Academic literature on the topic 'Dry-column flash chromatography'

Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles

Select a source type:

Consult the lists of relevant articles, books, theses, conference reports, and other scholarly sources on the topic 'Dry-column flash chromatography.'

Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.

You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.

Journal articles on the topic "Dry-column flash chromatography"

1

Shusterman, Alan J., Patrick G. McDougal, and Arthur Glasfeld. "Dry-Column Flash Chromatography." Journal of Chemical Education 74, no. 10 (1997): 1222. http://dx.doi.org/10.1021/ed074p1222.

Full text
APA, Harvard, Vancouver, ISO, and other styles
2

Cvetković, Mirjana, Ana Damjanović, Tatjana P. Stanojković, et al. "Integration of dry-column flash chromatography with NMR and FTIR metabolomics to reveal cytotoxic metabolites from Amphoricarpos autariatus." Talanta 206, Jan 1 (2020): 120248. https://doi.org/10.1016/j.talanta.2019.120248.

Full text
Abstract:
Metabolomics generate a profile of small molecules from plant extracts, which could be directly responsible for bioactivity effects. Using dry-column flash chromatography enabled a rapid and inexpensive method for the very efficient separation of plant extract with a high resolution. This separation method coupled to NMR and FTIR-based metabolomics is applied to identify bioactive natural products. OPLS multivariate analysis method, was used for correlation the chemical composition of the plant extracts, Amphoricarpos autariatus, with the results of cytotoxic activity against Human cervical adenocarcinoma cell line (HeLa) and epithelial lung cancer cell line (A549). In this way, the highest contribution to the cytotoxic activity was recorded for the guaianolide sesquiterpene lactones named amphoricarpolides. The compounds indicated as bioactive after metabolomics analysis were tested, and their cytotoxic activity were confirmed.
APA, Harvard, Vancouver, ISO, and other styles
3

Cvetković, Mirjana, Ana Damjanović, Tatjana P. Stanojković, et al. "Integration of dry-column flash chromatography with NMR and FTIR metabolomics to reveal cytotoxic metabolites from Amphoricarpos autariatus." Talanta 206 (January 2020): 120248. http://dx.doi.org/10.1016/j.talanta.2019.120248.

Full text
APA, Harvard, Vancouver, ISO, and other styles
4

de Avellar, Isa G. J., Taís A. P. G. Cotta, and Amarílis de V. Finageiv Neder. "Using Artificial Soil and Dry-Column Flash Chromatography To Simulate Organic Substance Leaching Process: A Colorful Environmental Chemistry Experiment." Journal of Chemical Education 89, no. 2 (2011): 248–53. http://dx.doi.org/10.1021/ed100728j.

Full text
APA, Harvard, Vancouver, ISO, and other styles
5

Santos-Rebelo, Ana, Catarina Garcia, Carla Eleutério, et al. "Development of Parvifloron D-loaded Smart Nanoparticles to Target Pancreatic Cancer." Pharmaceutics 10, no. 4 (2018): 216. http://dx.doi.org/10.3390/pharmaceutics10040216.

Full text
Abstract:
Pancreatic cancer is the eighth leading cause of cancer death worldwide. For this reason, the development of more effective therapies is a major concern for the scientific community. Accordingly, plants belonging to Plectranthus genus and their isolated compounds, such as Parvifloron D, were found to have cytotoxic and antiproliferative activities. However, Parvifloron D is a very low water-soluble compound. Thus, nanotechnology can be a promising delivery system to enhance drug solubility and targeted delivery. The extraction of Parvifloron D from P. ecklonii was optimized through an acetone ultrasound-assisted method and isolated by Flash-Dry Column Chromatography. Then, its antiproliferative effect was selectivity evaluated against different tumor cell lines (IC50 of 0.15 ± 0.05 μM, 11.9 ± 0.7 μM, 21.6 ± 0.5, 34.3 ± 4.1 μM, 35.1 ± 2.2 μM and 32.1 ± 4.3 μM for BxPC3, PANC-1, Ins1-E, MCF-7, HaCat and Caco-2, respectively). To obtain an optimized stable Parvifloron D pharmaceutical dosage form, albumin nanoparticles were produced through a desolvation method (yield of encapsulation of 91.2%) and characterized in terms of size (165 nm; PI 0.11), zeta potential (−7.88 mV) and morphology. In conclusion, Parvifloron D can be efficiently obtained from P. ecklonii and it has shown selective cytotoxicity to pancreatic cell lines. Parvifloron D nanoencapsulation can be considered as a possible efficient alternative approach in the treatment of pancreatic cancer.
APA, Harvard, Vancouver, ISO, and other styles
6

Mekarunothai, Amita, Markus Bacher, Raveevatoo Buathong та ін. "β-sitosterol isolated from the leaves of Trema orientalis (Cannabaceae) promotes viability and proliferation of BF-2 cells". PeerJ 12 (23 січня 2024): e16774. http://dx.doi.org/10.7717/peerj.16774.

Full text
Abstract:
Trema orientalis is a pioneer species in the cannabis family (Cannabaceae) that is widely distributed in Thai community forests and forest edges. The mature leaves are predominantly used as an anti-parasite treatment and feed for local freshwater fish, inspiring investigation of their phytochemical composition and bioactivity. The purpose of this work was to investigate the bioactive compounds in T. orientalis leaf extract and their cytotoxicity in the BF-2 fish cell line (ATCC CCL-91). Flash column chromatography was used to produce 25 mL fractions with a mixture solvent system comprised of hexane, diethyl ether, methanol, and acetone. All fractions were profiled with HPLC-DAD (mobile phase methanol:aqueous buffer, 60:40 v/v) and UV detection (wavelengths 256 and 365 nm). After drying, a yellowish powder was isolated from lipophilic leaf extract with a yield of 280 µg/g dry weight. Structure elucidation by nuclear magnetic resonance (NMR) indicated it to consist of pure β-sitosterol. The lipophilic extract and pure compound were evaluated for cytotoxicity using BF-2 cells. MTT assays showed both leaf extract and pure compound at 1 µg/mL to increase cell viability after 24 h treatment. The respective half maximal inhibitory concentration (IC50) values of leaf extract and β-sitosterol were 7,027.13 and 86.42 µg/ml, indicating a lack of toxicity in the BF-2 cell line. Hence, T. orientalis can serve as a source of non-toxic natural lipophilic compounds that can be useful as bioactive ingredients in supplement feed development.
APA, Harvard, Vancouver, ISO, and other styles
7

Fatharani, Deika Zhillan, Safri Ishmayana, Reginawanti Hindersyah, and Ukun M. S. Soedjanaatmadja. "Isolation and Characterization of Gibberellin (GA-3) from Banana Blossom of Musa balbisiana Colla and Effect of The Isolate Addition on The Growth of Soybean (Glycine max) Plant." Research Journal of Chemistry and Environment 26, no. 11 (2022): 28–37. http://dx.doi.org/10.25303/2611rjce28037.

Full text
Abstract:
There is a type of banana that is unique because the flesh is filled with black seeds locally known as “pisang batu” (Musa balbisiana Colla). The objective of research was directed to determine the content of GA3 in banana blossom of M. balbisiana Colla and its effect on the growth of soybean (Glycine max) plant in vegetative and generative phase. GA3 was prepared by maceration followed by extraction using ethyl acetate and further purified using adsorption column chromatography and preparative TLC. The isolated product was then characterized using FTIR and HPLC. Sprouts of soybeans with uniform height were planted on polybags containing a mixture of planting media (soil: husk: manure = 3:2:1). The sprout was divided into five groups. The first group is the control only spraying with water, while others groups were treated by spraying GA3 isolate, crude extract, commercial Biostimulant and 1% NPK fertilizer. The soybean was determined for chlorophyll content of the leaf, proximate content and α-amylase activity. The results of HPLC and FTIR on gibberelline isolates showed a retention time and wave number which is relatively similar to the GA3 standard. GA3 levels in banana blossom of M. balbisiana Colla were 2.55 mg/g dry weight. The results of research showed that the GA3 isolate, crude extract and commercial biostimulant have significant effects on the growth of soybean plant both in the vegetative and generative phases for each parameter determined, proximate content, chlorophyll content and α-amylase activity compared to the control and NPK fertilizer.
APA, Harvard, Vancouver, ISO, and other styles
8

Shankar Lankalapalli, Ravi, Ambili Sasikumar Athira, Ramkumar Kiruthika, et al. "NMR-based phytochemical profiling of palmyra palm syrup infused with dry ginger, black pepper, and long pepper." Current Nutraceuticals 04 (January 12, 2023). http://dx.doi.org/10.2174/2665978604666230112144757.

Full text
Abstract:
Background: Trikatu, a vital ingredient in many Indian Ayurvedic drugs, is a consortium of three spices, viz. dry ginger, black pepper, and long pepper, known for its peculiar pungency. To convert Trikatu into a widely acceptable palatable form, we blended these three spices in a decoction form and added them to syrup prepared from palmyra palm neera, which resulted in ‘Trikatu Syrup’ (TS). Recently, we reported in vivo immunomodulatory properties of TS [1]. Introduction: The immunomodulatory effects of spices are attributed largely to the presence of certain phytochemicals. The importance of phytochemicals in spices as immunomodulatory agents necessitate a thorough investigation of these bioactives in formulations comprising spices. In the present study, we have focused on understanding the retention of spice and syrup-based phytochemicals in the formulated product that assists in product standardization of TS. Methods: NMR serves as a highly reliable tool for explicit structural confirmation of phytochemicals when compared to HPLC or mass spectrometry tools. NMR spectra of a phytochemical, whether in pure form or when the phytochemical is a part of the mixture, enable qualitative and quantitative studies with a mixture of phytochemicals in organic extracts of food matrices. Hence, the NMR spectral comparison of compounds isolated from the organic extracts of TS is described here. Results: Fractionation of TS using Diaion® HP-20 resulted in the partitioning of compounds based on their polarity. Purification of the acetone fraction by column chromatography aided in the efficient isolation of compound 1 (pellitorine), compound 2 (piperine), compounds 3-5 (trienamides), and compound 6 (pipataline). Acetonitrile fraction yielded compound 7 (uridine) and compound 8 (3-O-methyl-myo-inositol), which were neither reported in the three spices nor palmyra palm. A qualitative display of the acetone fraction of TS with its phytochemicals 1-6 served as a fingerprint of TS. Conclusion: In summary, TS, a palatable spice-based nutraceutical in palmyra palm syrup with immunomodulatory potential 1, was thoroughly investigated for the phytochemical composition of its organic fractions. The process of fractionating TS using Diaion® HP-20, subsequent flash purification, and column chromatography facilitated the isolation of prominent phytochemicals. We report the utility of NMR as a reliable and efficient tool for fingerprinting phytochemicals in formulations, nutraceuticals, etc., which assists in ascertaining their authenticity.
APA, Harvard, Vancouver, ISO, and other styles
9

sprotocols. "Copolymerization preparation of cationic cyclodextrin chiral stationary phases for drug enantioseparation in chromatography." January 10, 2015. https://doi.org/10.5281/zenodo.13871.

Full text
Abstract:
Authors: Ren-Qi Wang, Teng-Teng Ong, Ke Huang, Weihua Tang & Siu-Choon Ng ### Abstract We described a facile and effective protocol wherein radical copolymerization is employed to covalently bond cationic β-cyclodextrin (β-CD) onto silica particles with extended linkage, resulting in a chiral stationary phase (IMPCSP) that can be used for the enantioseparation of racemic drugs in both high-performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC). Starting from commercially available chemicals, the IMPCSP is prepared in several steps: (i) reaction of β-CD with 1-(p-toluenesulfonyl)-imidazole to afford mono-6A-(p-toluenesulfonyl)-6A-deoxy-β-cyclodextrin (B); (ii) nucleophilic addition between B and 1-vinylimidazole and followed by treatment with anionic-exchange resin to give mono-vinylimidazolium-CD chloride (C); (iii) electrophilic addition between C and phenyl isocyanate to generate 6A-(3-vinylimidazolium)-6-deoxyperphenylcarbamate-β-CD chloride (D); (iv) reaction of silica gel with 3-methacryloxypropyltrimethoxysilane to engender vinylized silica (E); (v) immobilization of C onto vinylized silica via radical copolymerization with 2,3-dimethyl-1,3-butadiene in the presence of 2,2’-azobis(2-methylpropionitrile) (AIBN) to afford the desired chiral stationary phases. The overall IMPCSP preparation and column packing protocol requires ~2 weeks. ### Introduction Chromatographic methodologies have been extensively explored for accurate sample analyses, online monitoring of reaction progress and purifying of synthesized products (1). Especially, modern chromatographic techniques have been developed as powerful tools for chiral separation and preparation of enantiomers, most of which are of biological and pharmaceutical interests. Whatever chiral chromatographic techniques are used, chiral selectors either dissolved as mobile phases or mobilized onto supporting materials as stationary phases are crucial for successful and robust enantioseparations. Among the chiral selectors used so far, cyclodextrins (CDs) and their derivatives have been widely used in chiral chromatography since its first introduction by Armstrong et al. (2). In order to obtain a better solubility, the charged moieties are favorably introduced onto CD rims for capillary electrophoresis (CE). The self-mobility of CD in the ionized form enhances the separation ability when it is opposite to the electrophoretic mobility of the analytes (3). The hydrophobic moieties on the analytes could be included or adsorbed into the chiral cavity of CDs. Meanwhile, the analytes could also interact with the substituents on the CD rims (4,5). The native CDs and their chemically-modified derivatives afford fine-tuned hydrophobicities, charges and cone shapes etc., which ultimately result in different tightness and interactions for CD-analytes complexes selectors and thus chemically-manipulated diving forces for chiral recognitions (6-14). The charged CD-based chiral mobile phases and chiral stationary phases (CSPs) have been extensively explored in drug chiral analyses (15-20). A sulfated β-CD based CSP was developed which enabled great enantioseparations towards 33 racemic drugs in high-perfomace liquid chromatography (HPLC)9. Recently, ionic-liquids featured positively-charged CDs have been covalently bonded onto silica gels to prepare novel CSPs, which exhibited dual selectivity in HPLC for the enantioseparation of both polar and non-polar compounds (18). The additional electrostatic interactions are significant to achieve separations of polar analytes which may interact with the neutral CDs too weakly, where16 aromatic alcohol racemates and 2 drugs were achieved in polar organic mobile phases19. In our previous report, a series of coated CSPs based on fully derivatized cationic β-CD were prepared. These cationic β-CD CSPs have shown strong enantioseparation abilities and moderate retention times towards a series of α-phenyl alcohols in both supercritical fluid chromatography (SFC) and HPLC20. However, polar organic solvents in the mobile phase could cause damage to the coated CSPs. Besides, the efficiencies of the coated CSPs are usually low as the surface thickness of chiral selectors is hard to control (21). Figure-1 (R Wang) Syntheses of Ts-CD (B), compounds C and D. The immobilization of functionalized CD onto silica gel is generally achieved by chemical reactions between highly reactive substituent on CD and the functional groups on silica gel (14,16-18). Tedious synthetic approaches with possible protecting and de-protecting steps are usually required for successful grafting. Comparatively, the facile co-polymerization method is more accessible and reliable, universally employed in producing non-silica fillings for columns applicable for size-exclusion chromatography (SEC) or ion-exchange applications (22). The polymerized materials used for chromatography have wide applicable pH ranges and improved retentions for polar analytes, although lower mechanic strength and efficiency are often observed than the silica-based CSPs. The co-polymerization approach was also explored in the immobilizing of polysaccharide or enantiopure small molecules onto silica gel for the preparation of CSPs (23,24). Figure-2 (R Wang) Preparation of IMPCSP via radical copolymerization. In view of the our continuous and successful research endeavors in developing powerful cationic CDs as chiral selectors for both CE and HPLC (12,25,26), we recently developed the facile copolymerization methodology in preparing novel covalently-bonded cationic β-CD CSPs via co-polymerization approach (see Figs. 1 and 2). These as-prepared CSPs have successfully expanded the enatioseparation windows towards a broader range of chromatographic conditions for both HPLC and SFC application (27,28). The linkage between CD selector and silica-support was built by using diene (ca. 2,3-dimethyl-1,3-butadiene) as the third monomer for the dual copolymerization system with vinyllated CD and silica gel. The extended linkage can effectively improve the surface loading issue of CD onto silica gel, a challenge faced when direct immobilizing CD derivatives onto silica surface with short spacers due to steric hindrance (29). The as-developed CDs CSPs exhibited great potential in drug enantioseparations in both HPLC and SFC applications. The protocol describes herein the synthesis of imidazolium-based IMPCSP. This methodology can also be applied for the synthesis of other ammonium-based cationic CD CSPs (27,28), which may find wide applications for both drug enantioseparations and NOM assessments. ### Reagents 1. β-Cyclodextrin (β-CD; >95%; TCI, cat. no. C0900) - Imidazole (99%; Merck, cat. no. 436151) - Sodium hydroxide (NaOH, 97%; Sigma-Aldrich, cat. no. 138701) - Ammonium chloride (99.5%; Fluka, cat. no. 09725) - p-Toluenesulphonyl chloride (99%; Fluka, cat. no. 89730) - !CAUTION p-toluenesulfonyl chloride is very smelly and highly corrosive. It is recommended that it be weighed in a glovebox and transferred with sealed bottles or directly weighted out in reaction flask. Please refer to the MSD sheet of this compound for safety information. - Dichloromethane (CH2Cl2, 99.6%, ACS reagent; Sigma-Aldrich, cat. no. 443484) - 1-Vinylimidazole (≥ 99%; Sigma-Aldrich, cat. no. 235466) - Ethyl acetate (99.5%, ACS reagent; Sigma-Aldrich, cat. no. 141786) - n-Hexane (98.5%; Sigma-Aldrich, cat. no. 178918) - Phenyl isocyanate (≥ 98%; Sigma-Aldrich, cat. no. 185353) - Amberlite IRA-900 ion-exchange resin (Sigma-Aldrich, cat. no. 216585) - N,N-Dimethylformamide (DMF, 99.8%; Sigma-Aldrich, cat. no. 319937) - Pyridine (99.5%, Extra dry, Fisher, cat. no. AC33942) - ! CAUTION Pyridine is very harmful to eyes and skin. Goggle and gloves must be worn in handling pyridine and conduct experiments in well ventilated fumehood to avoid inhalation. - Chloroform (99.8%, ACS reagent; Fisher, cat. no. AC40463) - Magnesium sulphate (≥ 97%, anhydrous, reagent grade; Sigma-Aldrich, cat. no. 208094) - Nitrogen gas (ALPHAGAZ™; SOXAL) - Liquid nitrogen (SOXAL) - Paraffin oil (puriss.; Sigma-Aldrich, cat. no. 18512) - Silica gel (5 μm, Kromasil) - 3-Methacryloxypropyltri-methoxysilane (98%; Sigma-Aldrich, cat. no. 440159) - 2,2’-Azobis(2-methyl-propionitrile) (AIBN, ≥ 98%, purum; Sigma-Aldrich, cat. no. 11630) - Toluene (99.8%, anhydrous, reagent grade; Sigma-Aldrich, cat. no. 244511) - 2,3-Dimethyl-1,3-butadiene (98%; Sigma-Aldrich, cat. no. 145491) ### Equipment 1. Magnetic stirrer with thermal and speed controller (Heidolph) - Rotary evaporator (Büchi, R205) - Teflon-coated magnetic stirring bars - Vacuum pump - Balance - Round-bottomed flask - Conical flask - Pressure-equalizing addition funnel - Büchner funnel - Soxhlet extractor - Liebig condenser - Dewar dish - Glass and plastic syringes (polypropylene) - Disposable hypodermic syringe needles - NMR spectrometer (300 MHz; Brüker, cat. no. ACF300) - FTIR spectrometer (FTS165) - Vario EL universal CHNOS elemental analyzer - MALDI-TOF-MS (Shimadzu, AXIMA Confidence) - Stainless steel HPLC column (15 cm length, 2.1 mm in inner diameter; Isolation Technologies) - HPLC pump (LabAlliance-Scientific) - Filter membrane used for syringe (0.45 μm pore size; Millipore) - Membrane Filter (0.45 μm pore size; Millipore) - Agilent HPLC (HP 1100) equipped with a variable-wavelength detector (190–300 nm) - Jasco SFC (SF 2000) equipped with a variable-wavelength detector (190–900 nm) and a back pressure regulator (0-30 MPa) ### Procedure - **Synthesis of compound C** 1. Fit a 100-ml double-necked round-bottomed flask containing a Teflon-coated magnetic stir bar with a rubber septum and a Liebig condenser. Fit the condenser a rubber septum with inlet of dry N2 and an outlet towards a bubbler containing paraffin oil, in order to prevent the ingress of moisture and air. - Weigh out 6A-toluenesulfonyl-β-CD B 12.91 g (0.01 mol) into the flask. - Turn on the circulating water in the condenser. - Add 20 ml DMF into the flask and switch on the magnetic stirrer and heater. - Inject 1-vinylimidazole 3 ml (0.03 mol) into the flask though a plastic syringe. ? TROUBLESHOOTING - Allow the reaction to proceed at 90°C for 48 h under reflux. Cool down to room temperature. - Precipitate the product in acetone (200 ml). Collect the precipitate by filtration. Wash the raw product with acetone. - Dissolve the solid into water/methanol (200 ml/50 ml) - ! CAUTION The solution could be heated towards 50°C to afford a clear solution. - Fill a column (I.D. 30 × 250 mm) with Amberlite IRA-900 ion-exchange resin and washed with MilliQ water till the effluent pH going neutral. - Transfer the solution from step 8 into the column and let it hold for 1 h. Subsequently, collect the effluent drop by drop. Flush the column with equal volume MilliQ water and collect the effluent. - Distill off water on rotary evaporator to yield C as a light yellow solid (9.7 g, 78% yield) - PAUSE POINT Compound C can be stored in oven at 80°C for several weeks. - **Synthesis of compound D** - Fit a 250-ml double-necked, round-bottomed flask containing a Teflon-coated magnetic stir bar with a rubber septum and a Liebig condenser. Fit the condenser a rubber septum with inlet of dry N2 and an outlet towards a bubbler containing paraffin oil, in order to prevent the ingress of moisture and air. - Weigh out compound C 2.15 g (1.72 mmol) into the flask. - Add 20 ml dried pyridine into the flask and switch on the magnetic stirrer and heater. - ! CAUTION Pyridine is highly toxic solvent. All experiments dealing with pyridine should be operated in fumehood. Goggles, gloves and mask should be worn. - Inject phenyl isocyanate 12 ml (110.32 mmol) into the flask though a plastic syringe. - ! CAUTION Phenyl isocyanate has acute toxicity. It may cause severe skin burns and eye damage. It may cause allergy or asthma symptoms or breathing difficulties if inhaled. Adding 12 ml phenyl isocyanate as a whole would cause large extent of side reaction to produce triphenyl isocyanurate as a by-product. It is strongly recommended to add phenyl isocyanate dropwise with pressure equalizing funnel. Goggles, gloves and mask should be worn before experimental operation in fumehood. - ▲ CRITICAL STEP Phenyl isocyanate should be added with four equal portions. Add 12 ml as a whole would cause large extent of side reaction to produce triphenyl isocyanurate as a by-product. Add phenyl isocyanate drop by drop with pressure equalizing funnel would end up with its transformation in the funnel and the liquid colour changes to light yellow. - Allow the reaction to proceed at 85 °C for 20 h. Set up the vacuum distillation pipeline. - Distill off pyridine under reduced pressure at 85 °C. ? TROUBLESHOOTING - Dissolve the residue with chloroform 15 ml. - Add the solution into silica column. Flush the impurities with n-hexane/ethyl acetate (70:30 v:v). - ▲ CRITICAL STEP The ratio of n-hexane/ethyl acetate was determined by TLC analyses. The flash column separation progress was also monitored by TLC analyses. Lowering the ratio would result in an increase of the amount of solvents used for eluting the impurities completely. - Flush the product out of the column with MeOH. - Remove the solvent on rotary evaporator to yield D as a dark yellow solid (6.2 g, 66% yield). - PAUSE POINT Compound D can be stored in oven at 80 °C for several weeks. - **Synthesis of vinylized silica E** - Dry spherical silica gel particles (5 μm, 5g) in vacuum (10 mm Hg) at 150 °C for 24 h. Cool down to room temperature. - Fit a 250-ml double-necked, round-bottomed flask containing a Teflon-coated magnetic stir bar with a rubber septum and a Liebig condenser. Fit the condenser a rubber septum with inlet of dry N2 and an outlet towards a bubbler containing paraffin oil, in order to prevent the ingress of moisture and air. - Add dry toluene 100 ml into the flask. Switch on the magnetic stirrer and heater. - Inject 3-methacryloxypropyltrimethoxysilane (2.3 ml) into the flask. - Add dried silica gel from step 22 into the flask. ? TROUBLESHOOTING - Allow the reaction to stand at 90 °C for 18 h. Product was collected by filtration through 0.45 μm pore size membrane and washed with MeOH in Soxhlet apparatus overnight. - Collect the product by filtration through 0.45 μm pore size membrane and washed with MeOH in Soxhlet extractor overnight. - Dry the product overnight in an oven at 60°C in vacco to afford the vinylized silica E. - PAUSE POINT Vinylized silica E can be stored at room temperature for several months. - **Co-polymerization for preparation of IMPCSP** - Dissolve compound D (0.7 g) in chloroform (30 ml). Filter the solution through 0.45 μm pore size membrane. - Transfer the filtrate and drop onto vinylized silica gel E (1.4 g) evenly with a glass syringe. - ! CAUTION A glass syringe was preferred to avoid any introduction of siloxal impurities from plastic syringes. Chloroform could corrode the rubber piston of the plastic syringe. - Dry the colloid-like mixture in vacuum (10 mm Hg) at 25 °C. - Fit a 100-ml double-necked, round-bottomed flask containing a Teflon-coated magnetic stir bar with two rubber septums. - Add the solid from step 32 into the flask. Add AIBN (3 mg) into the flask. - ▲ CRITICAL STEP The amount of AIBN should be controlled. A less amount of AIBN would lead to slow reaction rate but a great amount resulted in rapid radical reactions and short chain growth. In both conditions, successful immobilized CD amounts were low. - Inject anhydrous toluene (20 ml) and 2,3-dimethyl-1,3-butadiene (2.6 ml) into the flask. - ▲ CRITICAL STEP 2,3-Dimethyl-1,3-butadiene should be added before heating started. It was initiated together with the vinyl groups in compound D and vinylized silica E. Therein, 2,3-dimethyl-1,3-butadiene could act as a interlink. A delayed addition led to a lower CD amount on IMPCSP, since the radicals formed on D and E could have been annihilated before 2,3-dimethyl-1,3-butadiene was added. - Freeze the mixture in liquid N2 in a Dewar dish and degas the reaction system in vacuum (10 mm Hg) for 0.5 h. - ! CAUTION Liquid N2 - Take the flask out of the Dewar dish and thaw at room temperature. - Repeat step 36 and 37 for three times. ? TROUBLESHOOTING - Fit the flask with a Liebig condenser. Fit the condenser a rubber septum with inlet of dry N2 and an outlet towards a bubbler containing paraffin oil, in order to prevent the ingress of moisture and air. - Switch on the magnetic stirrer and heater. Leave the reaction to proceed at 60 °C for 18 h. - Cool down the reaction mixture and collect the product by filtration through 0.45 μm pore size membrane. - Wrap the filter cake with filter paper. Extract the solid in a Soxhlet extractor overnight. - Stop heating and collect the silica from the Soxhlet extractor. Dry the product in an oven at 60 °C for 24 h. - Pack IMPCSP into an empty stainless steel column with MeOH at 8,000 psi for 30 min. - PAUSE POINT IMPCSP can be stored at room temperature for several months. ### Timing - Synthesis of B: ~40 h include synthesis of A. - Steps 1-11 Synthesis of C: Steps 1-5, 1 h; Step 6, 48 h; Steps 7-8, 1 h; Steps 9-10, 1 h; Step 11, 12 h. - Steps 12-21 Synthesis of D: Steps 12-14, 1 h; Step 15, 2 h; Step 16, 20 h; Step 17, 5 h; Steps 18-20, 12 h; Step 21, 12 h. - Steps 22-29 Preparation of E: Step 22, 12 h; Steps 23-25, 4 h; Steps 26-27, 18 h; Steps 28-29, 20 h. - Steps 30-44 Preparation of IMPCSP: Steps 30-32, 6 h; Steps 33-35, 1 h; Steps 36-38, 1 h; Steps 39-40, 20 h; Steps 41-42, 20 h; Step 43, 24 h; Step 44, 2 h. ### Troubleshooting **? TROUBLESHOOTING** Troubleshooting advices can be found in Table 1. ### Anticipated Results The success of immobilizing CD onto silica gel could be characterized by typical FT-IR vibration bands of phenyl-groups in phenylcarbamate substituents in CSP. The amount of immobilized CD derivatives on silica gel could be calculated based on elemental analyses results (%N exclusively from CD derivative). The representative analytical data of organic compounds: C, D, E and IMPCSP are given below. **Organic synthesis**: Compound C: m.p. 254-268°C. 1H NMR (300 MHz, DMSO-d6, δ ppm) 2.73 (m, 1H H-2), 2.88 (m, 1H, H-4), 3.00-3.15 (m,1H, H-5), 3.32-3.45 (overlap with solvent peak, 12H, H-2,4), 3.20-3.80 (overlap with solvent peak, 27H, H-3,5,6), 4.30-4.40 (m, 1H, OH-6), 4.48-4.59 (m, 6H, OH-6, =CH2vinyl) 4.84-4.86 (m, 6H, H-1) 5.00 (d, 1H, H-1) 5.40-5.50 (m, 1H, -CHvinyl) 5.64-5.84 (m, 13H, OH-2,3) 5.95-6.10 (d, 1H, OH-2), 7.87 (s, 1H, =CH-4im) 8.18 (s, 1H, CH-5im) 9.42 (s, 1H, =CH-2im) Compound D: m.p. 197-199°C. MALDI-TOF-MS [M+]: (expected) 3592.16; (found) 3592.07. 1H NMR (300 MHz, CDCl3, δ ppm) 3.00-6.00 (m, 52H, H-CD, H-vinyl) 6.00-7.80 (m, 100H, H-phenyl). Microanalysis for C187H175ClN22O54 (expected) C: 61.87%, H: 4.86%, N: 8.49%, (found) C: 60.25%, H: 5.13%, N: 9.11%. Surface modified silica gel E: Obvious vibration bands in FT-IR spectrum of 2964, 2855 cm-1 (C-H) 1705 cm-1 (C=O) 1635 cm-1 (C=C) and 1130 cm-1 (C-O and Si-O) represent the successful surface modification with mathacryloyl-groups. Microanalyses data give the surface double bond loading of 5 μm silica as 2.16 μmol/m2 based on the carbon content (Table 2) (30). **Prepared IMPCSP** The characteristic peaks in FT-IR spectrum at 1720 cm-1 (C=O), 1647, 1558, 1458 cm-1 (C=C phenyl group) and 1130 cm-1 (C-O and Si-O) show the CD derivative has been successfully bonded onto silica surface. The cyclodextrin derivatives’ grafting coverage was calculated based on the nitrogen content (%N), to be 0.09 μmol m-2 (Table 2)30. **Chromatographic separation results**: The packed column with IMPCSP was applied for enantiomeric separations in RP-LC, NP-LC and SFC respectively. The cationic β-CD exhibited good enantioselectivity and stability towards four representative racemic analytes in Figure 3. ### References 1. Ryan, J.F. *Chromatography: creating a central science*. (American Chemical Society, 2001). - Armstrong, D.W. et al. Separation of drug stereoisomers by the formation of beta-cyclodextrin inclusion complexes. *Science* 232, 1132-1135 (1986). - Amini, A. Recent developments in chiral capillary electrophoresis and applications of this technique to pharmaceutical and biomedical analysis. *Electrophoresis* 22, 3107-3130 (2001). - Gübitz, G. & Schmid, M.G. *Chiral separations: methods and protocols*. (Humana Press, Totowa, USA, 2004). - Cox, G.B. *Preparative enantioselective chromatography*. (Blackwell Pub., Oxford, UK, 2005). - Hinze, W.L. et al. Liquid chromatographic separation of enantiomers using a chiral beta-cyclodextrin-bonded stationary phase and conventional aqueous-organic mobile phases. *Anal. Chem*. 57, 237-242 (1985). - Lubda, D. et al. Monolithic silica columns with chemically bonded β-cyclodextrin as a stationary phase for enantiomer separations of chiral pharmaceuticals. *Anal. Bioanal. Chem*. 377, 892-901 (2003). - Guo, Z.M. et al. Synthesis, chromatographic evaluation and hydrophilic interaction/reversed-phase mixed-mode behavior of a “Click beta-cyclodextrin” stationary phase. *J. Chromatogr. A* 1216, 257-263 (2009). - Stalcup, A.M. & Gahm, K.H. A sulfated cyclodextrin chiral stationary phase for high-performance liquid chromatography. *Anal. Chem*. 68, 1369-1374 (1996). - Lai, X.H., Tang, W.H. & Ng, S.-C. Novel cyclodextrin chiral stationary phases for high performance liquid chromatography enantioseparation: effect of cyclodextrin type, *J. Chromatogr. A*, 1218, 5597-5601 (2011). - Lai, X.H., Tang, W.H. & Ng, S.-C. Novel -cyclodextrin chiral stationary phases with different length spacer for normal-phase high performance liquid chromatography enantioseparation, *J. Chromatogr. A*, 1218, 3496-3501 (2011). - Wang, Y. et al. Preparation of cyclodextrin chiral stationary phases by organic soluble catalytic ‘click’ chemistry. *Nat. Protoc*. 6, 935-942 (2011). - Poon, Y.F. et al. Synthesis and application of mono-2(A)-azido-2(A)-deoxyperphenyl-carbamoylated b-cyclodextrin and mono-2(A)-azido-2(A)-deoxyperacetylated beta-cyclodextrin as chiral stationary phases for high-performance liquid chromatography. *J. Chromatogr. A* 1101, 185-197 (2006). - Lai, X.H. & Ng, S.C. Enantioseparation on mono(6A-N-allylamino-6A-deoxy)permethylated -cyclodextrin covalently bonded silica gel. *J. Chromatogr. A* 1101, 53-59 (2004). - Cherkaoui, S. & Veuthey, J.L. Use of negatively charged cyclodextrins for the simultaneous enantioseparation of selected anesthetic drugs by capillary electrophoresis–mass spectrometry. *J. Pharm. Biomed. Anal*. 27, 615-626 (2002). - Wang, R.Q. et al. . Recent advances in pharmaceutical separations with supercritical fluid chromatography and chiral columns, *TrAC Trends Anal. Chem*., in press, DOI: 10.1016/j.trac.2012.02.012 (2012). - Zukowski, J., De Biasi, V. & Berthod, A. Chiral separation of basic drugs by capillary electrophoresis with carboxymethylcyclodextrins. *J. Chromatogr. A* 948, 331-342 (2002). - Armstrong, D.W. et al. Examination of ionic liquids and their interaction with molecules, when used as stationary phases in gas chromatography. *Anal. Chem*. 71, 3873-3876 (1999). - Zhou, Z. et al. Synthesis of ionic liquids functionalized β-cyclodextrin-bonded chiral stationary phases and their applications in high-performance liquid chromatography. *Anal. Chim. Acta* 678, 208-214 (2010). - Wang, R.Q. et al. Synthesis of cationic [beta]-cyclodextrin derivatives and their applications as chiral stationary phases for high-performance liquid chromatography and supercritical fluid chromatography. *J. Chromatogr. A* 1203, 185-192 (2008). - Glenn, K.M. & Lucy, C.A. Stability of surfactant coated columns for ion chromatography. *Analyst* 133, 1581-1586 (2008). - Walsh, G. *Pharmaceutical biotechnology: concepts and applications*. (John Wiley & Sons, Chichester, UK, 2007). - Chen, X.M. et al. Synthesis of chiral stationary phases with radical polymerization reaction of cellulose phenylcarbamate derivatives and vinylized silica gel. *J. Chromatogr. A* 1034, 109-116 (2004). - Gasparrini, F. et al. New hybrid polymeric liquid chromatography chiral stationary phase prepared by surface-initiated polymerization. *J. Chromatogr. A* 1064, 25-38 (2005). - Tang, W.H. & Ng, S.C. Synthesis of cationic single-isomer cyclodextrins for the chiral separation of amino acids and anionic pharmaceuticals. *Nat. Protoc*. 2, 3195-3200 (2007). - Tang, W.H. & Ng, S.C. Facile synthesis of mono-6-amino-6-deoxy-α-, -, γ-cyclodextrin hydrochlorides for molecular recognition, chiral separation and drug delivery, *Nat. Protoc*. 3, 691-697 (2008). - Wang, R.Q. et al. Cationic cyclodextrins chemically-bonded chiral stationary phases for high-performance liquid chromatography, *Anal. Chim. Acta* 718, 121-129 (2012). - Wang, R.Q. et al. Chemically bonded cationic -cyclodextrin derivatives and their applications in supercritical fluid chromatography, *J. Chromatogr. A* 1224, 97-203 (2012). - Liu, M. et al. Study on the preparation method and performance of a new β-cyclodextrin bonded silica stationary phase for liquid chromatography. *Anal. Chim. Acta* 533, 89-95 (2005). - Siles, B.A. et al. Retention and selectivity of flavanones on homopolypeptide-bonded stationary phases in both normal- and reversed-phase liquid chromatography. *J. Chromatogr. A* 704, 289-305 (1995). ### Acknowledgements We acknowledge funding from the A*STAR (SERC Grant No.: 0921010056) in support of this project. R.-Q.W. is grateful for the award of a research scholarship by NTU and helpful discussions with Dr A. Rajendran of NTU SCBE. ### Figures **Figure 1: Figure-1 (R Wang) Syntheses of Ts-CD (B), compounds C and D**. ![Fig 1](http://i.imgur.com/0zUfbJc.png "Fig 1") **Figure 2: Figure-2 (R Wang) Preparation of IMPCSP via radical copolymerization**. ![Fig 2](http://i.imgur.com/k4KD4M7.png "Fig 2") **Figure 3: Figure-3 Enantioseparations of racemic drugs using IMPCSP with multiple channel UV detector detection at 254 nm**. ![Fig 3](http://i.imgur.com/ExHrkWu.png "Fig 3") *Flow rate is 0.4 ml min-1 in normal-phase HPLC (NP-LC), 0.5 ml min-1 in reverse-phase HPLC (RP-LC) and 1.0 ml min-1 in SFC. Separation conditions are as follows: (a) 7-methoxyflavanone, reverse-phase HPLC buffer (0.1% TEAA pH 4.3)/MeOH (30/70); NP-LC n-hexane/2-propanol (97/3, v/v); SFC CO2/2-propanol (99/1, v/v); (b) 4’-hydroxyflavanone, RP-LC buffer (0.1% TEAA pH 4.3)/MeOH (40/60); NP-LC n-hexane/2-propanol (97/3, v/v); SFC CO2/2-propanol (99/1, v/v); (c) bendroflumethiazide, NP-LC n-hexane/2-propanol (85/15, v/v); SFC CO2/2-propanol (70/30, v/v); (d) althiazide, NP-LC n-hexane/2-propanol (70/30, v/v); SFC CO2/2-propanol (70/30, v/v)*. **Table 1: Troubleshooting table** [Download Table 1](http://www.nature.com/protocolexchange/system/uploads/2170/original/Table_1.pdf?1338951762) **Table 2: Microanalysis results** [Download Table 2](http://www.nature.com/protocolexchange/system/uploads/2171/original/Table_2.pdf?1338951848) **Full corrected version of the protocol: Copolymerization preparation of cationic cyclodextrin chiral stationary phases for drug enantioseparation in chromatography** [Download Full corrected version of the protocol](http://www.nature.com/protocolexchange/system/uploads/2178/original/protex.2012.023_-_full_text.doc?1339152323) ### Associated Publications 1. **Cationic cyclodextrins chemically-bonded chiral stationary phases for high-performance liquid chromatography**. Ren-Qi Wang, Teng-Teng Ong, Weihua Tang, and Siu-Choon Ng. *Analytica Chimica Acta* 718 () 121 - 129 [doi:10.1016/j.aca.2011.12.063](http://dx.doi.org/10.1016/j.aca.2011.12.063) - **Chemically bonded cationic β-cyclodextrin derivatives as chiral stationary phases for enantioseparation applications**. Ren-Qi Wang, Teng-Teng Ong, and Siu-Choon Ng. *Tetrahedron Letters* 53 (18) 2312 - 2315 [doi:10.1016/j.tetlet.2012.02.105](http://dx.doi.org/10.1016/j.tetlet.2012.02.105) - **Chemically bonded cationic β-cyclodextrin derivatives and their applications in supercritical fluid chromatography**. Ren-Qi Wang, Teng-Teng Ong, and Siu-Choon Ng. *Journal of Chromatography A* 1224 () 97 - 103 [doi:10.1016/j.chroma.2011.12.053](http://dx.doi.org/10.1016/j.chroma.2011.12.053) ### Author information **Ren-Qi Wang, Teng-Teng Ong, Ke Huang & Siu-Choon Ng**, Division of Chemical and Biomolecular Engineering, College of Engineering, Nanyang Technological University, 16 Nanyang Drive, Singapore 637722, Singapore **Weihua Tang**, Key Laboratory of Soft Chemistry and Functional Materials (Ministry of Education of China), Nanjing University of Science and Technology, Nanjing 210094, People’s Republic of China Correspondence to: Ren-Qi Wang (wangrenqi@gmail.com) Siu-Choon Ng (ngsc@ntu.edu.sg) *Source: [Protocol Exchange](http://www.nature.com/protocolexchange/protocols/2409) (2012) doi:10.1038/protex.2012.023. Originally published online 6 June 2012*.
APA, Harvard, Vancouver, ISO, and other styles
10

sprotocols. "Synthesis of nNOS-Capon interaction inhibitors: ZLc-002 and its derivatives." December 31, 2014. https://doi.org/10.5281/zenodo.13641.

Full text
Abstract:
Authors: Ting-You Li, Lei Chang, Yu Ma & Dong-Ya Zhu ### Abstract nNOS-Capon interaction is involved in anxiety disorder, disrupting the nNOS-Capon interaction has been demonstrated to show anxiolytic-like effect. In this protocol, N-(2-carbomethoxyacetyl)-D-valine methyl ester, a nNOS-Capon interaction inhibitor, we named it ZLc-002, was prepared by condensation of D-valine methyl ester hydrochloride with methyl malonyl chloride. The synthesis of some analogues of ZLc-002 was also described. ### Introduction Anxiety disorders are highly prevalent psychiatric diseases (1). Selective serotonin reuptake inhibitors (SSRIs) and benzodiazepines (BZDs) are the most commonly prescribed anxiolytics. However, severe side effects for BZDs and taking effect much slowly for SSRIs render their use problematic (2). Thus, there is need to develop novel anxiolytic agents that have quicker anxiolytic potential and are clinically well tolerated. Several lines of evidence suggest that nNOS plays a pivotal role in the pathogenesis of anxiety disorders (3). nNOS contains a PDZ domain that can interact with a variety of other proteins including post-synaptic density protein 95 (PSD-95) and carboxy-terminal PDZ ligand of nNOS (CAPON) (4), a scaffolding protein that regulates dendrite and synapse, and is associated with increased severity of posttraumatic stress disorder and depression (5). The N-terminal phosphotyrosine binding domain of CAPON binds to dexamethasone-induced ras protein 1 (Dexras1). Dexras1 is activated by S-nitrosylation induced by nNOS in response to N-methyl-D-aspartate receptors (NMDARs) stimulation in brain. It has been demonstrated that nNOS, Capon, and Dexras1 can form a ternary complex of nNOS-Capon-Dexras1. The nNOS–CAPON association facilitates nNOS activation of Dexras1 (6). In the brain, Dexras1 negatively regulates the phosphorylation of extracellular signal-regulated kinase (ERK) (7), a kinase substantially implicated in emotional behaviors. In a recent publication, we demonstrated that disruption of the interaction of nNOS and Capon exhibits significantly anxiolytic-like effect, furthermore, based on the structural feature of the nNOS PDZ domain that bonds to the C-terminal of Capon, a series of small molecular inhibitors of the PDZ domain were designed and synthesized, and especially, the N-(2-carbomethoxyacetyl)-D-valine methyl ester, we named it ZLc-002, showed significant in vitro and in vivo anxiolytic activities (8). In this protocol, the detailed synthetic procedures of the ZLc-002 and its analogues were described. ### Materials 1. N-Methylmorphinline (Sinopharm Chemical Reagent Co., Ltd (SCRC)) - D-Valine methyl ester hydrochloride (GL Biochem Ltd) - Methyl malonyl chloride (SCRC) - Citric acid (SCRC) - Methyl succinyl chloride (SCRC) - L-Valine methyl ester (GL Biochem) - Methyl 3-bromopropionate (SCRC) - D-Phenylalanine methyl ester hydrochloride (GL Biochem) - Cyanoacetic acid (SCRC) - Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP; GL Biochem) - Triethylamine (Et3N; SCRC) - N,N-Diisopropylethylamine (DIEA; SCRC) - Nitrobenzene (SCRC) - Methanol (CH3OH; SCRC) - Dichloromethane (CH2Cl2; SCRC) - Ehtyl acetate (EtOAc; SCRC) - Diethyl ether (Et2O; SCRC) - Sodium sulfate (Na2SO4; SCRC) - Sodium bicarbonate (NaHCO3; SCRC) - Sodium chloride (NaCl; SCRC) - Sodium hydroxide (NaOH; SCRC) - Hydrochloride (HCl; SCRC) - Silica gel (200-300 mesh; Qingdao Haiyang Chem. Co., Ltd) - Potassium iodide (KI; SCRC) - Sodium azide (NaN3; SCRC) ### Equipment 1. Analytical balance (G&G; Electronic, JJ124BC) - Heating and stirring mantles/heater - Water ring vacuum pump - Oil vacuum pump - Rotary evaporators - UV detector - Nuclear Magnetic Resonance (Bruker, AVANCE AV-300) - Agilent 6410B Triple Quadrupole LC/MS Electrospray System Bundle - Microwave apparatus ### Procedure Synthesis of N-(2-carbomethoxyacetyl)-D-valine methyl ester (ZLc-002) TIMING 20 h ![Figure 1](http://i.imgur.com/BLOX4nm.jpg "Figure 1") 1. Add N-methylmorphinline (2 ml, 18.45 mmol) dropwise to a solution of D-valine methyl ester hydrochloride (1.50 g, 9 mmol) in CH2Cl2 (35 ml) under -15°C. - Add methyl malonyl chloride (1 ml, 9.45 mmol) to the resulting reaction mixture while stirring for 30 min under -15°C. - CAUTION Methyl malonyl chloride is acrid. Carry out this reaction in a well-ventilated fume hood. Since methyl malonyl chloride is highly sensitive to moisture, the reaction should be carried out under anhydrous conditions. - Allow the reaction to warm to room temperature and stir at room temperature for 12 h. - Remove the solvent under vacuum, dilute the residue with water (8 ml), and transfer the solution into a 250-ml separation funnel. - CAUTION ZLc-002 is water soluble, if too much water is added, it makes the extraction difficult and results a low yield. - Extract the solution with EtOAc (4 × 50 mL) and combine the organic phases. - Wash the solution with 10% citric acid solution (10 mL), 5% NaHCO3 solution (10 mL), and saturated NaCl aqueous solution (2 × 50 mL) consecutively. - Dry the organic solution over anhydrous Na2SO4 for 0.5 h, filter the Na2SO4 using a fritted Buchner funnel into a 500-mL round-bottomed flask and concentrate under vacuum with a rotary evaporator. - Pack a chromatography column (2.8 × 40 cm) with a slurry of silica gel in EtOAc : PE = 1 : 2. - Transfer the crude product solution to the column with a Pasteur pipette. Wash the flask with 2 mL of solvent (EtOAc : PE = 1 : 2). - Elute the column with EtOAc : PE = 1 : 2. - Collect 25-mL fractions in test tubes. Identify the fractions containing the product using TLC (Rf = 0.5, EtOAc : PE = 1 : 2), and visualize it with iodine. Combine fractions which contain the product, and then remove the solvent under a rotary evaporator to yield the product as a crystalline solid. Synthesis of N-(3-carbomethoxypropionyl)-D-valine methyl ester (ZLc-004) TIMING 20 h ![Figure 2](http://i.imgur.com/ogYtdVM.jpg?1 "Figure 2") 1. Add Et3N (3.42 mL, 24.6 mmol) dropwise to a solution of D-valine methyl ester hydrochloride (2 g, 12 mmol) in CH2Cl2 (40 ml) under -15°C. - Add methyl succinyl chloride (1.55 ml, 12.6 mmol) to the resulting reaction mixture while stirring for 30 min under -15°C. - CAUTION Methyl succinyl chloride is acrid. Carry out this reaction in a well-ventilated fume hood. Since methyl succinyl chloride is highly sensitive to moisture, the reaction should be carried out under anhydrous conditions. - Allow the reaction to warm to room temperature and stir at room temperature for 12 h. - Remove the solvent under vacuum, dilute the residue with water (20 ml), and transfer the solution into a 250-ml separation funnel. - Extract the solution with EtOAc (4 × 60 mL) and combine the organic phases. - Wash the solution with 10% citric acid solution (2 × 20 mL), 5% NaHCO3 solution (2 × 20 mL), and saturated NaCl aqueous solution (2 × 50 mL) consecutively. - Dry the organic solution over anhydrous Na2SO4 for 0.5 h, filter the Na2SO4 using a fritted Buchner funnel into a 500-mL round-bottomed flask and concentrate under vacuum with a rotary evaporator. - Pack a chromatography column (2.8 × 40 cm) with a slurry of silica gel in EtOAc : PE = 2 : 3. - Transfer the crude product solution to the column with a Pasteur pipette. Wash the flask with 2 mL of solvent (EtOAc : PE = 2 : 3). - Elute the column with EtOAc : PE = 2 : 3. - Collect 25-mL fractions in test tubes. Identify the fractions containing the product using TLC (Rf = 0.65, EtOAc : PE = 1 : 2), and visualize it with iodine. - Combine fractions which contain the product, and then remove the solvent under a rotary evaporator to yield the product as a yellowish solid. Synthesis of N-(2-carboxyacetyl)-D-valine methyl ester (ZLc-002-1) TIMING 15 h ![Figure 3](http://i.imgur.com/22volH8.jpg?1 "Figure 3") 1. Weigh out 1.92 g of N-(2-carbomethoxyacetyl)-D-valine methyl ester (ZLc-002, 8.3 mmol) in a 100 mL round-bottomed flask, add 9 ml of methanol. - Cool down to about 0℃ with a ice-water bath. - Add 9.15 mL of NaOH solution (1 mol/l, 9.15 mmol), and stir at 0℃ for 15 min. - Remove the ice-water bath and allow the reaction to stir at room temperature for 6 h. - Remove the methanol under reduced pressure. - Adjust the pH to 2~3 by using concentrated HCl solution. - Extract the residue with EtOAc (4 × 50 mL) and combine the extracts. - Wash the solution with saturated NaCl aqueous solution (2 × 50 mL). - Dry the organic solution over anhydrous Na2SO4 for 0.5 h, filter the Na2SO4 using a fritted Buchner funnel into a 500-mL round-bottomed flask and concentrate under vacuum with a rotatory evaporator. - Pack a chromatography column (2.8 × 40 cm) with a slurry of silica gel in EtOAc : PE : HOAc = 15 : 5 : 1. - Transfer the crude product solution to the column with a Pasteur pipette. Wash the flask with 2 mL of solvent (EtOAc : PE : HOAc = 15 : 5 : 1). - Elute the column with EtOAc : PE : HOAc = 15 : 5 : 1. - Collect 25-mL fractions in test tubes. Identify the fractions containing the product using TLC (Rf = 0.45, EtOAc : PE : HOAc = 15 : 5 : 1), and visualize it with bromophenol blue. - Combine fractions which contain the product, and then remove the solvent under a rotary evaporator to yield the product as a yellowish liquid. Synthesis of N-(2-carboxyacetyl)-D-valine (ZLc-002-2) TIMING 15 h ![Figure 4](http://i.imgur.com/o3Ar6U0.jpg?1 "Figure 4") 1. Weigh out 1.16 g of N-(2-carbomethoxyacetyl)-D-valine methyl ester (ZLc-002, 5 mmol)) in a 100 mL round-bottomed flask, and add 10 ml of methanol. - Cool down to about 0℃ with an ice-water bath. - Add 11 mL of NaOH solution (1 mol/l, 11 mmol), and stir at 0℃ for 15 min. - Remove ice-water bath and allow the reaction to stir at 30℃ for 6 h. - Remove the methanol under reduced pressure. - Adjust the pH to 2~3 by using concentrated HCl solution. - Extract the residue with EtOAc (4× 50 mL) and combine the extracts. - Wash the solution with saturated NaCl aqueous solution (2 × 50 mL). - Dry the organic solution over anhydrous Na2SO4 for 0.5 h, filter the Na2SO4 using a fritted Buchner funnel into a 500-mL round-bottomed flask and concentrate under vacuum with a rotary evaporator. - Pack a chromatography column (2.8 × 40 cm) with a slurry of silica gel in EtOAc : PE : HOAc = 10 : 10 : 3. - Transfer the crude product solution to the column with a Pasteur pipette. Wash the flask with 2 mL of solvent (EtOAc : PE : HOAc = 10 : 10 : 3). - Elute the column with EtOAc : PE : HOAc = 10 : 10 : 3. - Collect 25-mL fractions in test tubes. Identify the fractions containing the product using TLC (Rf = 0.35, EtOAc : PE : HOAc = 10 : 10 : 3), and visualize it with bromophenol blue. - Combine fractions which contain the product, and then remove the solvent under a rotary evaporator to yield the product as a yellowish liquid. Synthesis of N-(2-carbomethoxyacetyl)-L-valine methyl ester (ZLc-034) TIMING 20 h ![Figure 5](http://i.imgur.com/RVaaywX.jpg?1 "Figure 5") This compound was synthesized by the method described for ZLc-002. Synthesis of N-(2-carbomethoxyethyl)-D-valine methyl ester (ZLc-006) TIMING 18 h ![Figure 6](http://i.imgur.com/Nh4Uckt.jpg?1 "Figure 6") 1. Add triethylamine (3.24 mL, 24.6 mmol) to a stirring solution of D-valine methyl ester hydrochloride (2.0 g, 12 mmol) in methanol (50 ml). - Add methyl 3-bromopropionate (1.6 ml, 14.4 mmol) to the reaction solution. - Add small amount of potassium iodide (10 mg) as catalyst. - Stir for 20 min at room temperature. - Heat the reaction mixture to refluxing while stirring for 12 h. - Cool down to room temperature. - Remove the solvent with a rotary evaporator. - Add ether (30 mL) to the residue to precipitate the produced triethylamine hydrogen bromide salt, wash the filtrate cake with ether (2 × 10 mL). - Remove the solvent with a rotary evaporator. - Pack a chromatography column (2.8 × 40 cm) with a slurry of silica gel in EtOAc : PE = 2 : 3. - Transfer the crude product solution to the column with a Pasteur pipette. Wash the flask with 2 mL of solvent (EtOAc : PE = 2 : 3). - Elute the column with EtOAc : PE = 2 : 3. - Collect 25-mL fractions in test tubes. Identify the fractions containing the product using TLC (Rf = 0.65, CHCl3: CH3OH : HOAc = 90 : 8 : 2), and visualize it with iodine. - Combine fractions which contain the product, and then remove the solvent under a rotary evaporator to yield the product as a reddish brown liquid. Synthesis of N-(2-carbomethoxyacetyl)-D-phenylalanine methyl ester (ZLc-011) TIMING 20 h ![Figure 7](http://i.imgur.com/eCFRPb0.jpg?1 "Figure 7") This compound was synthesized by the method described for ZLc-002. Synthesis of N-(2-cyanoacetyl)-D-valine methyl ester TIMING 22 h ![Figure 8](http://i.imgur.com/N3wLkjG.jpg?1 "Figure 8") 1. Add cyanoacetic acid (2.7 g, 59 mmol) to a solution of D-valine methy ester hydrochloride (5 g, 56 mmol) in methanol (60 ml) under 0℃. - Add DIEA (12 mL, 124 mmol) and stir for 5 mim. - Add PyBOP (18.6 g, 67 mmol) while stir for 30 min at 0℃. - Remove the ice-water bath, and stir the reaction mixture at room temperature for 12 h. - Remove the solvent with a rotary evaporator. - Dilute the residue with EtOAc 120 mL. - Transfer the solution to a 500-mL separation funnel, wash the solution with 10% citric acid solution (30 mL × 2), 5% NaHCO3 solution (30 mL × 2), and saturated NaCl aqueous solution (30 mL × 2) consecutively. - Dry the organic solution over anhydrous Na2SO4 for 0.5 h, filter the Na2SO4 using a fritted Buchner funnel into a 500-mL round-bottomed flask and concentrate under vacuum with a rotary evaporator. - Pack a chromatography column (2.8 × 40 cm) with a slurry of silica gel in EtOAc : PE = 1 : 1. - Transfer the crude product solution to the column with a Pasteur pipette. Wash the flask with 2 mL of solvent (EtOAc : PE = 1:1). - Elute the column with EtOAc : PE = 1 : 1. - Collect 25-mL fractions in test tubes. Identify the fractions containing the product using TLC (Rf = 0.62, EtOAc : PE = 1 : 1), and visualize it with iodine. - Combine fractions which contain the product, and then remove the solvent under a rotary evaporator to yield the product as a white solid. Synthesis of N-(2-tetrazoylacetyl)-D-valine methyl ester (ZLc-008) TIMING 12 h ![Figure 9](http://i.imgur.com/AB714yo.jpg?1 "Figure 9") 1. Add N-(2-cyanoacetyl)-D-valine methyl ester (4.0 g, 20 mmol) to nitrobenzene (30 mL). - Add sodium azide (1.69 g, 26 mmol) and triethylamine hydrochloride (3.56 g, 26 mmol) to the above solution. - Irradiate the reaction solution with a microwave apparatus at 100oC for 6 h. - Cool to room temperature, extract the solution with water (4 × 30 mL). - Combine the water extracts, wash the solution with ethyl ether (2 × 30 mL). - Acidify the solution with hydrochloride solution (2 M) to pH 2~3. - Extract the product with ethyl acetate (4 × 30 mL). - Combine the ethyl acetate extracts and wash the solution with saturated NaCl solution (2 × 30 mL). - Dry the organic solution over anhydrous Na2SO4 for 0.5 h, filter the Na2SO4 using a fritted Buchner funnel into a 500-mL round-bottomed flask and concentrate under vacuum with a rotary evaporator to about 10 mL. - Cool the solution in an ice-water bath, add petroleum ether (30 mL) to precipitate the product. - Collect the crystals by filtration, wash with small amount of petroleum ether and dry the product in a desiccator under vacuum for 2 h. ### Anticipated Results - N-(2-Carbomethoxyacetyl)-D-valine methyl ester (ZLc-002) - Yield 50%, white crystal. - TLC (EtOAc : PE= 1 : 2): Rf = 0.5. - 1H NMR (300 MHz,CDCl3) δ (ppm) : 0.96 (t, 6H, J = 6.48 Hz), 2.20 (s, 1H), 3.37 (s, 2H), 3.75 (s, 3H), 3.77 (s, 3H), 4.56 (s, 1H), 7.52 (s, 1H). - Mass (ESI) (m/z) [M+H]+ calculated for C10H18NO5, 232.25; found, 232.12. - N-(3-Carbomethoxypropionyl)-D-valine methyl ester (ZLc-004) - Yield 69%, yellowish solid. - TLC (EtOAc : PE = 2 : 3): Rf = 0.65. - 1H NMR (300 MHz,CDCl3) δ (ppm) : 0.91 (d, 3H, J = 6.84 Hz), 0.94 (d, 3H, J = 6.81 Hz), 2.09~2.21 (m, 1H), 2.53~2.59 (m, 2H), 2.61~2.73 (m, 2H), 3.69 (s, 3H), 3.74 (s, 3H), 4.53~4.58 (q, 1H), 6.13~6.16 (d, 1H, J = 8.22 Hz). - Mass (ESI) (m/z) [M+H]+ calculated for C11H20NO5, 246.27; found, 246.10. - N-(2-Carboxyacetyl)-D-valine methyl ester (ZLc-002-1) - Yield 62.7%, yellowish liquid. - TLC (EtOAc : PE : HOAc = 15 : 5 : 1): Rf = 0.45. - 1H NMR (500 MHz,CDCl3) δ (ppm) : 0.94 (d, 3H, J = 6.90 Hz), 0.96 (d, 3H J = 6.85 Hz), 2.19~2.24(m, 1H), 3.42 (s, 2H), 3.77 (s, 3H), 4.58~4.61 (q, 1H), 7.11 (s, 1H). - Mass (ESI) (m/z) [M+H]+ calculated for C10H18NO5, 232.25; found, 232.20. - N-(2-Carboxyacetyl)-D-valine (ZLc-002-2) - Yield 57.1%, yellowish solid. - TLC (EtOAc : PE : HOAc = 15 : 5 : 1): Rf = 0.35. - 1H NMR (500 MHz,DMSO-d6) δ (ppm) : 0.88 (d, 3H, J = 3.20 Hz), 0.89 (d, 3H, J = 3.25 Hz), 1.99~2.06 (m, 1H), 3.28~3.31 (m, 2H), 4.16~4.19 (m, 1H), 8.18 (d, 1H, J = 8.6 Hz), 12.50 (s, 2H). - Mass (ESI) (m/z) [M+H]+ calculated for C8H14NO5, 204.19; found, 204.22. - N-(2-Carbomethoxyacetyl)-L-valine methyl ester (ZLc-034) - Yield 37.7%, yellowish liquid. - TLC (EtOAc : PE = 1 : 2): Rf = 0.5. - 1H NMR (500 MHz,CDCl3) δ (ppm): 0.94 (d, 3H, J = 6.90 Hz), 0.97 (d, 3H, J = 6.90 Hz), 2.18~2.24 (m, 1H ), 3.38 (s, 2H), 3.75 (s, 3H), 3.77 (s, 3H), 4.55~4.57 (q, 1H), 7.53 (d, 1H, J = 6.85 Hz). - Mass (ESI) (m/z) [M+H]+ calculated for C10H18NO5, 232.25; found, 232.19. - N-(2-Carbomethoxyethyl)-D-valine methyl ester (ZLc-006) - Yield 90.6%, reddish brown liquid. - TLC (CHCl3 : CH3OH : HOAc = 90 : 8 : 2): Rf = 0.65. - 1H NMR (500 MHz,CDCl3) δ : 3.73 (s, 3H), 3.67 (s, 3H), 3.02 (d, 1H, J = 6.10 Hz), 2.97~2.93 (m, 1H), 2.75~2.70 (m, 1H), 2.52~2.47 (m, 2H), 2.11 (s,1H ), 1.93 (q, 1H, J = 6.80 Hz), 0.99~0.92 (m, 6H). - Mass (ESI) (m/z) [M+H]+ calculated for C10H20NO4, 218.26; found, 218.20. - N-(2-Carbomethoxyacetyl)-D-phenylalanine methyl ester (ZLc-011) - Yield 74.5%, yellowish liquid. - TLC (EtOAc : PE = 1 : 1): Rf = 0.7. - 1H NMR (300 MHz,CDCl3) δ : 7.38 (d, 1H, J = 6.42 Hz), 7.32~7.22 (m, 3H ), 7.13 (d, 2H, J = 7.32 Hz), 4.87 (q, 1H, J = 6.27 Hz), 3.72 (s, 6H), 3.30 (s, 2H), 3.21~3.05 (m, 2H)。 - Mass (ESI) (m/z) [M+H]+ calculated for C14H18NO5, 280.29; found, 280.33. - N-(2-Cyanoacetyl)-D-valine methyl ester - Yield 48.7%, white solid. - TLC (EtOAc : PE = 1 : 1): Rf = 0.62 - Mass (ESI) (m/z) [M+H]+ calculated for C9H15N2O3, 199.22; found, 199.23. - N-(2-Tetrazoylacetyl)-D-valine methyl ester (ZLc-008) - Yield 43.5%, white solid. - TLC (CHCl3 : CH3OH : HOAc = 90 : 8 : 2): Rf = 0.22 - 1H NMR (300 MHz,CDCl3) δ : 6.60 (s, 1H), 4.55 (dd, 1H, J1 = 8.61 Hz, J2 = 4.89 Hz), 3.77 (s, 3H), 2.71 (s, 1H), 2.27~2.16 (m, 1H), 0.96 (dd, 6H, J1 = 8.37 Hz, J2 = 7.11 Hz)。 - Mass (ESI) (m/z) [M+H]+ calculated for C9H16N5O3, 242.25; found, 242.30. ### References 1. Tye, K. M. et al. Amygdala circuitry mediating reversible and bidirectional control of anxiety. Nature 471, 358–362 (2011). - Rupprecht, R. et al. Translocator protein (18 kD) as target for anxiolytics without benzodiazepine-like side effects. Science 325, 490–493 (2009). - Zhou, Q. G. et al. Hippocampal neuronal nitric oxide synthase mediates the stress-related depressive behaviors of glucocorticoids by downregulating glucocorticoid receptor. J. Neurosci. 31, 7579–7590 (2011). - Jaffrey, S. R. et al. CAPON: a protein associated with neuronal nitric oxide synthase that regulates its interactions with PSD95. Neuron 20, 115–124 (1998). - Lawford, B. R. et al. NOS1AP is associated with increased severity of PTSD and depression in untreated combat veterans. J. Affect Disord. 147, 87–93 (2013). - Fang, M. et al. Dexras1: a G protein specifically coupled to neuronal nitric oxide synthase via CAPON. Neuron 28, 183–193 (2000). - Cheng, H. Y. et al. The molecular gatekeeper Dexras1 sculpts the photic responsiveness of the mammalian circadian clock. J Neurosci. 26, 12984–12995 (2006). - zhu, L. J. et al. CAPON–nNOS coupling can serve as a target for developing novel anxiolytics. Nat. Med. Diol: 10.1038/nm.3644 (2014). ### Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (91232304, 81222016, 81030023, 21303086), National Basic Research Program of China (973 Program) (2011CB504404). *Source: [Protocol Exchange (2014)](http://www.nature.com/protocolexchange/protocols/3427). Oiriginally published online 16 September 2014*
APA, Harvard, Vancouver, ISO, and other styles
We offer discounts on all premium plans for authors whose works are included in thematic literature selections. Contact us to get a unique promo code!