Diisopropyl ether

3-Methylbut-2-enal (97%), p-anisidine (99%), acetoxy acetyl chloride (97%), tert-butanol (≥99%), methanol (>99.5%), hexane (>99.5%),
ethyl
acetate (>99.5%), diisopropyl ether (≥98.5%), and immobilized
CAL-B (lipase B from Candida antarctica) on the acrylic resin (L4777) were purchased from Sigma-Aldrich
(St. Louis, MO). Ceric ammonium nitrate (99%) was purchased from Fisher
Scientific Company (Fair Lawn, NJ). Coenzyme A (95%) was obtained
from AmBeed (Arlington Hts, IL). Nickel-affinity chromatography resin
(HisPur Ni-NTA Resin) was purchased from Thermo Fisher Scientific
(Waltham, MA). ATP, DTT, isopropyl β-d-1-thiogalactopyranoside
(IPTG), kanamycin, and phenylmethylsulfonyl fluoride (PMSF) were purchased
from Gold Biotechnology (Olivette, MO). Baccatin III (>98%) and
10-O-deacetylBaccatin III (>98%) were purchased
from Natland
International Corporation (Research Triangle Park, NC). Triethylamine
(100%) was obtained from J. T. Baker (Center Valley, PA). C18 silica
gel resin (carbon 23%, 40–63 μm) was purchased from Silicycle,
Quebec City, Quebec, Canada. The following compounds (Table 1) were biocatalyzed in earlier
studies and were among the laboratory inventory.

N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% TMCS, tert-butyl methyl ether (MTBE), ethyl acetate, methanol, n-hexane, dichloromethane, diisopropyl ether, toluene, anhydrous pyridine, trifluoroacetic acid, sodium methanolate, silica gel (70–230 mesh, high purity), and standards of stigmasterol (95%), 5α-cholestane, and ethyl heptadecanoate were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Glyceryl heptadecanoate was obtained from Larodan (Sweden).

DMF (dimethylformamide), DCM (dichlormethane), diisopropylether, piperidine and TIPS (triisopropylsilane) were purchased from Sigma-Aldrich. NMM (4-methylmorpholine) and TFA (2,2,2-trifluoroacetic acid) were acquired from Fisher Scientific; Fmoc-protected Wang-resins, Fmoc-protected amino acids, and HCTU (O-(6-chloro-1-hydrocibenzotriazol-1-yl)-1,1,3,3-tetra -methyluronium hexafluorophosphate) were obtained from AAPPTEC. An Overture robotic solid phase peptide synthesizer (Protein Technologies, Tucson, USA) was used to synthesize peptides on a 20 µmol scale utilizing 10-fold excess of Fmoc-protected amino acids (200 mM) relative to the Fmoc-Wang-resin. Deprotection by 20% piperidine in DMF for 2×5 min. Double coupling was performed in a conservative manner of 2×15 min rotations with a ratio of 1∶1∶2 (200 mM amino acid, 200 mM HCTU, 400 mM NMM) in DMF. DMF washing was applied for 5×30 sec after deprotection and double coupling. Automated cleavage was applied for 2 h with 95% vol.+2.5% vol.+2.5% vol. (TFA, H₂O, TIPS), after multiple washing steps with DCM (3×30 sec). Using ice-cold diisopropylether, the peptides were precipitated from the final TFA-peptide solution and rewashed. All peptide products were analyzed on an LC-20A HPLC instrument (Shimadzu) using an rpC18, 110 Å, 5 µm, 150×3 mm column (Macherey-Nagel), with a linear gradient of 5–70% ACN/H₂O (0.1% TFA) over 25 min with a flow rate of 0.5 ml×min⁻¹; UV-VIS detection at 210 nm. Masses were detected between 300–1500 Da with a Shimadzu LCMS-2020 single-quad mass spectrometer (ESI+). UV²¹⁰ purity of all products: >98%. Peptide sequences are denoted with the calculated molecular weight (mw, unit: Da), observed retention times (Rt, unit: minutes) and masses (m+); some peptides exhibited water clusters (*): SII (mw = 331.40, Rt = 9.87, m+ = 332.15, 333.15, 663.4, 664.4), IIN (mw = 358.43, Rt = 6.86, m+ = 359.15, 360.15, 719.40, 717.45, 718.45), INF (mw = 392.45, Rt = 10.15, m+ = 393.15, 394.10, 786.30, 785.4, 1175.75), NFE (mw = 408.41, Rt = 7.66, m+ = 408.10, 409.95, 817.35), FEK (mw = 422.48, Rt = 6.45, m+ = 432.20, 424.20), EKL (mw = 388.46, Rt = 7.59, m+ = 389.15, 390.15, 777.45), SIIN (mw = 445.51, Rt = 8.86, m+ = 446.25, 447.25, 891.55, 892.50), IINF (mw = 505.61, Rt = 10.36, m+ = 527.35*, 528.25*, 529.30*, 957.30*, 958.40*), INFE (mw = 521.57, Rt = 9.77, m+ = 522.25, 523.20, 1043.55, 1044.55, 1045.50), NFEK (mw = 536.59, Rt = 6.93, m+ = 537.25, 538.25, 539.25), FEKL (mw = 534.63, Rt = 9.39, m+ = 536.35, 537.25, 538.20, 1071.65, 1072.60), SIINF (mw = 592.68, Rt = 11.99, m+ = 593.30, 594.35, 596.25, 1184.85, 1185.75, 1186.70, 1187.70), IINFE (mw = 634.72, Rt = 10.76, m+ = 635.35, 636.30, 637.30, 1269.70, 1270.80), INFEK (mw = 649.74, Rt = 8.84, m+ = 325.75, 650.35, 651.35, 652.35), NFEKL (mw = 649.74, Rt = 9.59, m+ = 650.35, 651.35, 652.30, 1300.85), SIINFE (mw = 721.79, Rt = 11.5, m+ = 722.40, 723.40, 724.30, 1443.9, 1444.90, 1445.90), IINFEK (mw = 762.89, Rt = 9.77, m+ = 382.35, 763.40, 764.40, 765.40), INFEKL (mw = 762.89, Rt = 10.84, m+ = 382.30, 763.45, 764.4, 957.45, 965.45, 965.45), SIINFEK (mw = 849.97, Rt = 10.51, m+ = 425.80, 850.50, 851.45, 852.45), IINFEKL (mw = 876.05, Rt = 11.51, m+ = 438.95, 876.55, 877.50, 878.50).