UniChemo Protection: A Novel Concept For Chemical Synthesis. Les P. Miranda and Morten Meldal

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1 Copyright WILEY-VCH Verlag GmbH, D Weinheim, Supporting Information for Angew. Chem. Int. Ed. Z17269 UniChemo Protection: A ovel Concept For Chemical Synthesis Les P. Miranda and Morten Meldal Synthesis of protecting groups. The Fmoc-derivatized protecting group unit was prepared in analogous to literature procedure from sec-butylamine and ethyl bromoacetate [J.A.W Kruijtzer, L.J.F Hofmeyer, W. Heerma, C. Versluis, RM.J. Liskamp, Eur. J. rg. Chem. 1998, 4, ]. Briefly, ethyl bromoacetate (50 mmol) in tetrahydrofuran (25 ml, THF) was added dropwise to a cooled solution of sec-butylamine (110 mmol) in THF (25 ml) over 5 min. After stirring for 4 h at room temperature, the reaction mixture was concentrated in vacuo and resuspended in dry diethyl ether. The mixture was filtered to remove sec-butylamine hydrobromide, the residue washed with ether, and the filtrate concentrated in vacuo. Yield 91% as a colourless oil. The product was then saponified with sodium hydroxide and derivatized with 9- fluorenylmethyloxycarbonyl chloride (Fmoc-Cl). The MR spectra clearly show the presence of stereoisomers. -sec-butyl-fluoren-9-ylmethoxycarbonyl-glycine, 1 H MR (250 MHz, 25 o C, CDCl 3, ppm): δ=0.83, 0.73 (t, 3H, J = 7.2 Hz), 1.00, 1.06 (d, 3H, J = 6.9 Hz), (m, 2H), , (m, 4H), (m, 2H), (m, 4 H ar ), (m, 2H ar ), (m, 2H ar ), (b, 1H); 13 C MR (62.90 MHz, 298 o K, CDCl 3 ) δ 10.93, 18.03, 18.32, 27.48, 27.73, 43.28, 43.99, 47.30, 53.35, 67.58, , , , , , , , , , , , Calc. for C 21 H (monoisotopic), Exp. ESMS: Da. Protecting groups were assembled through the side-chain ε - 1

2 amino group of α -Alloc-Lys-Wang resin using standard Fmoc/Bromo-trispyrrolidino-phosphonium hexafluorophosphate (PyBroP)-based coupling chemistry was employed as previously described [14]. Synthesis of Unichemo Protected Pentalysine Scaffold. First, five serial ε -oligo(-sec-butylglycyl) α -allyloxycarbonyl (Alloc) protected lysine building block (1.5 equiv) were coupled to 4-{4-[1-(9H-fluoren-9-ylmethoxycarbonylamino)- ethyl]-2-methoxy-5-nitro-phenoxy}-butanoic aminomethyl polystyrene resin (1.4 mmol/g; photolabile linker) with -(7- azabenzotriazol-1-yl)-,,','-tetramethyluronium hexafluorophosphate (HATU, 1.5 equiv) and,diisopropylethylamine (DIEA, 4 equiv) in,-dimethylformamide (DMF) at a concentration of 0.3 M at room temperature. Global Fmoc deprotection was carried out with 50% v/v piperidine in DMF to liberate the secondary amines of the protecting groups after scaffold assembly. Intermediate elutes as a single peak by RP-HPLC analysis. Calc. for C 125 H (monoisotopic), Exp. ESMS: Da. MALDI-TF: Da. General UCP deprotection procedure. The resin-bound protected scaffold was treated twice with an excess mixture of 25% v/v solution phenylisothiocyanate (PITC) plus 10% - methylmorpholine in,-dimethylformamide (DMF) at 55 o C for 30 min. After reaction, the resin was washed with DMF. The resin was incubated three times with anhydrous trifluoroacetic acid (TFA) at 55 o C for 30 min. The resin was then washed with DCM and then DMF. The efficiency of the deprotection step is >98% as determined by reversed-phase HPLC. General derivatization procedure with para-nitrphenyl (p) esters. Boc-Ala-p (15.5 mg, 0.05 mmol, 5 mole equivalents) 2

3 or another preformed p or Su ester derivative, is dissolved in anhydrous DMF to a concentration of 0.3 M. The solution is then added to a neutralized resin-bound primary amine (0.1 mmol equivalent) at room temperature and left for 1 h. Depending on the nature or reactivity of the p or Su ester and also the accessibility of the resin-bound primary amine the optimal reaction time may be shorter or longer. Characterization of product 2: The target compound was cleaved from the solid-support in methanol with UV irradiation for 3 hours, extracted with 70% (v/v) acetonitrile in water, filtered and then purified by reversed phase high-performance liquid chromatography (RP-HPLC). HPLC crude purity estimate 62%. Isolated yield 26%, from starting resin loading value and after reversed phase-hplc purification. RP-HPLC was performed on a Waters 110 solvent delivery and Wisp 710B system equipped with a Schimadzu UV absorbance and recorded on a PC computer using TurboChrom avigator 4.1 (Perkin Elmer). Analytical RP-HPLC was performed on Zorbax C 18 (5 µm, 0.46 cm 5 cm) column. Chromatographic separations were achieved using linear gradients of 0-80% buffer B in A (A=0.1% aqueous TFA; B=90% CH3C, 10% H2, 0.09% TFA) over 40 min at a flow rate of 1 ml/min. 1 H MR ( MHz, 25 o C, CD 3 D, ppm) δ Thymine moiety: 1.84, 4.37, 7.36; Shikimic moiety: 2.15, 2.71, 3.66, 4.35, 6.38; Thiophene moiety: 7.07, 7.59, 7.64; aphthyl moiety: 7.85, 7.92, 7.95, tetrahydroisoquinoline moiety: 4.54, 7.14, Due to spectral degeneracy and overlap of the lysine scaffold, only characteristic resonances of side chain groups are reported. Calc. for C 70 H S, (monoisotopic). Exp. ESMS: Da. MALDI-TF: Da. Mass Spectrometry. Electrospray mass spectra were acquired on a Hewlett-Packard HP1100-MSD mass spectrometer equipped with an atmospheric pressure ionization source. Samples dissolved in 3

4 50% aqueous acetonitrile (3 µl) were injected into a moving solvent (100 µl/min; 50:50 0.3% acetic acid in water/0.03% acetic acid in acetonitrile) coupled directly to the ionization source via a fused silica capillary interface (50 µm i.d. 25 cm length). Sample droplets were ionized at a positive potential of 5 kv and entered the analyzer through an interface plate and subsequently through an orifice ( µm diameter) with a capillary potential of 90 V. Full scan mass spectra were acquired over the mass range of Da with a scan step size of 0.1 Da. Molecular masses were derived from the observed m/z values using the HP LC/MSD Chemstation Rev A software packages (HP, USA). Matrix-assisted laser desorption ionization time of flight (MALDI-TF) mass spectrometry were acquired on a Bruker Reflex TM III MALDI-TF mass spectrometer. Spectra were obtained (1 100 pulses) using the lowest power required to facilitate desorption and ionization. Ions were accelerated toward the discrete dynode multiplier detector with an acceleration voltage of 20 kv. The matrix α-cyano-4- hydroxycinnamic acid (CHC, 10 mg in 1 ml of 70% acetonitrile) was used. Bradykin ( Da), renin ( Da), and mellitin ( Da) were used as the standards for internal calibration of the mass spectra. Beads were irradiated on stainless steel targets with a strong UV lamp for 60 min. The analyte was extracted on the target from the beads using 0.5 µl of 70% acetonitrile and then dried at room temperature (RT). The appropriate matrix was added, the sample dried at 40 C. 4

5 SUPPLEMETARY FIGURES A B 1. A. Low energy conformer of the pentalysine scaffold with -sec-butylglycyl protecting group units. The lysine scaffold is shown in orange. B. Molecular surface of a low energy conformer of penta(-sec-butylglycyl) amide as viewed from an axial perspective. The Connolly surface (r=1.4 Å) was calculated, and colored as follows: carbon/aliphatic (white), oxygen (red), and amine (blue). Conformations of the protecting group were generated by 3x100 ps annealing using molecular dynamics simulations. 5

6 UCP Deprotection Chemistry: As utlined for a Dimeric α -sec-butylglycyl UCP Group H FG = represents a functional group attached to a given molecule C S S CF 3 CH FG ph 8-9 H H FG FG S C S ph 8-9 H S FG CF 3 CH FG S 2. Schematic representation of UCP deprotection chemistry. 6

7 A. ES-MS ESI-MS and MALDI-TF Spectra of the Crude Protected Pentalysine Scaffold, 1, From Resin Cleavage S3 [M+4H] Exp. Mol. Wt. : Calc. Mol. Wt.: [M+5H] [M+3H] [M+2H] [M+H] B. MALDI-TF MS Exp. Mol. Wt. : Calc. Mol. Wt.: M M+a M+K 3. ESI-MS and MALDI-TF spectra of the crude protected pentalysine scaffold, 1. 7

8 S4 Reversed-Phase HPLC Analysis of the Crude UCP Product, 2. System : Waters 110 solvent delivery-wisp 710B system Detector: Schimadzu UV absorbance, 214 nm Recorder: Windows 95 PC using TurboChrom avigator 4.1 (Perkin Elmer). Column : Zorbax C 18 (5 µm, 0.46 cm 5 cm). Gradient: Linear gradient of 0-80% buffer B in A over 40 min Buffer A: 0.1% aqueous TFA Buffer B: 90% CH 3 C, 10% H 2, 0.09% TFA Flow : 1 ml/min. UCP Product 2 4. Reversed-phase HPLC analysis of crude UCP product, 2. 8

9 A. ES-MS ESI-MS and a MALDI-TF Spectra of the Purified UCP Product, 2, Cleaved from the Resin S5 [M+2H] Exp. Mol. Wt. : Calc. Mol. Wt.: [M+H] + B. MALDI-TF MS Exp. Mol. Wt. : M M+a M+K Calc. Mol. Wt.: ESI-MS and MALDI-TF spectra of purified UCP product, 2. 9

10 1 H-MR Spectrum of UCP Product, MHz 1 H MAS-MR 2048 Res. CD 3 H S H MR of UCP product, 2. 10