Alanine Unit Research Articles

Regulating the ctDNA binding interactions and in vitro antitumor activities by chiral amide-bonded H2Porphyrins containing zero- to three- L-alanine units

Herein, a series of four chiral amide-bonded H2porphyrins containing zero- to three- L-alanine units have been prepared and characterized. Also, the spectroscopic investigations and biocompatibility evaluations of these amphiphilic H2porphyrins were carried out to illustrate the relationship between the number of alanine units and antitumor behaviors. Interestingly, H2Porphyrin 3 with two L-alanine units has a stronger DNA interaction and cell membrane penetration ability that significantly enhances the antitumor behaviors.

Synthetic copolymers with natural product side‐chain pendents

AbstractDehydroabietic acid is a natural product in the class of rosins. Here we report the design, synthesis and characterization of copolymers containing dehydroabietate side‐chain pendents, derived from homologated dehydroabietic ethyl methacrylate and tert‐butoxy carbonyl (Boc) l‐alanine methacryloyloxyethyl ester with varying comonomer proportions by reversible addition fragmentation chain transfer polymerization. The cleavage of Boc groups from the copolymers resulted in water‐soluble copolymers with cationic surface charge. Since proper hydrophobic−hydrophilic balance in a single polymer chain is maintained, the copolymers self‐assembled in aqueous media with a critical aggregation concentration of 2.8–3.0 mg L−1, confirmed by fluorescence spectroscopy. 1H NMR spectroscopy, dynamic light scattering, SEM and AFM were used to further investigate the aqueous solution self‐assembly of random copolymers. The collective findings from 1H NMR, dynamic light scattering, SEM and AFM clearly revealed the existence of micellar particles in water with alanine units on the surface of the nanoaggregates and homologated dehydroabietate pendents along with the hydrophobic main‐chain backbone in the core. In addition, to get a clear insight into the nanoaggregates' potential for drug encapsulation in aqueous medium, Nile Red was incorporated as a model hydrophobic dye. © 2023 Society of Industrial Chemistry.

Oriented Fibers Cooperate with DFO to Prevent Tendon Adhesions by Improving the Repair Microenvironment

AbstractTo solve the problem of tendon adhesion after an operation, an anti‐adhesion membrane is designed, which can inhibit the exogenous healing of tendons and promote endogenous healing. Here, poly[3(S)‐methyl‐morpholine‐2,5‐dione‐co‐lactic acid] P(MMD‐co‐LA) containing alanine units is obtained by melt ring‐opening polymerization (ROP). It can effectively reduce the production of acidic degradation products that cause aseptic inflammation. Then a kind of oriented P(MMD‐co‐LA)/deferoxamine (DFO) anti‐adhesion membrane with good mechanical properties, cell adhesion properties, and induced macrophage polarization properties is constructed by electrospinning. In vitro and in vivo studies have shown that oriented fibrous membranes can down‐regulate the expression of inflammatory cytokines. In addition, the fibrous membrane can up‐regulate the expression of angiogenesis‐related genes, namely HIF1‐α, SDF‐1α, and VEGF, by releasing DFO in situ, thus promoting the revascularization of tendon defects and providing nutritional support for endogenous tendon healing. This method provides a new barrier strategy to reduce tendon adhesion through anti‐inflammation combined with vascularization to inhibit exogenous tendon healing while promoting tendon endogenous healing. The results provide essential insights into the corresponding regulation of the tendon healing microenvironment.

Complex Formation between Cytochrome c and a Tetra-alanino-calix[4]arene

Owing to their remarkable features, calix[n]arenes are being exploited to study different aspects of molecular recognition, including protein complexation. Different complexation modes have been described, depending on the moieties that complement the aromatic cavity, allowing for function regulation and/or controlled assembly of the protein target. Here, a rigid cone calix[4]arene, bearing four anionic alanine units at the upper rim, was tested as a ligand for cytochrome c. Cocrystallization attempts were unfruitful, preventing a solid-state study of the system. Next, the complex was studied using NMR spectroscopy, which revealed the presence of two binding sites at lysine residues with dissociation constants (Kd) in the millimolar range.

Investigating Peptide-Coenzyme A Conjugates as Chemical Probes for Proteomic Profiling of N-Terminal and Lysine Acetyltransferases.

Acetyl groups are transferred from acetyl‐coenzyme A (Ac‐CoA) to protein N‐termini and lysine side chains by N‐terminal acetyltransferases (NATs) and lysine acetyltransferases (KATs), respectively. Building on lysine‐CoA conjugates as KAT probes, we have synthesized peptide probes with CoA conjugated to N‐terminal alanine (α‐Ala‐CoA), proline (α‐Pro‐CoA) or tri‐glutamic acid (α‐3Glu‐CoA) units for interactome profiling of NAT complexes. The α‐Ala‐CoA probe enriched the majority of NAT catalytic and auxiliary subunits, while a lysine CoA‐conjugate bound only a subset of endogenous KATs. Interactome profiling with the α‐Pro‐CoA probe showed reduced NAT recruitment in favor of metabolic CoA binding proteins and α‐3Glu‐CoA steered the interactome towards NAA80 and NatB. These findings agreed with the inherent substrate specificities of the target proteins and showed that N‐terminal CoA‐conjugated peptides are versatile probes for NAT complex profiling in lysates of physiological and pathological backgrounds.

Oriented nanofibrous P(MMD-co-LA)/Deferoxamine nerve scaffold facilitates peripheral nerve regeneration by regulating macrophage phenotype and revascularization

Delayed injured nerve regeneration remains a clinical problem, partly ascribing to the lack of regulation of regenerative microenvironment, topographical cues, and blood nourishment. Functional electrospun conduits have been established as an efficacious strategy to facilitate nerve regeneration by providing structural guidance, regulating the regenerative immune microenvironment, and improving vascular regeneration. However, the synthetic polymers conventionally used to fabricate electrospinning scaffolds, such as poly(L-lactic acid), poly(glycolic acid), and poly(lactic-co-glycolic acid), can cause aseptic inflammation due to acidic degradation products. Therefore, a poly[3(S)-methyl-morpholine-2,5-dione-co-lactic] [P(MMD-co-LA)] containing alanine units with good mechanical properties and reduced acid degradation products, was obtained by melt ring-opening polymerization (ROP). Here, we aimed to explore the effect of oriented nanofiber/Deferoxamine (DFO, a hydrophilic angiogenic drug) scaffold in the rapid construction of a favorable regenerative microenvironment, including cell bridge, polarized vascular system, and immune microenvironment. In vitro studies have shown that the scaffold can sustainably release DFO, which accelerates the migration and tube formation of human umbilical vein endothelial cells (HUVECs), as well as the expression of genes related to angiogenesis. The physical clues provided by the arranged nanofibers can regulate the polarization of macrophages and reduce the expression of inflammatory factors. Furthermore, the in vivo results demonstrated a higher M2 polarization level of the oriented nanofibrous scaffold treatment group with reducedinflammation reaction in the injured nerve. Moreover, the in-situ release of DFO up-regulated the expression of HIF1-α and SDF-1α genes, as well as the expression of HIF1-α′s target gene VEGF, further promoting revascularization and enhancing nerve regeneration at the defect site. The obtained results provide essential insights on accelerating the creation of the nerve regeneration microenvironment by combining the physiological processes of nerve regeneration with topographical cues and chemical signal induction.

Heterochiral Dimer Formation of α-l- and α-d-Polyalanine Molecules on Surfaces

The self-assembly process of racemic mixtures of right(L)- and left(D)-handed α-polyalanine molecules (DL-PA) with a length of 4 nm (27 alanine units) and a helix pitch of λ=0.54 nm on HOPG was inv...

Transparent, High-Strength, and Shape Memory Hydrogels from Thermo-Responsive Amino Acid-Derived Vinyl Polymer Networks.

Herein, the formation of unique shape-memory hydrogels that are composed of thermo-responsive amino-acid-derived vinyl polymer networks is reported; these are readily prepared by radical copolymerization of N-acryloyl glycinamide with commercially available cross-linkers, namely, methylenebis(acrylamide) and poly(ethylene glycol) diacrylate. These hydrogels are transparent (>90% transmittance at 600 nm) and are comprised of 97-70 wt% water. Furthermore, these contain both chemical and physical cross-linkages that are based on the multiple hydrogen bonds attained via amino acid units; this composition is aimed at generating opposing stimuli-responsive characters, namely, chemically stable and thermo-sensitive properties. A cooperative interplay of these two networks enables the hydrogels to exhibit a decent mechanical toughness (breaking strength ≈0.3 MPa and breaking elongation >600%) and a shape fix/memory capability. The temporary shape is easily fixed by cooling at 4 °C after deformation at high temperature, and it instantly recovers its original shape through reheating. Furthermore, a multi-shape memory effect is achieved by incorporating the pH-responsive N-acryloyl alanine unit into the hydrogel system as a comonomer; in this system, three distinct shapes can be fixed through temperature and pH manipulations. This facilely attainable shape memory hydrogel has significant potential in various fields, such as soft actuators, sensors, and biomedical materials.

Thermal properties and degradation of enantiomeric copolyesteramides poly(lactic acid-co-alanine)s

Thermal properties and degradation of enantiomeric random copolyesteramides poly(l-lactic acid-co-l-alanine) [P(LLA-LAL)] and poly(d-lactic acid-co-d-alanine) [P(DLA-DAL)] copolymers with wide alanine unit content ranges from 0 to 21 and 22 mol% were investigated by differential scanning calorimetry and themogravimetry. The total enthalpy of cold crystallization enthalpy and melting enthalpy as an indicator of crystallinity decreased with a decrease in lactic acid unit content or an increase in alanine unit content. Equilibrium melting temperature values of α-form homo-crystallites for the unblended samples decreased with an increase in alanine unit content (157.3–179.6 °C) and those of δ-form homo-crystallites for the unblended L100 and D100 (190.7 and 185.1 °C) were much higher than the reported values (both 172.8 °C, Polymer 2017, 9, 625). Thermal stability was enhanced by enantiomeric polymer blending, although thermal degradation was accelerated by incorporated alanine units. The activation energy for thermal degradation (ΔEtd) of the blend samples decreased with an increase in alanine unit content, whereas that of the unblended samples showed complicated dependence on alanine unit content and became maximum at alanine unit contents of 21 and 22 mol%. The blend samples had higher ΔEtd values comparted to those of the unblended samples, except for the alanine unit contents of 21 and 22 mol%. The reason for the high ΔEtd values of the unblended samples compared with that of the blend sample at the alanine unit contents of 21 and 22 mol% is discussed.

Stereocomplex crystallization, homocrystallization, and polymorphism of enantiomeric copolyesteramides poly(lactic acid‐ co ‐alanine)s from the melt

Stereocomplex (SC) crystallization, homocrystallization, and polymorphism of poly(l-lactic acid-co-l-alanine) [P(LLA-LAL)] and poly(d-lactic acid-co-d-alanine) [P(DLA-DAL)] copolymers with wide alanine unit content ranges from 0 to 21 and 22 mol% are investigated for melt-crystallization. P(LLA-LAL)/P(DLA-DAL) blends crystallize for wide alanine unit content ranges compared to unblended P(LLA-LAL) and P(DLA-DAL) samples, due to facile SC crystallization compared to homocrystallization. The phase diagrams of the unblended samples [α- and δ-form homocrystallites] and the blend samples (SC and homocrystallites) are drawn. The transition crystallization temperature of unblended samples from α-form to δ-form decreases with an increase in alanine unit content. In the unblended and blend samples, alanine units are correspondingly incorporated in α-form homocrystalline regions and excluded from SC crystalline regions. The maximum radial growth rate values of spherulites are higher for the blend samples than for the unblended samples. The experimental crystallization half time [tc(1/2)(exp)] values of the unblended samples increase with an increase in alanine unit content, whereas the tc(1/2)(exp) values of the blend samples with alanine unit contents of 3 and 4 mol% and 6 and 6 mol% and of 12 and 13 mol% are respectively higher than and similar to those of poly(l-lactic acid)/poly(d-lactic acid) blend.

Spectroscopic and modeling analyses of bimolecular structure of corn silk

Corn silk (CS) is a protein/ cellulosic agricultural waste. Due to its documented beneficial medical applications; corn silk aqueous extract was greenly prepared. FTIR spectroscopic analyses are conducted for three fractions of CS, then a model is prepared to simulate CS molecular structure. Based on FTIR analyses, a model is described as composite of dehydrated alanine unit lignin unit and two cellulose units with metal oxides namely MgO and CuO. Some important descriptors were calculated with Quantitative structure–activity relationship QSAR calculations. Then quantum mechanical calculations were conducted. Molecular modeling data indicated the CS applicability for several biomedical applications due to its active sites proved by electrostatic potential. The results confirmed the suitability of CS as promising tool for many biological applications.

Synthesis, stereocomplex crystallization and homo-crystallization of enantiomeric poly(lactic acid-co-alanine)s with ester and amide linkages

α-Hydroxy acid and α-amino acid-derived poly(l-lactic acid-co-l-alanine)s and poly(d-lactic acid-co-d-alanine)s with different alanine unit contents were synthesized and their stereocomplex crystallization and homo-crystallization were investigated.

Preparation and evaluation of a chiral HPLC stationary phase based on cone calix[4]arene functionalized at the upper rim with l-alanine units.

Here we report a new chiral stationary phase (CSP) immobilized on silica gel based on cone calix[4]arene functionalized at the upper rim with two l-alanine units as new chiral selector that has been used in high-performance liquid chromatography. The CSP was prepared by covalently bonding the allyl groups at the lower rim of calix[4]arene to silica gel by thiol-ene click chemistry reaction. Elemental analysis of the CSP showed that 120 μmol of chiral selector bonded per gram of silica gel. 1-Hexene was used for end-capping of unreacted mercapto groups on silica gel. Since the CSP is chemically bonded to the silica, it can be used in the normal-phase and reversed-phase mode and with halogenated solvents as mobile phases, if desired. The chromatographic performance of the CSP was evaluated in the enantioseparation of the 3,5-dinitrobenzoyl derivatives of some amino acids, diclofop-methyl and dl-mandelic acid.

Artificial Metalloproteins for Binding and Stabilization of a Semiquinone Radical.

The interaction of a number of first-row transition-metal ions with a 2,2′-bipyridyl alanine (bpyA) unit incorporated into the lactococcal multidrug resistance regulator (LmrR) scaffold is reported. The composition of the active site is shown to influence binding affinities. In the case of Fe(II), we demonstrate the need of additional ligating residues, in particular those containing carboxylate groups, in the vicinity of the binding site. Moreover, stabilization of di-tert-butylsemiquinone radical (DTB-SQ) in water was achieved by binding to the designed metalloproteins, which resulted in the radical being shielded from the aqueous environment. This allowed the first characterization of the radical semiquinone in water by resonance Raman spectroscopy.

Chiral Indolylarylsulfone Non-Nucleoside Reverse Transcriptase Inhibitors as New Potent and Broad Spectrum Anti-HIV-1 Agents.

We designed and synthesized a series of chiral indolyarylsulfones (IASs) as new HIV-1 NNRTIs. The new IASs 8-37 showed potent inhibition of the HIV-1 WT NL4-3 strain and of the mutant K103N, Y181C, Y188L, and K103N-Y181C HIV-1 strains. Six racemic mixtures, 8, 23-25, 31, and 33, were separated at semipreparative level into their pure enantiomers. The (R)-8 enantiomer bearing the chiral (α-methylbenzyl) was superior to the (S)-counterpart. IAS derivatives bearing the (S) alanine unit, (S)-23, (S,R)-25, (S)-31, and (S)-33, were remarkably more potent than the corresponding (R)-enantiomers. Compound 23 protected hippocampal neuronal cells from the excitotoxic insult, while efavirenz (EFV) did not contrast the neurotoxic effect of glutamate. The present results highlight the chiral IASs as new NNRTIs with improved resistance profile against the mutant HIV-1 strains and reduced neurotoxic effects.

In silico studies on the role of mutant Y337A to reactivate tabun inhibited mAChE with K048

Organophosphorus compound (OP) tabun is resistant to reactivate by many oxime drugs after the formation of OP-conjugate with AChE. The reactivation of tabun-inhibited mAChE and site-directed mutants by bispyridinium oxime, K048 (N-[4-(4-hydroxyiminomethylpyridinio)butyl]-4-carbamoylpyridinium dibromide) showed that the mutations significantly poor the overall reactivation efficacy of K048. We have unravelled the lowered efficacy of K048 with the tabun-mutant mAChE(Y337A) using docking and steered molecular dynamics (SMD) simulations. The computed results showed some interesting features for the interaction of drug molecule K048 with tabun-mAChE(wild-type) and tabun-mutant mAChE(Y337A). The SMD simulations showed that the active pyridinium ring of K048 is directed towards the phosphorus atom conjugated to the active serine (SUN203) of tabun-mAChE(wild-type). The cradle shaped residues Tyr337-Phe338 present in the choline binding site stabilize the active pyridinium ring of K048 with π–π interaction and the residue Trp86 involved in T-shaped cation-π interaction. However, in the case of tabun-mutant mAChE(Y337A).K048 conjugate, the replacement of aromatic Tyr337 with the aliphatic alanine unit in the choline binding site, however, loses one of the π–π interaction between the active pyridinium ring of K048 and the Tyr337. The placement of aliphatic alanine unit resulted in the displacement of the side chain of Phe338 towards the His447. Such displacement is causing the inaccessibility of the drug towards the phosphorus atom conjugated to the active serine (SUN203) of tabun-mutant mAChE(Y337A). Furthermore, the unbinding of the K048 with SMD studies showed that the active pyridinium ring of the drug undergoes a complete turn along the gorge axis and is directed away from the phosphorus atom conjugated to the active serine of the tabun-mutant mAChE(Y337A). Such effects inside the gorge of tabun-mutant mAChE(Y337A) would lower the efficacy of the drug molecule (K048) for the reactivation process. The binding free energy computed for the tabun-mAChE(wild-type) and tabun-mutant mAChE(Y337A) with K048 showed that the drug molecule prefers to bind strongly with the former enzyme (∼30 kJ/mol) than the later one.

The stereoselective separation of serine containing peptides by zwitterionic ion exchanger type chiral stationary phases and the study of serine racemization mechanisms by isotope exchange and tandem mass spectrometry

The occurrence of d-amino acids (d-AAs) in higher-developed organisms in their free form, and within peptides and proteins, has been investigated with an increasing number of studies. Often the inversion of the stereochemical configuration of an individual amino acid drastically changes its biological activity. Alongside Asn and Asp, Ser is most prone to racemization within peptides. Specific enzymes catalyzing d-Ser generation and breakdown have been described. Hence, the applicability of enantioselective ZWIX(+)® and ZWIX(−)® chiral stationary phases (CSPs) to peptide separations was assessed and a set of 14 pairs of diastereomeric and enantiomeric Ser and Thr containing di-, tri- and tetra-peptides was chromatographically separated without prior hydrolysis to the individual amino acids. To a certain extent, RP chromatography also enabled the separation of peptide diastereomers. The ZWIX CSPs delivered chromatographic selectivities between 1.04 and 7.23, allowing a change of elution order by switching between the ZWIX(+) and the ZWIX(−) CSP. Coupling these highly selective chromatographic columns with an LTQ-Orbitrap XL™ mass spectrometer and performing high resolution MS2 measurements enabled us to investigate mechanistic aspects of chemically induced racemization of Ser embedded in short peptides. As reaction medium an alkaline aqueous solution (pH 12.3) was selected. Proton/deuterium exchange experiments provided evidence of a fast Cα proton exchange with simultaneous racemization. Additionally, 18O/16O exchange allowed the identification of an alternative, and somewhat retarded racemization via a reversible β-elimination and reintroduction of water at the hydroxymethyl side chain of Ser. This involved the intermediate generation of the prochiral didehydro alanine unit.

Preparation and release study of ibuprofen‐loaded porous matrices of a biodegradable poly(ester amide) derived from L‐alanine units

AbstractScaffolds of a biodegradable poly(ester amide) constituted of L‐alanine, sebacic acid, and 1,12‐dodecanediol units (abbreviated as PADAS) were prepared by the compression‐molding/particulate‐leaching method. The influence of the type, size, and percentage of salt on the scaffold porosity and morphology was evaluated. The thermal behavior and crystallinity were also studied for samples obtained under different processing conditions. PADAS scaffolds were not cytotoxic because they showed good cell viability and supported cell growth at a similar ratio to that observed for the biocompatible materials used as a reference. The use of PADAS scaffolds as a drug‐delivery system was also evaluated by the employment of ibuprofen, a drug with well known anti‐inflammatory effects. Different drug‐loading methods were considered, and their influence on the release in a Sörensen's medium was evaluated as well as the influence of the scaffold morphology. A sustained release of ibuprofen could be attained without the production of a negative effect on the cell viability. The release kinetics of samples loaded before melt processing was well described by the combined Higuchi/first‐order model. This allowed the estimation of the diffusion coefficients, which ranged between 3 × 10−14 and 5 × 10−13 m2/s. Samples loaded by immersion in ibuprofen solutions showed a rapid release that could be delayed by the addition of polycaprolactone to the immersion medium (i.e., the release rate decreased from 0.027 to 0.015 h−1). © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011.

Suppressed Native Hydrolytic Activity of a Lipase to Reveal Promiscuous Michael Addition Activity in Water

AbstractSuppression of the native hydrolytic activity of Pseudozyma antarctica lipase B (PalB) (formerly Candida antarctica lipase B) in water is demonstrated. By replacing the catalytic Ser 105 residue with an alanine unit, promiscuous Michael addition activity is favored. A Michael addition reaction between methyl acrylate and acetylacetone was explored as a model system. For the PalB Ser 105 Ala mutant, the hydrolytic activity was suppressed more than 1000 times and, at the same time, the Michael addition activity was increased by a factor of 100. Docking studies and molecular dynamics simulations revealed an increased ability of the PalB Ser 105 Ala mutant to harbor the substrates close to a catalytically competent conformation.

Peptide Brush—Ordered Mesoporous Silica Nanocomposite Materials

Organic−inorganic nanocomposites are synthesized through the surface-initiated polymerization of N-carboxyanhydrides from amine-functionalized ordered mesoporous silica (OMS). Composites with high densities of grafted peptide were obtained which possess dramatically decreased porosity compared to the parent OMS material. The effect of the amine loading, pore size, pore topology, and monomer identity on the peptide brush layer obtained is investigated. Nitrogen porosimetry, thermal gravimetric analysis, elemental analysis, infrared and NMR spectroscopy, and mass spectrometry verify the formation of the peptide brushes and indicate much of the organic phase formed is in the silica mesopores. The l-Lys(Z) peptide brushes can be deprotected on the solid surface as evidenced by thermal gravimetric analysis, infrared spectroscopy, and NMR spectroscopy. l-Ala peptide brushes may also be synthesized, and polymer growth confinement is evidenced by mass spectrometry as the number of alanine units per brush appears ...