Methionine ligation strategy in the biomimetic synthesis of parathyroid hormones

In biological systems, both proteolysis and aminolysis of amide bonds produce activated intermediates through acyl transfer reactions either inter- or intramolecularly. Protein splicing is an illustrative example that proceeds through a series of catalyzed acyl transfer reactions and culminates at an O- or S-acyl intermediate. This intermediate leads to an uncatalyzed acyl migration to form an amide bond in the spliced product. A ligation method mimicking the uncatalyzed final steps in protein splicing has been developed utilizing the acyl transfer amide-bond feature for the blockwise coupling of unprotected, free peptide segments at methionine (Met). The latent thiol moiety of Met can be exploited using homocysteine at the α-amino terminal position of a free peptide for transthioesterification with another free peptide containing an α-thioester to give an S-acyl intermediate. A subsequent, proximity-driven S- to N-acyl migration of this acyl intermediate spontaneously rearranges to form a homocysteinyl amide bond. S-methylation with excess p-nitrobenezensulfonate yields Met at the ligation site. The methionine ligation is selective and orthogonal, and is usually completed within 4 h when performed at slightly basic pH and under strongly reductive conditions. No side reactions due to acylation were observed with any other α-amines of both peptide segments as seen in the synthesis of parathyroid hormone peptides. Furthermore, cyclic peptide can also be obtained through the same strategy by placing both homocysteine at the amino terminus and the thioester at the carboxyl terminus in an unprotected peptide precursor. These biomimetic ligation strategies hold promise for engineering novel peptides and proteins. © 1998 John Wiley & Sons, Inc. Biopoly 46: 319–327, 1998     

Synthesis and anti-elastase properties of 6-amino-2-phenyl-4H-3,1-benzoxazin-4-one aminoacyl and dipeptidyl derivatives

A series of 6-amino-2-phenyl-4H-3,1-benzoxazin-4-one aminoacyl and dipeptidyl derivatives, in which aminoacids and dipeptides are linked to the benzoxazinone moiety via an amide bond, were synthesized and tested in vitro for their inhibitory activity towards human leukocyte elastase (HLE). When compared to their values without inhibitors, the residual enzymatic activities decrease with time, indicating a time-dependent inhibition. The most potent inhibitions were obtained when Z-Arg-(Pmc), Z-Val-Phe, Z-Ala-Val or Z-Val-Ala are linked to the 6-amino group. Twenty-five new compounds were synthesized.     

铽(Ⅲ)离子及其复合物:从发光特性到传感应用

稀土元素也称镧系元素,因其独特的发光性质和配位性质,其发光复合物被广泛研究于生物技术领域。其中稀土铽(Ⅲ)离子复合物因具有优异的光谱特性,关于其研究呈现出快速的发展趋势。主要从其发光特性的角度出发,探讨了其发光机理,并对铽(Ⅲ)离子与不同有机化合物结合形成的发光铽配合物以及铽(Ⅲ)离子及其配合物与不同纳米材料形成的复合物进行了分类综述。此外,还详细地阐述了铽离子及其复合物在荧光探针、生物传感器、药物递送、细胞成像、癌症治疗等相关领域的应用。最后,对其今后发展趋势和潜在的研究价值进行了展望。     

A derivative of diethylenetriaminepentaacetic acid for europium labeling of proteins.

Preparation of a reagent that will incorporate diethylenetriaminepentaacetic acid (DTPA) into proteins under mild conditions and make a strong europium chelate is described. Aminoacetaldehyde diethyl acetal was reacted with DTPA dianhydride, and mono- and disubstituted products as well as unsubstituted DTPA were separated by gel filtration. The monosubstituted product, after conversion into the corresponding aldehyde by mild acid hydrolysis, is conjugated to protein or other amino-containing compounds via reductive amination at neutral pH. Although the DTPA-Eu-labeled proteins are themselves not fluorescent, a strong fluorescence of europium can be generated easily by the dissociation-enhancement mechanism. A direct measurement of lectin-ligand interaction using Eu-labeled ligand and lectin immobilized on 96-well plate illustrates that the assay utilizing Eu fluorescence is as sensitive as the radioactive assays.     

Study of non-covalent interactions between MRI contrast agents and human serum albumin by NMR diffusometry

The NMR diffusometry technique, based on the measurement of the diffusion coefficient of a ligand in the absence and in the presence of its macromolecular partner, was used to study the affinity for human serum albumin (HSA) of four gadolinium complexes, potential or already used magnetic resonance imaging contrast agents. Diamagnetic lanthanum(III) ion or europium(III) ion, which has the advantage of shifting the NMR signals far away from those of the macromolecule, was used to avoid the excessive broadening of the NMR signals induced by the gadolinium(III) ion. Titration experiments, in which the HSA concentration was kept constant and the concentration of the europium or lanthanum chelate was varied, were performed to evaluate the association constant and the number of binding sites. Some additional information about the kinetics of the exchange between the free and the bound chelate was also obtained. Competition experiments with ibuprofen and salicylate, which are ligands with a known affinity for the macromolecule and for which the binding site is known, were also performed to get information about the binding site of the contrast agents.     

Terbium as a solid-state probe for RNA

This paper continues previous work on the analysis of nucleic acid-terbium complexes in the solid state. The fluorescence excitation and emission spectra of the RNA-terbium(III) complex is reported. The fluorescence excitation and emission spectra of both the RNA-terbium(III) and DNA-terbium(III) complexes as trapped on millipore filters is reported. One hundred percent of the DNA combined with terbium was trapped on millipore filters. Deoxyribonucleic acid was recovered from DNA-terbium(III) complexes trapped on millipore filters using SDS-extraction. Energy transfer was shown to occur from the bases in nucleic acids to the terbium ion, whereas the actual binding of terbium to nucleic acids was due to phosphate groups. The relative fluorescence of homopolyribonucleotide-terbium complexes showed that the guanine moiety was responsible for most of the observed fluorescence. Binding studies showed an equal affinity of radioactive terbium for all the homopolyribonucleotides. The fluorescence of solid-state DNA and RNA terbium complexes was used to measure picomole quantities of DNA or RNA.     

The effect of histidyl residues on the complexation of bis(imidazolyl) containing tripeptides with copper(II) ion

Copper(II) complexes of tripeptide derivatives of bis(imidazol-2-yl) group have been studied by potentiometric, UV-visible and EPR spectroscopic methods. The peptide molecules correspond to the amino acid sequence of collagen containing histidyl residues in different locations and were connected to the bis(imidazol-2-yl) group either on the C-termini (BOC-Pro-Leu-His-BIMA, BOC-His-Leu-Gly-BIMA) or on the N-termini (BIP-His-Ala-Gly-OEt, BIP-Ile-Ala-His-OMe). It was concluded that the imidazole nitrogen donor atoms of the bis(imidazol-2-yl) moiety are the primary metal binding sites, but the histidyl imidazole nitrogens in the side chains have also some effect on the stability and the coordination mode of the complexes. All ligands can coordinate tridentately to copper(II) ion forming a six-membered chelate and a macrochelate in the [CuL]2+ complexes, which results in a slight distortion in the coordination geometry of [CuL2]2+ complexes. The deprotonation and coordination of amide nitrogens, however, were not observed in any cases.     

Semi-synthesis of biologically active nisin hybrids composed of the native lanthionine ABC-fragment and a cross-stapled synthetic DE-fragment

The antimicrobial peptide nisin is a promising template for designing novel peptide-based antibiotics to improve its drug-like properties. First steps in that direction represent the synthesis of hybrid nisin derivatives that contain a native nisin ABC-part and synthesized cross-stapled DE-ring fragments and are described here. The biological activity of the newly synthesized nisin derivatives was evaluated in order to compare the bioactivity of the synthetic DE-ring containing mimic and native lanthionine-bridged DE-ring containing nisin. The native nisin ABC-ring system was obtained via chymotrypsin digestion of full-length nisin, and was subsequently functionalized at the C-terminal carboxylate with two different amino alkyne moieties. Next, nisin hybrids were successfully prepared using Cu(I)-catalyzed azide alkyne cycloaddition ‘click’ chemistry by chemo-selective ligation of the ABC-alkyne with the N-terminal azido functionalized dicarba-DE ring mimic. The newly synthesized compounds were active as potent lipid II binders and retained antimicrobial activity in a growth inhibition assay. However, pore formation was not observed, possibly either due to the different character of the ‘staples’ as compared to the parent sulfides, or due to the triazole moiety as a sub-optimal amide bond isostere.     

13C NMR of methylated lysines of fd gene 5 protein: evidence for a conformational change involving lysine 24 upon binding of a negatively charged lanthanide chelate

Helical complexes formed between fd DNA and reductively methylated fd gene 5 protein were indistinguishable by electron microscopy from complexes formed with the nonmethylated protein. 13C NMR spectroscopy of 13C-enriched N epsilon, N epsilon-dimethyllsyl residues of the protein showed that three of these residues (Lys-24, Lys-46, and Lys-69) were selectively perturbed by binding of the oligomer d(pA)7. These were the same lysyl residues that we previously found to be most protected from methylation by binding of the protein to poly[r(U)] [Dick, L. R., Sherry, A. D., Newkirk, M. M., & Gray D. M. (1988) J. Biol. Chem. 263, 18864-18872]. Thus, these lysines are probably directly involved in the nucleic acid binding function of the protein. Negatively charged chelates of lanthanide ions were used to perturb the 13C NMR resonances of labeled lysyl and amino-terminal residues of the gene 5 protein. The terbium chelate was found to bind tightly (Ka approximately 10(5) M-1) to the protein with a stoichiometry of 1 chelate molecule per protein dimer. 13C resonances of Lys-24, Lys-46, and Lys-69 were maximally shifted by the terbium chelate and were maximally relaxed by the gadolinium chelate. Also, the terbium chelate was excluded by the oligomer d(pA)7. Computer fits of the induced chemical shifts of 13C resonances with those expected for various positions of the terbium chelate failed to yield a possible chelate binding site unless the chemical shift for Lys-24 was excluded from the fitting process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Luminescent trimethoprim-polyaminocarboxylate lanthanide complex conjugates for selective protein labeling and time-resolved bioassays

Labeling proteins with long-lifetime emitting lanthanide (III) chelate reporters enables sensitive, time-resolved luminescence bioaffinity assays. Heterodimers of trimethoprim (TMP) covalently linked to various cs124-sensitized, polyaminocarboxylate chelates stably retain lanthanide ions and exhibit quantum yields of europium emission up to 20% in water. A time-resolved, luminescence resonance energy transfer (LRET) assay showed that TMP-polyaminocarboxylates bind to Escherichia coli dihydrofolate reductase (eDHFR) fusion proteins with nanomolar affinity in purified solutions and in bacterial lysates. The ability to selectively impart terbium or europium luminescence to fusion proteins in complex physiological mixtures bypasses the need for specific antibodies and simplifies sample preparation.     

Raman spectroscopic studies of NAD coenzymes bound to malate dehydrogenases by difference techniques

We report here on the Raman spectra of NADH, 3-acetylpyridine adenine dinucleotide, APAD+, and a fragment of these molecules, adenosine 5'-diphosphate ribose (ADPR) bound to the mitochondrial (mMDH) and cytoplasmic (or soluble, sMDH) forms of malate dehydrogenase. We observe changes in the Raman spectrum of the adenosine moiety of these cofactors upon binding to mMDH, indicating that the binding site is hydrophobic. On the other hand, there is little change in the spectrum of the adenosine moiety when it binds to sMDH. Such observations are in clear contrast with those results obtained in LDH and LADH, where there are significant changes in the spectrum of the adenosine moiety when it binds to these two proteins. A strong hydrogen bond is postulated to exist between amide carbonyl group of NAD+ and the enzyme in the binary complexes with both mMDH and sMDH on the basis of a sizable decrease in the frequency of the carbonyl double bond. The interaction energy for formation of a hydrogen bond is the same as found previously for LDH, and we estimate that it is 2.8 kcal/mol more favorable in the binary complex than in water. A hydrogen bond is also detected between the amide-NH2 group of NADH and sMDH that is stronger than that formed in water and is of the same size as found in LDH. Surprisingly, the hydrogen bond to the -NH2 group in mMDH is the same as that found for water.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solid-phase synthesis of chelate-labelled oligonucleotides: application in triple-color ligase-mediated gene analysis.

Oligonucleotides labelled with detectable groups are essential tools in gene detection. We describe here the synthesis of pyrimidine deoxynucleotide-building blocks, modified at their C-5 position with a protected form of a strongly chelating agent. These reagents can be used to introduce multiple metal ions into oligodeoxynucleotides during standard oligonucleotide synthesis. The chelating functions form strongly fluorescent complexes with europium ions, characterized by a wide separation between the excitation and emission spectra. Moreover, the long decay time of the fluorescence permits sensitive time-resolved fluorescence measurements. The chelates also have the stability required to function in triple-color assays involving europium, samarium, and terbium ions. We demonstrate the application of these reagents for ligase-based gene analysis reactions.     

A new label technology for the detection of specific polymerase chain reaction products in a closed tube

A novel signal generation principle suitable for real time and end-point detection of specific PCR products in a closed tube is described. Linear DNA probes were labeled at their 5′-ends with a stable, fluorescent terbium chelate. The fluorescence intensity of this chelate is lower when it is coupled to single-stranded DNA than when the chelate is free in solution. The synthesized probes were used in the real time monitoring of PCR using a prototype instrument that consisted of a fluorometer coupled to a thermal cycler. When the probe anneals to a complementary target amplicon, the 5′→3′ exonucleolytic activity of DNA polymerase detaches the label from the probe. This results in an enhanced terbium fluorescence signal. Since terbium has a long excited state lifetime, its fluorescence can be measured in a time-resolved manner, which results in a low background fluorescence and a 1000-fold signal amplification. The detection method is quantitative over an extremely wide linear range (at least 10–10⁷ initial template molecules). The label strategy can easily be combined with existing label technologies, such as TaqMan 5′-exonuclease assays, in order to carry out multiplex assays that do not suffer from overlapping emission peaks of the fluorophores.     

Diffusion-enhanced lanthanide energy-transfer study of DNA-bound cobalt(III) bleomycins: comparisons of accessibility and electrostatic potential with DNA complexes of ethidium and acridine orange

Energy transfer in the "rapid-diffusion" limit reflects the equilibrium properties of a donor-acceptor system. Rates of energy transfer from freely diffusing terbium chelates to DNA-binding chromophores change dramatically when DNA is added; energy transfer from an electrically neutral chelate is reduced because the energy acceptor becomes partially buried in DNA, while energy transfer from a positive chelate is increased because of electrostatic attraction. The rate constants for energy transfer to DNA-bound chromophores from a positively charged terbium chelate, relative to those from a neutral chelate, were used to estimate the following values for the electrostatic potential near the surface of each DNA-bound acceptor at 298 K in the presence of 1.0 mM added salt (in units of -e/kT): acridine orange, 4.54 +/- 0.11; ethidium, 4.66 +/- 0.07; green Co(III) bleomycin A2, 4.06 +/- 0.11; orange Co(III) bleomycin A2, 3.11 +/- 0.10. Smaller numbers indicate less negative potentials; these can be due to a combination of (1) positive charge on the chromophore, (2) location of the chromophore [particularly Co(III) bleomycin] away from the DNA phosphates, and/or (3) separation of DNA phosphate negative charges by an intercalator. The magnitudes of the individual rate constants indicate that all the DNA-bound chromophores can be directly encountered by the terbium probes. Energy-transfer rate constants from a neutral terbium chelate to DNA-bound and free acceptors can provide a measure of the accessibility of the terbium probe to each bound chromophore. The ratios of these rate constants were as follows: acridine orange, 0.17 +/- 0.01; ethidium, 0.27 +/- 0.02; green form of Co(III) bleomycin A2, 0.48 +/- 0.06; orange form of Co(III) bleomycin A2, 0.71 +/- 0.06. These results are consistent with the probable differences in binding mechanisms for the intercalating chromophores (ethidium and acridine orange) as compared to the Co(III) bleomycins (in which the relevant chromophores are nonintercalating metal centers). In addition, all the results imply that the green Co(III) bleomycin chromophore binds closer to DNA than the orange; this provides a first step toward understanding the structural basis for the different biological properties of these metallobleomycins. Control experiments and theoretical considerations necessary to establish the validity of the results are also presented.     

Using 3'-bridging phosphorothiolates to isolate the forward DNA cleavage reaction of human topoisomerase IIalpha

The ability to cleave DNA is critical to the cellular and pharmacological functions of human type II topoisomerases. However, the low level of cleavage at equilibrium and the tight coupling of the cleavage and ligation reactions make it difficult to characterize the mechanism by which these enzymes cut DNA. Therefore, to establish a system that isolates topoisomerase II-mediated DNA scission from ligation, oligonucleotide substrates were developed that contained a 3'-bridging phosphorothiolate at the scissile bond. Scission of these substrates generates a 3'-terminal -SH moiety that is a poor nucleophile relative to the normal 3'-terminal -OH group. Consequently, topoisomerase II cannot efficiently ligate phosphorothiolate substrates once they are cleaved. The characteristics of topoisomerase IIalpha-mediated cleavage of phosphorothiolate oligonucleotides were identical to those seen with wild-type substrates, except that no ligation was observed. This unidirectional accumulation of cleavage complexes provided critical information regarding coordination of the protomer subunits of topoisomerase IIalpha and the mechanism of action of topoisomerase II poisons. Results indicate that the two enzyme subunits are partially coordinated and that cleavage at one scissile bond increases the degree of cleavage at the other. Furthermore, anticancer drugs such as etoposide and amsacrine that strongly inhibit topoisomerase II-mediated DNA ligation have little effect on the forward scission reaction. In contrast, abasic sites that increase levels of cleavage complexes without affecting ligation stimulate the forward rate of scission. Phosphorothiolate substrates provide significant advantages over traditional "suicide substrates" and should be valuable for future studies on DNA scission and the topoisomerase II-DNA cleavage complex.     

Peptide Bond Synthesis by a Mechanism Involving an Enzymatic Reaction and a Subsequent Chemical Reaction

We recently reported that an amide bond is unexpectedly formed by an acyl-CoA synthetase (which catalyzes the formation of a carbon-sulfur bond) when a suitable acid and l-cysteine are used as substrates. DltA, which is homologous to the adenylation domain of nonribosomal peptide synthetase, belongs to the same superfamily of adenylate-forming enzymes, which includes many kinds of enzymes, including the acyl-CoA synthetases. Here, we demonstrate that DltA synthesizes not only N-(d-alanyl)-l-cysteine (a dipeptide) but also various oligopeptides. We propose that this enzyme catalyzes peptide synthesis by the following unprecedented mechanism: (i) the formation of S-acyl-l-cysteine as an intermediate via its “enzymatic activity” and (ii) subsequent “chemical” S → N acyl transfer in the intermediate, resulting in peptide formation. Step ii is identical to the corresponding reaction in native chemical ligation, a method of chemical peptide synthesis, whereas step i is not. To the best of our knowledge, our discovery of this peptide synthesis mechanism involving an enzymatic reaction and a subsequent chemical reaction is the first such one to be reported. This new process yields peptides without the use of a thioesterified fragment, which is required in native chemical ligation. Together with these findings, the same mechanism-dependent formation of N-acyl compounds by other members of the above-mentioned superfamily demonstrated that all members most likely form peptide/amide compounds by using this novel mechanism. Each member enzyme acts on a specific substrate; thus, not only the corresponding peptides but also new types of amide compounds can be formed.     

Enkephalin-degrading enzyme inhibitors. Crucial role of the C-terminal residue on the inhibitory potencies of retro-hydroxamate dipeptides

The retro-inversion of the amide bond in kelatorphan and analogs, the first series of complete inhibitors of enkephalin metabolism, led to compounds highly efficient only against the neutral endopeptidase 24-11 (NEP). In order to increase the recognition of the aminopeptidase N (APN) and dipeptidylaminopeptidase (DAP), without loss of affinity for NEP, the malonyl group of these retro-inhibitors was replaced by diversely substituted succinyl moieties. All the molecules synthesized are highly efficient NEP inhibitors with Ki's in the 0.2-1 nM range, indicating that NEP possesses a relatively large and not very selective S'2 subsite. In contrast, inhibition of DAP activity is crucially dependent on the size and the position of the substituent in the succinyl moiety. Inhibitory potencies in the nanomolar range are obtained with compounds containing a benzyl group in the alpha-position related to the retro amide bond. Finally, a relatively modest inhibition of APN was observed with Ki's in the 0.5-1 microM range for compounds with benzyl or cyclohexyl group in P'2 position. However, these data demonstrate that efficient and complete inhibition of enkephalin degradation can be obtained with hydroxamate dipeptides containing a retro amide bond. The analgesic potency of the most active inhibitors was measured using the hot plate test in mice. Significant antinociceptive responses were obtained but these effects were rather weaker than those expected from the in vitro inhibitory potencies of these compounds on the three enkephalin-degrading enzymes.     

Recent developments in the synthesis and applications of phosphinic peptide analogs

Synthetic pseudopeptides that fit well with the active site architecture allow the most effective binding to enzymes, similar to native substrates in high-energy transition states. Phosphinic acid peptide analogs that comprise the tetrahedral phosphorus moiety introduced to replace an internal amide bond exert such an isosteric or isoelectronic resemblance, combined with providing other advantageous features, for example, metal complexing properties. Accordingly, they are capable of inhibiting metal-dependent enzymes involved in biological functions in eukaryotic and prokaryotic cells. These enzymes are associated with notorious human diseases, such as cancer, e.g., matrix metalloproteinases, or are etiological factors of protozoal and bacterial infections, e.g., metalloaminopeptidases. The affinity and selectivity of these compounds can be conveniently adjusted, either by structural modification of dedicated side chains or by backbone elongation to enhance specific interactions with the corresponding binding pockets. Recent approaches to the synthesis of these compounds are illustrated by examples of the preparation of rationally designed structures of inhibitors of particular enzymes. Activity against appealing enzymatic targets is presented, along with the molecular mechanisms of action and therapeutic implications. Innovative aspects of phosphinic peptide application, e.g., as activity-based probes, and ligands of complexes of radioisotopes for nuclear medicine are also outlined.     

Synthesis of affinity-label chelates: a novel synthetic method of coupling ethylenediaminetetraacetic acid to amine functional groups

An affinity-label chelate for the enzyme trypsin was synthesized by a novel synthetic technique which takes advantage of the presence of a dangling carboxylate arm in the [Co(EDTA)Cl]2- complex anion. The dangling carboxylate group was coupled to the amino group of p-aminobenzamidine, an effective inhibitor of trypsin activity, via the carbodiimmide reaction to produce a trypsin affinity label at one end and a strong EDTA-like chelating agent at the other, coupled through an amide bond. The cobalt ion can be removed if desired by reduction with Fe2+ + ascorbate, and alternate metal ions inserted in its place. The reaction is general, and affinity labels which contain amino groups can be easily coupled via this procedure, allowing the introduction of a paramagnetic or fluorescent probe into a protein or nucleotide system. The same method has been used to prepare a highly effective chelating gel which is capable of removing calcium and lanthanide ions from the binding protein parvalbumin.     

Contributions of Ionic Interactions and Protein Dynamics to Cytochrome P450 2D6 (CYP2D6) Substrate and Inhibitor Binding

P450 2D6 contributes significantly to the metabolism of >15% of the 200 most marketed drugs. Open and closed crystal structures of P450 2D6 thioridazine complexes were obtained using different crystallization conditions. The protonated piperidine moiety of thioridazine forms a charge-stabilized hydrogen bond with Asp-301 in the active sites of both complexes. The more open conformation exhibits a second molecule of thioridazine bound in an expanded substrate access channel antechamber with its piperidine moiety forming a charge-stabilized hydrogen bond with Glu-222. Incubation of the crystalline open thioridazine complex with alternative ligands, prinomastat, quinidine, quinine, or ajmalicine, displaced both thioridazines. Quinine and ajmalicine formed charge-stabilized hydrogen bonds with Glu-216, whereas the protonated nitrogen of quinidine is equidistant from Asp-301 and Glu-216 with protonated nitrogen H-bonded to a water molecule in the access channel. Prinomastat is not ionized. Adaptations of active site side-chain rotamers and polypeptide conformations were evident between the complexes, with the binding of ajmalicine eliciting a closure of the open structure reflecting in part the inward movement of Glu-216 to form a hydrogen bond with ajmalicine as well as sparse lattice restraints that would hinder adaptations. These results indicate that P450 2D6 exhibits sufficient elasticity within the crystal lattice to allow the passage of compounds between the active site and bulk solvent and to adopt a more closed form that adapts for binding alternative ligands with different degrees of closure. These crystals provide a means to characterize substrate and inhibitor binding to the enzyme after replacement of thioridazine with alternative compounds.