Recent progress on polySarcosine as an alternative to PEGylation: Synthesis and biomedical applications.

Biotherapeutic PEGylation to prolong action of medications has gained popularity over the last decades. Various hydrophilic natural polymers have been developed to tackle the drawbacks of PEGylation, such as its accelerated blood clearance and non-biodegradability. Polypeptoides, such as polysarcosine (pSar), have been explored as hydrophilic substitutes for PEG. pSar has PEG-like physicochemical characteristics such as water solubility and no reported cytotoxicity and immunogenicity. This review discusses pSar derivatives, synthesis, characterization approaches, biomedical applications, in addition to the challenges and future perspectives of pSar based biomaterials as an alternative to PEG.

Reversible transformation of peptide assembly between densified-polysarcosine-driven kinetically and helix-orientation-driven thermodynamically stable morphologies.

Stimuli-responsive transformable biomaterials development can be manipulated practically by fine-tuning the built-in molecular design of their structural segments. Here, we demonstrate a peptide assembly by the bola-type amphiphilic polypeptide, glycolic acid-polysarcosine (PSar)13-b-(L-Leu-Aib)6-b-PSar13-glycolic acid (S13L12S13), which shows morphological transformations between hydrophilic chain-driven and hydrophobic unit-driven morphologies. The hydrophobic α-helical unit (L-Leu-Aib)6 precisely controls packing in the hydrophobic layer of the assembly and induces tubule formation. The densified, hydrophilic PSar chain on the assembly surface becomes slightly more hydrophobic as the temperature increases above 70 °C, starting to disturb the helix-helix interaction-driven formation of tubules. As a result, the S13L12S13 peptide assembly undergoes a reversible vesicle-nanotube transformation following a time course at room temperature and a heat treatment above 80 °C. Using membrane fluidity analysis with DPH and TMA-DPH and evaluating the environment surrounding the PSar side chain with NMR, we clarify that the vesicle was in a kinetically stable state driven by the dehydrated PSar chain, while the nanotube was in a thermodynamically stable state.

Glycine transporters in schizophrenia. A new hope or informational noise? / Transportery glicynowe w schizofrenii. Nowa nadzieja czy szum informacyjny?

Currently, we observe a huge number of publications describing the role of glycine transporter (GlyT1) inhibitors in schizophrenia treatment. The concept of application for these drugs derives from the glutamatergic theory of schizophrenia. This theory explains psychotic disturbances as the consequence of NMDA receptor functioning defect. The role of the mentioned receptor depends mostly on the presence of cofactors. One such cofactor is the simplest aminoacid, glycine. This amino acid affects the glycine-binding site, located on the NR1 subunit of NMDAR and enables activation of the receptor. Substances enhancing the access of glycine to the receptor could hypothetically improve neuroplasticity. Higher efficacy of these neuroplastic processes may protect from cognitive deterioration and negative symptoms in the course of schizophrenia. In this article we present a systematic review of current literature on the topic of GlyT1 inhibitors in schizophrenia treatment (the state of literature as of November 2019). Firstly, we described the preclinical reasons for glycine enhancement use. Next, we used CINAHL, EMBASE, EMCARE, Medline, PsycINFO, PubMed and Google Scholar databases to extract and analyze evidence from clinical trials. GlyT1 inhibitors seem to have a potential in searching for novel substances in the treatment of negative symptoms, but their capacity to reduce cognitive deficits is not evidenced. So far, the clinical efficacy of several substances was proven, including N-methylglycine (sarcosine), bitopertin and derivatives obtained with chemical synthesis. Some of these substances demonstrate a beneficial clinical effect, but the number of published reports in this area is disproportionate to the value of evidence.

Biodegradable Polysarcosine with Inserted Alanine Residues: Synthesis and Enzymolysis.

Polysarcosine (PSar), a water-soluble polypeptoid, is gifted with biodegradability via the random ring-opening copolymerization of sarcosine- and alanine-N-thiocarboxyanhydrides catalyzed by acetic acid in controlled manners. Kinetic investigation reveals the copolymerization behavior of the two monomers. The random copolymers, named PaS, with high molecular weights between 5.3 and 43.6 kg/mol and tunable Ala molar fractions varying from 6 to 43% can be degraded by porcine pancreatic elastase within 50 days under mild conditions (pH = 8.0 at 37 °C). Both the biodegradation rate and water solubility of PaS depend on the content of Ala residues. PaS with Ala fractions below 43% are soluble in water, while the one with 43% Ala self-assembles in water into nanoparticles. Moreover, PaS are noncytotoxic at the concentration of 5 mg/mL. The biodegradability and biocompatibility endow the Ala-containing PSar with the potential to replace poly(ethylene glycol) as a protective shield in drug-delivery.

Controlling the diversity of ion-induced fragmentation pathways by N-methylation of amino acids.

We present a combined experimental and theoretical study of the fragmentation of singly and doubly N-methylated glycine (sarcosine and N,N-dimethyl glycine, respectively) induced by low-energy (keV) O6+ ions. Multicoincidence mass spectrometry techniques and quantum chemistry simulations (ab initio molecular dynamics and density functional theory) allow us to characterise different fragmentation pathways as well as the associated mechanisms. We focus on the fragmentation of doubly ionised species, for which coincidence measurements provide unambiguous information on the origin of the various charged fragments. We have found that single N-methylation leads to a larger variety of fragmentation channels than in no methylation of glycine, while double N-methylation effectively closes many of these fragmentation channels, including some of those appearing in pristine glycine. Importantly, the closure of fragmentation channels in the latter case does not imply a protective effect by the methyl group.

An optimized SDS-PAGE protocol with a new blotting system for the initial testing procedure of ESAs in doping control.

Recombinant erythropoietins (rEPOs) are still among the substances endurance athletes use for doping. Detection methods are based on an electrophoretic separation of the proteins followed by a western blot and immunodetection with specific anti-EPO antibodies. In addition to IEF-PAGE, the SDS-PAGE method has been used to differentiate endogenous EPO from rEPOs by their molecular weight (MW). However, to adapt to new generations of rEPOs exhibiting higher MW, which were not well detected after SDS-PAGE, sodium lauroyl sarcosinate (SAR) is now used instead of sodium dodecyl sulfate (SDS) for the initial EPO testing procedure on doping control samples. The SAR-PAGE method is nevertheless expensive as it requires frequent buffer preparations using highly purified sarkosyl powder. In addition, this reagent needs to be handled with care due to acute toxicity by inhalation. The aim of this work was to improve the SDS-PAGE method by increasing its sensitivity and transfer of high-MW rEPOs. First, using a biotinylated primary anti-EPO antibody and avoiding the use of a secondary antibody increased the general sensitivity of both SDS-PAGE and SAR-PAGE to all rEPOs about four-fold. Then, by changing the buffer system during the protein transfer, with a CAPS buffer and a discontinuous buffer transfer system, high-MW rEPOs, EPO-Fc and CERA were transferred with higher efficiency and detected with high sensitivity. This optimized SDS-PAGE protocol could be adopted by anti-doping laboratories as an alternative to SAR-PAGE.

Tubular Assembly Formation Induced by Leucine Alignment along the Hydrophobic Helix of Amphiphilic Polypeptides.

The introduction of α-helical structure with a specific helix-helix interaction into an amphipathic molecule enables the determination of the molecular packing in the assembly and the morphological control of peptide assemblies. We previously reported that the amphiphilic polypeptide SL12 with a polysarcosine (PSar) hydrophilic chain and hydrophobic α-helix (l-Leu-Aib)6 involving the LxxxLxxxL sequence, which induces homo-dimerization due to the concave-convex interaction, formed a nanotube with a uniform 80 nm diameter. In this study, we investigated the importance of the LxxxLxxxL sequence for tube formation by comparing amphiphilic polypeptide SL4A4L4 with hydrophobic α-helix (l-Leu-Aib)2-(l-Ala-Aib)2-(l-Leu-Aib)2 and SL12. SL4A4L4 formed spherical vesicles and micelles. The effect of the LxxxLxxxL sequence elongation on tube formation was demonstrated by studying assemblies of PSar-b-(l-Ala-Aib)-(l-Leu-Aib)6-(l-Ala-Aib) (SA2L12A2) and PSar-b-(l-Leu-Aib)8 (SL16). SA2L12A2 formed nanotubes with a uniform 123 nm diameter, while SL16 assembled into vesicles. These results showed that LxxxLxxxL is a necessary and sufficient sequence for the self-assembly of nanotubes. Furthermore, we fabricated a double-layer nanotube by combining two kinds of nanotubes with 80 and 120 nm diameters-SL12 and SA2L12A2. When SA2L12A2 self-assembled in SL12 nanotube dispersion, SA2L12A2 initially formed a rolled sheet, the sheet then wrapped the SL12 nanotube, and a double-layer nanotube was obtained.

Therapeutic melanoma inhibition by local micelle-mediated cyclic nucleotide repression.

The acidic tumor microenvironment in melanoma drives immune evasion by up-regulating cyclic adenosine monophosphate (cAMP) in tumor-infiltrating monocytes. Here we show that the release of non-toxic concentrations of an adenylate cyclase (AC) inhibitor from poly(sarcosine)-block-poly(L-glutamic acid γ-benzyl ester) (polypept(o)id) copolymer micelles restores antitumor immunity. In combination with selective, non-therapeutic regulatory T cell depletion, AC inhibitor micelles achieve a complete remission of established B16-F10-OVA tumors. Single-cell sequencing of melanoma-infiltrating immune cells shows that AC inhibitor micelles reduce the number of anti-inflammatory myeloid cells and checkpoint receptor expression on T cells. AC inhibitor micelles thus represent an immunotherapeutic measure to counteract melanoma immune escape.

Amended Safety Assessment of Fatty Acyl Sarcosines and Sarcosinate Salts as Used in Cosmetics.

The Expert Panel for Cosmetic Ingredient Safety (Panel) assessed the safety of 5 acyl sarcosines and 9 sarcosinate salts as used in cosmetics; all of these ingredients are reported to function in cosmetics as hair conditioning agents and most also can function as surfactants-cleansing agents. The ingredients reviewed in this assessment are composed of an amide comprising a fatty acyl residue and sarcosine and are either free acids or simple salts thereof. The Panel relied on relevant new data, including concentration of use, and considered data from the previous Panel report, such as the reaction of sarcosine with oxidizing materials possibly resulting in nitrosation and the formation of N-nitrososarcosine. The Panel concluded that these ingredients are safe as used in cosmetics when formulated to be non-irritating, but these ingredients should not be used in cosmetic products in which N-nitroso compounds may be formed.

Trimethylamine N-oxide alters structure-function integrity of ß-casein: Structural disorder co-regulates the aggregation propensity and chaperone activity.

Intrinsically disordered proteins (IDPs), involved in the regulation and function of various cellular processes like transcription, translation, cell cycle etc., exist as ensembles of rapidly interconverting structures with functional plasticity. Among numerous cellular regulatory mechanisms involved in structural and functional regulation of IDPs, osmolytes are emerging as promising regulatory agents due to their ability to affect the structure-function integrity of IDPs. The present study investigated the effect of methylamine osmolytes on ß-casein, an IDP essential for maintaining the overall stability of casein complex in milk. It was observed that trimethylamine N-oxide induces a compact structural state in ß-casein with slightly decreased chaperone activity and insignificant aggregation propensity. However, the other two osmolytes from this group, i.e., sarcosine and betaine, had no significant effect on the overall structure and chaperone activity of the IDP. The present study hints towards the possible evolutionary selection of higher structural disorder in ß-casein, compared to α-casein, for stability of the casein complex and prevention of amyloidosis in the mammary gland.

Influence of Riboflavin Targeting on Tumor Accumulation and Internalization of Peptostar Based Drug Delivery Systems.

Riboflavin carrier protein (RCP) and riboflavin transporters (RFVTs) have been reported to be highly overexpressed in various cancer cells. Hence, targeting RCP and RFVTs using riboflavin may enhance tumor accumulation and internalization of drug delivery systems. To test this hypothesis, butyl-based 3-arm peptostar polymers were synthesized consisting of a lysine core (10 units per arm) and a sarcosine shell (100 units per arm). The end groups of the arms and the core were successfully modified with riboflavin and the Cy5.5 fluorescent dye, respectively. While in phosphate buffered saline the functionalized peptostars showed a bimodal behavior and formed supramolecular structures over time, they were stable in the serum maintaining their hydrodynamic diameter of 12 nm. Moreover, the polymers were biocompatible and the uptake of riboflavin targeted peptostars in A431 and PC3 cells was higher than in nontargeted controls and could be blocked competitively. In vivo, the polymers showed a moderate passive tumor accumulation, which was not significantly different between targeted and nontargeted peptostars. Nonetheless, at the histological level, internalization into tumor cells was strongly enhanced for the riboflavin-targeted peptostars. Based on these results, we conclude that passive accumulation is dominating the accumulation of peptostars, while tumor cell internalization is strongly promoted by riboflavin targeting.

Sarkosyl Preparation of Antigens from Bacterial Inclusion Bodies.

In many cases, solubility and proper folding of fusion proteins expressed in bacteria pose a major challenge in protein purification and crystallization. This is especially true when the fusion proteins are of eukaryotic origin. They form aggregates or become packaged into inclusion bodies, which makes protein purification extremely difficult. Sarkosyl is widely used to extract misfolded proteins from inclusion bodies in soluble form.

Iron doped graphitic carbon nitride with peroxidase like activity for colorimetric detection of sarcosine and hydrogen peroxide.

The successful synthesis is reported of Mn, Fe, Co, Ni, Cu-doped g-C3N4 nanoflakes via a simple one-step pyrolysis method, respectively. Among them, the Fe-doped g-C3N4 nanoflakes exhibited the highest peroxidase-like activity, which can be used for colorimetric detection of hydrogen peroxide (H2O2) and sarcosine (SA), within the detection ranges of 2-100 µM and 10-500 µM and detection limits of 1.8 µM and 8.6 µM, respectively. The catalytic mechanism of the Fe-doped g-C3N4 nanoflake was also explored and verified the generation of hydroxyl radical (•OH) through fluorescence method. It is believed that the Fe-doped g-C3N4 nanoflakes as enzyme mimics will greatly promote the practical applications in a variety of fields in the future including biomedical science, environmental governance, antibacterial agent, and bioimaging due to their extraordinary catalytic performance and stability. Graphical abstract.

Sarcosine Prostate Cancer Biomarker Detection by Controlling Oxygen in NiOx Membrane on Vertical Silicon Nanowires in Electrolyte-Insulator-Nanowire Structure.

Sarcosine prostate cancer biomarker with the low concentration of 1 pM has been detected by controlling oxygen from 1 to 15 sccm in a NiOx membrane on chemically etched vertical Si nanowires (SiNWs) in an electrolyte-insulator-nanowire (EIN) structure. The vertical Si nanowires with approximately 17 µm length and polycrystalline NiOx membrane are observed by both field-emission scanning electron microscope (FE-SEM) and high-resolution transmission electron microscope (HRTEM) images, respectively. The optimized NiOx membrane with oxygen content of 4 sccm on planar SiOx/Si substrate shows good pH sensitivity of approximately 50 mV/pH, low hysteresis of 3.4 mV, and low drift rate of 2.4 mV/h as compared to other oxygen content membranes of 1, 10, and 15 sccm. Further, uric acid with the concentration of 0.1 µM is detected directly by using the optimized NiOx membrane. In addition, repeatable H2O2 sensing with the low concentration of 10 pM as well as prostate cancer biomarker is detected, which is owing to the reduction-oxidation phenomena of the NiOx membranes. The sensing mechanism is owing to the Ni2+/Ni3+ oxidation states of the NiOx membrane, which is confirmed by X-ray photoelectron spectroscopy. The optimized NiOx membrane on vertical Si nanowire in the EIN structure shows a good drift rate of 3.84 mV/h and sarcosine detection with improvement of approximately 1000 times as compared to the planar Si in an electrolyte-insulator-semiconductor (EIS) structure. This sensor paves a way to detect early-stage diagnosis of prostate cancer rapidly in the near future.

Exploring the Scope of Tandem Palladium and Isothiourea Relay Catalysis for the Synthesis of α-Amino Acid Derivatives.

The scope and limitations of a tandem N-allylation/[2,3]-rearrangement protocol are investigated through the incorporation of a variety of functional groups within an allylic phosphate precursor. This method uses readily accessible N,N-dimethylglycine aryl esters and functionalized allylic phosphates, forming quaternary ammonium salts in situ in the presence of a palladium catalyst. Subsequent enantioselective [2,3]-sigmatropic rearrangement, promoted by the chiral isothiourea tetramisole, generates α-amino acid derivatives with two contiguous stereocenters. The incorporation of electron-withdrawing ester and amide groups gave the best results, furnishing the desired products in moderate to good yields (29-70%), with low diastereocontrol (typically 60:40 dr) but high enantioselectivity (up to 90:10 er). These results indicate that substrate-catalyst interactions in the proposed transition state are sensitive to the substitution pattern of the substrates.

The role of osmolytes in the temperature-triggered conformational transition of poly(N-vinylcaprolactam): an experimental and computational study.

Biomedical industries are widely exploring the use of thermo-responsive polymers (TRPs) in the advanced development of drug delivery and in many other pharmaceutical applications. There is a great need to investigate the use of less toxic and more (bio-)compatible TRPs employing several additives, which could modify the conformational transition behavior of TRPs in aqueous solution. To move forward in this aspect, we have chosen the less toxic bio-based polymer poly(N-vinylcaprolactam) (PVCL) and three different methylamine-based osmolytes, trimethylamine N-oxide (TMAO), betaine and sarcosine, in order to investigate their particular interactions with the polymer segments in PVCL and therefore the corresponding changes in the thermo-responsive conformational behavior. Several biophysical techniques, UV-visible spectroscopy, fluorescence spectroscopy, dynamic light scattering (DLS) and laser Raman spectroscopy, as well as classical computer simulation methods such as molecular dynamics are employed in the current work. All the studied methylamines are found to favor the hydrophobic collapse of the polymer thus stabilizing the globular state of PVCL. Sarcosine is observed to cause the maximum decrease in lower critical solution temperature (LCST) of PVCL followed by TMAO and then betaine. The differences observed in the LCST values of PVCL in the presence of these molecules can be attributed to the different polymer-osmolyte interactions. The less sterically hindered N atom in the case of sarcosine causes a significant difference in the phase transition temperature values of PVCL compared to betaine and TMAO, where the nitrogen atom is buried by three methyl groups attached to it.

05SAR-PAGE: Separation of protein dimerization and modification using a gel with 0.05% sarkosyl.

A protein gel electrophoresis procedure using 0.05% w/v sarkosyl, is reported. The method called 05SAR-PAGE can be used to identify the native masses, dimeric states and modification states of proteins, and also be suitable for pursuing native electroblotting and immunodetection. It has been demonstrated by NMR spectroscopy that 0.05% w/v SAR is much milder than SDS, so it has subtle effects on the native structure of proteins. Therefore, the non-covalent dimerization of PhoBN and PhoRcp can be identified by 05SAR-PAGE which cannot be observed by SDS-PAGE. It has also been demonstrated that 05SAR-PAGE can be used to identify the phosphorylated or methylated proteins. Besides, 05SAR-PAGE shows the advantages of simple operation and low cost, and can be easily adapted to diverse applications.

Improved Host Cell Protein Analysis in Monoclonal Antibody Products through Molecular Weight Cutoff Enrichment.

Host cell proteins (HCPs) are process-related impurities that are generated by the host organism and are typically present at low levels in recombinant biopharmaceutical products, such as therapeutic antibodies. While overall HCP levels are usually monitored by enzyme-linked immunosorbent assay (ELISA), liquid chromatography coupled to mass spectrometry (LC-MS) is emerging as a powerful tool that can provide both qualitative and quantitative information about HCP levels during purification process development. However, a major challenge for LC-MS-based methods is that there can be a more than 5 orders of magnitude difference in the concentration between HCPs and therapeutic antibody in solution, which precludes the effective identification of low abundance HCPs in antibody product. This work reports a simple and powerful strategy to identify HCPs in antibody drug substance by applying molecular weight cutoff (MWCO) filtration step followed by shotgun proteomic analysis. After dissociating the interaction between HCPs and antibody with an anionic detergent, the depletion of antibody from HCPs can be easily achieved with the MWCO filtration step. Using this method, we observed that the dynamic range across proteins in the HCP samples was significantly decreased up to 1000-fold. In addition, by spiking in known amounts of HCPs to purified antibody drug substance with low levels of HCPs, we demonstrated that our method could detect HCP with low molecular weight (11 kDa and 17 kDa) at a concentration as low as 1 ppm. When applying this methodology to the study of HCPs in NIST monoclonal antibody (NISTmAb), more than 150 HCPs were confidently identified, which doubles the number of identified HCPs that have been previously reported. Parallel reaction monitoring (PRM) results confirmed that the novel HCPs found using this method were present in very low abundance (0.01-8 ppm), highlighting that our method reduces the dynamic range by removing antibody interference and improving the sensitivity of HCP identification and quantification.

Preparation and characterization of copper and zinc adsorbed cetylpyridinium and N-lauroylsarcosinate intercalated montmorillonites and their antibacterial activity.

The main objective of the present study was the preparation and characterization of new cationic/anionic surfactants and Cu2+/Zn2+ modified montmorillonites and the evaluation of their potential applicability as antibacterial agents for topical applications. To evaluate the antibacterial activity of Cu2+ and Zn2+ by synergistic effect, as well as to reduce the well-known toxicity of these metal cations; cetylpyridinium (CP) and N-lauroylsarcosinate (SR) intercalated montmorillonite (Mt-CP-SR) was used as the host material. In addition to their role to capture the metal cations and inhibit their release in any contact medium, these surfactants also increase the efficacy of the material due to their antibacterial properties. The effect of surfactant loading on the adsorption behavior of the metal cations onto the Mt-CP was investigated using SR in two different concentrations, namely 0.7 and 1.0 CEC of sodium montmorillonite (Mt-Na). The samples prepared were characterized using SEM, ATR-FTIR, zeta potential, and XRD analyses and they were subjected to antibacterial tests using the "Standard Method Under Dynamic Contact Conditions" on the Gram positive S. aureus, and Gram negative E. coli. As confirmed with desorption and characterization studies, the addition of Cu2+/Zn2+ onto the Mt-CP-SR yielded double adsorbed amounts compared to that of the Mt-CP, which indicated that Cu2+/Zn2+ bound to SR- interacted with the Mt surface. In contrary of Zn2+ caused no considerable change in the antibacterial effect of the host, Cu2+ addition enhanced the antibacterial activity. The produced antibacterial agents have the potential use in dyes, polymer composites, personal care products, and topical medicinal applications.