisotropy factors (g), which provided the regions of maximum circular dichroism effect for each epimer.     

Chiral recognition of cyclic alpha-hydroxyketones by CD-sensitive zinc tetraphenylporphyrin tweezer

A combined chemical/chiroptical microscale protocol for the determination of absolute configurations of cyclic alpha-hydroxyketones is described. The hydroxyl group in cyclic alpha-hydroxyketones is converted into (3-aminopropylamino)acetate (NH2CH2CH2CH2NHCH2COOR), or more generally, according to a newly developed protocol, into (3-hydroxypropylamino)acetate group (HOCH2CH2CH2NHCH2COOR). The resultant conjugated compound forms a 1:1 host-guest complex with a dimeric zinc porphyrin tweezer, which exhibits exciton-coupled bisignate CD spectrum centered around the 420-nm porphyrin Soret band due to induced helicity between the two porphyrins in the complex. The absolute configurations of the alpha-stereogenic center is then determined by comparison of the sign of the observed CD exciton couplet of the complex with that of the preferred porphyrin twist predicted by the Merck Molecular Force Field (MMFFs) method.