> Chemicals & Drugs > Amino Acid > Histidine

Histidine

Most cited protocols related to «Histidine»

Engineered ArnA Mutations Optimize Protein Expression

Cited 61 times

Wild type arnA was PCR amplified from E. coli genomic DNA with NdeI and XhoI restriction site overhangs on the 5’ and 3’ ends, respectively, using primers 1F and 1R (See all primer details in Table S1), and cloned into the bacterial expression vector pColaDuet (EMD Millipore). Two serine point mutations were introduced at site 1 (H359S and H361S) using primers 2F and 2R. Two additional serine point mutations were introduced at site 2 (H592S and H593S) using primers 3F and 3R to generate the final arnA mutant containing a total of four histidine to serine mutations.
The arnA knockout strain was generated with the E. coli recombineering technique^{10 (link)}, using the pKD4 plasmid as a template for the selectable marker and BL21(DE3) as the parental strain. The forward and reverse primers, 4F and 4R, were designed to maintain the reading frame of arnB, which shares its start codon with the stop codon of arnA within the arn operon^{11 (link)} (also called pmrHFIJKLM operon^{12 (link)}). A slightly modified scheme was used to introduce the arnA mutant back into the arnA knockout strain at the original locus (Fig. S1). First, mutant arnA was amplified and combined with the amplified selectable marker in a second PCR step. The resulting PCR product containing mutated arnA and the selectable marker was transformed into the arnA knockout strain for recombination using the λ Red recombinase plasmid (pKD46). The selectable marker was eliminated using the FLP plasmid (pCP20). For the modification in slyD, the arnA mutant strain was transformed with a PCR product (generated using primers 5F and 5R) containing a selectable marker flanked by homologous overhangs that, after recombination, result in the elimination of the 46-residue C-terminal, histidine-rich segment of SlyD. Again, the selectable marker was later removed using pCP20. Proper genomic integration was confirmed by PCR and sequencing. The RIL plasmid (Agilent Technologies) encoding rare tRNAs was transformed into the final expression strain to improve the expression of our eukaryotic target proteins.
The binding affinity of wild type and mutant ArnA were assessed by immobilizing purified protein onto a 1 ml His-Trap FF column (GE Healthcare) equilibrated in 50 mM potassium phosphate pH 8.0, 300 mM NaCl, and 5 mM beta-mercaptoethanol. Protein was eluted with a linear gradient of 0–150 mM imidazole. The imidazole concentration at the elution peak of each protein was recorded and compared.
Growth analysis was performed at 18, 25 and 37°C for both LOBSTR and the BL21(DE3) strains carrying the same test expression plasmid (See table S2 for a list of all test constructs). Cultures of 1L were grown in LB medium supplemented with 0.4% (w/v) glucose and antibiotic selection at 37°C to OD600 ~0.7. Protein expression was induced with 0.2 mM IPTG 20 minutes after the cultures were shifted to the desired expression temperature. OD600 was measured from the initial synchronization time and until the cells were harvested ~20–22 hours after induction.
To test protein purification, BL21(DE3) and LOBSTR cultures were started at 37°C in LB medium supplemented with 0.4% (w/v) glucose and appropriate antibiotic selection. At OD600 ~0.7, cultures were shifted to 18°C and induced with 0.2 mM IPTG ~20 min later. Cultures were harvested after 18–20 hours. For each strain and construct tested, a total of ~3.5g of cells were resuspended in 50 mL of resuspension buffer (40 mM potassium phosphate pH 8.0, 150 mM NaCl, 40 mM imidazole, and 3mM beta-mercaptoethanol) and lysed with a cell disrupter (Constant Systems). Lysates were cleared for 25 min at 9500×g and the soluble fraction was incubated with 400 µl bed volume of Ni Sepharose 6 Fast Flow (GE Healthcare) resin for 1 hour while stirring at 4°C. The resin was collected and washed with 6 mL of resuspension buffer and eluted with 2 mL of elution buffer (40 mM potassium phosphate pH 8.0, 150 mM NaCl, 250 mM imidazole, and 3 mM beta-mercaptoethanol). Elution fractions were analyzed on a 4–15 % SDS-PAGE gradient gel (Bio-RAD) and stained with Coomassie Blue R250. Purifications using Ni-NTA (Qiagen) and Talon (Clontech) resins were performed using resuspension buffer containing 20 mM or 5 mM imidazole, respectively, following manufacturer’s recommendations.

Andersen K.R., Leksa N.C, & Schwartz T.U. (2013). Optimized E. coli expression strain LOBSTR eliminates common contaminants from His-tag purification. Proteins, 81(11), 1857-1861.

Publication 2013

2-Mercaptoethanol Antibiotics Autosomal Recessive Polycystic Kidney Disease Bacteria Buffers Cells Claw Cloning Vectors Codon, Initiator Coomassie blue Escherichia coli Eukaryotic Cells Genome Glucose Histidine imidazole Isopropyl Thiogalactoside Mutation Oligonucleotide Primers Parent Plasmids Point Mutation potassium phosphate Proteins Protein Targeting, Cellular Reading Frames Recombinase Recombination, Genetic Resins, Plant SDS-PAGE Sepharose Serine Sodium Chloride Strains Transfer RNA

Engineered β1-Adrenergic Receptor Structure

Cited 55 times

The β₁AR construct T34-424/His642 (link) was the starting point for the generation of the β₁AR36-m23 construct that crystallized. The C-terminus was further truncated after Leu367, and 6 histidines were added. Two segments, comprising residues 244-271 and 277-278 of CL3, were also deleted. The construct included the following 8 point mutations: C116^3.27L increased expression; C358A at the C-terminus of H8 removed palmitoylation and helped crystallisation; R68^1.59S, M90^2.53V, Y227^5.58A, A282^6.27L, F327^7.37A and F338^7.48M thermostabilised the receptor in the antagonist conformation15 (link). Baculovirus expression and receptor purification42 (link) were performed in decylmaltoside, with the detergent exchanged to octylthioglucoside on the alprenolol sepharose column. Crystals were obtained by vapour diffusion at 18°C with hanging drops after addition of an equal volume of reservoir solution, 0.1M N-(2-acetamido)iminodiacetic acid:NaOH pH 6.9-7.3 and 29-32% PEG600 to receptor concentrated to 6.0 mg/ml.

Warne T., Serrano-Vega M.J., Baker J.G., Moukhametzianov R., Edwards P.C., Henderson R., Leslie A.G., Tate C.G, & Schertler G.F. (2008). Structure of a β1-adrenergic G protein-coupled receptor. Nature, 454(7203), 486-491.

Publication 2008

Alprenolol Baculoviridae Crystallization Detergents Diffusion Histidine iminodiacetic acid octylthioglucoside Palmitoylation Point Mutation Sepharose

Production and Purification of SARS-CoV Spike Protein

Cited 51 times

Protocol full text hidden due to copyright restrictions

Open the protocol to access the free full text link

Publication 2020

A-130A Buffers Cells Cloning Vectors Cobalt Freezing Hexosaminidase A His-His-His-His-His-His Histidine Phosphoric Monoester Hydrolases Proteins SARS-CoV-2 SDS-PAGE Severe acute respiratory syndrome-related coronavirus Signal Peptides Sodium Chloride Transfection Tromethamine

Molecular Dynamics Simulations of μOR

Cited 51 times

Simulations of the μ opioid receptor (μOR) were based on both the antagonist-bound inactive-state crystal structure (PDB ID: 4DKL) and the agonist-bound active-state crystal structure described in this manuscript. Coordinates were prepared by first removing all non-ligand and non-receptor molecules except for the cholesterol neighboring TM7 and for crystallographic water molecules near the receptor. For inactive μOR simulations, the T4 lysozyme was removed and acetyl and methylamide capping groups were placed on R263^ICL3 and E270^ICL3. For active μOR simulations, the nanobody was removed. In both cases, Prime (Schrödinger, Inc.) was used to model missing side-chains, and capping groups were then added to the N- and C- termini of the receptor. Histidine residues were simulated as the neutral Nε tautomer. Other titratable residues were simulated in their dominant protonation state at pH 7 except for D114^2.50, which was charged in inactive simulations and neutral in active simulations. A sodium ion was placed adjacent to D114^2.50 in inactive simulations.
The μOR was simulated in seven distinct conditions. These include: (1) The unliganded, inactive μOR, prepared by deleting the covalently bound, co-crystallized ligand, β-FNA, and adding a proton in its place to K233^5.39; (2) The inactive μOR with the co-crystallized ligand β-FNA; (3) The inactive μOR with agonist β-FOA (which does not bind covalently); (4) The unliganded, active μOR, prepared by deleting the co-crystallized ligand, BU72; (5) The active μOR with the co-crystallized ligand BU72; (6) The active μOR with the co-crystallized ligand BU72, with the N-terminal peptide deleted; (7) The active μOR with the antagonist BU74, with the N-terminal peptide deleted. Simulations of the active μOR without N-terminal peptide were prepared by deleting residues 52 through 64 of the receptor. Simulations with β-FOA were prepared by docking β-FOA to the crystallographic pose of β-FNA. Simulations with BU74 were prepared by docking BU74 to the crystallographic pose of BU72 and rotating the torsion angle of the methylcyclopropyl group to agree with that of β-FNA's methylcyclopropyl group.
We performed three to six simulations per condition (Supplementary Section). Simulations in a given condition were initiated from identical structures, but with initial atom velocities assigned independently and randomly.
It should be noted that in all liganded simulations, including those with β-FNA, β-FOA, BU72, and BU74, the ligand's tertiary amine nitrogen was protonated and therefore the ligand was simulated as a cation. This is necessary for the ligand to form the conserved salt bridge with neighboring D147^3.32.
Each of the resulting prepared μOR receptor structures was then aligned to the Orientations of Proteins in Membranes (OPM)^{52 (link)} entry for the inactive μOR using MacPyMOL (Schrödinger). The μOR was modified with disulfide bridges and inserted into a hydrated, equilibrated palmitoyloleoylphosphatidylcholine (POPC) bilayer using the CHARMM-GUI interface^{53 (link)-56 (link)}. Sodium and chloride ions were added to neutralize the system, reaching a final concentration of approximately 150 mM. All simulations contained one μOR receptor embedded in a lipid bilayer with 160 POPC molecules.

Huang W., Manglik A., Venkatakrishnan A.J., Laeremans T., Feinberg E.N., Sanborn A.L., Kato H.E., Livingston K.E., Thorsen T.S., Kling R., Granier S., Gmeiner P., Husbands S.M., Traynor J.R., Weis W.I., Steyaert J., Dror R.O, & Kobilka B.K. (2015). Structural insights into μ-opioid receptor activation. Nature, 524(7565), 315-321.

Publication 2015

1-palmitoyl-2-oleoylphosphatidylcholine Amines Chlorides Cholesterol Crystallography Disulfides Histidine Ions Ligands Lipid A Lipid Bilayers Membrane Proteins Mental Orientation Muramidase Nitrogen Peptides Protons Receptors, Opioid, mu Sodium Sodium Chloride

Growth and Culturing of B. thetaiotaomicron

Cited 50 times

Protocol full text hidden due to copyright restrictions

Open the protocol to access the free full text link

Publication 2008

5-fluoro-2'-deoxyuridine Agar Antibiotics, Antitubercular Bacteria BLOOD Brain Carbon Cysteine Equus caballus Erythromycin Gentamicin Glucose Heart Hematin Histidine Magnesium Chloride Plasmids Polysaccharides Serum Sodium Chloride Tetracycline Vitamin B12 Vitamin K3 Yeast, Dried

Most recents protocols related to «Histidine»

Recombinant Chimeric Protein Purification

Not available on PMC !

Example 3

Recombinant Protein Purification

FIG. 5 shows the steps of one of the purifications carried out on the chimera. In the case of GRNLY, this process was shown in an earlier paper [Ibáñez, R., University of Zaragoza. 2015]. It can be seen in FIG. 5A that the P. Pastoris supernatant obtained after induction (lane 1) contains rather diluted proteins. After concentrating same with Pellicom, protein bands are not seen in the permeate (lane 3), but proteins that are much more concentrated than in the supernatant are seen in the concentrate (lane 2). After dialysis (lane 4), the band profile remains similar to the concentrate. Furthermore, protein bands are not seen in the buffer in which the dialysis bag (lane 5) was introduced. Upon addition of the nickel resin, the chimera binds to said resin as it has a histidine tag. After adding the resin (lane 6), the intensity of a band corresponding to a protein of about 40 kDa decreases with respect to the concentrate and dialysate. This band may correspond to the chimera. The fact that this band does not altogether disappear may indicate that the nickel resin was saturated. In the washes performed on the resin, particularly in the first wash (lane 7), it can be seen how the residues of other proteins are removed. Finally, after the elution of the nickel column, a major protein with a molecular weight of about 40 kDa corresponding to the molecular weight of the chimera (lane 11) is clearly observed. As shown in FIG. 5B, it was confirmed by means of immunoblot that this band of about 40 kDa corresponds to the chimera (lane 11). It is also confirmed that the resin was saturated because a band appears in the post-resin dialysis phase (lane 6).

FIG. 6 shows different elution fractions and the pooling of all of them with the exception of elution fraction 1. FIG. 6A shows several bands in the different elution fractions and in the total eluate. The band with the highest intensity has a molecular weight corresponding to the chimera. Furthermore, other bands having intermediate molecular weights are observed, which means that the chimera undergoes partial proteolysis. The band with the second highest intensity has a molecular weight of about 10 kDa, which corresponds to 9-kDa GRNLY, as its molecular weight increases since it is bound to a histidine tag. In FIG. 6B, it was confirmed by means of immunoblot that these bands of about 40 and 10 kDa correspond to the chimeric recombinant protein and to recombinant GRNLY, respectively.

Once the chimera is generated, its functionality must be assured, that is, on one hand the scFv still recognizes the CEA antigen, and on the other hand GRNLY is still cytotoxic.

US11858973B2. Granulysin, method of obtaining same, and uses (2024-01-02). UNIVERSIDAD DE ZARAGOZA [ES], FUNDACIÓN AGENCIA ARAGONESA PARA LA INVESTIGACIÓN Y EL DESARROLLO (ARAID) [ES], FUNDACIÓN PARA LA INVESTIGACIÓN BIOMÉDICA HOSPITAL UNIVERSITARIO PUERTA DE HIERRO MAJADAHONDA [ES]. Inventors: Luis Alberto Anel Bernal [ES], Raquel Ibañez Perez [ES], Patricia Guerrero Ochoa [ES], Luis Martinez Lostao [ES], Blanca Conde Guerri [ES], Ramón Hurtado Guerrero [ES], Ana Laura Sanz Alcober [ES], Rocio Navarro Ortiz [ES].

Full text: Click here

Patent 2024

Antigens Buffers Chimera Chimeric Proteins, Recombinant Dialysis Dialysis Solutions GNLY protein, human Histidine Immunoblotting Nickel One-Step dentin bonding system Proteins Proteolysis Recombinant Proteins Resins, Plant Staphylococcal Protein A Vision

Marker-Free Bacillus subtilis Strain Engineering

Not available on PMC !

Example 1

This example describes the generation of a marker-free B. subtilis strain expressing allulose epimerase. Briefly, in a first step, a B. subtilis strain was transformed with a cassette encoding the BMCGD1 epimerase and including an antibiotic resistance marker. This cassette recombined into the Bacillus chromosome and knocked out 8 kb of DNA, including a large sporulation gene cluster and the lysine biosynthesis gene lysA. In a second step, a second cassette was recombined into the B. subtilis chromosome, restoring the lysA gene and removing DNA encoding the antibiotic resistance. E. coli strain 39 A10 from the Keio collection was used to passage plasmid DNA prior to transformation of B. subtilis. The relevant phenotype is a deficiency in the DNA methylase HsdM in an otherwise wild-type K-12 strain of E. coli.

In detail, a cassette of 5120 bp (SEQ ID NO:1; synthetic DNA from IDT, Coralville, Iowa) was synthesized and cloned into a standard ampicillin resistant pIDT vector. The synthetic piece encoded 700 bp upstream of lysA on the B. subtilis chromosome, the antibiotic marker cat (651 bp), the DNA-binding protein lad (1083 bp), and the allulose epimerase (894 bp), and included 700 bp of homology in dacF. This vector was transformed into E. coli strain 39 A10 (Baba et al., 2006), and plasmid DNA was prepared and transformed into B. subtilis strains 1A751 and 1A976.

Transformants were selected on LB supplemented with chloramphenicol. The replicon for pIDT is functional in E. coli but does not work in Gram positive bacteria such as B. subtilis. The colonies that arose therefore represented an integration event into the chromosome. In strain 1A751, the colony morphology on the plates was used to distinguish between single and double recombination events. The double recombination event would knock out genes required for sporulation, whereas the single recombination would not. After three days on LB plates, colonies capable of sporulation were brown and opaque; sporulation-deficient colonies were more translucent.

B. subtilis strain 1A976 with the allulose epimerase cassette is auxotrophic for histidine and lysine and can achieve very high transformation efficiency upon xylose induction. A 1925 bp synthetic DNA (SEQ ID NO:2) was amplified by primers (SEQ ID NO:3, SEQ ID NO:4) and Taq polymerase (Promega). This PCR product encoded the lysA gene that was deleted by the dropping in the epimerase cassette and 500 bp of homology to lad. A successful double recombination event of this DNA should result in colonies that are prototrophic for lysine and sensitive to chloramphenicol; i.e., the entire cat gene should be lost.

Transformants were selected on Davis minimal media supplemented with histidine. Colonies that arose were characterized by PCR and streaking onto LB with and without chloramphenicol. Strains that amplified the introduced DNA and that were chloramphenicol sensitive were further characterized, and their chromosomal DNA was extracted.

Strain 1A751 containing the chloramphenicol resistant allulose was transformed with this chromosomal DNA and selected on Davis minimal media supplemented with histidine. Transformants were streaked onto LB with and without chloramphenicol and characterized enzymatically as described below.

US11859228B2. Epimerase enzymes and their use (2024-01-02). Archer Daniels Midland Company [US]. Inventors: William Schroeder [US], Padmesh Venkitasubramanian [US], Sridhar Govindarajan [US], Mark Welch [US].

Full text: Click here

Patent 2024

Ampicillin Anabolism Antibiotic Resistance, Microbial Antibiotics Bacillus Bacillus subtilis Chloramphenicol Chromosomes Cloning Vectors DNA, A-Form DNA-Binding Proteins Epimerases Escherichia coli Gene Clusters Gene Knockout Techniques Genes Gram-Positive Bacteria Histidine Lysine Methyltransferase Oligonucleotide Primers Phenotype Plasmids psicose Recombination, Genetic Replicon Strains Taq Polymerase Xylose

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)]
[Figure (not displayed)]
[Figure (not displayed)]

[Figure (not displayed)]
[Figure (not displayed)]

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].

Full text: Click here

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

Engineered SpyTag002 Peptide Improves Reaction Rate

Not available on PMC !

Example 5

The SpyTag002-MBP fusion has a reaction rate of 0.40 μM⁻¹min⁻¹with SpyCatcher002. We surprisingly determined that the reaction rate could be further improved by introducing additional modifications to the SpyTag002 peptide.

Substitution of the threonine residue at position 3 of SpyTag002 (SEQ ID NO: 3) with histidine, i.e. reversion to the residue at the equivalent position in SpyTag, resulted in a peptide (SEQ ID NO: 4) with a reaction rate of 0.53-0.55 μM⁻¹min⁻¹, i.e. about a 35% increase in activity (FIG. 15A).

Modification of the improved peptide to include arginine and glycine residues at the N-terminus (SEQ ID NO: 5) more than doubled the reaction rate to 1.21 μM⁻¹min⁻¹(FIG. 15B).

US11873323B2. Proteins and peptide tags with enhanced rate of spontaneous isopeptide bond formation and uses thereof (2024-01-16). Oxford University Innovation Limited [GB]. Inventors: Mark Howarth [GB], Anthony Keeble [GB].

Full text: Click here

Patent 2024

Arginine Glycine Histidine Peptides Threonine

Quantification of Peptide-PNA Reaction Ratio

Not available on PMC !

Example 9

The number of peptide Fmoc-GFFYGHY that has been reacted with PNA is estimated from FIG. 3a and is equivalent to 141 μmoles: this is the amount of PNA converted when 22, 28 and 41 mM of PNA is used. We postulate that one PNA has reacted with one histidine. To determine the ratio of peptides Fmoc-GFFYGHY that have reacted with PNA against peptides present in the CASH within the investigated tubular column (15 cm length and 4 mm diameter, see 4b in the manuscript), the supported gel was entirely dissolved using a flow of acetone. After the removal of this organic solvent under reduced pressure, the residue was first analyzed by 1H NMR and HPLC to confirm the solely presence of Fmoc-GFFYGHY, and then weight. A mass of 1.66 mg (1.49 mmoles) was isolated as a white solid. Thus, the ratio r of peptide involved in the catalytic process against the whole number of peptide Fmoc-GFFYGHY engaged in the CASH is: r=1490/141≈111.

US11879038B2. Supramolecular gel supported on open-cell polymer foam (2024-01-23). UNIVERSITÉ DE STRASBOURG [FR], CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE [FR], INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE (INSERM) [FR]. Inventors: Loïc Jierry [FR], Christophe Serra [FR], Pierre Schaaf [FR], Jennifer Rodon Fores [FR], Fouzia Boulmedais [FR].

Full text: Click here

Patent 2024

1H NMR Acetone Catalysis High-Performance Liquid Chromatographies Histidine Peptides Pressure R 111 Solvents

Top products related to «Histidine»

L histidine by Merck Group

Sourced in United States, Germany, United Kingdom, Italy, Israel, Sao Tome and Principe

L-histidine is an amino acid that can be used as a laboratory reagent. It is a white, crystalline powder that is soluble in water and has a neutral pH. L-histidine is commonly used in buffer solutions and as a building block for peptides and proteins in biochemical applications.

Histidine by Merck Group

Sourced in United States, Germany, United Kingdom, Spain, China, Croatia

Histidine is a naturally occurring amino acid that is used in various laboratory applications. It plays a crucial role in the functioning of numerous enzymes and serves as a buffer in biological systems. Histidine is a widely used component in buffer solutions and culture media for cellular and biomolecular studies.

Pgbkt7 by Takara Bio

Sourced in United States, Japan, China, Germany, United Kingdom, France

The PGBKT7 is a plasmid vector used for gene expression in yeast cells. It contains a yeast selectable marker and a multiple cloning site for the insertion of DNA sequences.

L lysine by Merck Group

Sourced in United States, Germany, United Kingdom, Israel, China, Macao

L-lysine is an essential amino acid that is commonly used in various lab equipment and applications. It serves as a building block for proteins and plays a crucial role in various biological processes. The core function of L-lysine is to provide the necessary amino acid for protein synthesis and maintenance.

Ni nta agarose by Qiagen

Sourced in Germany, United States, United Kingdom, Netherlands, China, Switzerland, Italy, Canada, Spain, India

Ni-NTA agarose is a solid-phase affinity chromatography resin designed for the purification of recombinant proteins containing a histidine-tag. It consists of nickel-nitrilotriacetic acid (Ni-NTA) coupled to agarose beads, which selectively bind to the histidine-tagged proteins.

Pgadt7 by Takara Bio

Sourced in United States, Japan, China, France, Canada, United Kingdom

The PGADT7 is a plasmid vector used in yeast two-hybrid systems for the detection of protein-protein interactions. It contains the GAL4 DNA-binding domain, a multiple cloning site, and various genetic markers for selection in yeast.

L arginine by Merck Group

Sourced in United States, Germany, Poland, France, United Kingdom, Italy, Sao Tome and Principe, Switzerland, Spain, Croatia, Australia, China, Brazil

L-arginine is an amino acid that plays a crucial role in various physiological processes. It serves as a substrate for the production of nitric oxide, which is essential for maintaining healthy blood flow and cardiovascular function. This lab equipment product can be utilized for research and scientific applications related to the study of L-arginine and its associated biological functions.

L phenylalanine by Merck Group

Sourced in United States, Germany, Poland, Israel, Spain, United Kingdom, Japan, China, Italy

L-phenylalanine is an essential amino acid that serves as a fundamental building block for proteins. It is a commonly used laboratory reagent in various applications, including biochemical research and analysis.

Leucine by Merck Group

Sourced in United States, Germany, China, Spain, Switzerland, Sao Tome and Principe

Leucine is an essential amino acid commonly used in biochemical and cell culture applications. It serves as a building block for proteins and plays a role in various metabolic processes. The core function of Leucine is to support cellular growth and development.

Methionine by Merck Group

Sourced in United States, Germany, China, Macao, United Kingdom, Sao Tome and Principe, Japan, Canada, Switzerland

Methionine is an essential amino acid used in laboratory settings. It is a colorless, crystalline compound that serves as a building block for proteins and other biomolecules. Methionine is a key component in various biochemical processes and is utilized in experimental procedures within the scientific community.

What are the key functions of Histidine in the body?

Histidine is an essential amino acid that plays a crucial role in various biological processes. It is involved in the regulation of pH, enzymatic reactions, and protein structure. Histidine helps maintain the proper pH balance in the body and is a component of many enzymes that catalyze important reactions. It also contributes to the stability and folding of proteins, making it essential for proper protein function and structure.

Where can Histidine be found in the diet?

Why is optimizing Histidine research important?

Optimizing Histidine research is important for understanding its impact on human health and developing targeted therapeutic interventions. Histidine plays a critical role in various physiological processes, and gaining a deeper understanding of its functions and potential applications can lead to advancements in areas such as pH regulation, enzyme functionality, and protein-based therapies.

How can PubCompare.ai assist in Histidine research?

PubCompare.ai's AI-driven protocol comparison tool can help researchers identify the most effective methods from literature, pre-prints, and patents, enhancing reproducibility and research accuracy for Histidine optimization. The platform allows you to screen protocol literature more efficiently, leverage AI to pinpoint critical insights, and help you choose the best protocols related to Histidine for your specific research goals. The AI-driven analysis can highlight key differences in protocol effectiveness, enabling you to make informed decisions and improve the quality of your Histidine research.

Are there any common usage challenges with Histidine that researchers should be aware of?

One common usage challenge with Histidine is its sensitivity to heat and oxidation. Histidine can be degraded by high temperatures and exposure to oxidizing agents, which can affect its stability and functionality in certain applications. Researchers should be mindful of these factors when designing experiments or formulations involving Histidine to ensure optimal results and reproducibility. Additionally, some individuals may have allergies or sensitivities to Histidine, so caution should be exercised when incorporating it into research protocols or products.

More about "Histidine"

Histidine is an essential amino acid that plays a crucial role in various biological processes.
It is involved in the regulation of pH, enzymatic reactions, and protein structure.
Histidine can be found in a wide range of food sources, including meats, dairy products, legumes, and grains.
Optimzing histidine research is important for understanding its impact on human health and developing targeted therapeutic interventions.
L-histidine is the naturally occurring form of histidine, an amino acid that is essential for human health.
It is involved in the production of histamine, a chemical that plays a role in immune function and inflammation.
Histidine is also important for the production of red blood cells and the maintenance of the myelin sheath, which insulates nerve fibers.
PGBKT7 and PGADT7 are vectors used in yeast two-hybrid systems to study protein-protein interactions.
These systems can be used to investigate the role of histidine and other amino acids in protein complex formation and biological pathways.
L-lysine, L-arginine, L-phenylalanine, leucine, and methionine are other essential amino acids that work together with histidine to support various physiological processes.
Ni-NTA agarose is a chromatography resin used to purify histidine-tagged proteins for research and production purposes.
Optimizing histidine research can lead to a better understanding of its effects on human health, such as its potential role in managing conditions like anemia, nerve disorders, and immune system dysfunction.
PubCompare.ai's AI-driven protocol comparison tool can help researchers identify the most effective methods from literature, pre-prints, and patents, enhancing reproducibility and research accuracy for histidine optimization.