D-form Amino Acids Research Articles

Cyclization Scaffolding for Improved Vaccine Immunogen Stability: Application to Tau Protein in Alzheimer's Disease.

Effective scaffolding of immunogens is crucial for generating conformationally selective antibodies through active immunization, particularly in the treatment of protein misfolding diseases such as Alzheimer's and Parkinson's disease. Previous computational work has revealed that a disorder-prone region of the tau protein, when in a stacked form, is predicted to structurally resemble a small, soluble protofibril, having conformational properties similar to those of experimental in vitro tau oligomers. Such an oligomeric structural mimic has the potential to serve as a vaccine immunogen design for Alzheimer's disease. In this study, we developed a cyclization scaffolding method in Rosetta, in which multiple cyclic peptides are stacked into a protofibril. Cyclization results in significant stabilization of protofibril-like structures by constraining the conformational space. Applying this method to the disorder-prone region of the tau fibril, we evaluated the metastability of the cyclized tau immunogen using molecular dynamics simulations, and we identified sequences of two cyclic constructs having high metastability in the protofibril. We then assessed their thermodynamic stability by computing the free energy required to separate a distal chain from the rest of the stacked structure. Our computational results, based on molecular dynamics simulations and free energy calculations, demonstrate that two cyclized constructs, cyclo-(VKSEKLDFKDRVQSKIFyN) and cyclo-(VKSEKLDFKDRVQSKIYvG) (lowercase letters indicate d-form amino acids), possess significantly increased thermodynamic stability in the protofibril over an uncyclized linear construct VKSEKLDFKDRVQSKI. The cyclization scaffolding approach proposed here holds promise as a means to effectively design immunogens for protein misfolding diseases, particularly those involving liposome-conjugated peptide constructs.

Enantiomer-Specific Colorimetric Tandem Assays for Salivary d-Alanine Associated with Gastric Cancer.

Salivary d-alanine (d-Ala) and d-proline (d-Pro) are of concern for their potential in the noninvasive diagnosis of gastric cancer (GC). Most reports have succeeded in determining the total concentration of d-Ala and d-Pro. However, for personalized diagnosis and better elucidation of the underlying specific correlation of d-Ala (or d-Pro) with GC, it is desirable to determine the specific concentration of d-Ala or d-Pro. Herein, we propose an enantiomer-specific tandem assay of d-Ala based on the colorimetric reaction between 2,4-dinitrophenylhydrazine and pyruvic acid generated from the deamination of d-Ala catalyzed by d-amino acid oxidase, which is easily distinguished from l-form amino acids, d-Pro, and many other species. A linear concentration range is established from 20 to 400 μmol/L with a limit of detection of 1.01 μmol/L. Real saliva sample tests reveal that the levels of d-Ala in GC cases are remarkably higher than those in healthy individuals, which offers a simple and low-cost strategy for GC diagnosis. Simultaneously, the total concentrations of d-Ala and d-Pro in saliva are determined. Hence, the concentration of d-Pro and the proportion of d-Ala could be calculated, which further provides more molecule- and individual-specific information. This research may offer a convenient method for noninvasive diagnosis of GC and pave a new route to explore the potentials of rare d-form amino acids in disease diagnosis and treatment.

Impact of wild fish faeces to deep sea dissolved organic carbon as revealed by laboratory incubations

Wild fish faeces play an important role in mediating marine organic carbon cycle, but the faeces released dissolved organic matter (DOM) and its potentials in contribution to refractory DOM is unclear. The mixed wild fish (including mesopelagic fish) faecal samples from the northern South China Sea were collected and incubated up to 75 days using the waters collected at 800 m depth of the same site, to reveal the stability of the faeces released DOM. Our result showed that the faeces had a good DOM-release potential and dissolved organic carbon (DOC) was up to 150 μM in the first day and reached its maximum (172 μM) on day 3, which was in comparison with the control group (DOC <70 μM). In our incubations, the faecal samples contributed net DOC of 110 μM, in which 42 μM persisted till the end of the 75 days dark incubation. This meant the faeces-released DOC had a refractory fraction of 38% over the incubation. Total hydrolysable amino acids concentration significantly dropped, and amino acids molecular indicator (DI, AA C yield and D/L ratio) changes over the incubation indicated the fast transformation of DOM from labile to degraded stage. Network analysis was applied to the amino acids and our result showed that D-form amino acids were almost positively coupled with L-form amino acids. Glycine (in mol%) showed consistent accumulation along with DOM degradation. Also, glycine was at the key node position of the amino acids network, showing negative relation with all other amino acids enantiomers in both faeces group and control group. Our results proved that glycine had the potential in tracing the refractory fraction over the DOM degradation.

In vitro and in vivo suppression of SARS-CoV-2 replication by a modified, short, cell-penetrating peptide targeting the C-terminal domain of the viral spike protein.

Peptides are promising therapeutic agents for COVID-19 because of their specificity, easy synthesis, and ability to be fine-tuned. We previously demonstrated that a cell-permeable peptide corresponding to the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) Spike C-terminal domain (CD)inhibits the interaction between viral spike and nucleocapsid proteins that results in SARS-CoV-2 replication in vitro. Here, we used docking studies to design R-t-Spike CD(D), a more potent short cell-penetrating peptide composed of all D-form amino acids and evaluated its inhibitory effect against the replication of SARS-CoV-2 S clade and other variants. R-t-Spike CD(D) was internalized into Vero cells and Calu-3 cells and suppressed the replication ofSARS-CoV-2 S clade, delta variant, and omicron variant with higher potency than the original peptide. In hemizygous K18-hACE2 mice, intratracheal administration of R-t-Spike CD(D) effectively delivered the peptide to the trachea and lungs, whereas intranasal administration delivered the peptide mostly to the upper respiratory system and stomach, and a small amount to the lungs. Administration by either route reduced viral loads in mouse lungs and turbinates. Furthermore, intranasally administered R-t-Spike CD(D) mitigated pathological change in the lungs and increased the survival of mice after infection with the SARS-CoV-2 S clade or delta variant. Our data suggest that R-t-Spike CD(D) has potential as a therapeutic agent against SARS-CoV-2 infection.

Peptide Self-Assemblies from Unusual α-Sheet Conformations Based on Alternation of d/l Amino Acids.

Peptide self-assembly is a hierarchical process during which secondary structures formed in the initial stages play a critical role in determining the subsequent assembling processes and final structural ordering. Unusual secondary structures hold promise as a source to develop novel supramolecular architectures with unique properties. In this work, we report the design of a new peptide self-assembly strategy based on unusual α-sheet secondary structures. In light of the strong propensity of leucine toward forming helical conformations and its high hydrophobicity, we design two short amphiphilic peptides Ac-LDLLDLK-NH2 and Ac-DLLDLLDK-NH2 with alternating l- and d-form amino acids. Microscopic imaging, neutron scattering, and spectroscopic measurements indicate that the two heterochiral peptides form highly ordered wide nanotubes and helical ribbons with monolayer thickness, in sharp contrast to twisted nanofibrils formed by the homochiral peptide Ac-LLLLK-NH2. Molecular dynamics simulations from monomers to trimers reveal that the two heteropeptides fold into α-sheets instead of β-sheets, which readily pack into tubular architectures in oligomer simulations. Simulated circular dichroism spectra based on α-sheet oligomers validate the proposed α-sheet secondary structures. These results form an important basis for the rational design of higher-order peptide assemblies with novel properties based on unusual α-sheet secondary structures.

Broad-Spectrum Antimicrobial Activity and Improved Stability of a D-Amino Acid Enantiomer of DMPC-10A, the Designed Derivative of Dermaseptin Truncates.

DMPC-10A (ALWKKLLKK-Cha-NH2) is a 10-mer peptide derivative from the N-terminal domain of Dermaseptin-PC which has shown broad-spectrum antimicrobial activity as well as a considerable hemolytic effect. In order to reduce hemolytic activity and improve stability to endogenous enzymes, a D-amino acid enantiomer (DMPC-10B) was designed by substituting all L-Lys and L-Leu with their respective D-form amino acid residues, while the Ala1 and Trp3 remained unchanged. The D-amino acid enantiomer exhibited similar antimicrobial potency to the parent peptide but exerted lower cytotoxicity and hemolytic activity. Meanwhile, DMPC-10B exhibited remarkable resistance to hydrolysis by trypsin and chymotrypsin. In addition to these advantages, DMPC-10B exhibited an outstanding antibacterial effect against Methicillin-resistant Staphylococcus aureus (MRSA) and Klebsiella pneumoniae using the Galleria mellonella larva model and displayed synergistic activities with gentamicin against carbapenem-resistant K. pneumoniae strains. This indicates that DMPC-10B would be a promising alternative for treating antibiotic-resistant pathogens.

Delivery of MSCs with a Hybrid β-Sheet Peptide Hydrogel Consisting IGF-1C Domain and D-Form Peptide for Acute Kidney Injury Therapy.

PurposeBy providing a stem cell microenvironment with particular bioactive constituents in vivo, synthetic biomaterials have been progressively successful in stem cell-based tissue regeneration by enhancing the engraftment and survival of transplanted cells. Designs with bioactive motifs to influence cell behavior and with D-form amino acids to modulate scaffold stability may be critical for the development and optimization of self-assembling biomimetic hydrogel scaffolds for stem cell therapy.Materials and MethodsIn this study, we linked naphthalene (Nap) covalently to a short D-form peptide (Nap-DFDFG) and the C domain of insulin-like growth factor-1 (IGF-1C) as a functional hydrogel-based scaffolds, and we hypothesized that this hydrogel could enhance the therapeutic efficiency of human placenta-derived mesenchymal stem cells (hP-MSCs) in a murine acute kidney injury (AKI) model.ResultsThe self-assembling peptide was constrained into a classical β-sheet structure and showed hydrogel properties. Our results revealed that this hydrogel exhibited increased affinity for IGF-1 receptor. Furthermore, cotransplantation of the β-IGF-1C hydrogel and hP-MSCs contributed to endogenous regeneration post-injury and boosted angiogenesis in a murine AKI model, leading to recovery of renal function.ConclusionThis hydrogel could provide a favorable niche for hP-MSCs and thereby rescue renal function in an AKI model by promoting cell survival and angiogenesis. In conclusion, by covalently linking the desired functional groups to D-form peptides to create functional hydrogels, self-assembling β-sheet peptide hydrogels may serve as a promising platform for tissue-engineering and stem cell therapy.

SUN-027 Delivery of MSCs with a Hybrid β-sheet Peptide Hydrogel Consisting IGF-1C Domain and D-form Peptide for Acute Kidney Injury Therapy

By providing an appropriate microenvironment, synthetic biomaterials have been progressively successful in stem cell-based tissue regeneration by enhancing the engraftment and survival of transplanted cells. The designs with bioactive motifs to influence cell behavior and with D-form amino acid to modulate scaffold stability will be critical for the development and optimization of self-assembling biomimetic hydrogel scaffolds for stem cell therapy. In this study, we linked naphthalene (Nap) covalently conjugated a short D-form peptide (Nap-DFDFG) to C domain of insulin-like growth factor-1 (IGF-1C) as a functional peptide-based scaffold. Our purpose is to provide a functional self-assembling peptide hydrogel with encapsulated human placenta derived mesenchymal stem cells (hP-MSCs) and thereby to enhance the therapeutic efficiency of hP-MSCs in murine acute kidney injury (AKI) model. Our results revealed that a typical β-sheet conformation was confirmed and this self-assembling peptide hydrogel exhibited higher affinity with IGF-1 receptor. Furthermore, this hydrogel could provide a favorable niche for hP-MSCs and thereby ameliorate renal function in an AKI model by promoting cell survival and angiogenesis. In conclusion, by covalently linking the desired functional group to D-form peptide to create a functional hydrogel, this self-assembling β-sheet peptide hydrogel will serve as a promising platform for future tissue-engineering and stem cell therapy. We prepared a supramolecular self-assembly peptide Nap-DFDFG-IGF-1C by standard solid phase peptide synthesis (SPPS), and profiled the characteristics by circular dichroism (CD) analysis, surface plasmon resonance (SPR), transmission electron microscope (TEM) and rheological analysis. For real-time tracing the cell fate post transplantation, we introduced dual fusion MSCs which express firefly luciferase and GFP simultaneously. Also we utilized the bioluminescence imaging (BLI) to longitudinally monitor the dynamics of cell retention and renal angiogenesis. Serial experiments have bee done to evaluate the putative therapeutic effect for AKI. We here reported a hybrid self-assembly peptide Nap-DFDFG-IGF-1C. CD analysis revealed the peptide was constrained to a typical β-sheet conformation via a heating-cooling pathway, leading to an enhanced affinity with recombinant human IGF-1 receptor (rhIGF-1R) comfirmed by SPR. A cross-linked nanofibrous structure was observed by TEM and the rheological analysis evidenced its hydrogel property. Our BLI results showed that delivery of MSCs with the IGF-1C hydrogel contribute to an enhanced cell retention and renal angiogenesis, which rsulted in repair and regeneration post AKI. In summary, we reported a cell enhancement strategy with a hybrid β-sheet peptide hydrogel which holds bioactive motif IGF-1C as functional backbone for the first time. This hydrogel favors cell retention, provides a pro-angiogenesis niche for the transplanted cells, and thereby restored the renal architecture and function post AKI. This study introduced a brand-new strategy for integration of self-assembly peptide and stem cell therapy and might shed light into the development of tissue engineering as well as regeneration medicine.

Delivery of MSCs with a Hybrid β-Sheet Peptide Hydrogel Consisting IGF-1C Domain and D-Form Peptide for Acute Kidney Injury Therapy

By providing an appropriate microenvironment, synthetic biomaterials have been progressively successful in stem cell-based tissue regeneration by enhancing the engraftment and survival of transplanted cells. The designs with bioactive motifs to influence cell behavior and with D-form amino acid to modulate scaffold stability will be critical for the development and optimization of self-assembling biomimetic hydrogel scaffolds for stem cell therapy. In this study, we linked naphthalene (Nap) covalently conjugated a short D-form peptide (Nap-DFDFG) to C domain of insulin-like growth factor-1 (IGF-1C) as a functional peptide-based scaffold. Our purpose is to provide a functional self-assembling peptide hydrogel with encapsulated human placenta derived mesenchymal stem cells (hP-MSCs) and thereby to enhance the therapeutic efficiency of hP-MSCs in murine acute kidney injury (AKI) model. Our results revealed that a typical β-sheet conformation was confirmed and this self-assembling peptide hydrogel exhibited higher affinity with IGF-1 receptor. Furthermore, this hydrogel could provide a favorable niche for hP-MSCs and thereby ameliorate renal function in an AKI model by promoting cell survival and angiogenesis. In conclusion, by covalently linking the desired functional group to D-form peptide to create a functional hydrogel, this self-assembling β-sheet peptide hydrogel will serve as a promising platform for future tissue-engineering and stem cell therapy.

Selective apoptosis-inducing activity of synthetic hydrocarbon-stapled SOS1 helix with d-amino acids in H358 cancer cells expressing KRASG12C

Lung cancer is one of the most malignant tumors with the highest morbidity and mortality. Most of them are non-small cell lung cancer (NSCLC). KRASG12C gene mutation is an important driving factor for NSCLC. However, the development of high-affinity inhibitors targeting KRASG12C mutants remains a daunting challenge. Here, we report the design and development of a series of hydrocarbon-stapled peptides containing d-amino acids to mimic the alpha helix of SOS1. D-hydrocarbon-stapled peptides maintain good alpha helix structure and bind to KRASG12C with high affinity. Subsequent anti-proliferation experiments indicated that D-hydrocarbon-stapled peptide 5 inhibited the proliferation of NSCLC H358 cells carrying KRASG12C. However, it showed no significant anti-proliferative effect on KRASG12S-positive A549 cells, suggesting that peptide 5 selectively inhibits KRASG12C-driven tumor cells. D-hydrocarbon-stapled peptide 5 could also cause the cell cycle of H358 cells to arrest in the G2/M phase and induce apoptosis. No significant cell arrest and apoptosis were observed in A549 cells treated by peptide 5. In summary, the introduction of d-amino acids could improve the affinity and cell selectivity of hydrocarbon peptides. We hope that peptides containing D-form amino acids can provide strategies for further optimization of the KRASG12C/SOS1 inhibitor.

Rational engineering of amide synthetase enables bioconversion to diverse xiamenmycin derivatives.

XimA is a unique amide synthetase that belongs to the ANL superfamily of adenylating enzymes, but with a special structural fold. In order to improve the enzyme promiscuity, we engineered XimA by site-directed mutagenesis at a specific position based on our theoretical model of XimA. Thus, we were able to produce diverse benzopyran derivatives with up to 15 different l-form and d-form amino acid substitutions, catalyzed by several XimA variants. Molecular docking and molecular dynamics simulations conducted for various XimA systems provide further structural insights into the substitution effects of the phenylalanine-201 as an active site residue on protein dynamics and enzyme catalysis.

Combining enzymatic hydrolysis with magnetic nanoparticles for resolution of chiral substances

At present, the separation of racemates is still a great challenge, since they are enantiomers, having similar chemical structures and physical properties. Here, we proposed a new strategy for efficiently separating chiral compounds, i.e., stereoselectively hydrolyzing the individual enantiomer of racemates via enzyme, followed by a simple magnetic separation using Fe3O4 nanoparticles, may implement a complete resolution of racemates. In this work, d-aminoacylase was selected as enzyme, and phenylalanine and methionine as chiral models. The use of d-aminoacylase can hydrolyze d-enantiomer, while l-enantiomer cannot. Magnetic nanoparticles of Fe3O4@SiO2-NH2-COOH composites were designed and synthesized for the amidation reaction with the hydrolyzed d-amino acids from the mixture. With the help of a magnetic field, d-form amino acids can be readily separated at a large scale and the resultant resolving performances indicated that this method exhibited a higher selectivity, compared with common chiral separation techniques. The enantiomeric excess (e.e.) values are 75% and 98% for racemic N-acyl-phenylalanine and methionine, respectively, which solely rely on the hydrolysis capability of used enzymes. This result suggests that our method is feasible, simple, easy to scale-up and high-efficient for the chiral resolution. These findings demonstrate that this strategy could provide new opportunities for improving enantioseparation performances for a host of enantio- and bio-substance with both high-yield and high-selectivity.

Ligand Size and Conformation Affect the Behavior of Nanoparticles Coated with in Vitro and in Vivo Protein Corona.

Protein corona is immediately established on the surface of nanoparticles upon their introduction into biological milieu. Several studies have shown that the targeting efficiency of ligand-modified nanoparticles is attenuated or abolished owing to the protein adsorption. Here, transferrin receptor-targeting ligands, including LT7 (CHAIYPRH), DT7 (hrpyiahc, all d-form amino acids), and transferrin, were used to identify the influence of the ligand size and conformation on protein corona formation. The results showed that the targeting capacity of ligand-modified nanoparticles was lost after incubation with plasma in vitro, whereas it was partially retained after in vivo corona formation. Results from sodium dodecyl sulfate polyacrylamide gel electrophoresis and liquid chromatography-mass spectrometry revealed the difference in the composition of in vitro and in vivo corona, wherein the ligand size and conformation played a critical role. Differences were observed in cellular internalization and exocytosis profiles on the basis of the ligand and corona source.

Distinguishing d- and l-aspartic and isoaspartic acids in amyloid β peptides with ultrahigh resolution ion mobility spectrometry.

While α-linked amino acids in the l-form are exclusively utilized in mammalian protein building, β-linked and d-form amino acids also have important biological roles. Unfortunately, the structural elucidation and separation of these different amino acid types in peptides has been analytically challenging to date due to the numerous isomers present, limiting our knowledge about their existence and biological roles. Here, we utilized an ultrahigh resolution ion mobility spectrometry platform coupled with mass spectrometry (IMS-MS) to separate amyloid β (Aβ) peptides containing l-aspartic acid, d-aspartic acid, l-isoaspartic acid, and d-isoaspartic acid residues which span α- and β-linked amino acids in both d- and l-forms. The results illustrate how IMS-MS could be used to better understand age-related diseases or protein folding disorders resulting from amino acid modifications.

Decreased levels of d-aspartate and NMDA in the prefrontal cortex and striatum of patients with schizophrenia

The potential implication of a decrease in the function of N-methyl-d-aspartate receptors (NMDARs) in the pathophysiology of schizophrenia has long been hypothesised. Accordingly, compounds that inhibit the glycine-1 transporter or target the glycine-binding site of NMDARs, including the co-agonists d-serine and glycine, have shown promise in treating the symptoms of schizophrenia. Clinical interest for d-serine has also been supported by evidence for its abnormal metabolism in schizophrenic patients. Together with d-serine, another d-form amino acid, d-aspartate, exists in the brain of mammals. Synthesised by the enzyme aspartate racemase, d-aspartate is highly concentrated in the prenatal brain; after birth, its levels sharply decrease due to the catabolising activity of the enzyme d-aspartate oxidase. d-aspartate is able to stimulate NMDAR-dependent neurotransmission through direct action at the glutamate-binding site of NMDARs, thus functioning as an endogenous agonist for this subclass of glutamate receptors. In this study, we evaluated for the first time the content of d-aspartate and of its derivative, NMDA, in the post-mortem prefrontal cortex and striatum of schizophrenic patients. Moreover, in the same brain samples, we analysed the expression levels of the subunits that form NMDARs, which are the in vivo targets of d-aspartate and NMDA. Interestingly, we found that d-aspartate and NMDA are consistently decreased in schizophrenia brains compared to control brains. In the prefrontal cortex, this decrease is correlated with a marked downregulation of NMDAR subunits. Overall, these results agree with the innovative therapeutic research in schizophrenia that is aimed at targeting glutamatergic transmission viad-amino acids.

Synthetic Coprisin Analog Peptide, D-CopA3 has Antimicrobial Activity and Pro-Apoptotic Effects in Human Leukemia Cells

Recently, we reported that the synthetic Coprisin analog peptide 9-mer dimer CopA3 (consisted of all-L amino acid sequence) was designed based on a defensin-like peptide, Coprisin isolated from Copris tripartitus. The 9-mer dimer CopA3 (L-CopA3) had antibacterial activity and induced apoptosis in human leukemia cells via a caspase-independent pathway. In this study, all of amino acid sequences of L-CopA3 were modified to all D-form amino acids (D-CopA3) to develop a more effective antimicrobial peptide. We investigated whether D-CopA3 had antimicrobial activities against pathogenic microorganisms and proapoptotic effects in human leukemia cells (U937, Jurkat, and AML-2). The synthetic peptide D-CopA3 had antimicrobial activities against various pathogenic bacteria and yeast fungus with MIC values in the 4~64 microM range. Moreover, D-CopA3 caused cell growth inhibition, and increased the chromosomal DNA fragmentation and the expression of inflammatory cytokines, TNF-alpha and IL1-beta, transcripts in human leukemia cells. The all-D amino acid peptide D-CopA3 proved as effective as the L-CopA3 reported previously. These results provide the basis for developing D-CopA3 as a new antibiotic peptide.

Definition of peptide inhibitors from a synthetic peptide library by targeting gelatinase B/matrix metalloproteinase-9 (MMP-9) and TNF-α converting enzyme (TACE/ADAM-17)

Gelatinase B/matrix metalloproteinase-9 (MMP-9) is a regulatory and effector metalloproteinase in inflammation. TNF-α is an important proinflammatory cytokine and is released by the action of a Zn2+-containing converting enzyme (TACE/ADAM-17). Both metallo-enzymes play important roles during the development of shock syndromes. Combinatorial chemical synthesis and subsequent library deconvolution were previously used to define a peptide inhibitor (Regasepin1) acting, almost to the same degree, on neutrophil collagenase/MMP-8 and MMP-9 in vitro, and protecting mice against lethal endotoxinemia in vivo. We have now extended this approach by incorporating D-form amino acids and residues preferred by TACE. A new peptide library was designed and synthesized, and by deconvolution new peptide inhibitors were defined. These included a TACE-specific inhibitor, an MMP-9- specific inhibitor, and inhibitors for both enzymes.

Stereoconversion of Amino Acids and Peptides in Uryl‐Pendant Binol Schiff Bases

(S)-2-Hydroxy-2'-(3-phenyluryl-benzyl)-1,1'-binaphthyl-3-carboxaldehyde (1) forms Schiff bases with a wide range of nonderivatized amino acids, including unnatural ones. Multiple hydrogen bonds, including resonance-assisted ones, fix the whole orientation of the imine and provoke structural rigidity around the imine C==N bond. Due to the structural difference and the increase in acidity of the alpha proton of the amino acid, the imine formed with an L-amino acid (1-l-aa) is converted into the imine of the D-amino acid (1-D-aa), with a D/L ratio of more than 10 for most amino acids at equilibrium. N-terminal amino acids in dipeptides are also predominantly epimerized to the D form upon imine formation with 1. Density functional theory calculations show that 1-D-Ala is more stable than 1-L-Ala by 1.64 kcal mol(-1), a value that is in qualitative agreement with the experimental result. Deuterium exchange of the alpha proton of alanine in the imine form was studied by (1)H NMR spectroscopy and the results support a stepwise mechanism in the L-into-D conversion rather than a concerted one; that is, deprotonation and protonation take place in a sequential manner. The deprotonation rate of L-Ala is approximately 16 times faster than that of D-Ala. The protonation step, however, appears to favor L-amino acid production, which prevents a much higher predominance of the D form in the imine. Receptor 1 and the predominantly D-form amino acid can be recovered from the imine by simple extraction under acidic conditions. Hence, 1 is a useful auxiliary to produce D-amino acids of industrial interest by the conversion of naturally occurring L-amino acids or relatively easily obtainable racemic amino acids.

WRC-213, an l-methionine-conjugated mitoxantrone derivative, displays anticancer activity with reduced cardiotoxicity and drug resistance: Identification of topoisomerase II inhibition and apoptotic machinery in prostate cancers

Anthracyclines and anthracenediones are well-known cancer chemotherapeutic agents but their uses are limited with cardiotoxicity and drug resistance. Several l- and d-form amino acids were introduced into the anthraquinone skeleton and numerous derivatives were synthesized for the evaluation of anticancer activity. The screening tests showed that WRC-213, an l-methionine conjugation, was the most effective derivative to inhibit proliferative effect of human androgen-independent prostate cancer PC-3 cells (IC50=50nM). In an extension evaluation, WRC-213 displayed a potent anti-proliferative activity in various cancer cell lines, including non-small cell lung cancer A549, androgen-independent prostate cancer DU145, colorectal cancer HT-29, breast cancer MCF-7 and hepatocellular carcinoma Hep3B and HepG2. It induced cell-cycle arrest at S and G2, but not mitotic phase, in PC-3 cells. The comet assay revealed that induction of DNA damage and inhibition of topoisomerase II were the primary insults. After the checkpoint arrest of the cell-cycle, WRC-213 induced the mitochondria-mediated intrinsic apoptotic pathway, including Mcl-1 cleavage, Bcl-2 down-regulation and activation of caspase-9/caspase-3 cascades. Survivin degradation and caspase-2 activation also contributed to WRC-213-induced apoptosis. Moreover, the assessment of cytotoxicity in H9c2 cardiomyocytes and drug resistance in NCI/ADR-RES cells demonstrated that WRC-213 showed much lower cardiotoxicity and P-glycoprotein-related resistance than those of mitoxantrone, etoposide and doxorubicin. In conclusion, it is suggested that WRC-213 is a potential topoisomerase II inhibitor with reduced cardiotoxicity and drug resistance. It inhibits topoisomerase II activity and induces chromosomal DNA strand breaks, leading to S and G2 arrest of the cell-cycle and activation of mitochondria-mediated apoptotic pathways.

Combination of Amino Acids Reduces Pigmentation in B16F0 Melanoma Cells

Amino acids, the building blocks of proteins, play significant roles in numerous physiological events in mammals. As the effects of amino acids on melanogenesis have yet to be demonstrated, the present study was conducted to identify whether amino acids, in particular alanine, glycine, isoleucine and leucine, influence melanogenesis in B16F0 melanoma cells. Glycine and L-isoleucine, but not D-isoleucine, reduced melanogenesis in a concentration-dependent manner without any morphological changes in B16F0 melanoma cells. L-Alanine and L-leucine, but not D-alanine and D-leucine, also reduced melanogenesis without any morphological changes in B16F0 melanoma cells. However these amino acids did not show a concentration-dependency. Combination of L-alanine and the other amino acids, particularly 4 amino acids combination, had an additive effect on the inhibition of melanogenesis compared with single treatment of L-alanine. None of the amino acids affected the activity of tyrosinase, a key enzyme in melanogenesis. These results suggest that L-alanine, glycine, L-isoleucine and L-leucine, but not the D-form amino acids, have a hypopigmenting effect in B16F0 melanoma cells, and that these effects are not due to the inhibition of tyrosinase activity. Combination of these 4 amino acids had the additive effect on hypopigmentation that was as similar as that of kojic acid.