Side Chain Hydroxyl Research Articles

In Situ Functionalization of Polar Polythiophene-Based Organic Electrochemical Transistor to Interface In Vitro Models.

Organic mixed ionic-electronic conductors are promising materials for interfacing and monitoring biological systems, with the aim of overcoming current challenges based on the mismatch between biological materials and convectional inorganic conductors. The conjugated polymer/polyelectrolyte complex poly(3,4-ethylenedioxythiophene):polystyrenesulfonate (PEDOT/PSS) is, up to date, the most widely used polymer for in vitro or in vivo measurements in the field of organic bioelectronics. However, PEDOT/PSS organic electrochemical transistors (OECTs) are limited by depletion mode operation and lack chemical groups that enable synthetic modifications for biointerfacing. Recently introduced thiophene-based polymers with oligoether side chains can operate in accumulation mode, and their chemical structure can be tuned during synthesis, for example, by the introduction of hydroxylated side chains. Here, we introduce a new thiophene-based conjugated polymer, p(g42T-T)-8% OH, where 8% of the glycol side chains are functionalized with a hydroxyl group. We report for the first time the compatibility of conjugated polymers containing ethylene glycol side chains in direct contact with cells. The additional hydroxyl group allows covalent modification of the surface of polymer films, enabling fine-tuning of the surface interaction properties of p(g42T-T)-8% OH with biological materials, either hindering or promoting cell adhesion. We further use p(g42T-T)-8% OH to fabricate the OECTs and demonstrate for the first time the monitoring of epithelial barrier formation of Caco-2 cells in vitro using accumulation mode OECTs. The conjugated polymer p(g42T-T)-8% OH allows organic-electronic-based materials to be easily modified and optimized to interface and monitor biological systems.

Exploring the reactivity and interactions of a poly(fluorene‐co‐tetrazine)‐conjugated polymer with Single‐walled Carbon Nanotubes

AbstractConjugated tetrazine‐containing polymers that undergo inverse electron demand Diels‐Alder (IEDDA) reactions with trans‐cyclooctenes are interesting not only for their intrinsic optoelectronic properties, but also their interactions with π‐conjugated surfaces. Here, we prepared a series of poly(fluorene‐co‐tetrazine) polymers and carried out IEDDA reactions to decorate them with hydroxyl, hexadecyl, or triethylene glycol side chains. The polymers were investigated pre‐ and post‐IEDDA coupling in terms of their ability to disperse single‐walled carbon nanotubes (SWNTs) in organic solvent. It was found that polymer molecular weight, side chain structure, and degree of conjugation all impacted the quality of SWNT dispersions. While the starting poly(fluorene‐co‐tetrazine) polymer produced concentrated dispersions, the post‐IEDDA polymer containing dihydropyridazine groups did not produce dispersions of equal concentration. However, upon oxidation to the fully aromatic pyridazines, the polymers regained their ability to form concentrated dispersions. Furthermore, the post‐IEDDA polymers exhibited increased selectivity toward metallic SWNTs relative to the starting polymer. Due to the efficiency of the IEDDA reaction, it was also possible to use this chemistry to derivatize the nanotube complex with poly(fluorene‐co‐tetrazine) post‐dispersion. Overall, this work demonstrates the first use of reactive polytetrazines to disperse SWNTs, allowing rapid modification of polymer‐nanotube complexes.

Utilizing Thermal Shift Assay to Probe Substrate Binding to Selenoprotein O.

Defining the biological importance of proteins with unknown functions poses a significant obstacle in understanding cellular processes. Although bioinformatic and structural predictions have contributed to the study of unknown proteins, in vitro experimental validations are often hampered by the optimal conditions and cofactors required for biochemical activity. Cofactor binding is not only essential for the activity of some enzymes but may also enhance the thermal stability of the protein. One practical application of this phenomenon lies in utilizing the change in thermal stability, as measured by alterations in the protein's melting temperature, to probe ligand binding. Thermal shift assay (TSA) can be used to analyze the binding of different ligands to the protein of interest or find a stabilizing condition to perform experiments such as X-ray crystallography. Here we will describe a protocol for TSA utilizing the pseudokinase, Selenoprotein O (SelO), for a simple and high-throughput method for testing metal and nucleotide binding. In contrast to canonical kinases, SelO binds ATP in an inverted orientation to catalyze the transfer of AMP to the hydroxyl side chains of proteins in a posttranslational modification known as protein AMPylation. By leveraging the shift in the melting temperatures, we provide crucial insights into the molecular interactions underlying SelO function.

Design and synthesis of novel and potent allosteric HIV-1 integrase inhibitors with a spirocyclic moiety

We report herein the design and discovery of novel allosteric HIV-1 integrase inhibitors. Our design concept utilized the spirocyclic moiety to restrain the flexibility of the conformation of the lipophilic part of the inhibitor. Compound 5 showed antiviral activity by binding to the nuclear lens epithelium-derived growth factor (LEDGF/p75) binding site of HIV-1 integrase (IN). The introduction of a lipophilic amide substituent into the central benzene ring resulted in a significant increase in antiviral activity against HIV-1 WT X-ray crystallography of compound 15 in complex with the integrase revealed the presence of a hydrogen bond between the oxygen atom of the amide of compound 15 and the hydroxyl group of the T125 side chain. Chiral compound 17 showed high antiviral activity, good bioavailability, and low clearance in rats.

Impact of single-residue mutations on protein thermal stability: The case of threonine 83 of BC2L-CN lectin

The thermal stability of trimeric lectin BC2L-CN was investigated and found to be considerably altered when mutating residue 83, originally a threonine, located at the fucose-binding loop. Mutants were analyzed using differential scanning calorimetry and isothermal microcalorimetry. Although most mutations decreased the affinity of the protein for oligosaccharide H type 1, six mutations increased the melting temperature (Tm) by >5 °C; one mutation, T83P, increased the Tm value by 18.2 °C(T83P, Tm = 96.3 °C). In molecular dynamic simulations, the investigated thermostable mutants, T83P, T83A, and T83S, had decreased fluctuations in the loop containing residue 83. In the T83S mutation, the side-chain hydroxyl group of serine formed a hydrogen bond with a nearby residue, suggesting that the restricted movement of the side-chain resulted in fewer fluctuations and enhanced thermal stability. Residue 83 is located at the interface and near the upstream end of the equivalent loop in a different protomer; therefore, fluctuations by this residue likely propagate throughout the loop. Our study of the dramatic change in thermal stability by a single amino acid mutation provides useful insights into the rational design of protein structures, especially the structures of oligomeric proteins.

Conformations of a highly expressed Z19 α-zein studied with AlphaFold2 and MD simulations.

α-zeins are amphiphilic maize seed storage proteins with material properties suitable for a multitude of applications e.g., in renewable plastics, foods, therapeutics and additive manufacturing (3D-printing). To exploit their full potential, molecular-level insights are essential. The difficulties in experimental atomic-resolution characterization of α-zeins have resulted in a diversity of published molecular models. However, deep-learning α-zein models are largely unexplored. Therefore, this work studies an AlphaFold2 (AF2) model of a highly expressed α-zein using molecular dynamics (MD) simulations. The sequence of the α-zein cZ19C2 gave a loosely packed AF2 model with 7 α-helical segments connected by turns/loops. Compact tertiary structure was limited to a C-terminal bundle of three α-helices, each showing notable agreement with a published consensus sequence. Aiming to chart possible α-zein conformations in practically relevant solvents, rather than the native solid-state, the AF2 model was subjected to MD simulations in water/ethanol mixtures with varying ethanol concentrations. Despite giving structurally diverse endpoints, the simulations showed several patterns: In water and low ethanol concentrations, the model rapidly formed compact globular structures, largely preserving the C-terminal bundle. At ≥ 50 mol% ethanol, extended conformations prevailed, consistent with previous SAXS studies. Tertiary structure was partially stabilized in water and low ethanol concentrations, but was disrupted in ≥ 50 mol% ethanol. Aggregated results indicated minor increases in helicity with ethanol concentration. β-sheet content was consistently low (∼1%) across all conditions. Beyond structural dynamics, the rapid formation of branched α-zein aggregates in aqueous environments was highlighted. Furthermore, aqueous simulations revealed favorable interactions between the protein and the crosslinking agent glycidyl methacrylate (GMA). The proximity of GMA epoxide carbons and side chain hydroxyl oxygens simultaneously suggested accessible reactive sites in compact α-zein conformations and pre-reaction geometries for methacrylation. The findings may assist in expanding the applications of these technologically significant proteins, e.g., by guiding chemical modifications.

Molecular engineering of π-extended viologens consisting of oxadiazoles-based bridges for highly stable electrochromic devices

Four π-extended viologen derivatives (Spr-PO/Sbu-PO/OHpr-PO/ OHet-PO) were synthesized by introducing oxadiazole group and side chain modification. Compared with viologen with bipyridines as the chromophore, these POs possessed extended conjugation lengths and optimized photoelectron conversion efficiency. The CMC gel electrochromic devices based on them could realize the color transformation from colorless to yellow-green under the working voltage of 1.1 V. Furthermore, due to different side chain groups, the diffusion rates of POs in the gel changed significantly. Compounds based on hydroxyl side chain groups showed higher response rates (<10 s) in the CMC gel system, while sulfonate side chain modified POs showed slower response characteristics (>10 s). All POs showed excellent stability, the optical contrasts were almost unchanged after continuous working for 100 cycles. This study provided an important method for obtaining efficient and stable electrochromic devices.

Effect of lignin modification on the selectivity of pyrolysis products from softwood kraft lignin

The synthesis of phenol via pyrolysis of lignin can be envisaged as a prospective approach for the high-value utilization of lignin, Herein, we describe an in-depth investigation of the selective modification of lignin to effectively adjust the distribution of lignin pyrolysis products. We found that hydroxypropyl modification of lignin can facilitate the elimination of methoxy from the lignin macromolecule and improve the selectivity of phenol products. In addition, increasing the carbon number of the lignin side chains causes polymerization of pyrolysis products, which is unfavorable for the synthesis of fine chemicals via lignin pyrolysis. Conversely, an increase in lignin side-chain hydroxyl content can lead to more liquid-phase products from lignin pyrolysis, and the selectivity of phenol and its congeners will also increase. As a result, the yield of liquid products obtained via pyrolysis of lignin modified via selective oxyalkylation can reach 52 %, and the relative content of phenol and its homologs can reach 45.2 %.

Protection against β-N-methylamino-l-alanineꟷinduced vesicular monoamine transporter 2 inhibition by hydroxyl-containing proteinogenic amino acids

β-N-methylamino-l-alanine (BMAA) has been shown to inhibit vesicular monoamine transporter 2 (VMAT2), thereby preventing the uptake of monoaminergic neurotransmitters into platelet dense granules and synaptic vesicles. The inhibition is hypothesized to be through direct association of BMAA with hydroxyl groupꟷcontaining amino acid residues in VMAT2. This study evaluated whether BMAA-induced inhibition of VMAT2 could be prevented directly by co-incubation of BMAA with amino acids, and if this protection was specific for BMAA inhibition of VMAT2. l-tyrosine, and to a lesser extent l-serine, was able to prevent BMAA-induced VMAT2 inhibition in a concentration-dependent manner, whereas neither l-threonine nor amino acids without side chain hydroxyl groups could reduce this inhibition. Reserpine-induced VMAT2 inhibition was unaffected by any of the amino acids. These data support the hypothesized interaction between BMAA and hydroxyl groupꟷcontaining amino acids and suggests that this interaction might be leveraged to protect against the toxicity of BMAA.

Construction methods and biomedical applications of PVA-based hydrogels.

Polyvinyl alcohol (PVA) hydrogel is favored by researchers due to its good biocompatibility, high mechanical strength, low friction coefficient, and suitable water content. The widely distributed hydroxyl side chains on the PVA molecule allow the hydrogels to be branched with various functional groups. By improving the synthesis method and changing the hydrogel structure, PVA-based hydrogels can obtain excellent cytocompatibility, flexibility, electrical conductivity, viscoelasticity, and antimicrobial properties, representing a good candidate for articular cartilage restoration, electronic skin, wound dressing, and other fields. This review introduces various preparation methods of PVA-based hydrogels and their wide applications in the biomedical field.

Synthesis and Identification of decarbamoyloxySaxitoxins in Toxic Microalgae and their Reactions with the Oxygenase, SxtT, Reveal Saxitoxin Biosynthesis.

Saxitoxin (STX, 1) is a representative compound of paralytic shellfish toxins (PSTs) that are produced by marine dinoflagellates and freshwater cyanobacteria. Although several pathways have been proposed for the biosynthesis of STX, the order of ring and side chain hydroxylation, and formation of the tricyclic skeleton have not been well established. In this study, 12,12-dideoxy-decarbamoyloxySTX (dd-doSTX, 2), the most reduced STX analogue having the tricyclic skeleton, and its analogues, 12β-deoxy-doSTX (12β-d-doSTX, 3), 12α-deoxy-doSTX (12α-d-doSTX, 4), and doSTX (5), were synthesized, and these compounds were screened in the toxic microalgae using high-resolution LCMSMS. dd-doSTX (2) and 12β-d-doSTX (3) were identified in the PSTs-producing dinoflagellates (Alexandrium catenella, A. pacificum, and/or Gymnodinium catenatum) and in the cyanobacterium Dolichospermum circinale (TA04). doSTX (5), previously isolated from the dinoflagellate G. catenatum, was also identified in D. circinale (TA04). Furthermore, the conversion of 2 to 3, and 4 to 5, by SxtT with VanB, a reported Rieske oxygenase and its redox partner in STX biosynthesis, was confirmed. These results support that 2 is a possible biosynthetic precursor of STX, and that ring and side-chain hydroxylations proceed after cyclization.

Three-Minute Enantioselective Amino Acid Analysis by Ultra-High-Performance Liquid Chromatography Drift Tube Ion Mobility-Mass Spectrometry Using a Chiral Core-Shell Tandem Column Approach.

Fast liquid chromatography (LC) amino acid enantiomer separation of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatives using a chiral core-shell particle tandem column with weak anion exchange and zwitterionic-type quinine carbamate selectors in less than 3 min was achieved. Enantiomers of all AQC-derivatized proteinogenic amino acids and some isomeric ones (24 in total plus achiral glycine) were baseline separated (Rs > 1.5 except for glutamic acid with Rs = 1.3), while peaks of distinct amino acids and structural isomers (constitutional isomers and diastereomers of leucine and threonine) of the same configuration overlapped to various degrees. For this reason, drift tube ion mobility-mass spectrometry was added (i.e., LC-IM-MS) as an additional selectivity filter without extending run time. The IM separation dimension in combination with high-resolution demultiplexing enabled confirmation of threonine isomers (threonine, allo-threonine, homoserine), while leucine, isoleucine, and allo-isoleucine have almost identical collisional cross-section (DTCCSN2) values and added no selectivity to the partial LC separation. Density functional theory (DFT) calculations show that IM separation of threonine isomers was possible due to conformational stabilization by hydrogen bond formation between the hydroxyl side chain and the urea group. Generally, the CCSN2 of protonated ions increased uniformly with addition of the AQC label, while outliers could be explained by consideration of intramolecular interactions and additional structural analysis. Preliminary validation of the enantioselective LC-IM-MS method for quantitative analysis showed compliance of accuracy and precision with common limits in bioanalytical methods, and applicability to a natural lipopeptide and a therapeutic synthetic peptide could be demonstrated.

In vitro retardation and modulation of human insulin amyloid fibrillation by Fe3+ and Cu2+ ions

Metal ions of Fe, Co, Ni, Cu, and Zn can form bonds through the carboxylate, hydroxyl, thiol, and imidazole side chains of proteins and those bonds are significantly more stable than those formed by non-transition metals.

Enhancing production and purity of 9-OH-AD from phytosterols by balancing metabolic flux of the side-chain degradation and 9-position hydroxylation in Mycobacterium neoaurum.

9α-Hydroxyandroster-4-ene-3,17-dione (9-OH-AD) is a representative steroid drug intermediate that can be prepared by phytosterols (PS) biotransformation with mycobacteria in a resting cell-cyclodextrin system. In this study, over-expression of 17β-hydroxysteroid dehydrogenase (Hsd4A)was testified to enhance the side-chain degradation of PS and to reduce the incomplete degradation by-products. Meanwhile, the complete degradation product 4-androstene-3,17-dione (AD)was increased due to the lack of 3-Ketosteroid 9α-Hydroxylase (KshA1) activities. To increase the production and purity of 9-OH-AD, the metabolic pathway of the side-chain degradation of PS and 9-position hydroxylationwas modulated by balancing the over-expression of Hsd4A and KshA1 in mycobacteria and reducing the bioconversion rate via lowering the ratio of PS and cyclodextrin. The production and purity of 9-OH-AD in brothwere improved from 22.18gL-1 and 77.13% to 28.27gL-1 and 87.84%, with a molar yield of 78.32%.

Microphase separation structures facilitated by dipole molecules and hydrogen bonding in poly (biphenyl piperidine-isatin) anion exchange membranes

The anion exchange membranes (AEMs) have the important function of blocking fuel and conducting OH− to make the cell form a circuit, so its alkali stability directly affects the performance of alkaline fuel cells. Currently, AEMs face a “trade-off” between ionic conductivity and stability. In order to solve this problem, we optimize the alkali resistance and conductivity of AEMs by introducing dipole-interacting side chains and hydrophilic hydroxyl side chains acting simultaneously on a rigid polymer backbone. By adopting the design strategy of “1 + 1>2″, the ion channel efficiency also makes a high-speed leap from a “two-wheeled bicycle” to a “four-wheeled car”. This effective microscopic modulation is clearly demonstrated by transmission electron microscope (TEM), where the AEMs after molecular design carry out a more obvious microphase separation structure at 200 nm. Through the PBP-54-IS- [OPD-100-OH-0] membrane, the maximum ionic conductivity is 100.62 mS cm−1 at 80 °C. After 1000 h at 80 °C, the retention rate of OH− conductivity of all AEMs was over 88.24 %. We also test its fuel cell performance for analysis. When the temperature of the two poles is 75 °C/72 °C, the power density is 147.28 mW cm−2. These are in line with the inherent requirements of AEMs for fuel cell applications.

Enzymatically Formed Oxysterols and Cell Death.

The side-chain hydroxylation of cholesterol by specific enzymes produces 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, and other products. These enzymatically formed side-chain oxysterols act as intermediates in the biosynthesis of bile acids and serve as signaling molecules that regulate cholesterol homeostasis. Besides these intracellular functions, an imbalance in oxysterol homeostasis is implicated in pathophysiology. Furthermore, growing evidence reveals that oxysterols affect cell proliferation and cause cell death. This chapter provides an overview of the pathophysiological role of side-chain oxysterols in developing human diseases. We also summarize our understanding of the molecular mechanisms underlying the induction of various forms of cell death by side-chain oxysterols.

Unraveling the structural and dynamic heterogeneities of hydroxyl-functionalized di-cationic ionic liquids (HFDILs): An integrated ab initio and molecular dynamics approach

This study comprehensively investigates the effects of hydroxyl groups in side chains and cation symmetry on intra- and inter-molecular structures, dynamics, and thermodynamic properties of two hydroxyl-functionalized di-cationic ionic liquids (HFDILs). Using a combination of molecular dynamics (MD) simulations and quantum chemistry (QC) calculations, we compare the influence of these effects on various properties with corresponding non-hydroxyl DILs reported in previous works. The structure of HFDILs is explored using angle distribution, radial distribution, spatial distribution, and combined distribution functions. Also, the averaged noncovalent interaction (aNCI) analysis is used to characterize the weak noncovalent interactions in fluctuating environment of HFDILs. Results showed that the oxygen atoms of anion have the strongest interaction with the hydrogen atom of hydroxyl group, followed by the hydrogen atom at the top of the imidazolium ring. Domain analysis and Voronoi calculations are performed to investigate the number of polar and non-polar subunits, revealing that symmetric HFDILs with two hydroxyl groups in their side chains have greater structural heterogeneity than asymmetric ones with only one hydroxyl group due to the larger accumulation of non-polar subunits. To investigate dynamical heterogeneity, we calculate the non-Gaussian parameter, van Hove correlation, hydrogen bond, ion pair, ion cage, and reorientation dynamics. Results indicated that symmetric HFDILs have slower dynamics, greater dynamic heterogeneity, and more stability than asymmetric ones. Finally, the structure and strength of ion triplet interactions are examined using quantum mechanical methods, revealing that symmetric HFDILs are more stable than asymmetric ones. This study provides valuable information for understanding DILs and their potential applications in various fields.

Copper catalyzed Shono-type oxidation of proline residues in peptide.

Since the initial report in 1975, the Shono oxidation has become a powerful tool to functionalize the α position of amines, including proline derivatives, by electrochemical oxidation. However, the application of electrochemical Shono oxidations is restricted to the preparation of simple building blocks and homogeneous Shono-type oxidation of proline derivatives remains challenging. The late-stage functionalization at proline residues embedded within peptides is highly important as substitutions about the proline ring are known to affect biological and pharmacological activities. Here, we show that homogenous copper-catalyzed oxidation conditions complement the Shono oxidation and this general protocol can be applied to a series of formal C-C coupling reactions with a variety of nucleophiles using a one-pot procedure. This protocol shows good tolerance toward 19 proteinogenic amino acids and was used to functionalize several representative bioactive peptides, including captopril, enalapril, Smac, and endomorphin-2. Last, peptide cyclization can also be achieved by using an appropriately positioned side-chain hydroxyl moiety.

Preparation of UV Debonding Acrylate Adhesives by a Postgrafting Reaction.

UV debonding acrylate adhesive (UDAA) plays a crucial role in the semiconductor industry, where its excellent adhesion is required to ensure the stability of silicon wafers and leave no residue on the surface after UV irradiation. The necessary UV debonding is achieved through the formation of rigid networks by the reactions of all the vinyl groups in the system. Acrylate copolymers with vinyl groups are typically obtained by the grafting reaction of isocyanate with a side-chain hydroxyl comonomer. However, these grafting reactions easily fail due to early cross-link formation. In this study, we illustrate a straightforward method for preparing UDAA by conducting a postgrafting reaction after one-step mixing of isocyanate functional monomer (IPDI-H) and hydroxyl acrylate copolymers (BA-H), thereby skipping the abovementioned vinyl grafting process. The chemical structures of the synthesized IPDI-H and BA-H were confirmed using Fourier transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (1H-NMR) analysis. Gel permeation chromatography (GPC) was employed to determine their molecular weights, while differential scanning calorimetry (DSC) was used to determine their glass transition temperatures. The postgrafting reactions successfully introduced vinyl groups onto the polyacrylate copolymer chains, resulting in high bonding strength during use and a significant decrease in peeling strength after UV irradiation. Rheological methods, including the three-interval thixotropy test (3ITT) and tack test modes, were employed to characterize a series of acrylate UV debonding adhesives. The recovery percentage of the storage modulus in the 3ITT mode indicated that a 0.6 wt% isocyanate curing agent made the UV debonding adhesives resistant to deformation. From the maximum normal force in the tack test mode, it was found that UDAA with 10 wt% PETA monomer and 30 wt% C5 tackifying resin exhibited excellent combined adhesion and debonding properties, which were further confirmed by peel strength tests. Microscope images of the wafer surfaces after removing the adhesive tapes demonstrated the excellent UV debonding properties achieved after 40 s of UV irradiation through the postgrafting reaction. The prepared UDAA has excellent properties; the peel strength can reach 15 N/25 mm before UV irradiation and can be reduced to 0.5 N/25 mm after ultraviolet irradiation. This research establishes a comprehensive method for understanding and applying UDAA in various applications.

Hydrogen bonds connecting the N-terminal region and the DE loop stabilize the monomeric structure of transthyretin.

Transthyretin (TTR) is a homo-tetrameric serum protein associated with sporadic and hereditary systemic amyloidosis. TTR amyloid formation proceeds by the dissociation of the TTR tetramer and the subsequent partial unfolding of the TTR monomer into an aggregation-prone conformation. Although TTR kinetic stabilizers suppress tetramer dissociation, a strategy for stabilizing monomers has not yet been developed. Here, we show that an N-terminal C10S mutation increases the thermodynamic stability of the TTR monomer by forming new hydrogen bond networks through the side chain hydroxyl group of Ser10. Nuclear magnetic resonance spectrometry and molecular dynamics simulation revealed that the Ser10 hydroxyl group forms hydrogen bonds with the main chain amide group of either Gly57 or Thr59 on the DE loop. These hydrogen bonds prevent the dissociation of edge strands in the DAGH and CBEF β-sheets during the unfolding of the TTR monomer by stabilizing the interaction between β-strands A and D and the quasi-helical structure in the DE loop. We propose that introducing hydrogen bonds to connect the N-terminal region to the DE loop reduces the amyloidogenic potential of TTR by stabilizing the monomer.