Supporting Information

Different reactivity of Sp and Rp isomers of phosphorothioate-modified oligonucleotides in a duplex structure

Md Ariful Islam a,b, Aki Fujisaka a,c, Junji Kawakami d, Takao Yamaguchi a and Satoshi Obika a*

[a] Graduate School of Pharmaceutical Sciences, Osaka University,1-6 Yamadaoka, Suita,

Osaka 565-0871 (Japan). E-mail: obika@phs.osaka-u.ac.jp

[b] Department of Pharmacy, Noakhali Science and Technology University, Sonapur, Noakhali-3814 (Bangladesh)

[c] Faculty of Pharmacy, Osaka Ohtani University, Nishikiori-Kita 3-11-1, Tondabayashi, Osaka 584-8540 (Japan)

[d] Faculty of Frontiers of Innovative Research in Science and Technology, Konan University, 7-1-20 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047 (Japan)

Experimental Section

1. Materials All starting materials and reactants were purchased and used without further purification. The reacted PS-ONs were analyzed using the reverse-phase on the HPLC (RP-HPLC, Shimadzu Inc.) with Xbridge OST C18 column 2.5 μm (4.6 × 50 mm) (Waters Corporation). MALDI-TOF mass spectra for reacted or unreacted PS-ONs were recorded on a MALDI-TOF mass spectrometry (Autoflex II TOF/TOF mass spectrometer, Bruker Daltonics K.K.). Thermal denaturation experiments were carried out on Shimadzu UV-1650 and UV-1800 spectrophotometers equipped with a Tm analysis accessory. Stereo-isomerically pure Sp and Rp PS-ONs bearing a polypyrimidine tract were purchased from Gene Design Inc. 2-Bromoethylamine hydrobromide was purchased from TCI.

2. Optimization of reaction conditions Various reaction conditions tested in this study are summarized in Table S1 and Figure S1. During the optimization stage, 2-bromoethylamine hydrobromide was used as an alkylating agent, and cDNA1 was used as a complementary strand for both stereoisomers (Sp and Rp). Initially, a reaction was performed in both 20 mM HEPES buffer (pH 6.5) and 20 mM PB (pH 7.0) in the presence or absence of 100 mM NaCl. After several trials, it was found that reaction did not progress at all in the presence of NaCl. HEPES Buffer (pH 6.5) was clearly better than PB (pH 7.0) considering the rate of reaction. To determine the optimal temperature, the reactions were performed at different temperatures (20, 25,

and 30 °C). We concluded that 25 °C was the best temperature for our experiment. This type of reaction yields heavily depended on the ratio between the alkylating agents and PS-ON. After several trials, we found 30 equiv. alkylating reagent was not sufficient for the expected reaction rate, whereas 50 equiv. (slow reactivity but good enough for experimental purposes) and 100 equiv. of alkylating reagents were a sufficient amount to evaluate the reaction rates.

Table S1. Summary of different reaction condition

Buffer RX

(Equiv.) Temp (°C)

NaCl (mM)

Time (h)

Remarks on Reaction

PB 100 30 100 24 No Reaction HEPES 100 30 100 24 No Reaction PB 100 30 - 24 Moderate HEPES 100 30 - 24 Good HEPES 30 25 - 24 Very slow PB 100 25 - 24 Moderate HEPES 100 20 - 24 No Reaction HEPES 100 25 - 24 Moderate HEPES 100 25 - 2,6,12 No Reaction HEPES 50 25 - 24 Slow Alkylation reaction of Sp and Rp isomer in duplex structure with cDNA1 and 2-bromoethylamine in different conditions.

Figure S1. HPLC analysis of Sp and Rp isomers in the presence of cDNA1 in HEPES buffer (100 equiv. of 2-bromoethylamine, 24 h). The samples were manually injected.

3. Reaction of PS-ON in diastereomeric pure form Figure S2 shows the HPLC peak surface area of Sp and Rp isomer and their alkylated product in the presence or absence of the complementary strands (cDNA1/mmDNA) in PB buffer (100 equiv. of 2-bromoethylamine) at 36, 60, or 84 h

Figure S2. Reaction HPLC analysis of Sp and Rp isomer in the presence or absence of the complementary strands (cDNA1/mmDNA) in PB buffer (100 equiv. of 2-bromoethylamine, 36, 60, or 84 h). Crude mixtures were analyzed by means of autosampler HPLC using a Waters XbridgeTM C18 column 2.5 μm (4.6 x 50 mm). Eluent A: 0.1 M TEAA buffer, eluent B: MeCN, gradient: 6-9% of MeCN over 35 min at 60 °C with a flow rate of 1 mL/min.

Again, Figure S3 shows the HPLC peak surface area of Sp and Rp isomer and their alkylated product in the presence or absence of the complementary strands (cDNA1/cDNA2) in HEPES buffer (100 equiv. of 2-bromoethylamine) at 36, 60, or 84 h.

4. Reaction of PS-ON in diastereomeric mixture form Figure S4 shows the degradation curve of alkylation reaction in diastereomeric mixture (Rp:Sp=1:1) form in different buffer conditions (HEPES buffer or PB) using 50 equiv. of 2-bromoethylamine.

Figure S3. Reaction HPLC analysis of Sp and Rp isomer in the presence or absence of the complementary strands (cDNA1/mcDNA) in PB buffer (100 equiv. of 2-bromoethylamine, 36, 60, or 84 h). Crude mixtures were analyzed by means of autosampler HPLC using a Waters XbridgeTM C18 column 2.5 μm (4.6 x 75 mm). Eluent A: 0.1 M TEAA buffer, eluent B: A/MeCN(1/1, v/v), gradient: 6–9% of MeCN over 35 min at 60 °C with a flow rate of 1 mL/min.

5. Methods and characterization

5.1. Tm measurement data The UV melting profiles were recorded by dissolving the oligonucleotide at 20 μM in 20 mM HEPES buffer (pH 6.5) or PB (pH 7.0), scanning at 0.5 °C/min, and detecting at 260 nm. The temperature at which half of the duplex was dissociated was considered the Tm. Figure S5 showed the UV melting temperature of PS-ON with its complementary strand (cDNA1/cDNA2) in PB or HEPES buffer.

Figure S5. UV melting curves of the duplexes of PS-ON with complementary DNA in Phosphate buffer (A) and HEPES buffer (B). Here, Rp/Sp=ccctttstttcct, cDNA1=AGgaaaaaagGG, cDNA2=AGGaaaaaaGGG; mmDNA= agcaaaaaacgc; Lower case = DNA; Upper case = LNA; s = PS linkage; Conditions: 20 mM HEPES buffer (pH 6.5) or 20 mM phosphate buffer (pH 7.0), 20 μM each strand with or without R-Br (100 equiv. 2-bromoethym amine). R-Br was added to a duplex of Rp/Sp and cDNA1 at room temperature just before the UV melting temperature experiment starts.

Figure S4: Alkylation kinetics and stereoselectivity of Sp and Rp isomers in the duplex structures with cDNA1 in PB (A&B) and HEPES buffer (C&D) with 50 equiv. of 2-bromoethylamine.

5.2. HPLC analysis method Diluted reaction mixtures were subjected to reverse-phase HPLC (RP-HPLC, Shimadzu Inc., Kyoto, Japan). An auto sampler for HPLC analysis with an XbridgeTM OST C18 column 2.5 μm (4.6 x 50/75 mm) (Waters Corporation, Milford, US) using 0.1 M TEAA (triethylammonium acetate) buffer (pH 7.0) containing 50% MeCN (gradient: 6–9% MeCN over 35 min at 60 °C) was used. Composition and purity were characterized by HPLC retention time and MALDI-TOF mass spectrometry. Table S2. HPLC, mass spectrum and melting temperature (Tm) data

a Crude mixtures were analyzed by means of HPLC using a Waters XbridgeTM C18 column 2.5 μm (4.6 x 50 mm). Eluent A: 0.1 M TEAA buffer, eluent B: A/MeCN(1/1, v/v), gradient: 6–9% of MeCN over 35 min at 60 °C with a flow rate of 1 mL/min.; The samples were manually into HPLC system.; ND: Not determined, bUV melting profiles measured in 20 mM HEPES buffer (pH 6.5) or 20 mM phosphate buffer (pH 7.0) at a scan rate of 0.5 °C/min at 260 nm. The concentration of oligonucleotide used was 20 μM for each strand. R=-CH2CH2NH2.* Tm measured in presence of 2-bromoethylamine (2.0 mM).

Table S2 lists the HPLC retention time and MALDI-TOF mass spectrum data of Rp, Sp, and alkylated Rp and Sp isomers (Rp-R and Sp-R). The duplex forming ability of PS-ONs was estimated by measuring the melting temperature (Tm) of the duplexes between the Rp or Sp isomers and complementary DNA strands (cDNA1 or cDNA2) in 20 mM HEPES (pH 6.5) buffer or PB (pH 7.0). Here, it is evident that the Tm value was significantly lower in the HEPES buffer, yet moderate in PB. 5.3. CD measurements CD spectra were recorded using a JASCO J-720W spectrophotometer. The spectra were recorded at 10 °C in a quartz cuvette with a 1 cm optical path length. The samples were prepared in the same manner as described for the UV melting experiments. The molar ellipticity was calculated from the equation [θ] = θ/cl, where θ is the relative intensity, c is the sample concentration, and l is the cell path length in centimeters.

Figure S6. CD spectra of duplex between Rp/cDNA1 (blue) and Sp/cDNA1 (red). Conditions: 20 mM phosphate buffer (pH 7.2), 100 mM NaCl, 4 μM each strand. Sp/Rp: 5’-ccctttstttcct-3’ cDNA1: 5’-AGgaaaaaagGG-3’, Lower case = DNA; Upper case = LNA; s = PS linkage

ONs

HPLC RT

(min)

a

MALDI TOF Mass (M-H)-

Tm (°C)b

HEPES buffer PB

Calculated Observed cDNA1 cDNA2 cDNA1 cDNA2

Rp 14.6 3529.41 3528.68 21.0 (28.8*) 30.9 46.6 58.4 Sp 16.4 3529.41 3528.75 20.0 (29.1*) 30.4 47.2 58.0 Rp-R 17.6 3572.40 3573.80 ND ND ND ND Sp-R 17.7 3572.40 3572.98 ND ND ND ND

5.4. Reaction of PS-ONs in duplex form

Equimolar mixtures of PS-ON (20.0 μM, Sp or Rp) and its complementary ONs (20.0 μM, cDNA1, cDNA2, or mmDNA) were mixed with 20.0 mM of HEPES buffer (pH 6.5) or PB (pH 7.0) into a reaction tube. The uniformly mixed ONs were allowed to hybridize by heating to 90 °C for 10 min, followed by slow cooling to room temperature. 2-Bromoethylamine hydrobromide in water was added to the reaction mixture at room temperature (with a final concentration of 2.0 mM (100 equiv.) or 1.0 mM (50 equiv.)). The final reaction mixture was incubated at a suitable temperature. Collected reaction mixture fraction was diluted with water, and the reaction rate was determined by HPLC analysis.