Thiocoumarin Caged Nucleotides: Synthetic Access and Their Photophysical Properties

Photocages have been successfully applied in cellular signaling studies for the controlled release of metabolites with high spatio-temporal resolution. Commonly, coumarin photocages are activated by UV light and the quantum yields of uncaging are relatively low, which can limit their applications in vivo. Here, syntheses, the determination of the photophysical properties, and quantum chemical calculations of 7-diethylamino-4-hydroxymethyl-thiocoumarin (thio-DEACM) and caged adenine nucleotides are reported and compared to the widely used 7-diethylamino-4-hydroxymethyl-coumarin (DEACM) caging group. In this comparison, thio-DEACM stands out as a phosphate cage with improved photophysical properties, such as red-shifted absorption and significantly faster photolysis kinetics.


Abbreviations
Ar: Argon gas TEAA: Triethylammonium acetate TLC: Thin layer chromatography NaIO4 (7.6 g, 35.7 mmol, 3.0 eq.) was added to a solution of enamine 2 (3.4 g, 11.9 mmol, 1.0 eq.) in THF/H2O (80 ml 1:1) and the resulting mixture was stirred for 1 h at room temperature. The formed precipitate was filtered off and washed with EtOAc (3 x 20 ml). The filtrate was concentrated under reduced pressure (to evaporate the THF and EtOAc) and saturated aqueous NaHCO3 solution was added. It was transferred into a separatory funnel, DCM (3 x 50ml) was used for extraction and the combined organic layers were dried over Na2SO4. The solvent was removed under reduced pressure and the residue was purified by silica gel chromatography (Cyclohexane/EtOAc, 10:1 to 5:1). Target compound 3 (2.6 g, 10.6 mmol, 89%) was obtained as a red solid. Analytical data are consistent with those reported in the literature.  Scheme S2. Synthesis of thio-DEACM 7.
The solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography (Cyclohexane/EtOAc, 10:1 to 6:1). Compound 6 (1.99 g, 6.5 mmol, 89%) was obtained as a yellow powder. Analytical data are consistent with those reported in the literature. Rf

((iPr2N)2P(OFm)) (8)
The synthesis of (iPr2N) (FmO)P-OThioDEACM (8) (9) was adapted from a previously reported procedure 9-Fluorenylmethanol (6.03 g, 30.6 mmol, 1.02 eq.) was dried for 1 h under high vacuum. Afterwards, it was dissolved in dry Et2O/THF 5:1 (v/v; 60 ml) under argon atmosphere, dry Et3N (4.2 ml, 30.6 mmol, 1 eq.) was added and the mixture was cooled to 0 ºC. After the addition of bis(diisopropylamino)-chlorophosphine (8.16 g, 30.6 mmol, 1.00 eq.), it was stirred at 0 ºC for 1.5 h and the formed precipitate was quickly filtered off over neutral Al2O3. The filtrate was concentrated under reduced pressure and immediately purified by recrystallization from pentane 20 ml. (The product containing pentane was heated till all product dissolved in it, then filtered it through filter paper, the filtrate containing flask was kept in -20 ºC freezer for 5h, the crystal formed inside.) After filtration, the crystal was dried under reduced pressure (0.3 mbar) for 5h.    was mixed with tetrabutylammonium hydroxide 30-hydrate (3.52 g, 4.4 mmol, 2.2 eq.). After lyophilization, the correct amount of TBA counter ion was determined by 1 H-NMR analysis, using PMe4Br as internal standard. A stock solution of AMP • 2 TBA was prepared as a 0.1 M solution in dry DMF. Therefore, the obtained solid after lyophilization was co-evaporated 3-times with MeCN (5 ml), dissolved in dry DMF (20 ml) and stored over activated molecular sieve (3 Å) under argon atmosphere.

Thio-DEACM-ATP
The synthesis of Thio-DEACM-ATP was adapted from a previously reported procedure by Alexandre Hofer [3]. With the developed method, ADP was coupled to thio-DEACM Fm phosphoramidite. Within 7 minutes, ADP was fully converted to a mixed P(III)-P(V) intermediate. Then mCPBA was used to oxidize P(III) to P(V), leading also to minor amounts of DEACM caged ATP, as mCPBA is able to convert the thio-carbonyl group into an oxo-carbonyl group.
Compound 9 (648 mg, 1.1 mmol, 1.1 eq.) was added to a solution of ADP • 2 TBA (5 ml, 1 mmol, 1.0 eq.) in dry DMF. ETT (390 mg, 3 mmol, 3.0 eq.) was added to the reaction mixture, after it was co-evaporated with dry CH3CN (3 x 1 ml). The mixture was stirred for 7 minutes at r.t. Afterwards, the solution was cooled to −10 °C, using a NaCl/ice mixture, and mCPBA (170 mg, 1 mmol, 1.0 eq.) was added. Afterwards, piperidine (500 μl, v/v=5%) was added and the mixture was stirred for 30 minutes. The product was then precipitated by adding the reaction mixture dropwise to a NaClO4 (0.5 M) acetone solution (30 ml). The precipitate was isolated via centrifugation (7000 rpm, 5 min), the solid was washed with acetone (3 x 5 ml) and then dried under high vacuum. The crude product was purified by strong ion-exchange chromatography using an Äktasystem (NH4HCO3buffer, VIS detection at 700 nm). The product containing fractions, were combined and lyophilized. The obtained solid was further purified by RP-C18-chromatography using

Thio-DEACM-AP4
The procedure is identical to the synthesis of thio-DEACM-ATP.

Absorption and Fluorescence Spectra
Absorption and fluorescence spectra of compounds were measured by Spark ® multimode microplate reader.
Fluorescence: black plates, concentration depends on fluorescence quantum yield for different samples.

Molar absorption coefficient
The absorption was measured on Thermo Scientific™ Genesys 10s UV-VIS Spectrophotometer. And then molar absorption coefficient was calculated according to Lambert-Beer law: = A * .  Figure S7. Fluorescence quantum yield of photocages and photocaged compounds.