5-aminopentanoic Acid Research Articles

The Ullmann Coupling between 2-Chlorobenzoic Acids and Amino Acids; A Valuable Reaction for Preparing 2-Substituted 1-Acetyl-1H-indol-3-yl Acetates­

Substituted 3-acetoxy-1-acetyl-1H-indoles were pre- pared by condensing 2-chlorobenzoic acids with amino acids under Ullmann conditions in good yields, and further cyclodecarboxyla- tion using the Rossing method in moderate to good yields. N,O-Diacetylindoxyls (1-acetyl-1H-indol-3-yl acetates, 3-acetoxy-1-acetyl-1H-indoles) are present as precursors of some aglicons in chromogenic compounds that are very useful in the identification of various microorganisms. 1-3 However, 2-substituted 3-acetoxy-1-acetyl-1H-indoles are rarely found in literature and there are no procedures available that describe the synthesis of these compounds from the corresponding unactivated amino acid and 2- chlorobenzoic acids by the Ullman procedure. Neverthe- less, 2-substituted 3-acetoxy-1-acetyl-1H-indoles have been used as the starting point for the preparation of 2- substituted 1-acetyl-1,2-dihydro-3H-indol-3-ones. 4 On the other hand, only one compound, 3-acetoxy-1- acetyl-2-methyl-1H-indole, has been prepared from the corresponding N-acetylated 2-((carboxymethyl)ami- no)benzoic acid, but it was not prepared using the Ullman conditions. 4 In previous work we reported a simple two-step procedure for the preparation of 3-acetoxy-1-acetyl-6-chloro-1H-in- dole from 2,4-dichlorobenzoic acid and an unactivated amino acid in good yield. 5 Encouraged by this result, we decided to carry out the preparation of other substituted 3- acetoxy-1-acetyl-1H-indoles starting from the corre- sponding 2-chlorobenzoic acids. 6 These papers made us think about the possibility of using the same procedure to prepare 2-substituted 3-acetoxy-1-acetyl-1H-indoles 6 from 2-chlorobenzoic acids 1 and 2-substituted amino acids 2 or 5-aminopentanoic acid (3). We started by using the same reaction conditions given in previous papers and using 2-chlorobenzoic acids 1a-d and amino acids 2a-c or 3 to give Ullman compounds, 2-((carboxymethyl)ami- no)benzoic acids 4a-g and 5a-c (Table 1) in very good yields. Subsequent Rossing cyclodecarboxylation of 4a-g allowed us to obtain 2-substituted 3-acetoxy-1-acetyl-1H- indoles 6a-g in moderate to good yields (Table 2).

A liquid chromatography/electrospray ionization mass spectrometry (LC–MS/MS) assay for the determination of irinotecan (CPT-11) and its two major metabolites in human liver microsomal incubations and human plasma samples

A sensitive, rapid LC–MS/MS assay has been developed and validated for the simultaneous quantification of CPT-11 and its two principal metabolites, 7-ethyl-10-hydroxycamptothecin (SN-38), and 7-ethyl-10-[4- N-(5-aminopentanoic acid)-1-piperidino]carbonyloxy-camptothecin (APC) in human liver microsomal fractions and plasma. The method was linear over the ranges of 1.56–100 ng/mL, 3.13–150 ng/mL, and 0.78–100 ng/mL for CPT-11, SN-38, and APC, respectively. The total run time was 7.0 min. This assay offers advantages in terms of expediency, recovery of analytes, and suitability for the analysis of CPT-11 and its metabolites in various biological fluids.

A high-throughput and generic assay method for the determination of substrate specificities of thermophilic α-aminotransferases

For the determination of substrate specificities of thermophilic α-aminotransferases (AATs), a novel high-throughput assay method was developed. In this method, a thermophilic ω-aminotransferase (OAT) and a thermophilic aldehyde dehydrogenase (ALDH) are coupled to the AAT reaction. Glutamic acid is used as an amino group donor for the AAT reaction yielding 2-oxoglutalic acid. 2-Oxoglutalic acid produced by the AAT reaction is used as an amino group acceptor in the OAT reaction regenerating glutamic acid. The amino group donor of the OAT reaction is 5-aminopentanoic acid yielding pentanedioic acid semialdehyde which is oxidized by ALDH to pentanedioic acid with concomitant reduction of NADP + to NADPH. NADPH thus produced then reduces colorless tetrazolium salt into colored formazan. To construct such a reaction system, the genes for a thermophilic AAT, a thermophilic OAT and a thermophilic ALDH were cloned and expressed in Escherichia coli. These enzymes were subsequently purified and used to determine the activities of AAT for the synthesis of unnatural amino acids. This method allowed the clear detection of the AAT activities as it measures the increase in the absorbance on a low background absorbance reading.

Heterologous Expression, Purification, and Characterization of an l -Ornithine N 5 -Hydroxylase Involved in Pyoverdine Siderophore Biosynthesis in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen that produces the siderophore pyoverdine, which enables it to acquire the essential nutrient iron from its host. Formation of the iron-chelating hydroxamate functional group in pyoverdine requires the enzyme PvdA, a flavin-dependent monooxygenase that catalyzes the N(5) hydroxylation of l-ornithine. pvdA from P. aeruginosa was successfully overexpressed in Escherichia coli, and the enzyme was purified for the first time. The enzyme possessed its maximum activity at pH 8.0. In the absence of l-ornithine, PvdA has an NADPH oxidase activity of 0.24 +/- 0.02 micromol min(-1) mg(-1). The substrate l-ornithine stimulated this activity by a factor of 5, and the reaction was tightly coupled to the formation of hydroxylamine. The enzyme is specific for NADPH and flavin adenine dinucleotide (FAD(+)) as cofactors, as it cannot utilize NADH and flavin mononucleotide. By fluorescence titration, the dissociation constants for NADPH and FAD(+) were determined to be 105.6 +/- 6.0 microM and 9.9 +/- 0.3 microM, respectively. Steady-state kinetic analysis showed that the l-ornithine-dependent NADPH oxidation obeyed Michaelis-Menten kinetics with apparent K(m) and V(max) values of 0.58 mM and 1.34 micromol min(-1) mg(-1). l-Lysine was a nonsubstrate effector that stimulated NADPH oxidation, but uncoupling occurred and hydrogen peroxide instead of hydroxylated l-lysine was produced. l-2,4-Diaminobutyrate, l-homoserine, and 5-aminopentanoic acid were not substrates or effectors, but they were competitive inhibitors of the l-ornithine-dependent NADPH oxidation reaction, with K(ic)s of 3 to 8 mM. The results indicate that the chemical nature of effectors is important for simulation of the NADPH oxidation rate in PvdA.

Hepatic transport, Metabolism and biliary excretion of irinotecan in a cancer patient with an external bile drain

Background: Irinotecan is metabolized by various enzymes, including carboxylesterases, cytochrome P450 3A isozymes (CYP3A), and uridine-diphosphate glucuronosyltransferase 1A isoforms (UGT1A). Here we report on the disposition of irinotecan and its metabolites in plasma, urine, bile and feces of a single cancer patient with an external bile drain. Methodology: Irinotecan (450 mg) was administered during a 90-minutes continuous infusion to a cancer patient with an external bile drain. Blood samples were collected up to 55 hours after infusion, while bile, feces and urine were collected during 6 consecutive days after administration. Samples were analyzed using high-performance liquid chromatography (HPLC)-assays with fluorescence detection. Results: Plasma pharmacokinetics were characterized by a relatively slow clearance of irinotecan and a relatively high exposure to 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxycamptothecin (APC). Exposures to the other metabolites were within the normal range. Overall, 83.4% of the administered dose was recovered in the excreta, with the majority excreted during the first 24 hours. Conclusions: Enterohepatic recirculation is of minor importance for the plasma disposition of irinotecan and its metabolites. The high percentage of the dose recovered in urine, the relatively slow clearance of irinotecan and the preferential urinary over biliary excretion of APC in this particular patient are likely related to reduced functioning of ABC-transporters. Circumstantial evidence was found that APC is subject to further intestinal biotransformation indicating a role in the etiology of irinotecan-induced diarrhea. Since intra-luminal exposure to SN-38 in patients with an external bile drain seems limited, neutropenia will be the dose-limiting toxicity. Based on presented data, although collected from only one patient, irinotecan therapy in patients with an external bile drain is feasible. No a priori dose adjustments are recommended, although depending on the absence of severe side-effects in previous courses, a higher dose may be considered.

Development of Antiviral Fusion Inhibitors: Short Modified Peptides Derived from the Transmembrane Glycoprotein of Feline Immunodeficiency Virus

Feline immunodeficiency virus (FIV) is a naturally occurring pathogen that causes an AIDS-like syndrome in domestic cats and is a valuable model system by which criteria for antiviral vaccines and drugs development can be tested. The cell-entry step of the lentivirus life cycle is regarded as a promising target for the development of new generation inhibitors. We have previously described potent in vitro anti-FIV activity associated with a synthetic octapeptide, termed C8 (Ac-Trp-Glu-Asp-Trp-Val-Gly-Trp-Ile-NH2), containing the Trp-rich motif of FIV transmembrane glycoprotein, which shares a common structural framework with the corresponding molecule of HIV and appears to play a similar role in cell entry. In this report, in an attempt to develop simpler potential fusion inhibitors to be tested in vivo, we describe further studies focused on synthetic peptide analogues of C8. Since C8 inhibitory activity is dependent upon the Trp motif, we systematically replaced these residues with bulky and/or aromatic natural and unnatural amino acids, in order to develop a rational structure-activity relationship. Furthermore, the amino acids located between the Trp residues, which are not crucial for inhibitory activity, were replaced by simple alkyl spacers of appropriate length. Design, NMR structural analysis, in vitro anti-FIV activity in lymphoid cell cultures, and serum stability of these new analogues are reported. The final results indicate that a simpler hexapeptide (Ac-Nal2-Ape-Nal2-Ape-Nal2-Ile-NH2; Nal2 = 3-naphthalen-2-yl-L-alanine, Ape = 5-aminopentanoic acid), almost entirely made up of unnatural amino acid residues, has markedly increased enzymatic stability, while maintaining strong antiviral potency in vitro.

Partial molar volumes and adiabatic compressibilities at infinite dilution of aminocarboxylic acids and glycylglycine in water and aqueous solutions of sodium sulphate at (288.15, 298.15 and 308.15) K

The partial molar volume and partial molar adiabatic compressibilities at infinite dilution of dl-aminobutanoic acid, dl-norvaline, β-alanine, 4-aminobutanoic acid, 5-aminopentanoic acid, 6-aminohexanoic acid and glycylglycine have been obtained in water and aqueous solutions of (0.5, 1.0 and 1.5) mol · kg −1 sodium sulphate at (288.15, 298.15 and 308.15) K from measurements of density and ultrasonic velocity. A qualitative interpretation of the results has been given using the Kirkwood model and nature of the interactions in solutions. A model, derived from Scaled Particle Theory, has been used for quantitative explanation of partial molar volumes and for the understanding the volumes of interaction. The results distinguish the behaviour of α-amino acids from that of α,ω-amino acids, and of the “less polar” 5-aminopentanoic acid from that of the analogous, but “more polar”, glycylglycine in solution. These findings are in agreement with previous studies in aqueous solutions and they support the water-structure making ability of sodium sulphate.

In vitro condensation of amino acids in aqueous media: A synthesis driven by catalytic carbon monoxide oxidation

A novel mechanism is proposed for amino acid condensations to oligopeptides in aqueous media. The palladium-catalyzed condensation appears to proceed through a mechanism involving the Pd(0)/Pd(II) redox cycle. The reaction requires the use of carbon monoxide and an oxidant, and it is proposed that the oxidation of carbon monoxide to carbon dioxide drives the otherwise thermodynamically uphill condensation. The reaction occurs for several amino acids including glycine, alanine, β-alanine, and 5-aminopentanoic acid. Competition studies between glycine and alanine reveal steric effects on product formation. Dipeptides are the predominant condensation products, though some longer chains, containing up to 4 amino acids, can be observed. First-order plots for dipeptide appearance show an inverse, secondary, isotope effect consistent with the rate determining carboxylic-carbamic anhydride formation.

Effect of Milk Thistle (Silybum marianum) on the Pharmacokinetics of Irinotecan

Milk thistle (Silybum marianum) is one of the most commonly used herbal therapies, and its principal constituent silybin significantly inhibits cytochrome P450 isoform 3A4 (CYP3A4) and UDP glucuronosyltransferase isoform 1A1 (UGT1A1) in vitro. Here, we investigated whether milk thistle affects the pharmacokinetics of irinotecan, a substrate for CYP3A4 and UGT1A1, in humans. Six cancer patients were treated with irinotecan (dose, 125 mg/m(2)) given as a 90-minute infusion once every week. Four days before the second dose, patients received 200 mg milk thistle, thrice a day, for 14 consecutive days. Pharmacokinetic studies of irinotecan and its metabolites 7-ethyl-10-hydroxycamptothecin (SN-38), 7-ethyl-10-[3,4,5-trihydroxy-pyran-2-carboxylic acid]-camptothecin (SN-38-glucuronide), and 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]-carbonyloxycamptothecin were done during the first three irinotecan administrations. Short-term (4 days) or more prolonged intake of milk thistle (12 days) had no significant effect on irinotecan clearance (mean, 31.2 versus 25.4 versus 25.6 L/h; P = 0.16). The area under the curve ratio of SN-38 and irinotecan was slightly decreased by milk thistle (2.58% versus 2.23% versus 2.17%; P = 0.047), whereas the relative extent of glucuronidation of SN-38 was similar (10.8 versus 13.5 versus 13.1; P = 0.64). Likewise, the area under the curve ratio of 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]-carbonyloxycamptothecin and irinotecan was unaffected by milk thistle (0.332 versus 0.285 versus 0.337; P = 0.53). The maximum plasma concentrations of silybin ranged between 0.0249 and 0.257 micromol/L. Silybin concentrations after intake of milk thistle are too low to significantly affect the function of CYP3A4 and UGT1A1 in vivo, indicating that milk thistle is unlikely to alter the disposition of anticancer drugs metabolized by these enzymes.

Gefitinib (Iressa) inhibits the CYP3A4-mediated formation of NPC, but activates that of APC from irinotecan

Gefitinib (Iressa) is an anticancer drug that selectively inhibits tyrosine kinases of epidermal growth factor receptor. Gefitinib might affect CYP3A4-mediated metabolism, since the drug is a substrate of human CYP3A. In this study, we evaluated the effects of gefitinib on drug metabolism catalyzed by human CYP3A4. The effects of gefitinib on the CYP3A4-mediated formation of NPC (7-ethyl-10-(4-amino-1-piperidino)carbonyloxycamptothecin) and that of APC (7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]carbonyloxycamptothecin) from irinotecan were examined with the use of human liver and small intestinal microsomes. Gefitinib inhibited the formation of NPC in liver and small intestinal microsomes. The apparent intrinsic metabolic clearance (CL(int)) in the presence of 40 microM gefitinib was equivalent to about 26% of control in liver microsomes and 45% of control in small intestinal microsomes. Gefitinib stimulated the formation of APC by CYP3A4. CL(int) in the presence of 20 microM gefitinib with human liver microsomes was about 1.9 times higher than control. In human small intestinal microsomes, APC formation was enhanced by the addition of gefitinib at concentrations 20 microM or higher. CL(int) in the presence of 40 microM gefitinib was 2.8 times higher than control. Thus, we discovered that gefitinib inhibited the formation of NPC but stimulated the formation of APC from irinotecan.

Loperamide inhibits the biliary excretion of irinotecan (CPT-11) in the rat isolated perfused liver

Patients treated with irinotecan (CPT-11) occasionally suffer from severe diarrhoea and aggressive treatment with loperamide at the first signs of loose stools is recommended. We have examined the effect of loperamide on the hepatic metabolism and biliary excretion of CPT-11 in the isolated perfused rat liver (IPRL). CPT-11 (0.5 mumol) was injected as a bolus into the IPRL reservoir, and perfusate and bile samples were collected over 3 h. Experiments were conducted using loperamide-free perfusate (n = 5) or perfusate containing 10 muM loperamide (n = 6). Perfusate and bile concentrations of total CPT-11 and the major metabolites SN-38 (7-ethyl-10-hydroxy-camptothecin), SN-38G (7-ethyl-10-hydroxy-camptothecin glucuronide) and APC (7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidine] carbonyloxycamptothecin) were determined by HPLC. The unchanged parent drug was the predominant species in bile, with approximately 4% of the dose recovered over 180 min as compared with only 1% for the metabolites. Loperamide significantly reduced the biliary excretion of CPT-11 by approximately 50% (2.0 +/- 0.9% dose compared with 3.8 +/- 1.0% in the control group, P = 0.019) over the same period. In contrast, the biliary excretion of SN-38, SN-38G and APC was not significantly affected by loperamide (P > 0.05). Furthermore, bile flow rate was not affected by loperamide. Loperamide appeared to selectively inhibit the biliary excretion of CPT-11, although the extent to which loperamide altered the disposition of CPT-11 in the clinical setting remains to be determined.

Hydrolysis of irinotecan and its oxidative metabolites, 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxycamptothecin and 7-ethyl-10-[4-(1-piperidino)-1-amino]-carbonyloxycamptothecin, by human carboxylesterases CES1A1, CES2, and a newly expressed carboxylesterase isoenzyme, CES3.

Carboxylesterases metabolize ester, thioester, carbamate, and amide compounds to more soluble acid, alcohol, and amine products. They belong to a multigene family with about 50% sequence identity between classes. CES1A1 and CES2 are the most studied human isoenzymes from class 1 and 2, respectively. In this study, we report the cloning and expression of a new human isoenzyme, CES3, that belongs to class 3. The purified recombinant CES3 protein has carboxylesterase activity. Carboxylesterases metabolize the carbamate prodrug 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin (CPT-11; irinotecan) to its active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38), a potent topoisomerase I inhibitor. CYP3A4 oxidizes CPT-11 to two major oxidative metabolites, 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxycamptothecin (APC) and 7-ethyl-10-[4-(1-piperidino)-1-amino]-carbonyloxycamptothecin (NPC). In this study, we investigate whether these oxidative metabolites, NPC and APC, can be metabolized to SN-38 by purified human carboxylesterases, CES1A1, CES2, and CES3. We find that CPT-11, APC, and NPC can all be metabolized by carboxylesterases to SN-38. CES2 has the highest catalytic activity of 0.012 min(-1) microM(-1) among the three carboxylesterases studied for hydrolysis of CPT-11. NPC was an equally good substrate of CES2 in comparison to CPT-11, with a catalytic efficiency of 0.005 min(-1) microM(-1). APC was a very poor substrate for all three isoenzymes, exhibiting a catalytic activity of 0.015 x 10(-3) min(-1) microM(-1) for CES2. Catalytic efficiency of CES3 for CPT-11 hydrolysis was 20- to 2000-fold less than that of CES1A1 and CES2. The relative activity of the three isoenzymes was CES2 > CES1A1 >> CES3, for all three substrates.

Clinical pharmacokinetics of irinotecan and its metabolites: a population analysis.

To build population pharmacokinetic (PK) models for irinotecan (CPT-11) and its currently identified metabolites. Seventy cancer patients (24 women and 46 men) received 90-minute intravenous infusions of CPT-11 in the dose range of 175 to 300 mg/m(2). The PK models were developed to describe plasma concentration profiles of the lactone and carboxylate forms of CPT-11 and 7-ethyl-10-hydroxycamptothecin (SN-38) and the total forms of SN-38 glucuronide (SN-38G), 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]-carbonyloxycamptothecin (APC), and 7-ethyl-10-[4-amino-1-piperidino]-carbonyloxycamptothecin (NPC) by using NONMEM. The interconversion between the lactone and carboxylate forms of CPT-11 was relatively rapid, with an equilibration half-life of 14 minutes in the central compartment and hydrolysis occurring at a rate five times faster than lactonization. The same interconversion also occurred in peripheral compartments. CPT-11 lactone had extensive tissue distribution (steady-state volume of distribution [Vss], 445 L) compared with the carboxylate form (Vss, 78 L, excluding peripherally formed CPT-11 carboxylate). Clearance (CL) was higher for the lactone form (74.3 L/h) compared with the carboxylate form (12.3 L/h). During metabolite data modeling, goodness of fit indicated a preference of SN-38 and NPC to be formed out of the lactone form of CPT-11, whereas APC could be modeled best by presuming formation from CPT-11 carboxylate. The interconversion between SN-38 lactone and carboxylate was slower than that of CPT-11, with the lactone form dominating at equilibrium. The CLs for SN-38 lactone and carboxylate were similar, but the lactone form had more extensive tissue distribution. Plasma data of CPT-11 and metabolites could be adequately described by this compartmental model, which may be useful in predicting the time courses, including interindividual variability, of all characterized substances after intravenous administrations of CPT-11.

Determination of drug interactions occurring with the metabolic pathways of irinotecan.

Irinotecan or CPT-11 [7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecine] is a derivative of camptothecine used in the treatment of advanced colorectal cancer. It requires activation to SN-38 (7-ethyl-10-hydroxycamptothecine) by carboxylesterase. Irinotecan and SN-38 are detoxified through two major metabolic pathways: the first one leads to oxidative degradation compounds, APC [7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]carbonyloxycamptothecine] and NPC [7-ethyl-10-(4-amino-1-piperidino)carbonyloxycamptothecine], and involves cytochrome P450 (3A4 isoform); the second one leads to SN-38 glucuronide (SN-38G) and involves UDP-glucuronosyltransferase (UGT). Using human hepatic microsomes, we studied the interactions of 15 drugs of common use in colorectal cancer patients on these metabolic pathways. Only nifedipine had a significant effect on SN-38 formation, decreasing carboxylesterase activity by 50% at 100 microM and 35% at 10 microM. Three drugs had a significant effect on SN-38G formation: clonazepam increased UGT activity by 50% at 100 microM and 30% at 10 microM, and nifedipine and vinorelbine inhibited the activity by 65 and 55% at 100 microM, respectively, with no effect at 10 microM. Five drugs exerted a significant inhibition on SN-38 formation at 100 microM: clonazepam (70%), methylprednisolone (50%), nifedipine (80%), omeprazole (85%), and vinorelbine (100%). Only omeprazole and vinorelbine still exerted a significant inhibition at 10 microM (30 and 90%, respectively), whereas only vinorelbine had a significant effect at 2 and 0.5 microM (70 and 40%, respectively). In conclusion, potential clinical interactions with the metabolism of irinotecan are likely to be important for vinorelbine, which strongly inhibits irinotecan catabolism by CYP3A4 at clinically relevant concentrations, but not for the other drugs, which exert an effect at concentrations not achievable in patients.

SYNTHESIS AND HYBRIDIZATION STUDIES OF A 5-AMINOPENTANOIC ACID NUCLEOBASE (APN) DIMER

ABSTRACT We have prepared a 5-aminopentanoic acid nucleobase (APN) dimer and investigated its hybridization capabilities to complementary DNA using both UV melting and NMR techniques.

An analytical method for irinotecan (CPT-11) and its metabolites using a high-performance liquid chromatography: parallel detection with fluorescence and mass spectrometry.

Irinotecan or 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11) is an anticancer pro-drug used in the treatment of many types of cancer. We describe here the validation of an analytical method for CPT-11 and its metabolites, including an active metabolite, 7-ethyl-10-hydroxycamptothecin (SN-38), its glucuronidated form, SN-38G, and several cytochrome P450 3A-mediated products such as 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]carbonyloxycamptothecin (APC) using a high-performance liquid chromatography connected to parallel fluorescence and mass spectrometry detection systems. This method is characterized as follows: (1) simple extraction of the analytes from biomaterials with perchloric acid/methanol; (2) sensitive quantitation of major metabolites (SN-38G, SN-38 and APC) with a fluorescence detector (FLD), where the limits of quantitation by FLD were 2.5 ng/mL for SN-38G and APC, 5 ng/mL for CPT-11 and 1 ng/mL for SN-38, respectively; (3) parallel selective monitoring of the metabolites including minor metabolites with a mass selected detector (MSD). There was no observed interference by other drugs expected to be co-administered. This method showed its usefulness by identifying a novel metabolite produced in human hepatic microsomes. The results indicate that this combination of FLD and MSD enables a highly selective analysis of CPT-11 and its metabolites, and is useful for studies both in vivo and in vitro.

Synthesis and activity on human neutrophil functions of fMLF-OMe analogs containing alkyl spacers at the central position

We report here the synthesis and activity of new analogs of the N-formyl and N- tert-butyloxycarbonyl (Boc) derivatives of the tripeptide Met-Leu-Phe-OMe containing an achiral ω-amino acid residue replacing the hydrophobic central leucine. The tripeptides HCO-Met-NH-(CH 2) n -CO-Phe-OMe and Boc-Met-NH-(CH 2) n -CO-Phe-OMe ( n=3–5) containing the central homomorphic residue of 5-aminopentanoic acid (δ-aminovaleric acid; δ-Ava; n=4) and the two non-homomorphic residues of 4-aminobutanoic acid (γ-aminobutyric acid; γ-Abu; n=3) and 6-aminohexanoic acid (ε-aminocaproic acid; ε-Aca; n=5) have been examined. The activity as agonists and antagonists in chemotaxis, lysozyme release, and superoxide anion production of the new analogs has been determined. The N-Boc derivatives 2a and 2b, incorporating the γ-Abu and the δ-Ava residues, show good and selective antagonist activity on superoxide anion production.

Solution structure and dynamics of the plasminogen kringle 2-AMCHA complex: 3(1)-helix in homologous domains.

The kringle 2 (K2) module of human plasminogen (Pgn) binds L-lysine and analogous zwitterionic compounds, such as the antifibronolytic agent trans-(aminomethyl)cyclohexanecarboxylic acid (AMCHA). Far-UV CD and NMR spectra reveal little conformational change in K2 upon ligand binding. However, retarded (1)H-(2)H isotope exchange kinetics induced by AMCHA indicate stabilization of the K2 conformation by the ligand. Assessment of secondary structure content from CD spectra yields approximately 26% beta-STRAND, approximately 13% beta-TURN, approximately 15% 3(1)-HELIX, and approximately 6% 3(10)-HELIX. The NMR solution conformation of the K2 domain complexed to AMCHA has been determined [heavy atom rmsd = 0.49 +/- 0.09A (BACKBONE) AND 1.02+/- 0.08 (ALL)]. The K2 molecule has overall dimensions of approximately 34.5A times approximately 33.4A times approximately 22.7A . Analogous with the polypeptide outline of homologous domains, K2 contains three short antiparallel beta-sheets (paired strands 15-16/20-21, 24-25/48-49, and 62-64/72-74) and four defined beta-turns (residues 6-9, 16-19, 53-56, AND 67-70). Consistent with the CD analysis, albeit novel in the context of kringle folding, the NMR structure reveals an unpaired beta-strand structured by residues 30-32, a turn of 3(10)-helix compromising residues 38-41, and a 3(1)-helix for residues 21-24 and 74-79. We also identify alignable 3(1)-helices in previously reported homologous kringle structures. Rather high order parameter S(2) values (<S(2)>= approximately 0.85 +/- 0.04) characterize the K2 backbone dynamics. The lowest flexibility is observed for the two inner loop segments of residues 51-63 AND 63-75 (<S(2)>= approximately 0.86-0.87 +/- 0.03). Overhauser connectivities reveal close hydrophobic contacts of the ligand ring with side chains of Tyr(36), Trp(62), Phe(64), Trp(72), AND Leu(74). In most K2 structures, the N atom of AMCHA places itself approximately 3.9 and 4.4A from the anionic groups of Glu(57) and Asp(55), respectively, while its carboxylate group, H-bonded to the Tyr(36) side chain OH(eta), ion-pairs the Arg(71) guanidinium group. Consistent with the preference of K2 for binding 5-aminopentanoic acid over 6-aminohexanoic acid, the positions of the ionic centers within the K2 binding site approach each other approximately 1A closer relative to what is observed in lysine binding sites of homologous Pgn modules.

Enhancement through Mutagenesis of the Binding of the Isolated Kringle 2 Domain of Human Plasminogen to ω-Amino Acid Ligands and to an Internal Sequence of a Streptococcal Surface Protein

In the background of the recombinant K2 module of human plasminogen (K2(Pg)), a triple mutant, K2(Pg)[C4G/E56D/L72Y], was generated and expressed in Pichia pastoris cells in yields exceeding 100 mg/liter. The binding affinities of a series of lysine analogs, viz. 4-aminobutyric acid, 5-aminopentanoic acid, epsilon-aminocaproic acid, 7-aminoheptanoic acid, and t-4-aminomethylcyclohexane-1-carboxylic acid, to this mutant were measured and showed up to a 15-fold tighter interaction, as compared with wild-type K2(Pg) (K2(Pg)[C4G]). The variant, K2(Pg)[C4G/E56D], afforded up to a 4-fold increase in the binding affinity to these same ligands, whereas the K2(Pg)[C4G/L72Y] mutant decreased the same affinities up to 5-fold, as compared with K2(Pg)[C4G]. The thermal stability of K2(Pg)[C4G/E56D/L72Y] was increased by approximately 13 degrees C, as compared with K2(Pg)[C4G]. The functional consequence of up-regulating the lysine binding property of K2(Pg) was explored, as reflected by its ability to interact with an internal sequence of a plasminogen-binding protein (PAM) on the surface of group A streptococci. A 30-mer peptide of PAM, containing its K2(Pg)-specific binding region, was synthesized, and its binding to each mutant of K2(Pg) was assessed. Only a slight enhancement in peptide binding was observed for K2(Pg)[C4G/E56D], compared with K2(Pg)[C4G] (K(d) = 460 nM). A 5-fold decrease in binding affinity was observed for K2(Pg)[C4G/L72Y] (K(d) = 2200 nM). However, a 12-fold enhancement in binding to this peptide was observed for K2(Pg)[C4G/E56D/L72Y] (K(d) = 37 nM). Results of these PAM peptide binding studies parallel results of omega-amino acid binding to these K2(Pg) mutants, indicating that the high affinity PAM binding by plasminogen, mediated exclusively through K2(Pg), occurs through its lysine-binding site. This conclusion is supported by the 100-fold decrease in PAM peptide binding to K2(Pg)[C4G/E56D/L72Y] in the presence of 50 mM 6-aminohexanoic acid. Finally, a thermodynamic analysis of PAM peptide binding to each of these mutants reveals that the positions Asp(56) and Tyr(72) in the K2(Pg)[C4G/E56D/L72Y] mutant are synergistically coupled in terms of their contribution to the enhancement of PAM peptide binding.

Expression, isolation and characterization of a mutated human plasminogen kringle 3 with a functional lysine binding site.

Each kringle of human plasminogen (HPg) except kringle 3 (K3) exhibits affinity for omega-aminocarboxylic acids. Assuming that the K3 domain contains a preformed but nonfunctional lysine binding site (LBS), Lys311 was altered by site-directed mutagenesis into Asp311 in accordance with the consensus sequence of the LBS. Cys297 involved in the interkringle disulfide bridge was mutated into Ser297 to minimize dimerization and aggregation. The mutated K3 TYQ[K3HPg/C297S/K311D]DS (r-K3mut) was expressed in Escherichia coli, isolated on an Ni2(+)-nitrilotriacetic acid-agarose column, refolded and purified on a lysine Bio-Gel column. Fluorescence titration indicates affinity of r-K3mut for omega-aminocarboxylic acids with the following association constants (Kass, mM-1): 5-aminopentanoic acid: 1.3; 6-aminohexanoic acid: 4.2; 7-aminoheptanoic acid: 0.5; trans-(aminomethyl)cyclohexanecarboxylic acid: 12.7; p-benzylaminesulfonic acid: 11.8. r-K3mut exhibits an affinity similar to native and mutated (R220G, E221D) K2. The results indicate the presence of a preformed but nonfunctional LBS in native K3 of HPg. We were able to demonstrate for the first time that an appropriate mutation in the LBS of a kringle produced a weak but distinct affinity for omega-aminocarboxylic acids.