* 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.
Reference：
[1] European Journal of Inorganic Chemistry, 2016, vol. 2016, # 35, p. 5427 - 5434
2
[ 503-74-2 ]
[ 68858-20-8 ]
[ 35661-39-3 ]
[ 158257-40-0 ]
[ 26305-03-3 ]
Yield
Reaction Conditions
Operation in experiment
46%
Stage #1: With N-ethyl-N,N-diisopropylamine In dichloromethane for 3 h; Stage #2: With piperidine In N,N-dimethyl-formamide for 0.1 h;
General procedure: 4.2 General procedure A: resin loading (0026) Solid phase peptide synthesis was conducted manually in a sinter-fitted polypropylene syringe. 2-Chlorotritylchloride (CTC) resin was preswelled in DCM (mL) for 15min and drained. The first amino acid in 0.4M DIPEA/DCM was added and the mixture was agitated for 3h. After draining the solvent, any free 2-CTC resin linkers were capped by treatment of the resin with a solution of 17:2:1 DCM/MeOH/DIPEA (3×3mL×5min), and subsequently with a solution of 8:1:1 DMF/DIPEA/acetic anhydride (2×3mL×10min). The resin was finally washed with DCM (2×3mL×1min), DMF (2×3mL×1min), DCM (2×3mL×1min) and DMF (2×3mL×1min). 4.3 General procedure B: Fmoc deprotection (0027) The resin was agitated with a solution of 10percent piperidine inDMF (2×3mL×3min) and subsequently washed with DMF(3×3mL×1min), DCM (3×3mL×1min), DMF (5×3mL×1min). The deprotected solutions were combined and diluted appropriately (100-fold for 0.05mmol resin loading). The resin loading was estimated by measuring the absorbance of the piperidine-fulvene adduct with 10percent piperidine in DMF as a reference (λ=301nm; ε=7800M−1cm−1). 4.4 General procedure C: peptide coupling with HBTU (0028) A solution was prepared of the appropriate Fmoc-protected amino acid (3 equiv. relative to resin loading) and HBTU (2.9 equiv. relative to resin loading) in minimum amount of DMF. DIPEA (6 equiv. relative to resin loading) was added and the resin was agitated for 1.5h. The resin was then drained and washed with DMF (3×3mL×1min), DCM (3×3mL×1min) and DMF (5×3mL×1min). 4.5 General procedure D: peptide coupling with HATU (0029) A solution was prepared of the appropriate Fmoc-protected amino acid (3 equiv. relative to resin loading) and HATU (2.9 equiv. relation to resin loading) in minimal DMF. DIPEA (6 equiv. relation to resin loading) was added to the solution and the mixture was immediately added to the resin and agitated. Reaction times were altered based on the residue being coupled: Phe(NMe) and Ala (2×2h); Thr and Sta (1×2h); Asn, Leu, D-Val and L-Val (2×2h); DMVal (3×3h). Once the reaction was complete, the resin was drained and washed with DMF (3×3mL×1min), DCM (3×3mL×1min) and DMF (3×3mL×1min). 4.6 General procedure E: peptide coupling with DIC (0030) A solution was prepared of the appropriate Fmoc-protected amino acid (1.5 equiv. relative to resin loading), HOBt (1.5 equiv. relative to resin loading) and DIC (1.5 equiv. relative to resin loading) in minimal DMF. This solution was stirred for 20min, then added to the resin and agitated overnight. The resin was drained and washed with DMF (3×3mL×1min), DCM (3×3mL×1min) and DMF (5×3mL×1min). Double coupling of the next amino acid after the coupling of the fluorinated amino acid was applied. 4.7 General procedure F: resin cleavage (0031) After the last Fmoc deprotection, the resin was washed with DMF (3×3mL×1min) and DCM (3×3mL×1min) then dried in vacuo. The resin was agitated with a solution of 95:2.5:2.5 TFA/TIS/H2O (3mL) for 2h. The resin was drained and washed with the same TFA mixture above (2×3mL×1min). The combined cleavage solutions were concentrated under a stream of nitrogen. Diethyl ether was added and the supernatant was decanted (3×). The residue was then dried in vacuo to provide the crude linear peptide
Reference：
[1] European Journal of Organic Chemistry, 2013, # 21, p. 4509 - 4513
[2] Tetrahedron, 2014, vol. 70, # 14, p. 2351 - 2358
4
[ 6066-82-6 ]
[ 68858-20-8 ]
[ 130878-68-1 ]
Yield
Reaction Conditions
Operation in experiment
89%
With dicyclohexyl-carbodiimide In dichloromethane at 20℃; for 3 h;
Dicyclocarbodiimide (1 .55 g, 7.52 mmol) and N-hydroxysuccinimide (762 mg, 6.63 mmol) are added at room temperature to a stirrer solution of Fmoc-Val-OH [9] (1 .5 g, 4.42 mmol) in anhydrous dichloromethane (25 ml_). The mixture is kept at room temperature for 3 hours. The white solid formed in this reaction is filtrated with dichloromethane to remove the dicyclohexylurea, the organic phase is washed with HCI 0.1 N and water, then dried over anhydrous sodium sulfate and the solvent removed by rotatory evaporation. The residue is subjected to a flash column chromatography in 1 percent methanol in dichloromethane to afford product [10] as a white solid, 1 .7 g (89percent yield). MS: m/z 459 [M+Na]+.1H NMR (400 MHz, CDCIs) δ 7.80 (d, J = 7.5 Hz, 2H), 7.68 - 7.55 (m, 2H), 7.43 (t, J = 7.4 Hz, 2H), 7.34 (dd, J = 15.9, 8.5 Hz, 2H), 4.70 (d, J = 4.6 Hz, 1 H), 4.56 - 4.40 (m, 3H), 4.28 (t, J = 6.6 Hz, 1 H), 2.85 (s, 4H), 2.37 (dd, J = 12.3, 6.5 Hz, 1 H), 1 .08 (dd, J = 1 1 .0, 6.9 Hz, 6H).
65%
With dicyclohexyl-carbodiimide In 1,2-dimethoxyethane at 20℃; for 23 h; Cooling with ice; Inert atmosphere
Fmoc-Valine (1.02 g, 3 mmol) and N-hydroxysuccinimide (0.345 g, 3 mmol) were dissolved in dimethoxy ethane (35 ml) and cooled in an ice bath, then DCC (0.681 g, 3.3 mmol) was added. The resulting mixture was stirred in the ice bath for 3 hours, then at room temperature for 20 hours. The precipitate formed was filtered off and the filtrate concentrated under vacuo. The crude product was further purified by flash chromatography (ethyl acetate/hexane, v:v, 4:6) to afford SI20B as a white solid. Isolated yield: 65percent. TLC (EtOAc:Hexane 3:2). Rf=0.57, irradiated by a UV lamp. HPLC: 0.1percent TFA (v/v) in water (solvent A):acetonitrile (solvent B); gradient 45-85percent in 30 min, flow rate=0.5 mL/min. Retention time (Rt)=15.77 min. 1H NMR (FIG. 26A): (400 MHz, CDCl3) δ 7.76-7.78 (d, J=7.20 Hz, 2H), 7.59-7.60 (d, J=7.20 Hz, 2H), 7.38-7.42 (t, J=7.20 Hz, 2H), 7.30-7.34 (m, 2H), 5.26-5.28 (d, J=9.20 Hz, 1H), 4.67-4.71 (dd, J=4.8, 5.2 Hz, 1H), 4.42-4.46 (dd, J=6.8, 6.4 Hz, 2H), 4.23-4.26 (t, J=6.8 Hz, 1H), 2.84 (s, 4H), 2.04-2.36 (m, 1H), 0.83-088 (m, 6H). 13C NMR (FIG. 26B): (101 MHz, CDCl3) δ 168.7, 141.5, 127.9, 127.3, 127.3, 125.3, 120.2, 120.2, 67.5, 57.7, 47.4, 31.9, 25.8, 18.9, 17.5.
26.51 g
With dicyclohexyl-carbodiimide In tetrahydrofuran at 0 - 20℃; for 6 h; Inert atmosphere
[00125] This compound is prepared according to R. A. Firestone et al, US 6,214,345. Fmoc-Val-OH (20.24 g; 59.64 mmol) and N-hydroxysuccinimide (6.86 g =1 .0 eq.) in tetrahydrofuran (200 ml) at 000 were treated with N,N’dicyclohexylcarbodiimide (12.30 g; 1.0 eq.). The mixture was stirred at RT under argon atmosphere for 6 h and then the solid dicyclohexyl urea (DCU) by-product was filtered off and washed with THF and the solvent was removed by rotavap. The residue was dissolved in 300 ml dichloromethane, cooled in an ice bath for 1 h and filtered again to remove additional DCU. The dichloromethane was evaporated and the solid foam (26.51 g) was used in the next step without further purification.
Reference：
[1] Journal of Pharmaceutical Sciences, 1994, vol. 83, # 7, p. 999 - 1005
[2] Langmuir, 2010, vol. 26, # 7, p. 4990 - 4998
[3] Patent: WO2018/178060, 2018, A1, . Location in patent: Page/Page column 64-66
[4] Journal of the American Chemical Society, 2015, vol. 137, # 21, p. 6932 - 6940
[5] Patent: US2017/247324, 2017, A1, . Location in patent: Paragraph 0284; 0285
[6] Tetrahedron, 2009, vol. 65, # 19, p. 3871 - 3877
[7] Organic and Biomolecular Chemistry, 2011, vol. 9, # 11, p. 4182 - 4187
[8] Journal of Controlled Release, 2012, vol. 160, # 3, p. 618 - 629
[9] Patent: WO2013/67597, 2013, A1, . Location in patent: Page/Page column 80
[10] Patent: WO2014/80251, 2014, A1, . Location in patent: Sheet 16/23
[11] Patent: WO2015/21092, 2015, A1, . Location in patent: Page/Page column 36
[12] Bioconjugate Chemistry, 2015, vol. 26, # 11, p. 2261 - 2278
[13] Patent: WO2017/149077, 2017, A1, . Location in patent: Paragraph 00128-00129
[14] Patent: CN107789630, 2018, A, . Location in patent: Paragraph 0067; 0068; 0069
[15] Patent: WO2018/115466, 2018, A1, . Location in patent: Paragraph 00103; 00124; 00125
Stage #1: With 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane-2,4,6-trioxide; N-ethyl-N,N-diisopropylamine In ethyl acetate at 0℃; for 0.166667 h; Stage #2: With sodium tetrahydroborate In water; ethyl acetate at 0℃; for 0.466667 h;
General procedure: To a solution of carboxylic acid (10 mmol) in THF (10 mL), DIPEA (11 mmol, 1.42 mL) and 50percent T3P in EtOAc (20 mmol, 6.36 mL) were added at 0 °C and the solution was stirred for about 10 min. Then aqueous solution of NaBH4 (10 mmol, 388 mg in 0.3 mL of H2O) was added to the reaction mixture at the same temperature and the reaction was allowed to stir till the completion of the reaction as indicated by TLC. After the completion of the reaction, the solvent was evaporated and the crude alcohol was extracted into EtOAc and the organic phase was washed with 5percent citric acid (10 mL .x. 2), 5percent Na2CO3 (10 mL .x. 2), water, and brine solution. The product was isolated after the evaporation of solvent under reduced pressure and dried over anhydrous Na2SO4.
Reference：
[1] Journal of Organic Chemistry, 2001, vol. 66, # 25, p. 8454 - 8462
[2] Tetrahedron Letters, 1999, vol. 40, # 23, p. 4395 - 4396
[3] Tetrahedron Letters, 2012, vol. 53, # 38, p. 5059 - 5063
[4] RSC Advances, 2014, vol. 4, # 87, p. 46947 - 46950
[5] Organic and Biomolecular Chemistry, 2011, vol. 9, # 11, p. 4182 - 4187
[6] Tetrahedron Asymmetry, 1998, vol. 9, # 11, p. 1855 - 1858
[7] Journal of Organic Chemistry, 2009, vol. 74, # 15, p. 5260 - 5266
[8] Synthetic Communications, 2009, vol. 39, # 19, p. 3555 - 3566
[9] Bioorganic and Medicinal Chemistry, 2011, vol. 19, # 7, p. 2397 - 2406
[10] Organic and Biomolecular Chemistry, 2011, vol. 9, # 11, p. 4182 - 4187
[11] ACS Combinatorial Science, 2017, vol. 19, # 3, p. 131 - 136
[12] Organic and Biomolecular Chemistry, 2018, vol. 16, # 26, p. 4874 - 4880
8
[ 68858-20-8 ]
[ 159858-22-7 ]
Reference：
[1] Journal of Controlled Release, 2012, vol. 160, # 3, p. 618 - 629
[2] Patent: WO2014/80251, 2014, A1,
[3] Bioconjugate Chemistry, 2015, vol. 26, # 11, p. 2261 - 2278
[4] Patent: US2016/271270, 2016, A1,
[5] International Journal of Molecular Sciences, 2017, vol. 18, # 9,
[6] Patent: CN107789630, 2018, A,
[7] Patent: WO2018/178060, 2018, A1,
Chelmical synthesis: Peptides were synthesized on a Rink amide resin, 0.45 mmol/g [Fmoc-Cys(Trityl)-Wang; Novabiochem, San Diego, Calif.] usinig N-(9-fluorenyl)methoxycarboxyl chemistry and standard side chain protection except on cysteine residues. Cysteine residues were protected in pairs with either S-trityl on the first and third cysteines or S-acetamidomethyl on the second and fourth cysteines. Amino acid derivatives were from Advanced Chemtech (Louisville, Ky.). The peptides were removed from the resin and precipitated, and a two-step oxidation protocol was used to selectively fold the peptides as described previously (Luo et al., 1999). Briefly, the first disulfide bridge was closed by dripping the peptide into an equal volume of 20 mM potassium feliicyanide and 0.1 M Tris, pH 7.5. The solution was allowed to react for 30 min, and the monocyclic peptide was purified by reverse-phase HPLC. Simultaneous removal of the S-acetamidomethyl groups and closure of the second disulfide bridge was carried out by iodine oxidation. The monocyclic peptide and HPLC eluent was dripped into an equal volume of iodine (10 mM) in H20/trifluoroacetic acid/acetonitrile (78:2:20 by volume) and allowed to react for 10 min. The reaction was terminated by the addition of ascorbic acid diluted 20-fold with 0.1percent trifluoroacetic acid and the bicyclic product purified by HPLC. Mass Spectrometry: Measurements were performed at the Salk Institute for Biological Studies (San Diego, Calif.) under the direction of Jean Rivier. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and liquid secondary ionization mass spectrometry were used.
Example 9: Preparing the Tripeptide Ac-(D-Msa)-Val-Nal-NH2; A syringe was provided with 100 mg (0.06 mmol) of Rink amide resin; it was conditioned with CH2CL2 (5 x 1 min) and DMF (5 x 1 min). The synthesis was carried out by means of a standard Fmoc/tBu strategy, using diisopropylcarbodiimide (DIPCDI) as a coupling agent and hydroxybenzotriazole (HOBt) as an additive. Fmoc-3-(1-naphthyl)-L-Ala-OH (Fmoc-Nal) (78.75 mg, 0.18 mmol, 3 eq), Fmoc-L-Val-OH (61.2 mg, 0.18 mmol, 3 eq) and Fmoc-D-Mesityl alanine-OH (Fmoc-D-Msa) (77.4 mg, 0.18 mmol, 3 eq) were used, and the successive incorporation of amino acids was corroborated with ninhydrin tests. After incorporating the third amino acid, the Fmoc group was removed with a mixture of piperidine in DMF and the free amino end was acetylated with Ac2O - diisopropyldiethylamine (DIEA). The resin was finally filtered and thoroughly washed with DMF (5 x 1 min), CH2CL2 (5 x 1 min) and methanol (5 x 1 min). To cleave the tripeptide, it was treated with a trifluoroacetic-water-triisopropylsilane (TFA-H2O-TIS) (95:2.5:2.5) solution for 1 hour and the resulting filtrate was evaporated. It was characterized by reverse-phase chromatography (HPLC) and by EM. After 9 synthesis steps and with no intermediate purification; the obtained crude product had a 39percent purity. It was purified with a semi-preparative HPLC (gradient 20-50 in 10 min and 50-100 in 15 min) obtaining 3.4 mg of the tripeptide with an 86percent purity (lambda = 220 nm). HPLC-MS: tr (H2O 0.1percent HCOOH; ACN 0.07percent HCOOH)=4.830 min. ES+:545.65 (calc. C32H40N4O4, 544.30).
Nα-(9-fluorenylmethyloxycarbonyl)-Nγ-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine[ No CAS ]
[ 268209-75-2 ]
(Boc-Ser(tBu))-Lys(Boc)-Glu(OtBu)-Pro-Leu-Arg(pbf)-Pro-Arg(pbf)-(S-(acetylaminomethyl)Cys)-Arg(pbf)-Pro-Ile-(O-(2-(isopropyl)phenyl)Asp)-Ala-(N,O-isopropylidene-Thr)-Leu-Ala-Val-Glu(OtBu)-Lys(Boc)-Glu(OtBu)-Gly-OH[ No CAS ]
Nα-(9-fluorenylmethyloxycarbonyl)-Nγ-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine[ No CAS ]
[ 268209-75-2 ]
(Boc-Ser(tBu))-Lys(Boc)-Glu(OtBu)-Pro-Leu-Arg(pbf)-(S-(acetylaminomethyl)Cys)-Arg(pbf)-Pro-Ile-(O-(2-(isopropyl)phenyl)Asp)-Ala-(N,O-isopropylidene-Thr)-Leu-Ala-Val-Glu(OtBu)-Lys(Boc)-Glu(OtBu)-Gly-(S-(acetylaminomethyl)Cys)-Pro-Val-(S-(acetylaminomethyl)Cys)-Ile-(N,O-isopropylidene-Thr)-Val-(O-(2-(isopropyl)phenyl)Asp)-Thr(tBu)-Thr(tBu)-Ile-(S-(acetylaminomethyl)Cys)-Ala-Gly-OH[ No CAS ]
Nα-(9-fluorenylmethyloxycarbonyl)-Nγ-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine[ No CAS ]
[ 268209-75-2 ]
Ser(Boc)-Arg(pbf)-Glu(OtBu)-Pro-Leu-Arg(pbf)-Pro-Trp(Boc)-(S-(acetylaminomethyl)Cys)-His(Trt)-Pro-Ile-Asn(Trt)-Ala-Ile-Leu-Ala-Val-Glu(OtBu)-Lys(Boc)-Glu(OtBu)-Gly-(S-(acetylaminomethyl)Cys)-Pro-Val-(S-(acetylaminomethyl)Cys)-Ile-(N,O-isopropylidene-Thr)-Val-(O-(2-(isopropyl)phenyl)Asp)-Thr(tBu)-Thr(tBu)-Ile-(S-(acetylaminomethyl)Cys)-Ala-Gly-OH[ No CAS ]
Y-(α-aminoisobutyroyl)-EGTFTSDYSIYLDKKAQRAFVNWLLA-(α-aminoisobutyroyl)-KYG-(β-(1-naphthyl)-alaninoyl)-LDF-NH2[ No CAS ]
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
Peptide monomers of the present invention were synthesized using the Merrifield solid phase synthesis techniques on Protein Technology's Symphony multiple channel synthesizer. The peptides were assembled using HBTU (0-Benzotriazole-N,N,N',N'-tetramethyl-uronium- hexafluoro-phosphate), Diisopropylethylamine(DIEA) coupling conditions. For some amino acid couplings PyAOP(7-Azabenzotriazol- 1 -yloxy)tripyrrolidinophosponium hexafluorophosphate) and DIEA conditions were used. Rink Amide MB HA resin (100-200 mesh, 0.57 mmol/g) was used for peptide with C-terminal amides and pre-loaded Wang Resin with N-a-Fmoc protected amino acid was used for peptide with C-terminal acids. The coupling reagents (HBTU and DIEA premixed) were prepared at lOOmmol concentration. Similarly amino acids solutions were prepared at 100 mmol concentration. Peptide inhibitors of the present invention were identified based on medical chemistry optimization and/or phage display and screened to identify those having superior binding and/or inhibitory properties.[00611] The peptides were assembled using standard Symphony protocols. The peptide sequences were assembled as follows: Resin (250 mg, 0.14 mmol) in each reaction vial was washed twice with 4ml of DMF followed by treatment with 2.5ml of 20percent 4-methyl piped dine (Fmoc de- protection) for lOmin. The resin was then filtered and washed two times with DMF (4ml) and re -treated with N-methyl piperifine for additional 30 minute. The resin was again washed three times with DMF (4 ml) followed by addition 2.5ml of amino acid and 2.5ml of HBTU-DIEA mixture. After 45min of frequent agitations, the resin was filtered and washed three timed with DMF (4 ml each). For a typical peptide of the present invention, double couplings were performed. After completing the coupling reaction, the resin was washed three times with DMF (4 ml each) before proceeding to the next amino acid coupling.
Peptide monomers of the present invention were synthesized using the Merrifield solid phase synthesis techniques on Protein Technology's Symphony multiple channel synthesizer. The peptides were assembled using HBTU (0-Benzotriazole-N,N,N',N'-tetramethyl-uronium- hexafluoro-phosphate), Diisopropylethylamine(DIEA) coupling conditions. For some amino acid couplings PyAOP(7-Azabenzotriazol- 1 -yloxy)tripyrrolidinophosponium hexafluorophosphate) and DIEA conditions were used. Rink Amide MB HA resin (100-200 mesh, 0.57 mmol/g) was used for peptide with C-terminal amides and pre-loaded Wang Resin with N-a-Fmoc protected amino acid was used for peptide with C-terminal acids. The coupling reagents (HBTU and DIEA premixed) were prepared at lOOmmol concentration. Similarly amino acids solutions were prepared at 100 mmol concentration. Peptide inhibitors of the present invention were identified based on medical chemistry optimization and/or phage display and screened to identify those having superior binding and/or inhibitory properties.[00611] The peptides were assembled using standard Symphony protocols. The peptide sequences were assembled as follows: Resin (250 mg, 0.14 mmol) in each reaction vial was washed twice with 4ml of DMF followed by treatment with 2.5ml of 20percent 4-methyl piped dine (Fmoc de- protection) for lOmin. The resin was then filtered and washed two times with DMF (4ml) and re -treated with N-methyl piperifine for additional 30 minute. The resin was again washed three times with DMF (4 ml) followed by addition 2.5ml of amino acid and 2.5ml of HBTU-DIEA mixture. After 45min of frequent agitations, the resin was filtered and washed three timed with DMF (4 ml each). For a typical peptide of the present invention, double couplings were performed. After completing the coupling reaction, the resin was washed three times with DMF (4 ml each) before proceeding to the next amino acid coupling.
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.
N-α-(9-fluorenylmethyloxycarbonyl)-N-γ-tert-butyloxycarbonyl-D-2,4-diaminobutyric acid[ No CAS ]
C68H118N22O18S2[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
1. Peptide synthesis 1.1 General synthetic procedures A general method for the synthesis of the peptidomimetics of the present invention is exemplified in the following. This is to demonstrate the principal concept and does not limit or restrict the present invention in any way. A person skilled in the art is easily able to modify these procedures, especially, but not limited to, choosing a different starting position within the ring system, to still achieve the preparation of the claimed cyclic peptidomimetic compounds of the present invention. Coupling of the first protected amino acid residue to the resin . In a dried flask, 2-chlorotritylchloride resin (polystyrene, 1percent crosslinked; loading: 1.4 mMol/g) was swollen in dry CH2CI2 for 30 min (7 mL CH2CI2 per g resin). A solution of 0.8 eq of the Fmoc-protected amino acid and 6 eq of DIPEA in dry CH2CI2/DMF (4/1) (10 mL per g resin) was added. After shaking for 2-4 h at rt the resin was filtered off and washed successively with CH2CI2, DMF, CH2CI2, DMF and CH2CI2. Then a solution of dry CH2CI2/MeOH/DIPEA (17:2:1) was added (10 mL per g resin). After shaking for 3 x 30 min the resin was filtered off in a pre-weighed sinter funnel and washed successively with CH2CI2, DMF, CH2CI2, MeOH, CH2CI2, MeOH, CH2CI2 (2x) and Et20 (2x). The resin was dried under high vacuum overnight. The final mass of resin was calculated before the qualitative control. Loading was typically 0.6 - 0.7 mMol/g. The following preloaded resins were prepared: Fmoc-Dab(Boc)-2-chlorotrityl resin, Fmoc-DDab(Boc)-2-chlorotrityl resin, Fmoc-Lys(Boc)-2-chlorotrityl resin, Fmoc- Trp(Boc)-2-chlortrityl resin, Fmoc-Phe-2-chlortrityl resin; Fmoc-Val-2-chlorotrityl resin, Fmoc-Pro-2-chlorotrityl resin, Fmoc-Arg(Pbf)-2-chlorotrityl resin and Fmoc-Glu(iBu)-2- chlorotrityl resin. Synthesis of the fully protected peptide fragment The synthesis was carried out on a Syro-peptide synthesizer (MultiSynTech GmbH) using 24 to 96 reaction vessels. In each vessel 0.04 mMol of the above resin were placed and the resin was swelled in CH2CI2 and DMF for 15 min, respectively. The following reaction cycles were programmed and carried out: Step Reagent Time 1 CH2CI2, wash and swell (manual) 1 x 3 min 2 DMF, wash and swell 2 x 30 min 3 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 4 DMF, wash 5 x 1 min 5 3.5 eq Fmoc amino acid/3.5 eq HOAt in DMF + 3.5 eq PyBOP/7 eq DIPEA or 3.5 eq DIC 1 x 40 min 6 3.5 eq Fmoc amino acid/DMF + 3.5 eq HATU or PyBOP or HCTU + 7 eq DIPEA 1 x 40 min 7 DMF, wash 5 x 1 min 8 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 9 DMF, wash 5 x 1 min 10 CH2CI2, wash (at the end of the synthesis) 3 x 1 min Steps 5 to 9 are repeated to add each amino-acid residue. After the termination of the synthesis of the fully protected peptide fragment, one of the procedures A - E, as described herein below, was adopted subsequently, depending on which kind of interstrand linkages, as described herein below, were to be formed. Finally, the peptides were purified by preparative reverse phase LC-MS, as described herein below. Procedure A: Cyclization and work up of a backbone cyclized peptide having no interstrand linkage Cleavage, backbone cyclization and deprotection After assembly of the linear peptide, the resin was suspended in 1 mL of 1percent TFA in CH2CI2 (v/v; 0.14 mMol) for 3 minutes. After filtration the filtrate was neutralized with 1 mL of 20percent DI PEA in CH2CI2 (v/v; 1.15 mMol). This procedure was repeated four times to ensure completion of the cleavage. An alternative cleavage method comprises suspension of the resin in lmL of 20percent HFIP in CH2CI2 (v/v; 1.9 mMol) for 30 minutes, filtration and repetition of the procedure. The resin was washed three times with 1 mL of CH2CI2. The CH2CI2 layers containing product were evaporated to dryness. The fully protected linear peptide was solubilised in 8 mL of dry DM F. Then 2 eq of HATU and 2 eq of HOAt in dry DM F (1-2 mL) and 4 eq of DIPEA in dry DM F (1-2 mL) were added to the peptide, followed by stirring for ca. 16 h. The volatiles were removed by evaporation. The crude cyclic peptide was dissolved in 7 mL of CH2CI2 and washed three times with 4.5 mL 10percent acetonitrile in water (v/v). The CH2CI2 layer was then evaporated to dryness. To fully deprotect the peptide, 7 mL of cleavage cocktail TFA/DODT/thioanisol/H20 (87.5 :2.5:5:5) or TFA/TIS/H20 (95:2.5 :2.5) were added, and the mixture was kept for 2.5-4 h at room temperature until the reaction was completed. The reaction mixture was evaporated close to dryness, the peptide precipitated with 7 mL of cold Et20/pentane and finally washed 3 times with 4 mL of cold Et20/pentane. Procedures Bl and B2: Cyclization and work up of a backbone cyclized peptide having a disulfide interstrand linkage Bl: Formation of a disulfide interstrand linkage using DMSO After cleavage, backbone cyclization and deprotection of the linear peptide, as described in the corresponding section of procedure A, th...
N-α-(9-fluorenylmethyloxycarbonyl)-N-γ-tert-butyloxycarbonyl-D-2,4-diaminobutyric acid[ No CAS ]
C76H115N21O19S2[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
1. Peptide synthesis 1.1 General synthetic procedures A general method for the synthesis of the peptidomimetics of the present invention is exemplified in the following. This is to demonstrate the principal concept and does not limit or restrict the present invention in any way. A person skilled in the art is easily able to modify these procedures, especially, but not limited to, choosing a different starting position within the ring system, to still achieve the preparation of the claimed cyclic peptidomimetic compounds of the present invention. Coupling of the first protected amino acid residue to the resin . In a dried flask, 2-chlorotritylchloride resin (polystyrene, 1percent crosslinked; loading: 1.4 mMol/g) was swollen in dry CH2CI2 for 30 min (7 mL CH2CI2 per g resin). A solution of 0.8 eq of the Fmoc-protected amino acid and 6 eq of DIPEA in dry CH2CI2/DMF (4/1) (10 mL per g resin) was added. After shaking for 2-4 h at rt the resin was filtered off and washed successively with CH2CI2, DMF, CH2CI2, DMF and CH2CI2. Then a solution of dry CH2CI2/MeOH/DIPEA (17:2:1) was added (10 mL per g resin). After shaking for 3 x 30 min the resin was filtered off in a pre-weighed sinter funnel and washed successively with CH2CI2, DMF, CH2CI2, MeOH, CH2CI2, MeOH, CH2CI2 (2x) and Et20 (2x). The resin was dried under high vacuum overnight. The final mass of resin was calculated before the qualitative control. Loading was typically 0.6 - 0.7 mMol/g. The following preloaded resins were prepared: Fmoc-Dab(Boc)-2-chlorotrityl resin, Fmoc-DDab(Boc)-2-chlorotrityl resin, Fmoc-Lys(Boc)-2-chlorotrityl resin, Fmoc- Trp(Boc)-2-chlortrityl resin, Fmoc-Phe-2-chlortrityl resin; Fmoc-Val-2-chlorotrityl resin, Fmoc-Pro-2-chlorotrityl resin, Fmoc-Arg(Pbf)-2-chlorotrityl resin and Fmoc-Glu(iBu)-2- chlorotrityl resin. Synthesis of the fully protected peptide fragment The synthesis was carried out on a Syro-peptide synthesizer (MultiSynTech GmbH) using 24 to 96 reaction vessels. In each vessel 0.04 mMol of the above resin were placed and the resin was swelled in CH2CI2 and DMF for 15 min, respectively. The following reaction cycles were programmed and carried out: Step Reagent Time 1 CH2CI2, wash and swell (manual) 1 x 3 min 2 DMF, wash and swell 2 x 30 min 3 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 4 DMF, wash 5 x 1 min 5 3.5 eq Fmoc amino acid/3.5 eq HOAt in DMF + 3.5 eq PyBOP/7 eq DIPEA or 3.5 eq DIC 1 x 40 min 6 3.5 eq Fmoc amino acid/DMF + 3.5 eq HATU or PyBOP or HCTU + 7 eq DIPEA 1 x 40 min 7 DMF, wash 5 x 1 min 8 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 9 DMF, wash 5 x 1 min 10 CH2CI2, wash (at the end of the synthesis) 3 x 1 min Steps 5 to 9 are repeated to add each amino-acid residue. After the termination of the synthesis of the fully protected peptide fragment, one of the procedures A - E, as described herein below, was adopted subsequently, depending on which kind of interstrand linkages, as described herein below, were to be formed. Finally, the peptides were purified by preparative reverse phase LC-MS, as described herein below. Procedure A: Cyclization and work up of a backbone cyclized peptide having no interstrand linkage Cleavage, backbone cyclization and deprotection After assembly of the linear peptide, the resin was suspended in 1 mL of 1percent TFA in CH2CI2 (v/v; 0.14 mMol) for 3 minutes. After filtration the filtrate was neutralized with 1 mL of 20percent DI PEA in CH2CI2 (v/v; 1.15 mMol). This procedure was repeated four times to ensure completion of the cleavage. An alternative cleavage method comprises suspension of the resin in lmL of 20percent HFIP in CH2CI2 (v/v; 1.9 mMol) for 30 minutes, filtration and repetition of the procedure. The resin was washed three times with 1 mL of CH2CI2. The CH2CI2 layers containing product were evaporated to dryness. The fully protected linear peptide was solubilised in 8 mL of dry DM F. Then 2 eq of HATU and 2 eq of HOAt in dry DM F (1-2 mL) and 4 eq of DIPEA in dry DM F (1-2 mL) were added to the peptide, followed by stirring for ca. 16 h. The volatiles were removed by evaporation. The crude cyclic peptide was dissolved in 7 mL of CH2CI2 and washed three times with 4.5 mL 10percent acetonitrile in water (v/v). The CH2CI2 layer was then evaporated to dryness. To fully deprotect the peptide, 7 mL of cleavage cocktail TFA/DODT/thioanisol/H20 (87.5 :2.5:5:5) or TFA/TIS/H20 (95:2.5 :2.5) were added, and the mixture was kept for 2.5-4 h at room temperature until the reaction was completed. The reaction mixture was evaporated close to dryness, the peptide precipitated with 7 mL of cold Et20/pentane and finally washed 3 times with 4 mL of cold Et20/pentane. Procedures Bl and B2: Cyclization and work up of a backbone cyclized peptide having a disulfide interstrand linkage Bl: Formation of a disulfide interstrand linkage using DMSO After cleavage, backbone cyclization and deprotection of the linear peptide, as described in the corresponding section of procedure A, th...
N-α-(9-fluorenylmethyloxycarbonyl)-N-γ-tert-butyloxycarbonyl-D-2,4-diaminobutyric acid[ No CAS ]
C76H115N23O19S2[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
1. Peptide synthesis 1.1 General synthetic procedures A general method for the synthesis of the peptidomimetics of the present invention is exemplified in the following. This is to demonstrate the principal concept and does not limit or restrict the present invention in any way. A person skilled in the art is easily able to modify these procedures, especially, but not limited to, choosing a different starting position within the ring system, to still achieve the preparation of the claimed cyclic peptidomimetic compounds of the present invention. Coupling of the first protected amino acid residue to the resin . In a dried flask, 2-chlorotritylchloride resin (polystyrene, 1percent crosslinked; loading: 1.4 mMol/g) was swollen in dry CH2CI2 for 30 min (7 mL CH2CI2 per g resin). A solution of 0.8 eq of the Fmoc-protected amino acid and 6 eq of DIPEA in dry CH2CI2/DMF (4/1) (10 mL per g resin) was added. After shaking for 2-4 h at rt the resin was filtered off and washed successively with CH2CI2, DMF, CH2CI2, DMF and CH2CI2. Then a solution of dry CH2CI2/MeOH/DIPEA (17:2:1) was added (10 mL per g resin). After shaking for 3 x 30 min the resin was filtered off in a pre-weighed sinter funnel and washed successively with CH2CI2, DMF, CH2CI2, MeOH, CH2CI2, MeOH, CH2CI2 (2x) and Et20 (2x). The resin was dried under high vacuum overnight. The final mass of resin was calculated before the qualitative control. Loading was typically 0.6 - 0.7 mMol/g. The following preloaded resins were prepared: Fmoc-Dab(Boc)-2-chlorotrityl resin, Fmoc-DDab(Boc)-2-chlorotrityl resin, Fmoc-Lys(Boc)-2-chlorotrityl resin, Fmoc- Trp(Boc)-2-chlortrityl resin, Fmoc-Phe-2-chlortrityl resin; Fmoc-Val-2-chlorotrityl resin, Fmoc-Pro-2-chlorotrityl resin, Fmoc-Arg(Pbf)-2-chlorotrityl resin and Fmoc-Glu(iBu)-2- chlorotrityl resin. Synthesis of the fully protected peptide fragment The synthesis was carried out on a Syro-peptide synthesizer (MultiSynTech GmbH) using 24 to 96 reaction vessels. In each vessel 0.04 mMol of the above resin were placed and the resin was swelled in CH2CI2 and DMF for 15 min, respectively. The following reaction cycles were programmed and carried out: Step Reagent Time 1 CH2CI2, wash and swell (manual) 1 x 3 min 2 DMF, wash and swell 2 x 30 min 3 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 4 DMF, wash 5 x 1 min 5 3.5 eq Fmoc amino acid/3.5 eq HOAt in DMF + 3.5 eq PyBOP/7 eq DIPEA or 3.5 eq DIC 1 x 40 min 6 3.5 eq Fmoc amino acid/DMF + 3.5 eq HATU or PyBOP or HCTU + 7 eq DIPEA 1 x 40 min 7 DMF, wash 5 x 1 min 8 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 9 DMF, wash 5 x 1 min 10 CH2CI2, wash (at the end of the synthesis) 3 x 1 min Steps 5 to 9 are repeated to add each amino-acid residue. After the termination of the synthesis of the fully protected peptide fragment, one of the procedures A - E, as described herein below, was adopted subsequently, depending on which kind of interstrand linkages, as described herein below, were to be formed. Finally, the peptides were purified by preparative reverse phase LC-MS, as described herein below. Procedure A: Cyclization and work up of a backbone cyclized peptide having no interstrand linkage Cleavage, backbone cyclization and deprotection After assembly of the linear peptide, the resin was suspended in 1 mL of 1percent TFA in CH2CI2 (v/v; 0.14 mMol) for 3 minutes. After filtration the filtrate was neutralized with 1 mL of 20percent DI PEA in CH2CI2 (v/v; 1.15 mMol). This procedure was repeated four times to ensure completion of the cleavage. An alternative cleavage method comprises suspension of the resin in lmL of 20percent HFIP in CH2CI2 (v/v; 1.9 mMol) for 30 minutes, filtration and repetition of the procedure. The resin was washed three times with 1 mL of CH2CI2. The CH2CI2 layers containing product were evaporated to dryness. The fully protected linear peptide was solubilised in 8 mL of dry DM F. Then 2 eq of HATU and 2 eq of HOAt in dry DM F (1-2 mL) and 4 eq of DIPEA in dry DM F (1-2 mL) were added to the peptide, followed by stirring for ca. 16 h. The volatiles were removed by evaporation. The crude cyclic peptide was dissolved in 7 mL of CH2CI2 and washed three times with 4.5 mL 10percent acetonitrile in water (v/v). The CH2CI2 layer was then evaporated to dryness. To fully deprotect the peptide, 7 mL of cleavage cocktail TFA/DODT/thioanisol/H20 (87.5 :2.5:5:5) or TFA/TIS/H20 (95:2.5 :2.5) were added, and the mixture was kept for 2.5-4 h at room temperature until the reaction was completed. The reaction mixture was evaporated close to dryness, the peptide precipitated with 7 mL of cold Et20/pentane and finally washed 3 times with 4 mL of cold Et20/pentane. Procedures Bl and B2: Cyclization and work up of a backbone cyclized peptide having a disulfide interstrand linkage Bl: Formation of a disulfide interstrand linkage using DMSO After cleavage, backbone cyclization and deprotection of the linear peptide, as described in the corresponding section of procedure A, th...
N-α-(9-fluorenylmethyloxycarbonyl)-N-γ-tert-butyloxycarbonyl-D-2,4-diaminobutyric acid[ No CAS ]
C76H115N23O19S2[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
1. Peptide synthesis 1.1 General synthetic procedures A general method for the synthesis of the peptidomimetics of the present invention is exemplified in the following. This is to demonstrate the principal concept and does not limit or restrict the present invention in any way. A person skilled in the art is easily able to modify these procedures, especially, but not limited to, choosing a different starting position within the ring system, to still achieve the preparation of the claimed cyclic peptidomimetic compounds of the present invention. Coupling of the first protected amino acid residue to the resin . In a dried flask, 2-chlorotritylchloride resin (polystyrene, 1percent crosslinked; loading: 1.4 mMol/g) was swollen in dry CH2CI2 for 30 min (7 mL CH2CI2 per g resin). A solution of 0.8 eq of the Fmoc-protected amino acid and 6 eq of DIPEA in dry CH2CI2/DMF (4/1) (10 mL per g resin) was added. After shaking for 2-4 h at rt the resin was filtered off and washed successively with CH2CI2, DMF, CH2CI2, DMF and CH2CI2. Then a solution of dry CH2CI2/MeOH/DIPEA (17:2:1) was added (10 mL per g resin). After shaking for 3 x 30 min the resin was filtered off in a pre-weighed sinter funnel and washed successively with CH2CI2, DMF, CH2CI2, MeOH, CH2CI2, MeOH, CH2CI2 (2x) and Et20 (2x). The resin was dried under high vacuum overnight. The final mass of resin was calculated before the qualitative control. Loading was typically 0.6 - 0.7 mMol/g. The following preloaded resins were prepared: Fmoc-Dab(Boc)-2-chlorotrityl resin, Fmoc-DDab(Boc)-2-chlorotrityl resin, Fmoc-Lys(Boc)-2-chlorotrityl resin, Fmoc- Trp(Boc)-2-chlortrityl resin, Fmoc-Phe-2-chlortrityl resin; Fmoc-Val-2-chlorotrityl resin, Fmoc-Pro-2-chlorotrityl resin, Fmoc-Arg(Pbf)-2-chlorotrityl resin and Fmoc-Glu(iBu)-2- chlorotrityl resin. Synthesis of the fully protected peptide fragment The synthesis was carried out on a Syro-peptide synthesizer (MultiSynTech GmbH) using 24 to 96 reaction vessels. In each vessel 0.04 mMol of the above resin were placed and the resin was swelled in CH2CI2 and DMF for 15 min, respectively. The following reaction cycles were programmed and carried out: Step Reagent Time 1 CH2CI2, wash and swell (manual) 1 x 3 min 2 DMF, wash and swell 2 x 30 min 3 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 4 DMF, wash 5 x 1 min 5 3.5 eq Fmoc amino acid/3.5 eq HOAt in DMF + 3.5 eq PyBOP/7 eq DIPEA or 3.5 eq DIC 1 x 40 min 6 3.5 eq Fmoc amino acid/DMF + 3.5 eq HATU or PyBOP or HCTU + 7 eq DIPEA 1 x 40 min 7 DMF, wash 5 x 1 min 8 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 9 DMF, wash 5 x 1 min 10 CH2CI2, wash (at the end of the synthesis) 3 x 1 min Steps 5 to 9 are repeated to add each amino-acid residue. After the termination of the synthesis of the fully protected peptide fragment, one of the procedures A - E, as described herein below, was adopted subsequently, depending on which kind of interstrand linkages, as described herein below, were to be formed. Finally, the peptides were purified by preparative reverse phase LC-MS, as described herein below. Procedure A: Cyclization and work up of a backbone cyclized peptide having no interstrand linkage Cleavage, backbone cyclization and deprotection After assembly of the linear peptide, the resin was suspended in 1 mL of 1percent TFA in CH2CI2 (v/v; 0.14 mMol) for 3 minutes. After filtration the filtrate was neutralized with 1 mL of 20percent DI PEA in CH2CI2 (v/v; 1.15 mMol). This procedure was repeated four times to ensure completion of the cleavage. An alternative cleavage method comprises suspension of the resin in lmL of 20percent HFIP in CH2CI2 (v/v; 1.9 mMol) for 30 minutes, filtration and repetition of the procedure. The resin was washed three times with 1 mL of CH2CI2. The CH2CI2 layers containing product were evaporated to dryness. The fully protected linear peptide was solubilised in 8 mL of dry DM F. Then 2 eq of HATU and 2 eq of HOAt in dry DM F (1-2 mL) and 4 eq of DIPEA in dry DM F (1-2 mL) were added to the peptide, followed by stirring for ca. 16 h. The volatiles were removed by evaporation. The crude cyclic peptide was dissolved in 7 mL of CH2CI2 and washed three times with 4.5 mL 10percent acetonitrile in water (v/v). The CH2CI2 layer was then evaporated to dryness. To fully deprotect the peptide, 7 mL of cleavage cocktail TFA/DODT/thioanisol/H20 (87.5 :2.5:5:5) or TFA/TIS/H20 (95:2.5 :2.5) were added, and the mixture was kept for 2.5-4 h at room temperature until the reaction was completed. The reaction mixture was evaporated close to dryness, the peptide precipitated with 7 mL of cold Et20/pentane and finally washed 3 times with 4 mL of cold Et20/pentane. Procedures Bl and B2: Cyclization and work up of a backbone cyclized peptide having a disulfide interstrand linkage Bl: Formation of a disulfide interstrand linkage using DMSO After cleavage, backbone cyclization and deprotection of the linear peptide, as described in the corresponding section of procedure A, th...
N-α-(9-fluorenylmethyloxycarbonyl)-N-γ-tert-butyloxycarbonyl-D-2,4-diaminobutyric acid[ No CAS ]
Nα-(9-fluorenylmethyloxycarbonyl)-Nγ-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine[ No CAS ]
C75H115N23O19S3[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
1. Peptide synthesis 1.1 General synthetic procedures A general method for the synthesis of the peptidomimetics of the present invention is exemplified in the following. This is to demonstrate the principal concept and does not limit or restrict the present invention in any way. A person skilled in the art is easily able to modify these procedures, especially, but not limited to, choosing a different starting position within the ring system, to still achieve the preparation of the claimed cyclic peptidomimetic compounds of the present invention. Coupling of the first protected amino acid residue to the resin . In a dried flask, 2-chlorotritylchloride resin (polystyrene, 1percent crosslinked; loading: 1.4 mMol/g) was swollen in dry CH2CI2 for 30 min (7 mL CH2CI2 per g resin). A solution of 0.8 eq of the Fmoc-protected amino acid and 6 eq of DIPEA in dry CH2CI2/DMF (4/1) (10 mL per g resin) was added. After shaking for 2-4 h at rt the resin was filtered off and washed successively with CH2CI2, DMF, CH2CI2, DMF and CH2CI2. Then a solution of dry CH2CI2/MeOH/DIPEA (17:2:1) was added (10 mL per g resin). After shaking for 3 x 30 min the resin was filtered off in a pre-weighed sinter funnel and washed successively with CH2CI2, DMF, CH2CI2, MeOH, CH2CI2, MeOH, CH2CI2 (2x) and Et20 (2x). The resin was dried under high vacuum overnight. The final mass of resin was calculated before the qualitative control. Loading was typically 0.6 - 0.7 mMol/g. The following preloaded resins were prepared: Fmoc-Dab(Boc)-2-chlorotrityl resin, Fmoc-DDab(Boc)-2-chlorotrityl resin, Fmoc-Lys(Boc)-2-chlorotrityl resin, Fmoc- Trp(Boc)-2-chlortrityl resin, Fmoc-Phe-2-chlortrityl resin; Fmoc-Val-2-chlorotrityl resin, Fmoc-Pro-2-chlorotrityl resin, Fmoc-Arg(Pbf)-2-chlorotrityl resin and Fmoc-Glu(iBu)-2- chlorotrityl resin. Synthesis of the fully protected peptide fragment The synthesis was carried out on a Syro-peptide synthesizer (MultiSynTech GmbH) using 24 to 96 reaction vessels. In each vessel 0.04 mMol of the above resin were placed and the resin was swelled in CH2CI2 and DMF for 15 min, respectively. The following reaction cycles were programmed and carried out: Step Reagent Time 1 CH2CI2, wash and swell (manual) 1 x 3 min 2 DMF, wash and swell 2 x 30 min 3 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 4 DMF, wash 5 x 1 min 5 3.5 eq Fmoc amino acid/3.5 eq HOAt in DMF + 3.5 eq PyBOP/7 eq DIPEA or 3.5 eq DIC 1 x 40 min 6 3.5 eq Fmoc amino acid/DMF + 3.5 eq HATU or PyBOP or HCTU + 7 eq DIPEA 1 x 40 min 7 DMF, wash 5 x 1 min 8 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 9 DMF, wash 5 x 1 min 10 CH2CI2, wash (at the end of the synthesis) 3 x 1 min Steps 5 to 9 are repeated to add each amino-acid residue. After the termination of the synthesis of the fully protected peptide fragment, one of the procedures A - E, as described herein below, was adopted subsequently, depending on which kind of interstrand linkages, as described herein below, were to be formed. Finally, the peptides were purified by preparative reverse phase LC-MS, as described herein below. Procedure A: Cyclization and work up of a backbone cyclized peptide having no interstrand linkage Cleavage, backbone cyclization and deprotection After assembly of the linear peptide, the resin was suspended in 1 mL of 1percent TFA in CH2CI2 (v/v; 0.14 mMol) for 3 minutes. After filtration the filtrate was neutralized with 1 mL of 20percent DI PEA in CH2CI2 (v/v; 1.15 mMol). This procedure was repeated four times to ensure completion of the cleavage. An alternative cleavage method comprises suspension of the resin in lmL of 20percent HFIP in CH2CI2 (v/v; 1.9 mMol) for 30 minutes, filtration and repetition of the procedure. The resin was washed three times with 1 mL of CH2CI2. The CH2CI2 layers containing product were evaporated to dryness. The fully protected linear peptide was solubilised in 8 mL of dry DM F. Then 2 eq of HATU and 2 eq of HOAt in dry DM F (1-2 mL) and 4 eq of DIPEA in dry DM F (1-2 mL) were added to the peptide, followed by stirring for ca. 16 h. The volatiles were removed by evaporation. The crude cyclic peptide was dissolved in 7 mL of CH2CI2 and washed three times with 4.5 mL 10percent acetonitrile in water (v/v). The CH2CI2 layer was then evaporated to dryness. To fully deprotect the peptide, 7 mL of cleavage cocktail TFA/DODT/thioanisol/H20 (87.5 :2.5:5:5) or TFA/TIS/H20 (95:2.5 :2.5) were added, and the mixture was kept for 2.5-4 h at room temperature until the reaction was completed. The reaction mixture was evaporated close to dryness, the peptide precipitated with 7 mL of cold Et20/pentane and finally washed 3 times with 4 mL of cold Et20/pentane. Procedures Bl and B2: Cyclization and work up of a backbone cyclized peptide having a disulfide interstrand linkage Bl: Formation of a disulfide interstrand linkage using DMSO After cleavage, backbone cyclization and deprotection of the linear peptide, as described in the corresponding section of procedure A, th...
N-α-(9-fluorenylmethyloxycarbonyl)-N-γ-tert-butyloxycarbonyl-D-2,4-diaminobutyric acid[ No CAS ]
C74H111N21O19S2[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
1. Peptide synthesis 1.1 General synthetic procedures A general method for the synthesis of the peptidomimetics of the present invention is exemplified in the following. This is to demonstrate the principal concept and does not limit or restrict the present invention in any way. A person skilled in the art is easily able to modify these procedures, especially, but not limited to, choosing a different starting position within the ring system, to still achieve the preparation of the claimed cyclic peptidomimetic compounds of the present invention. Coupling of the first protected amino acid residue to the resin . In a dried flask, 2-chlorotritylchloride resin (polystyrene, 1percent crosslinked; loading: 1.4 mMol/g) was swollen in dry CH2CI2 for 30 min (7 mL CH2CI2 per g resin). A solution of 0.8 eq of the Fmoc-protected amino acid and 6 eq of DIPEA in dry CH2CI2/DMF (4/1) (10 mL per g resin) was added. After shaking for 2-4 h at rt the resin was filtered off and washed successively with CH2CI2, DMF, CH2CI2, DMF and CH2CI2. Then a solution of dry CH2CI2/MeOH/DIPEA (17:2:1) was added (10 mL per g resin). After shaking for 3 x 30 min the resin was filtered off in a pre-weighed sinter funnel and washed successively with CH2CI2, DMF, CH2CI2, MeOH, CH2CI2, MeOH, CH2CI2 (2x) and Et20 (2x). The resin was dried under high vacuum overnight. The final mass of resin was calculated before the qualitative control. Loading was typically 0.6 - 0.7 mMol/g. The following preloaded resins were prepared: Fmoc-Dab(Boc)-2-chlorotrityl resin, Fmoc-DDab(Boc)-2-chlorotrityl resin, Fmoc-Lys(Boc)-2-chlorotrityl resin, Fmoc- Trp(Boc)-2-chlortrityl resin, Fmoc-Phe-2-chlortrityl resin; Fmoc-Val-2-chlorotrityl resin, Fmoc-Pro-2-chlorotrityl resin, Fmoc-Arg(Pbf)-2-chlorotrityl resin and Fmoc-Glu(iBu)-2- chlorotrityl resin. Synthesis of the fully protected peptide fragment The synthesis was carried out on a Syro-peptide synthesizer (MultiSynTech GmbH) using 24 to 96 reaction vessels. In each vessel 0.04 mMol of the above resin were placed and the resin was swelled in CH2CI2 and DMF for 15 min, respectively. The following reaction cycles were programmed and carried out: Step Reagent Time 1 CH2CI2, wash and swell (manual) 1 x 3 min 2 DMF, wash and swell 2 x 30 min 3 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 4 DMF, wash 5 x 1 min 5 3.5 eq Fmoc amino acid/3.5 eq HOAt in DMF + 3.5 eq PyBOP/7 eq DIPEA or 3.5 eq DIC 1 x 40 min 6 3.5 eq Fmoc amino acid/DMF + 3.5 eq HATU or PyBOP or HCTU + 7 eq DIPEA 1 x 40 min 7 DMF, wash 5 x 1 min 8 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 9 DMF, wash 5 x 1 min 10 CH2CI2, wash (at the end of the synthesis) 3 x 1 min Steps 5 to 9 are repeated to add each amino-acid residue. After the termination of the synthesis of the fully protected peptide fragment, one of the procedures A - E, as described herein below, was adopted subsequently, depending on which kind of interstrand linkages, as described herein below, were to be formed. Finally, the peptides were purified by preparative reverse phase LC-MS, as described herein below. Procedure A: Cyclization and work up of a backbone cyclized peptide having no interstrand linkage Cleavage, backbone cyclization and deprotection After assembly of the linear peptide, the resin was suspended in 1 mL of 1percent TFA in CH2CI2 (v/v; 0.14 mMol) for 3 minutes. After filtration the filtrate was neutralized with 1 mL of 20percent DI PEA in CH2CI2 (v/v; 1.15 mMol). This procedure was repeated four times to ensure completion of the cleavage. An alternative cleavage method comprises suspension of the resin in lmL of 20percent HFIP in CH2CI2 (v/v; 1.9 mMol) for 30 minutes, filtration and repetition of the procedure. The resin was washed three times with 1 mL of CH2CI2. The CH2CI2 layers containing product were evaporated to dryness. The fully protected linear peptide was solubilised in 8 mL of dry DM F. Then 2 eq of HATU and 2 eq of HOAt in dry DM F (1-2 mL) and 4 eq of DIPEA in dry DM F (1-2 mL) were added to the peptide, followed by stirring for ca. 16 h. The volatiles were removed by evaporation. The crude cyclic peptide was dissolved in 7 mL of CH2CI2 and washed three times with 4.5 mL 10percent acetonitrile in water (v/v). The CH2CI2 layer was then evaporated to dryness. To fully deprotect the peptide, 7 mL of cleavage cocktail TFA/DODT/thioanisol/H20 (87.5 :2.5:5:5) or TFA/TIS/H20 (95:2.5 :2.5) were added, and the mixture was kept for 2.5-4 h at room temperature until the reaction was completed. The reaction mixture was evaporated close to dryness, the peptide precipitated with 7 mL of cold Et20/pentane and finally washed 3 times with 4 mL of cold Et20/pentane. Procedures Bl and B2: Cyclization and work up of a backbone cyclized peptide having a disulfide interstrand linkage Bl: Formation of a disulfide interstrand linkage using DMSO After cleavage, backbone cyclization and deprotection of the linear peptide, as described in the corresponding section of procedure A, th...
N-α-(9-fluorenylmethyloxycarbonyl)-N-γ-tert-butyloxycarbonyl-D-2,4-diaminobutyric acid[ No CAS ]
C73H109N21O19S2[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
1. Peptide synthesis 1.1 General synthetic procedures A general method for the synthesis of the peptidomimetics of the present invention is exemplified in the following. This is to demonstrate the principal concept and does not limit or restrict the present invention in any way. A person skilled in the art is easily able to modify these procedures, especially, but not limited to, choosing a different starting position within the ring system, to still achieve the preparation of the claimed cyclic peptidomimetic compounds of the present invention. Coupling of the first protected amino acid residue to the resin . In a dried flask, 2-chlorotritylchloride resin (polystyrene, 1percent crosslinked; loading: 1.4 mMol/g) was swollen in dry CH2CI2 for 30 min (7 mL CH2CI2 per g resin). A solution of 0.8 eq of the Fmoc-protected amino acid and 6 eq of DIPEA in dry CH2CI2/DMF (4/1) (10 mL per g resin) was added. After shaking for 2-4 h at rt the resin was filtered off and washed successively with CH2CI2, DMF, CH2CI2, DMF and CH2CI2. Then a solution of dry CH2CI2/MeOH/DIPEA (17:2:1) was added (10 mL per g resin). After shaking for 3 x 30 min the resin was filtered off in a pre-weighed sinter funnel and washed successively with CH2CI2, DMF, CH2CI2, MeOH, CH2CI2, MeOH, CH2CI2 (2x) and Et20 (2x). The resin was dried under high vacuum overnight. The final mass of resin was calculated before the qualitative control. Loading was typically 0.6 - 0.7 mMol/g. The following preloaded resins were prepared: Fmoc-Dab(Boc)-2-chlorotrityl resin, Fmoc-DDab(Boc)-2-chlorotrityl resin, Fmoc-Lys(Boc)-2-chlorotrityl resin, Fmoc- Trp(Boc)-2-chlortrityl resin, Fmoc-Phe-2-chlortrityl resin; Fmoc-Val-2-chlorotrityl resin, Fmoc-Pro-2-chlorotrityl resin, Fmoc-Arg(Pbf)-2-chlorotrityl resin and Fmoc-Glu(iBu)-2- chlorotrityl resin. Synthesis of the fully protected peptide fragment The synthesis was carried out on a Syro-peptide synthesizer (MultiSynTech GmbH) using 24 to 96 reaction vessels. In each vessel 0.04 mMol of the above resin were placed and the resin was swelled in CH2CI2 and DMF for 15 min, respectively. The following reaction cycles were programmed and carried out: Step Reagent Time 1 CH2CI2, wash and swell (manual) 1 x 3 min 2 DMF, wash and swell 2 x 30 min 3 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 4 DMF, wash 5 x 1 min 5 3.5 eq Fmoc amino acid/3.5 eq HOAt in DMF + 3.5 eq PyBOP/7 eq DIPEA or 3.5 eq DIC 1 x 40 min 6 3.5 eq Fmoc amino acid/DMF + 3.5 eq HATU or PyBOP or HCTU + 7 eq DIPEA 1 x 40 min 7 DMF, wash 5 x 1 min 8 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 9 DMF, wash 5 x 1 min 10 CH2CI2, wash (at the end of the synthesis) 3 x 1 min Steps 5 to 9 are repeated to add each amino-acid residue. After the termination of the synthesis of the fully protected peptide fragment, one of the procedures A - E, as described herein below, was adopted subsequently, depending on which kind of interstrand linkages, as described herein below, were to be formed. Finally, the peptides were purified by preparative reverse phase LC-MS, as described herein below. Procedure A: Cyclization and work up of a backbone cyclized peptide having no interstrand linkage Cleavage, backbone cyclization and deprotection After assembly of the linear peptide, the resin was suspended in 1 mL of 1percent TFA in CH2CI2 (v/v; 0.14 mMol) for 3 minutes. After filtration the filtrate was neutralized with 1 mL of 20percent DI PEA in CH2CI2 (v/v; 1.15 mMol). This procedure was repeated four times to ensure completion of the cleavage. An alternative cleavage method comprises suspension of the resin in lmL of 20percent HFIP in CH2CI2 (v/v; 1.9 mMol) for 30 minutes, filtration and repetition of the procedure. The resin was washed three times with 1 mL of CH2CI2. The CH2CI2 layers containing product were evaporated to dryness. The fully protected linear peptide was solubilised in 8 mL of dry DM F. Then 2 eq of HATU and 2 eq of HOAt in dry DM F (1-2 mL) and 4 eq of DIPEA in dry DM F (1-2 mL) were added to the peptide, followed by stirring for ca. 16 h. The volatiles were removed by evaporation. The crude cyclic peptide was dissolved in 7 mL of CH2CI2 and washed three times with 4.5 mL 10percent acetonitrile in water (v/v). The CH2CI2 layer was then evaporated to dryness. To fully deprotect the peptide, 7 mL of cleavage cocktail TFA/DODT/thioanisol/H20 (87.5 :2.5:5:5) or TFA/TIS/H20 (95:2.5 :2.5) were added, and the mixture was kept for 2.5-4 h at room temperature until the reaction was completed. The reaction mixture was evaporated close to dryness, the peptide precipitated with 7 mL of cold Et20/pentane and finally washed 3 times with 4 mL of cold Et20/pentane. Procedures Bl and B2: Cyclization and work up of a backbone cyclized peptide having a disulfide interstrand linkage Bl: Formation of a disulfide interstrand linkage using DMSO After cleavage, backbone cyclization and deprotection of the linear peptide, as described in the corresponding section of procedure A, th...
N-α-(9-fluorenylmethyloxycarbonyl)-N-γ-tert-butyloxycarbonyl-D-2,4-diaminobutyric acid[ No CAS ]
Nα-(9-fluorenylmethyloxycarbonyl)-Nγ-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine[ No CAS ]
C75H115N23O18S2[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
1. Peptide synthesis 1.1 General synthetic procedures A general method for the synthesis of the peptidomimetics of the present invention is exemplified in the following. This is to demonstrate the principal concept and does not limit or restrict the present invention in any way. A person skilled in the art is easily able to modify these procedures, especially, but not limited to, choosing a different starting position within the ring system, to still achieve the preparation of the claimed cyclic peptidomimetic compounds of the present invention. Coupling of the first protected amino acid residue to the resin . In a dried flask, 2-chlorotritylchloride resin (polystyrene, 1percent crosslinked; loading: 1.4 mMol/g) was swollen in dry CH2CI2 for 30 min (7 mL CH2CI2 per g resin). A solution of 0.8 eq of the Fmoc-protected amino acid and 6 eq of DIPEA in dry CH2CI2/DMF (4/1) (10 mL per g resin) was added. After shaking for 2-4 h at rt the resin was filtered off and washed successively with CH2CI2, DMF, CH2CI2, DMF and CH2CI2. Then a solution of dry CH2CI2/MeOH/DIPEA (17:2:1) was added (10 mL per g resin). After shaking for 3 x 30 min the resin was filtered off in a pre-weighed sinter funnel and washed successively with CH2CI2, DMF, CH2CI2, MeOH, CH2CI2, MeOH, CH2CI2 (2x) and Et20 (2x). The resin was dried under high vacuum overnight. The final mass of resin was calculated before the qualitative control. Loading was typically 0.6 - 0.7 mMol/g. The following preloaded resins were prepared: Fmoc-Dab(Boc)-2-chlorotrityl resin, Fmoc-DDab(Boc)-2-chlorotrityl resin, Fmoc-Lys(Boc)-2-chlorotrityl resin, Fmoc- Trp(Boc)-2-chlortrityl resin, Fmoc-Phe-2-chlortrityl resin; Fmoc-Val-2-chlorotrityl resin, Fmoc-Pro-2-chlorotrityl resin, Fmoc-Arg(Pbf)-2-chlorotrityl resin and Fmoc-Glu(iBu)-2- chlorotrityl resin. Synthesis of the fully protected peptide fragment The synthesis was carried out on a Syro-peptide synthesizer (MultiSynTech GmbH) using 24 to 96 reaction vessels. In each vessel 0.04 mMol of the above resin were placed and the resin was swelled in CH2CI2 and DMF for 15 min, respectively. The following reaction cycles were programmed and carried out: Step Reagent Time 1 CH2CI2, wash and swell (manual) 1 x 3 min 2 DMF, wash and swell 2 x 30 min 3 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 4 DMF, wash 5 x 1 min 5 3.5 eq Fmoc amino acid/3.5 eq HOAt in DMF + 3.5 eq PyBOP/7 eq DIPEA or 3.5 eq DIC 1 x 40 min 6 3.5 eq Fmoc amino acid/DMF + 3.5 eq HATU or PyBOP or HCTU + 7 eq DIPEA 1 x 40 min 7 DMF, wash 5 x 1 min 8 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 9 DMF, wash 5 x 1 min 10 CH2CI2, wash (at the end of the synthesis) 3 x 1 min Steps 5 to 9 are repeated to add each amino-acid residue. After the termination of the synthesis of the fully protected peptide fragment, one of the procedures A - E, as described herein below, was adopted subsequently, depending on which kind of interstrand linkages, as described herein below, were to be formed. Finally, the peptides were purified by preparative reverse phase LC-MS, as described herein below. Procedure A: Cyclization and work up of a backbone cyclized peptide having no interstrand linkage Cleavage, backbone cyclization and deprotection After assembly of the linear peptide, the resin was suspended in 1 mL of 1percent TFA in CH2CI2 (v/v; 0.14 mMol) for 3 minutes. After filtration the filtrate was neutralized with 1 mL of 20percent DI PEA in CH2CI2 (v/v; 1.15 mMol). This procedure was repeated four times to ensure completion of the cleavage. An alternative cleavage method comprises suspension of the resin in lmL of 20percent HFIP in CH2CI2 (v/v; 1.9 mMol) for 30 minutes, filtration and repetition of the procedure. The resin was washed three times with 1 mL of CH2CI2. The CH2CI2 layers containing product were evaporated to dryness. The fully protected linear peptide was solubilised in 8 mL of dry DM F. Then 2 eq of HATU and 2 eq of HOAt in dry DM F (1-2 mL) and 4 eq of DIPEA in dry DM F (1-2 mL) were added to the peptide, followed by stirring for ca. 16 h. The volatiles were removed by evaporation. The crude cyclic peptide was dissolved in 7 mL of CH2CI2 and washed three times with 4.5 mL 10percent acetonitrile in water (v/v). The CH2CI2 layer was then evaporated to dryness. To fully deprotect the peptide, 7 mL of cleavage cocktail TFA/DODT/thioanisol/H20 (87.5 :2.5:5:5) or TFA/TIS/H20 (95:2.5 :2.5) were added, and the mixture was kept for 2.5-4 h at room temperature until the reaction was completed. The reaction mixture was evaporated close to dryness, the peptide precipitated with 7 mL of cold Et20/pentane and finally washed 3 times with 4 mL of cold Et20/pentane. Procedures Bl and B2: Cyclization and work up of a backbone cyclized peptide having a disulfide interstrand linkage Bl: Formation of a disulfide interstrand linkage using DMSO After cleavage, backbone cyclization and deprotection of the linear peptide, as described in the corresponding section of procedure A, th...
N-α-(9-fluorenylmethyloxycarbonyl)-N-γ-tert-butyloxycarbonyl-D-2,4-diaminobutyric acid[ No CAS ]
C73H113N21O17S2[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
1. Peptide synthesis 1.1 General synthetic procedures A general method for the synthesis of the peptidomimetics of the present invention is exemplified in the following. This is to demonstrate the principal concept and does not limit or restrict the present invention in any way. A person skilled in the art is easily able to modify these procedures, especially, but not limited to, choosing a different starting position within the ring system, to still achieve the preparation of the claimed cyclic peptidomimetic compounds of the present invention. Coupling of the first protected amino acid residue to the resin . In a dried flask, 2-chlorotritylchloride resin (polystyrene, 1percent crosslinked; loading: 1.4 mMol/g) was swollen in dry CH2CI2 for 30 min (7 mL CH2CI2 per g resin). A solution of 0.8 eq of the Fmoc-protected amino acid and 6 eq of DIPEA in dry CH2CI2/DMF (4/1) (10 mL per g resin) was added. After shaking for 2-4 h at rt the resin was filtered off and washed successively with CH2CI2, DMF, CH2CI2, DMF and CH2CI2. Then a solution of dry CH2CI2/MeOH/DIPEA (17:2:1) was added (10 mL per g resin). After shaking for 3 x 30 min the resin was filtered off in a pre-weighed sinter funnel and washed successively with CH2CI2, DMF, CH2CI2, MeOH, CH2CI2, MeOH, CH2CI2 (2x) and Et20 (2x). The resin was dried under high vacuum overnight. The final mass of resin was calculated before the qualitative control. Loading was typically 0.6 - 0.7 mMol/g. The following preloaded resins were prepared: Fmoc-Dab(Boc)-2-chlorotrityl resin, Fmoc-DDab(Boc)-2-chlorotrityl resin, Fmoc-Lys(Boc)-2-chlorotrityl resin, Fmoc- Trp(Boc)-2-chlortrityl resin, Fmoc-Phe-2-chlortrityl resin; Fmoc-Val-2-chlorotrityl resin, Fmoc-Pro-2-chlorotrityl resin, Fmoc-Arg(Pbf)-2-chlorotrityl resin and Fmoc-Glu(iBu)-2- chlorotrityl resin. Synthesis of the fully protected peptide fragment The synthesis was carried out on a Syro-peptide synthesizer (MultiSynTech GmbH) using 24 to 96 reaction vessels. In each vessel 0.04 mMol of the above resin were placed and the resin was swelled in CH2CI2 and DMF for 15 min, respectively. The following reaction cycles were programmed and carried out: Step Reagent Time 1 CH2CI2, wash and swell (manual) 1 x 3 min 2 DMF, wash and swell 2 x 30 min 3 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 4 DMF, wash 5 x 1 min 5 3.5 eq Fmoc amino acid/3.5 eq HOAt in DMF + 3.5 eq PyBOP/7 eq DIPEA or 3.5 eq DIC 1 x 40 min 6 3.5 eq Fmoc amino acid/DMF + 3.5 eq HATU or PyBOP or HCTU + 7 eq DIPEA 1 x 40 min 7 DMF, wash 5 x 1 min 8 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 9 DMF, wash 5 x 1 min 10 CH2CI2, wash (at the end of the synthesis) 3 x 1 min Steps 5 to 9 are repeated to add each amino-acid residue. After the termination of the synthesis of the fully protected peptide fragment, one of the procedures A - E, as described herein below, was adopted subsequently, depending on which kind of interstrand linkages, as described herein below, were to be formed. Finally, the peptides were purified by preparative reverse phase LC-MS, as described herein below. Procedure A: Cyclization and work up of a backbone cyclized peptide having no interstrand linkage Cleavage, backbone cyclization and deprotection After assembly of the linear peptide, the resin was suspended in 1 mL of 1percent TFA in CH2CI2 (v/v; 0.14 mMol) for 3 minutes. After filtration the filtrate was neutralized with 1 mL of 20percent DI PEA in CH2CI2 (v/v; 1.15 mMol). This procedure was repeated four times to ensure completion of the cleavage. An alternative cleavage method comprises suspension of the resin in lmL of 20percent HFIP in CH2CI2 (v/v; 1.9 mMol) for 30 minutes, filtration and repetition of the procedure. The resin was washed three times with 1 mL of CH2CI2. The CH2CI2 layers containing product were evaporated to dryness. The fully protected linear peptide was solubilised in 8 mL of dry DM F. Then 2 eq of HATU and 2 eq of HOAt in dry DM F (1-2 mL) and 4 eq of DIPEA in dry DM F (1-2 mL) were added to the peptide, followed by stirring for ca. 16 h. The volatiles were removed by evaporation. The crude cyclic peptide was dissolved in 7 mL of CH2CI2 and washed three times with 4.5 mL 10percent acetonitrile in water (v/v). The CH2CI2 layer was then evaporated to dryness. To fully deprotect the peptide, 7 mL of cleavage cocktail TFA/DODT/thioanisol/H20 (87.5 :2.5:5:5) or TFA/TIS/H20 (95:2.5 :2.5) were added, and the mixture was kept for 2.5-4 h at room temperature until the reaction was completed. The reaction mixture was evaporated close to dryness, the peptide precipitated with 7 mL of cold Et20/pentane and finally washed 3 times with 4 mL of cold Et20/pentane. Procedures Bl and B2: Cyclization and work up of a backbone cyclized peptide having a disulfide interstrand linkage Bl: Formation of a disulfide interstrand linkage using DMSO After cleavage, backbone cyclization and deprotection of the linear peptide, as described in the corresponding section of procedure A, th...
N-α-(9-fluorenylmethyloxycarbonyl)-N-γ-tert-butyloxycarbonyl-D-2,4-diaminobutyric acid[ No CAS ]
C71H111N21O16S2[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
1. Peptide synthesis 1.1 General synthetic procedures A general method for the synthesis of the peptidomimetics of the present invention is exemplified in the following. This is to demonstrate the principal concept and does not limit or restrict the present invention in any way. A person skilled in the art is easily able to modify these procedures, especially, but not limited to, choosing a different starting position within the ring system, to still achieve the preparation of the claimed cyclic peptidomimetic compounds of the present invention. Coupling of the first protected amino acid residue to the resin . In a dried flask, 2-chlorotritylchloride resin (polystyrene, 1percent crosslinked; loading: 1.4 mMol/g) was swollen in dry CH2CI2 for 30 min (7 mL CH2CI2 per g resin). A solution of 0.8 eq of the Fmoc-protected amino acid and 6 eq of DIPEA in dry CH2CI2/DMF (4/1) (10 mL per g resin) was added. After shaking for 2-4 h at rt the resin was filtered off and washed successively with CH2CI2, DMF, CH2CI2, DMF and CH2CI2. Then a solution of dry CH2CI2/MeOH/DIPEA (17:2:1) was added (10 mL per g resin). After shaking for 3 x 30 min the resin was filtered off in a pre-weighed sinter funnel and washed successively with CH2CI2, DMF, CH2CI2, MeOH, CH2CI2, MeOH, CH2CI2 (2x) and Et20 (2x). The resin was dried under high vacuum overnight. The final mass of resin was calculated before the qualitative control. Loading was typically 0.6 - 0.7 mMol/g. (0997) The following preloaded resins were prepared: Fmoc-Dab(Boc)-2-chlorotrityl resin, Fmoc-DDab(Boc)-2-chlorotrityl resin, Fmoc-Lys(Boc)-2-chlorotrityl resin, Fmoc- Trp(Boc)-2-chlortrityl resin, Fmoc-Phe-2-chlortrityl resin; Fmoc-Val-2-chlorotrityl resin, Fmoc-Pro-2-chlorotrityl resin, Fmoc-Arg(Pbf)-2-chlorotrityl resin and Fmoc-Glu(iBu)-2- chlorotrityl resin. Synthesis of the fully protected peptide fragment The synthesis was carried out on a Syro-peptide synthesizer (MultiSynTech GmbH) using 24 to 96 reaction vessels. In each vessel 0.04 mMol of the above resin were placed and the resin was swelled in CH2CI2 and DMF for 15 min, respectively. The following reaction cycles were programmed and carried out: Step Reagent Time 1 CH2CI2, wash and swell (manual) 1 x 3 min 2 DMF, wash and swell 2 x 30 min 3 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 4 DMF, wash 5 x 1 min 5 3.5 eq Fmoc amino acid/3.5 eq HOAt in DMF + 3.5 eq PyBOP/7 eq DIPEA or 3.5 eq DIC 1 x 40 min 6 3.5 eq Fmoc amino acid/DMF + 3.5 eq HATU or PyBOP or HCTU + 7 eq DIPEA 1 x 40 min 7 DMF, wash 5 x 1 min 8 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 9 DMF, wash 5 x 1 min 10 CH2CI2, wash (at the end of the synthesis) 3 x 1 min Steps 5 to 9 are repeated to add each amino-acid residue. After the termination of the synthesis of the fully protected peptide fragment, one of the procedures A - E, as described herein below, was adopted subsequently, depending on which kind of interstrand linkages, as described herein below, were to be formed. Finally, the peptides were purified by preparative reverse phase LC-MS, as described herein below. Procedure A: Cyclization and work up of a backbone cyclized peptide having no interstrand linkage Cleavage, backbone cyclization and deprotection After assembly of the linear peptide, the resin was suspended in 1 mL of 1percent TFA in CH2CI2 (v/v; 0.14 mMol) for 3 minutes. After filtration the filtrate was neutralized with 1 mL of 20percent DI PEA in CH2CI2 (v/v; 1.15 mMol). This procedure was repeated four times to ensure completion of the cleavage. An alternative cleavage method comprises suspension of the resin in lmL of 20percent HFIP in CH2CI2 (v/v; 1.9 mMol) for 30 minutes, filtration and repetition of the procedure. The resin was washed three times with 1 mL of CH2CI2. The CH2CI2 layers containing product were evaporated to dryness. The fully protected linear peptide was solubilised in 8 mL of dry DM F. Then 2 eq of HATU and 2 eq of HOAt in dry DM F (1-2 mL) and 4 eq of DIPEA in dry DM F (1-2 mL) were added to the peptide, followed by stirring for ca. 16 h. The volatiles were removed by evaporation. The crude cyclic peptide was dissolved in 7 mL of CH2CI2 and washed three times with 4.5 mL 10percent acetonitrile in water (v/v). The CH2CI2 layer was then evaporated to dryness. To fully deprotect the peptide, 7 mL of cleavage cocktail TFA/DODT/thioanisol/H20 (87.5 :2.5:5:5) or TFA/TIS/H20 (95:2.5 :2.5) were added, and the mixture was kept for 2.5-4 h at room temperature until the reaction was completed. The reaction mixture was evaporated close to dryness, the peptide precipitated with 7 mL of cold Et20/pentane and finally washed 3 times with 4 mL of cold Et20/pentane. Procedures Bl and B2: Cyclization and work up of a backbone cyclized peptide having a disulfide interstrand linkage Bl: Formation of a disulfide interstrand linkage using DMSO After cleavage, backbone cyclization and deprotection of the linear peptide, as described in the corresponding section of procedur...
1. Peptide synthesis 1.1 General synthetic procedures A general method for the synthesis of the peptidomimetics of the present invention is exemplified in the following. This is to demonstrate the principal concept and does not limit or restrict the present invention in any way. A person skilled in the art is easily able to modify these procedures, especially, but not limited to, choosing a different starting position within the ring system, to still achieve the preparation of the claimed cyclic peptidomimetic compounds of the present invention. Coupling of the first protected amino acid residue to the resin . In a dried flask, 2-chlorotritylchloride resin (polystyrene, 1percent crosslinked; loading: 1.4 mMol/g) was swollen in dry CH2CI2 for 30 min (7 mL CH2CI2 per g resin). A solution of 0.8 eq of the Fmoc-protected amino acid and 6 eq of DIPEA in dry CH2CI2/DMF (4/1) (10 mL per g resin) was added. After shaking for 2-4 h at rt the resin was filtered off and washed successively with CH2CI2, DMF, CH2CI2, DMF and CH2CI2. Then a solution of dry CH2CI2/MeOH/DIPEA (17:2:1) was added (10 mL per g resin). After shaking for 3 x 30 min the resin was filtered off in a pre-weighed sinter funnel and washed successively with CH2CI2, DMF, CH2CI2, MeOH, CH2CI2, MeOH, CH2CI2 (2x) and Et20 (2x). The resin was dried under high vacuum overnight. The final mass of resin was calculated before the qualitative control. Loading was typically 0.6 - 0.7 mMol/g. (0997) The following preloaded resins were prepared: Fmoc-Dab(Boc)-2-chlorotrityl resin, Fmoc-DDab(Boc)-2-chlorotrityl resin, Fmoc-Lys(Boc)-2-chlorotrityl resin, Fmoc- Trp(Boc)-2-chlortrityl resin, Fmoc-Phe-2-chlortrityl resin; Fmoc-Val-2-chlorotrityl resin, Fmoc-Pro-2-chlorotrityl resin, Fmoc-Arg(Pbf)-2-chlorotrityl resin and Fmoc-Glu(iBu)-2- chlorotrityl resin. Synthesis of the fully protected peptide fragment The synthesis was carried out on a Syro-peptide synthesizer (MultiSynTech GmbH) using 24 to 96 reaction vessels. In each vessel 0.04 mMol of the above resin were placed and the resin was swelled in CH2CI2 and DMF for 15 min, respectively. The following reaction cycles were programmed and carried out: Step Reagent Time 1 CH2CI2, wash and swell (manual) 1 x 3 min 2 DMF, wash and swell 2 x 30 min 3 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 4 DMF, wash 5 x 1 min 5 3.5 eq Fmoc amino acid/3.5 eq HOAt in DMF + 3.5 eq PyBOP/7 eq DIPEA or 3.5 eq DIC 1 x 40 min 6 3.5 eq Fmoc amino acid/DMF + 3.5 eq HATU or PyBOP or HCTU + 7 eq DIPEA 1 x 40 min 7 DMF, wash 5 x 1 min 8 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 9 DMF, wash 5 x 1 min 10 CH2CI2, wash (at the end of the synthesis) 3 x 1 min Steps 5 to 9 are repeated to add each amino-acid residue. After the termination of the synthesis of the fully protected peptide fragment, one of the procedures A - E, as described herein below, was adopted subsequently, depending on which kind of interstrand linkages, as described herein below, were to be formed. Finally, the peptides were purified by preparative reverse phase LC-MS, as described herein below. Procedure A: Cyclization and work up of a backbone cyclized peptide having no interstrand linkage Cleavage, backbone cyclization and deprotection After assembly of the linear peptide, the resin was suspended in 1 mL of 1percent TFA in CH2CI2 (v/v; 0.14 mMol) for 3 minutes. After filtration the filtrate was neutralized with 1 mL of 20percent DI PEA in CH2CI2 (v/v; 1.15 mMol). This procedure was repeated four times to ensure completion of the cleavage. An alternative cleavage method comprises suspension of the resin in lmL of 20percent HFIP in CH2CI2 (v/v; 1.9 mMol) for 30 minutes, filtration and repetition of the procedure. The resin was washed three times with 1 mL of CH2CI2. The CH2CI2 layers containing product were evaporated to dryness. The fully protected linear peptide was solubilised in 8 mL of dry DM F. Then 2 eq of HATU and 2 eq of HOAt in dry DM F (1-2 mL) and 4 eq of DIPEA in dry DM F (1-2 mL) were added to the peptide, followed by stirring for ca. 16 h. The volatiles were removed by evaporation. The crude cyclic peptide was dissolved in 7 mL of CH2CI2 and washed three times with 4.5 mL 10percent acetonitrile in water (v/v). The CH2CI2 layer was then evaporated to dryness. To fully deprotect the peptide, 7 mL of cleavage cocktail TFA/DODT/thioanisol/H20 (87.5 :2.5:5:5) or TFA/TIS/H20 (95:2.5 :2.5) were added, and the mixture was kept for 2.5-4 h at room temperature until the reaction was completed. The reaction mixture was evaporated close to dryness, the peptide precipitated with 7 mL of cold Et20/pentane and finally washed 3 times with 4 mL of cold Et20/pentane. Procedures Bl and B2: Cyclization and work up of a backbone cyclized peptide having a disulfide interstrand linkage Bl: Formation of a disulfide interstrand linkage using DMSO After cleavage, backbone cyclization and deprotection of the linear peptide, as described in the corresponding section of procedur...
Nα-(9-fluorenylmethyloxycarbonyl)-Nγ-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine[ No CAS ]
C75H109N21O17S4[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
1. Peptide synthesis 1.1 General synthetic procedures A general method for the synthesis of the peptidomimetics of the present invention is exemplified in the following. This is to demonstrate the principal concept and does not limit or restrict the present invention in any way. A person skilled in the art is easily able to modify these procedures, especially, but not limited to, choosing a different starting position within the ring system, to still achieve the preparation of the claimed cyclic peptidomimetic compounds of the present invention. Coupling of the first protected amino acid residue to the resin . In a dried flask, 2-chlorotritylchloride resin (polystyrene, 1percent crosslinked; loading: 1.4 mMol/g) was swollen in dry CH2CI2 for 30 min (7 mL CH2CI2 per g resin). A solution of 0.8 eq of the Fmoc-protected amino acid and 6 eq of DIPEA in dry CH2CI2/DMF (4/1) (10 mL per g resin) was added. After shaking for 2-4 h at rt the resin was filtered off and washed successively with CH2CI2, DMF, CH2CI2, DMF and CH2CI2. Then a solution of dry CH2CI2/MeOH/DIPEA (17:2:1) was added (10 mL per g resin). After shaking for 3 x 30 min the resin was filtered off in a pre-weighed sinter funnel and washed successively with CH2CI2, DMF, CH2CI2, MeOH, CH2CI2, MeOH, CH2CI2 (2x) and Et20 (2x). The resin was dried under high vacuum overnight. The final mass of resin was calculated before the qualitative control. Loading was typically 0.6 - 0.7 mMol/g. The following preloaded resins were prepared: Fmoc-Dab(Boc)-2-chlorotrityl resin, Fmoc-DDab(Boc)-2-chlorotrityl resin, Fmoc-Lys(Boc)-2-chlorotrityl resin, Fmoc- Trp(Boc)-2-chlortrityl resin, Fmoc-Phe-2-chlortrityl resin; Fmoc-Val-2-chlorotrityl resin, Fmoc-Pro-2-chlorotrityl resin, Fmoc-Arg(Pbf)-2-chlorotrityl resin and Fmoc-Glu(iBu)-2- chlorotrityl resin. Synthesis of the fully protected peptide fragment The synthesis was carried out on a Syro-peptide synthesizer (MultiSynTech GmbH) using 24 to 96 reaction vessels. In each vessel 0.04 mMol of the above resin were placed and the resin was swelled in CH2CI2 and DMF for 15 min, respectively. The following reaction cycles were programmed and carried out: Step Reagent Time 1 CH2CI2, wash and swell (manual) 1 x 3 min 2 DMF, wash and swell 2 x 30 min 3 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 4 DMF, wash 5 x 1 min 5 3.5 eq Fmoc amino acid/3.5 eq HOAt in DMF + 3.5 eq PyBOP/7 eq DIPEA or 3.5 eq DIC 1 x 40 min 6 3.5 eq Fmoc amino acid/DMF + 3.5 eq HATU or PyBOP or HCTU + 7 eq DIPEA 1 x 40 min 7 DMF, wash 5 x 1 min 8 20percent piperidine/DMF 1 x 5 min and 1 x 15 min 9 DMF, wash 5 x 1 min 10 CH2CI2, wash (at the end of the synthesis) 3 x 1 min Steps 5 to 9 are repeated to add each amino-acid residue. After the termination of the synthesis of the fully protected peptide fragment, one of the procedures A - E, as described herein below, was adopted subsequently, depending on which kind of interstrand linkages, as described herein below, were to be formed. Finally, the peptides were purified by preparative reverse phase LC-MS, as described herein below. Procedure A: Cyclization and work up of a backbone cyclized peptide having no interstrand linkage Cleavage, backbone cyclization and deprotection After assembly of the linear peptide, the resin was suspended in 1 mL of 1percent TFA in CH2CI2 (v/v; 0.14 mMol) for 3 minutes. After filtration the filtrate was neutralized with 1 mL of 20percent DI PEA in CH2CI2 (v/v; 1.15 mMol). This procedure was repeated four times to ensure completion of the cleavage. An alternative cleavage method comprises suspension of the resin in lmL of 20percent HFIP in CH2CI2 (v/v; 1.9 mMol) for 30 minutes, filtration and repetition of the procedure. The resin was washed three times with 1 mL of CH2CI2. The CH2CI2 layers containing product were evaporated to dryness. The fully protected linear peptide was solubilised in 8 mL of dry DM F. Then 2 eq of HATU and 2 eq of HOAt in dry DM F (1-2 mL) and 4 eq of DIPEA in dry DM F (1-2 mL) were added to the peptide, followed by stirring for ca. 16 h. The volatiles were removed by evaporation. The crude cyclic peptide was dissolved in 7 mL of CH2CI2 and washed three times with 4.5 mL 10percent acetonitrile in water (v/v). The CH2CI2 layer was then evaporated to dryness. To fully deprotect the peptide, 7 mL of cleavage cocktail TFA/DODT/thioanisol/H20 (87.5 :2.5:5:5) or TFA/TIS/H20 (95:2.5 :2.5) were added, and the mixture was kept for 2.5-4 h at room temperature until the reaction was completed. The reaction mixture was evaporated close to dryness, the peptide precipitated with 7 mL of cold Et20/pentane and finally washed 3 times with 4 mL of cold Et20/pentane. Procedures Bl and B2: Cyclization and work up of a backbone cyclized peptide having a disulfide interstrand linkage Bl: Formation of a disulfide interstrand linkage using DMSO After cleavage, backbone cyclization and deprotection of the linear peptide, as described in the corresponding section of procedure A, th...
General procedure: 100mg 2-chlorotrityl resin (0.5mmol/g) was swollen in dry DCM for 30min and treated with the first building block (2.0equiv) and DIEA (4.0equiv) in dry DCM. After it was shook for 1h, 80muL MeOH was added to cap the unreacted resin for another 20min. The loaded resin was washed by DCM (3×2mL) and DMF (3×2mL). Fmoc deprotection was achieved by shaken with 2mL 20percent solution of piperidine in DMF for 20min. The following Fmoc- or Boc-amino acids (4.0equiv) was coupled using 32 HATU (4.0equiv) as coupling reagent and DIEA (8.0 equiv) as base. The mixture was shaken in DMF for 1h. After each Fmoc deprotection and coupling reaction, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). The loaded resin was washed by DCM (3×2mL) and then a solution of Pd(PPh3)4 (1.0equiv) and phenylsilane (25equiv) in 2mL anhydrous DCM was added. The mixture was shaken for 1h under the protection of dry argon. After Alloc deprotection was completed, the resin was washed by DMF (3×2mL), DCM (3×2mL) and DMF (3×2mL). After coupling of the last building block, the resin was washed by DCM (3 2 mL), DMF (3 2 mL) and DCM (5 2 mL). Then a cocktail of DCM/AcOH/TFE (v/v/v = 8:1:1) was added to the resinand shaken for 1.5 h. Then the resin was filtrated off and rinsedwith DCM (5 2 mL). The combined filtrates were concentrated under low pressure and azeotroped several times with DCM to remove the Acetic acid. The side-chain-protected peptides were obtained as white solid.
General procedure: Solid-phase peptide synthesis was carried out on Fmoc-cappedpolystyrene rink amide MBHA resin (100-200 mesh, 0.05-0.15 mmol scale). The following amino acidderivatives suitable for Fmoc SPPS were used: Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(tBu)-OH,Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Val-OH, Fmoc-aPhe-OH, Fmoc-aVal-OH,Fmoc-aTyr(tBu)-OH, Fmoc-(N-Me)-Phe-OH, Fmoc-D-Ser(TBS)-OH, Fmoc-D-hSer(TBS)-OH, Boc-Gly-OH. Dry resin was washed with DMF 3x and allowed to swell in DMF for 2 h prior to use. Allreactions were carried out using gentle agitation. Fmoc deprotection steps were carried out by treating theresin with a solution of 20percent piperidine/DMF (15 min x 2). Coupling of Fmoc-protected amino acids aswell as (N2-Boc)-hydrazino acids was effected using 5 equiv. HATU (0.5 M in DMF), 10 equiv. DIEA(1.0 M in DMF), and 5 equiv. of the carboxylic acid in DMF at 50 oC (1 h). Coupling of residues Nterminalto the hydrazino acids was carried out with 30 equiv. collidine and 10 equiv. of pre-formed Fmocamino acid chlorides (or 10 equiv. of Fmoc amino acids with 3.3 equiv. triphosgene) in THF at rt (1 h x2).3 After each reaction the resin was washed with DMF 2x, DCM 1x, then DMF 1x. Peptides undergoingMitsunobu reactions were capped with Boc-Gly-OH, washed with DCM 3x, and treated with 5 equiv.TBAF in THF for 3 h at rt. After the reaction the resin was washed with DCM 3x and then treated with 5equiv. triphenylphosphine in THF followed by 5 equiv. of DIAD, then strirred overnight at rt. Peptideswere cleaved from the resin by incubating with gentle stirring in 2 mL of 95:5 TFA:H2O at rt for 2 h. Thecleavage mixture was filtered and the resin was rinsed with an additional 1 mL of cleavage solution. Thefiltrate was treated with 8 mL of cold Et2O to induce precipitation. The mixture was centrifuged and thesupernatant was removed. The remaining solid was washed 2 more times with Et2O and dried undervacuum. Cysteine-containing peptides were purified, lyophilized, dissolved in 10mM phosphate buffer(pH 8.9, 5percent v/v DMSO), stirred until analytical HPLC and MS showed complete conversion to the cyclicdisulfide (1-2 d), and then repurified. Peptides were analyzed and purified on C12 RP-HPLC columns(preparative: 4mu, 90A, 250 x 21.2 mm; analytical: 4mu, 90A, 150 x 4.6 mm) using linear gradients ofMeCN/H2O (with 0.1percent formic acid), then lyophilized to afford white powders. All peptides werecharacterized by LCMS (ESI), HRMS (ESI-TOF), and 1H NMR. Analytical HPLC samples for all purifiedpeptides were prepared as 1 mM in H2O containing 20 mM phosphate buffer at pH 7.0. Linear gradientsof MeCN in H2O (0.1percent formic acid) were run over 20 minutes and spectra are provided for lambda = 220 nm.
Nα-(9-fluorenylmethyloxycarbonyl)-Nγ-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine[ No CAS ]
[ 58-85-5 ]
NH2-NK(Biotin)VPNLRGDLQVLAQ[diaminobutyric acid]VART-OH[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
29%
(0234) To enable investigation of introducing non-proteinogenic amino acids (listed in Figure 1) on A20FMDV2 binding activity, biotinylated peptides 1-15 (see Table 1), except for peptide 6, were synthesised by standard Fmoc SPPS on the acid liable (0235) hydroxymethylphenoxypropionic acid linker (HMPP) which delivers a C- terminal carboxylic acid using to the conditions depicted in Scheme 1. The desired peptide sequences were assembled using 20percent (0236) piperidine/DMF to remove the Fmoc protecting group and 0- (0237) (benzotriazol-l-yl) -N, N, N ' , N '-tetramethyluronium hexafluorophosphate (HBTU) / DIPEA as coupling reagents. (0238) Since specific binding to the nubetabeta integrin was to be studied by flow cytometry, the native alanine at the second residue in A20FMDV2 (1) and all analogues thereof, were substituted with a biotinylated lysine residue. This substitution has previously been shown to be well tolerated [24,25] . We chose to install the D-biotin moiety by selective deprotection of a 1- ( 4 , 4-dimethyl-2 , 6-dioxocyclohex-l- ylidene ) ethyl (Dde) [19] group on the side chain group followed by condensation with D-biotin using HBTU/DIPEA. (0239) Trifluoroacetic acid (TFA) /H2O/3, 6-dioxa-l , 8-octanedithiol (0240) (DODT) /triisopropylsilane (TIPS) (94:2.5:2.5:1.0, v/v/v/v) effected cleavage of the synthesised peptides from the corresponding (0241) peptidyl-resins . Peptides 1-15 were obtained in good yields ranging from 2percent-50percent and purity exceeding 99percent (see peptide characterization data) . (0242) For the synthesis of peptide 6 containing an i\7-L-methyllysine modification we employed an on-resin i\7-methylation protocol [22] which furnished peptide 6 in good yield (30percent) following TFA-mediated peptide cleavage and RP-HPLC purification. (0243) The lead peptide, A20FMDV2, which contains all naturally-occurring amino acids would be susceptible to degradation by exopeptidases which act on the amino- and carboxy terminuses. To mitigate this, six N- and/or C-terminus-modified and biotinylated A20FDMV2 mimics were prepared wherein we systematically modified the amino and carboxy ends (peptides 16-18) and the N-terminal and C-terminal amino acids (Asnl and Thr20, respectively, peptides 19-21) . N- terminal/C-terminal modified peptides 16-18 were obtained by capping of the N-terminus with acetic anhydride (16) or by employing the Rink amide linker to afford the C-terminal carboxamide (17) or a combination of both (peptide 18) . (0244) Peptide 19, bearing the unnatural D-Asnl in place of the native Asnl at the N-terminus of biotinylated A20FMDV2 (1) was obtained using the synthetic route outlined in Scheme 1 except that the Fmoc-D- Asn(Trt)-OH building block was incorporated into the synthesis as the N-terminal residue. For the preparation of peptides 20 and 21, which contains the unnatural D-Thr at the C-terminus, HMP-anchored resin 27 (see Scheme 1, HMP = hydroxymethylphenoxyacetic acid) was first esterified with Fmoc-D-Thr (tBu) -OH using DIC/DMAP and the sequence then elongated by Fmoc SPPS . (0245) Table 1. List of prepared synthetic peptides [N-term] - XiK (Biotin) VPNLRGDLQVX2AQX3VARX4- [C-term] containing substitutions for the native Lysl6 (peptides 2-6) or Leul3 (peptides 7-15), C- terminal/N-terminal variants (peptides 16-21) and DTPA-modified peptides (22-26) . NB: nomenclature, particularly X position (0246) numbering used in this table is not the same as that used in the claims . (0247) Compound N- Xl X2 X3 X4 C- term. term. (0248) 1 NH2 Asn Leu Lys Thr C02H (0249) 2 NH2 Asn Leu D-Lys Thr C02H (0250) 3 NH2 Asn Leu L-Orn Thr C02H (0251) 1-2,4- (0252) 4 NH2 Asn Leu diaminobutyric Thr C02H acid (0253) 1-2,3- (0254) 5 NH2 Asn Leu diaminopropionic Thr C02H acid (0255) 6 NH2 Asn Leu ZV-L-meth llysine Thr C02H (0256) 7 NH2 Asn aminoisobutyric Lys Thr C02H acid (0257) 8 NH2 Asn L-norvaline Lys Thr C02H (0258) 9 NH2 Asn L-norleucine Lys Thr C02H (0259) 10 NH2 Asn L-allylglycine Lys Thr C02H (0260) L-tert- (0261) 11 NH2 Asn Lys Thr C02H butylalanine (0262) 12 NH2 Asn L-homoleucine Lys Thr C02H (0263) L-2-amino-3- (0264) 13 NH2 Asn ethylpentanoic Lys Thr C02H acid (0265) L- (0266) 14 NH2 Asn Lys Thr C02H cyclohexylalanine (0267) 15 L- (0268) NH2 Asn Lys Thr C02H adamantylglycine (0269) 16 Ac-NH Asn Leu Lys Thr C02H (0270) 17 NH2 Asn Leu Lys Thr CONH2 (0271) 18 Ac-NH Asn Leu Lys Thr CONH2 (0272) 19 D- (0273) NH2 Leu Lys Thr C02H (0274) Asn (0275) 20 D- (0276) NH2 Asn Leu Lys C02H (0277) Thr (0278) 21 D- D- (0279) NH2 Leu Lys C02H (0280) Asn Thr (0281) 22 DTPA- (0282) Asn Leu Lys Thr C02H NH (0283) 23 DTPA- (0284) Asn Leu Lys Thr C02H Gly-NH (0285) 24 DTPA- (0286) Asn Leu Lys Thr CONH2 NH (0287) 25 DTPA- D- (0288) Leu Lys Thr C02H NH Asn (0289) 26 DTPA- D- D- (0290) Leu Lys C02H NH Asn Thr (0291) 1 D -yrpercent \?Q ^ (0292) ? -^H Q ? (0293) (0294) Scheme 1. Synthetic protocol for the preparation of the biotinylated A20FMDV2 peptide variants. (0295) The results obtained from th...
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