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Late-stage diversification of structurally complex peptides has enormous potential for drug discovery and molecular imaging. We report a simple, metal-free, late-stage reductive C2 alkylation of tryptophan and tryptophan-containing peptides using readily available 1,3-dithianes. This alkylation protocol has a wide substrate scope and an excellent tolerance for reactive functional groups.

Due to its abundance in blood, a great deal of research has been undertaken to develop efficient biosensors for serum albumin and provide insight into the interactions that take place between these biosensing molecules and the protein. Near-infrared (NIR, >700 nm) organic dyes have been shown to be effective biosensors of serum albumin, but their effectiveness is diminished in whole blood. Herein, it is shown that an NIR sulfonate indolizine-donor-based squaraine dye, SO 3 SQ, can be strengthened as a biosensor of albumin through the addition of biocompatible ionic liquids (ILs). Specifically, the IL choline glycolate (1:1), at a concentration of 160 mM, results in the enhanced fluorescence emission ("switch-on") of the dye in the presence of blood. The origin of the fluorescence enhancement was investigated via methods, including DLS, ITC, and molecular dynamics. Further, fluorescence measurements were conducted to see the impact the dye-IL system had on the fluorescence of the tryptophan residue of human serum albumin (HSA), as well as to determine its apparent association constants in relation to albumin. Circular dichroism (CD) spectroscopy was used to provide evidence that the dye-IL system does not alter the secondary structures of albumin or DNA. Our results suggest that the enhanced fluorescence of the dye in the presence of IL and blood is due to diversification of binding sites in albumin, controlled by the interaction of the IL-dye-albumin complex.

Tryptophan (Trp) plays a unique role in peptides and proteins as its indole ring possesses an electron-rich character and an N1-H hydrogen-bond donor. Because of its non-rotationally symmetric structure, synthetic alterations of the orientation of the indole ring would modulate the intrinsic structures and functions of peptides and proteins. Here we developed synthetic routes to the five Trp isomers in which the C3-substitution of the indole ring was changed to the C2/4/5/6/7-substitutions, and applied the five monomers to Fmoc-based solid-phase peptide synthesis. Specifically, the five monomers were prepared via Negishi cross-coupling reactions of C2/4/5/6/7-iodoindoles. To demonstrate the applicability of the monomers to the solid-phase synthesis, the five Trp isomers of macrocyclic antibiotic lysocin E were selected as target molecules and synthesized through peptide elongation, on-resin macrocyclization, and global deprotection. The Trp isomers displayed markedly weaker antibacterial activity than the parent natural product, revealing the biological importance of the precise three-dimensional shape of the original Trp residue of lysocin E. The present methods for the preparation and application of these five Trp isomers provide a new strategy for analyzing and modifying the specific functions of numerous Trp-containing peptides and proteins beyond this study., (© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Liquid biopsy analysis represents a suitable alternative analysis procedure in several cases where no tumor tissue is available or in poor patient conditions. Amino acids can play a crucial role in aiding cancer diagnosis. Monitoring of tryptophan (Trp) catabolism can aid in tracking cancer progression. Therefore, a novel nanocomposite was fabricated using overoxidized polypyrrole film doped with nano-carbon dots (nano-CDs) on the pencil graphite electrode (PGE) surface for sensitive evaluation of Trp in human serum. Using square wave voltammetry (SWV), the overoxidized polypyrrole/carbon dots/pencil graphite electrode (Ov-Ox PPy/CDs/PGE) achieved excellent electrochemical catalytic activity for evaluating Trp. The modified electrode, known as Ov-Ox PPy/CDs/PGE, demonstrated superior electrochemical catalytic activity compared to bare PGE, CDs/PGE, PPy/PGE, and PPy/CDs/PGE for evaluation of Trp. The method's excellent sensitivity was confirmed by the low limits of detection (LOD = 0.003 μmol L -1 ) and limit of quantitation (LOQ = 0.009 μmol L -1 ). The biosensor that was developed can measure tryptophan (Trp) levels in the serum of both healthy individuals and female breast cancer patients with high accuracy and sensitivity. The results indicate that there is a significant difference, as shown by the F-test, between healthy individuals and those with breast cancer. This suggests that Trp amino acid could be an essential biomarker for cancer diagnosis. Consequently, liquid biopsy analysis presents a valuable opportunity for early disease detection, particularly for cancer., (© 2023. The Author(s).)

The acylated Repeats in ToXins (RTX) leukotoxins, the adenylate cyclase toxin (CyaA) or α-hemolysin (HlyA), bind β 2 integrins of leukocytes but also penetrate cells lacking these receptors. We show that the indoles of conserved tryptophans in the acylated segments, W876 of CyaA and W579 of HlyA, are crucial for β 2 integrin-independent membrane penetration. Substitutions of W876 by aliphatic or aromatic residues did not affect acylation, folding, or the activities of CyaA W876L/F/Y variants on cells expressing high amounts of the β 2 integrin CR3. However, toxin activity of CyaA W876L/F/Y on cells lacking CR3 was strongly impaired. Similarly, a W579L substitution selectively reduced HlyA W579L cytotoxicity towards cells lacking β 2 integrins. Intriguingly, the W876L/F/Y substitutions increased the thermal stability (T m ) of CyaA by 4 to 8 °C but locally enhanced the accessibility to deuteration of the hydrophobic segment and of the interface of the two acylated loops. W876Q substitution (showing no increase in T m ), or combination of W876F with a cavity-filling V822M substitution (this combination decreasing the T m closer to that of CyaA), yielded a milder defect of toxin activity on erythrocytes lacking CR3. Furthermore, the activity of CyaA on erythrocytes was also selectively impaired when the interaction of the pyrrolidine of P848 with the indole of W876 was ablated. Hence, the bulky indoles of residues W876 of CyaA, or W579 of HlyA, rule the local positioning of the acylated loops and enable a membrane-penetrating conformation in the absence of RTX toxin docking onto the cell membrane by β 2 integrins., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Across eukaryotic proteomes, tryptophan is the least abundant of the 20 canonical amino acids, which makes it an ideal chemical handle for the late-stage functionalization of peptide and protein scaffolds with minimal production of undesired isoforms. Herein, we report the photocatalytic C2-alkylation of tryptophan using bromodifluoroacetate/acetamide-derived radical precursors. This rapid visible-light-mediated reaction is additive-free, operationally simple, and tolerates diverse functionality. We demonstrate the late-stage modification of a variety of complex peptides, including examples of biological significance.

Gold nanoparticles were functionalized by amino acid tryptophan and vitamin riboflavin - a resonance energy transfer (RET) pair of biomolecules. The presence of the gold nanoparticles resulted in 65% increase in RET efficiency. Because of enhanced RET efficiency, the photobleaching dynamics of the fluorescent molecules at the surface of the nanoparticles is different from that of molecules in solution. The observed effect was used for detection of the functionalized nanoparticles within biological material rich with autofluorescent species. Synchrotron radiation deep-ultraviolet fluorescence microscopy is used to study the photobleaching dynamics of the fluorescence centers within human hepatocellular carcinoma Huh7.5.1 cells incubated with the nanoparticles. The fluorescent centers were classified according to their photobleaching dynamics, which enabled the discrimination of the cell areas where the accumulation of the nanoparticles takes place, even though the particles were smaller than the spatial resolution of the images., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

To understand the roles of D-amino acids, evaluating their chemical properties in living organisms is essential. Herein, D-amino acid recognition of peptides was investigated using a tandem mass spectrometer equipped with an electrospray ionization source and a cold ion trap. Ultraviolet (UV) photodissociation spectroscopy and water adsorption of hydrogen-bonded protonated clusters of tryptophan (Trp) enantiomers and tripeptides (SAA, ASA, and AAS, where S and A denote L-serine and L-alanine, respectively) were carried out at 8 K in the gas phase. In the UV photodissociation spectrum of H + (D-Trp)ASA, the bandwidth of the S 1 -S 0 transition, which corresponds to the ππ* state of the Trp indole ring, was narrower than those of the other five clusters, H + (D-Trp)SAA, H + (D-Trp)AAS, H + (L-Trp)SAA, H + (L-Trp)ASA, and H + (L-Trp)AAS. In the UV photoexcitation of H + (D-Trp)ASA(H 2 O) n , which were formed via water adsorption on gas-phase H + (D-Trp)ASA, the evaporation of water molecules was the main photodissociation pathway. An NH 2 CHCOOH-eliminated ion and H + ASA were observed in the product ion spectrum. By contrast, water molecules adsorbed on the other five clusters remained on the product ions for NH 2 CHCOOH elimination and Trp detachment after the UV photoexcitation. The results indicated that the indole ring of Trp was located on the surface of H + (D-Trp)ASA, and the amino and carboxyl groups of Trp formed hydrogen bonds in H + (D-Trp)ASA. For the other five clusters, the indole rings of Trp were hydrogen bonded in the clusters, and the amino and carboxyl groups of Trp were present on the cluster surfaces., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Heterodimeric tryptophan-containing diketopiperazines (HTDKPs) are an important class of bioactive secondary metabolites. Biosynthesis offers a practical opportunity to access their bioactive structural diversity, however, it is restricted by the limited substrate scopes of the HTDKPs-forming P450 dimerases. Herein, by genome mining and investigation of the sequence-product relationships, we unveiled three important residues (F387, F388 and E73) in these P450s that are pivotal for selecting different diketopiperazine (DKP) substrates in the upper binding pocket. Engineering these residues in Nas F5053 significantly expanded its substrate specificity and enabled the collective biosynthesis, including 12 self-dimerized and at least 81 cross-dimerized HTDKPs. Structural and molecular dynamics analysis of F387G and E73S revealed that they control the substrate specificity via reducing steric hindrance and regulating substrate tunnels, respectively., (© 2023 Wiley-VCH GmbH.)

The interaction dynamics between flurbiprofen (FBP) and tryptophan (Trp) has been studied in covalently linked dyads and within human serum albumin (HSA) by means of fluorescence and ultrafast transient absorption spectroscopy. The dyads have proven to be excellent models to investigate photoinduced processes such as energy and/or electron transfer that may occur in proteins and other biological media. Since the relative spatial arrangement of the interacting units may affect the yield and kinetics of the photoinduced processes, two spacers consisting of amino and carboxylic groups separated by a cyclic or a long linear hydrocarbon chain (1 and 2, respectively) have been used to link the ( S )- or ( R )-FBP with the ( S )-Trp moieties. The main feature observed in the dyads was a strong intramolecular quenching of the fluorescence, which was more important for the ( S,S )- than for the ( R,S )- diastereomer in dyads 1, whereas the reverse was true for dyads 2. This was consistent with the results obtained by simple molecular modelling (PM3). The observed stereodifferentiation in ( S,S )-1 and ( R,S )-1 arises from the deactivation of 1 Trp*, while in ( S,S )-2 and ( R,S )-2 it is associated with 1 FBP*. The mechanistic nature of 1 FBP* quenching is ascribed to energy transfer, while for 1 Trp* it is attributed to electron transfer and/or exciplex formation. These results are consistent with those obtained by ultrafast transient absorption spectroscopy, where 1 FBP* was detected as a band with a maximum at ca. 425 nm and a shoulder at ∼375 nm, whereas Trp did not give rise to any noticeable transient. Interestingly, similar photoprocesses were observed in the dyads and in the supramolecular FBP@HSA complexes. Overall, these results may aid to gain a deeper understanding of the photoinduced processes occurring in protein-bound drugs, which may shed light on the mechanistic pathways involved in photobiological damage.

The primary step in the mechanism by which migratory birds sense the Earth's magnetic field is thought to be the light-induced formation of long-lived magnetically sensitive radical pairs within cryptochrome flavoproteins located in the birds' retinas. Blue-light absorption by the non-covalently bound flavin chromophore triggers sequential electron transfers along a chain of four tryptophan residues toward the photoexcited flavin. The recently demonstrated ability to express cryptochrome 4a from the night-migratory European robin ( Erithacus rubecula ), Er Cry4a, and to replace each of the tryptophan residues by a redox-inactive phenylalanine offers the prospect of exploring the roles of the four tryptophans. Here, we use ultrafast transient absorption spectroscopy to compare wild type Er Cry4a and four mutants having a phenylalanine at different positions in the chain. We find that each of the three tryptophan residues closest to the flavin adds a distinct relaxation component (time constants: 0.5, 30, and 150 ps) in the transient absorption data. The dynamics of the mutant containing a phenylalanine at the fourth position, furthest from the flavin, are very similar to those of wild type Er Cry4a, except for a reduced concentration of long-lived radical pairs. The experimental results are evaluated and discussed in the framework of real-time quantum mechanical/molecular mechanical electron transfer simulations based on the density functional-based tight binding approach. This comparison between simulation results and experimental measurements provides a detailed microscopic insight into the sequential electron transfers along the tryptophan chain. Our results offer a route to the study of spin transport and dynamical spin correlations in flavoprotein radical pairs.

Biomimetic oxidative dimerization of tryptophan derivatives in aqueous media with oxygen as a bulk oxidant catalyzed by an iron octacarboxy phthalocyanine complex was established. The discovery of the extremely active iron catalyst enables aerobic enzyme-mimetic oxidation to be performed in a flask. This method was applicable to the oxidative dimerization of a wide range of tryptophan derivatives, including various dipeptides and oligopeptides, with remarkable functional-group tolerance without the protection of the amino acid residues. Furthermore, oxidative dimerization of tryptophan derivatives bearing dioxopiperazine units enabled the convergent total synthesis of five natural pyrroloindole compounds and unnatural congeners. The established chemical method provides facile access to a broad range of dimerized peptides with a unique scaffold to link two turn structures, which will serve as a powerful tool to create new small- and medium-sized-molecules as drug candidates., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Chiral materials have drawn the widespread attention for their its chiral recognition ability. The design and synthesis of chiral material are of importance owing to the unpredictability in controlling chirality during the synthesis process. To circumvent problems, a chiral MOF (D-His-ZIF-8) was synthesized by ligand exchange of 2-methylimidazole (Hmim) on ZIF-8 by D-histidine (D-His), which can be treated as chiral host to distinguish amino acid enantiomers. The obtained D-His-ZIF-8 can provide chiral nanochannels for amino acid guests. Meanwhile, polynary transition-metal ion (Co 2+ and Fe 3+ ) coordinating with polydopamine (PDA) wrapped on the surface of D-His-ZIF-8 can increase the active sites. The electrochemical chiral recognition behavior showed that D-His-ZIF-8@CoFe-PDA exhibited good recognition of the tryptophan enantiomer (L/D-Trp) (working potential of -0.2 V vs. Hg/HgCl 2 ). The LOD and LOQ of L-Trp were 0.066 mM and 0.22 mM, respectively, while the LOD and LOQ of D-Trp were 0.15 mM and 0.50 mM, respectively. Finally, the usefulness of D-His-ZIF-8@CoFe-PDA/GCE was evaluated with a recovery of 94.4-103%. The analysis of real  samples shows that D-His-ZIF-8@CoFe-PDA/GCE is a feasible sensing platform for the detection of L-Trp and D-Trp., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Chiral recognition of amino acid enantiomers is critical in enhancing drug efficacy, detecting disease markers, and understanding physiological processes. Enantioselective fluorescent identification has gained attention among researchers due to its nontoxicity, easy synthesis, and biocompatibility. In this work, chiral fluorescent carbon dots (CCDs) were produced through a hydrothermal reaction followed by chiral modification. The fluorescent probe, Fe 3+ -CCDs (F-CCDs), was constructed by complexing Fe 3+ with CCDs to differentiate between the enantiomers of tryptophan (Trp) and determine ascorbic acid (AA) through an "on-off-on" response. It is worth noting that l-Trp can greatly enhance the fluorescence of F-CCDs with a blue shift, whereas d-Trp does not have any effect on the fluorescence of F-CCDs. F-CCDs showed a low limit of detection (LOD) for l-Trp and l-AA, with an LOD of 3.98 and 6.28 μM, respectively. The chiral recognition mechanism of tryptophan enantiomers using F-CCDs was proposed based on the interaction force between the enantiomers and F-CCDs, as confirmed by UV-vis absorption spectroscopy and density functional theory calculations. The determination of l-AA by F-CCDs was also confirmed through the binding of l-AA to Fe 3+ to release CCDs, as seen in UV-vis absorption spectra and time-resolved fluorescence decays. In addition, AND and OR gates were constructed based on the different responses of CCDs to Fe 3+ and Fe 3+ -CCDs to l-Trp/d-Trp, demonstrating the significance of molecular-level logic gates in drug detection and clinical diagnosis.

Green spectrofluorimetric methods have been adopted for the determination of Mirabegron (MG) in pure drug and pharmaceutical dosage form. The developed methods based on fluorescence quenching of tyrosine and L-tryptophan amino acids fluorophores by the effect of Mirabegron as a quencher. Experimental conditions of the reaction were studied and optimized. The Fluorescence quenching (ΔF) values were proportional to the concentration range of MG 2-20 μg/ml for the tyrosine-MG system in buffered media pH 2 and 1-30 μg/ml for L-tryptophan-MG system pH 6. Good correlation coefficients with low detection limits of 0.163 and 0.234 μg/ml for the two systems respectively. Method validation was applied according to ICH guidelines. The cited methods were successively applied for MG determination in tablet formulation. No statistically significant difference between the results of the cited and the reference methods regarding t and F tests. The proposed spectrofluorimetric methods are simple, rapid, eco-friendly and can contribute to MG's methodologies in quality control labs. Stern-Volmer relationship, the effect of temperature, quenching constant (K q ), and UV spectra were studied to identify the mechanism by which the quenching might occur. The results demonstrated that fluorescence quenching of tyrosine was a dynamic quenching process and L-tryptophan was static. The double log plots were constructed to determine the binding constants and binding sites. The greenness profile of the developed methods has been assessed by Green Analytical procedure index (GAPI) and Analytical Greenness Metric Approach (AGREE)., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Tryptophan is one of important amino acids in the human body, therefore its detection is particularly important. The 3,5-bis(4-pyridyl)-4-amino-1,2,4-triazole (BPAT) organic molecule was designed to be used as fluorescence detectors to detect tryptophan molecules for the interaction between the host and the guest. BPAT shows good sensitivity and selectivity towards tryptophan compared with other amino acid molecules. The limit of detection obtained from formula 3δ/K SV is considered to be 5.43 × 10 -7  mol/L. We speculated that this change is mainly caused by the hydrogen bond between tryptophan and the host molecule BPAT. This conjecture was verified by the controlled experiments with other host molecules., (© 2023 John Wiley & Sons Ltd.)

In a broader scenario, the forced degradation studies provided by the ICH guidelines for Q1A, Q1B, and Q2B degradation studies allow to know the CQA of the molecule used as a drug product, to determine the appropriate analytical methods, excipients, and storage conditions ensuring the quality of the drug, its efficacy, and patient safety. In this study, we focused our attention on understanding how oxidative stress is performed by H 2 O 2 -impacted small synthetic peptides that do not contain residues susceptible to oxidation such as methionine. Among the amino acids susceptible to oxidation, methionine is the most reactive and depending on the structure of the protein where it is exposed, it tends to oxidize by converting into methionine sulfone or methionine sulfoxide by oxidation of its sulfur atom. Scouting experiments obtained by forced oxidative stress conditions are presented on two small synthetic peptides that do not contain any methionine residues spiked with different amounts of H 2 O 2 , and they are analyzed by LC-MS/MS. Less frequent oxidation products than those commonly observed on proteins/peptides-containing methionine have been characterized on both peptides. The study demonstrated that somatostatin, by means of one residue of tryptophan on the molecule, can generate traces of several oxidized products detected by UPLC-MS. Furthermore, even at a negligible level, oxidation on tyrosine and proline in cetrorelix that does not contain methionine nor tryptophan has been detected by UHPLC-MS/MS. Identification and quantification of oxidized species were achieved by high-resolution MS and MS/MS experiments. Thus, FDSs undoubtedly aid the evaluation of the CQAs as an important component of the characterization package as recommended by HAs and ICH, facilitating the understanding of unforeseen features of the studied molecule used as drugs., (© 2023 Merck Serono SpA. Journal of Mass Spectrometry published by John Wiley & Sons Ltd.)

The aromatic amino acids (AAA), phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp), were cationized with ZnCl + and CdCl + , and the complexes were evaluated using infrared multiple photon dissociation (IRMPD) action spectroscopy. Specifically, the ZnCl + (Phe), CdCl + (Phe), ZnCl + (Tyr), CdCl + (Tyr), and ZnCl + (Trp) species were examined because the CdCl + (Trp) IRMPD spectrum is available in the literature. Several low-energy conformers for all complexes were found using quantum chemical calculations, and their simulated vibrational spectra were compared to the experimental IRMPD spectra to identify dominant isomers formed. In the case of MCl + (Phe) and MCl + (Tyr), these comparisons indicated the dominant binding motif is a tridentate structure, where the metal atom coordinates with the backbone amino nitrogen and carbonyl oxygen, as well as the aryl ring. These observations are consistent with the predicted ground states at the B3LYP, B3P86, B3LYP-GD3BJ, and MP2 levels of theory. For the ZnCl + (Trp) system, the experimental spectrum indicates a similar binding motif, with the zinc atom coordinating with the backbone nitrogen and carbonyl oxygen and either the pyrrole ring or the benzene ring of the indole side chain. These observations are consistent with the predicted low-lying conformers identified by the aforementioned levels of theory, with the B3LYP and B3P86 levels predicting the metal-pyrrole ring interaction is more favorable than the metal-benzene ring interactions and the opposite at the B3LYP-GD3BJ and MP2 levels.

Here, we report a novel strategy for constructing maleimide-containing peptides and cyclic peptides using Rh(III)-catalyzed tryptophan (Trp) (C7) alkenylation, which is challenging due to the inherent reactivity of the indole benzenoid ring. This method is scalable and exhibits broad substrate scope. The utility of this protocol could further be demonstrated by the synthesis of peptide conjugates with natural products and amino acids as well as the construction of maleimide-braced cyclic peptides.

Aromatic prenyltransferases from cyanobactin biosynthetic pathways catalyse the chemoselective and regioselective intramolecular transfer of prenyl/geranyl groups from isoprene donors to an electron-rich position in these macrocyclic and linear peptides. These enzymes often demonstrate relaxed substrate specificity and are considered useful biocatalysts for structural diversification of peptides. Herein, we assess the isoprene donor specificity of the N1-tryptophan prenyltransferase AcyF from the anacyclamide A8P pathway using a library of 22 synthetic alkyl pyrophosphate analogues, of which many display reactive groups that are amenable to additional functionalization. We further used AcyF to introduce a reactive moiety into a tryptophan-containing cyclic peptide and subsequently used click chemistry to fluorescently label the enzymatically modified peptide. This chemoenzymatic strategy allows late-stage modification of peptides and is useful for many applications., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Nuclear magnetic resonance (NMR) spectroscopy is uniquely suited to study the dynamics of biomolecules in solution. Most NMR studies exploit the spins of proton, carbon and nitrogen isotopes, as these atoms are highly abundant in proteins and nucleic acids. As an alternative and complementary approach, fluorine atoms can be introduced into biomolecules at specific sites of interest. These labels can then be used as sensitive probes for biomolecular structure, dynamics or interactions. Here, we address if the replacement of tryptophan with 5-fluorotryptophan residues has an effect on the overall dynamics of proteins and if the introduced fluorine probe is able to accurately report on global exchange processes. For the four different model proteins (KIX, Dcp1, Dcp2 and DcpS) that we examined, we established that 15 N CPMG relaxation dispersion or EXSY profiles are not affected by the 5-fluorotryptophan, indicating that this replacement of a proton with a fluorine has no effect on the protein motions. However, we found that the motions that the 5-fluorotryptophan reports on can be significantly faster than the backbone motions. This implies that care needs to be taken when interpreting fluorine relaxation data in terms of global protein motions. In summary, our results underscore the great potential of fluorine NMR methods, but also highlight potential pitfalls that need to be considered., (© 2022. The Author(s).)

Reduction of transient carnosine (β-alanyl-L-histidine) radicals by L-tryptophan, N -acetyl tryptophan, and the Trp-Gly peptide in neutral and basic aqueous solutions was studied using the technique of time-resolved chemically induced dynamic nuclear polarization (TR CIDNP). Carnosine radicals were generated in the photoinduced reaction with triplet excited 3,3',4,4'-tetracarboxy benzophenone. In this reaction, carnosine radicals with their radical center at the histidine residue are formed. Modeling of CIDNP kinetic data allowed for the determination of pH-dependent rate constants of the reduction reaction. It was shown that the protonation state of the amino group of the non-reacting β-alanine residue of the carnosine radical affects the rate constant of the reduction reaction. The results were compared to those obtained previously for the reduction of histidine and N -acetyl histidine free radicals and to newly obtained results for the reduction of radicals derived from Gly-His, a homologue of carnosine. Clear differences were demonstrated.

The diheme bacterial cytochrome c peroxidase (bC c P)/MauG superfamily is a diverse set of enzymes that remains largely uncharacterized. One recently discovered member, MbnH, converts a tryptophan residue in its substrate protein, MbnP, to kynurenine. Here we show that upon reaction with H 2 O 2 , MbnH forms a bis -Fe(IV) intermediate, a state previously detected in just two other enzymes, MauG and BthA. Using absorption, Mössbauer, and electron paramagnetic resonance (EPR) spectroscopies coupled with kinetic analysis, we characterized the bis -Fe(IV) state of MbnH and determined that this intermediate decays back to the diferric state in the absence of MbnP substrate. In the absence of MbnP substrate, MbnH can also detoxify H 2 O 2 to prevent oxidative self damage, unlike MauG, which has long been viewed as the prototype for bis -Fe(IV) forming enzymes. MbnH performs a different reaction from MauG, while the role of BthA remains unclear. All three enzymes can form a bis -Fe(IV) intermediate but within distinct kinetic regimes. The study of MbnH significantly expands our knowledge of enzymes that form this species. Computational and structural analyses indicate that electron transfer between the two heme groups in MbnH and between MbnH and the target tryptophan in MbnP likely occurs via a hole-hopping mechanism involving intervening tryptophan residues. These findings set the stage for discovery of additional functional and mechanistic diversity within the bC c P/MauG superfamily.

Tryptophan (TRP) is an essential amino acid catabolized mainly through the kynurenine pathway, and part of it is catabolized in the brain. The abnormal depletion of TRP and production of kynurenine (KYN) by two enzymes, tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO), have been linked to various neurological diseases. The ratio of TRP/KYN in plasma is a valuable measure for IDO/TDO activity and the prognosis of disease conditions. The 4-vinylphenylboronic acid (4-VPBA) was evaluated as a novel stationary phase for OT-CEC-MS/MS. TRP, KYN, and 3-hydroxykynurenine were separated using optimum conditions of 15 mM (NH 4 ) 2 CO 3 at pH 8 as a background electrolyte and 25 kV separation voltage on a 90 cm column. The usefulness of the 4-VPBA column for simple, fast, repeatable, and sensitive CEC-ESI-MS/MS application was demonstrated for the quantitation of TRP and KYN in the plasma of healthy human subjects and neuroinflammation subjects. The plasma sample was extracted on a zirconia-based ion-exchange cartridge for simultaneous protein precipitation and phospholipid removal. The method of standard addition, in combination with the internal standards approach, was used to prepare the calibration curve to overcome matrix matching and eliminate procedural errors. The developed quantitation method was validated according to FDA guidelines for sensitivity, accuracy, precision, and extraction recovery. The measured plasma level of TRP and KYN in healthy humans is aligned with the human metabolome database for the same two metabolites., (© 2023 Wiley-VCH GmbH.)

Molecular self- and co-assembly allow the formation of diverse and well-defined supramolecular structures with notable physical properties. Among the associating molecules, amino acids are especially attractive due to their inherent biocompatibility and simplicity. The biologically active enantiomer of l-histidine (l-His) plays structural and functional roles in proteins but does not self-assemble to form discrete nanostructures. In order to expand the structural space to include l-His-containing materials, we explored the co-assembly of l-His with all aromatic amino acids, including phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp), all in both enantiomeric forms. In contrast to pristine l-His, the combination of this building block with all aromatic amino acids resulted in distinct morphologies including fibers, rods, and flake-like structures. Electrospray ionization mass spectrometry (ESI-MS) indicated the formation of supramolecular co-assemblies in all six combinations, but time-of-flight secondary-ion mass spectrometry (ToF-SIMS) indicated the best seamless co-assembly occurs between l-His and l-Phe while in the other cases, different degrees of phase separation could be observed. Indeed, isothermal titration calorimetry (ITC) suggested the highest affinity between l-His and l-Phe where the formation of co-assembled structures was driven by entropy. In accordance, among all the combinations, the co-assembly of l-His and l-Phe produced single crystals. The structure revealed the formation of a 3D network with nanocavities stabilized by hydrogen bonding between -N (l-His) and -NH (l-Phe). Taken together, using the co-assembly approach we expanded the field of amino acid nanomaterials and showed the ability to obtain discrete supramolecular nanostructures containing l-His based on its specific interactions with l-Phe.

A series of oxidized cysteinyl peptides ([P-Cys-X-OMe] 2 ; P = Boc or H; X = Trp or Glu) showed vesicular and fibrillar assemblies. The anatomy of the self-assembled vesicles from the water-soluble cystine peptide [Cys-Trp-OMe] 2 (1a) has been investigated by using various fluorescent probes such as ammonium 8-anilinonaphthalene-1-sulfonate, Nile Red and pyrene. The morphological characterization was carried out by fluorescence lifetime imaging microscopy (FLIM) and super resolution-structured illumination microscopy (SR-SIM) utilizing the autofluorescence of the vesicles stemming from the self-assembly. The self-assembled structures are also observed in solution as evident from the quantitative phase images obtained using a dual-mode digital holographic microscope (DHM) system. Present investigations show that the self-assembly is enthalpy- and entropy-driven in the aqueous medium. Based on the CD spectral studies, we proposed that 1a organizes into vesicles through the sequestration of indole units. We observed that the solutions of dipeptides 1a-b exhibit autofluorescence in the blue region upon excitation at a wavelength >350 nm. Detailed spectroscopic studies on the peptides lacking tryptophan 2a-b unequivocally showed that the autofluorescence stems exclusively from peptide aggregation. Our experimental results with appropriate controls revealed that the clustering of carbonyl chromophores is central to autofluorescence. Autofluorescence was also used to probe the vesicle formation without using any external fluorescent probe. To the best of our knowledge, this is the first report on autofluorescent vesicles formed by the spontaneous association of dipeptides. We also found that the vesicles formed by 1a can act as a host for guests like C 60 . The biocompatibility and biodegradability of these peptides along with the autofluorescent nature and guest binding ability of peptide-based vesicles offer numerous applications in the biomedical area.

Bioorthogonal late-stage diversification of structurally complex peptides bears enormous potential for drug discovery and molecular imaging, among other applications. Herein, we report on a palladium-catalyzed C-H arylation of tryptophan-containing peptides with readily accessible and modular arylthianthrenium salts. Under exceedingly mild reaction conditions, the late-stage diversification of structurally complex peptides was accomplished. The tunability and ease of preparation of arylthianthrenium salts allowed the expedient stitching of tryptophan-containing peptides with drug, natural product, and peptidic scaffolds by forging sterically congested biaryl linkages. The robustness of the palladium catalysis regime was reflected by the full tolerance of a plethora of sensitive and coordinating functional groups. Hence, our manifold enabled efficient access to highly decorated, labelled, conjugated, and ligated linear and cyclic peptides., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

An electrochemical chiral sensing platform based on a multi-substituted ferrocene-cuprous ion (Cu + ) complex is constructed for the discrimination of electroactive amino acid (AA) isomers. Due to the opposite configurations of the AA isomers, the developed multi-substituted ferrocene-Cu + can preferably combine with a right-handed AA (D-AA) isomer to form the ternary complex of multi-substituted ferrocene-Cu + -D-AA through π-π interactions, resulting in higher peak currents of D-AA. Therefore, the isomers of electroactive AA can be successfully discriminated. Among the tested electroactive AA isomers, the chiral sensing platform exhibits higher discrimination capability toward the isomers of tryptophan (Trp) than that of tyrosine (Tyr) and cysteine (Cys), which might be ascribed to the stronger π-π interactions between the benzene ring of the multi-substituted ferrocene and the indole ring of the Trp isomers.

Optical isomers of short peptide Lysine-Tryptophan-Lysine (Lys-{L/D-Trp}-Lys) and Lys-Trp-Lys with an acetate counter-ion were used to study photoinduced intramolecular and intermolecular processes of interest in photobiology. A comparison of L- and D-amino acid reactivity is also the focus of scientists' attention in various specialties because today, the presence of amyloid proteins with D-amino acids in the human brain is considered one of the leading causes of Alzheimer's disease. Since aggregated amyloids, mainly Aβ42, are highly disordered peptides that cannot be studied with traditional NMR and X-ray techniques, it is trending to explore the reasons for differences between L- and D-amino acids using short peptides, as in our article. Using NMR, chemically induced dynamic nuclear polarization (CIDNP) and fluorescence techniques allowed us to detect the influence of tryptophan (Trp) optical configuration on the peptides fluorescence quantum yields, bimolecular quenching rates of Trp excited state, and the photocleavage products formation. Thus, compared with the D-analog, the L-isomer shows a greater Trp excited state quenching efficiency with the electron transfer (ET) mechanism. There are experimental confirmations of the hypothesis about photoinduced ET between Trp and the CONH peptide bond, as well as between Trp and another amide group.

Autofluorescence of blood plasma has been broadly considered as a prospective disease screening method. However, the assessment of such intrinsic fluorescence is mostly phenomenological, and its origin is still not fully understood, complicating its use in the clinical practice. Here we present the detailed evaluation of analytical capabilities, variability, and formation of blood plasma protein fluorescence based on the open dataset of excitation-emission matrices measured for ∼300 patients with suspected colorectal cancer, and our supporting model experiments. Using high-resolution size-exclusion chromatography coupled with comprehensive spectral analysis, we demonstrate, for the first time, the dominant role of HSA in the formation of blood plasma fluorescence in the visible spectral range (excitation wavelength >350 nm), presumably caused by its oxidative modifications. Furthermore, the diagnostic value of the tryptophan emission, as well as of the tyrosine fluorescence and visible fluorescence of proteins is shown by building a tree-based classification model that uses a small subset of physically interpretable fluorescence features for distinguishing between the control group and cancer patients with >80% accuracy. The obtained results extend current understanding and approaches used for the analysis of blood plasma fluorescence and pave the way for novel autofluorescence-based disease screening methods., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

A chiral sensing platform was constructed via adsorptive functionalization of ammonium persulfate doped polyaniline (APS-DPANI) with bovine serum albumin (BSA). The novelty of this work is the construction of such chiral interface with adsorption principle. The material has been characterized by scanning electron microscope, Fourier transform infrared and X-ray photoelectron spectroscopy, and thermogravimetric and water contact angle analyses. It displayed considerable stability in multi-run cyclic voltammetric scanning. Moreover, the superior conductivity of APS-DPANI and the decent binding ability of BSA endowed this sensing platform with an excellent recognition effect for tryptophan (Trp) enantiomers in the differential pulse voltammetry (DPV) test. The recognition was highly reproducible, and the detection limits for L- and D-isomer were 0.071 and 0.0478 mM, respectively., (© 2022 Wiley Periodicals LLC.)

Using the ultraviolet autofluorescence of tryptophan amino acids offers fascinating perspectives to study single proteins without the drawbacks of fluorescence labeling. However, the low autofluorescence signals have so far limited the UV detection to large proteins containing several tens of tryptophan residues. This limit is not compatible with the vast majority of proteins which contain only a few tryptophans. Here we push the sensitivity of label-free ultraviolet fluorescence correlation spectroscopy (UV-FCS) down to the single tryptophan level. Our results show how the combination of nanophotonic plasmonic antennas, antioxidants, and background reduction techniques can improve the signal-to-background ratio by over an order of magnitude and enable UV-FCS on thermonuclease proteins with a single tryptophan residue. This sensitivity breakthrough unlocks the applicability of UV-FCS technique to a broad library of label-free proteins.

Electrostatic interactions are key determinants of protein structure, dynamics, and function. Since protein electrostatics are nonuniform, assessment of the internal electric fields (EFs) of proteins requires spatial resolution at the amino acid residue level. In this regard, vibrational Stark spectroscopy, in conjunction with various unnatural amino acid-based vibrational probes, has become a common method for site-specific interrogation of protein EFs. However, application of this method is often limited to proteins with relatively high solubility, due to the intrinsically low oscillator strength of vibrational transitions. Therefore, it would be useful to develop an alternative method that can overcome this limitation. To this end, we show that, using solvatochromic study and molecular dynamics simulations, the frequency of maximum emission intensity of the fluorophore of 4-cyanotryptophan (4CN-Trp), 3-methyl-1 H -indole-4-carbonitrile, exhibits a linear dependence on the local EF. Since the absorption and emission spectra of 4CN-Trp are easily distinguishable from those of naturally occurring aromatic amino acids, we believe that this linear relationship provides an easier and more sensitive means to determine the local EF of proteins.

The photodynamics of protonated tryptophan and its mono hydrated complex TrpH + -H 2 O has been revisited. A combination of steady-state IR and UV cryogenic ion spectroscopies with picosecond pump-probe photodissociation experiments sheds new lights on the deactivation processes of TrpH + and conformer-selected TrpH + -H 2 O complex, supported by quantum chemistry calculations at the DFT and coupled-cluster levels for the ground and excited states, respectively. TrpH + excited at the band origin exhibits a transient of less than 100 ps, assigned to the lifetime of the excited state proton transfer (ESPT) structure. The two experimentally observed conformers of TrpH + -H 2 O have been assigned. A striking result arises from the conformer-selective photodynamics of TrpH + -H 2 O, in which a single water molecule inserted in between the ammonium and the indole ring hinders the barrierless ESPT reaction responsible for the ultra-fast deactivation process observed in the other conformer and in bare TrpH + ., (© 2022 The Authors. ChemPhysChem published by Wiley-VCH GmbH.)

Heme-copper oxidases (HCOs) utilize tyrosine (Tyr) to donate one of the four electrons required for the reduction of O 2 to water in biological respiration, while tryptophan (Trp) is speculated to fulfill the same role in cyt bd oxidases. We previously engineered myoglobin into a biosynthetic model of HCOs and demonstrated the critical role that Tyr serves in the oxygen reduction reaction (ORR). To address the roles of Tyr and Trp in these oxidases, we herein report the preparation of the same biosynthetic model with the Tyr replaced by Trp and further demonstrate that Trp can also promote the ORR, albeit with lower activity. An X-ray crystal structure of the Trp variant shows a hydrogen-bonding network involving two water molecules that are organized by Trp, similar to that in the Tyr variant, which is absent in the crystal structure with the native Phe residue. Additional electron paramagnetic resonance measurements are consistent with the formation of a Trp radical species upon reacting with H 2 O 2 . We attribute the lower activity of the Trp variant to Trp's higher reduction potential relative to Tyr. Together, these findings demonstrate, for the first time, that Trp can indeed promote the ORR and provides a structural basis for the observation of varying activities. The results support a redox role for the conserved Trp in bd oxidase while suggesting that HCOs use Tyr instead of Trp to achieve higher reactivity.

Assemblies of proteins and charged macromolecules (polyelectrolytes) find important applications as pharmaceutical formulations, biocatalysts, and cell-contacting substrates. A key question is how the polymer component influences the structure and function of the protein. The present paper addresses the influence of charged polymers on the thermal stability of two model beta-hairpin-forming peptides through an all-atom, replica exchange molecular dynamics simulation. The (negatively charged) peptides consist of the terminal 16 amino acids of the B1 domain of Protein G (GB1) and a variant with three of the GB1 residues substituted with tryptophan (Tryptophan Zipper 4, or TZ4). A (cationic) lysine polymer is seen to thermally stabilize TZ4 and destabilize GB1, while a (also cationic) chitosan polymer slightly stabilizes GB1 but has essentially no effect on TZ4. Free energy profiles reveal folded and unfolded conformations to be separated by kinetic barriers generally acting in the direction of the thermodynamically favored state. Through application of an Ising-like statistical mechanical model, a mechanism is proposed based on competition between (indirect) entropic stabilization of folded versus unfolded states and (direct) competition for hydrogen-bonding and hydrophobic interactions. These findings have important implications to the design of polyelectrolyte-based materials for biomedical and biotechnological applications.

Tryptophan serves as an important redox-active amino acid in mediating electron transfer and mitigating oxidative damage in proteins. We previously showed a difference in electrochemical potentials for two tryptophan residues in azurin with distinct hydrogen-bonding environments. Here, we test whether reducing the side chain bulk at position Phe110 to Leu, Ser, or Ala impacts the electrochemical potentials ( E °) for tryptophan at position 48. X-ray diffraction confirmed the influx of crystallographically resolved water molecules for both the F110A and F110L tyrosine free azurin mutants. The local environments of W48 in all azurin mutants were further evaluated by UV resonance Raman (UVRR) spectroscopy to probe the impact of mutations on hydrogen bonding and polarity. A correlation between the frequency of the ω17 mode─considered a vibrational marker for hydrogen bonding─and E ° is proposed. However, the trend is opposite to the expectation from a previous study on small molecules. Density functional theory calculations suggest that the ω17 mode reflects hydrogen bonding as well as local polarity. Further, the UVRR data reveal different intensity/frequency shifts of the ω9/ω10 vibrational modes that characterize the local H-bonding environments of tryptophan. The cumulative data support that the presence of water increases E ° and reveal properties of the protein microenvironment surrounding tryptophan.

Previous studies reported significantly altered tryptophan catabolite concentrations in major depression. Thus, tryptophan catabolites were considered as potential biomarkers of depression and their modulators as potential targets for psychopharmacotherapy. However, the results were based mainly on studies with small sample sizes limiting their generalizability. Against this background, we investigated the relationship of peripheral tryptophan catabolites with depression in a population-based sample with n = 3,389 participants (with fasting status ≥ 8 h and C-reactive protein < 10 mg/L). N = 248 had clinically significant depression according to a PHQ-9 score of ≥ 10, n = 1,101 subjects had mild depressive symptoms with PHQ-9 scores between 5 and 9, and n = 2,040 had no depression. After multivariable adjustment, clinically significant depression was associated with lower kynurenine and kynurenic acid. Spearman correlation coefficients of the tryptophan catabolites with the severity of depression were very small (rho ≤ 0.080, p ≤ 0.015). None of the tryptophan catabolites could diagnostically separate depressed from not depressed persons. Concerning linear associations, kynurenine and kynurenic acid were associated only with the severity and the cognitive dimension of depression but not its somatic dimension. Tryptophan catabolites were not associated with persistence or recurrence of depression at the 5 year follow-up. The results replicated the association between kynurenine and kynurenic acid with depression. However, the associations were small raising doubts about their clinical utility. Findings underline the complexity of the relationships between depression and tryptophan catabolites. The search for subgroups of depression with a potentially higher impact of depression might be warranted., (© 2023. The Author(s).)

The chemical and physical properties of cold, gas-phase hydrogen-bonded clusters of L-alanine (L-Ala), L-trialanine (L-Ala 3 ), L-tetraalanine (L-Ala 4 ), and tryptophan (Trp) enantiomers were investigated using tandem mass spectrometry with an electrospray ionization source and cold ion trap. From the ultraviolet (UV) photodissociation spectra at 265-290 nm, the electronic structures of homochiral H + (L-Trp)(L-Ala) at 8 K were found to be different from those of heterochiral H + (D-Trp)(L-Ala) and protonated Trp. The number of water molecules adsorbed on the surface of gas-phase H + (D-Trp)(L-Ala) was larger than that of H + (L-Trp)(L-Ala), indicating stronger intermolecular interactions of L-Ala with H + (L-Trp) than those with H + (D-Trp). The product ion spectrum obtained by 265 nm photoexcitation of H + (L-Trp)(L-Ala 3 )(H 2 O) n formed via gas-phase water adsorption on H + (L-Trp)(L-Ala 3 ) showed that the evaporation of water molecules was the main photodissociation process. In the case of H + (L-Trp)(L-Ala 4 )(H 2 O) n , signals of H + (L-Ala 4 ) (H 2 O) n formed via L-Trp evaporation were observed in the product ion spectra, and the cross-section for UV photoinduced L-Trp evaporation became larger as the number of adsorbed water molecules increased. This observation indicates that water molecules were selectively adsorbed on the H + (L-Ala 4 ) side of H + (L-Trp)(L-Ala 4 ) and weakened the intermolecular interactions between L-Trp and H + (L-Ala 4 ) in the hydrogen-bonded cluster., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The development of rapid methods for the detection of virus particles based on their intrinsic fluorescence (the native auto-fluorescence that originates from the non-labeled analyte) is challenging. Pure viruses may be detected in filtered solutions, based on the strong fluorescence of the amino acid tryptophan (Trp) in their proteins. Nevertheless, Trp also exists in high quantities in the hosts and host cultivation media. In this work, we developed a new method for the detection of the naked φX-174 virus. We show that a separation of φX-174 from its Escherichia coli host (grown on the standard cultivation medium nutrient agar) by simple extraction and filtration is not sufficient for its detection based on the intrinsic fluorescence since ~ 70% of the Trp fluorescence is derived from impurities. We formulate a new cultivation medium with a very low Trp concentration. We apply synchronous fluorescence measurements to show that no Trp fluorescence is detected in the extract solution upon incubation of this medium substrate with ammonium acetate extraction buffer. Finally, we apply synchronous fluorescence to detect φX-174 based on the spectral fingerprint of its native Trp content. Such a method is more rapid than usual traditional separation and detection methods which can take several hours and does not require any addition of labeling agents such as fluorescent dyes or antibodies for the detection. As other virus species contain Trp as one of the amino acids presents in their proteins, this method has the potential to apply to the detection of other viral species., (© 2022. Springer-Verlag GmbH Germany, part of Springer Nature.)

Chemical properties of gas-phase hydrogen-bonded clusters were investigated as a model for interstellar molecular clouds. Cold gas-phase hydrogen-bonded clusters of tryptophan (Trp) enantiomers and disaccharide isomers, including d-maltose and d-cellobiose, were generated by electrospray ionization and collisional cooling in an ion trap at 8 K. Product ion spectra in the 265-290 nm wavelength range were obtained using tandem mass spectrometry. NH 2 CHCOOH loss via the C α -C β bond cleavage of Trp occurred frequently in homochiral H + (d-Trp)(d-maltose) compared with heterochiral H + (l-Trp)(d-maltose) at 278 nm, indicating that an enantiomeric excess of l-Trp was formed via the enantiomer-selective photodissociation. The photoreactivity differed between the enantiomers and isomers contained in the clusters at the photoexcitation of 278 nm. A calibration curve for the quantification of disaccharide isomers in solution was constructed by photoexcitation of the hydrogen-bonded clusters of disaccharide isomers with H + (l-Trp) at 278 nm. A linear relationship between the natural logarithm of the relative product ion abundance and the mole fraction of d-maltose to d-cellobiose ratio in the solution was obtained, indicating that the mole fraction could be determined from a single product ion spectrum. A calibration curve, for quantification of Trp enantiomers, was also obtained using d-maltose as a chiral auxiliary., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Cyano-tryptophan is an unnatural fluorescent amino acid that emits in the visible region. Along with the structural similarity with tryptophan, the unique photophysical properties of this fluorophore make it an ideal probe for biophysical research. Herein, combining fluorescence spectroscopy, infrared spectroscopy, and molecular dynamics simulations, we show that the cyano-tryptophan's emission energy quantifies the underlying bond-specific noncovalent interactions in terms of the electric field. We further report the use of fluorophore's emission energy to predict its hydrogen bond characteristics. We demonstrate that combining experiments with molecular dynamics simulations can provide the hydrogen bonding status of the nitrile moiety. In addition, we report a method to differentiate between aqueous and nonaqueous hydrogen-bonding partners. Using a phenomenological approach, we demonstrate that the presence of the cyano-indole moiety is responsible for the distinct correlations between the fluorophore's emission and the electrostatic forces on the nitrile bond. As indole is a privileged scaffold for both native amino acids and nucleobases, cyano-indoles will have many multifaceted applications.

The magnetic compass of migratory birds is thought to rely on a radical pair reaction inside the blue-light photoreceptor protein cryptochrome. The sensitivity of such a sensor to weak external magnetic fields is determined by a variety of magnetic interactions, including electron-nuclear hyperfine interactions. Here, we investigate the implications of thermal motion, focusing on fluctuations in the dihedral and librational angles of flavin adenine dinucleotide (FAD) and tryptophan (Trp) radicals in cryptochrome 4a from European robin ( Erithacus rubecula , ErCry4a) and pigeon ( Columba livia , ClCry4a) and cryptochrome 1 from the plant Arabidopsis thaliana (AtCry1). Molecular dynamics simulations and density functional theory-derived hyperfine interactions are used to calculate the quantum yield of radical pair recombination dependent on the direction of the geomagnetic field. This quantity and various dynamical parameters are compared for [FAD •- Trp •+ ] in ErCry4a, ClCry4a, and AtCry1, with TrpC or TrpD being the third and fourth components of the tryptophan triad/tetrad in the respective proteins. We find that (i) differences in the average dihedral angles in the radical pairs are small, (ii) the librational motions of TrpC •+ in the avian cryptochromes are appreciably smaller than in AtCry1, (iii) the rapid vibrational motions of the radicals leading to strong fluctuations in the hyperfine couplings affect the spin dynamics depending on the usage of instantaneous or time-averaged interactions. Future investigations of radical pair compass sensitivity should therefore not be based on single snapshots of the protein structure but should include the ensemble properties of the hyperfine interactions.

This article reports the interaction of rifampicin, one of the important antituberculosis drugs, with Bovine Serum Albumin (BSA). Herein, we have monitored the fluorescence properties of tryptophan (Trp) residue in BSA to understand the interactions between protein and rifampicin. Fluorescence intensity of BSA was quenched tremendously upon interacting with the drug. Using steady state and time-resolved spectroscopic tools the static and dynamic nature of quenching have been characterised. Time correlated single photon counting technique confirmed that out of two lifetime components ∼6.2 ns and ∼2.8 ns of BSA, the rifampicin has affected only the shorter lifetime component a lot that was assigned to Trp-213 residue. Hence, it was thought that the drug must have been located near to the amino acid residue. Molecular docking studies have revealed the structural information of drug-protein complex which supported the above conjecture, confirming the nearest tryptophan as Trp-213 to the complexing rifampicin molecule., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

In the presence of vitamin C, cyanidin and cyanidin glycosides are degraded during the processing and storage of food products. To solve this issue, we investigated the protective effects and mechanism of action of five amino acids on the stability of cyanidin and its glycosides from chokeberry. The results showed that 0.3% tryptophan most effectively inhibited the degradation of cyanidin and its glycosides in the presence of vitamin C, under ultraviolet, dark, and sucrose-rich conditions. Fluorescence spectrum analysis showed that tryptophan could form noncovalent binding complexes with cyanidin-3-O-galactoside and cyanidin through hydrophobic and electrostatic forces and hydrogen bonds. Molecular docking results showed that the indole structure of tryptophan could form hydrophobic interactions with cyanidin-3-O-galactoside and cyanidin via hydrogen bonding, resulting in greater protection. Therefore, tryptophan could effectively protect cyanidin and its glycosides in cyanidin- and cyanidin glycoside-rich food products., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Objectives: In this study we describe the development and validation of a liquid chromatography mass spectrometry method (LC-MS/MS) to quantify five tryptophan (TRP) metabolites within the kynurenine- and serotonin pathway and apply the method to serum samples of women in the first trimester of pregnancy. A secondary aim was to investigate the correlation between body mass index (BMI) and the five analytes., Methods: A LC-MS/MS was developed for the analysis of TRP, kynurenine (KYN), 5-hydroxytryptophan (5-HTP), hydroxytryptamine (5-HT), and 5-hydroxyindole acetic acid (5-HIAA). Serum samples (n=374) were analyzed of pregnant women (median gestational age: 8 ± 2 weeks) participating in a subcohort of the Rotterdam Periconceptional Cohort (Predict study)., Results: The LC-MS/MS method provided satisfactory separation of the five analytes (7 min run). For all analytes R 2 was >0.995. Within- and between-run accuracies were 72-97% and 79-104%, and the precisions were all <15% except for the between-run precisions of the low QC-samples of 5-HTP and 5-HT (both 16%). Analyte concentrations were determined in serum samples of pregnant women (median (IQR)); TRP (µmol/L): 57.5 (13.4), KYN (µmol/L): 1.4 (0.4), 5-HTP (nmol/L): 4.1 (1.2), 5-HT (nmol/L): 615 (323.1), and 5-HIAA (nmol/L): 39.9 (17.0). BMI was negatively correlated with TRP, 5-HTP, and 5-HIAA (TRP: r=-0.18, p<0.001; 5-HTP: r=-0.13, p=0.02; natural log of 5-HIAA: r=-0.11, p=0.04), and positively with KYN (r=0.11, p=0.04)., Conclusions: The LC-MS/MS method is able to accurately quantify kynurenine- and serotonin pathway metabolites in pregnant women, providing an opportunity to investigate the role of the TRP metabolism in the (patho)physiology of pregnancy., (© 2022 the author(s), published by De Gruyter, Berlin/Boston.)

Autoinhibition of p53 binding to MDMX requires two short-linear motifs (SLiMs) containing adjacent tryptophan (WW) and tryptophan-phenylalanine (WF) residues. NMR spectroscopy was used to show the WW and WF motifs directly compete for the p53 binding site on MDMX and circular dichroism spectroscopy was used to show the WW motif becomes helical when it is bound to the p53 binding domain (p53BD) of MDMX. Binding studies using isothermal titration calorimetry showed the WW motif is a stronger inhibitor of p53 binding than the WF motif when they are both tethered to p53BD by the natural disordered linker. We also investigated how the WW and WF motifs interact with the DNA binding domain (DBD) of p53. Both motifs bind independently to similar sites on DBD that overlap the DNA binding site. Taken together our work defines a model for complex formation between MDMX and p53 where a pair of disordered SLiMs bind overlapping sites on both proteins., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Here, with the aim of developing a novel anti-cancer treatment, seven dipeptides were designed that contained methylated tryptophan and/or methylated arginine and were produced using Fmoc solid-phase peptide synthesis. Overexpression of the Src tyrosine kinase enzyme has been implicated in the development of different cancers. Dipeptides containing unnatural amino acids such as methylated arginine (RCH3), dimethylated arginine (R(CH3)2), and/or methylated tryptophan (WCH3) residues have earlier been shown to inhibit Src kinase. In this study, three such dipeptides, W-RCH3, WCH3-RCH3, and W-R(CH3)2, were tested using acellular assays and were found to have IC50 values (the concentration at which 50% inhibition occurs) of 510 nM, 916 nM, and 1 µM, respectively. These values were comparable to those obtained for cyclic penta- to nano-W-R peptides ([W-R]5-[W-R]9) synthesized in previous studies. However, the unmethylated versions of the dipeptides did not show any inhibitory activity against Src kinase. All of these dipeptides (50 µM) did not show any cytotoxicity after incubation up to 72 h with three different cancer cell lines, including leukemia (CCRF-CEM), breast adenocarcinoma (MDA-MB-231), and ovarian adenocarcinoma (SK-OV-3) cell lines, indicating the limited permeability of the peptides through the cell membrane. Therefore, further study is needed to improve the permeability of these peptides for anticancer applications, such as by using a peptide carrier or additional peptide functionalization. In summary, this study provides a protocol to synthesize and test peptides that inhibit Src kinase activity, and thus possess promising anticancer ability, as demonstrated using acellular and cellular assays.

Biomolecular binding relies on specific attractive interactions between two partner molecules, including electrostatics, dispersion, hydrophobicity, and solvation. Assessing the contributions of electrostatic interactions to binding is key to the understanding of ligand binding mechanisms and the design of improved biomolecular binders. For example, nicotine is a well-known agonist of nicotinic acetylcholine receptors (nAChRs), but the molecular mechanisms for the differential action of nicotine on brain and muscle nAChRs remain elusive. In this work, we have chosen the acetylcholine binding protein (AChBP) in complex with nicotine as a model system to interrogate the electrostatic contributions to nicotine binding. Our absolute binding free energy simulations confirm that nicotine binds AChBP predominantly in its protonated (charged) form. By comparing energetic contributions from decomposed interactions for either neutral or charged nicotine, our calculations shed light on the nature of the binding of nicotine to the AChBP. The preferred binding of charged nicotine over neutral nicotine originates from its stronger electrostatic interactions with AChBP, a cation-π interaction to a tryptophan residue and a hydrogen bond between nicotine and the backbone carbonyl of the tryptophan, whereas the major force driving the binding process appears to be van der Waals interactions. The various nonelectrostatic terms can also indirectly modulate the electrostatic interactions through fine-tuning the binding pose of the ligand in the binding site, providing an explanation of why the binding specificity of nicotine to the brain versus muscle nAChRs is driven by electrostatic interaction, given that the immediate binding site residues, including the key tryptophan residue, are identical in the two receptors.

A continuous-flow quaternization reaction of gramines with MeI (<1 min) followed by a substitution reaction with a chiral nucleophilic glycine-derived Ni-complex ( S )-2 (<1 min) has successfully been developed to afford the corresponding alkylated Ni-complexes 3 in good yields with excellent diastereoselectivity, based on the results of a one-pot quaternization-substitution reaction of gramines with ( S )-2 in a batch process. The continuous-flow process allowed the safe and efficient scale-up synthesis of 3j (84% yield, 99% de, 540 g h -1 ) to give 7-azatryptophan derivative ( S )-4j readily by an acid-catalyzed hydrolysis reaction followed by protection with an Fmoc group. The present method for the rapid and efficient syntheses of enantiopure unnatural tryptophan derivatives from various gramines and ( S )-2 will be useful to further promote peptide and protein drug discovery and development research.