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The BI-3802 was designed by Boehringer Ingelheim and could be obtained free of charge through the Boehringer Ingelheim open innovation portal opnMe.com, associated with its negative control.
Fmoc-D-Glu-OH is a D-glutamic acid derivative, commonly used in biosynthesis.
Synonyms: D-Fmoc-glutamic acid
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Batch number can be found on the product's label following the word 'Batch'.
Search for reports by entering the product batch number.
Batch number can be found on the product's label following the word 'Batch'.
Search for reports by entering the product batch number.
Batch number can be found on the product's label following the word 'Batch'.
Search for reports by entering the product batch number.
Batch number can be found on the product's label following the word 'Batch'.
Search for reports by entering the product batch number.
Batch number can be found on the product's label following the word 'Batch'.
CAS No. : | 104091-09-0 |
Formula : | C20H19NO6 |
M.W : | 369.37 |
SMILES Code : | O=C(O)CC[C@H](C(O)=O)NC(OCC1C2=C(C3=C1C=CC=C3)C=CC=C2)=O |
Synonyms : |
D-Fmoc-glutamic acid
|
MDL No. : | MFCD09037362 |
InChI Key : | QEPWHIXHJNNGLU-QGZVFWFLSA-N |
Pubchem ID : | 40501223 |
GHS Pictogram: |
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Signal Word: | Warning |
Hazard Statements: | H302-H315-H319-H335 |
Precautionary Statements: | P261-P305+P351+P338 |
* All experimental methods are cited from the reference, please refer to the original source for details. We do not guarantee the accuracy of the content in the reference.
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Synthesis of Compound 1 (ZJ-MCC-Ahx-dEdEdEG): The peptide Fmoc-Ahx-dGlu-dGlu-dGlu-G was assembled on a Wang resin. The three glutamates (dGlu) are of D-isoform. Peptide synthesis was carried out manually by Fmoc chemistry with HCTU (2-(6-Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate) activation. Generally, peptides were synthesized at a 0.01 mmol scale starting from the C-terminal amino acid on solid support. Fmoc-deprotection at each cycle was carried out using 20percent piperidine in DMF. Coupling reactions were carried out using 3.3 eq. of Fmoc-amino acids in DMF activated with 3.3 eq. of HCTU and 5 equivalents of diisopropylethylamine (DIPEA) in DMF. These steps were repeated each time with an amino acid added. After the peptide sequence was built on the resin, the Fmoc group of the N-terminal amino acid was deprotected. Coupling of 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) to the N-terminal amine group was achieved with 3.3 equivalents of SMCC in DMF. Coupling of Cys-C(O)-Glu was performed using 3.3 equivalents of Cys-C(O)-Glu in DMF after coupling SMCC to the peptide. The final peptide resin was washed with DMF and then dichloromethane and dried. Cleavage and deprotection were carried out using TFA/water/triisopropylsilane (950:25:25) for 1 h, the resin was removed by filtration and washed with TFA. The combined filtrate was dried under nitrogen. The synthesized peptide was precipitated by the addition of diethyl ether and collected by centrifugation. The cleaved peptide was purified by preparative HPLC. The products were ascertained by high resolution matrix-assisted laser desorption/ionization mass (MALDI-MS) spectra. Then Fmoc was deprotected followed by coupling of SMCC and Cys-C(O)-Glu. The product has retention time of 11.9 minutes on analytical HPLC with 0-55percent gradient over 45 minutes (flow rate 1 ml/min; A: 10 mM triethylammonium acetate TEAA, pH 7.0; B was acetonitrile.) The mass was verified by MALDI/TOF mass spectrometry?Calculated: 1088.4 (C44H64N8O22S). Found m/z: 1089.4 (M+1). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Solid phase peptide synthesis was performed on a CEM Liberty Peptide Synthesizer using standard Fmoc chemistry. TentaGel S Ram resin (1 g; 0.25 mmol/g) was swelled in NMP (10 ml) prior to use and transferred between tube and reaction vessel using DCM and NMP. Coupling (0148) An Fmoc-amino acid in NMP/DMF/DCM (1:1:1; 0.2 M; 5 ml) was added to the resin in a CEM Discover microwave unit together with HATU/DMF or COMU/DMF (0.5 M; 2 ml) and DIPEA/NMP (2.0 M; 1 ml). The coupling mixture was heated to 75° C. for 5 min while nitrogen was bubbled through the mixture. The resin was then washed with NMP (4×10 ml). Deprotection (0149) Piperidine/DMF (20percent; 10 ml) was added to the resin for initial deprotection and the mixture was heated by microwaves (30 sec; 40° C.). The reaction vessel was drained and a second portion of piperidine/NMP (20percent; 10 ml) was added and heated (75° C.; 3 min.) again. The resin was then washed with DMF (6×10 ml). Side Chain Acylation (0150) Fmoc-Lys(ivDde)-OH or alternatively another amino acid with an orthogonal side chain protective group was introduced at the position of the acylation. The N-terminal of the peptide backbone was then Boc-protected using Boc2O or alternatively by using a Boc-protected amino acid in the last coupling. While the peptide was still attached to the resin, the orthogonal side chain protective group was selectively cleaved using freshly prepared hydrazine hydrate (2-4percent) in NMP for 2×15 min. The unprotected lysine side chain was first coupled with Fmoc-Glu-OtBu or another spacer amino acid, which was deprotected with piperidine and acylated with a lipophilic moiety using the peptide coupling methodology as described above. Alternatively, the acylation moiety was introduced as a premade building block e.g. Fmoc-Lys(hexadecanoyl-gamma-Glu)-OH where gamm-Glu is the coupling of Glutamic acid through the side-chain. Abbreviations employed are as follows: COMU: 1-[(1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethylamino-morpholinomethylene)]methanaminium hexaflourophosphate ivDde: 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)3-methyl-butyl Dde: 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-ethyl DCM: dichloromethane DMF: N,N-dimethylformamide (0151) DIPEA: diisopropylethylamine EtOH: ethanol Et2O: diethyl ether HATU: N-[(dimethylamino)-1H-1,2,3-triazol[4,5-b]pyridine-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide MeCN: acetonitrile NMP: N-methylpyrrolidone (0152) TFA: trifluoroacetic acid TIS: triisopropylsilane Cleavage (0153) The resin was washed with EtOH (3×10 ml) and Et2O (3×10 ml) and dried to constant weight at room temperature (r.t.). The crude peptide was cleaved from the resin by treatment with TFA/TIS/water (95/2.5/2.5; 40 ml, 2 h; r.t.). Most of the TFA was removed at reduced pressure and the crude peptide was precipitated and washed three times with diethylether and dried to constant weight at room temperature. HPLC Purification of the Crude Peptide (0154) The crude peptide was purified to greater than 90percent by preparative reverse phase HPLC using a PerSeptive Biosystems VISION Workstation equipped with a C-18 column (5 cm; 10 mum) and a fraction collector and run at 35 ml/min with a gradient of buffer A (0.1percent TFA, aq.) and buffer B (0.1percent TFA, 90percent MeCN, aq.). Fractions were analyzed by analytical HPLC and MS and relevant fractions were pooled and lyophilized. The final product was characterized by HPLC and MS. (0155) The synthesized compounds are shown in Table 1 and Table 2 |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: tGLP-1 and its analogues 2?13 were all synthesized using general solid-phase peptide synthesis of N-Fmoc/tBu chemistry. 63Fmoc Rink Amide-MBHA resin (0.1 mmol) was added to a 25 ml peptide synthetic vessel and swollen with DMF for 40 min. After deprotected by 25percent piperidine in DMF, a solution of Fmoc-AA-OH (0.4 mmol), HATU (0.4 mmol), HoAt (0.4 mmol) and DIPEA (0.8 mmol) in DMF was added to the vessel. After reacted for 1 h, the resin was washed three times with DMF and three times with CH2Cl2, then qualitative ninhydrin testing was performed to monitor whether some free amino groups still existed on the resin ornot. If not, the resin was washed three times with DMF again and repeated the procedures of deprotection and coupling. Forthe coupling of some unnatural amino acids, NMM instead of DIPEA and NMP instead of DMF were used. Besides, the reaction time was prolonged to 4 h. Following the final deprotection of N-terminus, the target peptide was cleaved from resin with Reagent K (TFA/thioanisole/water/phenol/EDT, 82.5:5:5:5:2.5) for 2 h atroom temperature. After filtration, the residue solution was concentrated, precipitated with cold diethyl ether and centrifuged for three times. The residue was dissolved in water and purified by Waters 2545 preparative RP-HPLC system. Sephadex G-25 was used for the further purification to remove some short peptide impurities. The molecular mass of the target peptide was confirmed by MALDI-TOF. The purity of peptide was tested with analytical RP-HPLC, and the conditions were as follows: a linear gradient of 20percent mobile phase A and 80percent mobile phase B to 80percent mobile phase A and 20percent mobile phase B (A: acetonitrile containing 0.1percent TFA; B: H2O containing 0.1percent TFA) in 30 min, at a flow rate of 1 mL/minute with UV detection at 214 nm. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
80% | General procedure: Peptides 4 and 5 were synthesized using standard SPPS on Rink Amide high yield resin (loading=0.45mmol/g). The beads were swollen in DMF for 1 hr prior to coupling. Each peptide bond formation was done with 3eq. of the Fmoc protected amino acid, 3eq. of COMU coupling reagent, and 6eq. of DIEA in DMF. Reaction was mixed constantly at room temperature for 30-60min and monitored by Kaiser test. Beads were washed with DMF (3×) MeOH (3×) DMF (3×) after coupling. The N-terminus Fmoc deprotection was done in 1:4 piperidine:DMF mixed constantly for 30min at room temperature. Beads were washed with DMF (6×) after deprotection. The peptide was cleaved from the bead using the cleavage cocktail TFA:H2O:TIPS=95:2.5:2.5 by volume. The TFA was evaporated and cold diethyl ether was added to precipitate peptide. The solution was centrifuged at 4000rpm at 4C for 10min and decanted (3× in cold ether). The peptide was dissolved in water for reverse-phase HPLC purification (ACN/H2O). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: Sufficient quantity (50-100 mg) of Fmoc-PAL-PEG-PS resin or Fmoc-Rink amide MB HA resin, loading: 0.5-0.6 mmol/g was swelled in DMF (1-10 ml /100 mg of resin) for 2-10 minutes. The Fmoc-group on resin was removed by incubation of resin with 10-30 % piperidine in DMF (10- 30 ml / 100 mg of resin), for 10-30 minutes. Deprotected resin was filtered and washed excess of DMF, DCM and ether (50 ml X 4). Washed resin was incubated in freshly distilled DMF (1 ml / 100 mg of resin), under nitrogen atmosphere for 5 minutes. A 0.5 M solution of first Fmoc- protected amino acid (1-3 eq.), pre-activated with HOBt (1-3 eq.) and DIPCDI (1-2 eq.) in DMF was added to the resin, and the resin was then shaken for 1-3 hrs, under nitrogen atmosphere. Coupling completion was monitored using a qualitative ninhydrin test. After the coupling of first amino acid, the resin was washed with DMF, DCM and Diethyl ether (50 ml X 4). For the coupling of next amino acid, firstly, the Fmoc-protection on first amino acid, coupled with resin was deprotected, using a 10-20% piperidine solution, followed by the coupling the Fmoc- protected second amino acid, using a suitable coupling agents, and as described above. The repeated cycles of deprotection, washing, coupling and washing were performed until the desired peptide chain was assembled on resin, as per general (Scheme 1) above. Finally, the Fmoc- protected peptidyl-resin prepared above was deprotected by 20% piperidine treatment as described above and the peptidyl-resins were washed with DMF, DCM and Diethyl ether. Resin containing desired peptide was dried under nitrogen pressure for 10-15 minutes and subjected for cleavage/ deprotection. Using above protocol and suitable variations thereof which are within the scope of a person skilled in the art, the short chain peptides designed in the present invention were prepared, using Fmoc-SPPS approach. Furthermore, resin bound short chain peptides were cleaved and deprotected, purified and characterized using following protocol. CLEAVAGE AND DEPROTECTION: The desired short chain peptides were cleaved and deprotected from their respective peptidyl- resins by treatment with TFA cleavage mixture as follows. A solution of TFA / Water / Triisopropylsilane (95: 2.5: 2.5) (10 ml / 100 mg of peptidyl-resin) was added to peptidyl-resins and the mixture was kept at room temperature with occasional starring. The resin was filtered, washed with a cleavage mixture and the combined filtrate was evaporated to dryness. Residue obtained was dissolved in 10 ml of water and the aqueous layer was extracted 3 times with ether and finally the aqueous layer was freeze-dried. Crude peptide obtained after freeze-drying was purified by preparative HPLC as follows: PREPARATIVE HPLC PURIFICATION OF THE CRUDE SHORT CHAIN PEPTIDES: Preparative HPLC was carried out on a Shimadzu LC-8A liquid chromatography. A solution of crude peptide dissolved in DMF or water was injected into a semi-Prep column (Luna 10mu; C18; 100 A0), dimension 250 X 50 mm and eluted with a linear gradient of ACN in water, both buffered with 0.1 % TFA, using a flow rate of 15 -50 ml / min, with effluent monitoring by PDA detector at 220 nm. A typical gradient of 20 % to 70 % of water- ACN mixture, buffered with 0.1 % TFA was used, over a period of 50 minutes, with 1% gradient change per minute. The desired product eluted were collected in a single 10-20 ml fraction and pure short chain peptides were obtained as amorphous white powders by lyophilisation of respective HPLC fractions. HPLC ANALYSIS OF THE PURIFIED SHORT-CHAIN PEPTIDES After purification by preparative HPLC as described above, each peptide was analyzed by analytical RP-HPLC on a Shimadzu LC-10AD analytical HPLC system. For analytical HPLC analysis of short chain peptides, Luna 5mu; C18; 100 A , dimension 250 X 4.6 mm column was used, with a linear gradient of 0.1 % TFA and ACN buffer and the acquisition of chromatogram was carried out at 220 nm, using a PDA detector. CHARACTERIZATION BY MASS SPECTROMETRY Each peptide was characterized by electrospray ionisation mass spectrometry (ESI-MS), either in flow injection or LC/MS mode. Triple quadrupole mass spectrometers (API-3000 (MDS-SCIES, Canada) was used in all analyses in positive and negative ion electrospray mode. Full scan data was acquired over the mass range of quadrupole, operated at unit resolution. In all cases, the experimentally measured molecular weight was within 0.5 Daltons of the calculated monoisotopic molecular weight. Quantification of the mass chromatogram was done using Analyst 1.4.1 software. Following table l(i) is the list of short chain peptides synthesized using the SPPS approach as described above. Mentioned Seq. ID. No 1 in the list was taken as a reference from WO 2011027257. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
76.5% | With diphenylphosphoranyl azide; N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 50℃; for 18h; | (1) Add 100 g (0.27) of N-fluorenylmethoxycarbonyl-L-glutamic acid to the reaction flask, and then add 500 ml of DMF.Further, 52.5 g (0.41 mol) of DIEA and 111.5 g (0.41 mol) of DPPA were added and stirred, and heated to 50 C for 18 h.The progress of the reaction was monitored by TLC. After the reaction was completed, 6N hydrochloric acid was added to adjust the pH of the system to 3.And the reaction was completed, and then the mixture was treated with ethyl acetate to give fmoc-dab-OH 70.5 g (0.21 mol).Yield 76.5%, placed for use; |