1 

 

Chemical Communications Electronic Supplementary Information (ESI) Enabling [18F]-bicyclo[6.1.0]nonyne for oligonucleotide conjugation for positron emission tomography applications: [18F]-anti-microRNA-21 as an example Xiang-Guo Li,a,b Anne Roivainen,a,c Jörgen Bergman,a,b Arttu Heinonen,d Frank Bengel,e Thomas Thum,f Juhani Knuutia a Turku PET Centre, University of Turku and Turku University Hospital, FI-20521 Turku, Finland. E-mail: xiali@utu.fi b Accelerator Laboratory, Åbo Akademi University, Porthansgatan 3, FI 20500 Turku, Finland c Turku Center for Disease Modeling, University of Turku, FI-20014 Turku, Finland d Proteomics Facility, Turku Centre for Biotechnology, FI-20014 Turku, Finland. e Department of Nuclear Medicine, Hannover Medical School, Hannover, Germany f Institute of Molecular and Translational Therapeutic Strategies (IMTTS), Hannover Medical School, Hannover, Germany General remarks, material and methods All radiation work complies with the regulations of the Radiation and Nuclear Safety Authority of Finland (STUK). Compound 6 was prepared in our lab or custom synthesized by SynAffix, The Netherlands. Oligonucleotide 8 was custom synthesized by Sigma and the sequence of 8 was 5´-mUmCmAmAmCmAmUmCmAmGmUmCmUmGmAmUmAmAmGmCmUmA-3´ (m denotes backbone modification with 2´-O-methylation). Compound 11 was purchased from SynAffix, The Netherlands. Other chemicals (including compound 9) and reagents were purchased from Sigma-Aldrich-Fluka. Petroleum ether fraction 40–60 ºC (Sigma-Aldrich catalogue number 32299-2.5 L) was used for silica gel purification. Triethylammonium acetate (TEAA) buffer was prepared in our lab. Flash chromatography was performed using silica gel (60 Å, 230-400 mesh, enriched with 0.1% Ca, Merck). Nuclear magnetic resonance (NMR) spectra were recorded with a Bruker Avance 500 MHz spectrometer. High resolution mass spectrometry (HRMS) were performed by using QSTAR Elite quadrupole/time-of-flight mass spectrometer (Applied Biosystems/MDS Sciex, Canada) equipped with a nano-electrospray ionization source (Proxeon, Odense, Denmark). The samples were loaded into offline nanospray emitters (Thermo Scientific) and analyzed in a negative or positive electrospray ionisation mass spectrometry (ESI-MS) mode. An Analyst 2.0 software (Applied Biosystems/MDS Sciex, Canada) was used as a mass spectrometer control and data acquisition software. The mass spectra of oligonucleotides were deconvoluted by using both charge

Electronic Supplementary Material (ESI) for Chemical Communications.This journal is © The Royal Society of Chemistry 2015

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state and isotopic deconvolution in order to confirm an average mass of oligonucleotide samples. For small molecule samples, monoisotopic masses were measured. Preparation of compound 2-(2-(((((1R, 8S, 9s)-bicyclo[6.1.0]non-4-yn-9-yl)methoxy)carbonyl)amino)ethoxy)ethyl 4-methylbenzenesulfonate (6)

  To a solution of compound 11 (50 mg, 0.18 mmol) in dry pyridine (120 µL) was added 4-toluenesulfonic chloride (85 mg, 0.45 mmol) at 0 ºC. The reaction mixture was stirred for 40 min at 0 ºC. Subsequently, the solvent was evaporated off under vacuum and the residue was applied onto a silica gel column for purification. A mixture of ethyl acetate in petroleum ether (33% by volume) was used as the eluent. The tosylated compound 6 (21 mg, 0.05 mmol) was isolated as an oil in 27% yield. 1H-NMR (500 MHz, CDCl3): δ 7.74 (d, J = 8.3 Hz, 2H), 7.29 (d, J = 8.3 Hz, 2H), 4.93 (broad s, 1H), 4.08 (m, 4H), 3.57 (t, J = 4.5 Hz, 2H), 3.41 (t, J = 5.3 Hz, 2H), 3.24 (q, J = 5.2 Hz, 2H), 2.39 (s, 3H), 2.18 (m, 6H), 1.53 (m, 2H), 1.30 (m, 1H), 0.88 (m, 2H). 13C-NMR (126 MHz, CDCl3): δ 156.73, 144.95, 133.02, 129.87, 127.98, 98.84, 70.24, 69.07, 68.43, 62.83, 40.66, 29.07, 21.67, 21.44, 20.13, 17.79. HRMS (ESI) for [M + H]+, C22H30NO6S 436.1595 (Observed), 436.1794 (Calculated).  Preparation of compound ((1R, 8S, 9s)-bicyclo[6.1.0]non-4-yn-9-yl)methyl (2-(2-fluoroethoxy)ethyl)carbamate ([19F]BCN)

  To a solution of 11 (56 mg, 0.20 mmol) in dry CH2Cl2 (200 µL) at 0 ºC, Deoxofluor (66 mg, 0.30 mmol) in toluene (111 µL) was added. Then the reaction mixture was kept for 16 hours at r.t. Silica gel column purification was performed with a mixture of ethyl acetate in petroleum ether (40% by volume) as the eluent, affording [19F]BCN (6 mg, 0.02 mmol) in 10% yield. 1H-NMR (500 MHz, CDCl3): δ 5.08 (broad s, 1H), 4.60 (t, J = 4.0 Hz, 1H), 4.50 (t, J = 4.0 Hz, 1H), 4.13 (d, J = 8.0 Hz, 2H), 3.72 (t, J = 4.0 Hz, 1H), 3.66 (t, J = 4.0 Hz, 1H), 3.57 (t, J = 5.0 Hz, 2H), 3.37 (m, 2H), 2.20 (m, 5H), 1.56 (m, 2H), 1.33 (m, 1H), 1.20 (m, 1H), 0.93 (m, 2H). 13C-NMR (500 MHz, CDCl3): δ 156.76, 98.83, 83.64, 82.30, 70.32, 70.22, 70.06, 62.81, 40.76, 29.05, 21.42, 20.11, 17.75. HRMS (ESI) for [M + K]+, C15H22FKNO3 322.1278 (Observed), 322.1216 (Calculated). Preparation of compound 10

 

NH2-mUmCm

5'-end

N N

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A solutionprepared a0.6 M) at mixture wexclusion cwater (700and the pC251H330N8

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UV detection

Time (min)

10

15

n of compoun

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0

2

nd 10

15

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Supplimentary Figure 2. HRMS analysis of compound 10.

Preparation of [19F]-anti-miR-21

-TOF MS: 30 MCA scans from Sample 1 (140813_OL21_5) of 140813_OL21_5.wiffa=3.59785313826233120e-004, t0=-3.91464525288029110e+000 R; (Ion Spray)

Max. 9785.0 counts.

650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500m/z, Da

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acetonitrile (2.0 mL) for drying was from the elution of QMA cartridge, and it was not necessary to add additional acetonitrile for drying the fluoride.1 A solution of compound 6 (4.7 mg, 10.8 µmol) in acetonitrile (0.5 mL) was added to the dried K18F-K222 complex. The mixture was kept without stirring at 90 ºC for 15 min in a sealed reaction vessel. Subsequently, the reaction mixture was cooled down to 30 ºC and diluted with acetonitrile in water (40% by volume, 0.8 mL). The resulted mixture was injected into a semi-preparative HPLC column (Phenomenex Jupiter Proteo C18, 250 × 10 mm) for purification. [18F]BCN was eluted with acetonitrile in water (gradient 40-50% during 20 min, flow rate 4 mL/min) and collected into a Falcon tube which was pre-loaded with water (25 mL). The solution in the Falcon tube was passed through C18 cartridges (two 360 mg cartridges connected together). The C18 cartridges were washed with clinical grade ethanol (1.1 mL) and the eluent was discarded. [18F]BCN was eluted with clinical grade ethanol (1.0 mL) into a sterile vessel. The total preparation time of [18F]BCN was about 85 min and the decay-corrected radiochemical yield (RCY) starting from end of bombardment (EOB) was 20 ± 3% (n = 6). The radiochemical purity of [18F]BCN was higher than 98%. The obtained [18F]BCN was stable for 6 hours (longer time was not tested) and ready for next step conjugation reactions. Supplementary Figure 4. Quality control of [18F]BCN. HPLC conditions: Phenomenex Jupiter Proteo C18 column (250 × 4.6 mm), UV detection at 215 nm, flow rate 1 mL/min, solvent A was water and solvent B was CH3CN, gradient elution from 50% B to 80% of B during 15 min. (a) Radioactive detection of [18F]BCN. (b) The HPLC trace of a [18F]BCN sample under UV detection. (c) [19F]BCN as a reference. (d) Radioactive detection of a [18F]BCN sample spiked with [19F]BCN. (e) UV detection of a [18F]BCN sample spiked with [19F]BCN. (a).

‐10

0

10

20

30

40

50

60

0 5 10 15Time (min)Time (min)

Intensity (mV)

Radioactive detection of [18F]BCN

8 

 

(b).

(c).

(d).

‐0.02

0

0.02

0.04

0.06

0.08

0.1

0 5 10 15Time (min)

UV detection of [18F]BCN

Intensity (AU)

‐0.02

0

0.02

0.04

0.06

0.08

0.1

0.12

0 5 10 15

Time (min)

Intensity (AU)

UV detection of [19F]BCN as the reference

‐1

1

3

5

7

9

11

13

15

17

‐1 1 3 5 7 9 11 13 15Time (min)

Radioactive detection of [18F]BCN spiked with [19F]BCN

Intensity (mV)

9 

 

(e).

Supplementary Figure 5. Radio-TLC analysis of [18F]BCN. TLC conditions: normal phase silica gel TLC plates, eluent for TLC development was water in acetonitrile (2% by volume).

Supplementary Figure 6. HPLC analysis of reaction mixture in [18F]BCN synthesis. HPLC conditions: Phenomenex Jupiter Proteo C18 column (250 × 4.6 mm), UV detection at 215 nm, flow rate 1 mL/min, solvent A was water and solvent B was CH3CN, gradient elution from 50% B to 80% of B during 15 min.

‐0.02

0

0.02

0.04

0.06

0.08

0.1

0 5 10 15Time (min)

UV detection of [18F]BCN spiked with [19F]BCN

Intensity (AU)

Intensity (PSL) 

Length (mm)

10 

 

Radiosynthesis of [18F]-anti-miR-21

To above obtained [18F]BCN solution (1 mL, 1.2 – 1.4 GBq) was added a solution of compound 10 (10-23 nmol) in PBS (1.3 mL, pH 7.4) containing PPG (175 µL). After 3 min at r.t, the reaction mixture was diluted with PBS (7.5 mL, pH 7.4) containing PPG (7%). [18F]-anti-miR-21 was obtained in quantitative yield in this step. Starting from 18F-fluoride at EOB, [18F]-anti-miR-21was obtained in 90-95 min and the radiochemical purity was >95%. The specific radioactivity was 52–

‐0.2

0

0.2

0.4

0.6

0.8

1

‐1 1 3 5 7 9 11 13 15

side product 7

[18F]BCN

UV detection  of the reaction mixture in [18F]BCN synthesis

Time (min)

Intensity (AU)

11 

 

61 GBq/µmol (n = 6). Specific radioactivity was calculated by dividing the amount of radioactivity with the molar amount of 10 added to the reactions. The amount of 10 was measured with a Biospec-nano photometer. Supplementary Figure 7. HPLC monitoring of the conjugation reaction between [18F]BCN and compound 10, and quality control of [18F]-anti-miR-21. HPLC conditions: Phenomenex Jupiter Proteo C18 column (250 × 4.6 mm), UV detection at 260 nm and radioactive detection, flow rate 1 mL/min, solvent A was TEAA (0.1 M, pH 7.0) and solvent B was CH3CN, the gradient was 20-50% B during 0-10 min and 50-95% B during 10-20 min. (a) Radioactive detection of [18F]BCN. (b) [18F]BCN conjugation with compound 10 in the absence of PPG. (c) [18F]BCN conjugation with compound 10 in the presence of PPG (7 % by volume). (d) HPLC trace of [18F]-anti-miR-21 under UV detection. (e) [19F]-anti-miR-21 as the reference. (a).

(b). 

‐5

0

5

10

15

20

25

30

35

40

0 5 10 15 20

Time (min)

Radioactive detection of [18F]BCN

Intensity (mV)

‐2

0

2

4

6

8

10

12

0 5 10 15 20Time (min)

Intensity (mV)

Radioactive detection

[18F]‐anti‐miR‐21

side product

 

(c).

(d).

(e).

‐2

0

2

4

6

8

0

Intensity (mV)

‐0.3

‐0.1

0.1

0.3

0.5

0.7

0.9

Intensity (AU)

‐0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Intensity (mV)

Rad

3

1

1

3

5

7

9

0

0

UV dete

5

dioactive de

5

UV detec

5

ection of [19F

10

tection of [1

10

Time (min)

ction of [18F]

10

Time (mi

F]‐anti‐miR‐2

18F]‐anti‐miR

)

‐anti‐miR‐21

0

in)

21 as the ref

15

R‐21

15

1

15

ference

20

20

20

122 

13 

 

References:

1. X.-G. Li, K. Helariutta, A. Roivainen, S. Jalkanen, J. Knuuti and A. J. Airaksinen, Nat. Protoc., 2014, 9, 138-145.

2. X.-G. Li, A. Autio, H. Ahtinen, K. Helariutta, H. Liljenbäck, S. Jalkanen, A. Roivainen and A. J. Airaksinen, Chem. Commun., 2013, 49, 3682-3684.