The effect of casein glycomacropeptide versus free synthetic amino acids for early treatment of phenylketonuria in a mice model.

INTRODUCTION: Management of phenylketonuria (PKU) is mainly achieved through dietary control with limited intake of phenylalanine (Phe) from food, supplemented with low protein (LP) food and a mixture of free synthetic (FS) amino acids (AA) (FSAA). Casein glycomacropeptide (CGMP) is a natural peptide released in whey during cheese making by the action of the enzyme chymosin. Because CGMP in its pure form does not contain Phe, it is nutritionally suitable as a supplement in the diet for PKU when enriched with specific AAs. Lacprodan® CGMP-20 (= CGMP) used in this study contained only trace amounts of Phe due to minor presence of other proteins/peptides. OBJECTIVE: The aims were to address the following questions in a classical PKU mouse model: Study 1, off diet: Can pure CGMP or CGMP supplemented with Large Neutral Amino Acids (LNAA) as a supplement to normal diet significantly lower the content of Phe in the brain compared to a control group on normal diet, and does supplementation of selected LNAA results in significant lower brain Phe level?. Study 2, on diet: Does a combination of CGMP, essential (non-Phe) EAAs and LP diet, provide similar plasma and brain Phe levels, growth and behavioral skills as a formula which alone consist of FSAA, with a similar composition?. MATERIAL AND METHODS: 45 female mice homozygous for the Pahenu2 mutation were treated for 12 weeks in five different groups; G1(N-CGMP), fed on Normal (N) casein diet (75%) in combination with CGMP (25%); G2 (N-CGMP-LNAA), fed on Normal (N) casein diet (75%) in combination with CGMP (19,7%) and selected LNAA (5,3% Leu, Tyr and Trp); G3 (N), fed on normal casein diet (100%); G4 (CGMP-EAA-LP), fed on CGMP (70,4%) in combination with essential AA (19,6%) and LP diet; G5 (FSAA-LP), fed on FSAA (100%) and LP diet. The following parameters were measured during the treatment period: Plasma AA profiles including Phe and Tyr, growth, food and water intake and number of teeth cut. At the end of the treatment period, a body scan (fat and lean body mass) and a behavioral test (Barnes Maze) were performed. Finally, the brains were examined for content of Phe, Tyr, Trp, dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT) and 5-hydroxyindole-acetic acid (5-HIAA), and the bone density and bone mineral content were determined by dual-energy x-ray absorptiometry. RESULTS: Study 1: Mice off diet supplemented with CGMP (G1 (N-CGMP)) or supplemented with CGMP in combination with LNAA (G2 (N-CGMP-LNAA)) had significantly lower Phe in plasma and in the brain compared to mice fed only casein (G3 (N)). Extra LNAA (Tyr, Trp and Leu) to CGMP did not have any significant impact on Phe levels in the plasma and brain, but an increase in serotonin was measured in the brain of G2 mice compared to G1. Study 2: PKU mice fed with mixture of CGMP and EAA as supplement to LP diet (G4 (CGMP-EAA-LP)) demonstrated lower plasma-Phe levels but similar brain- Phe levels and growth as mice fed on an almost identical combination of FSAA (G5 (FSAA-LP)). CONCLUSION: CGMP can be a relevant supplement for the treatment of PKU.

Stereoselective and Divergent Construction of ß-Thiolated/Selenolated Amino Acids via Photoredox-Catalyzed Asymmetric Giese Reaction.

Sulfur and selenium occupy a distinguished position in biology owing to their redox activities, high nucleophilicity, and acyl transfer capabilities. Thiolated/selenolated amino acids, including cysteine, selenocysteine, and their derivatives, play critical roles in regulating the conformation and function of proteins and serve as an important motif for peptide design and bioconjugation. Unfortunately, a general and concise method to attain enantiopure ß-thiolated/selenolated amino acids remains an unsolved problem. Herein, we present a photoredox-catalyzed asymmetric method for the preparation of enantiopure ß-thiolated/selenolated amino acids using a simple chiral auxiliary, which controls the diastereoselectivity of the key alkylation step and acts as an orthogonal protecting group in the subsequent peptide synthesis. Our protocol can be used to prepare a wide range of ß-thiolated/selenolated amino acids on a gram scale, which would otherwise be difficult to obtain using conventional methods. The effect of our chemistry was further highlighted and validated through the preparation of a series of peptidyl thiol/selenol analogues, including cytochrome c oxidase subunit protein 7C and oxytocin.

Isolation from Stevia rebaudiana of DMDP acetic acid, a novel iminosugar amino acid: synthesis and glycosidase inhibition profile of glycine and ß-alanine pyrrolidine amino acids.

DMDP acetic acid [N-carboxymethyl-2,5-dideoxy-2,5-imino-D-mannitol] 5 from Stevia rebaudiana is the first isolated natural amino acid derived from iminosugars bearing an N-alkyl acid side chain; it is clear from GCMS studies that such derivatives with acetic and propionic acids are common in a broad range of plants including mulberry, Baphia, and English bluebells, but that they are very difficult to purify. Reaction of unprotected pyrrolidine iminosugars with aqueous glyoxal gives the corresponding N-acetic acids in very high yield; Michael addition of both pyrrolidine and piperidine iminosugars and that of polyhydroxylated prolines to tert-butyl acrylate give the corresponding N-propionic acids in which the amino group of ß-alanine is incorporated into the heterocyclic ring. These easy syntheses allow the identification of this new class of amino acid in plant extracts and provide pure samples for biological evaluation. DMDP N-acetic and propionic acids are potent α-galactosidase inhibitors in contrast to potent ß-galactosidase inhibition by DMDP.

Direct Catalytic Asymmetric Synthesis of Trifluoromethylated γ-Amino Esters/Lactones via Umpolung Strategy.

Enabled by the discovery of new cinchonium salts and coadditives, a direct and efficient asymmetric access to trifluoromethylated γ-amino esters/lactones has been realized through the enantioselective and diastereoselective umpolung reaction of trifluoromethyl imines with acrylates or α,ß-unsaturated lactones as carbon electrophiles. At 0.5-5.0 mol % catalyst loadings, the newly developed catalytic system activates a variety of imine substrates as unconventional nucleophiles to mediate highly chemo-, regio-, diastereo-, and enantioselective C-C bond forming reactions. The developed synthetic protocol represents an excellent strategy to target a series of versatile and enantiomerically enriched γ-amino esters/lactones in good to excellent yields from the readily available starting materials. Additionally, we found that the epi-vinyl catalysts based on cinchonidine and quinine promote a similarly high enantioselective reaction generating the opposite configuration of chiral products in a highly efficient manner, which allows convenient access to either the R- or S-enantiomer of the chiral amine products in high yields and excellent enantioselectivities.

Regiodivergent Ring-Opening Cross-Coupling of Vinyl Aziridines with Phosphorus Nucleophiles: Access to Phosphorus-Containing Amino Acid Derivatives.

Catalytic ring-opening phosphonation and phosphatation of vinyl aziridines have been developed in a regiodivergent fashion, giving linear and branched products. Generation of P-centered radicals enables SN2'-type ring-opening reactions of vinyl aziridines to afford δ-amino alkylphosphorus products at room temperature. On the other hand, in situ generated phosphate anions via the Ag-catalyzed aerobic oxidation of phosphonyl reactants underwent SN2 reaction to provide branched phosphorus-containing amine products. Furthermore, this divergent methodology serves as a powerful tool for the stereospecific synthesis of phosphorus-containing amino acid derivatives.

Amino acid and peptide prodrugs of diphenylpropanones positive allosteric modulators of α7 nicotinic receptors with analgesic activity.

α7 Nicotinic acetylcholine receptors (nAChRs) are ion channels implicated in a number of CNS pathological processes, including pain and psychiatric, cognitive and inflammatory diseases. Comparing with orthosteric agonism, positive allosteric modulation of these channels constitutes an interesting approach to achieve selectivity versus other nicotinic receptors. We have recently described new chalcones and 1,3-diphenylpropanones as positive allosteric modulators (PAMs) of α7 nAChRs, which proved to have good analgesic activities but poor pharmacokinetic properties. Here we report the preparation of amino acid and peptide derivatives as prodrugs of these modulators with the aim of improving their in vivo biological activity. While the valine derivative showed very short half life in aqueous solutions to be considered a prodrug, Val-Val and Val-Pro-Val are suitable precursors of the parent 1,3-diphenylpropanones, via chemical and enzymatic transformation, respectively. Compounds 19 (Val-Val) and 21 (Val-Pro-Val), prodrugs of the 2',5',4-trihydroxy-1,3-diphenylpropan-1-one 3, showed significant antinociceptive activity in in vivo assays. The best compound, 21, displayed a better profile in the analgesia test than its parent compound 3, exhibiting about the same potency but long-lasting effects.

Catalytic amidolysis of amino acid p-nitroanilides using transition state analogue imprinted artificial enzymes: Cooperative effect of pyridine moiety.

Enzyme-like polymer catalysts with the imprints of phosphonate transition state analogue (TSA) lined along with imidazole and pyridine moieties were synthesized using methacryloyl-l-histidine and 4-vinylpyridine as the functional monomers and phenyl-1-(N-benzyloxycarbonylamino)-2-(phenyl)ethyl phosphonate - the TSA of hydrolytic reaction as the template for the amidolysis of N-benzyloxycarbonyl-l-phenylalanine p-nitroanilide (Z-l-Phe-PNA). Polymers containing different functional groups can act together to provide catalytic activity and selectivity superior to what can be obtained from monofunctional analogues. The higher rate acceleration exhibited by the bifunctional polymer over the monofunctional polymers indicates cooperative catalysis of imidazole and pyridine moieties. The optimum catalytic competence is shown by the bifunctional polymer containing imidazole and pyridine moieties in 2:1M ratio which may be due to alignment of the functional groups in proper H-bond distance. In addition to the non-covalent interactions like hydrogen bonding or π-stacking interactions between the functional groups of the polymer and the template, 3D-microcavities complementary to the geometry of the template are necessary for effective shape selective binding. Michaelis-Menten kinetics implies that only the catalysts with imidazole moieties act as enzyme-like catalysts and imidazole is the key catalytic function of the enzyme mimics.

Present Global Situation of Amino Acids in Industry.

At present, amino acids are widely produced and utilized industrially. Initially, monosodium glutamate (MSG) was produced by extraction from a gluten hydrolysate. The amino acid industry started using the residual of the lysate. The discovery of the functions of amino acids has led to the expansion of their field of use. In addition to seasoning and other food use, amino acids are used in many fields such as animal nutrients, pharmaceuticals, and cosmetics. On the other hand, the invention of the glutamate fermentation process, followed by the development of fermentation methods for many other amino acids, is no less important. The supply of these amino acids at a low price is very essential for their industrial use. Most amino acids are now produced by fermentation. The consumption of many amino acids such as MSG or feed-use amino acids is still rapidly increasing.

Design and synthesis of chiral 2H-chromene-N-imidazolo-amino acid conjugates as aldose reductase inhibitors.

Aldose reductase (ALR2) inhibitors provide a viable mode to fight against diabetic complications. ALR2 exhibit plasticity in the active site vicinities and possible shifts in the nearby two supporting alpha helices. Therefore, a novel series of amino acid conjugates of chromene-3-imidazoles (13-15) were designed and synthesized based on natural isoflavonoids. The compounds were identified on the basis of spectral (1H NMR, 13C NMR and MS) data and tested in vitro for ALR2 inhibitory activity with an IC50 value ranges from 0.031 ± 0.082 µM to 4.29 ± 0.55 µM. Our in silico and biochemical studies confirmed that 15e has the best inhibition activity among the synthesized compounds with a high selective index against the Aldehyde reductase (ALR1). Supplementation of 15e to STZ induced rats decreased the blood glucose levels and delayed the progression of cataract in a dose-dependent manner. The present study thus provides novel series of compounds with a promising inhibitor to prevent or delay the cataract progression.

The Synthesis and Evaluation of Arctigenin Amino Acid Ester Derivatives.

The use of arctigenin (ARG), a traditional medicine with many pharmacological activities, has been restricted due to its poor solubility in water. Five amino acid derivatives of ARG have been synthesized using glycine, o-alanine, valine, leucine, and isoleucine, which have t-butyloxy carbonyl (BOC) as a protective group. In this study, we examined the effects of removing these protective groups. The results showed that the amino acid derivatives have better solubility and nitrite-clearing ability than ARG. Among the compounds tested, the amino acid derivatives without protective group were the best. Based on these results, ARG and its two amino acid derivatives without protective group (ARG8, ARG10) were selected to evaluate their anti-tumor activity in vivo at a dosage of 40 mg/kg. The results indicated that ARG8 and ARG10 both exhibit more anti-tumor activity than ARG in H22 tumor-bearing mice. The tumor inhibition rates of ARG8 and ARG10 were 69.27 and 43.58%, which was much higher than ARG. Furthermore, the mice treated with these compounds exhibited less damage to the liver, kidney and immune organs compared with the positive group. Furthermore, ARG8 and ARG10 improved the serum cytokine levels significantly compared to ARG. In brief, this study provides a method to improve the water solubility of drugs, and we also provide a reference basis for new drug development.

Syntheses and Transformations of α-Amino Acids via Palladium-Catalyzed Auxiliary-Directed sp(3) C-H Functionalization.

α-Amino acids (αAA) are one of the most useful chiral building blocks for synthesis. There are numerous general strategies that have commonly been used for αAA synthesis, many of which employ de novo synthesis focused on enantioselective bond construction around the Cα center and others that consider conversion of existing αAA precursors carrying suitable functional groups on side chains (e.g., serine and aspartic acid). Despite significant advances in synthetic methodology, the efficient synthesis of enantiopure αAAs carrying complex side chains, as seen in numerous peptide natural products, remains challenging. Complementary to these "conventional" strategies, a strategy based on the selective functionalization of side chain C-H bonds, particularly sp(3) hybridized C-H bonds, of various readily available αAA precursors may provide a more straightforward and broadly applicable means for the synthesis and transformation of αAAs. However, many hurdles related to the low reactivity of C(sp(3))-H bonds and the difficulty of controlling selectivity must be overcome to realize the potential of C-H functionalization chemistry in this synthetic application. Over the past few years, we have carried out a systematic investigation of palladium-catalyzed bidentate auxiliary-directed C-H functionalization reactions for αAA substrates. Our strategies utilize two different types of amide-linked auxiliary groups, attached at the N or C terminus of αAA substrates, to exert complementary regio- and stereocontrol on C-H functionalization reactions through palladacycle intermediates. A variety of αAA precursors can undergo multiple modes of C(sp(3))-H functionalization, including arylation, alkenylation, alkynylation, alkylation, alkoxylation, and intramolecular aminations, at the ß, γ, and even δ positions to form new αAA products with diverse structures. In addition to transforming αAAs at previously unreachable positions, these palladium-catalyzed C-H functionalization strategies enable new retrosynthetic logic for the synthesis of many basic αAAs from a common alanine precursor. This approach reduces the synthetic difficulty for many αAAs by bypassing the requirement for stereocontrol at Cα and relies on straightforward and convergent single-bond coupling transformations at the ß-methyl position of alanine to access a wide range of ß-monosubstituted αAAs. Moreover, these ß-monosubstituted αAAs can undergo further C-H functionalization at the ß-methylene position to generate various ß-branched αAAs in a stereoselective and programmable fashion. These new strategies offer readily applicable methods for synthesis of challenging αAAs and may facilitate the efficient total synthesis of complex peptide natural products.

Synthesis and Anti-microbial Activity of Novel Phosphatidylethanolamine-N-amino Acid Derivatives.

The study involved synthesis of five novel amino acid derivatives of phosphatidylethanolamine isolated from egg yolk lecithin employing a three step procedure i) N-protection of L-amino acids with BOC anhydride in alkaline medium ii) condensation of - CO2H group of N-protected amino acid with free -NH2 of PE by a peptide linkage and iii) deprotection of N-protected group of amino acids to obtain phosphatidylethanolamine-N-amino acid derivatives in 60-75% yield. The five L-amino acids used were L glycine, L-valine, L-leucine, L-isoleucine and L-phenylalanine. The amino acid derivatives were screened for anti-baterial activity against B. subtilis, S. aureus, P. aeroginosa and E. coli taking Streptomycin as reference compound and anti-fungal activity against C. albicans, S. cervisiae, A. niger taking AmphotericinB as reference compound. All the amino acid derivatives exhibited extraordinary anti-bacterial activities about 3 folds or comparable to Streptomycin and moderate or no anti-fungal activity against Amphotericin-B.

Synthesis and characterization of amino acid conjugates of oleanolic acid and their in vitro cytotoxic effect on HCC cell lines.

Oleanolic acid (3ß-hydroxy-olean-12-en-28-oic acid; OA-01), a pentacyclic triterpene, exhibit a wide range of pharmacological and biological activities. We have isolated oleanolic acid from methanolic extract of Periploca aphylla, collected from surroundings of Karachi in the month of February. Furthermore, four known and two new C-28 amino acid conjugates of oleanolic acid were prepared to explore potential of these compounds on HCCs and one breast cancer cell line. Cytotoxic effects revealed that as compare to parent compound (OA-01), two derivatives OA-04 (p<0.0001) and OA-06 (p<0.01) showed significantly increased/higher inhibition rates.

Amino acid-nucleotide-lipids: effect of amino acid on the self-assembly properties.

Hybrid amphiphiles composed of a lipid covalently linked to biomolecules are attracting considerable attention, owing to their unique physicochemical and biological properties. Herein, we have synthesized novel amino acid-nucleotide-lipids (ANLs), presenting phenylalanine and thymidine residues and saturated or unsaturated diacyl glycerol lipid moieties to investigate the effect of the specific aminoacid moieties on both aggregation properties and interactions of ANLs with single strand polyA RNA. Physicochemical studies (DLS, cryo-TEM, and small angle X-ray scattering) indicate that phenylanaline amino acids inserted at the 5' position of the nucleotide-lipids stabilize multilamellar systems, whereas unilamellar vesicles are formed preferentially in the case of nucleotide-lipids (NLs). Both NLs and ANLs exhibit weak interactions with complementary polyA RNA as revealed by isothermal titration calorimetry investigations. The multilamellar vesicles obtained with ANLs could be used as a versatile carrier, suitable for both hydrophobic and hydrophilic therapeutic molecules.

Synthesis, characterization and antibacterial activity of mixed ligand dioxouranium complexes of 8-hydroxyquinoline and some amino acids.

Mixed ligand complexes of dioxouranium(VI) of the type [UO2(Q)(L)-2H2O] have been synthesized using 8-hydroxyquinoline (HQ) as a primary ligand and N- and/or O- donor amino acids (HL) such as L-lysine, L-aspartic acid and L-cysteine as secondary ligands. The metal complexes have been characterized on the basis of elemental analysis, electrical conductance, room temperature magnetic susceptibility measurements, spectral and thermal studies. The electrical conductance studies of the complexes in DMF in 10(-3) M concentration indicate their non-electrolytic nature. Room temperature magnetic susceptibility measurements revealed diamagnetic nature of the complexes. Electronic absorption spectra of the complexes show intra-ligand and charge transfer transitions, respectively. Bonding of the metal ion through N- and O- donor atoms of the ligands is revealed by IR studies and the chemical environment of the protons is also confirmed by NMR studies. The thermal analysis data of the complexes indicate the presence of coordinated water molecules. The agar cup and tube dilution methods have been used to study the antibacterial activity of the complexes against the pathogenic bacteria S. aureus, C. diphtherinae, S. typhi and E. coli.

Synthesis of a new trifluoromethylketone analogue of l-arginine and contrasting inhibitory activity against human arginase I and histone deacetylase 8.

As part of our continuing search for new amino acid inhibitors of metalloenzymes, we now report the synthesis and biological evaluation of the trifluoromethylketone analogue of L-arginine, (S)-2-amino-8,8,8-trifluoro-7-oxo-octanoic acid (10). While this novel amino acid was initially designed as a potential inhibitor of human arginase I, it exhibits no measurable inhibitory activity against this enzyme. Surprisingly, however, 10 is a potent inhibitor of human histone deacetylase 8, with IC(50)=1.5 ± 0.2 µM. Additionally, 10 weakly inhibits the related bacterial enzyme, acetylpolyamine amidohydrolase, with IC(50)=110 ± 30 µM. The lack of inhibitory activity against human arginase I may result from unfavorable interactions of the bulky trifluoromethyl group of 10 in the constricted active site. Since the active site of histone deacetylase 8 is less constricted, we hypothesize that it accommodates 10 as the gem-diol, which mimics the tetrahedral intermediate and its flanking transition states in catalysis. Therefore, we suggest that 10 represents a new lead in the design of an amino acid or peptide-based inhibitor of histone deacetylases with simpler structure than previously studied trifluoromethylketones.

Gastroprotective effect and cytotoxicity of labdeneamides with amino acids.

Semisynthetic aromatic amides from ARAUCARIA ARAUCANA diterpene acids have been shown to display a relevant gastroprotective effect with low cytotoxicity. The aim of this work was to assess the gastroprotective effect of amino acid amides from imbricatolic acid and its 8(9)-en isomer in the ethanol/HCl-induced gastric lesions model in mice as well as to determine the cytotoxicity of the obtained compounds on the following human cell lines: normal lung fibroblasts (MRC-5), gastric adenocarcinoma (AGS), and liver hepatocellular carcinoma (Hep G2). The diterpenes 15-acetoxyimbricatolic acid, its 8(9)-en isomer, 15-hydroxyimbricatolic acid, and the 8(9)-en derivative, bearing a COOH function at C-19, were used as starting compounds. New amides with C-protected amino acids were prepared. The study reports the effect of a single oral administration of either compound 50 min before the induction of gastric lesions by ethanol/HCl. Some 20 amino acid monoamides were obtained. Dose-response experiments on the glycyl derivatives showed that at a single oral dose of 100 mg/kg, the compounds presented an effect comparable to the reference drug lansoprazole at 20 mg/kg and at 50 mg/kg reduced gastric lesions by about 50%. All derivatives obtained in amounts > 30 mg were compared at a single oral dose of 50 mg/kg. The best gastroprotective effect was observed for the exomethylene derivatives bearing a valine residue at C-19 either with an acetoxy or free hydroxy group at C-15. The tryptophanyl derivative from the acetate belonging to the 8,9-en series presented selective cytotoxicity against hepatocytes. The glycyl amide of 15-acetoxyimbricatolic acid was the most cytotoxic and less selective compound with IC50 values between 47 and 103 µM for the studied cell lines. This is the first report on the obtention of semisynthetic amino acid amides from labdane diterpenes.

Unexplored nucleophilic ring opening of aziridines.

The reactivity of dianions of carboxylic acids towards aziridines has been studied. Although, a similar reactivity to that of enolates from ketones, esters or amides has been observed, the method directly yields γ-aminoacids in one step. The method is complementary of previous results of enenediolate reactivity with other electrophiles. A comparative study with the reactivity of this enediolates with epoxides is included.

Enantioselective organocatalytic addition of oxazolones to 1,1-bis(phenylsulfonyl)ethylene: a convenient asymmetric synthesis of quaternary alpha-amino acids.

A new, easy, and highly enantioselective method for the synthesis of quaternary alpha-alkyl-alpha-amino acids based on organocatalysis is reported. The addition of oxazolones to 1,1-bis(phenylsulfonyl)ethylene is efficiently catalyzed by simple chiral bases or thioureas. The reaction affords alpha,alpha-disubstituted alpha-amino acid derivatives with complete C4 regioselectivity and with excellent yields and enantioselectivities. This methodology is complementary to previously reported enantioselective approaches to quaternary alpha-amino acids and allows the synthesis of alpha-phenyl-alpha-alkyl-alpha-amino acids and alpha-tert-butyl-alpha-alkyl-alpha-amino acids. It has distinct advantages in terms of operational simplicity, enviromentally friendly conditions, and suitability for large-scale reactions.