2-chlorotrityl Chloride Resin Research Articles

Total Synthesis and Anticancer Evaluation of BZR-cotoxin IV

Anticancer peptides (ACPs) is a potential alternative for future cancer therapy. ACPs specifically inhibit cancer cells through a non-enzymatic membranolysis mechanism. One potential anticancer peptide worthy of investigation is BZR-cotoxin IV. Currently, research on BZR-cotoxin IV is limited to isolation, making it an interesting area for further development. One approach to developing BZR-cotoxin IV is through chemical synthesis techniques. This compound belongs to the group of cyclodepsipeptides that are naturally produced by the fungus Bipolaris zeicola Race 3. The synthesis of BZR cotoxin IV involves 3 steps: (1) Synthesis of the hydroxy acid precursor by conversion of L-valine amino acid to (2R)-hydroxyisovaleric acid, (2) synthesis of the linear depsipeptide using solid-phase peptide synthesis on 2-chlorotrityl chloride resin and (3) cyclization of the linear depsipeptide using solution phase synthesis to produce the BZR cotoxin IV compound with a purity of 11.7 %. BZR-cotoxin IV was characterized using HR-TOF-MS and 1H and 13C-NMR to validate the desired product. Anticancer activity testing was performed using the Resazurin assay on the HeLa cancer cell line, resulting in an IC50 of 187.50 μg/mL (214.014 μM) categorized as moderate. HIGHLIGHTS The chemical synthesis of BZR-cotoxin IV compounds has not been firstly reported. The synthesis of BZR-cotoxin IV was accomplished through a combination of solid-phase and solution-phase peptide synthesis. The objective was to synthesize and evaluate BZR-cotoxin IV for its cancer properties. GRAPHICAL ABSTRACT

Synthesis, Characterization, and In vitro Evaluation of Short Cationic Peptides for Gene Delivery Vehicle Candidate

Background: Amongst gene delivery vehicles, peptide-based vectors have drawn the intensive attraction of experts globally due to their simplicity and many advantages due to ease in design, biocompatibility, and safety. Rationally designed peptides are capable of condensing DNA molecules efficiently and facilitating gene expression in the target cells. Objective: This study aims to design, synthesize and evaluate short cationic peptides composed of several positively charges amino acids of lysine (K) and arginine (R) for gene delivery vehicle candidates. Methods: The short cationic peptides of PKKKRKV (P1), CHSPKKKRKV (P2), and YGRKKRRQRRR (P3) were synthesized using a solid-phase method on 2-chlorotrityl chloride resin. The crude peptides were purified using RP-HPLC and characterized by HR-TOF-ESI-MS and 1H-NMR. The capability of the peptides to condense DNA was evaluated by ethidium bromide exclusion assay. Cytotoxicity study of the peptides was carried out in HEK-293T, CHO-K1, and HepG2 cells using MTT assay. Gene expression facilitated by the peptides was determined in the HEK-293T. Results: The peptides were successfully synthesized with high purity (> 90%) and in a high consistency with the synthetic products, as shown by the spectroscopic data. Physicochemical and biological evaluation showed that the cationic peptides are capable of condensing DNA molecule and have low cytotoxicity to the cells of HEK-293T, CHO-K1, and HepG2. Moreover, the cationic peptides facilitated gene delivery of green fluorescence protein more efficiently compared to PLL. Conclusion: The short cationic peptides rich in lysine and arginine have been successfully synthesized using solid-phase peptide synthesis method. They were found to be capable of condensing DNA, have low cytotoxicity, and facilitate gene delivery. However, structure modification or formulation of cationic peptide with lipid components to form cationic liposome is still needed to enhance transgene expression by these peptides.

Cyclic Peptide Conjugate Vaccines and Physically Mixed Cyclic Peptide Vaccines for Subcutaneous Immunization.

Immune stimulants (adjuvants) enhance immune system recognition to provide an effective and individualized immune response when delivered with an antigen. Synthetic cyclic deca-peptides, co-administered with a toll-like receptor targeting lipopeptide, have shown self-adjuvant properties, dramatically boosting the immune response in a murine model as a subunit peptide-based vaccine containing group A Streptococcus peptide antigens.Here, we designed a novel peptide and lipid adjuvant system for the delivery of group A Streptococcus peptide antigen and a T helper peptide epitope. Following linear peptide synthesis on 2-chlorotrityl chloride resin, the linear peptide was cleaved and head-to-tail cyclized in solution. The selective arrangement of amino acids in the deca-peptide allowed for selective conjugation of lipids and/or peptide antigens following cyclisation. Using both solution-phase peptide chemistry and copper-catalyzed azide-alkyne cycloaddition reaction were covalently (and selectively) ligated lipid and/or peptide antigens onto the cyclic deca-peptide core. Subcutaneous administration of the vaccine design to mice resulted in the generation of a large number of serum immunoglobulin (Ig) G antibodies.

Protocol for Facile Synthesis of Fmoc-N-Me-AA-OH Using 2-CTC Resin as Temporary and Reusable Protecting Group.

One approach to enhance the bioavailability and half-life of peptides in vivo is through N-methylation of one or more of the amino acids within the peptide sequence. However, commercially available Fmoc-N-Me-AA-OHs are limited and often expensive. In this study, a solid-phase synthesis method for Fmoc-N-Me-AA-OH was developed using a 2-chlorotrityl chloride (2-CTC) resin as a temporary protective group for the carboxylic acid strategy. Two strategies for the alkylation step were compared, employing either dimethyl sulfate or methyl iodide in the Biron-Kessler method. In this work we tested the protocol with two amino acids: Fmoc-Thr(tBu)-OH and Fmoc-βAla-OH. The first one is an alpha amino acid, very hindered and with the amine group directly influenced by the electronic effects of the carboxy group, whereas in Fmoc-βAla-OH, the presence of a methylene group weakens this influence due to the intervening carbon atoms. The desired amino acids, Fmoc-N-Me-Thr(tBu)-OH and Fmoc-N-Me-βAla-OH, were synthesized by both strategies with high yield and purity.

Improving 2-Chlorotrityl Chloride (2-CTC) Resin Activation.

Used in solid-phase peptide synthesis (SPPS) for peptides with an acid termination, the 2-chlorotrityl chloride (2-CTC) resin is highly susceptible to moisture, leading to reduced resin loading and lower synthetic yields. It is therefore recommended that the resin be activated with thionyl chloride (SOCl2) before peptide assembly. Here we present an optimized procedure for resin activation that minimizes the use of SOCl2 as the activation reagent and reduces the activation time. Additionally, we demonstrate the feasibility of reusing the 2-CTC resin when following the activation protocol, achieving comparable results to the first usage of the resin. Moreover, we achieved different degrees of resin activation by varying the amount of SOCl2. For instance, the use of 2% SOCl2 in anhydrous dichloromethane (DCM) allowed up to 44% activation of the resin, thereby making it suitable for the synthesis of longer peptides. Alternatively, employing 25% SOCl2 in anhydrous DCM resulted in up to 80% activation with a reaction time of only 5 min in both cases.

Greener Cleavage of Protected Peptide Fragments from Sieber Amide Resin.

Following the successful introduction of two benign solvents for cleaving protected acid peptide fragments from 2-chlorotrityl chloride (2-CTC) resin, there is a need to green the cleavage process for obtaining protected peptide amide fragments. In this work, p-xylene and toluene are introduced as greener alternates to dichloromethane (DCM) for preparing protected peptide amide fragments from a Sieber amide resin. The N-dealkylation is a demanding chemical reaction, requiring that the cleavage protocol be optimised to ensure complete cleavage from the resin. After a 30 min reaction time, only 66 % of the final peptide product was retrieved even with the conventional dichloromethane solvent. Therefore, 120 min was considered sufficient to fully cleave the peptide from the Sieber amide resin. This work reaffirms the fact that greening strategies are far from detrimental, with green alternatives often outperforming their replaced counterparts.

Gram-scale preparation of DAMGO by typical solid phase synthesis

DAMGO ([D-Ala2, Nα-MePhe4, Gly5-ol]-enkephalin), a high-efficacy µ-opioid receptor agonist, as a tool compound, is extensively used in the preclinical analgesia studies. Due to the large amount of DAMGO required in the research and the high market price, we developed a simple and efficient solid-phase synthesis method. DAMGO has a special Gly-ol residue at C terminus, its attachment to the resin is a challenge. We chose 2-chlorotritylchloride resin as the solid support, and explored two loading methods, an amino loading method followed by a finally N-O exchange after cleavage, and a direct hydroxyl loading method. Although each has its own drawbacks, both methods obtained good yields. The direct hydroxyl loading method is more convenient and can be achieved on the gram-scale with an overall 40% yield. The synthesized DAMGO was characterized by HPLC and mass spectrometry, and GPI functional assay was further used to confirm the biological effect.

Total Synthesis of a Reversed-Bacicyclin Using a Combination of Solid- and Solution-Phase Methods

Bacicyclin is a cyclic hexapeptide with antibacterial activity against Enterococcus faecalis and Staphylococcus aureus with minimum inhibition concentration (MIC) values of 8 and 12 µM, respectively. Studies on a reversed sequence of bacicyclin were conducted to investigate how the reversed peptide sequence affects its biological properties. A reversed-bacicylin, cyclo-(Gly-Leu-Val-Ile-Ala-Phe), was successfully synthesized by constructing the linear precursor on 2-chlorotrityl chloride resin using a Fmoc-based strategy. The HATU/HOAt reagent was applied in all peptidic bond formations, and the desired linear hexapeptide (82% yield) was cleaved off the resin using a mixture of trifluoroacetic acid:dichloromethane (2:8). The linear peptide was cyclized using 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxidehexa-fluorophosphate (HATU) as a coupling agent and diisopropylethylamine (DIPEA) as the base in a very dilute solution (0.001 M) in dichloromethane, then purified by octadecyl silica gel (ODS) column chromatography to obtain the reversed-bacicyclin (43.7% yield). The purity of the cyclic product was analyzed using analytical RP-HPLC (tR = 20.01 min), and characterized by HR-TOF-MS, 1H-NMR, and 13C-NMR. The biological activity of the reversed-bacicyclin is much lower compared to bacicyclin, indicating that the amino acid sequence of the cyclopeptide dictates the antibacterial activity. This finding gives additional information on the relationship between peptide sequence and biological properties.

Missing Link: Enabling Loading of 2-Chlorotrityl Chloride Resin inN-Butylpyrrolidinone as a Green Solvent

Missing Link: Enabling Loading of 2-Chlorotrityl Chloride Resin in<i>N</i>-Butylpyrrolidinone as a Green Solvent

Three Methods for Peptide Cyclization Via Lactamization.

Lactamization is the key step in the synthesis of many compounds with macrocyclic structure. As the interest for these types of molecules grows in various fields such as drug discovery and nanomaterials, different methodologies to access them are being developed. Three different strategies to obtain cyclic peptides via lactamization are described in this chapter: solution-phase macrocyclization following solid-phase peptide synthesis (SPPS) of the linear precursor, SPPS and on-resin cyclization on the 2-chlorotrityl chloride (2-CTC) resin, and SPPS and on-resin cyclization by native chemical ligation on the amino-PEGA resin.

Discovery of Dolastatinol: A Synthetic Analog of Dolastatin 10 and Low Nanomolar Inhibitor of Tubulin Polymerization.

We developed a highly potent anticancer agent, dolastatinol, which is a methylene hydroxyl derivative of dolastatin 10. Dolastatinol is a synthetic analog of dolastatin 10, synthesized by a solid-phase peptide Fmoc chemistry protocol on 2-chlorotrityl chloride resin utilizing a pH-triggering self-immolative monosuccinate linker. The introduction of the C-terminus hydroxyl methylene functionality preserves the anticancer properties of the parent dolastatin 10, including strong suppression of the cell proliferation, migration, high cytotoxicity. Our research establishes a new facile route toward the further development of C-terminus-modified dolastatin-10-based microtubule inhibitors for anticancer treatment.

The Total Chemical Synthesis and Biological Evaluation of the Cationic Antimicrobial Peptides, Laterocidine and Brevicidine

Antimicrobial resistance is a significant threat to public health systems worldwide, prompting immediate attention to develop new therapeutic agents with novel mechanisms of action. Recently, two new cationic non-ribosomal peptides (CNRPs), laterocidine and brevicidine, were discovered from Brevibacillus laterosporus through a global genome-mining approach. Both laterocidine and brevicidine exhibit potent antimicrobial activity toward Gram-negative bacteria, including difficult-to-treat Pseudonomas aeruginosa and colistin-resistant Escherichia coli, and a low risk of resistance development. Herein, we report the first total syntheses of laterocidine and brevicidine via an efficient and high-yielding combination of solid-phase synthesis and solution-phase macrolactamization. The crucial depsipeptide bond of the macrolactone rings of laterocidine and brevicidine was established on-resin between the side-chain hydroxy group of Thr9 with Alloc-Gly-OH or Alloc-Ser(tBu)-OH, respectively. A conserved glycine residue within the lactone macrocycle is exploited for the initial immobilization onto the hyper acid-labile 2-chlorotrityl chloride resin, subsequently enabling an efficient solution-phase macrocyclization to yield laterocidine and brevicidine in 36% and 10% overall yields, respectively (with respect to resin loading). A biological evaluation against both Gram-positive and Gram-negative bacteria demonstrated that synthetic laterocidine and brevicidine possessed a potent and selective antimicrobial activity toward Gram-negative bacteria, in accordance with the isolated compounds.

Synthesis of a Novel PTH1–34 Analog with Increased Human Serum Albumin Affinity

Parathyroid hormone (PTH)1–34 is an effective peptide drug for osteoporosis therapy. However, the half-life of PTH1–34 in vivo is short, leading to the need for frequent injections of this drug during its treatment. To prolong the half-life of PTH1–34, a novel PTH1–34 analog was generated based on fatty acid generation, and its synthesis process included recombinant protein expression, side-chain modification, and peptide decoration. The PTH1–34 variant was expressed in Escherichia coli, with a single Lys (position 27) retained as a modification site. The side chain, –AEEA-γGlu-C18 diacid, was synthesized using 2-chlorotrityl chloride resin as a solid support, and then was conjugated to the PTH1-34 variant to form PTH-Lys27-AGC. Reversed-phase chromatography confirmed a high final purity (&gt;98%) of the target compound; in vitro bioactivity tests showed that PTH-1 receptor potency of PTH-Lys27-AGC was comparable to that of the native PTH1–34. A competitive human serum albumin binding test demonstrated a high albumin affinity of PTH-Lys27-AGC in comparison to PTH1–34. In summary, we developed a novel PTH1–34 analog, PTH-Lys27-AGC, which may be a long-acting agent for osteoporosis treatment in the future.

A versatile resin for the generation of thioether-bonded head-to-tail cyclized peptides

Thioether-bonded cyclic peptide is widely present in current peptide-based drug discovery, and the diversification of its topological structure is conducive to the optimization of its biological activity. In this paper, we report a facile strategy for the synthesis of thioether-bonded head-to-tail cyclized peptides containing cross-linking arms of different length. The key of our strategy is the development of AMDMBT-based 2-chlorotrityl resins, which are prepared by the treatment of 2-chlorotrityl chloride resin with aminoethanethiol, dibromomethylbenzene and dithiol. The AMDMBT 2-chlorotrityl resin allows us to conveniently obtain peptides containing bromoacetyl group at the N-terminus and structurally diverfied sulfhydryl group at the C-terminus. The peptides can undergo head-to-tail cyclization in solution to form thioether-bonded head-to-tail cyclized peptides. The reported method will facilitate the in-depth optimization of the structure and activity of thioether-bonded head-to-tail cyclized peptides.

Antioxidant and anti-aging potential of a peptide formulation (Gal2-Pep) conjugated with gallic acid.

Skin is highly vulnerable to premature aging due to external stress, therefore, in this study, a peptide formulation, (galloyl)2–KTPPTTP (Gal2–Pep) was synthesized by combining TPPTTP peptide, and gallic acid (GA). All peptides were synthesized on 2-chlorotrityl chloride resin using solid-phase peptide synthesis (SPPS), and analyzed on an electrospray ionization (ESI)/quadrupole-time-of-flight (Q-TOF) tandem mass spectroscopy (MS) system. Initially, Gal2–Pep showed no toxicity below the concentration 100 μM with cell survival rate of 88% for keratinocytes and fibroblasts. The reactive oxygen species (ROS) scavenging activity of Gal2–Pep was more stable compared to GA alone; and after four weeks at room temperature, its ROS scavenging activity remained higher than 50%. Moreover, the peptide formulation, Gal2–Pep also exhibited elastase inhibitory effect in CCD-1064Sk fibroblast cells. Based on the results of RT-qPCR, it was proved in this study that Gal2–Pep increased the expression of PGC-1α to prevent oxidative stress, and validated its potential as an anti-aging agent through increasing the expression of type I collagen and by decreasing the expression of matrix metalloproteinase-1 (MMP1). The findings obtained reinforce the suggestion that the peptide formulation synthesized in this study could be used as a natural antioxidant and anti-aging agent for its cosmetic applications.

Rapid, High-Yielding Solid-Phase Synthesis of Cathepsin-B Cleavable Linkers for Targeted Cancer Therapeutics.

Antibody-drug conjugates (ADCs) constitute an emerging class of anticancer agents that deliver potent payloads selectively to tumors while avoiding systemic toxicity associated with conventional chemotherapeutics. Critical to ADC development is a serum-stable linker designed to decompose inside targeted cells thereby releasing the toxic payload. A protease-cleavable linker comprising a valine-citrulline (Val-Cit) motif has been successfully incorporated into three FDA-approved ADCs and is found in numerous preclinical candidates. Herein, we present a high-yielding and facile synthetic strategy for a Val-Cit linker that avoids extensive protecting group manipulation and laborious chromatography associated with previous syntheses and provides yields that are up to 10-fold higher than by standard methods. This method is easily scalable and takes advantage of cost-effective coupling reagents and high loading 2-chlorotrityl chloride (2-CTC) resin. Modularity allows for introduction of various conjugation handles in final stages of the synthesis. Facile access to such analogues serves to expand the repertoire of available enzymatically cleavable linkers for ADC generation. This methodology empowers a robust and facile library generation and future exploration into linker analogues containing unnatural amino acids as a selectivity tuning tool.

Design and Synthesis of Near-Infrared Mechanically Interlocked Molecules for Specific Targeting of Mitochondria.

The entrapment of squaraine (SQ) within a molecular container to form rotaxane has been shown to improve the dye stability and the fluorescence proficiency inside the mitochondria. The macrocycle provides shelter and protects the near-infrared (NIR) SQ chromophore from nucleophilic attacks made by the exposed thiol of Cys-containing mitochondrial proteins and mitochondrial glutathione. Herein a microwave-assisted template-directed clipping reaction on low-loading 2-chlorotrityl chloride resin is used to develop an NIR unsymmetrical squaraine rotaxane in high quantum yield.

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.

Synthesis of Tetrapeptides and Screening of their Antioxidant Properties

Background: Tetrapeptide Pro-Ala-Gly-Tyr (PAGY) is an antioxidant peptide that was isolated by Nikoo et al. (2014) from hydrolysate of skin gelatin of amur sturgeon fish (Acipenser schrenckii). This research aims to synthesize PAGY and its analogues by a solid-phase method, and to screen their antioxidant activities. Methods: PAGY and its analogues, namely, Pro-Ser-Gly-Tyr (PSGY), Pro-Ala-Phe-Tyr (PAFY), Pro- Phe-Phe-Tyr (PFFY) and Pro-Ala-Ile-Tyr (PAIY), were synthesized via a solid phase peptide synthesis method with the Fmoc/t-Bu strategy. The synthesis was undertaken on 2-chlorotritylchloride resin as solid support, and all coupling reactions were facilitated by a combination of HBTU and HOBt reagents. All peptides were cleaved from the resin by using 95% TFA in water. Results: Through the solid-phase synthesis method, all peptides were obtained in 50-85% yields. Pure peptides were analysed by analytical RP-HPLC, and were characterized by HR-TOF-ESI-MS and 1HNMR. DPPH inhibition assay was applied to all of the peptides with IC50 of 1.750; 1.116; 1.177 ;1.437; and 1.642 mg/mL for PAGY, PSGY, PAFY, PFFY, and PAIY, respectively. Conclusion: PAGY and its four analogs were successfully synthesized by solid-phase method. PSGY compound was found to have the highest activity of all in DPPH assay.

Convergent Synthesis of the Rat Galanin and Study of Its Biological Activity

The full-length rat galanin (GWTLNSAGYLLGPHAIDNHRSFSDKHGLT-NH2, G29) was prepared by the solid phase peptide synthesis using the Fmoc-strategy. The peptide chain was elongated both by one amino acid and by a fragment condensation. Fragments with the C-terminal glycine residue were synthesized by the solid phase method on the 2-chlorotrityl chloride resin or by the method of conventional peptide synthesis in solution. After the reversed phase HPLC, galanin had the correct molecular weight and 98% purity. We studied the G29 cardioprotective properties on a model of acute myocardial infarction in rats. The G29 administration reduced the infarct size by 40% and decreased the activity of necrosis markers CK-MB (Creatine Kinase-MB) and LDH (Lactate Dehydrogenase) in the blood plasma. The peptide improved metabolic state of the infarcted heart, increased the ATP content, the total adenine nucleotide pool, phosphocreatine, and total creatine, and decreased the lactate level compared to a control. These results indicated the possibility of the G29 use as a drug for reducing the myocardial reperfusion injury. In addition, the mechanisms of the G29 action should be studied.