S1 Fully Automated Sequential Solid Phase Approach Towards Viral RNA-Nucleopeptides Gerbrand J. van der Heden van Noort, Pieter van Delft, Nico J. Meeuwenoord, Herman S. Overkleeft, Gijsbert A. van der Marel and Dmitri V. Filippov Supporting Information General: 3’-O-DMT-2’-O-TBDMS-N 9 -benzoyladenosine-5’-(N,N-di-isopropylamino-2-cyanoethoxy) phosporamidite, 3’-O- DMT-2’-O-TBDMS-uridine-5’-(N,N-di-isopropylamino-2-cyanoethoxy) phosporamidite, -O-DMT-2’-O-TBDMS – N 4 - acetylcytidine-5’-(N,N-di-isopropylamino-2-cyanoethoxy) phosporamidite and 3’-O-DMT-2’-O-TBDMS – N 2 - isobutyrylguanosine-5’-(N,N-di-isopropylamino-2-cyanoethoxy) phosporamidite were purchased from ChemGenes Corporation, Wilmington, USA and used as received. HCP Custom Primer Support TM Amino 200 resin was purchased from GE Healthcare. LC/MS analysis was performed on A) Jasco HPLC system (UV detection simultaneously at 214 and 254 nm) coupled to a PE/SCIEX API 165 single quadruple mass spectrometer (Perkin-Elmer) using an analytical Gemini C 18 column (Phenomex, 50 x 4.60 mm, 3 micron) in combination with eluents A: H 2 O; B: MeCN and C: 0.1 M aq. NH 4 OAc B) Thermo Finnigan LCQ Advantage MAX ion-trap mass spectrometer with an electrospray ion source coupled to Surveyor HPLC system (Thermo Finnegan) using an analytical Gemini C 18 column (Phenomex, 50 x 4.60 mm, 3 micron) in combination with eluents A: H 2 O; B: MeCN and C: 1% aq. TFA as the solvent system. High resolution mass spectra spectra were recorded by direct injection (2 μL of a 2 μM solution in water/acetonitrile; 50/50; v/v and 0.1% formic acid) on a mass spectrometer (Thermo Finnigan LTQ Orbitrap) equipped with an electrospray ion source in positive mode (source voltage 3.5 kV, sheath gas flow 10, capillary temperature 250 °C) with resolution R = 60000 at m/z 400 (mass range m/z = 150‐2000) and dioctylpthalate (m/z = 391.2842) as a “lock mass”. The high resolution mass spectrometer was calibrated prior to measurements with a calibration mixture (Thermo Finnigan). HCP-HMBA resin Amino HCP resin (1 g, 200 μmol) was treated with 4-hydroxymethylbenzoic acid (HMBA) (92 mg, 600 μmol), BOP (266 mg, 600 μmol), HOBt (82 mg, 600 μmol) and DiPEA (200 μl, 1.2 mmol) in 10 ml DMF for 5 hours at room temperature. After washing with DCM three times, the unreacted amines were capped using 230 μl acetic anhydride, 270 μl DiPEA, 10 mg HOBt in 5 mL NMP for 5 minutes. The resin was then washed three times with DMF, DCM and subsequently air dried. Fmoc-Gly-Ala-Tyr-Thr-Gly-HMBA-HCP resin (3) Starting from HCP-HMBA resin (500 mg, 100 μmol) Fmoc-Gly-OH (150 mg, 500 μmol) was coupled using DIC (101 μL, 500 μmol) and catalytic DMAP in DCM (5 mL) for 4 hours at room temperature. After washing with DCM the unreacted resin was capped using the procedure used for oligonucleotide capping as described below. The resin was transferred to an automated peptide synthesizer (ABI-433A, Applied Biosystems, Perkin-Elmer) and the peptide was elongated with Fmoc-Thr(OTBDMS)- OH, Fmoc-Tyr-OH, Fmoc-Ala-OH and Fmoc-Gly-OH using the following repetitive steps: - Fmoc cleavage using 20% piperidine in DMF. - Coupling of the appropriate amino acid applying a five-fold excess, activation by 5 eq. HCTU in NMP (0.25M) and 12.5 eq. DiPEA in NMP (1.25 M) for 1 hour. An analytical sample of resin 3 (ca. 5 μmol) was treated with a saturated solution of NH 3 in trifluoroethanol (1 mL) for 16 hours at room temperature after which the reaction mixture was filtrated and concentrated. LCMS (0 – 90 % MeCN, conditions B, 15 Electronic Supplementary Material (ESI) for Chemical Communications This journal is © The Royal Society of Chemistry 2012