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Ornithine

Ornithine is a non-proteinogenic amino acid that plays a key role in the urea cycle, facilitating the conversion of ammonia into urea for excretion.
It is also involved in the production of polyamines, which are essential for cell growth and development.
Ornithine research explores its biochemical functions, potential therapeutic applications, and its impact on various physiological processes.
PubCompare.ai can optimize your Ornithine studies by helping you locate the best research protocols from literature, preprints, and patents, providing accurate comparisons and enhancing reproducibility.
Leverage the power of this AI-driven platfrom to take your Ornithine research to the next level and achieve your goals.

Most cited protocols related to «Ornithine»

Neural Induction and Differentiation of hESCs/iPSCs

Cited 66 times

hESCs or iPSCs were isolated from MEFs following dissociation to single cells with Accutase (Innovative Cell Technologies) by a 1 hr pre-plate on gelatin-coated dishes in hESC medium supplemented with 10 ng/ml FGF2 and 10 μM ROCK inhibitor (Calbiochem). The non-adherent pluripotent stem cells were harvested and plated on Matrigel (BD) coated 12-well plates in MEF-conditioned hESC medium with 10 ng/ml FGF2. Once the cell culture reached 95% confluence, neural induction was initiated by changing the culture medium to a culture medium that supports neural induction, neurogenesis and neuronal differentiation (referred to as 3N medium), a 1:1 mixture of N2- and B27-containing media. N2 medium: DMEM/F12, N2 (GIBCO), 5 μg/ml Insulin, 1mM L-Glutamine, 100 μm non-essential amino acids, 100 μM 2-mercaptoethanol, 50 U/ml Penicillin and 50 mg/ml Streptomycin; B27 medium: Neurobasal (Invitrogen), B27 with or without vitamin A (GIBCO), 200 mM Glutamine, 50 U/ml Penicillin and 50 mg/ml Streptomycin. 3N medium was supplemented with either 1 μm Dorsomorphin (Tocris) or 500 ng/ml mouse Noggin-CF chimera (R&D Systems), and 10 μm SB431542 (Tocris) to inhibit TGFβ signaling during neural induction ^{19 (link)}. Cells were maintained in this medium for 8-11 days, during which time the efficiency of neural induction was monitored by the appearance of cells with characteristic neuroepithelial cell morphology. Neuroepithelial cells were harvested by dissociation with Dispase and replated in 3N medium including 20 ng/ml FGF2 on poly-ornithine and laminin-coated plastic plates. After a further 2 days, FGF2 was withdrawn to promote differentiation. Cultures were passaged once more with Accutase, replated at 50,000 cells/cm² on poly-ornithine and laminin-coated plastic plates in 3N medium and maintained for up to 100 days with a medium change every other day.
For quantitative RT-PCR, total RNA was isolated from three cultures at each timepoint (days 5, 10, 15, 20 and 25) (Trizol, Sigma). Total RNA was reverse-transcribed and used for quantitative RT-PCR with primers specific to Foxg1 and Tbr2 using the Applied Biosystems 7000 system. Semi-quantitative RT-PCR with primers for Emx1, Dlx1, Nkx2.1, HoxB4 and Isl1 was carried out according to standard techniques on first strand, random-primed cDNA generated from total RNA extracted from cultures grown in the presence or absence of purmorphamine.

Shi Y., Kirwan P., Smith J., Robinson H.P, & Livesey F.J. (2012). Human cerebral cortex development from pluripotent stem cells to functional excitatory synapses. Nature neuroscience, 15(3), 10.1038/nn.3041.

Publication 2012

2-Mercaptoethanol 4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide accutase Amino Acids, Essential Cardiac Arrest Cell Culture Techniques Cells Chimera Culture Media, Conditioned dispase DNA, Complementary dorsomorphin Fibroblast Growth Factor 2 Gelatins Glutamine Human Embryonic Stem Cells Hyperostosis, Diffuse Idiopathic Skeletal Induced Pluripotent Stem Cells Insulin Laminin matrigel Mus Nervousness Neuroepithelial Cells Neurogenesis Neurons NKX2-1 protein, human noggin protein Oligonucleotide Primers Ornithine Penicillins Pluripotent Stem Cells Poly A purmorphamine Reverse Transcriptase Polymerase Chain Reaction Streptomycin Transforming Growth Factor beta trizol Vitamin A

Establishment and Differentiation of Mouse ESCs

Cited 25 times

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Publication 2014

activin A Cells Chir 99021 Fibroblast Growth Factor Receptors Fibronectins GSK3B protein, human Hyperostosis, Diffuse Idiopathic Skeletal Laminin Mus Ornithine PD-0325901 Psychological Inhibition Serum Signal Transduction Pathways Tissues

Comprehensive Peptide Modifications in PEPstrMOD

Cited 13 times

The updated web server PEPstrMOD (PEPstr with modified residues) can handle natural as well as modified peptides. Apart from incorporation of these modifications, the simulations are performed using AMBER v11.0 instead of old v6.0. The GROMACS (version 4.6.5) molecular dynamics software package is also used for implementing the SwissSideChain force field library. Table 1 shows different modifications, which can be handled using PEPstrMOD and the resources used to handle such modifications. The modifications incorporated in the PEPstrMOD are described below.Table 1

Types of peptides that can be handled with different modifications and the resources used to handle such modifications

Module name	Brief description	Resources used
Natural peptides	Prediction of peptides having natural residue.	PEPstr algorithm using AMBER11.
D-amino acids	Incorporation of D amino acids in a peptide.	Using inbuilt ‘flip’ command in AMBER11
Terminal modifications	Acetylation at N-terminus and/or amidation/N-methylamide group at C-terminus.	Using existing force field parameters in AMBER11.
Peptide cyclization	N-C cyclization of peptides or peptides having disulfide bridges.	Using inbuilt ‘bond’ command in AMBER11.
Non-natural modification	Incorporation of any of the 147 non-natural residues. (e.g. Homoserine, N-alkylated residues, β-substituted residues etc.).	FFNCAA library comprising 147 non-natural residues compatible with AMBER11.
Non-natural modification	Incorporation of any of the 210 non-natural residues. (e.g. Ornithine, Norvaline, Halogenated residues etc.).	SwissSideChain library comprising 210 non-natural residues compatible with GROMACS.
PTMs of residue	Peptides with any of the 32 diverse PTMs. (e.g. phosphorylation, palmitoylation, hydroxylation etc.).	FFPTM library compatible with AMBER11.
Advance modification	Combination of all the above six modules to provide facility to incorporate multiple modifications in one step.	All the resources used in the above modules.
Structure simulations	Facility to provide extended simulations.	All the resources used in the above modules.

Singh S., Singh H., Tuknait A., Chaudhary K., Singh B., Kumaran S, & Raghava G.P. (2015). PEPstrMOD: structure prediction of peptides containing natural, non-natural and modified residues. Biology Direct, 10, 73.

Publication 2015

Amber Amino Acids cDNA Library Cyclization Disulfides Homoserine Hydroxylation Molecular Dynamics norvaline Ornithine Palmitoylation Peptides Phosphorylation polypeptide C Post-Translational Protein Processing

Differentiation of hESCs to Spinal GABA Neurons

Cited 13 times

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Publication 2012

accutase Amino Acids, Essential Dietary Supplements Embryo Erinaceidae factor A Feeder Cell Layers Fibroblasts GABAergic Neurons GDNF protein, human Human Embryonic Stem Cells IGF1 protein, human Laminin Mice, Laboratory Nervousness Neurons Neurotrophic Factor, Brain-Derived Ornithine Poly A purmorphamine Tretinoin

Differentiating iPSCs into Glutamatergic and GABAergic Neurons

Cited 12 times

iPSCs were grown and induced to differentiate into neurons using two different protocols: A and B, which are described in detail in Methods S1. Briefly, in protocol A, iPSCs were maintained on irradiated mouse embryo fibroblasts supplemented with FGF2 (10 ng/ml). Colonies were subsequently detached and grown as embryoid bodies (EBs) on non-adherent plates in the absence of FGF2. After 4 days, EBs were plated on laminin, which resulted in the development of clusters of neurons [37] . These were manually dissected after 10 days. The neurons derived from this protocol are primarily glutatmatergic (∼90%) [37] . In protocol B, iPSCs were maintained on matrigel and mTeSR1^® (StemCell Technologies, Vancouver, Canada). EBs were formed and neural rosettes were cultivated using standard techniques [34] , [36] . Neurons emerged after rosettes were isolated and grown on Poly-dL-Ornithine/laminin coated plates. Neurons (generally an equal mix of glutamatergic and GABAergic neuorns) were harvested after 14 days.
RNA-Seq was carried out using line iPSC-1, which was derived from a control female. Validation by Sanger sequencing was carried out on iPSC-2 (a control male), and 3 lines derived from male subjects with SZ (SZ39, SZ97 and iPSC-15, the latter of which has a 22q11.2 deletion).

Lin M., Hrabovsky A., Pedrosa E., Wang T., Zheng D, & Lachman H.M. (2012). Allele-Biased Expression in Differentiating Human Neurons: Implications for Neuropsychiatric Disorders. PLoS ONE, 7(8), e44017.

Publication 2012

Deletion Mutation Embryo Embryoid Bodies Females Fibroblast Growth Factor 2 Fibroblasts Induced Pluripotent Stem Cells Laminin LINE-1 Elements Males matrigel Mice, Laboratory Nervousness Neurons Ornithine Poly A RNA-Seq Stem Cells

Most recents protocols related to «Ornithine»

Synthesis of Cyclic Peptide Analogs

Not available on PMC !

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.

[Figure (not displayed)]

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.

[Figure (not displayed)]
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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.

[Figure (not displayed)]

Cyclic azapeptide GHRP-6 analogs were synthesized by the A³-macrocyclization method in yields and purities suitable for biological evaluation (Table 1).

TABLE 1

Yields and purity of the cyclic azapeptide GHRP-6 analogs

CyclicSyntheticIsolated

AnalogApproachYield (%)Purity^[a]HRMS

8I3.5 99%809.4201(809.4206)

9I2.4 99%924.4627(924.4628)

17I and (II)0.5(1.5)99%835.4376(835.4362)

18I and (II)0.4(1.1)99%950.4787(950.4784)

19I0.5 94%884.4549(884.4566)

24II0.9 99%769.4140(769.4144)

25II1.1 97%955.4942(955.4937)

26II2.0%99%997.5031(997.5043)

27II1.6%99%926.4658(926.4671)

31I1.5 96%826.4718(826.4723)

32I1.2 97%828.4875(828.4879)

33II0.9 94%897.5092(897.5094)

34II2.5%98%939.5186(939.5199)

35II1.4%96%868.4804(868.4828)

^[a]Determined by LCMS analysis as described above.

Synthesis of Cyclic Analogs MPE-110, MPE-111, MPE-074 and MPE-048

Solution-Phase Chemistry

Ornithine Building Block Synthesis

[Figure (not displayed)]

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 MgSO₄and 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) R_t=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

[Figure (not displayed)]

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.

[Figure (not displayed)]

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) R_t=8.65 min. ESI-MS m/z calcd for C₃₀H₃₃N₄O₇S⁺ [M+H]⁺593.2, found 593.2.

[Figure (not displayed)]

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) R_t=5.73 min. ESI-MS m/z calcd for C₄₆H₅₇N₁₀O₁₀S⁺ [M−2Boc+H]⁺941.4, found 941.4.

[Figure (not displayed)]

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) R_t=5.77 min. ESI-MS m/z calcd for C₄₆H₅₇N₁₀O₁₀S⁺ [M−2Boc+H]⁺941.4, found 941.4.

[Figure (not displayed)]

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) R_t=6.48 min. ESI-MS m/z calcd for C₅₇H₆₇N₁₂O₁₁S⁺ [M−3Boc+H]⁺1127.5, found 1127.5.

[Figure (not displayed)]

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) R_t=6.66 min. ESI-MS m/z calcd for C₅₇H₆₇N₁₂O₁₁S⁺ [M−3Boc+H]⁺1127.5, found 1127.5.

[Figure (not displayed)]

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) R_t=1.50 min. ESI-MS m/z calcd for C₄₀H₅₄N₉O₆⁺ [M−2Boc+H]⁺756.4, found 756.4.

[Figure (not displayed)]

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) R_t=1.51 min. ESI-MS m/z calcd for C₄₀H₅₄N₉O₆⁺ [M−2Boc+H]⁺756.4, found 756.4.

[Figure (not displayed)]

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) R_t=4.79 min. ESI-MS m/z calcd for C₅₁H₆₄N₁₁O₇⁺ [M−3Boc+H]⁺942.5, found 942.5.

[Figure (not displayed)]

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) R_t=5.05 min. ESI-MS m/z calcd for C₅₁H₆₄N₁₁O₇⁺ [M−3Boc+H]⁺942.5, found 942.5.

[Figure (not displayed)]

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, R_t=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, R_t=1.70 min; HRMS m/z. calcd for C₄₁H₅₄N₉O₆⁺ [M+H]⁺768.4192, found 768.4176.

[Figure (not displayed)]

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, R_t=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, R_t=2.03 min; HRMS m/z. calcd for C₄₁H₅₄N₉O₆⁺ [M+H]⁺ 768.4192, found 768.4172.

[Figure (not displayed)]

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, R_t=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, R_t=4.24 min; HRMS m/z calcd for C₅₂H₆₃N₁₁NaO₇⁺ [M+Na]⁺976.4804, found 976.4817.

[Figure (not displayed)]

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, R_t=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, R_t=4.47 min; HRMS m/z calcd for C₅₂H₆₄N₁₁O₇⁺ [M+H]⁺ 954.4985, found 954.4973.

Ornithine as AA1

[Figure (not displayed)]

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.

[Figure (not displayed)]

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) R_t=8.47 min. ESI-MS m/z calcd for C₂₉H₃₁N₄O₇S⁺ [M+H]⁺579.2, found 579.2.

[Figure (not displayed)]

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) R_t=6.13 min. ESI-MS m/z calcd for C₄₈H₅₅N₁₀O₉S⁺ [M-Fmoc-2Boc+H]⁺947.4, found 947.3.

[Figure (not displayed)]

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) R_t=8.26 min. ESI-MS m/z calcd for C₅₂H₅₉N₁₂O₁₀S⁺ [M-Fmoc-2Boc+H]⁺1043.4, found 1043.3.

[Figure (not displayed)]

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) R_t=6.39 min. ESI-MS m/z calcd for C₅₅H₆₄N₁₃O₁₁S⁺ [M−3Boc+H]⁺1114.5, found 1114.4.

[Figure (not displayed)]

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) R_t=4.49 min. ESI-MS m/z calcd for C₄₉H₆₁N₁₂O₇⁺ [M−3Boc+2Na]²⁺487.2, found 487.3.

[Figure (not displayed)]

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, R_t=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, R_t=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

[Figure (not displayed)]

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: R_f0.42 (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.

US11879021B2. Cyclic peptides and uses thereof (2024-01-23). UNIVERSITÉ DE MONTRÉAL [CA]. Inventors: William D. Lubell [CA], Huy Ong [CA], Jinqiang Zhang [CN], Dilan Mukandila Mulumba [CA], Sylvie Marleau [CA], Ragnhild Gaard Ohm [DK], Ahsanullah [CA], Samy Omri [CA], Ramesh Chingle [US].

Patent 2024

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

Mitochondrial ROS Response to L-Asparaginase

*+pRS and *+pRS-shHAP1 cells seeded on poly-ornithine coated coverslips and pre-treated with RuR (3 μM), CsA (1 μM) or Mito-Tempo (5 μM) then stimulated with 100 mIU L-asparaginase for 12 h were stained with MitoSOX red (5 μM) and MitoTracker green (200 nM) or DCFDA (5 μM) for 30 min at 37°C. MitoTracker green was used to label mitochondria in live cells. Cell images were acquired using an Olympus 1 × 71 inverted microscope (Tokyo, Japan) at 160 to ×360 magnification. Fluorescence intensity of captured images (from a field with at least 200 cells) were measured using the ImageJ software. Values from cells stimulated with L-asparaginase alone were normalized to 1.

Lee J.K., Rosales J.L, & Lee K.Y. (2023). Requirement for ER-mitochondria Ca2+ transfer, ROS production and mPTP formation in L-asparaginase-induced apoptosis of acute lymphoblastic leukemia cells. Frontiers in Cell and Developmental Biology, 11, 1124164.

Publication 2023

Asparaginase Cells diacetyldichlorofluorescein Fluorescence Microscopy Mitochondria Mitomycin Ornithine Poly A

Isolation and Differentiation of Fetal Neural Stem Cells

All protocols described herein were carried out according to the European Community Council Directives (86/609/EEC) and comply with the guidelines published in the NIH Guide for the Care and Use of Laboratory Animals.
Fetal NSCs were isolated from E.13.5 forebrain as already described in a detailed methodological publication, including the culture characterization, the OGD, and the high content screening-based analysis of cell death and differentiation (Baldassarro, 2021 (link); Figure 1A). In brief, tissues were incubated in non-enzymatic dissociation buffer (Sigma-Aldrich, Saint Louis, MO, USA) at 37°C for 15 min, then mechanically dissociated by pipetting. Cells were resuspended in serum-free medium (DMEM/F12 GlutaMAX; 8 mmol/L HEPES; 100 U/100 μg Penicillin/Streptomycin; 1 × B27; 1 × N2; 20 ng/mL bFGF; 20 ng/mL EGF; Thermo Fisher Scientific, Waltham, MA, USA) and plated in suspension, at a density of 10 cells/μl in flasks (Nunc, Roskilde, DK) kept in vertical to avoid cell adhesion. Half medium was changed every 3 days, centrifuging the cell suspension at 300 × g for 5 min and gently resuspending the cellular pellet in fresh medium. Neurospheres were allowed to proliferate until they attained a diameter of about 100 μm.
To obtain the oligospheres, primary neurospheres were centrifuged at 300 × g for 5 min. The pellet was mechanically dissociated by pipetting, and cells were counted and plated again at a density of 10 cells/μl in OPC medium (DMEM/F12 GlutaMAX; 8 mmol/L HEPES; 100 U/100 μg Penicillin/Streptomycin; 1 × B27; 1 × N2; 20 ng/mL bFGF; 20 ng/mL PDGF; Thermo Fisher Scientific, Waltham, MA, USA). The oligospheres were centrifuged and the pellet mechanically dissociated to obtain a single cell suspension. Following cell count, cells were plated at a density of 3,000 cells/cm² on poly-D,L-ornithine (50 μg/ml)/laminin (5 μg/ml; Sigma-Aldrich) coating, in OPC medium.
To induce oligodendrocyte differentiation and maturation, the OPC medium was replaced with the oligodendrocyte differentiation medium (DMEM/F12 GlutaMAX; 8 mmol/L HEPES; 100 U/100 μg Penicillin/Streptomycin; 1 × B27; 1 × N2; 50 nM T3; 10 ng/ml CNTF; 1 × N-acetyl-L-cysteine – NAC; Thermo Fisher Scientific, Waltham, MA, USA) following 3 DIVs.
To characterize the responsiveness of OPCs to NGF in the mixed cultures, in a set of experiments we treated cultures with vehicle or NGF (100 ng/ml) at DIV 0 for 24 h (Figure 2A).

Baldassarro V.A., Cescatti M., Rocco M.L., Aloe L., Lorenzini L., Giardino L, & Calzà L. (2023). Nerve growth factor promotes differentiation and protects the oligodendrocyte precursor cells from in vitro hypoxia/ischemia. Frontiers in Neuroscience, 17, 1111170.

Publication 2023

Acetylcysteine Animals, Laboratory Buffers Cell Adhesion Cells Ciliary Neurotrophic Factor Enzymes Fetus HEPES Laminin Oligodendroglia Ornithine Penicillins Platelet-Derived Growth Factor Poly A Prosencephalon Serum Streptomycin Tissues

Quantification of Amino Acids by UPLC-MS/MS

The concentrations of all 20 proteinogenic amino acids as well as those of 4-aminobutyric acid, citrulline, and ornithine were quantified with external calibration, in triplicate, by ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS), using an Acquity system equipped with an HSS T3 column (Waters, Milford, MA, United States), as described previously (Van der Veken et al., 2022 (link)). Minor modifications included a constant flow rate of 0.35 mL/min for the mobile phase and the following gradient: 0.0 to 1.0 min, 99% A and 1% B; 1.0 to 8.0 min, 30% A and 70% B; 8.1 min to 10.0 min, 100% B; and 10.1 to 25.0 min, 99% A and 1% B. Samples were prepared by the addition of 300 μL of acetonitrile (Merck) to 100 μL of aqueous extract, followed by microcentrifugation at 18,000 × g for 15 min. Then, 900 μL of ultrapure water with 0.10% formic acid and 8.0 mg/L of 2-aminobutyric acid (IS; Sigma-Aldrich) was added to 100 μL of supernatant, and the mixture was filtered with a 0.2-μm LG H-PTFE filter (Millex; Merck) before injection (10 μL) into the column.

Decadt H., Weckx S, & De Vuyst L. (2023). The rotation of primary starter culture mixtures results in batch-to-batch variations during Gouda cheese production. Frontiers in Microbiology, 14, 1128394.

Publication 2023

acetonitrile Amino Acids Aminobutyric Acid Citrulline formic acid Liquid Chromatography Ornithine Polytetrafluoroethylene Tandem Mass Spectrometry

Targeted Metabolomics Profiling of Amino Acids in UK Biobank

Cited 1 time

In EPIC, targeted metabolomics profiling was performed at the International Agency for Research on Cancer (Biocrates AbsoluteIDQ^TM p180 kit) and the Helmholtz Centre in Munich (Biocrates AbsoluteIDQ^TM p150 kit). The samples were prepared as per the Biocrates kit instructions [25 (link), 26 (link)]. Assay preparation steps were carried out on 96 well plates and a volume of 10 μL plasma was prepared. The p150 kit allows the quantification of up to 13 amino acids and the p180 kit up to 21 amino acids (Additional file 1: Supplemental Methods) [25 (link), 27 (link)]. Liquid chromatography–mass spectrometry (LC-MS) was used to quantify the levels of the amino acids in accordance with the kit manufacturer’s instructions. All 21 amino acids included were fully quantified in μmol/L. The amino acids quantified were arginine, glutamine, glycine, histidine, methionine, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine (p150 and p180 kits); and alanine, asparagine, aspartate, citrulline, glutamate, isoleucine, leucine, and lysine (p180 kit only). See Additional file 1: Supplemental Methods for full details of sample preparation. Coefficients of variation for amino acids are given in Table S1.
Analysis of plasma from around 118,000 participants of the UK Biobank was performed using nucleic magnetic resonance (NMR) spectroscopy on the Nightingale metabolic biomarker platform (Nightingale Health Ltd, Finland), which comprises 249 metabolic measures, among which are concentrations of 9 amino acids. In brief, stored plasma samples prepared in 96-well plates were thawed, mixed gently, and centrifuged for 3 min at 3400 g to remove the precipitate. Aliquots of each sample were mixed with phosphate buffer, loaded onto a cooled sample changer, and analyzed by NMR spectroscopy. Metabolic biomarkers were identified and quantified from two separate spectra, a pre-saturated proton NMR spectrum, and a T2-relaxation-filtered spectrum. Six identical Bruker AVANCE IIIHD instruments were employed in parallel. The amino acids quantified were alanine, glutamine, glycine, histidine, isoleucine, leucine, valine, phenylalanine, and tyrosine. See Additional file 1: Supplemental Methods for further details.

Rothwell J.A., Bešević J., Dimou N., Breeur M., Murphy N., Jenab M., Wedekind R., Viallon V., Ferrari P., Achaintre D., Gicquiau A., Rinaldi S., Scalbert A., Huybrechts I., Prehn C., Adamski J., Cross A.J., Keun H., Chadeau-Hyam M., Boutron-Ruault M.C., Overvad K., Dahm C.C., Nøst T.H., Sandanger T.M., Skeie G., Zamora-Ros R., Tsilidis K.K., Eichelmann F., Schulze M.B., van Guelpen B., Vidman L., Sánchez M.J., Amiano P., Ardanaz E., Smith-Byrne K., Travis R., Katzke V., Kaaks R., Derksen J.W., Colorado-Yohar S., Tumino R., Bueno-de-Mesquita B., Vineis P., Palli D., Pasanisi F., Eriksen A.K., Tjønneland A., Severi G, & Gunter M.J. (2023). Circulating amino acid levels and colorectal cancer risk in the European Prospective Investigation into Cancer and Nutrition and UK Biobank cohorts. BMC Medicine, 21, 80.

Publication 2023

Alanine Amino Acids Arginine Asparagine Aspartate Biological Assay Biological Markers Blood Plasma Volume Buffers Cell Nucleus Citrulline Elp1 protein, human Glutamates Glutamine Glycine Histidine Isoleucine Leucine Liquid Chromatography Lysine Magnetic Resonance Magnetic Resonance Spectroscopy Malignant Neoplasms Mass Spectrometry Methionine Ornithine Phenylalanine Phosphates Plasma Proline Protons Serine Threonine Tryptophan Tyrosine Valine

Top products related to «Ornithine»

Poly ornithine by Merck Group

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Poly-ornithine is a synthetic polymer consisting of repeating units of the amino acid ornithine. It is commonly used as a cell culture substrate to promote cell adhesion and growth. Poly-ornithine provides a positively charged surface that enhances the attachment of various cell types.

Neurobasal medium by Thermo Fisher Scientific

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Neurobasal medium is a cell culture medium designed for the maintenance and growth of primary neuronal cells. It provides a defined, serum-free environment that supports the survival and differentiation of neurons. The medium is optimized to maintain the phenotypic characteristics of neurons and minimizes the growth of non-neuronal cells.

B27 supplement by Thermo Fisher Scientific

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B27 supplement is a serum-free and animal component-free cell culture supplement developed by Thermo Fisher Scientific. It is designed to promote the growth and survival of diverse cell types, including neurons, embryonic stem cells, and other sensitive cell lines. The core function of B27 supplement is to provide a defined, optimized combination of vitamins, antioxidants, and other essential components to support cell culture applications.

Glutamax by Thermo Fisher Scientific

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GlutaMAX is a chemically defined, L-glutamine substitute for cell culture media. It is a stable source of L-glutamine that does not degrade over time like L-glutamine. GlutaMAX helps maintain consistent cell growth and performance in cell culture applications.

Poly dl ornithine by Merck Group

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Poly-DL-ornithine is a synthetic polyamino acid composed of equal parts of the D- and L-enantiomers of the amino acid ornithine. It is commonly used as a coating material in cell culture applications to promote cell adhesion and growth.

Laminin by Thermo Fisher Scientific

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Laminin is a protein found in the extracellular matrix of various tissues. It plays a key role in the structural support and organization of the basement membrane. Laminin serves as a substrate for cell attachment and migration, and is involved in various cellular processes.

Laminin by Merck Group

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Laminin is a protein component found in the extracellular matrix of cells. It plays a key role in cell attachment, differentiation, and migration processes.

Penicillin streptomycin by Thermo Fisher Scientific

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Penicillin/streptomycin is a commonly used antibiotic solution for cell culture applications. It contains a combination of penicillin and streptomycin, which are broad-spectrum antibiotics that inhibit the growth of both Gram-positive and Gram-negative bacteria.

N2 supplement by Thermo Fisher Scientific

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The N2 supplement is a laboratory-grade nitrogen enrichment solution used to support the growth and development of cell cultures. It provides an additional source of nitrogen to cell culture media, which is essential for cellular metabolism and protein synthesis.

What is Ornithine and what are its key functions?

Ornithine is a non-proteinogenic amino acid that plays a crucial role in the urea cycle. It facilitates the conversion of ammonia into urea, which can be safely excreted from the body. Ornithine is also involved in the production of polyamines, which are essential for cell growth and development. Researchers investigate the biochemical functions of Ornithine, its potential therapeutic applications, and its impact on various physiological processes.

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What are some common challenges in working with Ornithine?

One common challenge in Ornithine research is ensuring the optimal use and comparison of different protocols and methods. Variations in experimental conditions, reagents, and measurement techniques can lead to inconsistencies in results. PubCompare.ai can assist in navigating these challenges by providing accurate comparisons of protocols from literature, preprints, and patents, helping researchers identify the most effective approaches for their Ornithine studies.

What are the different variations or types of Ornithine?

Ornithine can exist in different forms, such as L-Ornithine and D-Ornithine. L-Ornithine is the naturally occurring form and is the more commonly studied variant. D-Ornithine is the enantiomer, which has some distinct biochemical properties and potential applications. Researchers may investigate the specific effects and uses of these different Ornithine variations in their studies.

What are some key applications of Ornithine?

Ornithine has several important applications in both basic research and potential therapeutic areas. It is widely studied for its role in the urea cycle and its involvement in polyamine synthesis, which are crucial for cellular processes. Ornithine research also explores its potential use in the management of certain metabolic disorders, liver disease, and even athletic performance enhancment. PubCompare.ai can help researchers identify the most relevant protocols and compare their effectiveness for these various Ornithine applications.

More about "Ornithine"

Ornithine is a non-proteinogenic amino acid that plays a crucial role in the urea cycle, facilitating the conversion of ammonia into urea for excretion.
This essential compound is also involved in the production of polyamines, which are vital for cell growth and development.
Ornithine research explores its diverse biochemical functions, potential therapeutic applications, and its impact on various physiological processes.
Poly-ornithine, a derivative of ornithine, is commonly used in cell culture applications as a cell adhesion substrate.
Neurobasal medium, often supplemented with B27 and GlutaMAX, is a popular cell culture medium that supports the growth and differentiation of neuronal cells.
Poly-DL-ornithine, another form of the compound, can also be utilized as a cell culture coating to enhance cell attachment and proliferation.
Laminin, a key extracellular matrix protein, is often used in conjunction with ornithine-based coatings to promote neuronal growth and development.
Penicillin/streptomycin, a common antibiotic combination, is frequently added to cell culture media to prevent bacterial contamination.
N2 supplement, a mixture of essential nutrients, is also commonly used to support the survival and differentiation of neuronal cells.
Leveraging the power of PubCompare.ai, a cutting-edge AI-driven platform, researchers can optimize their ornithine studies by locating the best research protocols from literature, preprints, and patents.
This tool provides accurate comparisons and enhances the reproducibility of their experiments, empowering them to take their ornithine research to new heights and achieve their goals.