Microwave-assisted Solid-phase Synthesis Research Articles

Microwave-assisted solid-phase synthesis of lactosylated peptides for food analytical application

The lactosylation of whey proteins affects their properties, especially their allergenicity and nutritional value, which matters in infant feeding. The quantification of lactosylated peptides requires analytically pure standards which are not commercially available. Herein, we proposed a fast, simple, and efficient protocol for the synthesis of lactosylated peptides on solid support based on microwave-assisted synthesis combined with boronate affinity chromatography utilizing the functionalized resin developed in our research group. We have used our method to synthesize identified modified peptides derived from β-lactoglobulin in tryptic digestion. Thus, the standard peptides contain the dabcyl moiety for determination of the amount in a sample and a cleavable linker to obtain tryptic analogs of β-lactoglobulin. Moreover, for the first time, we applied the functionalized resin to enrich the sample of human serum albumin lactosylated in vitro, into lactosylated peptides.

Microwave-Assisted Solid Phase Synthesis of Different Peptide Bonds: Recent Advancements

Abstract: Peptides are important as drugs and biologically active molecules. The synthesis of pep-tides has gathered considerable attention in recent years due to their various attractive properties. Conventional peptide synthesis is tedious and requires hazardous reagents and solvents. Micro-wave-assisted solid-phase peptide synthesis has several advantages compared with conventional batch synthesis. Herein, we have discussed various microwave-assisted solid-phase peptide bond synthesis methods developed over the last five years. Peptides are categorized into four groups - small, medium, large, and cyclic based on their length and structural characteristics to make it easier to understand. This review article also discusses the scope and limitations of microwave-assisted solid-phase peptide synthesis.

Large-Scale Synthesis of Tunable Fluorescent Carbon Dots Powder for Light-Emitting Diodes and Fingerprint Identification.

The emergence and fast development of carbon dots (CDs) provide an unprecedented opportunity for applications in the field of photoelectricity, but their practicability still suffers from complicated synthesis procedures and the substrate dependence of solid-state fluorescence. In this study, we design a unique microwave-assisted solid-phase synthesis route for preparing tunable fluorescent CD powders with yellow, orange, and red fluorescence (Y-CDs, O-CDs, R-CDs) by simply adjusting the mass ratio of reactants, a method which is suitable for the large-scale synthesis of CDs. The Y-/O-/R-CDs were systematically characterized using physics and spectroscopy techniques. Based on the perfect solid-state fluorescence performance of the proposed fluorescent CD powders, the Y-/O-/R-CDs were successfully applied for the construction of multi-color and white light-emitting diode devices at low cost. Furthermore, the Y-CDs displayed much higher yield and luminous efficiency than the O-CDs and R-CDs and were further used for fingerprint identification on the surfaces of glass sheets and tinfoil. In addition, the R-CD aqueous solution fluorescence is sensitive to pH, suggesting its use as a pH indicator for monitoring intracellular pH fluctuations. The proposed series of fluorescent powders composed of CDs may herald a new era in the application of optical components and criminal investigation fields.

Chemical Synthesis of Alpha-Synuclein Proteins via Solid-Phase Peptide Synthesis and Native Chemical Ligation.

Alpha-Synuclein (α-Synuclein) is a 140 amino acid protein implicated in neurodegenerative disorders known as synucleino-pathies, where it accumulates in proteinaceous inclusions in the brain. The normal physiological function of α-Synuclein remains obscure, as it exists in several non-neuronal cells in which its function has not been studied. Given the tremendous interest in studying α-Synuclein, and the existing limitations in the production of modified forms of the protein, we developed a method for the chemical synthesis of α-Synuclein by combining peptide fragment synthesis via automated microwave-assisted solid-phase peptide synthesis and ligation strategies. Our synthetic pathway enables the synthesis of protein variants of interest, carrying either mutations or posttranslational modifications, for further investigations of the effects on the structure and aggregation behavior of the protein. Ultimately, our study forms the foundation for future syntheses and studies of other custom-made α-Synuclein variants with a single or several modifications, as necessary.

Solid-phase synthesis and pathological evaluation of pyroglutamate amyloid-β3-42 peptide

Pyroglutamate amyloid-β3-42 (AβpE3-42) is an N-terminally truncated and pyroglutamate-modified Aβ peptide retaining highly hydrophobic, amyloidogenic, and neurotoxic properties. In Alzheimer’s disease (AD) patients, AβpE3-42 peptides accumulate into oligomers and induce cellular toxicity and synaptic dysfunction. AβpE3-42 aggregates further seed the formation of amyloid plaques, which are the pathological hallmarks of AD. Given that AβpE3-42 peptides play critical roles in the development of neurodegeneration, a reliable and reproducible synthetic access to these peptides may support pathological and medicinal studies of AD. Here, we synthesized AβpE3-42 peptides through the microwave-assisted solid-phase peptide synthesis (SPPS). Utilizing thioflavin T fluorescence assay and dot blotting analysis with anti-amyloid oligomer antibody, the amyloidogenic activity of synthesized AβpE3-42 peptides was confirmed. We further observed the cytotoxicity of AβpE3-42 aggregates in cell viability test. To examine the cognitive deficits induced by synthetic AβpE3-42 peptides, AβpE3-42 oligomers were intracerebroventricularly injected into imprinting control region mice and Y-maze and Morris water maze tests were performed. We found that AβpE3-42 aggregates altered the expression level of postsynaptic density protein 95 in cortical lysates. Collectively, we produced AβpE3-42 peptides in the microwave-assisted SPPS and evaluated the amyloidogenic and pathological function of the synthesized peptides.

TIGER 17 PEPTIDE LOADED POLYCAPROLACTONE/CELLULOSE ACETATE ELECTROSPUN NANOFIBROUS MATS FOR APPLICATIONS IN CUTANEOUS WOUND DRESSINGS

A skin wound if not properly treated can result in a chronic wound susceptible to widespread infections, which can result in the patient's death. Currently, tissue engineering is described as an interdisciplinary field that combines principles of engineering, chemistry and biology to generate solutions that allow to repair, restore and/or improve the functions of injured tissues. In the same sense, the textile area addresses solutions based on polymeric fibers, produced from a wide range of polymers, which allow the generation of structures with a large surface area, porosity and mechanical resistance that can be used as bioactive dressings that promote a healing and efficient antimicrobial activity. This research work focused on the synthesis of Tiger 17, through microwave-assisted solid-phase synthesis methodologies, and Tiger 17 commercially obtained, respective structural characterization and evaluation of the antimicrobial capacity. Simultaneously, nanofibrous polymer matrices were produced using the electrospinning technique with the aim of immobilizing the developed biomolecule and thus creating potential vehicles for a local and sustainable antimicrobial action (controlled release). In order to verify its physical and chemical properties, advanced characterization techniques were used: proton nuclear magnetic resonance, high performance liquid chromatography, optical microscopy, scanning electron microscopy, fourier transform infrared spectroscopy–attenuated total reflectance, thermogravimetry, differential scanning calorimetry, contact angle and surface energy and determination of porosity and hydration.

Synthesis and biological evaluation of glucagon-like peptide-1 analogs with the C-terminal helix 3 of albumin-binding domain 3

Based on peptide 6 (Ser8-GLP-1 [7–35]-GVKALIDEILAA-NH2; GVKALI-DEILAA is the C-terminal helix 3 of albumin-binding domain 3 of protein G from bacterial Streptococcal G strain 148 [G148-ABD3]), a series of its analogs (compounds 0-VI: Aib8-GLP-1 [7–35]-linkers-GVKALIDEILAA-NH2) were designed and synthesized using microwave-assisted solid-phase synthesis. First, to monitor the reaction process and reduce potential risks, the synthesis process of compounds 0-VI was divided into three stages. Next, to explore the effect of these linkers on their albumin-binding rates, albumin-binding assays were performed. Finally, to evaluate their biological activities in vitro and in vivo, receptor potency, surface plasmon resonance (SPR), weight-loss, and glucose-lowering assays were carried out. These results indicated the linkers of different polarities between Aib8-GLP-1 (7–35) and the C-terminal helix 3 of ABD3 can significantly affect the albumin-binding rate of the C-terminal helix 3 of ABD3. And compound IV had the highest albumin-binding rates, weight-loss, and glucose-lowering effects among them.

An Optimized Safe Process from Bench to Pilot cGMP Production of API Eptifibatide Using a Multigram-Scale Microwave-Assisted Solid-Phase Peptide Synthesizer

A growing industrial interest toward the peptide drug market fueled the need for the development of effective and cGMP compliant manufacturing methods for these complex molecules. Solid-phase strategies are considered methods of election for medium-length peptide syntheses not only on the research scale but for multigram-scale production, as well. The possibility to use microwave-assisted technology on the multigram scale, recently introduced, prompted us to evaluate the possibility to conveniently set up a safe and fully cGMP-compliant pilot process to produce eptifibatide, a generic peptide active pharmaceutical ingredient. Accordingly, we developed an optimized process on the laboratory scale (1–5 mmol), which was subsequently successfully scaled up to 70 mmol, obtaining all the information required by regulatory agencies to validate the process and qualify the pilot scale plant. The process consists of 5 steps: (1) automated microwave-assisted solid-phase synthesis of eptifibatide linear precursor; (2) cleavage from the resin with concomitant amino acid side-chains deprotection; (3) disulfide-bond formation in solution; (4) purification by flash column chromatography; (5) ion-exchange solid-phase extraction. Since the direct scale-up of a multigram-scale cGMP compliant peptide API production procedure is a challenge that requires an accurate understanding of each involved step, we initially performed a quality management risk assessment, which enabled a smooth and effective achievement of a successful final result.

Microwave-assisted solid-phase synthesis of nitrogen-doping carbon dot with good solvent compatibility and its sensing of sunitinib.

Microwave-assisted solid-phase synthesis method was simple, convenient, and fast, and herein adopted to produce nitrogen-doping carbon dots (N-CDs) in only 3min. The N-CDs possessed high fluorescence quantum yield up to 15.9% with satisfactory stability to the environmental pH, ionic strength, and ultraviolet radiation. Particularly, the N-CDs had excellent dispersibility in both water and water-compatible organic solvents with similar fluorescence properties. Sunitinib, a small-molecule tyrosine inhibitor effective for some solid tumors, was found to quench the fluorescence of N-CDs in these media via the inner-filter effect. Hence, it was convenient to combine the proper sample pretreatment with the N-CD probe for sensing sunitinib avoiding the medium incompatibility problem. For rat plasma sample, salting-out liquid-liquid extraction was employed to minimize the sample matrix and concentrate the target sunitinib from aqueous to acetonitrile. The fluorescence detection of sunitinib was then achieved in acetonitrile by the addition of the proper amount of N-CDs. The method provided a good linearity of 0.1μg/mL to 7μg/mL with a limit of detection of 30ng/mL, which met the requirement of the therapeutic drug monitoring of sunitinib. The developed method was potential for on-site detection of sunitinib.

Design, synthesis and biological evaluation of double fatty chain-modified glucagon-like peptide-1 conjugates

Twelve double fatty chains and Aib8-Arg34-GLP-1 (7–37) were designed and obtained by microwave-assisted solid-phase synthesis. Then, twelve conjugates of Aib8-Arg34-GLP-1 (7–37) were synthesized in 1% triethylamine aqueous solution. Conjugates 2, 3, 6, 7, 10 and 11 showed better GLP-1 receptor activation potency than semaglutide. However, conjugates 2, 6 and 10 showed slightly worse glucose-lowering effects in vivo than semaglutide but better effects than conjugates 3, 7 and 11. The CD spectra of conjugates 2, 6 and 10 indicated that they had the same secondary structure as liraglutide and semaglutide. The receptor affinity results for conjugates 2, 6 and 10 measured by SPR (surface plasmon resonance) showed that conjugate 2 had higher receptor affinity than conjugates 6 and 10. In addition, albumin binding assays indicated that double fatty acid chains had obvious synergistic effects compared with single fatty acid chains. In conclusion, the structure–activity relationship of different side chains was summarized and one candidate, conjugate 2, was screened.

An Optimized Scalable Fully Automated Solid-Phase Microwave-Assisted cGMP-Ready Process for the Preparation of Eptifibatide

We investigated several strategies, based on the use of microwave-assisted solid-phase peptide synthesis (MW-SPPS) and scalable to kilogram-scale manufacturing, for the preparation of Eptifibatide,...

A Rapid and Efficient Building Block Approach for Click Cyclization of Peptoids.

Cyclic peptide-peptoid hybrids possess improved stability and selectivity over linear peptides and are thus better drug candidates. However, their synthesis is far from trivial and is usually difficult to automate. Here we describe a new rapid and efficient approach for the synthesis of click-based cyclic peptide-peptoid hybrids. Our methodology is based on a combination between easily synthesized building blocks, automated microwave assisted solid phase synthesis and bioorthogonal click cyclization. We proved the concept of this method using the INS peptide, which we have previously shown to activate the HIV-1 integrase enzyme. This strategy enabled the rapid synthesis and biophysical evaluation of a library of cyclic peptide-peptoid hybrids derived from HIV-1 integrase in high yield and purity. The new cyclic hybrids showed improved biological activity and were significantly more stable than the original linear INS peptide.

Improved Fmoc‐solid‐phase peptide synthesis of an extracellular loop of CFTR for antibody selection by the phage display technology

Cystic fibrosis (CF), a life-shortening genetic disease, is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene that codes for the CFTR protein, the major chloride channel expressed at the apical membrane of epithelial cells. The development of an imaging probe capable of non-invasively detect CFTR at the cell surface could be of great advantage for the management of CF. With that purpose, we synthesized the first extracellular loop of CFTR protein (ECL1) through fluorenylmethyloxycarbonyl (Fmoc)-based microwave-assisted solid-phase peptide synthesis (SPPS), according to a reported methodology. However, aspartimide formation, a well-characterized side reaction in Fmoc-SPPS, prompted us to adopt a different side-chain protection strategy for aspartic acid residues present in ECL1 sequence. The peptide was subsequently modified via PEGylation and biotinylation, and cyclized through disulfide bridge formation, mimicking the native loop conformation in CFTR protein. Herein, we report improvements in the synthesis of the first extracellular loop of CFTR, including peptide modifications that can be used to improve antigen presentation in phage display for selection of novel antibodies against plasma membrane CFTR.

Supramolecular β-Sheet Forming Peptide Conjugated with Near-Infrared Chromophore for Selective Targeting, Imaging, and Dysfunction of Mitochondria.

Herein, conjugation of the amyloid-β (Aβ) peptide fragment, Lys-Leu-Val-Phe-Phe (KLVFF, fragment of Aβ16-20), with an unsymmetrical near-infrared (NIR) cyanine-5 (Cy-5) chromophore is achieved using microwave-assisted solid phase synthesis on 2-chlorotrityl chloride resin. Selective mitochondria tracking and staining in human carcinoma cells are accomplished by the KLVFF/Cy-5 conjugate containing triphenylphosphonium functionality, and this is compared to a control molecule KLVFF/Cy-5c. Mitochondrial target specificity of KLVFF/Cy-5 is established by the colocalization assay using mitochondria selective probe MitoTracker Red, which is monitored by confocal laser scanning microscope and shows a high Pearson's correlation coefficient. The KLVFF/Cy-5 conjugate has high photostability, NIR absorption/emission, high molar extinction coefficient, narrow absorption/emission band, high fluorescence lifetime, and high fluorescence quantum yield. Moreover, mitochondria targeting KLVFF/Cy-5 conjugate reaches the critical aggregation concentration inside the mitochondria of cancer cells due to the strong negative inner mitochondrial membrane potential [(ΔΨm)cancer -220 mV] and self-assembles to form amyloid fibrils at the target site, which is responsible for the mitochondrial dysfunction and cytotoxicity. Annexin V-FITC/PI apoptosis detection assay is used to determine the signal pathway of mitochondria targeted cellular dysfunction.

An Isodipeptide Building Block for Microwave-Assisted Solid-Phase Synthesis of Difficult Sequence-Containing Peptides.

Microwave technology, in conjunction with the isopeptide strategy including Fmoc-based solid-phase peptide synthesis (SPPS), was used to establish a methodology for time-efficient synthesis of peptides containing difficult sequences. A model difficult sequence-containing peptide (8QSer) was synthesized through this method in 1day, representing a tenfold reduction in synthesis time compared to the isopeptide method combined with classical SPPS.

Microwave-assisted solid-phase synthesis of antisense acpP peptide nucleic acid-peptide conjugates active against colistin- and tigecycline-resistant E. coli and K. pneumoniae

Recent discovery of potent antibacterial antisense PNA-peptide conjugates encouraged development of a fast and efficient synthesis protocol that facilitates structure-activity studies. The use of an Fmoc/Boc protection scheme for both PNA monomers and amino acid building blocks in combination with microwave-assisted solid-phase synthesis proved to be a convenient procedure for continuous assembly of antisense PNA-peptide conjugates.A validated antisense PNA oligomer (CTCATACTCT; targeting mRNA of the acpP gene) was linked to N-terminally modified drosocin (i.e., RXR-PRPYSPRPTSHPRPIRV; X = aminohexanoic acid) or to a truncated Pip1 peptide (i.e., RXRRXR-IKILFQNRRMKWKK; X = aminohexanoic acid), and determination of the antibacterial effects of the resulting conjugates allowed assessment of the influence of different linkers as well as differences between the L- and D-forms of the peptides.The drosocin-derived compound without a linker moiety exhibited highest antibacterial activity against both wild-type Escherichia coli and Klebsiella pneumoniae (MICs in the range 2–4 μg/mL ∼ 0.3–0.7 μM), while analogues displaying an ethylene glycol (eg1) moiety or a polar maleimide linker also possessed activity toward wild-type K. pneumoniae (MICs of 4–8 μg/mL ∼ 0.6–1.3 μM). Against two colistin-resistant E. coli strains the linker-deficient compound proved most potent (with MICs in the range 2–4 μg/mL ∼ 0.3–0.7 μM).The truncated all-L Pip1 peptide had moderate inherent activity against E. coli, and this was unaltered or reduced upon conjugation to the antisense PNA oligomer. By contrast, this peptide was 8-fold less potent against K. pneumoniae, but in this case some PNA-peptide conjugates exhibited potent antisense activity (MICs of 2–8 μg/mL ∼ 0.3–1.2 μM). Most interestingly, the antibacterial activity of the D-form peptide itself was 2- to 16-fold higher than that of the L-form, even for the colistin- and tigecycline-resistant E. coli strains (MIC of 1–2 μg/mL ∼ 0.25–0.5 μM). Low activity was found for conjugates with a two-mismatch PNA sequence corroborating an antisense mode of action. Conjugates containing a D-form peptide were also significantly less active. In conclusion, we have designed and synthesized antisense PNA-drosocin conjugates with potent antibacterial activity against colistin- and tigecycline-resistant E. coli and K. pneumonia without concomitant hemolytic properties. In addition, a truncated D-form of Pip1 was identified as a peptide exhibiting potent activity against both wild-type and multidrug-resistant E. coli, P. aeruginosa, and A. baumannii (MICs within the range 1–4 μg/mL ∼ 0.25–1 μM) as well as toward wild-type Staphylococcus aureus (MIC of 2–4 μg/mL ∼ 0.5–1.0 μM).

Microwave-assisted solid-phase synthesis of nicotinyl hydrazones for use in radiochemistry of technetium-99m

Microwave-assisted solid-phase synthesis of nicotinyl hydrazones for use in radiochemistry of technetium-99m

Microwave-assisted solid-phase synthesis of highly fluorescent carbon nanoparticles and its application in intracellular pH sensing

Fluorescent carbon nanoparticles (FCNPs) have been deeply researched and widely applied in recent years due to their good optics performance, chemical stability and biocompatibility. Herein, a green and rapid microwave-assisted solid-phase synthesis (solvent-free) approach was proposed for the fabrication of highly FCNPs in a very short period of time, 4 min. The as-prepared FCNPs can emit a blue emission with quantum yield of up to 63.2% in water solution and show yellow fluorescence in the solid state. The FCNPs also exhibit special solvent effect that the fluorescence emission can be adjusted by controlling the solvent ratio of ethanol and water. Most importantly, the FCNPs possess a narrow-range pH response. The probe responds linearly and rapidly to minor pH fluctuations within the range of 3.47–5.10 and the correlation coefficient is above 0.99. The proposed FCNPs also exhibit high photostability and reusability. As expected, the cell imaging and intracellular pH monitoring was achieved successfully in living SMMC 7721 hepatoma cells by this probe. The FCNPs is promising as a convenient and general fluorescent pH sensor for bioimaging applications.

Hydrolysis of a Ni-Schiff-Base Complex Using Conditions Suitable for Retention of Acid-labile Protecting Groups

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide sequences. Synthesis of some unnatural amino acids often includes the use of a precursor consisting of a Schiff-base stabilized by a nickel cation. Unnatural side-chains can be installed on an amino acid backbone found in this Schiff-base complex. The resulting unnatural amino acid can then be isolated from this complex using hydrolysis of the Schiff-base, typically by employing reflux in strongly acidic solution. These highly acidic conditions may remove acid-labile side-chain protecting groups necessary for the unnatural amino acids to be used in microwave-assisted solid-phase peptide synthesis. In this work, we present an efficient hydrolysis and subsequent Fmoc protection of an amino acid isolated from a Ni-Schiff base complex. Hydrolysis conditions presented in this work are suitable for retention of acid-labile side-chain protecting groups and may be adaptable to a variety of unnatural amino acid substrates.

Hydrolysis of a Ni-Schiff-Base Complex Using Conditions Suitable for Retention of Acid-labile Protecting Groups.

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide sequences. Synthesis of some unnatural amino acids often includes the use of a precursor consisting of a Schiff-base stabilized by a nickel cation. Unnatural side-chains can be installed on an amino acid backbone found in this Schiff-base complex. The resulting unnatural amino acid can then be isolated from this complex using hydrolysis of the Schiff-base, typically by employing reflux in strongly acidic solution. These highly acidic conditions may remove acid-labile side-chain protecting groups necessary for the unnatural amino acids to be used in microwave-assisted solid-phase peptide synthesis. In this work, we present an efficient hydrolysis and subsequent Fmoc protection of an amino acid isolated from a Ni-Schiff base complex. Hydrolysis conditions presented in this work are suitable for retention of acid-labile side-chain protecting groups and may be adaptable to a variety of unnatural amino acid substrates.