L-phenylalanine Methyl Ester Research Articles

Phenylalanine containing hydrophobic nanospheres for antibody purification

In this study, novel hydrophobic nanospheres with an average size of 158 nm utilizing N-methacryloyl-(L)-phenylalanine methyl ester (MAPA) as a hydrophobic monomer were produced by surfactant free emulsion polymerization of 2-hydroxyethyl methacrylate (HEMA) and MAPA conducted in an aqueous dispersion medium. MAPA was synthesized using methacryloyl chloride and L-phenylalanine methyl ester. Specific surface area of the nonporous nanospheres was found to be 1874 m2/g. Poly(HEMA-MAPA) nanospheres were characterized by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Average particle size, size distribution, and surface charge measurements were also performed. Elemental analysis of MAPA for nitrogen was estimated as 0.42 mmol/g polymer. Then, poly(HEMA-MAPA) nanospheres were used in the adsorption of immunoglobulin G (IgG) in batch system. Higher adsorption values (780 mg/g) were obtained when the poly (HEMA-MAPA) nanospheres were used from both aqueous solutions and human plasma. The adsorption phenomena appeared to follow a typical Langmuir isotherm. It was observed that IgG could be repeatedly adsorbed and desorbed without significant loss in adsorption amount. These findings show considerable promise for this material as a hydrophobic support in industrial processes.

The hydration/dehydration behavior of aspartame revisited

Aspartame, l-aspartyl- l-phenylalanine methyl ester, has two hydrates (IA and IB), a hemi-hydrate (IIA) and an anhydrate (IIB). The hydration/dehydration behavior of aspartame was investigated using hot-humidity stage X-ray powder diffraction (XRPD) and molecular mechanics modeling in combination with differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). The results of this study are compared to earlier studies on aspartame as described in literature. It is shown that earlier transition studies were hampered by incomplete conversions and wrong assignment of the forms. The combination of the techniques applied in this study now shows consistent results for aspartame and yields a clear conversion scheme for the hydration/dehydration behavior of the four forms.

Improved synthesis of no-carrier-added p-[ 124I]iodo- l-phenylalanine and p-[ 131I]iodo- l-phenylalanine for nuclear medicine applications in malignant gliomas

This work describes the synthesis and the tumor affinity testing of no-carrier-added (n.c.a.) p-[ 124I]iodo- l-phenyalanine ([ 124I]IPA) and n.c.a. p-[ 131I]iodo- l-phenyalanine ([ 131I]IPA) as radiopharmaceuticals for imaging brain tumors with PET and for radionuclid-based therapy, respectively. Parameters for labeling were optimized with regard to the amount of precursor, temperature and time. Thereafter, n.c.a. [ 124I]IPA and n.c.a. [ 131I]IPA were investigated in rat F98 glioma and in primary human A1207 and HOM-T3868 glioblastoma cells in vitro, followed by an in vivo evaluation in CD1 nu/nu mice engrafted with human glioblastoma. No-carrier-added [ 124I]IPA and n.c.a. [ 131I]IPA were obtained in 90±6% radiochemical yield and >99% radiochemical purity by iododestannylation of N-Boc-4-(tri- n-butylstannyl)- l-phenylalanine methylester in the presence of chloramine-T, followed by hydrolysis of the protecting groups. The total synthesis time, including the HPLC separation and pharmacological formulation, was less than 60 min and compatible with a clinical routine production. Both amino acid tracers accumulated intensively in rat and in human glioma cells. The radioactivity incorporation in tumor cells following a 15-min incubation at 37 °C/pH 7.4 varied from 25% to 42% of the total loaded activity per 10 6 tumor cells (296–540 cpm/1000 cells). Inhibition experiments confirmed that n.c.a. [ 124I]IPA and n.c.a. [ 131I]IPA were taken up into tumor by the sodium-independent L- and ASC-type transporters. Biodistribution and whole-body imaging by a gamma-camera and a PET scanner demonstrated a high targeting level and a prolonged retention of n.c.a. [ 124I]IPA and n.c.a. [ 131I]IPA within the xenotransplanted human glioblastoma and a primarily renal excretion. However, an accurate delineation of the tumors in mice was not possible by our imaging systems. Radioactivity accumulation in the thyroid and in the stomach as a secondary indication of deiodination was less than 1% of the injected dose at 24 h p.i., confirming the high in vivo stability of the radiopharmaceuticals. In conclusion, n.c.a. [ 124I]IPA and n.c.a. [ 131I]IPA are new promising radiopharmaceuticals, which can now be prepared in high radiochemical yields and high purity for widespread clinical applications. The specific and high-level targeting of n.c.a. [ 124I]IPA and n.c.a. [ 131I]IPA to glioma cells in vitro and to glioblastoma engrafts in vivo encourages further in vivo validations to ascertain their clinical potential as agent for imaging and quantitation of gliomas with PET, and for radionuclid-based therapy, respectively.

Aspartame decreases evoked extracellular dopamine levels in the rat brain: An in vivo voltammetry study

Conflicting reports exist concerning the effect aspartame (APM, l-aspartyl- l-phenylalanine methyl ester) has upon brain biogenic amines. In the following study, in vivo voltammetry was utilized to measure evoked extracellular dopamine (DA) levels in the striatum of rats in order to assess APM's effect. Time-course experiments revealed a significant decline in evoked extracellular DA levels within 1 h of a single systemic dose (500 mg/kg i.p.) when compared to vehicle-injected controls. The effect was frequency dependent and showed a significant decrease utilizing high frequency stimulation parameters (50 and 60 Hz). In order to further determine APM's potential to alter evoked extracellular DA levels, extended stimulation periods were employed to deplete releasable stores both before and after APM administration in intact and 6-OHDA partially lesioned animals. The extended stimulation periods were applied at 60 Hz for 2,5,10 and 20 s durations. APM decreased DA levels under these conditions in both intact and 6-OHDA partially lesioned animals by an average of 34% and 51%, respectively. Kinetic analysis performed on frequency series indicated that the diminished DA levels corresponded to a significant reduction in DA release. These findings suggest that APM has a relatively potent effect of decreasing evoked extracellular DA levels when administered systemically under the conditions specified.

Improving the activity and stability of thermolysin by site-directed mutagenesis

In previous site-directed mutagenesis study on thermolysin, mutations which increase the catalytic activity or the thermal stability have been identified. In this study, we attempted to generate highly active and stable thermolysin by combining the mutations so far revealed to be effective. Three mutant enzymes, L144S (Leu144 in the central α-helix located at the bottom of the active site cleft is replaced with Ser), G8C/N60C/S65P (Gly8, Asn60, and Ser65 in the N-terminal region are replaced with Cys, Cys, and Pro, respectively, to introduce a disulfide bridge between the positions 8 and 60), and G8C/N60C/S65P/L144S, were constructed by site-directed mutagenesis. In the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl- l-leucine amide (FAGLA) and N-carbobenzoxy- l-aspartyl- l-phenylalanine methyl ester (ZDFM), the k cat/ K m values of L144S and G8C/N60C/S65P/L144S were 5- to 10-fold higher than that of the wild-type enzyme. The rate constants for thermal inactivation at 70 °C and 80 °C of G8C/N60C/S65P and G8C/N60C/S65P/L144S decreased to 50% of that of the wild-type enzyme. These results indicate that G8C/N60C/S65P/L144S is more active and stable than the wild-type thermolysin. Thermodynamic analysis suggests that the single mutation of Leu144 → Ser and the triple mutation of Gly8 → Cys, Asn60 → Cys, and Ser65 → Pro are independent.

Peptide Synthesis of Aspartame Precursor Using Organic-Solvent-Stable PST-01 Protease in Monophasic Aqueous-Organic Solvent Systems

The PST-01 protease is a metalloprotease that has zinc ion at the active center and is very stable in the presence of water-soluble organic solvents. The reaction rates and the equilibrium yields of the aspartame precursor N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester (Cbz-Asp-Phe-OMe) synthesis from N-carbobenzoxy-L-aspartic acid (Cbz-Asp) and L-phenylalanine methyl ester (Phe-OMe) in the presence of water-soluble organic solvents were investigated under various conditions. Higher reaction rate and yield of Cbz-Asp-Phe-OMe were attained by the PST-01 protease when 30 mM Cbz-Asp and 500 mM Phe-OMe were used. The maximum reaction rate was obtained pH 8.0 and 37 degrees C. In the presence of dimethylsulfoxide (DMSO), glycerol, methanol, and ethylene glycol, higher reaction rates were obtained. The equilibrium yield was the highest in the presence of DMSO. The equilibrium yield of Cbz-Asp-Phe-OMe using the PST-01 protease attained 83% in the presence of 50% (v/v) DMSO.

Reversible adsorption of lipase on novel hydrophobic nanospheres

Novel hydrophobic nanospheres with an average size of 158 nm utilizing N-methacryloyl-( l)-phenylalanine methyl ester (MAPA) as a hydrophobic monomer were prepared by surfactant free emulsion polymerization of 2-hydroxyethyl methacrylate (HEMA) and MAPA conducted in an aqueous dispersion medium. MAPA was synthesized using methacryloyl chloride and l-phenylalanine methyl ester. Specific surface area of the nonporous nanospheres was found to be 1874 m 2/g. Poly(HEMA–MAPA) nanospheres were characterized by Fourier transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM). Average particle size, size distribution, and surface charge measurements were also performed. Elemental analysis of MAPA for nitrogen was estimated as 0.42 mmol/g polymer. Then, poly(HEMA–MAPA) nanospheres were used in the adsorption of lipase in batch system. Using an optimized adsorption protocol, a very high loading of 329 mg lipase/g nanosphere was obtained. The adsorption phenomena appeared to follow a typical Langmuir isotherm. The K m values for adsorbed lipase (8.0 mM) was higher than that of free enzyme (4.6 mM). Storage stability was found to increase with adsorption. It was observed that enzyme could be repeatedly adsorbed and desorbed without significant loss in adsorption amount or enzyme activity. These findings show considerable promise for this material as an adsorption matrix in industrial processes.

Ortho Palladation and Functionalization of l-Phenylalanine Methyl Ester

The ortho-metalated complex (S,S)-[Pd2{κ2(C,N)-C6H4CH2CH(CO2Me)NH2-2}2(μ-Br)2] (1b) can be prepared by refluxing in acetonitrile equimolecular amounts of Pd(OAc)2 and l-phenylalanine methyl ester hydrochloride, followed by addition of an excess of NaBr. Complex 1b reacts with 4-picoline to give the mononuclear derivative (S)-[Pd{κ2(C,N)-C6H4CH2CH(CO2Me)NH2-2}2Br(NC5H4Me-4)] (2), whose crystal structure has been determined by X-ray diffraction. The precursor of 1b, (S,S)-[Pd2{κ2(C,N)-C6H4CH2CH(CO2Me)NH2-2}2(μ-Cl)2] (1a), could not be isolated in a pure form, but it can be used as the starting material for the synthesis of functionalized derivatives of the phenylalanine methyl ester. Thus, CO and RNC (R = Xy, tBu) insert into the Pd−C bond of 1a to afford, after depalladation, (S)-1-oxo-3-(methoxycarbonyl)-1,2,3,4-tetrahydroisoquinoline (3) and (S)-1-R-3-(methoxycarbonyl)-3,4-dihydroisoquinolinium triflate (R = tBu (4), Xy (5)), respectively. Reaction of complex 1b with bromine or iodine affords trans-(S,S)-[PdBr2{NH2CH(CO2Me)CH2C6H4-X-2}2] (X = Br (6), I (7)), which further reacts with 1,10-phenanthroline (phen) to give [PdBr2(phen)] and (S)-2-X-phenylalanine methyl ester (X = Br (8), I (9)).

Preparation of Optically Active (2RS,3SR)-2-Amino-3-hydroxy-3-phenylpropanoic Acid (threo-.BETA.-Phenylserine) via Optical Resolutions by Replacing and Preferential Crystallization

To obtain optically active threo-2-amino-3-hydroxy-3-phenylpropanoic acid (1) via optical resolutions by replacing and preferential crystallization, the racemic structure of (2RS,3SR)-1 hydrochloride [(2RS,3SR)-1.HCl] was examined based on the melting point, solubility, and infrared spectrum. (2RS,3SR)-1.HCl was indicated to exist as a conglomerate at room temperature, although it forms a racemic compound at the melting point. When, in optical resolution by replacing crystallization, L-phenylalanine methyl ester hydrochloride (L-2) was used as the optically active co-solute, (2R,3S)-1.HCl was preferentially crystallized from the supersaturated racemic solution; the use of D-2 as the co-solute afforded (2S,3R)-1.HCl with an optical purity of 95%. In addition, optical resolution by preferential crystallization was successfully achieved to give successively (2R,3S)- and (2S,3R)-1.HCl with optical purities of 90-92%. The (2R,3S)- and (2S,3R)-1.HCl purified by recrystallization from 1-propanol were treated with triethylamine in methanol to give optically pure (2R,3S)- and (2S,3R)-1.

Extracellular production of recombinant thermolysin expressed in Escherichia coli, and its purification and enzymatic characterization

Thermolysin is a representative zinc metalloproteinase derived from Bacillus thermoproteolyticus and a target in protein engineering to understand the catalytic mechanism and thermostability. Extracellular production of thermolysin has been achieved in Bacillus, but not in Escherichia coli, although it is the most widely used as a host for the production of recombinant proteins. In this study, we expressed thermolysin as a single polypeptide pre-proenzyme in E. coli under the original promoter sequences in the npr gene, the gene from B. thermoproteolyticus, which encodes thermolysin. Active mature thermolysin (34.6 kDa) was secreted into the culture medium. The recombinant thermolysin was purified to homogeneity by sequential column chromatography procedures of the supernatant with hydrophobic-interaction chromatography followed by affinity chromatography. The purified recombinant product is indistinguishable from natural thermolysin from B. thermoproteolyticus as assessed by hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl- l-leucine amide and N-carbobenzoxy- l-asparatyl- l-phenylalanine methyl ester. The results demonstrate that our expression system should be useful for structural and functional analysis of thermolysin.

A Method for Quantitating the Intracellular Metabolism of AZT Amino Acid Phosphoramidate Pronucleotides by Capillary High-Performance Liquid Chromatography−Electrospray Ionization Mass Spectrometry

A methodology has been developed for the analysis of the intracellular metabolism of 3'-azido-3'-deoxythymidine (AZT) amino acid phosphoramidates utilizing reverse-phase high-performance liquid chromatography interfaced with negative ion electrospray ionization mass spectrometry (LC/ESI(-) -MS). The presented work demonstrates the potential of capillary LC/MS and LC/MS/MS to identify and quantitate the cellular uptake and metabolism of nucleoside phosphoramidate. Significant intracellular amounts of D- and L-phenylalanine methyl ester or D- and L-tryptophan methyl ester AZT phosphoramidates were observed for human T-lymphoblastoid leukemia (CEM) cells incubated for 2 and 4 h with the prodrugs. AZT-MP was the primary metabolite observed for human T-lymphoblastoid leukemia (CEM) cells. In this paper, the details of using LC/MS to analyze AZT amino acid phosphoramidates in biological samples are discussed. LC/MS is an efficient method for analyzing multiple samples containing several analytes in a short period of time. The method also provides high selectivity and sensitivity, and requires minimal sample preparation. This approach should be broadly applicable for the analysis of the intracellular metabolism of nucleoside prodrugs and pronucleotides.

Enrichment of phenylalanine ammonia lyase activity of Rhodotorula yeast

An improved method for the enrichment of phenylalanine ammonia lyase (E.C.4.3.1.5-PAL) activity of Rhodotorula yeast is described. Whole cells of various Rhodotorula yeast strains showed low levels of PAL activity on account of membrane permeability barriers for the substrate, l-phenylalanine. Different methods of enriching PAL activity including ultra-sonication, detergent and enzyme solubilization were investigated in this study in an effort to overcome these permeability barriers. Ultra-sonication of the yeast cells was found to be the most effective of the methods and yielded over 10-fold higher enzyme activity as compared to untreated whole cells. Solubilization of the yeast cells involving a synergistic treatment with Triton-X-100 and glucuronidase gave about nine-fold enrichment while cells treated individually with Triton-X-100, glucuronidase and cetyl trimethyl ammonium bromide gave 6.8-, 5.9- and 5.8-fold higher activity, respectively. Homogenization; treatment with chloroform, isopropanol, and toluene; freezing and thawing were ineffective in enhancing the enzyme activity. Of the six Rhodotorula yeast strains employed in this study, Rhodotorula glutinis, NCYC 61 exhibited maximum PAL activity. This simple, rapid and improved method of PAL activity enrichment could serve as a rich enzyme source, especially in the biosynthesis of l-phenylalanine and l-phenylalanine methyl ester.

Effect of 3,4‐di(OH)‐cinnamate synthetic derivative on plasma and hepatic cholesterol level and antioxidant enzyme activities in high cholesterol‐fed rats

The effect of 3,4-di(OH)-phenylpropionic acid (L-phenylalanine methyl ester) amide (SL-1063), a synthetic derivative of 3,4-di(OH)-cinnamate, on the cholesterol metabolism and antioxidant enzyme system was examined in rats. Diets that included either SL-1063 (0.046%, w/w) or lovastatin (0.02%, w/w) as a supplement, plus 1 g cholesterol/100 g diet were fed to rats ad libitum for 5 weeks. The total plasma cholesterol and triglyceride levels were significantly lowered by the SL-1063 supplement compared to the control group. Meanwhile, the levels of plasma HDL-cholesterol and ratio of HDL-cholesterol/total cholesterol (%) were significantly higher in the SL-1063 group than in the control group. However, the lovastatin supplement did not affect the plasma lipid level. The hepatic cholesterol level and 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity were significantly lowered in the lovastatin group compared to the SL-1063 group; however, the hepatic triglyceride level did not differ among the groups. The activity of hepatic acyl CoA: cholesterol acyltransferase (ACAT), the enzyme that catalyzes hepatic cholesterol esterification, was significantly lower in the lovastatin and SL-1063 groups than in the control group. Furthermore, the SL-1063 supplement elevated the excretion of fecal sterols. As regards the hepatic antioxidant enzyme system, the superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and glutathione reductase (GR) activities were all significantly higher in the SL-1063 group compared to the control group, whereas only the GR activity was significantly increased by the lovastatin supplement. No marked difference in the GSH levels and glucose-6-phosphate dehydrogenase (G6PD) activities was observed among the groups. The levels of plasma and hepatic thiobarbituric acid reactive substances (TBARS) were lowered by the SL-1063 supplement compared to the control group. Accordingly, the current results suggest that SL-1063, a synthetic derivative of 3,4-di(OH)-cinnamate, is effective in lowering the plasma lipids and improving the antioxidant enzyme system.

13C, 15N NMR and CP-MAS as well as FT-IR and PM5 studies of Schiff base of gossypol with l-phenylalanine methyl ester in solution and solid

The Schiff bases of racemic gossypol with l-phenylalanine amino acid methyl ester (GSBA) was studied by FT-IR, 13C and 15N NMR spectroscopy as well as by the PM5 semiempirical method. The spectroscopic methods have provided clear evidence that GSBA exists in the solid state and in solution as enamine–enamine tautomer. The existence of diastereoisomers is very well evidenced in the 13C NMR spectra in the solution by the double character of all carbon atoms' signals of the naphthalene rings. The structure of GSBA and the hydrogen bonds within this structure are discussed.

Synthesis of cinnamic acid derivatives and their inhibitory effects on LDL-oxidation, acyl-CoA:cholesterol acyltransferase-1 and -2 activity, and decrease of HDL-particle size

A series of cinnamic acid derivatives were synthesized and their biological abilities on lipoprotein metabolism were examined. Among the tested compounds, 4-hydroxycinnamic acid ( l-phenylalanine methyl ester) amide ( 1) and 3,4-dihydroxyhydrocinammic acid ( l-aspartic acid dibenzyl ester) amide ( 2) inhibited human acyl-CoA:cholesterol acyltransferase-1 and -2 activities with apparent IC 50 around 60 and 95 μM, respectively. Compounds 1 and 2 also served as an antioxidant against copper mediated low-density lipoproteins (LDL) oxidation with apparent IC 50 = 52 and 3 μM, compound 1 and 2, respectively. Additionally, decrease of HDL-particle size under presence of LDL was inhibited by the 1 at 307 μM of final concentration. Treatment of the 1 or 2 did not influence normal growth of RAW264.7 without detectable cytotoxic activity from a cell viability test. These results suggest that the new cinnamic acid derivatives possess useful biological activity as an anti-atherosclerotic agent with inhibition of cellular cholesterol storage and transport by the both ACAT, inhibition of LDL-oxidation, HDL particle size rearrangement.

The complexes between Schiff base of gossypol with l-phenylalanine methyl ester and some monovalent cations studied by 1H NMR, ESI MS, FT-IR as well as PM5 semi-empirical methods

The Schiff base of racemic gossypol with l-phenylalanine methyl ester and its complexes with H + and monovalent metal cations was synthesized and studied by FT-IR, 1H NMR spectroscopy and Electrospray Ionization mass spectrometry as well as by the PM5 semi-empirical method. The studied Schiff base exists in acetonitrile solution in the enamine–enamine tautomeric form. After the protonation, the tautomeric form changes into the imine–imine one. For this form of the Schiff base the existence of diastereoisomers was very well visible in the 1H NMR spectra. The structures of the protonated Schiff base and its 1:1 complex with K + cation as well as the respective hydrogen bonds within these structures are discussed.

The effect of phenylacetate on the expansion and cytotoxic activity of adherent lak cells from patients with hepatocellular carcinoma

Objective: To improve the preparation of adherent lymphokine-activated killer (A-LAK) cells and study the synergistic anti-tumor effect of phenylacetate (PA) and A-LAK cells. Methods: A-LAK cells were obtained from peripheral blood mononuclear cells (PBMC) of patients with hepatocellular carcinoma (HCC) by using L-phenylalanine methyl ester (PME) to deplete immunosuppressive monocytes. The proliferation of SMMC7721 cell line treated with PA was studied. A-LAK cells were treated with the supernatant of SMMC7721 cells which had been pretreated with PA and the changes of the proliferation and anti-tumor activity of A-LAK cells were investigated. Results: The expansion of A-LAK cells was significantly higher than that of non-adherent LAK (NA-LAK) cells as well as regular LAK cells. The growth of SMMC7721 cells was significantly suppressed by PA. The supernatant of cultured tumor cells intensively suppressed the proliferation and cytotoxicity of A-LAK cells, but the suppressive effect of supernatant treated with PA previously was decreased. Conclusion: A-LAK cells could be simply prepared by using PME, and showed a synergistic anti-tumor effect with the combination of PA.

Effects of cryopreservation and phenylacetate on biological characters of adherent LAK cells from patients with hepatocellular carcinoma.

To improve the preparation of adherent lymphokine-activated killer (A-LAK) cells and to study the effects of cryopreservation and phenylacetate (PA) on biological characters of A-LAK cells. A-LAK cells were obtained from peripheral blood mononuclear cells (PBMCs) of the patients with hepatocellular carcinoma (HCC) by using L-phenylalanine methyl ester (PME) to deplete immunosuppressive monocytes. Proliferative activity of SMMC7721 cell line after treatment with phenylacetate (PA) was observed. A-LAK cells were treated with the supernatant of SMMC7721 cells that had been pretreated with PA. The changes of proliferation, cytotoxicity and phenotype of A-LAK cells were investigated after cryopreservation. The expansion of A-LAK cells (96.79 +/- 69.10 folds on Day 14) was significantly higher than that of non-adherent LAK (NA-LAK) cells (22.77 +/- 13.20) as well as conventional LAK cells (4.64 +/- 0.91). PA significantly suppressed the growth of SMMC7721 cells, and the inhibitor ratio was 46%. The supernatant of cultured tumor cells intensively suppressed the proliferation and cytotoxicity of A-LAK cells, but the suppressive effect of the supernatant was previously decreased after treatment with PA. Impairments in proliferation and cytotoxicity of A-LAK cells immediately after thawing of cryopreservation and recovery after reincubation with IL-2 were observed. The cytotoxicity of thawed A-LAK cells on Day 5 was significantly higher than that of fresh A-LAK before freezing (54.8 +/- 10.2% vs 40.5 +/- 6.4%). No significant change in the percentage of lymphocyte subsets was identified in frozen A-LAK cells as compared with that in the fresh control cells. A-LAK cells can be simply prepared by using PME, and showed a synergistic anti-tumor effect with the combination of PA. Cryopreservation can increase the immunoactivities of A-LAK cells from the patients with hepatocellular carcinoma.

Enzymic synthesis of aspartame precursors from eutectic substrate mixtures

The precursor of aspartame N-benzyloxycarbonyl- l-aspartic acid- l-phenylalanine methyl ester ( N-CBZ- l-Asp- l-PheOMe) was synthesized using thermolysin from eutectic mixtures of the substrates N-benzyloxycarbonyl- l-aspartic acid ( N-CBZ- l-Asp) and l-phenylalanine methyl ester ( l-PheOMe). Eutectic mixtures were easily formed by mixing the substrates in the presence of water and/or organic solvents as adjuvants. Among the tested solvents the hydrophilic solvents dimethyl sulfoxide (DMSO) and 2-methoxy ethyl acetate (MEA) were the best adjuvants for enzymic reactions. A maximum conversion yield of 73.8% was obtained for a combination of 6% DMSO and 6% MEA. In addition to benzyloxycarbonyl (CBZ), l-Asp derivatives with other protecting groups were used. Reactions with N-CBZ- l-Asp or N- tert-butyloxycarbonyl (Boc)- l-Asp formed eutectic mixtures at approximately 0 °C, whereas reactions with hydrophobic N-Ac- l-Asp and N-9-fluorenylmethyloxycabronyl (Fmoc)- l-Asp formed mixtures at 25 and 40 °C, respectively. High conversion yields of 74 and 78% were achieved for N-CBZ- l-Asp and N-Boc- l-Asp but low yields of 56 and 35% were obtained for N-Ac- l-Asp and N-Fmoc- l-Asp. Thermolysin was most effective with a yield of 72% compared to fungal protease, subtilsin, α-chymotrypsin, papain, and pepsin with yields of 50% or less.

Aspartame Degradation in Solutions at Various pH Conditions

ABSTRACT: This study of aspartame degradation in phosphate‐citrate buffer solutions of pH2,4,6, 7,8,10, and 12 using reverse‐phase high‐performance liquid chromatography (HPLC) showed different major degradation products for different pH ranges. The major degradation product found at pH 2 to 6 was L‐phenylalanine methyl ester (PME). The major degradation product found at pH 7 to 10 was 3,6‐dioxo‐5‐phenylmethylpiperazine acetic acid (diketopiperazine, DKP). L‐aspartyl‐phenylalanine (Asp‐Phe) was the major product detected at pH 12.