The E. coli K12 strain was grown in standard LB medium, harvested, washed in PBS, and lysed in 4% SDS, 100 mm Tris, pH 8.5. Lysates were briefly boiled and DNA sheared using a Sonifier (Branson Model 250). Lysates were cleared by centrifugation at 15,000 × g for 15 min and precipitated with acetone. Proteins were resuspended in 8 m urea, 25 mm Tris, pH 8.5, 10 mm DTT. After 30 min of incubation, 20 mm iodoacetamide was added for alkylation. The sample was then diluted 1:3 with 50 mm ammonium bicarbonate buffer, and the protein concentration was estimated via tryptophan fluorescence emission assay. After 5 h of digestion with LysC (Wako Chemicals) at room temperature, the sample was further diluted 1:3 with ammonium bicarbonate buffer, and trypsin (Promega) digestion was performed overnight (protein-to-enzyme ratio of 60:1 in each case). E. coli peptides were then purified by using a C18 Sep Pak cartridge (Waters, Milford, MA) according to the manufacturer's instructions. UPS1 and UPS2 standards (Sigma-Aldrich) were resuspended in 30 μl of 8 m urea, 25 mm Tris, pH 8.5, 10 mm DTT and reduced, alkylated, and digested in an analogous manner, but with a lower protein-to-enzyme ratio (12:1 for UPS1 and 10:1 for UPS2, both LysC and trypsin). UPS peptides were then purified using C18 StageTips. E. coli and UPS peptides were quantified based on absorbance at 280 nm using a NanoDrop spectrophotometer (Fisher Scientific). For each run, 2 μg of E. coli peptides were then spiked with 0.15 μg of either UPS1 or UPS2 peptides, and about 1.6 μg of the mix was then analyzed via liquid chromatography combined with mass spectrometry on a Q Exactive (Thermo Fisher). Data were analyzed with MaxQuant as described above for the proteome dataset. All files were searched against the E. coli complete proteome sequences plus those of the UPS proteins and common contaminants.
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Tryptophan
Tryptophan
Tryptophan is an essential amino acid that plays a crucial role in various physiological processes.
It is a precursor to the neurotransmitter serotonin, which regulates mood, sleep, and appetite.
Tryptophan is also involved in protein synthesis and has been studied for its potential benefits in areas such as mood disorders, sleep disturbances, and immune function.
Resesrchers can optimize their tryptophan studies by leveraging the AI-driven platform PubCompare.ai, which enhances reproducibility and accuracy by providing access to protocols from literature, pre-prints, and patents, as well as AI-driven comparisons to identify the best protocols and products for their research.
It is a precursor to the neurotransmitter serotonin, which regulates mood, sleep, and appetite.
Tryptophan is also involved in protein synthesis and has been studied for its potential benefits in areas such as mood disorders, sleep disturbances, and immune function.
Resesrchers can optimize their tryptophan studies by leveraging the AI-driven platform PubCompare.ai, which enhances reproducibility and accuracy by providing access to protocols from literature, pre-prints, and patents, as well as AI-driven comparisons to identify the best protocols and products for their research.
Most cited protocols related to «Tryptophan»
Acetone
Alkylation
ammonium bicarbonate
Biological Assay
Buffers
Centrifugation
Digestion
Enzymes
Escherichia coli
Escherichia coli K12
Fluorescence
Iodoacetamide
Liquid Chromatography
Mass Spectrometry
peptide E (adrenal medulla)
Peptides
Promega
Proteins
Proteome
Staphylococcal Protein A
Strains
Tromethamine
Trypsin
Tryptophan
Urea
The RSCU value of a codon [38 (link)] is its observed frequency divided by its expected frequency in the absence of usage bias (which is the average frequency of all codons for that amino acid). RSCU values are not affected by sequence length and amino acid frequency since these factors are eliminated during the computation. Codons used less than average, at average level (no bias) and more than average have RSCU values, respectively of <1, 1 and >1 [37 (link),40 (link),41 (link)]. Codons with RSCU value >1.6 were regarded as over-represented, while codons with RSCU values <0.6) were said to be under-represented. Stop codons and codons that uniquely code for an amino acid (ATG - methionine and TGG - tryptophan), are not relevant to an RSCU analysis. For each sequence in the datasets, RSCU values were calculated for the 59 relevant codons by a PERL script (available upon request).
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Amino Acids
Codon
Codon, Terminator
Methionine
Tryptophan
The fluorescent dye ATTO-425 from ATTO-TEC (Germany), N-acetyl tryptophan amide (NATA) from Sigma (USA) and PBS buffer from Sigma (USA) were used without further purification. ATTO-425 was dissolved in PBS buffer (pH 7.4), and NATA was dissolved in distilled water.
Fluorescence measurements were performed in a homemade spectrofluorimeter [20] (link) and Fluorolog-3 (Horiba, Japan), which have vertical slits and a Cary Eclipse spectrofluorimeter (Agilent Technologies, Australia), which has horizontal slits. Fluorescence measurements in the Fluorolog-3 (Horiba, Japan) were performed in the Resource Center for Optical and Laser Materials Research of St. Petersburg State University, Russia. The fluorescence emission and excitation spectra of NATA were recorded at λex = 280 nm and λem = 350 nm, respectively. The fluorescence excitation spectra of ATTO-425 were recorded at different wavelengths of emission in the range from 470 nm to 550 nm with a step of 10 nm. The fluorescence spectra of ATTO-425 were recorded with λex = 436 nm, corresponding to the maximum of the long-wavelength band of the dye.
All the concentrational dependences for NATA and ATTO-425 were measured at constant experimental conditions (the excitation slit and emission slit values, the scan speed and the setting of PMT Detector Voltage). In particular for Cary Eclipse spectrofluorimeter excitation and emission slits were 5 nm, the scan speed was 600 nm/min, PMT Detector Voltage was in the range 410–530 V. Once chosen it was unchanged for measurement all spectra for plotting the dependence of F(λex) on AFL. All experiments were performed in a cell of dimensions 10×10×4 mm Starna Cells, Inc (USA).
The fluorescence quantum yield of ATTO-425 was taken as 0.9 (Product cataloque 2013/2015, ATTO-TEC GmbH, Germany), and the fluorescence quantum yield of NATA was taken as 0.14 [21] . The absorption spectra were recorded using a U-3900H spectrophotometer (Hitachi, Japan) in cells from Hellma GMBH & Co. (Germany) with different optical path lengths: 10, 1, 0.1 and 0.01 mm (100-QS 10, 100-QS 1, 106-QS 0.1 and 106-QS 0.01 with cell holder 013.000).
Fluorescence measurements were performed in a homemade spectrofluorimeter [20] (link) and Fluorolog-3 (Horiba, Japan), which have vertical slits and a Cary Eclipse spectrofluorimeter (Agilent Technologies, Australia), which has horizontal slits. Fluorescence measurements in the Fluorolog-3 (Horiba, Japan) were performed in the Resource Center for Optical and Laser Materials Research of St. Petersburg State University, Russia. The fluorescence emission and excitation spectra of NATA were recorded at λex = 280 nm and λem = 350 nm, respectively. The fluorescence excitation spectra of ATTO-425 were recorded at different wavelengths of emission in the range from 470 nm to 550 nm with a step of 10 nm. The fluorescence spectra of ATTO-425 were recorded with λex = 436 nm, corresponding to the maximum of the long-wavelength band of the dye.
All the concentrational dependences for NATA and ATTO-425 were measured at constant experimental conditions (the excitation slit and emission slit values, the scan speed and the setting of PMT Detector Voltage). In particular for Cary Eclipse spectrofluorimeter excitation and emission slits were 5 nm, the scan speed was 600 nm/min, PMT Detector Voltage was in the range 410–530 V. Once chosen it was unchanged for measurement all spectra for plotting the dependence of F(λex) on AFL. All experiments were performed in a cell of dimensions 10×10×4 mm Starna Cells, Inc (USA).
The fluorescence quantum yield of ATTO-425 was taken as 0.9 (Product cataloque 2013/2015, ATTO-TEC GmbH, Germany), and the fluorescence quantum yield of NATA was taken as 0.14 [21] . The absorption spectra were recorded using a U-3900H spectrophotometer (Hitachi, Japan) in cells from Hellma GMBH & Co. (Germany) with different optical path lengths: 10, 1, 0.1 and 0.01 mm (100-QS 10, 100-QS 1, 106-QS 0.1 and 106-QS 0.01 with cell holder 013.000).
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Amides
Buffers
Cells
Fluorescence
Fluorescent Dyes
Radionuclide Imaging
Tryptophan
GCaMPs in pRSETa were transformed into chemically competent BL21(DE3)-pLysS, and purified via the N-terminal His tag. Protein concentration was determined by intrinsic tryptophan fluorescence. Calcium clamping was performed at pH 7.2 with 10 mM blends of K2H2EGTA and Ca2EGTA from the Calcium Calibration Kit #1 (Invitrogen). Free [Ca2+] levels were calculated using MAXCHELATOR (maxchelator.stanford.edu). Fluorescence spectra were recorded on a Safire2 (link) fluorescence plate reader (Tecan). The dynamic range here is calculated as Fmax/Fmin. Fmax is the fluorescence intensity at saturating [Ca 2+] and Fmin is the fluorescence intensity at zero [Ca 2+].
Calcium, Dietary
Fluorescence
Proteins
Tryptophan
Alexa Fluor 647
Alopecia, Androgenetic, 2
anti-IgG
Antibodies, Anti-Idiotypic
Bacteriophages
Biotin
Chickens
Clone Cells
Cloning Vectors
Digestion
DNA, A-Form
DNA Library
Edetic Acid
Gene Expression
Gene Fusion
Gene Library
Genes
Homologous Recombination
Isopropyl Thiogalactoside
Mutagenesis
neutravidin
Phage Display Techniques
Proteins
Rabbits
Saccharomyces cerevisiae
Strains
Streptavidin
Tryptophan
Most recents protocols related to «Tryptophan»
Example 2
A. Seed Treatment with Isolated Microbe
In this example, an isolated microbe from Tables 1-3 will be applied as a seed coating to seeds of corn (Zea mays). Upon applying the isolated microbe as a seed coating, the corn will be planted and cultivated in the standard manner.
A control plot of corn seeds, which did not have the isolated microbe applied as a seed coating, will also be planted.
It is expected that the corn plants grown from the seeds treated with the seed coating will exhibit a quantifiably higher biomass than the control corn plants.
The biomass from the treated plants may be about 1-10% higher, 10-20% higher, 20-30% higher, 30-40% higher, 40-50% higher, 50-60% higher, 60-70% higher, 70-80% higher, 80-90% higher, or more.
The biomass from the treated plants may equate to about a 1 bushel per acre increase over the controls, or a 2 bushel per acre increase, or a 3 bushel per acre increase, or a 4 bushel per acre increase, or a 5 bushel per acre increase, or more.
In some aspects, the biomass increase is statistically significant. In other aspects, the biomass increase is not statistically significant, but is still quantifiable.
B. Seed Treatment with Microbial Consortia
In this example, a microbial consortium, comprising at least two microbes from Tables 1-3 will be applied as a seed coating to seeds of corn (Zea mays). Upon applying the microbial consortium as a seed coating, the corn will be planted and cultivated in the standard manner.
A control plot of corn seeds, which did not have the microbial consortium applied as a seed coating, will also be planted.
It is expected that the corn plants grown from the seeds treated with the seed coating will exhibit a quantifiably higher biomass than the control corn plants.
The biomass from the treated plants may be about 1-10% higher, 10-20% higher, 20-30% higher, 30-40% higher, 40-50% higher, 50-60% higher, 60-70% higher, 70-80% higher, 80-90% higher, or more.
The biomass from the treated plants may equate to about a 1 bushel per acre increase over the controls, or a 2 bushel per acre increase, or a 3 bushel per acre increase, or a 4 bushel per acre increase, or a 5 bushel per acre increase, or more.
In some aspects, the biomass increase is statistically significant. In other aspects, the biomass increase is not statistically significant, but is still quantifiable.
C. Treatment with Agricultural Composition Comprising Isolated Microbe
In this example, an isolated microbe from Tables 1-3 will be applied as an agricultural composition, administered to the corn seed at the time of sowing.
For example, it is anticipated that a farmer will apply the agricultural composition to the corn seeds simultaneously upon planting the seeds into the field. This can be accomplished, for example, by applying the agricultural composition to a hopper/bulk tank on a standard 16 row planter, which contains the corn seeds and which is configured to plant the same into rows. Alternatively, the agricultural composition can be contained in a separate bulk tank on the planter and sprayed into the rows upon planting the corn seed.
A control plot of corn seeds, which are not administered the agricultural composition, will also be planted.
It is expected that the corn plants grown from the seeds treated with the agricultural composition will exhibit a quantifiably higher biomass than the control corn plants.
The biomass from the treated plants may be about 1-10% higher, 10-20% higher, 20-30% higher, 30-40% higher, 40-50% higher, 50-60% higher, 60-70% higher, 70-80% higher, 80-90% higher, or more.
The biomass from the treated plants may equate to about a 1 bushel per acre increase over the controls, or a 2 bushel per acre increase, or a 3 bushel per acre increase, or a 4 bushel per acre increase, or a 5 bushel per acre increase, or more.
In some aspects, the biomass increase is statistically significant. In other aspects, the biomass increase is not statistically significant, but is still quantifiable.
D. Treatment with Agricultural Composition Comprising Microbial Consortia
In this example, a microbial consortium, comprising at least two microbes from Tables 1-3 will be applied as an agricultural composition, administered to the corn seed at the time of sowing.
For example, it is anticipated that a farmer will apply the agricultural composition to the corn seeds simultaneously upon planting the seeds into the field. This can be accomplished, for example, by applying the agricultural composition to a hopper/bulk tank on a standard 16 row planter, which contains the corn seeds and which is configured to plant the same into rows. Alternatively, the agricultural composition can be contained in a separate bulk tank on the planter and sprayed into the rows upon planting the corn seed.
A control plot of corn seeds, which are not administered the agricultural composition, will also be planted.
It is expected that the corn plants grown from the seeds treated with the agricultural composition will exhibit a quantifiably higher biomass than the control corn plants.
The biomass from the treated plants may be about 1-10% higher, 10-20% higher, 20-30% higher, 30-40% higher, 40-50% higher, 50-60% higher, 60-70% higher, 70-80% higher, 80-90% higher, or more.
The biomass from the treated plants may equate to about a 1 bushel per acre increase over the controls, or a 2 bushel per acre increase, or a 3 bushel per acre increase, or a 4 bushel per acre increase, or a 5 bushel per acre increase, or more.
In some aspects, the biomass increase is statistically significant. In other aspects, the biomass increase is not statistically significant, but is still quantifiable.
A. Seed Treatment with Isolated Microbe
In this example, an isolated microbe from Tables 1-3 will be applied as a seed coating to seeds of corn (Zea mays). Upon applying the isolated microbe as a seed coating, the corn will be planted and cultivated in the standard manner.
A control plot of corn seeds, which did not have the isolated microbe applied as a seed coating, will also be planted.
It is expected that the corn plants grown from the seeds treated with the seed coating will exhibit a quantifiable and superior ability to tolerate drought conditions and/or exhibit superior water use efficiency, as compared to the control corn plants.
The drought tolerance and/or water use efficiency can be based on any number of standard tests from the art, e.g leaf water retention, turgor loss point, rate of photosynthesis, leaf color and other phenotypic indications of drought stress, yield performance, and various root morphological and growth patterns.
B. Seed Treatment with Microbial Consortia
In this example, a microbial consortium, comprising at least two microbes from Tables 1-3 will be applied as a seed coating to seeds of corn (Zea mays). Upon applying the microbial consortium as a seed coating, the corn will be planted and cultivated in the standard manner.
A control plot of corn seeds, which did not have the microbial consortium applied as a seed coating, will also be planted.
It is expected that the corn plants grown from the seeds treated with the seed coating will exhibit a quantifiable and superior ability to tolerate drought conditions and/or exhibit superior water use efficiency, as compared to the control corn plants.
The drought tolerance and/or water use efficiency can be based on any number of standard tests from the art, e.g leaf water retention, turgor loss point, rate of photosynthesis, leaf color and other phenotypic indications of drought stress, yield performance, and various root morphological and growth patterns.
C. Treatment with Agricultural Composition Comprising Isolated Microbe
In this example, an isolated microbe from Tables 1-3 will be applied as an agricultural composition, administered to the corn seed at the time of sowing.
For example, it is anticipated that a farmer will apply the agricultural composition to the corn seeds simultaneously upon planting the seeds into the field. This can be accomplished, for example, by applying the agricultural composition to a hopper/bulk tank on a standard 16 row planter, which contains the corn seeds and which is configured to plant the same into rows. Alternatively, the agricultural composition can be contained in a separate bulk tank on the planter and sprayed into the rows upon planting the corn seed.
A control plot of corn seeds, which are not administered the agricultural composition, will also be planted.
It is expected that the corn plants grown from the seeds treated with the with the agricultural composition will exhibit a quantifiable and superior ability to tolerate drought conditions and/or exhibit superior water use efficiency, as compared to the control corn plants.
The drought tolerance and/or water use efficiency can be based on any number of standard tests from the art, e.g leaf water retention, turgor loss point, rate of photosynthesis, leaf color and other phenotypic indications of drought stress, yield performance, and various root morphological and growth patterns.
D. Treatment with Agricultural Composition Comprising Microbial Consortia
In this example, a microbial consortium, comprising at least two microbes from Tables 1-3 will be applied as an agricultural composition, administered to the corn seed at the time of sowing.
For example, it is anticipated that a farmer will apply the agricultural composition to the corn seeds simultaneously upon planting the seeds into the field. This can be accomplished, for example, by applying the agricultural composition to a hopper/bulk tank on a standard 16 row planter, which contains the corn seeds and which is configured to plant the same into rows. Alternatively, the agricultural composition can be contained in a separate bulk tank on the planter and sprayed into the rows upon planting the corn seed.
A control plot of corn seeds, which are not administered the agricultural composition, will also be planted.
It is expected that the corn plants grown from the seeds treated with the with the agricultural composition will exhibit a quantifiable and superior ability to tolerate drought conditions and/or exhibit superior water use efficiency, as compared to the control corn plants.
The drought tolerance and/or water use efficiency can be based on any number of standard tests from the art, e.g leaf water retention, turgor loss point, rate of photosynthesis, leaf color and other phenotypic indications of drought stress, yield performance, and various root morphological and growth patterns.
A. Seed Treatment with Isolated Microbe
In this example, an isolated microbe from Tables 1-3 will be applied as a seed coating to seeds of corn (Zea mays). Upon applying the isolated microbe as a seed coating, the corn will be planted and cultivated in the standard manner.
A control plot of corn seeds, which did not have the isolated microbe applied as a seed coating, will also be planted.
It is expected that the corn plants grown from the seeds treated with the seed coating will exhibit a quantifiable and superior ability to utilize nitrogen, as compared to the control corn plants.
The nitrogen use efficiency can be quantified by recording a measurable change in any of the main nitrogen metabolic pool sizes in the assimilation pathways (e.g., a measurable change in one or more of the following: nitrate, nitrite, ammonia, glutamic acid, aspartic acid, glutamine, asparagine, lysine, leucine, threonine, methionine, glycine, tryptophan, tyrosine, total protein content of a plant part, total nitrogen content of a plant part, and/or chlorophyll content), or where the treated plant is shown to provide the same or elevated biomass or harvestable yield at lower nitrogen fertilization levels compared to the control plant, or where the treated plant is shown to provide elevated biomass or harvestable yields at the same nitrogen fertilization levels compared to a control plant.
B. Seed Treatment with Microbial Consortia
In this example, a microbial consortium, comprising at least two microbes from Tables 1-3 will be applied as a seed coating to seeds of corn (Zea mays). Upon applying the microbial consortium as a seed coating, the corn will be planted and cultivated in the standard manner.
A control plot of corn seeds, which did not have the microbial consortium applied as a seed coating, will also be planted.
It is expected that the corn plants grown from the seeds treated with the seed coating will exhibit a quantifiable and superior ability to utilize nitrogen, as compared to the control corn plants.
The nitrogen use efficiency can be quantified by recording a measurable change in any of the main nitrogen metabolic pool sizes in the assimilation pathways (e.g., a measurable change in one or more of the following: nitrate, nitrite, ammonia, glutamic acid, aspartic acid, glutamine, asparagine, lysine, leucine, threonine, methionine, glycine, tryptophan, tyrosine, total protein content of a plant part, total nitrogen content of a plant part, and/or chlorophyll content), or where the treated plant is shown to provide the same or elevated biomass or harvestable yield at lower nitrogen fertilization levels compared to the control plant, or where the treated plant is shown to provide elevated biomass or harvestable yields at the same nitrogen fertilization levels compared to a control plant.
C. Treatment with Agricultural Composition Comprising Isolated Microbe
In this example, an isolated microbe from Tables 1-3 will be applied as an agricultural composition, administered to the corn seed at the time of sowing.
For example, it is anticipated that a farmer will apply the agricultural composition to the corn seeds simultaneously upon planting the seeds into the field. This can be accomplished, for example, by applying the agricultural composition to a hopper/bulk tank on a standard 16 row planter, which contains the corn seeds and which is configured to plant the same into rows. Alternatively, the agricultural composition can be contained in a separate bulk tank on the planter and sprayed into the rows upon planting the corn seed.
A control plot of corn seeds, which are not administered the agricultural composition, will also be planted.
It is expected that the corn plants grown from the seeds treated with the agricultural composition will exhibit a quantifiable and superior ability to utilize nitrogen, as compared to the control corn plants.
The nitrogen use efficiency can be quantified by recording a measurable change in any of the main nitrogen metabolic pool sizes in the assimilation pathways (e.g., a measurable change in one or more of the following: nitrate, nitrite, ammonia, glutamic acid, aspartic acid, glutamine, asparagine, lysine, leucine, threonine, methionine, glycine, tryptophan, tyrosine, total protein content of a plant part, total nitrogen content of a plant part, and/or chlorophyll content), or where the treated plant is shown to provide the same or elevated biomass or harvestable yield at lower nitrogen fertilization levels compared to the control plant, or where the treated plant is shown to provide elevated biomass or harvestable yields at the same nitrogen fertilization levels compared to a control plant.
D. Treatment with Agricultural Composition Comprising Microbial Consortia
In this example, a microbial consortium, comprising at least two microbes from Tables 1-3 will be applied as an agricultural composition, administered to the corn seed at the time of sowing.
For example, it is anticipated that a farmer will apply the agricultural composition to the corn seeds simultaneously upon planting the seeds into the field. This can be accomplished, for example, by applying the agricultural composition to a hopper/bulk tank on a standard 16 row planter, which contains the corn seeds and which is configured to plant the same into rows. Alternatively, the agricultural composition can be contained in a separate bulk tank on the planter and sprayed into the rows upon planting the corn seed.
A control plot of corn seeds, which are not administered the agricultural composition, will also be planted.
It is expected that the corn plants grown from the seeds treated with the agricultural composition will exhibit a quantifiable and superior ability to utilize nitrogen, as compared to the control corn plants.
The nitrogen use efficiency can be quantified by recording a measurable change in any of the main nitrogen metabolic pool sizes in the assimilation pathways (e.g., a measurable change in one or more of the following: nitrate, nitrite, ammonia, glutamic acid, aspartic acid, glutamine, asparagine, lysine, leucine, threonine, methionine, glycine, tryptophan, tyrosine, total protein content of a plant part, total nitrogen content of a plant part, and/or chlorophyll content), or where the treated plant is shown to provide the same or elevated biomass or harvestable yield at lower nitrogen fertilization levels compared to the control plant, or where the treated plant is shown to provide elevated biomass or harvestable yields at the same nitrogen fertilization levels compared to a control plant.
The inoculants were prepared from isolates grown as spread plates on R2A incubated at 25° C. for 48 to 72 hours. Colonies were harvested by blending with sterile distilled water (SDW) which was then transferred into sterile containers. Serial dilutions of the harvested cells were plated and incubated at 25° C. for 24 hours to estimate the number of colony forming units (CFU) in each suspension. Dilutions were prepared using individual isolates or blends of isolates (consortia) to deliver 1×105 cfu/microbe/seed and seeds inoculated by either imbibition in the liquid suspension or by overtreatment with 5% vegetable gum and oil.
Seeds corresponding to the plants of table 15 were planted within 24 to 48 hours of treatment in agricultural soil, potting media or inert growing media. Plants were grown in small pots (28 mL to 200 mL) in either a controlled environment or in a greenhouse. Chamber photoperiod was set to 16 hours for all experiments on all species. Air temperature was typically maintained between 22-24° C.
Unless otherwise stated, all plants were watered with tap water 2 to 3 times weekly. Growth conditions were varied according to the trait of interest and included manipulation of applied fertilizer, watering regime and salt stress as follows:
-
- Low N—seeds planted in soil potting media or inert growing media with no applied N fertilizer
- Moderate N—seeds planted in soil or growing media supplemented with commercial N fertilizer to equivalent of 135 kg/ha applied N
- Insol P—seeds planted in potting media or inert growth substrate and watered with quarter strength Pikovskaya's liquid medium containing tri-calcium phosphate as the only form phosphate fertilizer.
- Cold Stress—seeds planted in soil, potting media or inert growing media and incubated at 10° C. for one week before being transferred to the plant growth room.
- Salt stress—seeds planted in soil, potting media or inert growing media and watered with a solution containing between 100 to 200 mg/L NaCl.
Untreated (no applied microbe) controls were prepared for each experiment. Plants were randomized on trays throughout the growth environment. Between 10 and 30 replicate plants were prepared for each treatment in each experiment. Phenotypes were measured during early vegetative growth, typically before the V3 developmental stage and between 3 and 6 weeks after sowing. Foliage was cut and weighed. Roots were washed, blotted dry and weighed. Results indicate performance of treatments against the untreated control.
The data presented in table 15 describes the efficacy with which a microbial species or strain can change a phenotype of interest relative to a control run in the same experiment. Phenotypes measured were shoot fresh weight and root fresh weight for plants growing either in the absence of presence of a stress (assay). For each microbe species, an overall efficacy score indicates the percentage of times a strain of that species increased a both shoot and root fresh weight in independent evaluations. For each species, the specifics of each independent assay is given, providing a strain ID (strain) and the crop species the assay was performed on (crop). For each independent assay the percentage increase in shoot and root fresh weight over the controls is given.
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Ammonia
Asparagine
Aspartic Acid
Biological Assay
Bosea thiooxidans
Calcium Phosphates
Capsicum
Cells
Chlorophyll
Cold Shock Stress
Cold Temperature
Crop, Avian
Dietary Fiber
DNA Replication
Droughts
Drought Tolerance
Embryophyta
Environment, Controlled
Farmers
Fertilization
Glutamic Acid
Glutamine
Glycine
Growth Disorders
Herbaspirillum
Herbaspirillum huttiense
Leucine
Lolium
Lycopersicon esculentum
Lysine
Maize
Massilia niastensis
Methionine
Microbial Consortia
Nitrates
Nitrites
Nitrogen
Novosphingobium rosa
Paenibacillus
Paenibacillus amylolyticus
Pantoea agglomerans
Pantoea vagans
Phenotype
Phosphates
Photosynthesis
Plant Development
Plant Embryos
Plant Leaves
Plant Proteins
Plant Roots
Plants
Polaromonas ginsengisoli
Pseudoduganella violaceinigra
Pseudomonas
Pseudomonas fluorescens
Rahnella
Rahnella aquatilis
Retention (Psychology)
Rhodococcus erythropolis
Rosa
Salt Stress
Sodium Chloride
Sodium Chloride, Dietary
Stenotrophomonas chelatiphaga
Stenotrophomonas maltophilia
Stenotrophomonas rhizophila
Stenotrophomonas terrae
Sterility, Reproductive
Strains
Technique, Dilution
Threonine
Triticum aestivum
Tryptophan
Tyrosine
Vegetables
Zea mays
Example 3
Effectiveness of Newly Evolved TpH Background Strain Using Schistosoma mansoni TpH
One of the 7 evolved high 5HTP-producers was selected to further evaluate if the mutations identified were only specifically beneficial to hsTpH2 or could be widely applicable to others. The chosen evolved strain was first cured to lose the evolution plasmid (e.g. the hsTpH gene) and this was immediately followed by re-introducing the E. coli tyrA gene. Upon restoration of the strain's tyrosine auxotrophy, the resulting strain was transformed with pHM2, which is identical to pHM1 used in the earlier evolution study except that the hsTpH gene was replaced with a Schistosoma mansoni TpH gene (SEQ ID NO:9). The 5HTP production of the resulting strain was compared to a wild-type strain carrying pHM2 in the presence of 100 mg/l tryptophan. Results showed the wild-type transformants could only produce ˜0.05 mg/l 5HTP while the newly evolved background strain transformants accumulated >20 mg/l. These production results demonstrated that the mutations acquired in the evolved background strain were not only beneficial to hsTpH but also to other TpHs; possibly applicable also to other aromatic amino acid hydroxylases (e.g. tyrosine hydroxylase).
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5-Hydroxytryptophan
Aromatic Amino Acids
Biological Evolution
Cells
Escherichia coli
Genes
Melatonin
Mixed Function Oxygenases
Mutation
Plasmids
Schistosoma mansoni
Strains
Tryptophan
Tyrosine
Tyrosine 3-Monooxygenase
Example 2
Directed TpH Engineering
It was found that Homo sapiens TpH2, i.e., the fragment set forth as SEQ ID NO:13; hsTpH2, was sensitive to p-chlorophenylalanine. However, mutations at residues N97 and/or P99 were found to confer resistance to p-chlorophenylalanine and to exhibit improved 5HTP biosynthesis after growing cells in the presence of 100 mg/l of tryptophan overnight at 3TC. A further, saturated mutagenesis, study found that isoleucine (I) was a beneficial amino acid change at residue N97, while cysteine (C), aspartic acid (D), leucine (L) and glutamine (Q) were shown to be beneficial at residue P99. In particular, the combined changes 1\197I/P99D in hsTpH2 showed a >15% increase in 5HTP production in the presence of 100 mg/l tryptophan and the combined changes N97I/P99C in hsTpH2 showed a >25% increase in 5HTP biosynthesis, over the parent TPH2 sequence (SEQ ID NO:13) after acquiring the E2K mutation.
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5-Hydroxytryptophan
Amino Acids
Anabolism
Aspartic Acid
Cells
Cysteine
Fenclonine
Glutamine
Homo sapiens
Isoleucine
Leucine
Melatonin
Mutagenesis
Mutation
Parent
Tryptophan
Example 2
Tertiary propargylamine bridges were introduced into the peptide by initial incorporation of aza-propargylglycine and ε-N-alkyl-lysine residues into the GHRP-6 peptide sequence, followed by copper-catalyzed macrocyclization using an aldehyde linchpin. The A3-macrocyclization was examined immediately after introduction of the azapropargylglycine residue, as well as after completing the peptide sequence. To seek a diversity-oriented synthesis, two strategies were employed, in which an ε-N-alkyl-lysine residue was introduced respectively at the C-terminal and a central residue of the peptide sequence. With the ε-N-alkyl-lysine residue at the C-terminal, the macrocycle ring-size diversity was varied by azapropargyiglycine position scanning, in which the azapropargylglycyl residue was marched systematically to the N-terminal of the GHRP-6 sequence prior to macrocyclization with formaldehyde. With the ε-N-alkyl-lysine residue centred in the sequence, the influence of various & amino substituents was examined on macrocyclization.
The important step for the effective diversity-oriented synthesis of cyclic azapeptides by A3-macrocyclization was development of solid-phase methods to install the azapropargyiglycine residue and ε-N-alkyl-lysine residue into the peptide sequence prior to the copper-catalyzed macrocyclization using an aldehyde linchpin. The azapropargyiglycine can be inserted by submonomer synthesis of azapeptides on solid phase.[13] The ε-N-alkylated lysine was prepared in solution and then coupled to the resin-bound peptide; however, solid-phase ε-N-alkylation of lysine was also performed by Mitsunobu chemistry on the corresponding ε-N-o-nitrobenzenesulfonyl (o-NBS) amine.[20]
As a proof-of-concept of the A3-macrocyclization, cyclic azatripeptide 8 was pursued by placing ε-N-methyl lysine at the peptide C-terminal and inserting aza-propargyiglycine at the i+2 position. Prior to attachment to Rink amide resin, Fmoc-Lys(methyl, o-NBS)—OH 1 was synthesized from Boc-Lys-OH in solution. After Fmoc group removals and elongation with Fmoc-D-Phe-OH using DIC and HOBt, dipeptide 2a was acylated by the active carbazate prepared from benzophenone hydrazone and N,N′-disuccinimidyl carbonate (DSC) to provide semicarbazone 3a.[14] Propargylation was performed using Cs2CO3 (300 mol %) and proparyl bromide (600 mol %) to furnish the aza-propargyiglycine 4a in good purity as accessed by LCMS analysis of a cleaved aliquot. After removal of the o-NBS-group with 2-mercaptoethanol and DBU, secondary ε-N-methylamine 5a was ready to test the A3-macrocyclization. Macrocycle 6a was prepared successfully by treating aza-peptide 5a with CuI (20 mol %) and 37% aqueous formaldehyde (600 mol %) in DMSO at rt for 24 h, as verified by LCMS analysis. Elongation of macrocycle 6a to cyclic GHRP-6 analog 8 was accomplished by removal of the semicarbazone with hydroxylamine hydrochloride in pyridine, acylation of the resulting semicarbazide 7a using the symmetric anhydride from treating Fmoc-Ala-OH with DIC, and standard solid-phase peptide synthesis, deprotection and resin cleavage. GHRP-6 macrocycle 8 was isolated in 3.5% overall yield after purification by preparative HPLC. Employing the same strategy, macrocycle 9 was obtained in 2.4% overall yield.
With macrocyclic GHRP-6 analogs 8 and 9 in hand, ring-size scope was investigated by systematically moving the azapropargylglycine residue towards the N-terminal of the sequence. Moreover, the ε-N-alkyl-lysine residue was prepared on solid phase by a method designed to expand the diversity of the ε-amine substituent. After coupling Fmoc-Lys(o-NBS)—OH 10[19] to RINK amide resin and peptide elongation, semicarbazones 11a-d were synthesized. Chemoselective modification of the ε-N-o-(NBS)amine nitrogen was achieved by employing Mitsunobu chemistry to alkylate the former. Treatment of sulphonamide 11a-d with allyl alcohol, PPh3, and diisopropyl azodicarboxylate (DIAD) provided selectively ε-N-(allyl)lysinyl peptides 12a-d as verified by LCMS analysis of cleaved aliquots. Subsequently, propargylation of semicarbazone was performed using Cs2CO3 (300 mol %) and proparyl bromide (600 mol %) to yield aza-propargylglycine peptides 13a-d. A3-Macrocyclization was then performed using the same conditions as discussed above to provide respectively 16-, 19, 21, and 24-membered macrocycles 15a-d as verified by LCMS analysis. After cyclization, semicarbazone removal, semicarbazide acylation, peptide elongation and resin cleavage were performed as described above to afford cyclic GHRP-6 analogs 17 and 18 after purification by preparative HPLC (Table 1). Coupling to semicarbazide macrocycles 16c and 16d was however unsuccessful in the syntheses of the corresponding cyclic GHRP-6 analogs. Steric hindrance inhibited apparently, the coupling to the semicarbazide of the larger ring-sizes. Semicarbazide 16d was however cleaved from resin to give cyclic aza-hexapeptide 19 with a N-terminal semicarbazide after purification by preparative HPLC.
Failure to elongate semicarbazides 16c and 16d after cyclization promoted investigation of a strategy featuring elongation of the complete linear peptide prior to A3-macrocyclization as the penultimate step before simultaneous deprotection and resin cleavage. Semicarbazone 13a was thus treated with hydroxylamine hydrochloride to liberate the semicarbazide 20a, and the linear peptide was elongated as described for its cyclic counterpart above. Aza-hexapeptide 21a was treated with DBU and 2-mercaptoethanol to selectively remove the o-NBS group. Subsequently, aza-hexapeptide 23a was effectively converted to macrocycle 17 using the standard A3-macrocyclization conditions. Resin cleavage gave cyclic azapeptide 17 in about 2-fold higher yield (1.2%) than the earlier approach, involving peptide elongation after cyclization.
Employing the peptide elongation/A3-macrocyclization approach, linear peptides 22b-d were also successfully converted into macrocyclic aza-GHRP-6 analogs 18, 24 and 25. Cyclic azapeptides 24 and 25 were respectively prepared with N-terminal alanine residues to avoid racemization during coupling to the semicarbazide with histidine, and to add an N-terminal basic amine that may favor biological activity.
The diversity of the ε-amine substituent was explored by the synthesis of cyclic azatetrapeptides 30-32 employing different alcohols as electrophiles in the Mitsunobu reaction: methanol, allyl alcohol and isopropyl alcohol. An ε-N-alkylated lysine was inserted in the peptide sequence to replace the tryptophan residue and an azapropargylglycine was placed at the i+3 position to replace the histidine residue in the GHRP-6 sequence. Cyclic analog 33 was synthesized with an additional alanine in the N-terminal for comparison with analog 31 to study the importance of the N-terminal basic amine.
Cyclic azapeptide GHRP-6 analogs were synthesized by the A3-macrocyclization method in yields and purities suitable for biological evaluation (Table 1).
Solution-Phase Chemistry
Ornithine Building Block Synthesis
Fmoc-Orn(o-NBS)—OH (RGO1):
Fmoc-Orn(Boc)-OH (2.02 g, 4.44 mmol) was dissolved in CH2Cl2 (30 mL) treated with TFA (20 mL) stirred at room temperature for 3 hours, and the volatiles were removed by rotary evaporation. The resulting yellow oil was co-evaporated with toluene to give a residue that was dissolved in THF (40 mL) and water (40 mL) and treated with iPr2NEt (7.70 mL, 44.2 mmol) and o-NBSCl (1.13 g, 5.08 mmol) in one portion. The reaction was stirred at room temperature for 3 hours, diluted with EtOAc (100 mL) and sequentially washed with aqueous HCl (1 M, 100 mL×3), water (100 mL) and brine (100 mL). The organic layer was dried over MgSO4 and the volatiles were removed by rotary evaporation to give sulfonamide RGO1 (2.4 g, quant.) as a light yellow solid. The amino acid was used without further purification.
1H NMR (300 MHz, DMSO) δ 8.10 (t, J=5.6 Hz, 1H), 8.03-7.92 (m, 2H), 7.92-7.81 (m, 4H), 7.72 (d, J=7.4 Hz, 2H), 7.61 (d, J=8.0 Hz, 1H), 7.41 (t, J=7.2 Hz, 2H), 7.32 (t, J=7.1 Hz, 2H), 4.34-4.16 (m, 3H), 3.89 (td, J=8.7, 4.6 Hz, 1H), 2.90 (q, J=6.3 Hz, 2H), 1.73 (s, 1H), 1.65-1.43 (m, 3H). 13C NMR (75 MHz, DMSO) δ 173.7, 156.1, 147.8, 143.8, 140.7, 134.0, 132.7, 132.6, 129.4, 127.7, 127.1, 125.3, 124.4, 120.1, 65.6, 53.5, 46.7, 42.3, 27.9, 26.0. LCMS (10-90% MeOH containing 0.1% formic acid over 10 min) Rt=11.04 min. ESI-MS m/z calcd for C26H26N3O8S+ [M+H]+ 540.1, found 540.1. Melting point: 108-110° C.
Solid-Phase Chemistry
Fmoc-based peptide synthesis was performed using standard conditions (W. D. Lubell, J. W. Blankenship, G. Fridkin, and R. Kaul (2005) “Peptides.” Science of Synthesis 21.11, Chemistry of Amides. Thieme, Stuttgart, 713-809) on an automated shaker using polystyrene Rink amide resin. The loading was calculated from the UV absorbance for Fmoc-deprotection after the coupling of the first amino acid. Couplings of amino acids (3 equiv.) were performed in DMF using DIC (3 equiv.) and HOBt (3 equiv.) for 3-6 hours. Fmoc-deprotections were performed by treating the resin with 20% piperidine in DMF for 30 min. The resin was washed after each coupling and deprotection step sequentially with DMF (×3), MeOH (×3) THF (×3) and CH2Cl2 (×3).
Lysine as AA1
Fmoc-Lys(o-NBS)-Rink Amide Resin RGO7:
On Rink amide resin (3.00 g) in a syringe fitted with a Teflon™ filter, Fmoc removal was performed by treating the resin with a solution of 20% piperidine in DMF over 30 min. The resin was filtered and washed sequentially with DMF (×3), MeOH (×3) and CH2Cl2 (×3). Fmoc-Lys(o-NBS)—OH (1.62 g, 2.93 mmol) was dissolved in DMF (20 mL) and treated with DIC (0.7 mL, 4.52 mmol) and HOBt (611 mg, 4.52 mmol), stirred for 3 min. and added to the syringe containing the resin. The mixture was shaken for 14 hours. The resin was then filtered and sequentially washed with DMF (×3), MeOH (×3) and CH2Cl2 (×3). The resin was dried and the loading was measured at 0.345 mmol/g resin.
Fmoc-Lys(o-NBS, Allyl)-Rink Amide Resin RGO8:
Vacuum dried Fmoc-Lys(o-NBS)-resin RGO7 (0.441 mmol) was placed in a syringe fitted with a Teflon™ filter, suspended in THF (dry, 5 mL) and treated sequentially with solutions of allyl alcohol (206 μL, 3.03 mmol) in THF (dry, 1 mL), PPh3 (397 mg, 1.51 mmol) in THF (dry, 1 mL), and DIAD (298 μL, 1.51 mmol) in THF (dry, 1 mL). The mixture in the syringe was shaken for 90 min. The resin was filtered and sequentially washed with DMF (×3), MeOH (×3), THF (×3) and CH2Cl2 (×3). Examination by LCMS of a cleaved resin sample (5 mg) showed complete allylation: LCMS (30-95% MeOH containing 0.1% formic acid in water containing 0.1% formic acid over 10 min) Rt=8.65 min. ESI-MS m/z calcd for C30H33N4O7S+ [M+H]+ 593.2, found 593.2.
Boc-Ala-D-Pra-Ala-Trp(Boc)-D-Phe-Lys(o-NBS, Allyl)-Rink Amide Resin RGO99:
LCMS (30-95% MeOH containing 0.1% formic acid in water containing 0.1% formic acid over 10 min) Rt=5.73 min. ESI-MS m/z calcd for C46H57N10O10S+ [M−2Boc+H]+941.4, found 941.4.
Boc-Ala-L-Pra-Ala-Trp(Boc)-D-Phe-Lys(o-NBS, Allyl)-Rink Amide Resin RGO100:
LCMS (30-95% MeOH containing 0.1% formic acid in water containing 0.1% formic acid over 10 min) Rt=5.77 min. ESI-MS m/z calcd for C46H57N10O10S+ [M−2Boc+H]+941.4, found 941.4.
Boc-Ala-D-Pra-D-Trp(Boc)-Ala-Trp-D-Phe-Lys(o-NBS, Allyl)-Rink Amide Resin RGO65:
LCMS (30-95% MeOH containing 0.1% formic acid in water containing 0.1% formic acid over 10 min) Rt=6.48 min. ESI-MS m/z calcd for C57H67N12O11S+ [M−3Boc+H]+1127.5, found 1127.5.
Boc-Ala-L-Pra-D-Trp(Boc)-Ala-Trp-D-Phe-Lys(o-NBS, Allyl)-Rink Amide Resin RGO66:
LCMS (30-95% MeOH containing 0.1% formic acid in water containing 0.1% formic acid over 10 min) Rt=6.66 min. ESI-MS m/z calcd for C57H67N12O11S+ [M−3Boc+H]+1127.5, found 1127.5.
Boc-Ala-D-Pra-Ala-Trp(Boc)-D-Phe-Lys(Allyl)-Rink Amide Resin RGO104:
o-NBS-protected hexapeptide RGO99 (˜600 mg, 0.156 mmol) in a syringe fitted with a Teflon™ filter was swollen in DMF (5 mL) and treated with DBU (210 μL, 1.40 mmol) and 2-mercaptoethanol (50 μL, 0.71 mmol). The mixture in the syringe was shaken for 1 h. The resin was filtered and sequentially washed with DMF (×3), MeOH (×3), THF (×3) and CH2Cl2 (×3). Examination by LCMS of a cleaved resin sample (5 mg) showed complete o-NBS-removal: LCMS (30-95% MeOH containing 0.1% formic acid in water containing 0.1% formic acid over 10 min) Rt=1.50 min. ESI-MS m/z calcd for C40H54N9O6+ [M−2Boc+H]+ 756.4, found 756.4.
Boc-Ala-L-Pra-Ala-Trp(Boc)-D-Phe-Lys(Allyl)-Rink Amide Resin RGO105:
o-NBS-protected hexapeptide RGO100 (˜600 mg, 0.14 mmol) in a syringe fitted with a Teflon™ filter was swollen in DMF (5 mL) and treated with DBU (210 μL, 1.40 mmol) and 2-mercaptoethanol (50 μL, 0.71 mmol). The mixture in the syringe was shaken for 1 h. The resin was filtered and sequentially washed with DMF (×3), MeOH (×3), THF (×3) and CH2Cl2 (×3). Examination by LCMS of a cleaved resin sample (5 mg) showed complete o-NBS-removal: LCMS (30-95% MeOH containing 0.1% formic acid in water containing 0.1% formic acid over 10 min) Rt=1.51 min. ESI-MS m/z calcd for C40H54N9O6+ [M−2Boc+H]+ 756.4, found 756.4.
Boc-Ala-D-Pra-D-Trp(Boc)-Ala-Trp(Boc)-D-Phe-Lys(allyl)-Rink Amide Resin RGO69:
o-NBS-protected heptapeptide RGO65 (˜300 mg, 0.10 mmol) in a syringe fitted with a Teflon™ filter was swollen in DMF (6 mL) and treated with DBU (150 μL, 1.00 mmol) and 2-mercaptoethanol (35 μL, 0.50 mmol). The mixture in the syringe was shaken for 1 h. The resin was filtered and sequentially washed with DMF (×3), MeOH (×3), THF (×3) and CH2Cl2 (×3). Examination by LCMS of a cleaved resin sample (5 mg) showed complete o-NBS-removal: LCMS (20-80% MeOH containing 0.1% formic acid in water containing 0.1% formic acid over 10 min) Rt=4.79 min. ESI-MS m/z calcd for C51H64N11O7+ [M−3Boc+H]+ 942.5, found 942.5.
Boc-Ala-L-Pra-D-Trp(Boc)-Ala-Trp(Boc)-D-Phe-Lys(Allyl)-Rink Amide Resin RGO70:
o-NBS-protected heptapeptide RGO66 (˜300 mg, 0.09 mmol) in a syringe fitted with a Teflon™ filter was swollen in DMF (6 mL) and treated with DBU (130 μL, 0.87 mmol) and 2-mercaptoethanol (30 μL, 0.43 mmol). The mixture in the syringe was shaken for 1 h. The resin was filtered and sequentially washed with DMF (×3), MeOH (×3), THF (×3) and CH2Cl2 (×3). Examination by LCMS of a cleaved resin sample (5 mg) showed complete o-NBS-removal: LCMS (20-80% MeOH containing 0.1% formic acid in water containing 0.1% formic acid over 10 min) Rt=5.05 min. ESI-MS m/z calcd for C51H64N11O7+ [M−3Boc+H]+ 942.5, found 942.5.
Cyclic Peptide MPE-110:
Hexapeptide resin RGO104 (˜600 mg, 0.156 mmol) was swollen in DMSO (6 mL) for 30 min in a syringe tube equipped with Teflon™ filter, and stopper, treated with CuI (5.0 mg, 0.03 mmol) and aqueous formaldehyde (70 μL, 0.94 mmol, 37% in H2O), shaken on an automated shaker for 30 h, and filtered. After filtration, the resin was washed sequentially with AcOH/H2O/DMF (5:15:80, v/v/v, ×3), DMF (×3), THF (×3), MeOH (×3), and DCM (×3). Examination by LCMS of a cleaved resin sample (5 mg) showed complete conversion, and a peak with molecular ion consistent with cyclic hexapeptide MPE-110 was observed: MS m/z calcd for Ca41H54N9O6+ [M+H]+ 768.4, found 768.4.
Resin-bound cyclic peptide MPE-110 was deprotected and cleaved from the support using a freshly made solution of TFA/H2O/TES (95:2.5:2.5, v/v/v, 5 mL) at rt for 2 h. The resin was filtered and rinsed with TFA (5 mL). The filtrate and rinses were concentrated until a crude oil persisted, from which a precipitate was obtained by addition of cold ether (10 mL). After centrifugation (1200 rpm for 10 min), the supernatant was removed and the crude peptide precipitate was taken up in aqueous MeOH (10% v/v) and freeze-dried prior to purification. The resulting light brown fluffy material was purified by preparative HPLC to give cyclic pentapeptide MPE-110 (2.0 mg, 2%) as white fluffy material.
LCMS analysis of cyclic peptide MPE-110 was performed using a linear gradient of a) 10-90% of MeOH containing 0.1% formic acid in H2O (0.1% formic acid) over 10 min, then at 10% MeOH (0.1% formic acid) for 5 min, Rt=4.24 min; b) 10-90% MeCN containing 0.1% formic acid in H2O containing 0.1% formic acid over 10 min, then at 10% MeCN (0.1% formic acid) for 5 min, Rt=1.70 min; HRMS m/z. calcd for C41H54N9O6+ [M+H]+ 768.4192, found 768.4176.
Cyclic Peptide MPE-111:
Hexapeptide resin RGO105 (˜600 mg, 0.14 mmol) was swollen in DMSO (6 mL) for 30 min in a syringe tube equipped with Teflon™ filter, and stopper, treated with CuI (5.0 mg, 0.03 mmol) and aqueous formaldehyde (60 μL, 0.84 mmol, 37% in H2O), shaken on an automated shaker for 30 h, and filtered. After filtration, the resin was washed sequentially with AcOH/H2O/DMF (5:15:80, v/v/v, ×3), DMF (×3), THF (×3), MeOH (×3), and DCM (×3). Examination by LCMS of a cleaved resin sample (5 mg) showed complete conversion, and a peak with molecular ion consistent with cyclic hexapeptide MPE-111 was observed: MS m/z. calcd for C41H54N9O6+ [M+H]+ 768.4, found 768.4.
Resin-bound cyclic peptide MPE-111 was deprotected and cleaved from the support using a freshly made solution of TFA/H2O/TES (95:2.5:2.5, v/v/v, 5 mL) at rt for 2 h. The resin was filtered and rinsed with TFA (5 mL). The filtrate and rinses were concentrated until a crude oil persisted, from which a precipitate was obtained by addition of cold ether (10 mL). After centrifugation (1200 rpm for 10 min), the supernatant was removed and the crude peptide precipitate was taken up in aqueous MeOH (10% v/v) and freeze-dried prior to purification. The resulting light brown fluffy material was purified by preparative HPLC to give cyclic hexapeptide MPE-111 (2.9 mg, 3%) as white fluffy material.
LCMS analysis of cyclic peptide MPE-111 was performed using a linear gradient of a) 10-90% of MeOH containing 0.1% formic acid in H2O (0.1% formic acid) over 10 min, then at 10% MeOH (0.1% formic acid) for 5 min, Rt=4.50 min; b) 10-90% MeCN containing 0.1% formic acid in H2O containing 0.1% formic acid over 10 min, then at 10% MeCN (0.1% formic acid) for 5 min, Rt=2.03 min; HRMS m/z. calcd for C41H54N9O6+ [M+H]+ 768.4192, found 768.4172.
Cyclic Peptide MPE-074:
Heptapeptide resin RGO69 (˜300 mg, 0.10 mmol) was swollen in DMSO (5 mL) for 30 min in a syringe tube equipped with Teflon™ filter, and stopper, treated with CuI (4.0 mg, 0.02 mmol) and aqueous formaldehyde (50 μL, 0.69 mmol, 37% in H2O), shaken on an automated shaker for 29 h, and filtered. After filtration, the resin was washed sequentially with AcOH/H2O/DMF (5:15:80, v/v/v, ×3), DMF (×3), THF (×3), MeOH (×3), and DCM (×3). Examination by LCMS of a cleaved resin sample (5 mg) showed complete conversion, and a peak with molecular ion consistent with cyclic heptapeptide MPE-074 was observed: MS m/z calcd for C52H63N11NaO7+ [M+Na]+ 976.5, found 976.4.
Resin-bound cyclic peptide MPE-074 was deprotected and cleaved from the support using a freshly made solution of TFA/H2O/TES (95:2.5:2.5, v/v/v, 5 mL) at rt for 2 h. The resin was filtered and rinsed with TFA (5 mL). The filtrate and rinses were concentrated until a crude oil persisted, from which a precipitate was obtained by addition of cold ether (10 mL). After centrifugation (1200 rpm for 10 min), the supernatant was removed and the crude peptide precipitate was taken up in aqueous MeOH (10% v/v) and freeze-dried prior to purification. The resulting light brown fluffy material was purified by preparative HPLC to give cyclic heptapeptide MPE-074 (0.7 mg, 1%) as white fluffy material.
LCMS analysis of cyclic peptide MPE-074 was performed using a linear gradient of a) 10-90% of MeOH containing 0.1% formic acid in H2O (0.1% formic acid) over 10 min, then at 10% MeOH (0.1% formic acid) for 5 min, Rt=1.72 min; b) 10-90% MeCN containing 0.1% formic acid in H2O containing 0.1% formic acid over 10 min, then at 10% MeCN (0.1% formic acid) for 5 min, Rt=4.24 min; HRMS m/z calcd for C52H63N11NaO7+ [M+Na]+ 976.4804, found 976.4817.
Cyclic Peptide MPE-075:
Heptapeptide resin RGO69 (˜300 mg, 0.09 mmol) was swollen in DMSO (5 mL) for 30 min in a syringe tube equipped with Teflon™ filter, and stopper, treated with CuI (3.0 mg, 0.02 mmol) and aqueous formaldehyde (50 μL, 0.69 mmol, 37% in H2O), shaken on an automated shaker for 29 h, and filtered. After filtration, the resin was washed sequentially with AcOH/H2O/DMF (5:15:80, v/v/v, ×3), DMF (×3), THF (×3), MeOH (×3), and DCM (×3). Examination by LCMS of a cleaved resin sample (5 mg) showed complete conversion, and a peak with molecular ion consistent with cyclic heptapeptide MPE-075 was observed: MS m/z calcd for C52H64N11O7+ [M+H]+ 954.5, found 954.5.
Resin-bound cyclic peptide MPE-075 was deprotected and cleaved from the support using a freshly made solution of TFA/H2O/TES (95:2.5:2.5, v/v/v, 5 mL) at rt for 2 h. The resin was filtered and rinsed with TFA (5 mL). The filtrate and rinses were concentrated until a crude oil persisted, from which a precipitate was obtained by addition of cold ether (10 mL). After centrifugation (1200 rpm for 10 min), the supernatant was removed and the crude peptide precipitate was taken up in aqueous MeOH (10% v/v) and freeze-dried prior to purification. The resulting light brown fluffy material was purified by preparative HPLC to give cyclic heptapeptide MPE-075 (1.5 mg, 2%) as a white fluffy material.
LCMS analysis of cyclic peptide MPE-075 was performed using a linear gradient of a) 10-90% of MeOH containing 0.1% formic acid in H2O (0.1% formic acid) over 10 min, then at 10% MeOH (0.1% formic acid) for 5 min, Rt=1.89 min; b) 10-90% MeCN containing 0.1% formic acid in H2O containing 0.1% formic acid over 10 min, then at 10% MeCN (0.1% formic acid) for 5 min, Rt=4.47 min; HRMS m/z calcd for C52H64N11O7+ [M+H]+ 954.4985, found 954.4973.
Ornithine as AA1
Fmoc-Orn(o-NBS)-Rink Amide Resin RGO3:
Rink amide resin (2.51 g) was placed in a syringe fitted with a Teflon™ filter. The Fmoc group was removed by treating the resin with a solution of 20% piperidine in DMF over 30 min. The resin was filtered and washed sequentially with DMF (×3), MeOH (×3) and CH2Cl2 (×3). Fmoc-Orn(o-NBS)—OH (1.33 g, 2.46 mmol) was dissolved in DMF (20 mL) and treated with DIC (0.57 mL, 3.68 mmol) and HOBt (494 mg, 3.66 mmol) and stirred for 3 min, before being transferred to the syringe containing the swollen resin, and the mixture was shaken for 14 hours. The resin was filtered and washed sequentially with DMF (×3), MeOH (×3) and CH2Cl2 (×3). The resin was dried and the loading was measured to 0.187 mmol/g resin.
Fmoc-Orn(o-NBS, Allyl)-Rink Amide Resin RGO4:
Vacuum dried Fmoc-Orn(o-NBS)-resin (0.362 mmol) was placed in a syringe fitted with a Teflon™ filter, suspended in THF (dry, 20 mL) and treated sequentially with solutions of allyl alcohol (250 μL, 3.68 mmol) in THF (dry, 1 mL), PPh3 (482 mg, 1.84 mmol) in THF (dry, 2 mL) and DIAD (360 μL, 1.83 mmol) in THF (dry, 1 mL). The resin mixture in the syringe was shaken for 90 min. The resin was filtered and washed sequentially with DMF (×3), MeOH (×3), THF (×3) and CH2Cl2 (×3). Examination by LCMS of a cleaved resin sample (5 mg) showed complete allylation: LCMS (30-95% MeOH containing 0.1% formic acid in water containing 0.1% formic acid over 10 min) Rt=8.47 min. ESI-MS m/z calcd for C29H31N4O7S+ [M+H]+ 579.2, found 579.2.
Fmoc-D-Trp(Boc)-Ala-Trp(Boc)-D-Phe-Orn(o-NBS, Allyl)-Rink Amide Resin RGO22:
LCMS (30-95% MeOH containing 0.1% formic acid in water containing 0.1% formic acid over 10 min) Rt=6.13 min. ESI-MS m/z calcd for C48H55N10O9S+ [M-Fmoc-2Boc+H]+ 947.4, found 947.3.
Fmoc-azaPra-D-Trp(Boc)-Ala-Trp(Boc)-D-Phe-Orn(o-NBS, Allyl)-Rink Amide Resin RGO79:
N′-Propargyl-fluorenylmethylcarbazate (248 mg, 0.849 mmol, prepared by alkylation of fluorenylmethylcarbazate with propargyibromide as —N(R10)— described below) was dissolved in CH2Cl2 (dry, 40 mL) under argon atmosphere. The solution was cooled to 0° C., treated with a 20% solution of phosgene in toluene (1 mL, 1.87 mmol), warmed to rt, stirred 50 min, and the volatiles were removed by rotary evaporation. The residue was re-dissolved in CH2Cl2 (10 mL) and the volatiles were once again removed by rotary evaporation. The resulting white solid was dissolved in CH2Cl2 (dry, 7 mL) and added to the Fmoc-deprotected pentapeptide RGO22 in a syringe fitted with a Teflon™ filter. The mixture in the syringe was shaken for 28 h. The resin was filtered and washed sequentially with DMF (×3), MeOH (×3), THF (×3) and CH2Cl2 (×3). Examination by LCMS of a cleaved resin sample (5 mg) showed complete coupling: LCMS (30-95% MeOH containing 0.1% formic acid in water containing 0.1% formic acid over 10 min) Rt=8.26 min. ESI-MS m/z calcd for C52H59N12O10S+ [M-Fmoc-2Boc+H]+ 1043.4, found 1043.3.
Boc-Ala-azaPra-D-Trp(Boc)-Ala-Trp(Boc)-D-Phe-Orn(o-NBS, Allyl)-Rink Amide RGO29:
Coupling onto the semicarbazide RGO79 was performed by using amino acid symmetric anhydrides that were generated in situ (J. Zhang, C. Proulx, A. Tomberg, W. D. Lubell, Org. Lett. 2013, 16, 298-301). The procedure was repeated twice on semicarbazide RGO79. Examination by LCMS of a cleaved resin sample (5 mg) showed complete coupling: LCMS (30-95% MeOH containing 0.1% formic acid in water containing 0.1% formic acid over 10 min) Rt=6.39 min. ESI-MS m/z calcd for C55H64N13O11S+ [M−3Boc+H]+ 1114.5, found 1114.4.
Boc-Ala-azaPra-D-Trp(Boc)-Ala-Trp(Boc)-D-Phe-Orn(Allyl)-Rink Amide RGO30:
o-NBS-protected hetapeptide RGO29 (˜1 g, 0.2 mmol) in a syringe fitted with a Teflon™ filter was swollen in DMF (6 mL) and DBU (300 μL, 2.01 mmol) and treated with 2-mercaptoethanol (70 μL, 1.00 mmol). The mixture in the syringe was shaken for 1 h. The resin was filtered and washed sequentially with DMF (×3), MeOH (×3), THF (×3) and CH2Cl2 (×3). Examination by LCMS of a cleaved resin sample (5 mg) showed complete o-NBS-removal: LCMS (30-95% MeOH containing 0.1% formic acid in water containing 0.1% formic acid over 10 min) Rt=4.49 min. ESI-MS m/z calcd for C49H61N12O7+ [M−3Boc+2Na]2+ 487.2, found 487.3.
Cyclic Azapeptide MPE-048:
Azaheptapeptide resin RGO30 (˜1 g, 0.2 mmol) was swollen in DMSO (8 mL) for 30 min in a syringe tube equipped with Teflon™ filter, and stopper, treated with CuI (7.0 mg, 0.04 mmol) and aqueous formaldehyde (90 μL, 1.2 mmol, 37% in H2O), shaken on an automated shaker for 31 h, and filtered. After filtration, the resin was washed sequentially with AcOH/H2O/DMF (5:15:80, v/v/v, ×3), DMF (×3), THF (×3), MeOH (×3), and DCM (×3). Examination by LCMS of a cleaved resin sample (5 mg) showed complete conversion, and a peak with molecular ion consistent with cyclic azaheptapeptide MPE-048 was observed: MS m/z calcd for C50H61N12O7+ [M+H]+ 941.5, found 941.4.
Resin-bound cyclic azapeptide MPE-048 was deprotected and cleaved from the support using a freshly made solution of TFA/H2O/TES (95:2.5:2.5, v/v/v, 5 mL) at rt for 2 h. The resin was filtered and rinsed with TFA (5 mL). The filtrate and rinses were concentrated until a crude oil persisted, from which a precipitate was obtained by addition of cold ether (10 mL). After centrifugation (1200 rpm for 10 min), the supernatant was removed and the crude peptide precipitate was taken up in aqueous MeOH (10% v/v) and freeze-dried prior to purification. The resulting light brown fluffy material was purified by preparative HPLC to give cyclic azaheptapeptide MPE-048 (1.3 mg, 1%) as white fluffy material.
LCMS analysis of cyclic peptide MPE-048 was performed using a linear gradient of a) 10-90% of MeOH containing 0.1% formic acid in H2O (0.1% formic acid) over 10 min, then at 10% MeOH (0.1% formic acid) for 5 min, Rt=1.80 min; b10-90% MeCN containing 0.1% formic acid in H2O containing 0.1% formic acid over 10 min, then at 10% MeCN (0.1% formic acid) for 5 min, Rt=4.30 min; HRMS m/z calcd for C50H60N12O7Na+ [M+Na]+ 963.4600, found 963.4573.
Synthesis of Cyclic Analogs MPE-189, MPE-201, MPE-202, and MPE-203
Synthesis of Carbazates 2 and 3
To a well-stirred solution of fluorenylmethyl carbazate (1, 1 eq., 2.8 g, 11 mmol, prepared according to reference 1) and DIEA (2 eq., 2.85 g, 3.64 mL, 22 mmol) in dry DMF (280 mL) at 0° C., a solution of 3-bromopropyne (0.9 eq., 1.47 g, 1.07 mL, 9.91 mmol, 80 wt. % in toluene) in dry DMF (10 mL) was added drop-wise by cannula over 30 min. The cooling bath was removed. The reaction mixture was allowed to warm to room temperature and stirred for 16 h. The volatiles were evaporated. The residue was partitioned between EtOAc and brine. The aqueous layer was separated and extracted with EtOAc. The combined organic layer was dried over Na2SO4, filtered, and evaporated. The residue was purified by silica gel chromatography eluting with 40% EtOAc in hexane as solvent system to obtain N′-propargyl-fluorenylmethylcarbazate 3 (1.8 g, 62%), as white solid: Rf 0.42 (60% EtOAc); mp 148-149° C.; 1H NMR (500 MHz, DMSO-d6) δ 8.82 (s, 1H), 7.89 (d, J=7.5 Hz, 2H), 7.70 (d, J=7.4 Hz, 2H), 7.50-7.43 (m, 2H), 7.37-7.28 (m, 2H), 4.89 (q, J=4.6 Hz, 1H), 4.29 (d, J=6.9 Hz, 2H), 4.22 (t, J=6.1 Hz, 1H), 3.48 (s, 2H), 3.09 (t, J=2.3 Hz, 1H); 13C NMR (125 MHz, DMSO-d6) δ 156.7, 143.8, 140.7, 127.7 (2C), 127.1 (2C), 125.3 (2C), 120.1 (2C), 81.2, 74.2, 65.6 (2C), 46.6, 39.6 (2C). IR (neat) vmax/cm-1 3304, 3290, 2947, 1699, 1561, 1489, 1448, 1265, 1159, 1021; HRMS m/z calculated for C18H17N2O2 [M+H]+ 293.1285; found 293.1275.
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1-hydroxybenzotriazole
1H NMR
2-Mercaptoethanol
5A peptide
Acylation
Alanine
Alcohols
Aldehydes
Alkylation
allyl alcohol
Amides
Amines
Amino Acids
Anabolism
Anhydrides
Argon
Atmosphere
Bath
benzophenone
Biopharmaceuticals
brine
Bromides
Cannula
carbamylhydrazine
carbazate
Carbon-13 Magnetic Resonance Spectroscopy
Carbonates
Cardiac Arrest
Centrifugation
Chromatography
Cold Temperature
Copper
Cyclic Peptides
Cyclization
Cytokinesis
Dipeptides
Ethers
Filtration
Formaldehyde
formic acid
Freezing
Gel Chromatography
growth hormone releasing hexapeptide
H 1285
Hexanes
High-Performance Liquid Chromatographies
Histidine
Hydrazones
Hydroxylamine
Hydroxylamine Hydrochloride
Isopropyl Alcohol
Light
Lincomycin
Methanol
methylamine
N,N-diisopropylethylamine
N-propargyl
Nitrogen
Ornithine
Peptide Biosynthesis
Peptides
Petroleum
Phosgene
piperidine
polypeptide C
Polystyrenes
propargylamine
propargylglycine
pyridine
pyridine hydrochloride
Resins, Plant
Rink amide resin
Semicarbazides
Semicarbazones
Silica Gel
Silicon Dioxide
Solvents
Sulfate, Magnesium
Sulfonamides
Sulfoxide, Dimethyl
Syringes
Teflon
tert-butoxycarbonylalanine
Toluene
Training Programs
Tryptophan
Vacuum
NB is a traditional lowland indica cultivar that originated in India and is resistant to BSR caused by Burkholderia glumae. KO is a modern lowland rice cultivar released in Japan and is susceptible to BSR27 (link). To analyse RBG1res, by crossing SL535 with KO and using marker-assisted selection to remove nontarget DNA regions, we successfully developed a NIL homozygous for RBG1res. The resulting RBG1res-NIL contains approximately 380 kb from NB on the short arm of chromosome 10 (between simple sequence repeat (SSR) markers RM474 and RM7361-1; Supplementary Table S3 ). By screening 3072 M2 lines of KO mutagenized with N-methyl-N-nitrosourea according to a method described previously31 (link), we identified a null mutant (Mut-W56*) whose sequence encoding tryptophan at position 56 was changed such that a stop codon was introduced that produces a truncated protein (5.5 kD). Genomic DNA of the M2 plants was screened with the NB51 primer set listed in Supplementary Table S3 by the targeting induced local lesions in genomes (TILLING) method as described earlier52 (link). All of the experimental research and field studies on plants (either cultivated or wild), including transgenic plant materials, complied with relevant institutional, national, and international guidelines and legislation.
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Burkholderia glumae
Chromosomes, Human, Pair 10
Codon, Terminator
DNA, Plant
Genome
Homozygote
Nitrosourea Compounds
Oligonucleotide Primers
Plants
Plants, Transgenic
Proteins
Rice
Short Tandem Repeat
Tryptophan
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More about "Tryptophan"
Tryptophan is an essential amino acid that plays a crucial role in various physiological processes.
It is a precursor to the neurotransmitter serotonin, which regulates mood, sleep, and appetite.
Tryptophan is also involved in protein synthesis and has been studied for its potential benefits in areas such as mood disorders, sleep disturbances, and immune function.
Researchers can optimize their tryptophan studies by leveraging the AI-driven platform PubCompare.ai, which enhances reproducibility and accuracy by providing access to protocols from literature, pre-prints, and patents, as well as AI-driven comparisons to identify the best protocols and products for their research.
L-tryptophan, also known as 5-hydroxy-L-tryptophan (5-HTP), is a naturally occurring amino acid that is a precursor to the neurotransmitter serotonin.
It has been studied for its potential benefits in treating conditions such as depression, anxiety, and insomnia.
Prometheus NT.48 is a supplement that contains L-tryptophan and has been used to support healthy sleep and mood.
L-phenylalanine is another essential amino acid that is often studied in conjunction with tryptophan.
It is a precursor to the neurotransmitters dopamine and norepinephrine, which are involved in mood, cognition, and motivation.
The Matchmaker Gold Yeast Two-Hybrid System is a tool used in research to study protein-protein interactions, which can include the interactions between tryptophan, L-phenylalanine, and other related compounds.
Formic acid and methanol are common solvents used in the analysis and purification of tryptophan and other amino acids.
The PGBKT7 and PGADT7 vectors are also used in yeast two-hybrid systems to study protein-protein interactions, including those involving tryptophan and related compounds.
By leveraging the insights and tools provided by PubCompare.ai, researchers can enhance the reproducibility and accuracy of their tryptophan studies, leading to more robust and impactful findings in the fields of mood, sleep, and immune function.
It is a precursor to the neurotransmitter serotonin, which regulates mood, sleep, and appetite.
Tryptophan is also involved in protein synthesis and has been studied for its potential benefits in areas such as mood disorders, sleep disturbances, and immune function.
Researchers can optimize their tryptophan studies by leveraging the AI-driven platform PubCompare.ai, which enhances reproducibility and accuracy by providing access to protocols from literature, pre-prints, and patents, as well as AI-driven comparisons to identify the best protocols and products for their research.
L-tryptophan, also known as 5-hydroxy-L-tryptophan (5-HTP), is a naturally occurring amino acid that is a precursor to the neurotransmitter serotonin.
It has been studied for its potential benefits in treating conditions such as depression, anxiety, and insomnia.
Prometheus NT.48 is a supplement that contains L-tryptophan and has been used to support healthy sleep and mood.
L-phenylalanine is another essential amino acid that is often studied in conjunction with tryptophan.
It is a precursor to the neurotransmitters dopamine and norepinephrine, which are involved in mood, cognition, and motivation.
The Matchmaker Gold Yeast Two-Hybrid System is a tool used in research to study protein-protein interactions, which can include the interactions between tryptophan, L-phenylalanine, and other related compounds.
Formic acid and methanol are common solvents used in the analysis and purification of tryptophan and other amino acids.
The PGBKT7 and PGADT7 vectors are also used in yeast two-hybrid systems to study protein-protein interactions, including those involving tryptophan and related compounds.
By leveraging the insights and tools provided by PubCompare.ai, researchers can enhance the reproducibility and accuracy of their tryptophan studies, leading to more robust and impactful findings in the fields of mood, sleep, and immune function.