Photoredox-Catalyzed Labeling of Hydroxyindoles with Chemoselectivity (PhotoCLIC) for Site-Specific Protein Bioconjugation.

We have developed a novel visible-light-catalyzed bioconjugation reaction, PhotoCLIC, that enables chemoselective attachment of diverse aromatic amine reagents onto a site-specifically installed 5-hydroxytryptophan residue (5HTP) on full-length proteins of varied complexity. The reaction uses catalytic amounts of methylene blue and blue/red light-emitting diodes (455/650 nm) for rapid site-specific protein bioconjugation. Characterization of the PhotoCLIC product reveals a unique structure formed likely through a singlet oxygen-dependent modification of 5HTP. PhotoCLIC has a wide substrate scope and its compatibility with strain-promoted azide-alkyne click reaction, enables site-specific dual-labeling of a target protein.

Association between cord blood metabolites in tryptophan pathway and childhood risk of autism spectrum disorder and attention-deficit hyperactivity disorder.

Alterations in tryptophan and serotonin have been implicated in various mental disorders; but studies are limited on child neurodevelopmental disabilities such as autism spectrum disorder (ASD) and attention-deficit hyperactivity disorder (ADHD). This prospective cohort study examined the associations between levels of tryptophan and select metabolites (5-methoxytryptophol (5-MTX), 5-hydroxytryptophan (5-HTP), serotonin, N-acetyltrytophan) in cord plasma (collected at birth) and physician-diagnosed ASD, ADHD and other developmental disabilities (DD) in childhood. The study sample (n = 996) derived from the Boston Birth Cohort, which included 326 neurotypical children, 87 ASD, 269 ADHD, and 314 other DD children (mutually exclusive). These participants were enrolled at birth and followed-up prospectively (from October 1, 1998 to June 30, 2018) at the Boston Medical Center. Higher levels of cord 5-MTX was associated with a lower risk of ASD (aOR: 0.56, 95% CI: 0.41, 0.77) and ADHD (aOR: 0.79, 95% CI: 0.65, 0.96) per Z-score increase, after adjusting for potential confounders. Similarly, children with cord 5-MTX ≥ 25th percentile (vs. <25th percentile) had a reduction in ASD (aOR: 0.27, 95% CI: 0.14, 0.49) and ADHD risks (aOR: 0.45, 95% CI: 0.29, 0.70). In contrast, higher levels of cord tryptophan, 5-HTP and N-acetyltryptophan were associated with higher risk of ADHD, with aOR: 1.25, 95% CI: 1.03, 1.51; aOR: 1.32, 95% CI: 1.08, 1.61; and aOR: 1.27, 95% CI: 1.05, 1.53, respectively, but not with ASD and other DD. Cord serotonin was not associated with ASD, ADHD, and other DD. Most findings remained statistically significant in the sensitivity and subgroup analyses.

Solid-Phase Total Synthesis of Dehydrotryptophan-Bearing Cyclic Peptides Tunicyclin B, Sclerotide A, CDA3a, and CDA4a using a Protected ß-Hydroxytryptophan Building Block.

A new approach to the synthesis of Z-dehydrotryptophan (ΔTrp) peptides is described. This approach uses Fmoc-ß-HOTrp(Boc)(TBS)-OH as a building block, which is readily prepared in high yield and incorporated into peptides using solid-phase Fmoc chemistry. The tert-butyldimethylsilyl-protected indolic alcohol is eliminated during global deprotection/resin cleavage to give ΔTrp peptides exclusively as the thermodynamically favored Z isomer. This approach was applied to the solid-phase synthesis of tunicyclin B, sclerotide A, CDA3a, and CDA4a.

Synthesis of redox-active fluorinated 5-hydroxytryptophans as molecular reporters for biological electron transfer.

Fluorinated 5-hydroxytryptophans (Fn-5HOWs) were synthesized in gram scale quantities and incorporated into a ß-hairpin peptide and the protein azurin. The redox-active Fn-5HOWs exhibit unique radical spectroscopic signatures that expand the function of as probes for biological electron transfer.

Beta Cell Imaging-From Pre-Clinical Validation to First in Man Testing.

There are presently no reliable ways to quantify human pancreatic beta cell mass (BCM) in vivo, which prevents an accurate understanding of the progressive beta cell loss in diabetes or following islet transplantation. Furthermore, the lack of beta cell imaging hampers the evaluation of the impact of new drugs aiming to prevent beta cell loss or to restore BCM in diabetes. We presently discuss the potential value of BCM determination as a cornerstone for individualized therapies in diabetes, describe the presently available probes for human BCM evaluation, and discuss our approach for the discovery of novel beta cell biomarkers, based on the determination of specific splice variants present in human beta cells. This has already led to the identification of DPP6 and FXYD2ga as two promising targets for human BCM imaging, and is followed by a discussion of potential safety issues, the role for radiochemistry in the improvement of BCM imaging, and concludes with an overview of the different steps from pre-clinical validation to a first-in-man trial for novel tracers.

Physicochemical Properties of Zwitterionic Drugs in Therapy.

In solution, amphoteric compounds exist in anionic, uncharged, zwitterionic and cationic forms. The importance of zwitterionic drugs is currently under-represented in the literature. Herein, the acid-base parameters, lipophilicity and solubility of such compounds are discussed to deepen the molecular-level understanding of their pharmacokinetic and pharmacodynamic behaviour. Our recent studies show there are many drug molecules, including thyroid hormones and 5-hydroxytryptophan, the precursor of the neurotransmitter serotonin, for which the contribution of the zwitterionic microspecies to the overall lipophilicity exceeds that of the uncharged one, which is of higher individual lipophilicity, but occurs in much lower concentration. The second part of the minireview highlights the most important zwitterionic compounds in therapy, grouped into therapeutic classes. The importance of the charge of the molecules is emphasized in their binding to the target molecules.

Ternary copper(II) complex of 5-hydroxytryptophan and 1,10-phenanthroline with several pharmacological properties and an adequate safety profile.

We report the synthesis and biological evaluation of a ternary copper complex, [Cu(5HTP)(phen)(H2O)](NO3).2H2O, with the antioxidant agent 5-hydroxytryptophan (5-HTP) and phenanthroline (phen, added to improve its lipophilicity and membrane transport). The crystal structure of the complex was determined by X-ray diffraction methods. The complex showed antioxidant, antimicrobial, antitumor and antimetastatic properties with an adequate safety profile. The interaction of the metal with phen promotes cellular copper accumulation and cytotoxicity on human lung A549 cell line (IC50 = 3.6 µM). Furthermore, the viability of the normal human fetal lung fibroblast cell line (MRC-5) is not altered by the complex. An oxidative stress mechanism for the anticancer effect has been determined: cellular increase of reactive oxygen species (ROS), decrease of the glutathione (GSH) and oxidized GSH (GSSG) ratio and alteration of the mitochondrial potential. The complex also displays antimetastatic activities with inhibition of cell adhesion, invasion and migration. It has not mutagenic behavior and no toxicity on Artemia salina indicating its potential to act as an effective and safety antimicrobial and antitumor drug.

In silico and in vitro antioxidant activity profiles of urea and thiourea derivatives of 5-hydroxytryptophan.

A series of new urea and thiourea derivatives of 5-hydroxy tryptophan 3a-l was designed and synthesized from 5-hydroxy tryptophan (1) by treating various isocyanates 2a-g and isothiocyanates 2h-l in the presence of triethylamine (TEA) as a base. The antioxidant activities of the newly synthesized compounds were evaluated by using different in vitro assays such as 1, 1-diphenyl-2-picrylhydrazyl (DPPH), hydrogen peroxide (H2O2) and superoxide. The results obtained from the in vitro studies revealed that the compounds 3a, 3g, 3h, and 3l exhibited the significant high content of antioxidant activity. Besides, molecular docking studies indicated that, all the compounds 3a-l have formed higher binding energies with 3MNG protein than the reference compound 1. Among all the synthesized compounds, 3a, 3l, 3g, 3b, 3c and 3h have exhibited the highest dock scores than the rest of the compounds including the reference compound. Hence, it is concluded that the title compounds will open new vistas for the discovery of strong antioxidants and will stand as remarkable antioxidant moieties in future.

Labeling Proteins at Site-Specifically Incorporated 5-Hydroxytryptophan Residues Using a Chemoselective Rapid Azo-Coupling Reaction.

Chemoselective protein labeling is a valuable tool in the arsenal of modern chemical biology. The unnatural amino acid mutagenesis technology provides a powerful way to site-specifically introduce nonnatural chemical functionalities into recombinant proteins, which can be subsequently functionalized in a chemoselective manner. Even though several strategies currently exist to selectively label recombinant proteins in this manner, there is considerable interest for the development of additional chemoselective reactions that are fast, catalyst-free, use readily available reagents, and are compatible with existing conjugation chemistries. Here we describe a method to express recombinant proteins in E. coli site-specifically incorporating 5-hydroxytryptophan, followed by the chemoselective labeling of this residue using a chemoselective rapid azo-coupling reaction.

Chemical Characterization and DNA Fingerprinting of Griffonia simplicifolia Baill.

BACKGROUND: Griffonia simplicifolia Baill. (Caesalpiniaceae) is a medicinal plant whose seeds are widely used in traditional medicine for their high content of 5-hydroxy-l-tryptophan (5-HTP), a direct precursor and enhancer of the activity of the brain hormone serotonin (5-HT). The plant extracts are used in dietary supplements aimed to alleviate serotonin-related disorders. METHODS: In order to characterize the chemical components of G. simplicifolia seeds and their identity, we used a combined methodology by using HPLC-DAD-ESI-MS/MS for the qualitative and quantitative determination of the N-containing compounds, GC-FID and GC-MS for the characterization of the major fatty acids, and DNA fingerprinting based on PCR⁻RFLP for the unequivocal identification of the plant. RESULTS: 5-HTP was the most representative compound, followed by lower percentages of the ß-carboline alkaloid derivative griffonine and other alkaloids. Fatty acids were dominated by the unsaturated fatty acids linoleic acid and oleic acid, followed by the saturated fatty acids stearic and palmitic acids. PCR analysis of the internal transcribed spacer amplified sequence showed a major band at about 758 bp, whereas the PCR⁻RFLP analysis of this sequence using three different restriction enzymes (MspI, HhaI, and HaeIII) generated a specific fingerprinting useful for the plant identification. CONCLUSIONS: The combined chemical and molecular analysis of G. simplicifolia provided an interesting integrated approach for the unequivocal identification of commercial G. simplicifolia seeds.

A "Quenchergenic" Chemoselective Protein Labeling Strategy.

Dynamic changes in protein structure can be monitored by using a fluorescent probe and a dark quencher. This approach is contingent upon the ability to precisely introduce a fluorophore/quencher pair into two specific sites of a protein of interest. Despite recent advances, there is continued demand for new and convenient approaches to site-selectively label proteins with such optical probes. We have recently developed a chemoselectively rapid azo-coupling reaction (CRACR) for site-specific protein labeling; it relies on rapid coupling between a genetically encoded 5-hydroxytryptophan residue and various aromatic diazonium ions. Herein, it is reported that the product of this conjugation reaction, a highly chromophoric biarylazo group, is a potent fluorescence quencher. The absorption properties of this azo product can be tuned by systematically altering the structure of the aryldiazonium species. A particular "quenchergenic" aryldiazonium has been identified that, upon conjugation, efficiently quenches the fluorescence of green fluorescent protein, which is a widely used genetically encoded fluorescent probe that can be terminally attached to target proteins. This fluorophore/quencher pair was used to evaluate the protein-labeling kinetics of CRACR, as well as to monitor the proteolysis of a fusion protein.

Properties of Electropolymerized Dopamine and Its Analogues.

This work reports a study of electropolymerization kinetics, film thickness, stability, and antifouling properties of polydopamine (PDA) and its three analogues: poly(3-(3,4-dihydroxyphenyl)-l-alanine) (PL-DOPA), poly(5-hydroxytryptophan) (PL-5-HTP), and poly(Adrenalin) (PAdrenalin). It was observed that the number of the hydroxyl groups on the benzene ring and the type (primary vs secondary) of amine group significantly affect the electropolymerization kinetics and thus the thickness of the obtained polymer films. Monomers with two hydroxyl groups (except Adrenalin) resulted in films that were thicker (∼10-15 nm) than the one with only one hydroxyl group (PL-5-HTP) (∼5-8 nm) under similar conditions. Adrenalin containing a secondary amino group could not be deposited onto the ITO substrate, while the other three compounds containing a primary amino group completely covered the ITO. The PDA films had better electrochemical stability than the other films. No film showed stable antifouling surfaces against protein.

An Oxidative Bioconjugation Strategy Targeted to a Genetically Encoded 5-Hydroxytryptophan.

Approaches that enable the chemoselective, covalent modification of proteins in a site-specific manner have emerged as a powerful technology for a wide range of applications. The electron-rich unnatural amino acid 5-hydroxytryptophan was recently genetically encoded in both Escherichia coli and eukaryotes, thereby allowing its site-specific incorporation into virtually any recombinant protein. Herein, we report the chemoselective conjugation of various aromatic amines to full-length proteins under mild, oxidative conditions that target this site-specifically incorporated 5-hydroxytryptophan residue.

A Chemoselective Rapid Azo-Coupling Reaction (CRACR) for Unclickable Bioconjugation.

Chemoselective modification of complex biomolecules has become a cornerstone of chemical biology. Despite the exciting developments of the past two decades, the demand for new chemoselective reactions with unique abilities, and those compatible with existing chemistries for concurrent multisite-directed labeling, remains high. Here we show that 5-hydroxyindoles exhibit remarkably high reactivity toward aromatic diazonium ions and this reaction can be used to chemoselectively label proteins. We have previously genetically encoded the noncanonical amino acid 5-hydroxytryptophan in both E. coli and eukaryotes, enabling efficient site-specific incorporation of 5-hydroxyindole into virtually any protein. The 5-hydroxytryptophan residue was shown to allow rapid, chemoselective protein modification using the azo-coupling reaction, and the utility of this bioconjugation strategy was further illustrated by generating a functional antibody-fluorophore conjugate. Although the resulting azo-linkage is otherwise stable, we show that it can be efficiently cleaved upon treatment with dithionite. Our work establishes a unique chemoselective "unclickable" bioconjugation strategy to site-specifically modify proteins expressed in both bacteria and eukaryotes.

Enzymatic Synthesis of Psilocybin.

Psilocybin is the psychotropic tryptamine-derived natural product of Psilocybe carpophores, the so-called "magic mushrooms". Although its structure has been known for 60 years, the enzymatic basis of its biosynthesis has remained obscure. We characterized four psilocybin biosynthesis enzymes, namely i) PsiD, which represents a new class of fungal l-tryptophan decarboxylases, ii) PsiK, which catalyzes the phosphotransfer step, iii) the methyltransferase PsiM, catalyzing iterative N-methyl transfer as the terminal biosynthetic step, and iv) PsiH, a monooxygenase. In a combined PsiD/PsiK/PsiM reaction, psilocybin was synthesized enzymatically in a step-economic route from 4-hydroxy-l-tryptophan. Given the renewed pharmaceutical interest in psilocybin, our results may lay the foundation for its biotechnological production.

PHOXI: A High Quantum Yield, Solvent-Sensitive Blue Fluorescent 5-Hydroxytryptophan Derivative Synthesized within Ten Minutes under Aqueous, Ambient Conditions.

Multiple tryptophan (Trp) proteins are not amenable to fluorescence study because individual residue emission is not resolvable. Biosynthetic incorporation of an indole analogue such as 5-hydroxyindole has not provided sufficient spectroscopic resolution because of low quantum yield and small emission shift. Here, 5-hydroxyindole is used as the starting framework for building a blue emitting fluorophore of high quantum yield, 2-phenyl-6H-oxazolo[4,5-e]indole (PHOXI). This is a three reagent reaction completed in 10 min under ambient conditions in borate buffer at pH 8. Reaction conditions have been optimized using 5-hydroxyindole. Derivatization is demonstrated on tryptophanyl 5-hydroxytryptophan (5-HTP) and a stable ß-hairpin "zipper" peptide with four tryptophan residues, TrpZip2, where Trp 4 has been replaced with 5-HTP, W4 → 5-HTP. Reaction optimization yields a PHOXI fluorophore that is essentially free of byproducts. Reaction specificity is demonstrated by the lack of reaction with N-acetyl-cysteine and amyloid ß-40, a peptide containing all amino acids except tryptophan, proline, and cysteine and lacking 5-HTP. Fluorescence study of PHOXI-derivatized 5-hydroxyindole in different solvents reveals the sensitivity of PHOXI to solvent polarity with a remarkable 87 nm red-shift in water relative to cyclohexane while maintaining high quantum yield. Thus, PHOXI joins the ranks of solvatochromic fluorophores such as PRODAN. Surprisingly, DFT calculations reveal coplanarity of the oxazolo/indole extended ring system and the phenyl substituent for both the HOMO and LUMO orbitals. Despite the crowded environment of three additional Trps in TrpZip2, CD spectroscopy shows that the TrpZip2 ß-hairpin structure is partially retained upon PHOXI incorporation. In an environment of smaller residues, PHOXI incorporation can be less disruptive of protein secondary structure, especially at molecular interfaces and other environments where there is typically less steric hindrance.

Serotonin modulates a depression-like state in Drosophila responsive to lithium treatment.

Major depressive disorder (MDD) affects millions of patients; however, the pathophysiology is poorly understood. Rodent models have been developed using chronic mild stress or unavoidable punishment (learned helplessness) to induce features of depression, like general inactivity and anhedonia. Here we report a three-day vibration-stress protocol for Drosophila that reduces voluntary behavioural activity. As in many MDD patients, lithium-chloride treatment can suppress this depression-like state in flies. The behavioural changes correlate with reduced serotonin (5-HT) release at the mushroom body (MB) and can be relieved by feeding the antidepressant 5-hydroxy-L-tryptophan or sucrose, which results in elevated 5-HT levels in the brain. This relief is mediated by 5-HT-1A receptors in the α-/ß-lobes of the MB, whereas 5-HT-1B receptors in the γ-lobes control behavioural inactivity. The central role of serotonin in modulating stress responses in flies and mammals indicates evolutionary conserved pathways that can provide targets for treatment and strategies to induce resilience.

Passive Membrane Penetration of the Serotonin Precursor 5-Hydroxytryptophan is Controlled by Its Zwitterion.

Species-specific partition coefficients in the octanol/water system were determined for the neurotransmitter serotonin (5-HT) and its precursor 5-hydroxytryptophan (5-HTP). The pH-independent partition coefficients (p) of the individual microspecies were determined by combination of experimentally measured distribution constants and a custom-tailored evaluation method, using highly similar auxiliary compounds. Experimental microscopic partition coefficients for triprotic molecules have only been reported before for thyroxine and its derivatives. The parabolic pH-distribution profile of 5-HT shows the dominance of the lipophilic non-charged microspecies, with a log p of 0.66. However, the most lipophilic non-charged form of 5-HTP, with a log p of 0.31, has no significant contribution to the distribution coefficient at any pH value. Instead, the less lipophilic zwitterionic protonation isomer dominates the distribution in the pH range 2.10 - 11.11. Although the non-charged microspecies of 5-HTP is 151 times more lipophilic than its zwitterionic protonation isomer, the overwhelming dominance of the zwitterionic form ensures that its contribution to the overall lipophilicity exceeds 1320 times that of the non-charged one. This fact is another counter-example of the widespread belief that passive diffusion into lipophilic media is predominated by the non-charged species. The lipophilicity profile of 5-HT and 5-HTP is depicted in terms of species-specific lipophilicities.

Sensitive detection of L-5-hydroxytryptophan based on molecularly imprinted polymers with graphene amplification.

A novel electrochemical sensor was presented for the determination of L-5-hydroxytryptophan (L-5-HTP) based on a graphene-chitosan molecularly imprinted film modified on the surface of glassy carbon electrode (GR-MIP/GCE). The morphology and composition of the imprinted film were observed in field emission scanning electron microscopy (FESEM), raman spectroscopy and fourier transform infrared (FTIR). The properties of the sensor were evaluated by electrochemical techniques. Under the optimal conditions, the peak currents of L-5-HIP were found to be linear in the concentration range of 0.05-7.0 µM, while the sensor also exhibited great features such as low detection limit of 6.0 nM (S/N = 3), superb selectivity against the structural analogues, good antidisturbance ability among coexisting components, excellent repeatability and stability. Moreover, the proposed method had been applied to the detection of L-5-HTP in human blood serum with a satisfactory recoveries ranging from 90.6% to 105.6%.

Recurrent RNA motifs as scaffolds for genetically encodable small-molecule biosensors.

Allosteric RNA devices are increasingly being viewed as important tools capable of monitoring enzyme evolution, optimizing engineered metabolic pathways, facilitating gene discovery and regulators of nucleic acid-based therapeutics. A key bottleneck in the development of these platforms is the availability of small-molecule-binding RNA aptamers that robustly function in the cellular environment. Although aptamers can be raised against nearly any desired target through in vitro selection, many cannot easily be integrated into devices or do not reliably function in a cellular context. Here, we describe a new approach using secondary- and tertiary-structural scaffolds derived from biologically active riboswitches and small ribozymes. When applied to the neurotransmitter precursors 5-hydroxytryptophan and 3,4-dihydroxyphenylalanine, this approach yielded easily identifiable and characterizable aptamers predisposed for coupling to readout domains to allow engineering of nucleic acid-sensory devices that function in vitro and in the cellular context.