Stable Europium(III) Complexes with Short Linkers for Site‐Specific Labeling of Biomolecules

Abstract In this study, two new terpyridine‐based EuIII complexes were synthesized, the structures of which were optimized for luminescence resonance energy‐transfer (LRET) experiments. The complexes showed high quantum yields (32 %); a single long lifetime (1.25 ms), which was not influenced by coupling to protein; very high stability in the presence of chelators such as ethylenediamine‐N,N,N′,N′‐tetraacetate and ethylene glycol‐bis(2‐aminoethylether)‐N,N,N′,N′‐tetraacetic acid; and no interaction with cofactors such as adenosine triphosphate and guanosine triphosphate. A special feature is the short length of the linker between the EuIII ion and the maleimide or hydrazide function, which allows for site‐specific coupling of cysteine mutants or unnatural keto amino acids. As a consequence, the new complexes appear particularly suited for accurate distance measurements in biomolecules by LRET.


Supporting information
Estimation of the labeling efficiency of BSA: The coupling reaction of 14-Eu with BSA results in almost complete labeling of all available cysteine residues. It is known that on average every second BSA molecule possesses a reactive sulfhydryl-group. [1] The extinction coefficient of free 14-Eu at 335 nm was determined to be 11900 ᴍ -1 cm -1 . The formal extinction coefficient of labeled BSA at 280 nm is composed of the extinction coefficient of two BSA molecules (2  44300 ᴍ -1 cm -1 ) [2] and the absorbance of one 14-Eu at 280 nm (8200 ᴍ -1 cm -1 ), which sums up to 96800 ᴍ -1 cm -1 . This theoretical extinction coefficient is valid for 100% labeling of the available free 0.5 thiol groups per BSA molecule; it is shown in Table S1. In this maximally labeled complex the ratio of (335 nm)/(280 nm) has the value 0.123. In the actual sample of 14-Eu-labeled BSA, the ratio of (335 nm)/(280 nm) was 0.113, which means that approx. 91% of the available thiol groups of BSA were labeled with 14-Eu.

Determination of the luminescence quantum yield:
The quantum yields were calculated with the following formula: [3] D is the quantum yield of the sample dye, s is the quantum yield of the reference standard, ED/AD is the slope of emission intensity area vs. the absorption of the sample dye at 335 nm and Es/As is the corresponding slope of the reference standard, nD = 1.334 is the refractive index of HBS 7.3 buffer used to dissolve the sample dye and ns = 1.3638 is the refractive index of ethanol used to dissolve the reference standard. Table S2: Determination of the luminescence quantum yield by relative method with rhodamine 6G as standard. [3,4] Compound 2.24 · 10 9 28.8±2.3 0.024 6.55 · 10 7 0.015 4.44 · 10 7 0.0095 3.43 · 10 7 0.0069 2.62 · 10 7 0.0033 1.67 · 10 7 Figure S1: Measurements of luminescence quantum yield by relative method, using rhodamine 6G as standard. [3,4] Determination of the number of water molecules in the inner coordinationsphere: Figure S2: Lifetime measurement of 14-Eu-labeled BSA in HBS 7.3 buffer (dark blue solid line) and in deuterated HBS 7.3 buffer (red solid line). Essentially the same lifetimes were observed for 14-Eu in water (blue dashed line) and in deuterated water (pink dashed line).

Eu-complex stability in presence of competitors:
In Figure S3 the luminescence intensity of 17-Eu (3 µᴍ) in HBS 7.3 was monitored in the presence of competitors for more than 24 h. The luminescence intensity was not influenced upon addition of 2 mᴍ EDTA, 2 mᴍ EGTA, 2 mᴍ EGTA-Ca-buffer, 10 µᴍ zinc(II) or 200 µᴍ zinc(II). This compares well with the observations that neither EDTA nor EGTA can extract the Eu-ion from the chelate, and that zinc(II) or calcium(II) are not able to cause transmetalation. Control experiments were performed with 3 µᴍ EuCl3 in the identical HBS 7.3 buffer and 2 mᴍ EDTA or 2 mᴍ EGTA, in order to ensure that neither Eu-EDTA, Eu-EGTA nor any other buffer contents could cause luminescence, which would lead to misinterpretation of the data. Figure S3: The luminescence intensity of 17-Eu (3 µᴍ) and 17-Eu in presence of 2 mᴍ EDTA, 2 mᴍ EGTA, 2 mᴍ Ca-EGTA buffer, 10 µᴍ Zn II and 200 µᴍ Zn II was monitored for more than 24 h. As control experiment the luminescence intensity of 3 µᴍ europium(III) chloride in HBS 7.3 buffer with 2 mᴍ EDTA or 2 mᴍ EGTA was monitored, in order to control if luminescence from buffer components can be observed.

Influence of nucleotides on the luminescence properties of 14-Eu labeled BSA:
The effect of nucleotide present in the solution was strongly dependent on the wavelength used to excite the Eu-complex. For measurements in a fluorimeter cuvette, the samples were excited at 335 nm (slit 10 nm). Hereby the nucleotides (or a common impurity of the nucleotides) caused a significant 335 nm absorption at a nucleotide concentration of approx. 25 mᴍ (see Figure S6). When monitoring the luminescence intensity of 14-Eu labeled BSA incubated with nucleotide solutions over a period of 10 min, the intensity was not changed (see Figure S4). Figure S4B displays the luminescence intensity corrected for the dilution which had been caused by the addition of the nucleotide stock solution. In this case a prominent filter-effect was observed for GTP only. This experiment allows the conclusion, that the nucleotides do not perturb the emission of the Eu-complex when corrected for the "filter effect" ( Figure 5B and Figure S6). Figure S4: Stability of 14-Eu labeled BSA in the presence of different nucleotides or adenosine. The luminescence intensity was monitored at 1 min intervals. A) The emission intensities as measured is shown. B) The data from panel A were corrected for the dilution caused by the addition of the 100 mᴍ stock solution (the stock solution of adenosine was 10 mᴍ). The reduced emission intensity of 14-Eu in presence of 19 mᴍ GTP in panel B is the consequence of much higher absorbance of GTP or a common impurity of GTP at 335 nm, as shown in Figure S6.   Synthesis of 2,2':6',2''-terpyridine-6,6''-dicarbonitrile (2): 1 (15.6 mg, 58.8 µmol) was dissolved in DCM (706 µL) and trimethylsilyl cyanide (77 µL, 588 µmol, 10 equiv.) was added. The solution was stirred for 20 min at room temperature and subsequently PhCOCl (28 µL, 235 µmol, 4 equiv.) was added dropwise during a time period of 20 min. The mixture was stirred overnight at room temperature. The reaction mixture was concentrated to half of the volume, 10% K2CO3 (1.4 mL) was added and the suspension was stirred for 1 h at r.t. The resulting precipitate was filtered and washed with water and cold DCM. The remaining solid was dried in vacuum to obtain 2 (11.7 mg, 41.3 µmol, 70%). 1  Synthesis of dimethyl 2,2':6',2''-terpyridine-6,6''-dicarboxylate (3): 2 (145 mg, 512 µmol) was dissolved in water (2.3 mL), acetic acid (2.3 mL), and conc. sulfuric acid (512 µL). The mixture was heated to 90 -100°C and stirred for 24 h. Afterwards the solution was poured into ice-water (20.5 mL) and the precipitate was filtered off.

Material and methods
The solid was washed with cold water and acetonitrile. The carboxylic acid was dried 10 -100 Pa. MeOH (16 mL) was cooled in an ice bath and thionyl chloride (390 µL, 5.3 mmol, 10 equiv.) were added dropwise. The solution was stirred for 15 min at room temperature and was then added to the solid carboxylic acid (170 mg, 529 µmol). The mixture was heated to reflux for 18 h and subsequently the solvent was removed under reduced pressure. The solid was dissolved in CHCl3 (27 mL) and washed three times with 5% NaHCO3 (13 mL). The organic phase was dried with Na2SO4 and the solvent was removed in vacuum. The crude product was recrystallized from toluene (28 mL) to obtain 3 (126 mg, 445 µmol, 84%). 1  Synthesis of 6,6''-dihydroxymethyl-2,2':6',2''-terpyridine (4): 3 (120 mg, 344 µmol) was dissolved in dry EtOH (10 mL) and sodium borohydride (77 mg, 1.96 mmol, 5.7 equiv.) was added under argon atmosphere. The mixture was stirred for 3 h at r.t. and subsequently heated to reflux for 1 h. The organic solvent was removed under reduced pressure, sat. NaHCO3 (4.9 mL) was added and the mixture was shortly heated to boiling. After cooling to r.t. the precipitate was filtered off and washed with water to obtain 4 (87 mg, 297 µmol, 86%). 1

Synthesis of tert-butyl 2-(benzyl(2-hydroxyethyl)amino)acetate (8):
N-Benzylethanolamine (2.50 mL, 16.5 mmol) was placed in a round bottom flask, DIPEA (2.13 mL, 16.5 mmol) and dry DMF (20 mL) were added. The solution was stirred at 0°C and tert-butyl bromoacetate (2.44 mL, 16.5 mmol) was added dropwise. The mixture was allowed to warm up to room temperature and was stirred over night at r.t. The solvent was removed under reduced pressure and the residue was dissolved in DCM (250 mL). The organic solution was washed four times with water (100 mL), dried with Na2SO4 and concentrated in vacuum to obtain 8 (4.40 g, 16.5 mmol, 100%). 1 2-(benzyl(2-bromoethyl)amino)acetate (9): 8 (750 mg, 2.83 mmol) and triphenylphosphine (871 mg, 3.32 mmol, 1.18 equiv.) were combined and dried three times by addition and evaporation of dry toluene. To remove residual toluene, dry DCM was added and evaporated three times. The mixture was immediately dissolved in dry DCM (9.5 mL) under argon atmosphere. N-Bromosuccinimide (587 mg, 3.30 mmol, 1.17 equiv., dried in vacuum at 10 -100 Pa) was added in portions over a period of 1 h at 0°C. The mixture was stirred for another 0.5 h at 0°C and subsequently the solution was allowed to warm up to r.t. Reaction controls were performed by TLC (silica gel; CHCl3:MeOH:acetic acid = 9:1:0.1, Rf=0.87). After 4 h of stirring at r.t. the solvent was removed under reduced pressure. The crude product was purified by silica gel chromatography (gradient elution starting with CHCl3:n-heptane = 2:3 and ending with CHCl3:MeOH = 9:1) to obtain 9 (418 mg, 1.27 mmol, 45%). 1 Figure S8: High resolution mass-spectrum of compound 1.
[M+H] + [M+Na] + Figure S45: 1 H-NMR of compound 13. Figure S46: 13 C-NMR of compound 13.        [a] The aromatic signals originates from the terpyridine unit, but it cannot be distinguished between the different pyridine rings. With a moderately COSY respectively TOCSY it was possible to assign each three protons which are belonging to the same pyridine ring.
[b] The numbered signals are CH2-groupes which could not be distinguished from each other (They were continuously numbered without further relation to the chemical structure). Due to the paramagnetism of the europium, each proton of one CH2group has a different shift and the signals with the same number (distinguished by an apostrophe) belong to the same CH2-group.
[c] With a COSY and TOCSY experiment the signals α, β and γ could be assigned.
[d] Most of these signals are quaternary carbons, but some of them are CH2-groups which are exposed to an outstanding high paramagnetism and therefore the relaxation is too fast to detect any C-H or H-H correlations (e.g. in HSQC or COSY).