O-(1,1-Dimethylethyl)-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-threonine

White to light yellow powder

General procedure: Tetrapeptides 3a-d were synthesized by manual solid-phase peptide synthesis (SPPS) using 2-chlorotritylchloride polymer resin (2-CTC, 1.63mmol/g) in a 10-mL disposable syringe fitted with a porous polyethylene filter and N-terminal fluorenylmethyloxycarbonyl-protected amino acids. Coupling of the first amino acid: Fmoc-propargyl-Gly-OH (0.8 eq.) to the 2-CTC resin (1.0 eq.) was performed in anhydrous DCM (7.5mL) in the presence of DIEA (3.0 eq.) with shaking at room temperature for 2h. Then, MeOH (2mL) was added to cap the unreacted resin and the mixture was shaken for 30min. The resin was filtered, washed with several portions of DCM, DMF and again DCM and dried under high vacuum, overnight. Loading of the starting material was calculated via increase in dry weight. Next, the resin swelling was performed in DMF for 20min, Fmoc protecting group was removed by treatment with 20% piperidine in DMF (2×3mL, 5+20min) and the polymer was washed with DMF (5×3mL, 5×2min). Then, coupling of the appropriate amino acid: Fmoc-AA-OH (4.0 eq.) was performed in DMF (3mL) using HBTU (4.0 eq.), in the presence of DIEA (8.0 eq.) with shaking at room temperature. In the case of Fmoc-Ala-OH for 4h, with Fmoc-Orn(Boc)-OH, Fmoc-Thr (tBu)-OH and Fmoc-N-Me-Thr (tBu)-OH overnight and with Fmoc-Arg (Pbf)-OH and Fmoc-His (Trt)-OH the double coupling was performed (after 2h the resin was washed with DMF and treated with a freshly made solution of Fmoc-AA-OH, HBTU and DIEA in DMF and shaken for 2h). After each coupling, the resin was washed with DMF (4×4mL) and subsequently de-protected and washed as described above. At the end of the coupling cycle the resin was washed with several portions of DMF, MeOH and DCM and dried under high vacuum, overnight. Method (A) for coupling of the tail via HBTU activation: The coupling of the tails at the N-terminus of the synthesized tetrapeptides was performed using the acid (4 eq.) in DMF (2mL), in the presence of DIEA (8 eq.) and HBTU (4 eq.) at room temperature, shaking overnight. The resin was then washed extensively with several portions of DMF, MeOH and DCM and dried under high vacuum. Method (B) for coupling of the tail via mixed anhydride: Step 1. Heterocyclic acid derivative (0.52mmol, 1 eq.) was reacted with Boc anhydride (2.11mmol, 4 eq.) in dry tetrahydrofuran in presence of pyridine (2.64mmol, 5 eq.) at r. t. overnight. After full consumption of starting material volatiles were removed in vacuo. Aqueous 0.5M hydrochloric acid solution was added and the product was extracted with ethyl acetate. The organic layer was washed with water, separated and dried over sodium sulfate. Volatiles were removed in vacuo yielding a crude product as yellowish oil, which was used in the next step without further purification. Step 2. The 2-CTC resin containing 3a (0.13mmol, 1 eq.) was suspended in DMF (3mL) containing mixed anhydride prepared in Step 1 (presumed 0.52mmol, 4 eq.) and DIPEA (1.82mmol, 14 eq.). The reaction mixture was shaken at r. t. overnight. The resin was filtered, washed with DMF (5×2mL), DCM (5×2mL) and THF (5×4mL). General method for cleavage from the solid support. 2-CTC resin containing oligopeptides was added into a mixture of HFIP/DCM 1:4 (4mL) and the reaction mixture was shaken at room temperature for 1.5h. The resin was filtered off and washed with DCM and THF. The volatiles were evaporated in vacuo. Purification: The crude peptides were dissolved in THF (2.5mL) and H2O/MeCN (1:1, 2.5mL), filtered through a 0.45mum nylon membrane filter and purified in 1-3 runs by preparative RP-HPLC. The reported yields are based on calculated loading of the oligopeptide on 2-CTC resin.Prep.93 To a solution of the object compound of Prep.3 (1.20 g), Prep.95 To a solution of the object compound of Prep.166 below (1.20 g), (2S, 3R)-3-tert-butoxy-2-([(9H-fluoren-9-ylmethoxy)carbonyl](methyl)amino}butanoic acid (527 mg), and HOAt (174 mg) in CH2Cl2 (20 ml) was added a solution of WSCD (199 mg) in CH2Cl2 (1 ml) under ice-bath cooling. After being stirred at the same temperature for 1 hr and at room temperature for 4 hrs, the reaction solution was concentrated in vacuo. The residue was dissolved in AcOEt (50 ml) and the solution was washed with 0.5N HCl, 1M NaHCO3, and brine, dried over MgSO4, and concentrated in vacuo. The resulting residue was purified by Silica-gel column (eluent: 2% MeOH in CHCl3) to give methyl (5S, 8S, 11S, 14S, 17R, 20S, 23S, 26S, 29S, 32S)-5-(1-tert-butoxyethyl)-1-(9H-fluoren-9-yl)-32-[(1R)-1-hydroxyethyl]-29-[(1R, 2R, 4E)-1-hydroxy-2-methyl-4-hexen-1-yl]-8, 11, 20, 23-tetraisobutyl-26-isopropyl-4, 10, 14, 17, 19, 25, 28, 34-octamethyl-3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33-undecaoxo-2-oxa-4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34-undecaazahexatriacontan-36-oate (1.58 g) as an amorphous powder.

Molecular Weight:411.5
XLogP3:4.1
Hydrogen Bond Donor Count:1
Hydrogen Bond Acceptor Count:5
Rotatable Bond Count:8
Exact Mass:411.20457303
Monoisotopic Mass:411.20457303
Topological Polar Surface Area:76.1
Heavy Atom Count:30
Complexity:593
Defined Atom Stereocenter Count:2
Covalently-Bonded Unit Count:1
Compound Is Canonicalized:Yes