ABSTRACT
Two tryptophan compound classes 5- and 6-borono PEGylated boronotryptophan derivatives have been prepared for assessing their aqueous solubility as formulation of injections for boron neutron capture therapy (BNCT). The PEGylation has improved their aqueous solubility thereby increasing their test concentration in 1 mM without suffering from toxicity. In-vitro uptake assay of PEGylated 5- and 6-boronotryptophan showed that the B-10 concentration can reach 15-50 ppm in U87 cell whereas the uptake in LN229 cell varies. Shorter PEG compound 6-boronotryptophanPEG200[18F] was obtained in 1.7 % radiochemical yield and the PET-derived radioradioactivity percentage in 18 % was taken up by U87 tumor at the limb of xenograft mouse. As high as tumor to normal uptake ratio in 170 (T/N) was obtained while an inferior radioactivity uptake of 3 % and T/N of 8 was observed in LN229 xenografted mouse.
Subject(s)
Boron Neutron Capture Therapy , Brain Neoplasms , Fluorine Radioisotopes , Polyethylene Glycols , Positron-Emission Tomography , Animals , Mice , Humans , Fluorine Radioisotopes/chemistry , Polyethylene Glycols/chemistry , Cell Line, Tumor , Brain Neoplasms/diagnostic imaging , Brain Neoplasms/radiotherapy , Brain Neoplasms/metabolism , Boron Compounds/chemistry , Boron Compounds/pharmacokinetics , Boron Compounds/chemical synthesis , Tryptophan/chemistry , Tryptophan/analogs & derivatives , Tryptophan/pharmacokinetics , Tryptophan/chemical synthesis , Molecular StructureABSTRACT
Alzheimer's disease (AD) is a multifactorial neurological disease, and the multitarget directed ligand (MTDL) strategy may be an effective approach to delay its progression. Based on this strategy, 27 derivatives of l-tryptophan, 3a-1-3d-1, were designed, synthesized, and evaluated for their biological activity. Among them, IC50 (inhibitor concentration resulting in 50% inhibitory activity) values of compounds 3a-18 and 3b-1 were 0.58 and 0.44 µM for human serum butyrylcholinesterase (hBuChE), respectively, and both of them exhibited more than 30-fold selectivity for human serum acetylcholinesterase. Enzyme kinetics studies showed that these two compounds were mixed inhibitors of hBuChE. In addition, these two derivatives possessed extraordinary antioxidant activity in OH radical scavenging and oxygen radical absorption capacity fluorescein assays. Meanwhile, these compounds could also prevent ß-amyloid (Aß) self-aggregation and possessed low toxicity on PC12 and AML12 cells. Molecular modeling studies revealed that these two compounds could interact with the choline binding site, acetyl binding site, and peripheral anionic site to exert submicromolar BuChE inhibitory activity. In the vitro blood-brain barrier permeation assay, compounds 3a-18 and 3b-1 showed enough blood-brain barrier permeability. In drug-likeness prediction, compounds 3a-18 and 3b-1 showed good gastrointestinal absorption and a low risk of human ether-a-go-go-related gene toxicity. Therefore, compounds 3a-18 and 3b-1 are potential multitarget anti-AD lead compounds, which could work as powerful antioxidants with submicromolar selective inhibitory activity for hBuChE as well as prevent Aß self-aggregation.
Subject(s)
Acetylcholinesterase , Alzheimer Disease , Amyloid beta-Peptides , Antioxidants , Blood-Brain Barrier , Butyrylcholinesterase , Cholinesterase Inhibitors , Drug Design , Tryptophan , Alzheimer Disease/drug therapy , Humans , Antioxidants/pharmacology , Antioxidants/chemical synthesis , Antioxidants/chemistry , Cholinesterase Inhibitors/pharmacology , Cholinesterase Inhibitors/chemical synthesis , Cholinesterase Inhibitors/chemistry , Structure-Activity Relationship , Blood-Brain Barrier/metabolism , Butyrylcholinesterase/metabolism , Animals , Tryptophan/pharmacology , Tryptophan/chemistry , Tryptophan/analogs & derivatives , Tryptophan/chemical synthesis , Amyloid beta-Peptides/antagonists & inhibitors , Amyloid beta-Peptides/metabolism , Rats , Acetylcholinesterase/metabolism , Molecular Structure , PC12 Cells , Dose-Response Relationship, Drug , Models, MolecularABSTRACT
We have recently described a new generation of potent human immunodeficiency virus (HIV) and EV-A71 entry inhibitors. The prototypes contain three or four tryptophan (Trp) residues bearing an isophthalic acid moiety at the C2 position of each side-chain indole ring. This work is now extended by both shifting the position of the isophthalic acid to C7 and synthesizing doubly arylated C2/C7 derivatives. The most potent derivative (50% effective concentration (EC50) HIV-1, 6 nM; EC50 EV-A71, 40 nM), 33 (AL-518), is a C2/C7 doubly arylated tetrapodal compound. Its superior anti-HIV potency with respect to the previous C2-arylated prototype is in consonance with its higher affinity for the viral gp120. 33 (AL-518) showed comparable antiviral activities against X4 and R5 HIV-1 strains and seems to interact with the tip and base of the gp120 V3 loop. Taken together, these findings support the interest in 33 (AL-518) as a useful new prototype for anti-HIV/EV71 drug development.
Subject(s)
Anti-HIV Agents/pharmacology , Enterovirus A, Human/drug effects , HIV Fusion Inhibitors/pharmacology , HIV-1/drug effects , Indoles/pharmacology , Tryptophan/pharmacology , Anti-HIV Agents/chemical synthesis , Anti-HIV Agents/chemistry , Dose-Response Relationship, Drug , HIV Fusion Inhibitors/chemical synthesis , HIV Fusion Inhibitors/chemistry , Indoles/chemistry , Microbial Sensitivity Tests , Molecular Structure , Structure-Activity Relationship , Tryptophan/chemical synthesis , Tryptophan/chemistryABSTRACT
The enzyme 2-methylerythritol 2,4-cyclodiphosphate synthase, IspF, is essential for the biosynthesis of isoprenoids in most bacteria, some eukaryotic parasites, and the plastids of plant cells. The development of inhibitors that target IspF may lead to novel classes of anti-infective agents or herbicides. Enantiomers of tryptophan hydroxamate were synthesized and evaluated for binding to Burkholderia pseudomallei (Bp) IspF. The L-isomer possessed the highest potency, binding BpIspF with a KD of 36 µM and inhibited BpIspF activity 55% at 120 µM. The high-resolution crystal structure of the L-tryptophan hydroxamate (3)/BpIspF complex revealed a non-traditional mode of hydroxamate binding where the ligand interacts with the active site zinc ion through the primary amine. In addition, two hydrogen bonds are formed with active site groups, and the indole group is buried within the hydrophobic pocket composed of side chains from the 60 s/70 s loop. Along with the co-crystal structure, STD NMR studies suggest the methylene group and indole ring are potential positions for optimization to enhance binding potency.
Subject(s)
Bacterial Proteins/antagonists & inhibitors , Burkholderia pseudomallei/enzymology , Enzyme Inhibitors/pharmacology , Tryptophan/analogs & derivatives , Bacterial Proteins/metabolism , Binding Sites/drug effects , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Models, Molecular , Molecular Structure , Structure-Activity Relationship , Tryptophan/chemical synthesis , Tryptophan/chemistry , Tryptophan/pharmacologyABSTRACT
We report here a one-step aqueous method for the synthesis of isolated and purified polysaccharide-amino acid conjugates. Two different types of amino acid esters: glycine methyl ester and L-tryptophan methyl ester, as model compounds for peptides, were conjugated to the polysaccharide carboxymethylcellulose (CMC) in water using carbodiimide at ambient conditions. Detailed and systematic pH-dependent charge titration and spectroscopy (infrared, nuclear magnetic resonance: 1H, 13C- DEPT 135, 1H- 13C HMBC/HSQC correlation), UV-vis, elemental and ninhydrin analysis provided solid and direct evidence for the successful conjugation of the amino acid esters to the CMC backbone via an amide bond. As the concentration of amino acid esters increased, a conjugation efficiency of 20-80% was achieved. Activated charcoal aided base-catalyzed deprotection of the methyl esters improved the solubility of the conjugates in water. The approach proposed in this work should have the potential to tailor the backbone of polysaccharides containing di- or tri-peptides.
Subject(s)
Carbodiimides/chemistry , Carboxymethylcellulose Sodium/analogs & derivatives , Glycine/analogs & derivatives , Indicators and Reagents/chemistry , Tryptophan/analogs & derivatives , Carboxymethylcellulose Sodium/chemical synthesis , Glycine/chemical synthesis , Molecular Structure , Tryptophan/chemical synthesisABSTRACT
The fluorescent reporters commonly used to visualize proteins can perturb both protein structure and function. Recently, we found that 4-cyanotryptophan (4CN-Trp), a blue fluorescent amino acid, is suitable for one-photon imaging applications. Here, we demonstrate its utility in two-photon fluorescence microscopy by using it to image integrins on cell surfaces. Specifically, we used solid-phase peptide synthesis to generate CHAMP peptides labeled with 4-cyanoindole (4CNI) at their N-termini to image integrins on cell surfaces. CHAMP (computed helical anti-membrane protein) peptides spontaneously insert into membrane bilayers to target integrin transmembrane domains and cause integrin activation. We found that 4CNI labeling did not perturb the ability of CHAMP peptides to insert into membranes, bind to integrins, or cause integrin activation. We then used two-photon fluorescence microscopy to image 4CNI-containing integrins on the surface of platelets. Compared to a 4CNI-labeled scrambled peptide that uniformly decorated cell surfaces, 4CNI-labeled CHAMP peptides were present in discrete blue foci. To confirm that these foci represented CN peptide-containing integrins, we co-stained platelets with integrin-specific fluorescent monoclonal antibodies and found that CN peptide and antibody fluorescence coincided. Because 4CNI can readily be biosynthetically incorporated into proteins with little if any effect on protein structure and function, it provides a facile way to directly monitor protein behavior and protein-protein interactions in cellular environments. In addition, these results clearly demonstrate that the two-photon excitation cross section of 4CN-Trp is sufficiently large to make it a useful two-photon fluorescence reporter for biological applications.
Subject(s)
Integrins/metabolism , Microscopy, Fluorescence, Multiphoton/methods , Tryptophan/analogs & derivatives , Amino Acids/metabolism , Blood Platelets/metabolism , Cell Membrane/metabolism , Integrins/physiology , Peptides/chemical synthesis , Peptides/chemistry , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Protein Binding/physiology , Protein Domains/physiology , Tryptophan/chemical synthesis , Tryptophan/chemistryABSTRACT
Root parasitic plant germination is induced by the host-derived chemical, strigolactone (SL). We found that a major microbial culture broth component, tryptone, inhibits the SL-inducible germination of a root parasitic plant, Orobanche minor. l-tryptophan (l-Trp) was isolated as the active compound from tryptone. We further found that l-Trp related compounds (1b-11), such as a major plant hormone auxin (8, indole-3-acetic acid; IAA), also inhibit the germination and post-radicle growth of O. minor. We designed a hybrid chemical (13), in which IAA is attached to a part of SL, and found that this synthetic analog induced the germination of O. minor, and also inhibited post-radicle growth. Moreover, contrary to our expectations, we found that N-acetyl Trp (9) showed germination stimulating activity, and introduction of a substitution at C-5 position increased its activity (12a-12f). Our data, in particular, the discovery of a structurally hybrid compound that has two activities that induce spontaneous germination and inhibit subsequent radical growth, would provide new types of germination regulators for root parasitic plants.
Subject(s)
Orobanche/drug effects , Plant Roots/drug effects , Seeds/drug effects , Tryptophan/pharmacology , Dose-Response Relationship, Drug , Germination/drug effects , Molecular Structure , Plant Roots/growth & development , Seeds/growth & development , Structure-Activity Relationship , Tryptophan/chemical synthesis , Tryptophan/chemistryABSTRACT
A generalized synthetic strategy is proposed here for the synthesis of asymmetric ß-indoylated amino acids by 8-aminoquinoline (8AQ)-directed C(sp3)-H functionalization of suitably protected precursors. Peptides containing one of the four stereoisomers of (indol-3-yl)-3-phenylalanine at position 2 of the parent peptide KwFwLL-NH2 (w=d-Trp) cover a wide range of activities as ghrelin receptor inverse agonists, among them the most active described until now. This application exemplarily shows how ß-indoylated amino acids can be used for the systematic variation of the position of an indole group in a bioactive peptide.
Subject(s)
Tryptophan/chemistry , Indoles/chemistry , Molecular Structure , Peptides/chemistry , Tryptophan/analogs & derivatives , Tryptophan/chemical synthesisABSTRACT
Human serum albumin is widely used in clinical practice, and the development of new ligands with high affinity is beneficial to improve its separation efficiency. The Site II of human serum albumin is an active binding site of various molecules such as l-tryptophan, which was studied with molecular simulation to obtain insights for the design of new ligands. The results showed that the carboxyl and indolyl groups of l-tryptophan were critical for the binding on Site II. Seven ligands containing carboxyl groups and indolyl groups were designed, and molecular simulation showed that indole-3-pentanoic acid was the best ligand. A new ligand combined indole-3-acetic acid and cysteine was designed for easier resin preparation, and molecular simulation also indicated that the new ligand bound strongly to Site II. Resins with the new ligand designed was prepared and static adsorption experiments indicated that the new resin had high adsorption capacity of human serum albumin and strong salt tolerance. Finally, recombinant human serum albumin was separated from yeast broth with high purity of 90.4% and recovery of 94.2%, which indicated that the new resin had good adsorption selectivity and strong potential for applications.
Subject(s)
Cysteine/chemistry , Drug Design , Indoleacetic Acids/chemistry , Serum Albumin, Human/isolation & purification , Tryptophan/chemistry , Binding Sites , Cysteine/chemical synthesis , Humans , Indoleacetic Acids/chemical synthesis , Ligands , Molecular Dynamics Simulation , Molecular Structure , Serum Albumin, Human/chemistry , Tryptophan/chemical synthesisABSTRACT
A series of different prodrugs of indoximod, including estesrs and peptide amides were synthesized with the aim of improving its oral bioavailability in humans. The pharmacokinetics of prodrugs that were stable in buffers, plasma and simulated gastric and intestinal fluids was first assessed in rats after oral dosing in solution or in capsule formulation. Two prodrugs that produced the highest exposure to indoximod in rats were further tested in Cynomolgus monkeys, a species in which indoximod has oral bioavailability of 6-10% and an equivalent dose-dependent exposure profile as humans. NLG802 was selected as the clinical development candidate after increasing oral bioavailability (>5-fold), Cmax (6.1-3.6 fold) and AUC (2.9-5.2 fold) in monkeys, compared to equivalent molar oral doses of indoximod. NLG802 is extensively absorbed and rapidly metabolized to indoximod in all species tested and shows a safe toxicological profile at the anticipated therapeutic doses. NLG802 markedly enhanced the anti-tumor responses of tumor-specific pmel-1 T cells in a melanoma tumor model. In conclusion, NLG802 is a prodrug of indoximod expected to increase clinical drug exposure to indoximod above the current achievable levels, thus increasing the possibility of therapeutic effects in a larger fraction of the target patient population.
Subject(s)
Antineoplastic Agents/chemical synthesis , Neoplasms/drug therapy , Prodrugs/chemical synthesis , Tryptophan/analogs & derivatives , Administration, Oral , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacokinetics , Apoptosis/drug effects , Biological Availability , Cell Line, Tumor , Dose-Response Relationship, Drug , Drug Compounding , Drug Screening Assays, Antitumor , Haplorhini , Humans , Intestinal Absorption/physiology , Mice , Molecular Conformation , Prodrugs/administration & dosage , Prodrugs/pharmacokinetics , Rats , Tryptophan/administration & dosage , Tryptophan/chemical synthesis , Tryptophan/pharmacokineticsABSTRACT
Maleimide is widely applied in many fields, especially in antibody-drug conjugations and peptide drugs. Herein, we develop a strategy for the C-H alkenylation of tryptophan and tryptophan-containing peptides, providing a synthetic route of decorating maleimide on peptides. The method has a high tolerance of functional groups and protecting groups. Furthermore, this method was applied to prepare peptide conjugation with molecules such as drugs and fluorescence probes. Moreover, macrocyclic peptides were obtained via this reaction.
Subject(s)
Alkenes/chemistry , Maleimides/chemical synthesis , Peptides/chemical synthesis , Rhodium/chemistry , Tryptophan/chemical synthesis , Catalysis , Maleimides/chemistry , Molecular Structure , Peptides/chemistry , Tryptophan/chemistryABSTRACT
Most natural biomolecules may exist in either of two enantiomeric forms. Although in nature, amino acid biopolymers are characterized by l-type homochirality, incorporation of d-amino acids in the design of self-assembling peptide motifs has been shown to significantly alter enzyme stability, conformation, self-assembly behavior, cytotoxicity, and even therapeutic activity. However, while functional metabolite assemblies are ubiquitous throughout nature and play numerous important roles including physiological, structural, or catalytic functions, the effect of chirality on the self-assembly nature and function of single amino acids is not yet explored. Herein, we investigated the self-assembly mechanism of amyloid-like structure formation by two aromatic amino acids, phenylalanine (Phe) and tryptophan (Trp), both previously found as extremely important for the nucleation and self-assembly of aggregation-prone peptide regions into functional structures. Employing d-enantiomers, we demonstrate the critical role that amino acid chirality plays in their self-assembly process. The kinetics and morphology of pure enantiomers is completely altered upon their coassembly, allowing to fabricate different nanostructures that are mechanically more robust. Using diverse experimental techniques, we reveal the different molecular arrangement and self-assembly mechanism of the dl-racemic mixtures that resulted in the formation of advanced supramolecular materials. This study provides a simple yet sophisticated engineering model for the fabrication of attractive materials with bionanotechnological applications.
Subject(s)
Phenylalanine/chemical synthesis , Tryptophan/chemical synthesis , Macromolecular Substances/chemical synthesis , Macromolecular Substances/chemistry , Molecular Dynamics Simulation , Particle Size , Phenylalanine/chemistry , Stereoisomerism , Surface Properties , Tryptophan/chemistryABSTRACT
The structural optimization of the molecules making them to fit into the active site pocket of COX-2 occupying the same space as covered by the natural substrate arachidonic acid helped in the emergence of compound 10 as an efficacious anti-inflammatory agent. Selective for COX-2 over COX-1, compound 10 exhibited IC50 0.02 µM for COX-2 and reversed acetic acid induced inflammation in rats by 73% when used at 10 mg kg-1 dose and the same dose of the compound also rescued the animals from inflammatory phase of formalin induced hyperalgesia. As evidenced by the results of molecular modeling studies supported by the nuclear Overhauser enhancement data, the appropriate geometry of the molecule in the active site pocket of COX-2 contributing to its H-bond/hydrophobic interactions with Ser530, Trp387 and Tyr385 seems responsible for the enzyme inhibitory activity of the compound.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Cyclooxygenase 2 Inhibitors/pharmacology , Hyperalgesia/drug therapy , Inflammation/drug therapy , Acetic Acid , Animals , Anti-Inflammatory Agents, Non-Steroidal/chemical synthesis , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Cyclooxygenase 2/metabolism , Cyclooxygenase 2 Inhibitors/chemical synthesis , Cyclooxygenase 2 Inhibitors/chemistry , Dose-Response Relationship, Drug , Formaldehyde , Humans , Hyperalgesia/chemically induced , Hyperalgesia/metabolism , Inflammation/chemically induced , Inflammation/metabolism , Molecular Docking Simulation , Molecular Structure , Piperidines/chemical synthesis , Piperidines/chemistry , Piperidines/pharmacology , Rats , Rats, Wistar , Structure-Activity Relationship , Triazines/chemical synthesis , Triazines/chemistry , Triazines/pharmacology , Tryptophan/chemical synthesis , Tryptophan/chemistry , Tryptophan/pharmacologyABSTRACT
Blending synthetic biology and synthetic chemistry represents a powerful approach to diversity complex molecules. To further enable this, compatible synthetic tools are needed. We report the first Buchwald Hartwig amination reactions with unprotected halotryptophans under aqueous conditions and demonstrate this methodology is applicable also to the modification of unprotected tripeptides and the natural product barettin.
Subject(s)
Oligopeptides/chemistry , Peptides, Cyclic/chemistry , Tryptophan/analogs & derivatives , Water/chemistry , Amination , Aniline Compounds/chemistry , Catalysis , Halogens/chemistry , Oligopeptides/chemical synthesis , Palladium/chemistry , Tryptophan/chemical synthesisABSTRACT
We describe two synthetic amino acids with inverted side chain stereochemistry, which induce opposite biological activity. Phe4 is an important part of the activation motif of ghrelin, and in short peptide inverse agonists such as KwFwLL-NH2 , the aromatic core is necessary for inactivation of the receptor. To restrict indole/phenyl mobility and simultaneously strengthen the interaction between peptide and receptor, we exchanged the natural monoaryl amino acids for diaryl amino acids derived from tryptophan. By standard solid-phase peptide synthesis, each of them was inserted into ghrelin or in the aromatic core of the inverse agonist. Both ghrelin analogues showed nanomolar activity, indicating sufficient space to accommodate the additional side chain. In contrast, diaryl amino acids in the inverse agonist had considerable influence on receptor signaling. Whereas the introduction of Wsf maintains inverse agonism of the peptide, Wrf shifts the receptor more to active states and can induce agonism depending on its introduction site.
Subject(s)
Ghrelin/pharmacology , Receptors, Ghrelin/agonists , Tryptophan/pharmacology , Dose-Response Relationship, Drug , Ghrelin/chemical synthesis , Ghrelin/chemistry , Molecular Structure , Solid-Phase Synthesis Techniques , Structure-Activity Relationship , Tryptophan/chemical synthesis , Tryptophan/chemistryABSTRACT
There has recently been great concern regarding antibiotics due to potential drug resistance and the impact of antibiotics on the environment. Antimicrobial peptides are believed to have potential as novel antimicrobial agents to address the problems of antibiotics. Herein, we report a set of Trp-rich dodecapeptides derived from PMAP-36 that are based on the peptide folding principle and the amino acid characteristics. An effective peptide design template, (WXYX)3, where X represents Arg or Lys and Y represents hydrophobic or neutral amino acid, was summarized with the distribution of Trp at H-bond formation sites along the α-helical structure. The template peptide 6 (3W-2), with low amphipathicity, displayed strong antimicrobial activity against laboratory strains and clinical isolates while showing no cytotoxicity. Furthermore, 6 was able to suppress the emergence of antimicrobial resistance. Membrane permeabilization assays and microscope observations revealed the potent membrane-disruptive mechanism of 6. Overall, this study diminishes the randomness in peptide design and provides a strategy for generating effective antibiotic alternatives to overcome antibiotic resistance.
Subject(s)
Anti-Infective Agents/chemical synthesis , Bacterial Outer Membrane/drug effects , Drug Design , Oligopeptides/chemical synthesis , Tryptophan/chemical synthesis , Anti-Infective Agents/pharmacology , Antimicrobial Cationic Peptides/chemical synthesis , Antimicrobial Cationic Peptides/pharmacology , Bacterial Outer Membrane/physiology , Escherichia coli/drug effects , Escherichia coli/physiology , Humans , Microbial Sensitivity Tests/methods , Oligopeptides/pharmacology , Peptide Fragments/chemical synthesis , Peptide Fragments/pharmacology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Staphylococcus aureus/drug effects , Staphylococcus aureus/physiology , Tryptophan/pharmacologyABSTRACT
We synthesized [18 F]trifluoromethyl-l-tryptophan ([18 F]CF3 -l-Trp) using Cu(I)-mediated [18 F]trifluoromethylation to image serotonergic system. Radiochemical yield was 6 ± 1.5% (n = 9), and radiochemical purity was over 99%. The molar activity was 0.44 to 0.76 GBq/µmol. [18 F]CF3 -l-Trp was stable for up to 6 hours in mouse and human sera at 37°C. Protein-binding was 0.26 ± 0.03% and 0.34 ± 0.02% in human and mouse serum at 60 minutes, respectively. In conclusion, enantiopure [18 F]CF3 -l-Trp was synthesized as a feasible imaging agent for the serotonergic system.
Subject(s)
Copper/chemistry , Drug Design , Fluorine Radioisotopes/chemistry , Molecular Imaging/methods , Serotonin/metabolism , Tryptophan/chemistry , Tryptophan/chemical synthesis , Animals , Blood Proteins/metabolism , Catalysis , Chemistry Techniques, Synthetic , Halogenation , Humans , Isotope Labeling , Methylation , Mice , Radiochemistry , Stereoisomerism , Tryptophan/metabolismABSTRACT
Recently, l-4-cyanotryptophan has been shown to be an efficient blue fluorescence emitter, with the potential to enable novel applications in biological spectroscopy and microscopy. However, lack of facile synthetic routes to this unnatural amino acid limits its wide use. Herein, we describe an expedient approach to synthesize Fmoc protected l-4-cyanotryptophan with high optical purity (>99%). Additionally, we test the utility of this blue fluorophore in imaging cell-membrane-bound peptides and in determining peptide-membrane binding constants.
Subject(s)
Fluorescent Dyes/chemistry , Tryptophan/analogs & derivatives , Tryptophan/chemistry , Amino Acid Sequence , Cell Membrane/metabolism , Fluorescence , Fluorescence Resonance Energy Transfer/methods , Fluorescent Dyes/chemical synthesis , HeLa Cells , Humans , Membrane Proteins/chemical synthesis , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Microscopy, Fluorescence/methods , Phosphatidylcholines/chemistry , Phosphatidylcholines/metabolism , Protein Binding , Tryptophan/chemical synthesis , Unilamellar Liposomes/chemistry , Unilamellar Liposomes/metabolismABSTRACT
Abiotic hydrocarbons and carboxylic acids are known to be formed on Earth, notably during the hydrothermal alteration of mantle rocks. Although the abiotic formation of amino acids has been predicted both from experimental studies and thermodynamic calculations, its occurrence has not been demonstrated in terrestrial settings. Here, using a multimodal approach that combines high-resolution imaging techniques, we obtain evidence for the occurrence of aromatic amino acids formed abiotically and subsequently preserved at depth beneath the Atlantis Massif (Mid-Atlantic Ridge). These aromatic amino acids may have been formed through Friedel-Crafts reactions catalysed by an iron-rich saponite clay during a late alteration stage of the massif serpentinites. Demonstrating the potential of fluid-rock interactions in the oceanic lithosphere to generate amino acids abiotically gives credence to the hydrothermal theory for the origin of life, and may shed light on ancient metabolisms and the functioning of the present-day deep biosphere.
Subject(s)
Models, Chemical , Origin of Life , Tryptophan/analysis , Tryptophan/chemical synthesis , Aluminum Silicates/chemistry , Atlantic Ocean , Clay/chemistry , Evolution, Chemical , Fluorescence , Iron/chemistryABSTRACT
The radiosynthesis and GMP validation of [11 C]AMT for human use are described. Three consecutive batches were produced giving 940-3790 MBq (4%-17% RCY, decay corrected, based on [11 C]CO2 ) of the tracer. The molar activity at the end of synthesis was 19 to 35 GBq/µmol, the radiochemical purity was ≥98%, and the enantiomeric purity was >99%. While the synthesis method was automated using a new generation of synthesis equipment, tracer production system developed in house, the method should be readily applicable to other synthesis platforms with minor modifications.