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PubMed1985

Clostridium histolyticum collagenase: development of new thio ester, fluorogenic, and depsipeptide substrates and new inhibitors.

A new series of thio ester, depsipeptide, and peptide substrates have been synthesized for the bacterial enzyme Clostridium histolyticum collagenase. The hydrolysis of the depsipeptide substrate was followed on a pH stat, and thio ester hydrolysis was measured by inclusion of the chromogenic thiol reagent 4,4'-dithiopyridine in the assay mixture. The best thio ester substrate, Boc-Abz-Gly-Pro-Leu-SCH2CO-Pro-Nba, had a kcat/KM of 63 000 M-1 s-1, while several shorter thio ester sequences were inactive as substrates. In general, the peptide analogues of all the reactive thio ester substrates were shown to be hydrolyzed 5-10 times faster by collagenase. In one case (Z-Gly-Pro-Leu-Gly-Pro-NH2) where a comparison was made, the peptide substrate was respectively 8- and 106-fold more readily hydrolyzed than the corresponding thio ester and ester substrates. Cleavages of the two fluorescence-quench substrates Abz-Gly-Pro-Leu-Gly-Pro-Nba and Abz-Gly-Pro-Leu-SCH2CO-Pro-Nba could be easily followed fluorogenically since a 5-10-fold increase in fluorescence occurred upon hydrolysis. The fluorescent peptide substrate is the best synthetic substrate known for C. histolyticum collagenase with a kcat/KM value of 490 000 M-1 s-1. A series of new reversible inhibitors were developed by the attachment of zinc ligating groups (hydroxamic acid, carboxymethyl, and thiol) to various peptide sequences specific for C. histolyticum collagenase. The shorter peptides designed to bind to either the P3-P1 or P1'-P3' subsites were poor to moderate inhibitors. The thiol HSCH2CH2CO-Pro-Nba had the lowest K1 (0.02 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

Clostridium↗
PubMed1989

Beta-lactamase-catalyzed aminolysis of depsipeptides: proof of the nonexistence of a specific D-phenylalanine/enzyme complex by double-label isotope trapping.

The steady-state kinetics of the Enterobacter cloacae P99 beta-lactamase-catalyzed aminolysis of the depsipeptide m-[[(phenylacetyl)glycyl]oxy]benzoic acid by D-phenylalanine were consistent with an ordered sequential mechanism with D-phenylalanine binding first [Pazhanisamy, S., Govardhan, C. P., & Pratt, R. F. (1989) Biochemistry (first of three papers in this issue)]. In terms of this mechanism, the kinetics data required that in 20 mM MOPS buffer, pH 7.5, the dissociation constant of the initially formed enzyme/D-phenylalanine complex be around 1.3 mM; at pH 9.0 in 0.1 M carbonate buffer, the complex should be somewhat more stable. Attempts to detect this complex in a binary mixture by spectroscopic methods (fluorescence, circular dichroic, and nuclear magnetic resonance spectra) failed. Kinetic methods were also unsuccessful--the presence of 20 mM D-phenylalanine did not appear to affect beta-lactamase activity nor inhibition of the enzyme by phenylmethanesulfonyl fluoride, phenylboronic acid, or (3-dansylamidophenyl)boronic acid. Equilibrium dialysis experiments appeared to indicate that the dissociation constant of any binary enzyme/D-phenylalanine complex must be somewhat higher than the kinetics allowed (greater than 2 mM). Since the kinetics also required that, at high depsipeptide concentrations, and again with the assumption of the ordered sequential mechanism, the reaction of the enzyme/D-phenylalanine complex to aminolysis products be faster than its reversion to enzyme and D-phenylalanine, a double-label isotope-trapping experiment was performed.(ABSTRACT TRUNCATED AT 250 WORDS)

Circular Dichroism↗
PubMed1982

Enniatin synthetase, a novel type of multifunctional enzyme catalyzing depsipeptide synthesis in Fusarium oxysporum.

Enniatin synthetase, a multifunctional enzyme catalyzing depsipeptide formation in Fusarium oxysporum was purified to 98% homogeneity as judged by analytical disc gel electrophoresis. The enzyme consists of a single polypeptide chain of a molecular weight of about 250 000. Similar to a number of peptide synthetases and to fatty acid synthetase the enzyme contains 4'-phosphopantetheine as a prosthetic group. Studies on substrate specificity revealed that the enzyme is capable of synthesizing enniatins A--C and also mixed-type enniatins containing more than one species of amino acid. A linear dependence of rate of enniatin synthesis on enzyme concentration was observed, indicating that depsipeptide formation is an intramolecular process. Omission of the methyl donor S-adenosyl-L-methionine resulted in the formation of unmethylated enniatins with a reaction rate of about 10% of that observed in the case of enniatins. Sulfhydryl-directed reagents generally had an inhibitory influence on enniatin synthesis. However, inhibition studies with iodoacetamide revealed that it behaves differently toward the hydroxy acid site(s) and amino acid site(s). This indicates the presence of chemically distinct thiol groups in the active sites for the two substrates.

Amino Acids↗
PubMed1996

Kinetics and mechanism of the hydrolysis of depsipeptides catalyzed by the beta-lactamase of Enterobacter cloacae P99.

The steady-state kinetics and mechanism of the hydrolysis and aminolysis of a series of acyclic depsipeptides, catalyzed by the class C beta-lactamase of Enterobacter cloacae P99, have been studied in order to more firmly establish the nature of the transition states involved. The class C beta-lactamase of Enterobacter cloacae P99 was employed. The depsipeptide substrates contained a constant acyl group, (phenylacetyl)glycyl, and chemically different leaving groups, m-carboxyphenoxide, m-carboxythiophenoxide, 3-carboxyl-4-nitrophenoxide, lactate, and thiolactate. Evaluation of the steady-state kinetic parameters and the effect of the alternative nucleophile methanol on these parameters and on the product distribution showed that deacylation was largely rate-determining to turnover of the aryl esters under conditions of substrate saturation, while acylation was rate-determining to the alkyl esters. The earlier conclusion [Govardhan & Pratt (1987) Biochemistry 26, 3385-3395] that acylation largely limited the turnover of the aryl esters was shown to be an artifact of phosphate buffer inhibition. The aminolysis of both the aryl the alkyl esters by D-phenylalanine was influenced by binding of the substrate at a second binding site on the acyl-enzyme intermediate. A study of inhibiton of the hydrolysis of (phenylacetyl)-glycyl-D-thiolactate by the aminolysis product (phenylacetyl)glycyl-D-phenylalanine indicated that the second binding site is also available for ligands to bind the free enzyme and to the noncovalent Michaelis complex with this substrate. It is likely that penicillin-recognizing enzymes in general, both beta-lactamases and DD-peptidases, possess an extended substrate-binding site into which a variety of small ligands may bind at any point along the reaction coordinate and, to a greater or lesser extent depending on circumstances, affect catalysis.

Acylation↗
PubMed1991

Evidence from a mutant beta-lactamase for the mechanism of beta-lactamase-catalysed depsipeptide aminolysis.

The Ser-70----Gly mutant of the TEM-1 beta-lactamase, where the active-site serine hydroxy group has been lost, does not catalyse the hydrolysis of either benzylpenicillin or N-(phenylacetyl)glycyl depsipeptides. This is as would be expected for a double-displacement mechanism where the Ser-70 becomes acylated at an intermediate stage. Further, however, the mutant enzyme, unlike the wild-type, does not catalyse aminolysis of depsipeptides by D-phenylalanine. If the active site is not structurally disrupted by the mutation, this result shows that Ser-70 is necessary for the aminolysis reaction and implies that this reaction, like the hydrolysis, proceeds by way of an acyl-(serine)-enzyme intermediate. Although physical evidence suggests that the mutant enzyme does not have a structure in solution identical with that of the wild-type, the mutant does still bind beta-lactam substrates. The latter result suggests sufficient conservation of the active-site structure for the major conclusion above to hold.

Amines↗
PubMed2000

Genes encoding synthetases of cyclic depsipeptides, anabaenopeptilides, in Anabaena strain 90.

Anabaena strain 90 produces three hepatotoxic heptapeptides (microcystins), two seven-residue depsipeptides called anabaenopeptilide 90A and 90B, and three six-residue peptides called anabaenopeptins. The anabaenopeptilides belong to a group of cyanobacterial depsipeptides that share the structure of a six-amino-acid ring with a side-chain. Despite their similarity to known cyclic peptide toxins, no function has been assigned to the anabaenopeptilides. Degenerate oligonucleotide primers based on the conserved amino acid sequences of other peptide synthetases were used to amplify DNA from Anabaena 90, and the resulting polymerase chain reaction (PCR) products were used to identify a peptide synthetase gene cluster. Four genes encoding putative anabaenopeptilide synthetase domains were characterized. Three genes, apdA, apdB and apdD, contain two, four and one module, respectively, encoding a total of seven modules for activation and peptide bond formation of seven L-amino acids. Modules five and six also carry methyltransferase-like domains. Before the first module, there is a region similar in amino acid sequence to formyltransferases. A fourth gene (apdC), between modules six and seven, is similar in sequence to halogenase genes. Thus, the order of domains is co-linear with the positions of amino acid residues in the finished peptide. A mutant of Anabaena 90 was made by inserting a chloramphenicol resistance gene into the apdA gene. DNA amplification by PCR confirmed the insertion. Mass spectrometry analysis showed that anabaenopeptilides are not made in the mutant strain, but other peptides, such as microcystins and anabaenopeptins, are still produced by the mutant.

Amino Acid Sequence↗
PubMed1999

Synthesis and enzymatic degradation of optically active depsipeptide copolymers.

This paper describes the synthesis and biodegradation of copolymers of cyclic depsipeptide with epsilon-caprolactone (CL) or lactide (LA). Optically active cyclic depsipeptides, 3,6-dimethyl-2,5-morpholinediones (DMOs), were prepared by the reaction of an amino acid (D-, L-, or DL-alanine) with a hydroxy acid derivative (DL-2-bromopropionyl bromide). These isomers are abbreviated as D-DMO, L-DMO and DL-DMO respectively, according to the names of alanine isomers. Then, we have prepared the copolymers of DMO isomers with CL using tin(II) octylate as a catalyst. The NMR spectra and thermal properties of DMO/CL copolymers revealed that these copolymers exist randomly. The enzymatic degradation of the copolymers has been examined using Rhizopus delemar lipase, cholesterol esterase (from Pseudomonas sp.), and Proteinase K (from Tritirachium album). Cholesterol esterase and Proteinase K show high degradability, while the lipase shows little degradation. Among the enzymes used, only Proteinase K could recognize the isomerism of DMO, resulting in the following order of degradability: copoly(L-DMO/CL) > copoly(DL-DMO/CL) > copoly(D-DMO/CL), i.e. this enzyme has the highest substrate specificity for naturally occurring L-alanine. Further, we have prepared the random copolymers of L-DMO with lactide (L-LA or DL-LA), and evaluated the enzymatic degradation of the copolymers by Proteinase K. The introduction of a small amount (up to c. 10 mol%) of L-DMO unit into LA homopolymers brought about greater degradability compared with LA homopolymers. In particular, L-DMO/L-LA copolymers with high degradability have been obtained without significant decrease in the mechanical and thermal properties of L-LA homopolymer.

Endopeptidase K↗
PubMed1979

The identification of depsipeptides by chemical ionisation mass spectroscopy.

The chemical ionisation mass spectra of seven naturally occurring depsipeptides and some of their permethylated derivatives have been measured. The primary ionisation process involves an ester group and not an amide or other functionality. It probably occurs randomly when the molecule contains more than one ester link. Unlike electron impact mass spectra, those obtained under chemical ionisation conditions gave sequence information for all of the depsipeptides examined or their permethylated derivatives. A mechanism for the primary fragmentation is proposed.

Anti-Bacterial Agents↗
PubMed2007

Simulation of beta-depsipeptides: the effect of missing hydrogen-bond donors on their folding equilibria.

beta-Depsipeptides are beta-peptides in which one or more peptide linkages are replaced by ester linkages, resulting in a loss of a hydrogen-bond donor (N--H) and weakening of the corresponding carbonyl hydrogen-bond acceptor moiety. The effects of three of such peptide by ester substitutions in a hepta-beta-peptide upon its (un)folding equilibrium in methanol solution are investigated using molecular dynamics simulations and compared to experimental data from NMR spectroscopy. The simulated conformational ensembles largely reproduce the experimentally measured NOE and 3J-coupling constant data for the three different hepta-beta-peptides, and confirm the relative stabilities of the 3(14)-helical conformation, which is most weakened by substitution of the 4th peptide linkage and least by substitution of the 6th peptide linkage. The simulations are complementary to the experimental data by providing detailed insight into the conformational distributions that are compatible with the experimentally measured average values of observables.

Computational Biology↗
PubMed2004

Liquid chromatographic assay for the cyclic depsipeptide aplidine, a new marine antitumor drug, in whole blood using derivatization with trans-4'-hydrazino-2-stilbazole.

A sensitive bio-analytical assay for the depsipeptide aplidine in plasma has been modified and tested for human whole blood samples. The adapted method is based on reversed-phase liquid chromatography and fluorescence detection of the trans-4'-hydrazino-2-stilbazole derivative of the analyte. Aplidine is isolated from the matrix by solid-phase extraction on an octadecyl modified silica stationary phase. After evaporation of the acetone eluate, the derivatization with the hydrazino reagent is performed in a water-acetonitrile mixture at pH = 4. The reaction mixture is injected directly into the chromatograph and the analyte is quantified by fluorescence detection at 410 and 560 nm for excitation and emission, respectively. The method has been validated in the 2-100 ng/mL range, with 2 ng/mL being the lower limit of quantification. Precision and accuracy both meet the current requirements for a bioanalytical assay. The stability of aplidine in whole blood at ambient temperature and at 37 degrees C is limited; recoveries in the range 60-85% were observed after 7 h. Further, adequate stability of aplidine in plasma at -80 and -20 degrees C for 35 months could now be demonstrated.

Antineoplastic Agents↗
PubMed2006

Effect of the solvent on the conformation of a depsipeptide: NMR-derived solution structure of hormaomycin in DMSO from residual dipolar couplings in a novel DMSO-compatible alignment medium.

The macrocyclic compound hormaomycin has been investigated by NMR spectroscopy and by restrained molecular-dynamics simulations. Measurement of residual dipolar couplings induced by dissolving the depsipeptide in a polyacrylamide gel compatible with DMSO and their incorporation into the structure calculation of the title compound improved the precision of the family of structures. In DMSO the macrocyclic ring shows two beta-turns, whose positions in the sequence differ from those found in the CDCl3 solution structure and in the crystal structure obtained from hexylene glycol/H2O (50:50). The bulky side chain consisting of a 3-(2-nitrocyclopropyl)alanine and a chlorinated N-hydroxypyrrole moiety is flexible in DMSO.

Bacterial Proteins↗
PubMed2006

Self-assembly of chiral depsipeptide dendrimers.

The self-assembly of chiral depsipeptide dendrons 4, which contain a cyanuric acid building block at their focal point, with the homotritopic Hamilton receptor 1 is reported. The 1:3 compositions of the resulting chiral supramolecular dendrimers, the association constants K(n), and the cooperativity of binding expressed by Scatchard plots and the Hill coefficients n(H) was determined by NMR titration experiments. The most pronounced positive cooperativity was found for the complexes 1 L(3) with L being the second-generation dendrons 4 c-e. The least stable complexes are formed with the bulky third-generation dendrons 4 f-h. Similar results are obtained by the corresponding complexation of the achiral Frechét-type first- to third-generation dendrons 3 with 1. Chiroptical investigations of 1:3 complexes of 1 and 4 reveal chirality transfer from the dendron to the Hamilton receptor as demonstrated by the appearance of new CD absorption bands at 310 nm.

Binding Sites↗
PubMed2006

Asymmetric total syntheses of marine cyclic depsipeptide halipeptins A-D.

Halipeptins A-D (1 a-d) are a family of natural cyclic depsipeptides isolated from marine sponges. Total syntheses of these four compounds are detailed in this report. The key elements in this synthesis include the elaboration of the polysubstituted decanoic acid parts by two asymmetric aldol reactions, assembly of the N-methyl-delta-hydroxyisoleucine residue by using either aza-Claisen rearrangement or methylation of aspartates as the key steps, and macrocyclization at the polysubstituted decanoic acid alanine site.

Alanine↗
PubMed2007

Tandem mass spectrometry of kahalalides: identification of two new cyclic depsipeptides, kahalalide R and S from Elysia grandifolia.

Spectra obtained using electrospray ionization mass spectrometry (ESI-MS) of the mollusk Elysia grandifolia showed a cluster of molecular ion peaks centered at a molecular mass of 1478 Da (kahalalide F, an anticancer agent). Two new molecules, kahalalide R (m/z 1464) and S (m/z 1492) were characterized using tandem mass spectrometry. The mass differences of 14 Da suggest that they are homologous molecules. In addition, previously identified kahalalide D and kahalalide G are also reported. However, the ESI-MS of the mollusk's algal diet Bryopsis plumosa showed the presence of only kahalalide F. The amino acid sequences of kahalalide R and S are proposed using collision-induced dissociation (CID) experiments of singly and doubly charged molecular ions and by comparison with the amino acid sequence of kahalalide F. The pathway is presented for the loss of amino acid residues in kahalalide F. It is observed that there is sequential loss of amino acids in the linear peptide chain, but in the cyclic part the ring opens at the amide bond rather than at the lactone linkage, and the loss of amino acid residues is not sequential. The CID experiment of the alkali-metal-cationized molecular ions shows that the sodium and potassium ions coordinate to the amide nitrogen/oxygen in the linear peptide chain of the molecule and not to the lactone oxygen of the lactone. In the case of kahalalide D, CID of the protonated peptide opens the depsipeptide ring to form a linear peptide with acylium ion, and fragment ion signals indicate losses of amino acids in sequential order. In this study, tandem mass spectrometry has provided the detailed information required to fully characterize the new peptides.

Animals↗
PubMed2002

Quantitative analysis of the novel depsipeptide anticancer drug Kahalalide F in human plasma by high-performance liquid chromatography under basic conditions coupled to electrospray ionization tandem mass spectrometry.

Kahalalide F (KF) is a novel cyclic depsipeptide anticancer drug, which has shown anticancer activity both in vitro and in vivo especially against human prostate cancer cell lines. To characterize the pharmacokinetics of KF during a phase I clinical trial in patients with androgen refractory prostate cancer, a method was developed and validated for the quantitative analysis of KF in human plasma using high-performance liquid chromatography (HPLC) coupled to positive electrospray ionization tandem mass spectrometry (ESI-MS/MS). Microbore reversed-phase liquid chromatography (LC) performed with mobile phases containing trifluoroacetic acid, an additive commonly used for separating peptides, resulted in substantial suppression of the signal for KF on ESI-MS/MS. An alternative approach employing a basic mobile phase provided an excellent response for KF when detected in the positive ion mode. Plasma samples were prepared for LC MS/MS by solid-phase extraction on C(18) cartridges. The LC separation was performed on a Zorbax Extend C(18) column (150 x 2.1 mm i.d., particle size 5 micro m) with acetonitrile -10 mM aqueous ammonia (85 : 15, v/v) as the mobile phase, at a flow-rate of 0.20 ml min(-1). A butyric acid analogue of KF was used as the internal standard. The lower limit of quantitation (LLQ) using a 500 micro l sample volume was 1 ng ml(-1) and the linear dynamic range extended to 1000 ng ml(-1). The inter-assay accuracy of the assay was -15.1% at the LLQ and between -2.68 and -9.05% for quality control solutions ranging in concentration from 2.24 to 715 ng ml(-1). The inter-assay precision was 9.91% or better at these concentrations. The analyte was stable in plasma under all relevant conditions evaluated and for a period of 16 h after reconstituting plasma extracts for LC analysis at ambient temperature.

Antineoplastic Agents↗
PubMed2003

Identification of thiols and glutathione conjugates of depsipeptide FK228 (FR901228), a novel histone protein deacetylase inhibitor, in the blood.

Four glutathione (GSH) conjugates and two thiols were detected when depsipeptide FK228, formerly FR901228, a naturally occurring potent histone deacetylase (HDAC) inhibitor, was incubated in rat or human plasma in the presence of GSH. Their structures were elucidated by the high-performance liquid chromatography/electrospray ionization multi-stage mass spectrometry (HPLC/ESI-MS(n)) technique, and in some cases confirmed by accurate mass measurement. These products were also detected in rat and human blood homogenates following their incubation with FK228, but were not detected in GSH solution alone. A possible scheme for its formation is proposed. One of the thiols has recently been found to be more active as a histone deacetylase inhibitor than the parent compound.

Animals↗
PubMed2000

Callipeltin A, a cyclic depsipeptide inhibitor of the cardiac sodium-calcium exchanger and positive inotropic agent.

Callipeltin A, a cyclic depsipeptide from the New Caledonian Lithistida sponge Callipelta sp., is a macrocyclic lactone containing four amino acids in the L configuration, Ala, Leu, Thr (2 residues); one (Arg) in the D configuration; two N-methyl amino acids, N-MeAla and N-MeGln; a methoxy tyrosine, a 3, 4-dimethyl-l-glutamine; and a 4-amino-7-guanidino-2,3 dihydroxypentanoic acid (AGDHE), formally derived from L-Arg. In cardiac sarcolemmal vesicles Callipeltin A induces a powerful (IC(50) = 0.85 microM) and selective inhibition of the Na(+)/Ca(2+) exchanger. In electrically driven guinea-pig atria, at concentrations ranging between 0.7 and 2.5 microM, Callipeltin A induces a positive inotropic effect, which at the highest concentrations is accompanied by a rise in resting tension. It is suggested that the positive inotropic effect is linked to the inhibition of the Na(+)/Ca(2+) exchanger and that Callipeltin A may be an useful tool to study the role of the cardiac Na(+)/Ca(2+) exchanger in physiological and pathological conditions.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2006

Total synthesis of bioactive peptides and depsipeptides from marine opisthobranch molluscs.

This chapter covers the synthetic aspects of both linear or cyclic peptides and depsipeptides isolated from opisthobranch molluscs. In many cases, synthetic effort not only determined the absolute stereostructure of these compounds but also made it possible to supply sufficient amounts for the evaluation of pharmacological activities. A summary of the synthetic work associated with each compound is reported after a short description of its natural source and biological properties. Discussion in the text concentrates on key reactions and synthetic efficiency.

Animals↗