Exceptional Photochemical Stability of the Co–C Bond of Alkynyl Cobalamins, Potential Antivitamins B12 and Core Elements of B12-Based Biological Vectors

Alkynylcorrinoids are a class of organometallic B12 derivatives, recently rediscovered for use as antivitamins B12 and as core components of B12-based biological vectors. They feature exceptional photochemical and thermal stability of their characteristic extra-short Co–C bond. We describe here the synthesis and structure of 3-hydroxypropynylcobalamin (HOPryCbl) and photochemical experiments with HOPryCbl, as well as of the related alkynylcobalamins: phenylethynylcobalamin and difluoro-phenylethynylcobalamin. Ultrafast spectroscopic studies of the excited state dynamics and mechanism for ground state recovery demonstrate that the Co–C bond of alkynylcobalamins is stable, with the Co–N bond and ring deformations mediating internal conversion and ground state recovery within 100 ps. These studies provide insights required for the rational design of photostable or photolabile B12-based cellular vectors.


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In a 5 ml round bottom flask, 100.0 mg of H2OCbl*Cl (72.4 µmol) were dissolved in 2.5 ml of MeOH and the mixture was degassed with argon for 10 min. To this solution 50 µl formic acid (1.3 mmol) and 187 µl triethyl amine (1.3 mmol) were added under Ar and the solution was stirred for 30 min at RT. To the now brown solution 61.8 µl 3-iodoprop-2-yn-1-ol (300.0 µmol) were added under argon and the mixture was stirred for 24 hours. The reaction mixture was poured in 15 ml of ethyl acetate to precipitate the raw product. The mother liquor was removed and the red precipitate was dried under high vacuum for 2 hours. The raw product was purified by RP-18 column chromatography using MeOH/phosphate buffer pH 8 (10 mM), solvent system (5-50% MeOH, in 10% steps). H2OCbl*Cl eluted first (at 30 % MeOH) followed by the red fraction of the product HOPryCbl (at 40 % MeOH). The product fraction was collected and the solvents were evaporated on rotary evaporator at room temperature. Raw red corrin HOPryCbl was dissolved in water (0.5 ml) and crystallized after addition of 5 ml of acetone. The crystals of HOPryCbl were first washed with (1:9) mixture of water: acetone, then with acetone. The sample of HOPryCbl was dried under high vacuum overnight, to give 60.6 mg (43.7 µmol, 60.4% yield) of red crystalline solid, which was characterized as follows:

Crystal structure of Coβ-3-hydroxypropynyl-cobalamin
Crystals of Coβ-3-hydroxypropynyl-cobalamin (HOPryCbl) were grown from H2O/acetone. Diffraction experiments were carried out with a Nonius Kappa CCD diffractometer at a temperature of 233 K. Diffraction data extended to a resolution of 0.91 Å. The asymmetric unit of the orthorhombic crystal contained B12 molecule and well-ordered water molecules.
Indexing of diffraction images, intensity integration, and data scaling were performed with programs DENZO and SCALEPACK. 2 The crystal was orthorhombic (space group P212121) with unit cell constants a=16.2120(10) Ǻ, b=21.1070(10) Ǻ, and c=24.6140(10) Ǻ. The structure was solved by direct methods and refined against F 2 -values using the program SHELXL. 3 Full matrix least-squares anisotropic refinement converged at R1=0.0718 for all data. No absorption correction was applied to the data. The solvent region was modeled using 17 water molecules with anisotropic atomic displacement parameters (adp). H-Atom positions were calculated and refined as 'riding' on their respective non-H-atom. For methyl-and hydroxyl-groups the torsion angle around the C-C or C-O bond was also refined (omitted for water molecules). The isotropic adp for each H-atom was set to 1.5 times (for methyl-and hydroxyl-groups) and 1.2 times (for all other hydrogen atoms) the equivalent isotropic atomic displacement parameters of the adjacent non-H-atom. Data pertaining to diffraction data collection and structure refinement are summarized in Table S2.   S10 Figure S6. Atom numbering used for 3-hydroxypropynylcobalamin

Hydrolysis of Coβ-3-hydroxypropynylcobalamin (HOPryCbl) in acidic aqueous solutions:
3 mg (2.17 µmol) of HOPryCbl were dissolved in 1 ml H2O dist. and 50 µl of the resulting solution were diluted with 2.5 ml 100 mM HCl pH 1. The sample was stored under air at RT in the dark. UV/Vis spectra were recorded at regular time intervals in the course of 200 min. After 200 min the UV/Vis spectrum was similar to that of aquocobalamin (see Figure S7).

Photolytic stability of Coβ-3-hydroxypropynylcobalamin:
In a UV/Vis cell a solution of Coβ-3-hydroxypropynylcobalamin in H2O (ca. 3.5 × 10 -5 M) was exposed to daylight. UV/Vis spectra were recorded at regular intervals for 48 h. Figure S9. UV/Vis spectra of an aerated aqueous solution of Coβ-3-hydroxypropynylcobalamin before (black) and after (red) exposure to bright daylight at room temperature

Thermal stability of Coβ-3-hydroxypropynylcobalamin (HOPryCbl):
In polypropylene vials 10µl aliquots of a solution of 1mM HOPryCbl in DMSO were heated to 100°C. Samples, taken at five time points, were diluted with 190µl aq. 10mM K-phosphate buffer pH 7 and subjected to HPLC analysis. Significant decomposition of HOPryCbl could be detected, but not significant amounts of aquocobalamin.      Figure S17. Temperature dependence of the long-lived excited state of HOPryCbl in ethanol. Note that there is an increased absorption between 420 nm and 450 nm at higher temperatures, but the differences are not as pronounced as in water. Pump-scatter prevented characterization of the excited state absorption at shorter wavelengths.