Supporting Information. Copyright Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2006

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1 Supporting Information Copyright Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2006

2 Copyright Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2006 Supporting Information for Microtiter Plate-Based Screening for the Optimization of DNA- Protein Conjugate Synthesis by Means of Expressed Protein Ligation Marina Lovrinovic and Christof M. Niemeyer* Experimental Section General. All compounds were obtained from Sigma Aldrich apart of cysteine derivaives which were obtained from Novabiochem. Cy3 mono-reactive NHS ester was purchased from Molecular Probes. MALDI MS data were obtained using PerSeptive Biosystems Voyager-DE Biospectrometry Workstation. The 1 H and 13 C NMR spectra were taken on a Bruker DRX 400 and DRX 500 spectrometer. Chemical shifts are reported in parts per million referenced to internal standard ((CH 3 ) 4 Si = 0.00 ppm). Synthesis of a cysteine-modifier. The synthesis of a hexa-ethylenglycol modifier 9 and the cysteine NHS-ester 10, used for the cysteine modification of amino-modified oligonucleotides was performed as previously described. [1, 2] The cysteine modifier 8 containing a tris-ethylenglycol chain was synthesized according to protocols used for the synthesis of the hexa-ethylenglycol linker 9, using a triethylenglycol as the starting material (Scheme S1). Briefly, the corresponding azidoalcohol 11, prepared according to the method of Svedhem et al., [3] was treated with tert-butyl bromoacetate to give a tert-butylester azide. Staudinger reduction of the azide group yielded 89% of amino-compound 12, which was reacted with 2.5 equiv of dicyclohexyl carbodiimide (DCC)/1-hydroxy- S1

3 benzotriazole (HOBt) activated N-? -Fmoc S-tert-butylsulfenylcysteine in THF. After stirring overnight at room temperature, the solvent was evaporated and the residue was dissolved in dichlormethane. The organic phase was washed with a saturated solution of NaHCO 3, dried (MgSO 4 ) and concentrated in vacuo. Flash column chromatography of the residue (ethyl acetat/cyclohexane 5:1) yielded 74 % of H NMR (500 MHz, CDCl 3 ): δ = 7.57 (d, 2H), 7.40 (d, 2H), 7.39 (t, 2H), 7.31 (dt, 2H), 4.42 (t, 2H), 4.35 (t, 1H), 4.25 (t, 1H) 4.00 (s, 2H) 3.62 (bm, 10H), 3.49 (t, 1H), 3.11 (d, 2H), 1.49 (s, 9H), 1.32 (s, 9H); 13 C NMR (125 MHz, CDCl 3 ) δ = 170.4, 143.7, 141.2, 127.6, 127.0, 125.1, 119.8, 81.8, 72.5, 70.32, 70.30, 70.10, 69.4, 68.8, 67.1,60.6, 42.7, 39.6, 29.7, Scheme S1: Synthesis of a tris-ethylenglycol cysteine modifier 8. The tert-butylester was removed by treatment with trifluoroacetic acid (TFA) for 2 h at RT. The resulting acid was activated with dicyclohexyl carbodiimide (DCC) and reacted with 1.1 equivalents of N-hydroxysuccinimide (NHS) to yield 62% of cysteine modifier 8 as a yellow oil. 1 H NMR (500 MHz, CDCl 3 ) δ = 7.59 (d, 2H), 7.39 (d, 2H), S2

4 7.38 (t, 2H), 7.29 (dt, 2H), 4.48 (s, 2H), 4.41, (m, 1H), 4.24 (m, 2H), (bm, 10H), 3.46 (m, 1H), 3.07 (d, 2H), 2.68 (s, 4H), 1.32 (s, 9H); 13 C NMR (125 MHz, CDCl 3 ) δ = 172.4, 170.6, 169.0, 143.7, 141.2, 127.6, 127.0, 125.1, 119.9, 71.0, 70.3, 70.0, 69.4, 67.9, 67.5, 66.3, 41.7, 39.6, 29.7, 25.4; FAB-HRMS (m/z): calcd for C 34 H 43 O 10 N 3 S ([M+H] + ) and ([M+Na] + ), found and , respectively. Synthesis and purification of the cysteine-oligonucleotide conjugates was carried out from the corresponding cysteine modifier, as described previously. [1, 2] In brief, a solution of 100 µl of a cysteine modifier 8-10 (10 mm in DMF) was added to a solution of the amino-modified DNA 14 (5 -NH 2 -CCT GTG TGA AAT TG-3, Thermo Electron) in 100 µl PBS buffer (100 mm, ph 7.3). After incubation for 12 h at RT, the solvent was removed in vacuo and the resulting residue was dissolved in water. Subsequent to purification by gel filtration chromatography (NAP 5 and NAP 10 columns, Pharmacia) the products were characterized by MALDI-TOF mass spectrometry. MALDI-TOF MS (Matrix: 3-hydroxypicolinic acid): 2a: calcd [M+H] +, found a: calcd [M+H] +, found a: calcd [M+H] +, found The amino protection group Fmoc was removed by treatment with aqueous ammonia at 37 C for 6 h. The crude product was dissolved in water and purified by gel filtration chromatography (NAP 5) and reversed-phase HPLC (Nucleodur Gravity C 18, Macherey-Nagel) using a gradient of 5-95% acetonitrile in 1 M triethylammonium acetate over 40 min at a flow rate of 1 ml/min. The conjugates obtained from HPLC were characterized by MALDI-TOF MS (Figure S1). S3

5 Figure S1: Reversed-phase HPLC analysis and MALDI-TOF mass spectra of the reaction between corresponding cysteine modifier and alkylamino-dna. a) DNA 2, without linker moiety; b) DNA 3, tris-ethylenglycol linker; c) DNA 4, hexa-ethylenglycol linker. MALDI-TOF MS (Matrix: 3-hydroxypicolinic acid): 2: calcd [M+H] +, found S4

6 3: calcd [M+H] +, found : calcd [M+H] +, found Optimization of ph for the ligation reaction of EYFP-thioeseter with cysteineoligonucleotide 2. The optimization of the ph value of the Tris-HCl buffer for the ligation reaction between enhanced yellow fluorescence protein (EYFP) and cysteinoligonucleotide 2 was performed as described for MBP (see Experimental Section). In brief, 500 pmol protein solution in ligation buffer (20 mm Tris-HCl, 150 mm NaCl, 4% MESNA, 3% Ethanthiol) with ph between were mixed with 250 pmol cysteine-modified DNA 2 and the ligation mixture was incubated for 48 h at 4 C. Subsequently, a 3 µl aliquot was diluted with hybridization buffer (20 mm Tris-HCl, 150 mm NaCl, 5 mm EDTA,0.05% Tween, ph 7.5) and analyzed using DDI-based screening assay. For the detection of immobilized conjugates mouse-anti-eyfp (100 nm, Sigma) and alkaline phosphatase labelled goat anti-mouse conjugate (Sigma) were applied, followed by incubation of fluorogenic substrate for alkaline phosphatase (Attophos, Roche). As expected, the efficiency of the ligation reaction between EYFP and cysteinemodified DNA 2 strongly depended on the ph of the ligation buffer (Figure S2). In contrast to the MBP, the best results were obtained at ph 7.5. Likely, this is due to the increased hydrolysis of the EYFP-thioester at higher ph values as observed in previous experiments. [4] Figure S2. Influence of the ph of the ligation buffer on the EPL of the EYFP-Thioester with cysteine-dna oligonucleotide 2. The absolute fluorescence signals obtained from the wells S5

7 containing either complementary 5 or non-complementary 6 capture oligonucleotides are represented by black and grey bars, respectively. Synthesis of Cy3-b-cyclodextrin 19. Mono-6-O-(p-tolylsulfonyl)-b-cyclodextrin (16). 16 was prepared by a modified method of Brady at al. (Scheme S2). [5] To this end 5.0 g (4.3 mmol) β-cyclodextrin 15 were dissolved in a solution of 2.5 g NaOH in 150 ml of water and the solution was cooled to 0 C. Subsequently, 5.0 g (27 mmol) p-toluenesulfonyl chloride were added and the reaction mixture was stirred vigorously for 2 h at 0 C. After addition of 3.0 g (15.7 mmol) of p-toluenesulfonyl chloride the reaction was carried out at 0 C for an additional 3 h. The reaction mixture was filtered through Celite and cooled at 0 C while 35 ml of 10% aqueous HCl were added. The resulting solution was stored overnight at 4 C. The product was filtered, dried to constant weight and recrystallized from water as a white powder (1. 37 g, 25%). 1 H NMR (500 MHz, [D 6 ]DMSO) δ = 7.70 (d, 2H), 7.38 (d, 2H), (m, 14H), 4.80 (m, 5H), 4.72 (d, 2H), 4.48 (t, 3H,), 4.41 (m, 2H), 4.33 (t, 1H), (m, 1H), 4.13 (m, 1H), (m, 40H), 2.38 ppm (s, 2H); 13 C NMR (125 MHz, [D 6 ]DMSO) δ = 147.1, 145.1, 132.7, 131.8, 130.2, 129.6, 127.9, 125.7, 124.2, 102.2, 81.8, 81.5, 81.0, 73.3, 73.0, 72.7, 72.5, 72.3, 72.1, 70.0, 69.2, 60.2, 59.5, 21.5 ppm. S6

8 Scheme S2. Synthesis of a Cy3-derivatized β-cyclodextrine 19. Mono-6-deoxy-6-azido-b-cyclodextrin (17). The synthesis of 17 was performed as previously reported. [6] In brief, 4.15 g (3.2 mmol) 16 were dissolved in 50 ml water at 80 C. Subsequently, 2.9 g (48 mmol) sodium azide were added and the reaction mixture was stirred overnight at 80 C. After being cooled to room temperature, the solution was poured into 270 ml aceton. The resulting precipitate was filtered and dried in vacuo to give the azide 17 as a white powder (3.57 g, 96%). 1 H NMR (500 MHz, [D 6 ]DMSO) δ = (m, 7H), 4.82 (m, 1H), 4.77 (d, 3H), (m, 1H,), 4.43 (s, 2H), (m, 40H), 2.04 ppm (s, 2H). Mono-6-deoxy-6-amino-b-cyclodextrin (18). [6] 2.0 g (1.7 mmol) azido-β-cyclodextrine 17 were dissolved in 35 ml DMF. After addition of 1.0 g (3.8 mmol) triphenylphosphine, 10 ml of concentrated ammonia were added and the reaction S7

9 mixture was stirred at room temperature for 4 h. Subsequently, 200 ml aceton were added, yielding a crude product as a white precipitate. This product was purified by cation-exchange column chromatography using 1 M ammonia solution as the solvent. The collected fractions were evaporated to give 1.9 g (99%) 18 as a white powder. 1 H NMR (500 MHz, DMSO-d 6 ) δ = (m, 8H), 4.42 (m, 4H), 4.43 (m, 3H), (m, 40H), 2.87 (s, 1H), 2.71 ppm (s, 1 H); MALDI-TOF MS (Matrix: 2.5- dihydroxybenzoic acid): calcd for C 42 H 71 O 34 N ([M+H] + ), found Mono-6-deoxy-6-Cy3-b-cyclodextrin (19). Amino-β-cyclodextrin 18 (16.3 mg, 13.1 mmol) was dissolved in 500 µl of anhydrous DMSO. 1 mg (1.31 mmol) Cy3-NHS ester in 1 ml anhydrous DMSO was added and the reaction mixture was incubated in the dark for 3 h at RT. Subsequently, 7 ml cold aceton were added and the solution was cooled to 20 C. The resulting precipitate was separated by centrifugation (5 min, 3500 rpm), washed with aceton and dried in vacuo. The crude product was purified by cation-exchange column chromatography using a 0.1 M ammonia solution as eluent. Product fractions were pooled and evaporated to dryness to yield 19. The product identity was confirmed by mass spectrometric analysis. MALDI-TOF MS (Matrix: 2.5-dihydroxybenzoic acid): calcd for C 73 H 108 O 41 N 3 S ([M+H] + ) and ([M+Na] + ), found and , respectively. Chromatographic assay for the determination of the biological activity of DNA- MBP conjugates. For the determination of biological activity of the MBP in the MBP- DNA conjugates the specific binding of the Cy3-derivatized cyclodextrin 19 was analysed. To this end DNA-MBP conjugate 2-MBP, generated by EPL of the directly cysteine-modified oligonucleotide 2 with the MBP-thioester was incubated for 15 min with a tenfold excess of 19 in Tris-buffer (20 mm Tris-HCl, 150 mm NaCl,pH 8.0). The analysis of the β-cyclodextrin conjugate mixture was carried out using an Agilent 1100 Series HPLC system and Poroshell 300SB-C18 column (Agilent). A gradient of 0-95% acetonitrile in water over 10 min at a flow rate of 1 ml/min was used. The absorbance of the DNA-MBP conjugate was detected at 280 nm, while the fluorescence of the free and bound β-cyclodextrin was detected using a 550 nm excitation and a 570 nm emmision filter (Figure S3). S8

10 Figure S3. Determination of biological activity of MBP in MBP-DNA conjugate by the specific binding of a Cy3-derivatized cyclodextrin 19. Shown is the absorbance at 280 nm (dashed) and fluorescence at 570 nm (black). The fluorescent signal for 2-MBP conjugate (retention time 3.8 min) clearly indicates the binding of the β-cyclodextrin and thus the biological activity of the protein component. Note that the height of the CD-Cy3 peak is due to the tenfold excess of CD-Cy3 used for the analysis. [1] M. Lovrinovic, C. M. Niemeyer, Biochem Biophys Res Commun 2005, 335, 943. [2] M. Lovrinovic, C. M. Niemeyer, Angew Chem Int Ed Engl 2005, 44, [3] S. Svedhem, C.-Ä. Hollander, J. Shi, P. Konradsson, B. Liedberg, S. C. T. Svensson, J. Org. Chem. 2001, 66, [4] M. Lovrinovic, Dissertation, University of Dortmund (Germany), [5] B. Brady, N. Lynam, T. O'Sullivan, C. Ahern, R. Darcy, Organic Syntheses 2000, 77, 220. [6] K. Hamasaki, H. Ikeda, A. Nakamura, A. Ueno, F. Toda, I. Suzuki, T. Osa, J. Am. Chem. Soc. 1993, 115, S9