DOI: 10.1002/chem.201302037
4-Trifluoromethyl-Substituted Coumarins with Large Stokes Shifts: Synthesis, Bioconjugates, and Their Use in Super-Resolution Fluorescence Microscopy Heiko Schill,[a] Shamil Nizamov,[a] Francesca Bottanelli,[b] Jakob Bierwagen,[a] Vladimir N. Belov,*[a] and Stefan W. Hell*[a]
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FULL PAPER Abstract: Bright and photostable fluorescent dyes with large Stokes shifts are widely used as sensors, molecular probes, and light-emitting markers in chemistry, life sciences, and optical microscopy. In this study, new 7-dialkylamino-4-trifluoromethylcoumarins have been designed for use in bioconjugation reactions and optical microscopy. Their synthesis was based on the Stille reaction of 3-chloro-4-trifluoromethylcoumarins and available (hetACHTUNGREero)aryl- or (hetero)arylethenyltin derivatives. Alternatively, the acylation of 2-trifluoroacetyl-5-dialkylaminophenols with available (hetACHTUNGREero)aryl- or (hetACHTUNGREero)arylethenylacetic acids followed by intramolecular condensation afforded coumarins with 3-(hetero)aryl or 3-[2-
(hetero)aryl]ethenyl groups. Hydrophilic properties were provided by the introduction of a sulfonic acid residue or by phosphorylation of a primary hydroxy group attached at C-4 of the 2,2,4-trimethyl-1,2-dihydroquinoline fragment fused to the coumarin fluorophore. For use in immunolabeling procedures, the dyes were decorated with an (activated) carboxy group. The positions of the absorption and emission maxima vary in the ranges 413–480 and 527–668 nm, respectively. The phosphorylated dye, 9,CH=CH-2-py,H, with Keywords: bioconjugation · chromophores · fluorescence · imaging agents · light microscopy
Introduction Fluorescence microscopy is one of the most important techniques in physical, chemical, and biological studies of (living) cells, tissues, or even whole organisms.[1] Indeed, the cells, or the organelles inside them, can be specifically labeled in a versatile manner with diverse fluorescent markers.[2] Various classes of dyes have been used for staining and bioimaging, and coumarins are quite prominent among them. Coumarins have a long history as laser dyes,[3] and nowadays they are widely used as fluorescent chemosensors and labels for proteins, nucleic acids, lipids, carbohydrates, toxins, hormones, and other biomolecules.[4, 5] The parent compound at room temperature shows no fluorescence and only a weak absorption at 312 nm with e = 5370 dm3 mol1 cm1.[3o] 7-Hydroxy-[4] and 7-aminocoumarins,[5] however, emit light in the blue-green region of the visible spectrum due to the batho- and hyperchromic shifts induced by the push–pull system of the electron-donor group at C-7 and the electron-withdrawing lactone moiety. Electron donors at C-6 and/or electron-acceptors at C-3
[a] Dr. H. Schill, Dr. S. Nizamov, Dipl.-Chem. J. Bierwagen, Dr. V. N. Belov, Prof. S. W. Hell Department of NanoBiophotonics Max Planck Institute for Biophysical Chemistry Am Fassberg 11, 37077 Gçttingen (Germany) Fax: (+ 49) 551-2012505 E-mail: [email protected] [email protected] [b] Dr. F. Bottanelli Department of Cell Biology, Yale School of Medicine 333 Cedar Street, New Haven, Connecticut 06510 (USA) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201302037.
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the 1-(3-carboxypropyl)-4-hydroxymethyl-2,2-dimethyl-1,2-dihydroquinoline fragment fused to the coumarin fluorophore bearing the 3-[2-(2-pyriACHTUNGREdyl)ACHTUNGREethenyl] residue (absorption and emission maxima at 472 and 623 nm, respectively) was used in super-resolution light microscopy with stimulated emission depletion and provided an optical resolution better than 70 nm with a low background signal. As a result of their large Stokes shifts, good fluorescence quantum yields, and adequate photostabilities, phosphorylated coumarins enable two-color imaging (using several excitation sources and a single depletion laser) to be combined with subdiffractional optical resolution.
and/or C-4 shift the positions of the absorption and emission bands further into the red region. Extension of the p system attached to the 3-position by introduction of an unsaturated substituent may yield hybrid dyes and provide an additional bathofluoric shift of the fluorescence band.[6] Combinatorial studies on coumarin “libraries” have exemplified their structure–spectra relationships.[2e, 4a, 7] In general, these dyes are known to be brightly fluorescent, possess relatively large absorption coefficients, high fluorescence quantum yields, low degree of triplet state formation, and large Stokes shifts (separation between the absorption and emission maxima measured in nm or cm1). Apart from large Stokes shifts, the common features of coumarin dyes are moderate photostability (compared with xanthene dyes) and moderate fluorescence quantum yields in polar solvents. These drawbacks limit their use as fluorescent markers in light microscopy and, in particular, in stimulated emission depletion (STED), one of the most powerful diffraction-unlimited imaging techniques.[8] For more than a century, the optical resolution that could be achieved with a light microscope seemed limited by Abbes law, which states that the resolution cannot be better than (approximately) half the wavelength of the applied light.[9] For microscopy in the visible range (400–800 nm), this law limits the resolution to less than 200 nm. However, in many cases, a better optical resolution is desirable or even necessary. Although electron microscopy provides higher resolution, it is incompatible with live specimens and requires special preparation and fixation techniques, which can be tedious and even destructive towards the features of interest. As a result of recent innovations, several new super-resolution methods have been implemented in light microscopy.[8] They break Abbes law, leading to various diffraction-unlimited imaging techniques. These new proce-
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dures often require novel fluorescent dyes with superior photostability, greater brightness, and the ability to switch between bright and dark states. In the case of multicolor experiments, the spectral separation of different labels into two or more excitation or acquisition channels is essential. For example, a pair of dyes emitting approximately at the same wavelength with well-separated absorption bands may be used together provided that the excitation with two light sources does not result in cross-talk. In this respect, dyes with large Stokes shifts are beneficial as they allow the use of different fluorescence channels and only one excitation source (or vice versa). Under the more severe conditions of STED, two lasers are required for each fluorescent dye: One for the excitation and the other for depletion.[8c] In this case, dyes with large Stokes shifts allow the same wavelength to be used for depletion while maintaining two different excitation windows in which to distinguish the labels.[10] Unfortunately, photostable and brightly fluorescent dyes with large Stokes shifts are rare and only a few of them are commercially available.[11] Most of them contain a coumarin fragment as the fluorophore. For example, “Mega Stokes” dyes from Dyomics are coumarins that absorb at about 500– 520 nm and emit in the region of 590–670 nm.[11a,b] Another practically useful coumarin dye is AlexaFluorTM 430 with an absorption maximum at 434 nm and emission band at 539 nm (Life Technologies).[12] However, the chemical structures of many commercially available fluorescent dyes are unknown.[11c] For example, the structures of Pacific Orange (absorption 390 nm, emission 540 nm; Life Technologies), V500 (absorption 415 nm, emission 500 nm; BD Horizon), and Chromeo 494 (absorption 494 nm, emission 628 nm; Active Motif) have not been published. Surprisingly, among the disclosed coumarin fluorophores, there are only a few examples of dyes with perfluoroalkyl substituents even though it is known that a CF3 group, especially at the 4-position of the coumarin system, increases photostability.[13] Moreover, the introduction of a strong electronacceptor, for example, a perfluoroalkyl group, at C-4 should provide a pronounced redshift of the absorption and fluorescence bands and, because the bathofluoric shift is expected to be larger, increase the Stokes shifts. In view of the apparent lack of fluorescent dyes with large Stokes shifts suitable for STED microscopy,[10] we have designed and screened a new set of coumarins with very large Stokes shifts (> 100 nm), prepared their conjugates with proteins, and evaluated their performance in two-color STED super-resolution imaging (by using a red-emitting rhodamine dye with a small Stokes shift as a benchmark).
Results and Discussion Synthesis: A trifluoromethyl group at the 4-position of coumarin reduces the bleaching coefficient of a dye by about 20 %[13] and increases the Stokes shift (compared with the 4unsubstituted derivative).[3f] An unsaturated substituent at the 3-position is also known to increase the Stokes shift of
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a coumarin.[4a] Therefore we started the synthesis of variously substituted 7-amino-4-trifluoromethylcoumarins with unsaturated substituents at the 3-position by using a modular convergent approach to create a range of different dyes by a short synthetic route. In addition, an electron-acceptor group was introduced at the 3-position (directly or through a CH=CH connector) to provide an additional redshift of the absorption and emission bands so that the emission process can be efficiently stimulated with a “red” laser at 750– 770 nm. Because the halogenation of 7-aminocoumarins occurs selectively at C-3, we first attempted this reaction under various conditions with the commercially available Coumarin 152 (1; Figure 1). If these reactions were successful, 3-
Figure 1. Model compounds for the convergent synthesis of coumarin dyes.
haloACHTUNGREcoumarins 2-X could then be used in transition-metalcatalyzed cross-coupling transformations.[14] However, the presence of the 4-trifluoromethyl group seemed to prevent clean halogenation reactions. Halogenation was possible only under drastic conditions (e.g., by heating with N-bromosuccinimide in DMF) and mixtures of halogenated and/ or N-dealkylated compounds were always obtained. Similarly, attempted formylation reactions under Vilsmeier–Haack conditions[6, 15] failed to afford aldehyde 3 (Figure 1), which could be used in a Knoevenagel-type condensation reaction. Oxidation of the methyl group in the (unknown) 3-CH3 analogue or reduction of the carboxylic acid residue in compound 7 a,COOH (Scheme 1) could also have led to aldehyde 3. Acid 7 a,COOH was prepared from ester 7 a,COOEt (Scheme 1) according to the known method.[3f] Reduction of the acid chloride (prepared from 7 a,COOH and oxalyl or thionyl chloride) with hydrogen over the Lindlar catalyst failed to produce aldehyde 3, but we were able to detect the formation of aldehyde 3 by using LiAlHACHTUNGRE(OBut)3. However, we failed to isolate compound 3 in a pure state or to condense it in situ with N-methylpyridinium salts. Other synthetic approaches to coumarins 7 a–c,R4 proved to be more successful (see Scheme 1). Eventually, the useful 3-chlorocoumarins 6 a (2-Cl in Figure 1) and 6 b were obtained by the Pechmann reaction[16] of ethyl 2-chloro-4,4,4-trifluoro-3-oxobutyrate (5-Cl)[17] with an appropriate phenol 4 (Scheme 1, pathway i). The more reactive bromo derivative 5-Br proved to be too unstable and decomposed under these reaction conditions.[18] However, chlorocoumarins 6 a,b were found to be sufficiently active to undergo Stille coupling reactions to furnish the desired coumarins 7 a,b,R4, although in variable yields (Scheme 1, pathway ii and Table S1 in the Supporting Infor-
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Scheme 1. Synthetic routes to coumarins 7 a–c,R4 and their N-alkylated derivatives. Reagents and conditions: i) ZnCl2, EtOH, reflux; ii) R4SnBu3, catalyst, toluene, 120 8C (see Table S1 in the Supporting Information); iii) (CF3CO)2O, diethyl ether, reflux; iv) R4CH2COOH, N,N’-dicyclohexylcarbodiimide, Et3N, CH2Cl2, reflux (see Table S3 in the Supporting Information) or PhOP(O)Cl2, Et3N, DCE (for 7 a,CO2Et); v) 6bromo- or 6-iodohexanoic acid (products labeled with A) or 1,3-propanesultone (products labeled with B), acetonitrile, heating (see Table S2 in the Supporting Information); vi) aq. NaOH, EtOH, RT, 3 h. *The configuration of the CH=CH bond is always trans.
mation). Moreover, the yields often varied even in the same reaction, especially on changing the scale of the reaction. In view of the unpredictability of these cross-coupling reactions, we sought another method for obtaining the target coumarins 7 a,b,R4, as well as compounds 7 c,R4 (Scheme 1). The Knoevenagel-type cyclization reactions of o-keto-phenols with substituted acetic acids are known to furnish coumarins in high yields.[2e, 19] The selective acylation of 3-diACHTUNGREmethACHTUNGREylaminophenol (4 a), protected as its acetate, with trifluoroacetic anhydride (TFAA) followed by deprotection is known to afford compound 8 a.[19b] This result suggested the possibility of obtaining similar compounds by using phenols 4 b and 4 c. The removal of the acetate protecting group turned out to be surprisingly difficult in these cases, but fortunately the reactions of unprotected 3-(dialkylamino)phenols 4 a–c with an excess of TFAA directly furnished the desired trifluoroacetophenones 8 a–c in moderate-to-good yields (Scheme 1, pathways iii and Table S3 in the Support-
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FULL PAPER ing Information). The acylation/cyclization reaction between the 2-acetylphenols 8 a–c and arylACHTUNGRE(ethenyl)acetic acids was plagued by the incomplete conversion of the starting ketophenols (even in the presence of an excess of the substituted acetic acids), but nevertheless provided the target compounds in preparatively useful yields (Scheme 1, pathway iv and Table S3). In the case of pyridine-containing substituents (R4), quaternization of the pyridine nitrogen in coumarins 7 a–c,R4 was performed by reaction with either 6-bromoor 6-iodohexanoic acids (A) or 1,3-propanesultone (B) to prepare seven new compounds—7a-c,R4-A/B shown at the bottom of Scheme 1 (pathway v and Table S2). An additional free carboxylic acid group (in compounds with label “A”; Scheme 1) serves as a reactive site that can be directly used for the preparation of active esters and other derivatives for conjugation procedures. Quaternization with 6-halohexanoic acids is particularly useful in the case of compounds 7 a,R4, which do not have an ester group. On the other hand, introduction of a sulfonic acid residue by means of propanesultone strongly increases the solubility in aqueous solutions, preventing the undesirable aggregation of dye molecules, and is especially useful for labels already endowed with a reactive group. As expected, all coumarins with a quaternized pyridinium ring displayed even more enhanced bathochromic and -fluoric shifts due to the presence of the full positive charge in the fluorophore. However, the fluorescence quantum yields, with one exception (compare f values for 7 b,4-py and 7 b,4-py-B in Table 1), decreased significantly as a result of this transformation. By trying to increase the quantum yields of the new dyes (and their bioconjugates) in aqueous solutions, we improved their water solubility by hydroxylation and phosphorylation of the allylic methyl groups in compounds 7 c,H and 7 c,CH= CH-2-py according to a previously described methodology (Scheme 2).[20] These compounds contain the 2,2,4-trimethyl1,2-dihydroquinoline fragment in which the allylic position is prone to sulfonation[21] or oxidation. Oxidation (to aldehydes) was achieved by using selenium dioxide in dioxane (Scheme 2) and the aldehydes were converted in situ into alcohols 7 d,R4 by reduction with NaBH4. Alcohols 7 d,R4 were phosphorylated as described in the literature.[22] The analogue of compound 9,H,H with a sulfonic acid residue instead of the primary phosphate group is commercially available as Alexa FluorTM 430 (Invitrogen). Spectral properties of dyes, their bioconjugates, and their use in light microscopy: Twenty four newly obtained coumarins were characterized spectroscopically (Table 1). Three types of coumarins were prepared that differ in the substituent at the 3-position and the nature of the secondary amino group at the 7-position: compounds 7 a,R4 with an N,N-dimethylamino group at the 7-position (type “a”), 7 b,R4 with an N-(3-ethoxycarbonylpropyl)-1,2,3,4-tetrahydroquinoline fragment (type “b”), and 7 c,R4, 7 d, 7 e, and 9 (type “c”) with an N-(3-ethoxycarbonylpropyl)-2,2,4-trimethyl-1,2-dihydroquinoline fragment. Comparison of the spectral data of the different types of compounds but with the same sub-
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Table 1. Physical properties of the new coumarin dyes (Scheme 1) in ethanolic solution. Compound
labs [nm]
lem [nm]
e [103 m1 cm1]
f [%]
t [ns]
Compound
labs [nm]
lem [nm]
e [103 m1 cm1]
f [%]
t [ns]
7 a,2-th 7 a,CH=CH-2-py 7 a,CH=CH-2-py-A[a] 7 a,CH=CH-4-py 7 a,CH=CH-4-py-A[a] 7 a,4-py 7 a,4-py-A[a] 7 a,CH=CH-ph 7 b,4-py 7 b,4-py-B[b] 7 b,2-py 7 b,2-py-B[b]
416 444 475 449 500 413 439 438 430 459 429 462
611 578 646 580 668 588 644 576 593 655 578 607
24.4 36.6 9.9 28.5 13.3 18.8 11.8 27.6 13.7 14.8 16.4 19.2
13 64 37 71 47 47 27 49 19 34 20 11
1.2 2.9 1.4 3.0 1.6 1.1 1.3 4.0 1.0 1.3 1.6 –
7 c,4-py 7 c,2-py 7 c,2-py-B[b] 7 c,4-py-B[b] 7 c,2-th 7 c,CH=CH-ph 7 c,CH=CH-2-py 7 c,CH=CH-4-py 7 c,H 7 d,H 9,H,Et 9,CH=CH-2-py,H[c]
440 438 479 474 445 467 475 480 424 420 422 472
611 591 632 687 636 598 602 609 527 523 526 624
18.1 17.9 22.4 12.2 17.4 20.0 30.4 32.1 19.6 8.0 10.5 4.9
– 19 9 12 16 58 80 68 45 46 40 12
– 1.1 1.1 – 1.1 2.1 3.9 4.0 4.3 4.5 4.1 0.8
[a] A: The pyridine nitrogen is alkylated with an w-carboxypentyl residue. [b] B: The pyridine nitrogen is alkylated with an w-sulfopropyl group. [c] In aqueous phosphate buffer at pH 7.4.
Scheme 2. Hydroxylation and phosphorylation of coumarins 7 c,R4. Reagents and conditions: i) SeO2, dioxane, 100 8C, 1 h; NaBH4, THF/ MeOH, 0 8C, 15 min; ii) iPr2NP(OR)2, 1H-tetrazole/CH2Cl2, 40 8C, 1 h; MCPBA/CH2Cl2, 0 8C, 15 min; iii) [PdACHTUNGRE(Ph3P)4], Et3N, HCOOH, THF, 40 8C; iv) aq. NaOH, MeOH, 50 8C; then CF3COOH; v) N-hydroxysuccinimide, HATU, DMF, RT, 2 h. DMF = N,N-dimethylformamide, HATU = 1-[bisACHTUNGRE(dimethylamino)methylene]-1H-1,2,3-triazoloACHTUNGRE[4,5-b]pyridinium 3-oxide hexafluorophosphate, MCPBA = meta-chloroperbenzoic acid.
stituent at the 3-position (7 a,4-py and 7 b,4-py, 7 b,2-py and 7 c,2-py) led to the conclusion that, as expected, the transition from the 7-(N,N-dimethylamino) group (a) to the 1,2,3,4-tetrahydroquinoline system (b) and then to the Nalkyl-2,2,4-trimethyl-1,2-dihydroquinolines (c) provides redshifts of 17 and 9 nm in the absorption bands and small redshifts of 5 and 13 nm in the emission bands, respectively. The substituents attached at C-3 have a stronger influence on the positions of the absorption and emission bands than substitution at C-6 and C-7 of the coumarin fluorophore.
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The absorption maxima of the type “c” compounds increase in the order 7 c,H < 7 c,2-py (ffi7 c,4-py) < 7 c,2-th < 7 c,CH= CH-ph < 7 c,CH=CH-2-py (ffi7 c,4-py-B) < 7 c,CH=CH-4-py (ffi7 c,2-py-B). Thus, these changes correspond to increasing bathochromic shifts in the following order of substituents at C-3: H < 2-pyridyl (ffi4-pyridyl) < 2-thienyl < trans-2-phenylethenyl < trans-2-pyridylethenyl [ffiN-(3-sulfopropyl)-4-pyridinio] < trans-4-pyridylethenyl [ffiN-(3-sulfopropyl)-2-pyridinio]. These trends in the absorption bands are also valid for the coumarin types “a” and “b” (Table 1). Consider, for example, the order of increase of the absorption maxima: 7 a,4py < 7 a,2-th < 7 a,CH=CH-ph < 7 a,4-py-A < 7 a,CH=CH-2py < 7 a,CH=CH,4-py < 7 a,CH=CH-4-py-A. The C-3 unsubstituted compound is absent from this list, and two new Npyridinium groups (N-(5-carboxypentyl)-4-pyridinio and N[(5-carboxypentyl)-2-pyridinio]ethenyl) are included, but the sequence of the other substituents at C-3 is exactly the same as above: 4-pyridyl 2-thienyl trans-2-phenylethenyl < N(5-carboxypentyl)-4-pyridinio < trans-2-pyridylethenyl trans-4-pyridylethenyl < [ffiN-[(5-carboxypentyl)-2-pyridinio]ethenyl. For the emission bands, the bathofluoric shifts increase in a similar order, with the 2-thienyl and 4-[N-(3sulfopropyl)pyridinium] groups representing two exceptions: They shift the emission bands very strongly to the red spectral region and show very high Stokes shifts of around 200 and 230 nm in compounds 7 a,2-th and 7 c,4-py-B, respectively. In the former case, this effect is probably due to the strong electron-donating properties of the 2-thienyl group in coumarin 7 a,2-th, but it is not clear why the 4-[N-(3-sulfopropyl)pyridinium] group in compound 7 c,4-py-B provides a much larger Stokes shift (230 nm) than the structurally similar 2-[N-(3-sulfopropyl)pyridinium] group (145–150 nm; compounds 7 b,2-py-B and 7 c,2-py-B). Perhaps, the larger dipole moments of compounds 7 b,4-py-B and 7 c,4-py-B (compared with 7 b,2-py-B and 7 c,2-py-B, respectively) account for the larger Stokes shifts. Similarly, 4-pyridyl substituents shift the emission bands more to the red compared with 2-pyridyl groups (emission maxima for 7 b,4-py/7 b,2-py and 7 c,4-py/7 c,2-py are 593/578 and 611/591 nm, respective-
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Trifluoromethyl-Substituted Coumarins
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ly). However, dyes with huge Stokes shifts are not particudye 7 c,4-py-B. Its absorption and emission maxima are shiftlarly useful because, as a rule, they possess low fluorescence ed in opposite directions in conjugates with sheep antiquantum yields. Ethanol was used as the solvent for measmouse antibodies: The absorption is redshifted (+ 18 nm) urements of the fluorescence quantum yields of free dyes and the emission maximum blueshifted (32 nm). For the (Table 1) because in this solvent the emission efficiencies other dyes in Table 2, these deviations are considered to be are high enough to allow the differences between the dyes moderate because the absorption and emission bands in to be detected. In aqueous solutions (even in the presence coumarins are broad. The “long” components of the fluoresof bovine serum albumin (BSA), in the unbound state), the cence lifetimes of the antibody conjugates also deviate from fluorescence quantum yields of many of the dyes in this the values recorded for the free dyes. Interestingly, higher study are too low to allow detection and interpretation of and lower values of t1 were found, with the absolute deviaany pronounced and reliable variation in the fluorescence tions in the range of 1.1 to + 2.2 ns. A decrease in the lifequantum yields with any certainty. The emission efficiencies time of the excited state (t) may be associated with a diminof the compounds fluctuate in a wide range (9–80 %), and ished fluorescence quantum yield (f) if non-emissive transithe fluorescence lifetimes also vary in a relatively broad tions from the excited state (S1) become more pronounced. range (1–4.5 ns; Table 1). In this case, the radiative rate constants (kr = f/t) do not These results were obtained for solutions in ethanol, and change very much in the series of compounds. Alternatively, in this respect they are not directly relevant to the choice of these two parameters (f and t) may be independent of each the “best” dyes for use as labels for biomolecules. All the other, and the quantum yield may stay constant or even inbioconjugates were investigated in aqueous solutions. After crease, whereas the lifetime decreases (when the probability derivatization and in aqueous buffers, the fluorescence quantum yields may change unpredictably (most often deTable 2. Properties of the coumarin dyes conjugated with sheep anticrease). Nevertheless, the data from Table 1 helped us to mouse antibodies.[a] select dye candidates suitable for conjugation with antibodlem f t1 Dye conjugate DOL[b] labs t2 ies and immunofluorescence microscopy (Table 2). To ach[nm] [nm] [%] [ns][c] [ns][c] ieve this, we subjected the selected dyes to saponification 7 a,CH=CH-4-py-A 3.6 491 645 2 2.1 (16) 0.57 (9.4) and transformed the free acids into N-hydroxysuccinimidyl 7 b,4-py-B 0.6 463 638 8 2.2 (16) 1.0 (19) 7 b,2-py 10 440 576 5 3.8 (4.4) 1.1 (2.8) (NHS) esters according to standard protocols.[20] These 10 473 609 5 3.1 (51) 0.55 (11) 7 c,CH=CH-2-py[d] active esters were coupled with polyclonal (secondary) anti7 c,4-py-B 0.9 492 655 12 – – bodies and the physical parameters of the conjugates were – 9,CH=CH-2-py,H 11.6 479 607 4[e] 1.5[f] studied (see Figure 2 and Table 2). The potential of the dyes 7 c,H 3.5 423 544 8 3.2 (4.9) 0.25 (0.4) in confocal and STED microscopy were assessed by stan7 d,H 14 426 539 10 3.5 (5.0) 0.24 (0.2) 9,H,H 1.3 425 541 21 3.0 (5.7) 0.22 (0.3) dard immunofluorescence staining by using these conjugate[23a] s. Although most of the new dyes do not carry polar [a] Measured in PBS buffer solutions at pH 7.4; for the structures and spectral properties of the free dyes, see Scheme 1 and Table 1, respectivecharged groups or many hydrophilic residues, coupling to ly. [b] Degree of labeling (DOL): the average number of dye residues atantibodies according to the standard protocol[20] very often tached to one protein molecule with M ~ 150 000. [c] Lifetimes of the exprovided values for the degree of labeling (DOL) in the cited states with values in parentheses denoting the amplitudes of the range of 10 (see Table 2). As a rule, higher DOL values lead “long” and “short” components, respectively. [d] Goat anti-rabbit. [e] The same fluorescence quantum yield was found for the conjugate to lower fluorescence quantum yields of the conjugates due with goat anti-rabbit antibodies with DOL = 5.6. [f] Monoexponential fit; to selfquenching of the emission (due to the formation of t = 1.7 ns for the conjugate with goat anti-rabbit antibodies (DOL = 5.6). [23b] non-emitting dimers of the dye residues). This is also true for most of the coumarin dyes shown in Table 1. Smaller amounts of NHS esters provide conjugates with lower values of DOL (1–4), but higher fluorescence quantum yields (21–8 %). The absorption and emission maxima of the dye conjugates (measured in phosphate buffer saline (PBS)) do not agree very precisely with those of the corresponding free dyes (measured in ethanol). The deviations for the absorption maxima are 9 to + 18 nm, and Figure 2. Normalized absorption (left) and emission (right) spectra of coumarin 9,CH=CH-2-py (Star470SX + , green line) and rhodamine dye (Abberior Star635, red line) attached to antibodies in aqueous PBS buffer at for the emission maxima 32 to pH 7.4. Convenient excitation sources provide light of 488–532 nm (absorbed by Star470SX +) and 633– + 17 nm. The highest deviations 640 nm (absorbed by Star635), these regions are shown in green and red, respectively. For structures, see were found for the red-emitting Figure 4.
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of the radiative transition S1!S0 increases). In this case the radiative rate increases and the nonradiative rate (knr = (1f)/t) decreases.[24] The values of kr and knr for the dyes and their conjugates were calculated from the data in Tables 1 and 2 and are given in Table S4 in the Supporting Information. In the case of bioconjugates, nearly all the fluorescence quantum yields are lower than those of the free dyes (except for compound 9,CH=CH-2-py,H-AB, which displays higher t1 values in the conjugated states). And indeed, for most dye conjugates in Table 2, the t1 values are, as expected, smaller than those of the free dyes, and the relative amplitudes of the “short” (t1) components are nearly negligible. In three cases, 7 a,CH=CH-4-py-A, 7 b,4-py-B, and 7 b,2-py, the t1 values are larger and the t2 values are smaller than the fluorescence lifetimes of the free dyes. In these cases only the biexponential fits with comparable amplitudes of the “long” (t1) and “short” (t2) components gave a good approximation to the fluorescence decay curves (Table 2). For two-color imaging with the commercial Leica TCS STED microscope, it is necessary to have red-emitting dyes that can be depleted with powerful 750–770 nm light. The imaging performance of the dyes and the scope of their applicability depend on their photostability, the fluorescence quantum yields of their conjugates, the degree of labeling (DOL), intersystem crossing rates, excited-state lifetimes, and the absence of unspecific labeling. The photostabilities of coumarin dyes 7 a,CH=CH-2-py-A, 7 b,CH=CH-4-py-B, 7 c,2-py-B, and 9,CH=CH-2-py,H were evaluated by using 488 nm laser light, which is typically applied as an excitation source (Figure 3). For comparison, we used fluorescein and the new Atto490LS (long Stokes shift) dye. All these dyes were found to have similar resistance against photobleaching (comparable to that of fluorescein). Fluorescein is known to be less photostable than the spectrally identical RhoACHTUNGREdamine 110 and in general fluorescein derivatives bleach much faster than rhodamines. These results indicate that the coumarins in this study (and Atto490LS) are only moderately photostable under illumination with the excitation light (488 nm). Compound 7 a,CH=CH-2-py-A did not bleach totally, which might be explained by the formation of a product with a new and more photoresistant fluorophore. It is also important to note that STED microscopy requires dyes that do not bleach very quickly under irradiation with very strong red light (750–770 nm). In this respect, moderate photostabilities under the excitation conditions do not preclude the use of these dyes in super-resolution microscopy with depletion of the excited state by stimulated emission. As a fluorescent marker with a small Stokes shift, we chose the photostable and hydrophilic rhodamine dye Abberior Star635 (Figures 2 and 4; absorption and emission maxima at 635 and 655 nm, respectively), which was designed according to the features mentioned above and was shown to perform very well under STED conditions.[25] The need for some absorption at 750–770 nm suggests that redemitting compounds with emission maxima at wavelengths
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Figure 3. Photostability measurements showing the decrease in the emission intensity of selected coumarins and two commercially available dyes (Fluorescein and Atto490LS) in a PVA matrix exposed to constant excitation with laser light of 488 nm (50 W cm2).
greater than 580 nm may be depleted very efficiently (e.g., 7 a,2-th, 7 a,CH=CH-2-py-A, 7 a,CH=CH-4-py-A, 7 a,4-py-A, 7 a,4-py-B (prepared from 7 a,4-py and 1,3-propanesultone),
Figure 4. Structures of the fluorescent dyes Star470SX + and Star635 used in two-color STED microscopy.
7 b,4-py-B, 7 c,2-py, 7 c,2-py-B, 7 c,4-py-B, 7 c,2-th, 7 c,CH= CH-2-py, and 9,CH=CH-2-py,H). However, this is not a severe limitation because the emission bands of coumarins are broad and, in fact, all dyes from Table 1, except for 7 c,H, 7 d,H, and 9,H,Et, may undergo stimulated emission under irradiation with very powerful 750 nm light. Compound 7 c,CH=CH-2-py (Table 1) possesses the highest fluorescence quantum yield (80 %), and it was possible to obtain its conjugates with antibodies with a DOL value of 10 and an emission efficiency of 5 % (Table 2). In immunofluorescence microscopy, bioconjugates are employed under physiological conditions, and the absence of a fluorescence background (due to unspecific staining) is particularly important. In this respect, hydrophilic compounds are advantageous as they possess polar (ionic) groups that provide high solubility in aqueous buffers (enabling to increase the values of DOL), prevent aggregation and self-quenching of the dye residues, reduce unspecific binding, and increase the fluorescence quantum yields.[26] Therefore, as a test substance, we chose the phosphorylated coumarin 9,CH=CH-2-
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py,H (Table 1 and Figure 4) obtained from the nonpolar precursor 7 c,CH=CH-2-py (Scheme 2) and evaluated the possibility of using it in two-color STED microscopy together with the Star635 dye as reference (for spectra and structures, see Figures 2 and 4). The results are illustrated in Figure 5 and Figure S1 in the Supporting Information, which present the image of the Golgi apparatus in a HeLa cell. A ribbon-like structure localized in the perinuclear region is typical of Golgi staining. Golgi cisternae are impossible to image in sufficient detail by using a conventional confocal microscope with a lateral resolution of around 250 nm, which is much larger than the thickness of a single Golgi cisterna (around 100 nm). The transmembrane protein GPP130 is localized to the cisGolgi,[27] whereas p230 is associated with the trans-Golgi network.[28] These two Golgi markers were imaged by using indirect immunofluorescence with secondary antibodies labeled with Star470SX + and Star635 dyes (with a Stokes shift of 128 and 20 nm, respectively). For this purpose, one
FULL PAPER mentary dye with a large Stokes shift that can be used in multicolor confocal and STED microscopy without appreciable cross-talk. In this respect, it is a valuable addition to the family of red-emitting fluorescent dyes with long Stokes shifts (e.g., ChromeoTM 494)[29] intended for use in STED microscopy. The absence of cross-talk is particularly important because it provides an additional “degree of freedom” and enables two-color imaging and co-localization of biological objects without using any linear unmixing schemes, which require supplementary experiments and complicate the acquisition and procession of data.
Conclusion
Coumarins with a CF3 group attached at C-4 and 3-(hetero)aryl or 3-[2-(hetero)arylethenyl] groups have been synthesized. Aminophenols 4 a–c were used as starting materials and various coumarins with large Stokes shifts were prepared by the synthetic routes given in Scheme 1. This approach enACHTUNGREabled us to modify the positions of the absorption and emission bands and, if necessary, shift them to the red spectral region in which the autofluorescence of cells and their organelles is negligible. Dyes with diverse polarities were synthesized by fusion of the 2,2,4-trimethyl-1,2-dihydroquinoline fragment to the coumarin fluorophore (providing the basic nonpolar scaffold), subsequent decoration with a primary hydroxy group (giving a dye of medium polarity), and phosFigure 5. STED (upper panel) and confocal (lower panel) two-color imaging of the Golgi apparatus. Localizaphorylation (providing a highly tion of Gpp130 (cis-Golgi) and p230 (trans-Golgi) in HeLa cells by indirect immunofluorescence using antiGpp130 and anti-p230 antibodies. Cells were then stained with Star470SX + and Star635 conjugated secondary polar dye). This approach alantibodies as indicated in the figure. Scale bars: 1 mm. lowed the synthesis of 9,CH= CH-2-py,H (Star470SX +), which performed well in twotunable STED depletion laser (750–770 nm) and two excitacolor confocal and STED microscopy when used together tion sources (532 and 640 nm pulsed diode lasers) were with the reference red-emitting rhodamine dye Star635, used. Under these conditions, both dyes provide an optical which has a small Stokes shift. Other interesting dyes were lateral resolution of 61–66 nm (see Figure S2), which is near identified, for example, compound 7 c,4-py-B (see Tables 1 the spatial resolution of the Leica TCS STED microscope. and 2) also emits in the red region, possesses a huge Stokes The imaging results obtained with the Star470SX + dye are shift of 213 nm (free dye as ethyl ester in ethanol) and 163 nm (in antibody conjugates), as well as acceptable fluocomparable to the imaging performance of Star635: Both rescence quantum yields (e.g., 12 % in protein conjugates dyes are bright and provide an excellent signal-to-noise with DOL = 0.9). The properties of this dye may be further ratio with almost no detectable background (unspecific improved by introducing a polar sulfonic or phosphoric acid binding is very low). Taking into account the fact that residue into the 2,2,4-trimethyl-1,2-dihydroquinoline moiety. Star635 was shown to be a reference dye (with a small Other interesting dyes emitting orange or green light, comStokes shift) in immunofluorescence assays,[25] we may conpounds 7 b,2-py and 7 c,H, respectively, also deserve further clude that the new coumarin dye Star470SX + is a comple-
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improvement by “hydrophilization”, which is expected to increase their solubility in aqueous solutions, fluorescence quantum yields, and brightness in optical microscopy.
Acknowledgements The authors are grateful to Nina Ohm, Marianne Pulst, and Maxim Sednev for recording optical spectra and calculating the DOL values and fluorescence quantum yields. We appreciate the assistance of E. Rothermel in the cell culture. We thank Dr. Haisen Ta for measurements of the fluorescence lifetimes of the Star470SX + dye and its bioconjugates. We are indebted to Bundesministerium fr Bildung und Forschung (BMBF 513) for financial support within the program Optische Technologien fr Biowissenschaften und Gesundheit (FKZ 13N11066). F.B. was supported by a Post-Doctoral Research Fellowship from the American-Italian Cancer Foundation. We are thankful to J. Jethwa for his critical reading of the manuscript.
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[17] Compound 5-Cl was obtained by direct chlorination of commercially available ethyl 4,4,4-trifluoro-3-oxobutyrate: H. M. Walborsky, M. E. Baum, J. Am. Chem. Soc. 1958, 80, 187 – 192. [18] a) V. I. Saloutin, Z. E. Skryabina, M. N. Rudaya, K. I. Pashkevich, Izv. Akad. Nauk SSSR, Ser. Khim. 1984, 1106 – 1114; Russ. Chem. Bull. 1984, 1016 – 1022; compare with, however b) E. Cherbuliez, G. Weber, J. Rabinowitz, Helv. Chim. Acta 1965, 48, 1423 – 1426; and c) K. Tanaka, K. Nomura, H. Oda, S. Yoshida, K. Mitsuhashi, J. Heterocycl. Chem. 1991, 28, 907 – 911. [19] a) X. Zhang, Z. Sui, Synthesis 2006, 2568 – 2572; b) see ref. [3f]. [20] S. Nizamov, K. I. Willig, M. V. Sednev, V. N. Belov, S. W. Hell, Chem. Eur. J. 2012, 18, 16339 – 16348. [21] a) F. Mao, W.-Y. Leung, R. P. Haugland (Molecular Probes Inc.), US 6130101 A; b) A. Zilles, J. Arden-Jacob, K.-H. Drexhage, N. U. Kemnitzer, M. Hammers-Schneider (Atto-Tec GmbH), WO 2005/ 003086 A2; US 2006/0179585 A1. [22] For representative examples of phosphorylation reactions involving primary alcohols, see: a) G. G. Durgam, T. Virag, M. D. Walker, R. Tsukahara, S. Yasuda, K. Liliom, L. A. van Meeteren, W. H. Moolenaar, N. Wilke, W. Siess, G. Tigyi, D. D. Miller, J. Med. Chem. 2005, 48, 4919 – 4930; b) J. W. Perich, R. B. Johns, Synthesis 1988, 142 – 144. [23] a) C. A. Wurm, D. Neumann, R. Schmidt, A. Egner, S. Jakobs, in Live Cell Imaging: Methods and Protocols (Ed.: D. B. Papkovsky),
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4-Trifluoromethyl-Substituted Coumarins with Large Stokes Shifts: Synthesis, Bioconjugates, and Their Use in Super-Resolution Fluorescence Microscopy Heiko Schill,[a] Shamil Nizamov,[a] Francesca Bottanelli,[b] Jakob Bierwagen,[a] Vladimir N. Belov,*[a] and Stefan W. Hell*[a]
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FULL PAPER Abstract: Bright and photostable fluorescent dyes with large Stokes shifts are widely used as sensors, molecular probes, and light-emitting markers in chemistry, life sciences, and optical microscopy. In this study, new 7-dialkylamino-4-trifluoromethylcoumarins have been designed for use in bioconjugation reactions and optical microscopy. Their synthesis was based on the Stille reaction of 3-chloro-4-trifluoromethylcoumarins and available (hetACHTUNGREero)aryl- or (hetero)arylethenyltin derivatives. Alternatively, the acylation of 2-trifluoroacetyl-5-dialkylaminophenols with available (hetACHTUNGREero)aryl- or (hetACHTUNGREero)arylethenylacetic acids followed by intramolecular condensation afforded coumarins with 3-(hetero)aryl or 3-[2-
(hetero)aryl]ethenyl groups. Hydrophilic properties were provided by the introduction of a sulfonic acid residue or by phosphorylation of a primary hydroxy group attached at C-4 of the 2,2,4-trimethyl-1,2-dihydroquinoline fragment fused to the coumarin fluorophore. For use in immunolabeling procedures, the dyes were decorated with an (activated) carboxy group. The positions of the absorption and emission maxima vary in the ranges 413–480 and 527–668 nm, respectively. The phosphorylated dye, 9,CH=CH-2-py,H, with Keywords: bioconjugation · chromophores · fluorescence · imaging agents · light microscopy
Introduction Fluorescence microscopy is one of the most important techniques in physical, chemical, and biological studies of (living) cells, tissues, or even whole organisms.[1] Indeed, the cells, or the organelles inside them, can be specifically labeled in a versatile manner with diverse fluorescent markers.[2] Various classes of dyes have been used for staining and bioimaging, and coumarins are quite prominent among them. Coumarins have a long history as laser dyes,[3] and nowadays they are widely used as fluorescent chemosensors and labels for proteins, nucleic acids, lipids, carbohydrates, toxins, hormones, and other biomolecules.[4, 5] The parent compound at room temperature shows no fluorescence and only a weak absorption at 312 nm with e = 5370 dm3 mol1 cm1.[3o] 7-Hydroxy-[4] and 7-aminocoumarins,[5] however, emit light in the blue-green region of the visible spectrum due to the batho- and hyperchromic shifts induced by the push–pull system of the electron-donor group at C-7 and the electron-withdrawing lactone moiety. Electron donors at C-6 and/or electron-acceptors at C-3
[a] Dr. H. Schill, Dr. S. Nizamov, Dipl.-Chem. J. Bierwagen, Dr. V. N. Belov, Prof. S. W. Hell Department of NanoBiophotonics Max Planck Institute for Biophysical Chemistry Am Fassberg 11, 37077 Gçttingen (Germany) Fax: (+ 49) 551-2012505 E-mail: [email protected] [email protected] [b] Dr. F. Bottanelli Department of Cell Biology, Yale School of Medicine 333 Cedar Street, New Haven, Connecticut 06510 (USA) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201302037.
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the 1-(3-carboxypropyl)-4-hydroxymethyl-2,2-dimethyl-1,2-dihydroquinoline fragment fused to the coumarin fluorophore bearing the 3-[2-(2-pyriACHTUNGREdyl)ACHTUNGREethenyl] residue (absorption and emission maxima at 472 and 623 nm, respectively) was used in super-resolution light microscopy with stimulated emission depletion and provided an optical resolution better than 70 nm with a low background signal. As a result of their large Stokes shifts, good fluorescence quantum yields, and adequate photostabilities, phosphorylated coumarins enable two-color imaging (using several excitation sources and a single depletion laser) to be combined with subdiffractional optical resolution.
and/or C-4 shift the positions of the absorption and emission bands further into the red region. Extension of the p system attached to the 3-position by introduction of an unsaturated substituent may yield hybrid dyes and provide an additional bathofluoric shift of the fluorescence band.[6] Combinatorial studies on coumarin “libraries” have exemplified their structure–spectra relationships.[2e, 4a, 7] In general, these dyes are known to be brightly fluorescent, possess relatively large absorption coefficients, high fluorescence quantum yields, low degree of triplet state formation, and large Stokes shifts (separation between the absorption and emission maxima measured in nm or cm1). Apart from large Stokes shifts, the common features of coumarin dyes are moderate photostability (compared with xanthene dyes) and moderate fluorescence quantum yields in polar solvents. These drawbacks limit their use as fluorescent markers in light microscopy and, in particular, in stimulated emission depletion (STED), one of the most powerful diffraction-unlimited imaging techniques.[8] For more than a century, the optical resolution that could be achieved with a light microscope seemed limited by Abbes law, which states that the resolution cannot be better than (approximately) half the wavelength of the applied light.[9] For microscopy in the visible range (400–800 nm), this law limits the resolution to less than 200 nm. However, in many cases, a better optical resolution is desirable or even necessary. Although electron microscopy provides higher resolution, it is incompatible with live specimens and requires special preparation and fixation techniques, which can be tedious and even destructive towards the features of interest. As a result of recent innovations, several new super-resolution methods have been implemented in light microscopy.[8] They break Abbes law, leading to various diffraction-unlimited imaging techniques. These new proce-
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dures often require novel fluorescent dyes with superior photostability, greater brightness, and the ability to switch between bright and dark states. In the case of multicolor experiments, the spectral separation of different labels into two or more excitation or acquisition channels is essential. For example, a pair of dyes emitting approximately at the same wavelength with well-separated absorption bands may be used together provided that the excitation with two light sources does not result in cross-talk. In this respect, dyes with large Stokes shifts are beneficial as they allow the use of different fluorescence channels and only one excitation source (or vice versa). Under the more severe conditions of STED, two lasers are required for each fluorescent dye: One for the excitation and the other for depletion.[8c] In this case, dyes with large Stokes shifts allow the same wavelength to be used for depletion while maintaining two different excitation windows in which to distinguish the labels.[10] Unfortunately, photostable and brightly fluorescent dyes with large Stokes shifts are rare and only a few of them are commercially available.[11] Most of them contain a coumarin fragment as the fluorophore. For example, “Mega Stokes” dyes from Dyomics are coumarins that absorb at about 500– 520 nm and emit in the region of 590–670 nm.[11a,b] Another practically useful coumarin dye is AlexaFluorTM 430 with an absorption maximum at 434 nm and emission band at 539 nm (Life Technologies).[12] However, the chemical structures of many commercially available fluorescent dyes are unknown.[11c] For example, the structures of Pacific Orange (absorption 390 nm, emission 540 nm; Life Technologies), V500 (absorption 415 nm, emission 500 nm; BD Horizon), and Chromeo 494 (absorption 494 nm, emission 628 nm; Active Motif) have not been published. Surprisingly, among the disclosed coumarin fluorophores, there are only a few examples of dyes with perfluoroalkyl substituents even though it is known that a CF3 group, especially at the 4-position of the coumarin system, increases photostability.[13] Moreover, the introduction of a strong electronacceptor, for example, a perfluoroalkyl group, at C-4 should provide a pronounced redshift of the absorption and fluorescence bands and, because the bathofluoric shift is expected to be larger, increase the Stokes shifts. In view of the apparent lack of fluorescent dyes with large Stokes shifts suitable for STED microscopy,[10] we have designed and screened a new set of coumarins with very large Stokes shifts (> 100 nm), prepared their conjugates with proteins, and evaluated their performance in two-color STED super-resolution imaging (by using a red-emitting rhodamine dye with a small Stokes shift as a benchmark).
Results and Discussion Synthesis: A trifluoromethyl group at the 4-position of coumarin reduces the bleaching coefficient of a dye by about 20 %[13] and increases the Stokes shift (compared with the 4unsubstituted derivative).[3f] An unsaturated substituent at the 3-position is also known to increase the Stokes shift of
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a coumarin.[4a] Therefore we started the synthesis of variously substituted 7-amino-4-trifluoromethylcoumarins with unsaturated substituents at the 3-position by using a modular convergent approach to create a range of different dyes by a short synthetic route. In addition, an electron-acceptor group was introduced at the 3-position (directly or through a CH=CH connector) to provide an additional redshift of the absorption and emission bands so that the emission process can be efficiently stimulated with a “red” laser at 750– 770 nm. Because the halogenation of 7-aminocoumarins occurs selectively at C-3, we first attempted this reaction under various conditions with the commercially available Coumarin 152 (1; Figure 1). If these reactions were successful, 3-
Figure 1. Model compounds for the convergent synthesis of coumarin dyes.
haloACHTUNGREcoumarins 2-X could then be used in transition-metalcatalyzed cross-coupling transformations.[14] However, the presence of the 4-trifluoromethyl group seemed to prevent clean halogenation reactions. Halogenation was possible only under drastic conditions (e.g., by heating with N-bromosuccinimide in DMF) and mixtures of halogenated and/ or N-dealkylated compounds were always obtained. Similarly, attempted formylation reactions under Vilsmeier–Haack conditions[6, 15] failed to afford aldehyde 3 (Figure 1), which could be used in a Knoevenagel-type condensation reaction. Oxidation of the methyl group in the (unknown) 3-CH3 analogue or reduction of the carboxylic acid residue in compound 7 a,COOH (Scheme 1) could also have led to aldehyde 3. Acid 7 a,COOH was prepared from ester 7 a,COOEt (Scheme 1) according to the known method.[3f] Reduction of the acid chloride (prepared from 7 a,COOH and oxalyl or thionyl chloride) with hydrogen over the Lindlar catalyst failed to produce aldehyde 3, but we were able to detect the formation of aldehyde 3 by using LiAlHACHTUNGRE(OBut)3. However, we failed to isolate compound 3 in a pure state or to condense it in situ with N-methylpyridinium salts. Other synthetic approaches to coumarins 7 a–c,R4 proved to be more successful (see Scheme 1). Eventually, the useful 3-chlorocoumarins 6 a (2-Cl in Figure 1) and 6 b were obtained by the Pechmann reaction[16] of ethyl 2-chloro-4,4,4-trifluoro-3-oxobutyrate (5-Cl)[17] with an appropriate phenol 4 (Scheme 1, pathway i). The more reactive bromo derivative 5-Br proved to be too unstable and decomposed under these reaction conditions.[18] However, chlorocoumarins 6 a,b were found to be sufficiently active to undergo Stille coupling reactions to furnish the desired coumarins 7 a,b,R4, although in variable yields (Scheme 1, pathway ii and Table S1 in the Supporting Infor-
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Scheme 1. Synthetic routes to coumarins 7 a–c,R4 and their N-alkylated derivatives. Reagents and conditions: i) ZnCl2, EtOH, reflux; ii) R4SnBu3, catalyst, toluene, 120 8C (see Table S1 in the Supporting Information); iii) (CF3CO)2O, diethyl ether, reflux; iv) R4CH2COOH, N,N’-dicyclohexylcarbodiimide, Et3N, CH2Cl2, reflux (see Table S3 in the Supporting Information) or PhOP(O)Cl2, Et3N, DCE (for 7 a,CO2Et); v) 6bromo- or 6-iodohexanoic acid (products labeled with A) or 1,3-propanesultone (products labeled with B), acetonitrile, heating (see Table S2 in the Supporting Information); vi) aq. NaOH, EtOH, RT, 3 h. *The configuration of the CH=CH bond is always trans.
mation). Moreover, the yields often varied even in the same reaction, especially on changing the scale of the reaction. In view of the unpredictability of these cross-coupling reactions, we sought another method for obtaining the target coumarins 7 a,b,R4, as well as compounds 7 c,R4 (Scheme 1). The Knoevenagel-type cyclization reactions of o-keto-phenols with substituted acetic acids are known to furnish coumarins in high yields.[2e, 19] The selective acylation of 3-diACHTUNGREmethACHTUNGREylaminophenol (4 a), protected as its acetate, with trifluoroacetic anhydride (TFAA) followed by deprotection is known to afford compound 8 a.[19b] This result suggested the possibility of obtaining similar compounds by using phenols 4 b and 4 c. The removal of the acetate protecting group turned out to be surprisingly difficult in these cases, but fortunately the reactions of unprotected 3-(dialkylamino)phenols 4 a–c with an excess of TFAA directly furnished the desired trifluoroacetophenones 8 a–c in moderate-to-good yields (Scheme 1, pathways iii and Table S3 in the Support-
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FULL PAPER ing Information). The acylation/cyclization reaction between the 2-acetylphenols 8 a–c and arylACHTUNGRE(ethenyl)acetic acids was plagued by the incomplete conversion of the starting ketophenols (even in the presence of an excess of the substituted acetic acids), but nevertheless provided the target compounds in preparatively useful yields (Scheme 1, pathway iv and Table S3). In the case of pyridine-containing substituents (R4), quaternization of the pyridine nitrogen in coumarins 7 a–c,R4 was performed by reaction with either 6-bromoor 6-iodohexanoic acids (A) or 1,3-propanesultone (B) to prepare seven new compounds—7a-c,R4-A/B shown at the bottom of Scheme 1 (pathway v and Table S2). An additional free carboxylic acid group (in compounds with label “A”; Scheme 1) serves as a reactive site that can be directly used for the preparation of active esters and other derivatives for conjugation procedures. Quaternization with 6-halohexanoic acids is particularly useful in the case of compounds 7 a,R4, which do not have an ester group. On the other hand, introduction of a sulfonic acid residue by means of propanesultone strongly increases the solubility in aqueous solutions, preventing the undesirable aggregation of dye molecules, and is especially useful for labels already endowed with a reactive group. As expected, all coumarins with a quaternized pyridinium ring displayed even more enhanced bathochromic and -fluoric shifts due to the presence of the full positive charge in the fluorophore. However, the fluorescence quantum yields, with one exception (compare f values for 7 b,4-py and 7 b,4-py-B in Table 1), decreased significantly as a result of this transformation. By trying to increase the quantum yields of the new dyes (and their bioconjugates) in aqueous solutions, we improved their water solubility by hydroxylation and phosphorylation of the allylic methyl groups in compounds 7 c,H and 7 c,CH= CH-2-py according to a previously described methodology (Scheme 2).[20] These compounds contain the 2,2,4-trimethyl1,2-dihydroquinoline fragment in which the allylic position is prone to sulfonation[21] or oxidation. Oxidation (to aldehydes) was achieved by using selenium dioxide in dioxane (Scheme 2) and the aldehydes were converted in situ into alcohols 7 d,R4 by reduction with NaBH4. Alcohols 7 d,R4 were phosphorylated as described in the literature.[22] The analogue of compound 9,H,H with a sulfonic acid residue instead of the primary phosphate group is commercially available as Alexa FluorTM 430 (Invitrogen). Spectral properties of dyes, their bioconjugates, and their use in light microscopy: Twenty four newly obtained coumarins were characterized spectroscopically (Table 1). Three types of coumarins were prepared that differ in the substituent at the 3-position and the nature of the secondary amino group at the 7-position: compounds 7 a,R4 with an N,N-dimethylamino group at the 7-position (type “a”), 7 b,R4 with an N-(3-ethoxycarbonylpropyl)-1,2,3,4-tetrahydroquinoline fragment (type “b”), and 7 c,R4, 7 d, 7 e, and 9 (type “c”) with an N-(3-ethoxycarbonylpropyl)-2,2,4-trimethyl-1,2-dihydroquinoline fragment. Comparison of the spectral data of the different types of compounds but with the same sub-
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Table 1. Physical properties of the new coumarin dyes (Scheme 1) in ethanolic solution. Compound
labs [nm]
lem [nm]
e [103 m1 cm1]
f [%]
t [ns]
Compound
labs [nm]
lem [nm]
e [103 m1 cm1]
f [%]
t [ns]
7 a,2-th 7 a,CH=CH-2-py 7 a,CH=CH-2-py-A[a] 7 a,CH=CH-4-py 7 a,CH=CH-4-py-A[a] 7 a,4-py 7 a,4-py-A[a] 7 a,CH=CH-ph 7 b,4-py 7 b,4-py-B[b] 7 b,2-py 7 b,2-py-B[b]
416 444 475 449 500 413 439 438 430 459 429 462
611 578 646 580 668 588 644 576 593 655 578 607
24.4 36.6 9.9 28.5 13.3 18.8 11.8 27.6 13.7 14.8 16.4 19.2
13 64 37 71 47 47 27 49 19 34 20 11
1.2 2.9 1.4 3.0 1.6 1.1 1.3 4.0 1.0 1.3 1.6 –
7 c,4-py 7 c,2-py 7 c,2-py-B[b] 7 c,4-py-B[b] 7 c,2-th 7 c,CH=CH-ph 7 c,CH=CH-2-py 7 c,CH=CH-4-py 7 c,H 7 d,H 9,H,Et 9,CH=CH-2-py,H[c]
440 438 479 474 445 467 475 480 424 420 422 472
611 591 632 687 636 598 602 609 527 523 526 624
18.1 17.9 22.4 12.2 17.4 20.0 30.4 32.1 19.6 8.0 10.5 4.9
– 19 9 12 16 58 80 68 45 46 40 12
– 1.1 1.1 – 1.1 2.1 3.9 4.0 4.3 4.5 4.1 0.8
[a] A: The pyridine nitrogen is alkylated with an w-carboxypentyl residue. [b] B: The pyridine nitrogen is alkylated with an w-sulfopropyl group. [c] In aqueous phosphate buffer at pH 7.4.
Scheme 2. Hydroxylation and phosphorylation of coumarins 7 c,R4. Reagents and conditions: i) SeO2, dioxane, 100 8C, 1 h; NaBH4, THF/ MeOH, 0 8C, 15 min; ii) iPr2NP(OR)2, 1H-tetrazole/CH2Cl2, 40 8C, 1 h; MCPBA/CH2Cl2, 0 8C, 15 min; iii) [PdACHTUNGRE(Ph3P)4], Et3N, HCOOH, THF, 40 8C; iv) aq. NaOH, MeOH, 50 8C; then CF3COOH; v) N-hydroxysuccinimide, HATU, DMF, RT, 2 h. DMF = N,N-dimethylformamide, HATU = 1-[bisACHTUNGRE(dimethylamino)methylene]-1H-1,2,3-triazoloACHTUNGRE[4,5-b]pyridinium 3-oxide hexafluorophosphate, MCPBA = meta-chloroperbenzoic acid.
stituent at the 3-position (7 a,4-py and 7 b,4-py, 7 b,2-py and 7 c,2-py) led to the conclusion that, as expected, the transition from the 7-(N,N-dimethylamino) group (a) to the 1,2,3,4-tetrahydroquinoline system (b) and then to the Nalkyl-2,2,4-trimethyl-1,2-dihydroquinolines (c) provides redshifts of 17 and 9 nm in the absorption bands and small redshifts of 5 and 13 nm in the emission bands, respectively. The substituents attached at C-3 have a stronger influence on the positions of the absorption and emission bands than substitution at C-6 and C-7 of the coumarin fluorophore.
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The absorption maxima of the type “c” compounds increase in the order 7 c,H < 7 c,2-py (ffi7 c,4-py) < 7 c,2-th < 7 c,CH= CH-ph < 7 c,CH=CH-2-py (ffi7 c,4-py-B) < 7 c,CH=CH-4-py (ffi7 c,2-py-B). Thus, these changes correspond to increasing bathochromic shifts in the following order of substituents at C-3: H < 2-pyridyl (ffi4-pyridyl) < 2-thienyl < trans-2-phenylethenyl < trans-2-pyridylethenyl [ffiN-(3-sulfopropyl)-4-pyridinio] < trans-4-pyridylethenyl [ffiN-(3-sulfopropyl)-2-pyridinio]. These trends in the absorption bands are also valid for the coumarin types “a” and “b” (Table 1). Consider, for example, the order of increase of the absorption maxima: 7 a,4py < 7 a,2-th < 7 a,CH=CH-ph < 7 a,4-py-A < 7 a,CH=CH-2py < 7 a,CH=CH,4-py < 7 a,CH=CH-4-py-A. The C-3 unsubstituted compound is absent from this list, and two new Npyridinium groups (N-(5-carboxypentyl)-4-pyridinio and N[(5-carboxypentyl)-2-pyridinio]ethenyl) are included, but the sequence of the other substituents at C-3 is exactly the same as above: 4-pyridyl 2-thienyl trans-2-phenylethenyl < N(5-carboxypentyl)-4-pyridinio < trans-2-pyridylethenyl trans-4-pyridylethenyl < [ffiN-[(5-carboxypentyl)-2-pyridinio]ethenyl. For the emission bands, the bathofluoric shifts increase in a similar order, with the 2-thienyl and 4-[N-(3sulfopropyl)pyridinium] groups representing two exceptions: They shift the emission bands very strongly to the red spectral region and show very high Stokes shifts of around 200 and 230 nm in compounds 7 a,2-th and 7 c,4-py-B, respectively. In the former case, this effect is probably due to the strong electron-donating properties of the 2-thienyl group in coumarin 7 a,2-th, but it is not clear why the 4-[N-(3-sulfopropyl)pyridinium] group in compound 7 c,4-py-B provides a much larger Stokes shift (230 nm) than the structurally similar 2-[N-(3-sulfopropyl)pyridinium] group (145–150 nm; compounds 7 b,2-py-B and 7 c,2-py-B). Perhaps, the larger dipole moments of compounds 7 b,4-py-B and 7 c,4-py-B (compared with 7 b,2-py-B and 7 c,2-py-B, respectively) account for the larger Stokes shifts. Similarly, 4-pyridyl substituents shift the emission bands more to the red compared with 2-pyridyl groups (emission maxima for 7 b,4-py/7 b,2-py and 7 c,4-py/7 c,2-py are 593/578 and 611/591 nm, respective-
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ly). However, dyes with huge Stokes shifts are not particudye 7 c,4-py-B. Its absorption and emission maxima are shiftlarly useful because, as a rule, they possess low fluorescence ed in opposite directions in conjugates with sheep antiquantum yields. Ethanol was used as the solvent for measmouse antibodies: The absorption is redshifted (+ 18 nm) urements of the fluorescence quantum yields of free dyes and the emission maximum blueshifted (32 nm). For the (Table 1) because in this solvent the emission efficiencies other dyes in Table 2, these deviations are considered to be are high enough to allow the differences between the dyes moderate because the absorption and emission bands in to be detected. In aqueous solutions (even in the presence coumarins are broad. The “long” components of the fluoresof bovine serum albumin (BSA), in the unbound state), the cence lifetimes of the antibody conjugates also deviate from fluorescence quantum yields of many of the dyes in this the values recorded for the free dyes. Interestingly, higher study are too low to allow detection and interpretation of and lower values of t1 were found, with the absolute deviaany pronounced and reliable variation in the fluorescence tions in the range of 1.1 to + 2.2 ns. A decrease in the lifequantum yields with any certainty. The emission efficiencies time of the excited state (t) may be associated with a diminof the compounds fluctuate in a wide range (9–80 %), and ished fluorescence quantum yield (f) if non-emissive transithe fluorescence lifetimes also vary in a relatively broad tions from the excited state (S1) become more pronounced. range (1–4.5 ns; Table 1). In this case, the radiative rate constants (kr = f/t) do not These results were obtained for solutions in ethanol, and change very much in the series of compounds. Alternatively, in this respect they are not directly relevant to the choice of these two parameters (f and t) may be independent of each the “best” dyes for use as labels for biomolecules. All the other, and the quantum yield may stay constant or even inbioconjugates were investigated in aqueous solutions. After crease, whereas the lifetime decreases (when the probability derivatization and in aqueous buffers, the fluorescence quantum yields may change unpredictably (most often deTable 2. Properties of the coumarin dyes conjugated with sheep anticrease). Nevertheless, the data from Table 1 helped us to mouse antibodies.[a] select dye candidates suitable for conjugation with antibodlem f t1 Dye conjugate DOL[b] labs t2 ies and immunofluorescence microscopy (Table 2). To ach[nm] [nm] [%] [ns][c] [ns][c] ieve this, we subjected the selected dyes to saponification 7 a,CH=CH-4-py-A 3.6 491 645 2 2.1 (16) 0.57 (9.4) and transformed the free acids into N-hydroxysuccinimidyl 7 b,4-py-B 0.6 463 638 8 2.2 (16) 1.0 (19) 7 b,2-py 10 440 576 5 3.8 (4.4) 1.1 (2.8) (NHS) esters according to standard protocols.[20] These 10 473 609 5 3.1 (51) 0.55 (11) 7 c,CH=CH-2-py[d] active esters were coupled with polyclonal (secondary) anti7 c,4-py-B 0.9 492 655 12 – – bodies and the physical parameters of the conjugates were – 9,CH=CH-2-py,H 11.6 479 607 4[e] 1.5[f] studied (see Figure 2 and Table 2). The potential of the dyes 7 c,H 3.5 423 544 8 3.2 (4.9) 0.25 (0.4) in confocal and STED microscopy were assessed by stan7 d,H 14 426 539 10 3.5 (5.0) 0.24 (0.2) 9,H,H 1.3 425 541 21 3.0 (5.7) 0.22 (0.3) dard immunofluorescence staining by using these conjugate[23a] s. Although most of the new dyes do not carry polar [a] Measured in PBS buffer solutions at pH 7.4; for the structures and spectral properties of the free dyes, see Scheme 1 and Table 1, respectivecharged groups or many hydrophilic residues, coupling to ly. [b] Degree of labeling (DOL): the average number of dye residues atantibodies according to the standard protocol[20] very often tached to one protein molecule with M ~ 150 000. [c] Lifetimes of the exprovided values for the degree of labeling (DOL) in the cited states with values in parentheses denoting the amplitudes of the range of 10 (see Table 2). As a rule, higher DOL values lead “long” and “short” components, respectively. [d] Goat anti-rabbit. [e] The same fluorescence quantum yield was found for the conjugate to lower fluorescence quantum yields of the conjugates due with goat anti-rabbit antibodies with DOL = 5.6. [f] Monoexponential fit; to selfquenching of the emission (due to the formation of t = 1.7 ns for the conjugate with goat anti-rabbit antibodies (DOL = 5.6). [23b] non-emitting dimers of the dye residues). This is also true for most of the coumarin dyes shown in Table 1. Smaller amounts of NHS esters provide conjugates with lower values of DOL (1–4), but higher fluorescence quantum yields (21–8 %). The absorption and emission maxima of the dye conjugates (measured in phosphate buffer saline (PBS)) do not agree very precisely with those of the corresponding free dyes (measured in ethanol). The deviations for the absorption maxima are 9 to + 18 nm, and Figure 2. Normalized absorption (left) and emission (right) spectra of coumarin 9,CH=CH-2-py (Star470SX + , green line) and rhodamine dye (Abberior Star635, red line) attached to antibodies in aqueous PBS buffer at for the emission maxima 32 to pH 7.4. Convenient excitation sources provide light of 488–532 nm (absorbed by Star470SX +) and 633– + 17 nm. The highest deviations 640 nm (absorbed by Star635), these regions are shown in green and red, respectively. For structures, see were found for the red-emitting Figure 4.
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of the radiative transition S1!S0 increases). In this case the radiative rate increases and the nonradiative rate (knr = (1f)/t) decreases.[24] The values of kr and knr for the dyes and their conjugates were calculated from the data in Tables 1 and 2 and are given in Table S4 in the Supporting Information. In the case of bioconjugates, nearly all the fluorescence quantum yields are lower than those of the free dyes (except for compound 9,CH=CH-2-py,H-AB, which displays higher t1 values in the conjugated states). And indeed, for most dye conjugates in Table 2, the t1 values are, as expected, smaller than those of the free dyes, and the relative amplitudes of the “short” (t1) components are nearly negligible. In three cases, 7 a,CH=CH-4-py-A, 7 b,4-py-B, and 7 b,2-py, the t1 values are larger and the t2 values are smaller than the fluorescence lifetimes of the free dyes. In these cases only the biexponential fits with comparable amplitudes of the “long” (t1) and “short” (t2) components gave a good approximation to the fluorescence decay curves (Table 2). For two-color imaging with the commercial Leica TCS STED microscope, it is necessary to have red-emitting dyes that can be depleted with powerful 750–770 nm light. The imaging performance of the dyes and the scope of their applicability depend on their photostability, the fluorescence quantum yields of their conjugates, the degree of labeling (DOL), intersystem crossing rates, excited-state lifetimes, and the absence of unspecific labeling. The photostabilities of coumarin dyes 7 a,CH=CH-2-py-A, 7 b,CH=CH-4-py-B, 7 c,2-py-B, and 9,CH=CH-2-py,H were evaluated by using 488 nm laser light, which is typically applied as an excitation source (Figure 3). For comparison, we used fluorescein and the new Atto490LS (long Stokes shift) dye. All these dyes were found to have similar resistance against photobleaching (comparable to that of fluorescein). Fluorescein is known to be less photostable than the spectrally identical RhoACHTUNGREdamine 110 and in general fluorescein derivatives bleach much faster than rhodamines. These results indicate that the coumarins in this study (and Atto490LS) are only moderately photostable under illumination with the excitation light (488 nm). Compound 7 a,CH=CH-2-py-A did not bleach totally, which might be explained by the formation of a product with a new and more photoresistant fluorophore. It is also important to note that STED microscopy requires dyes that do not bleach very quickly under irradiation with very strong red light (750–770 nm). In this respect, moderate photostabilities under the excitation conditions do not preclude the use of these dyes in super-resolution microscopy with depletion of the excited state by stimulated emission. As a fluorescent marker with a small Stokes shift, we chose the photostable and hydrophilic rhodamine dye Abberior Star635 (Figures 2 and 4; absorption and emission maxima at 635 and 655 nm, respectively), which was designed according to the features mentioned above and was shown to perform very well under STED conditions.[25] The need for some absorption at 750–770 nm suggests that redemitting compounds with emission maxima at wavelengths
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Figure 3. Photostability measurements showing the decrease in the emission intensity of selected coumarins and two commercially available dyes (Fluorescein and Atto490LS) in a PVA matrix exposed to constant excitation with laser light of 488 nm (50 W cm2).
greater than 580 nm may be depleted very efficiently (e.g., 7 a,2-th, 7 a,CH=CH-2-py-A, 7 a,CH=CH-4-py-A, 7 a,4-py-A, 7 a,4-py-B (prepared from 7 a,4-py and 1,3-propanesultone),
Figure 4. Structures of the fluorescent dyes Star470SX + and Star635 used in two-color STED microscopy.
7 b,4-py-B, 7 c,2-py, 7 c,2-py-B, 7 c,4-py-B, 7 c,2-th, 7 c,CH= CH-2-py, and 9,CH=CH-2-py,H). However, this is not a severe limitation because the emission bands of coumarins are broad and, in fact, all dyes from Table 1, except for 7 c,H, 7 d,H, and 9,H,Et, may undergo stimulated emission under irradiation with very powerful 750 nm light. Compound 7 c,CH=CH-2-py (Table 1) possesses the highest fluorescence quantum yield (80 %), and it was possible to obtain its conjugates with antibodies with a DOL value of 10 and an emission efficiency of 5 % (Table 2). In immunofluorescence microscopy, bioconjugates are employed under physiological conditions, and the absence of a fluorescence background (due to unspecific staining) is particularly important. In this respect, hydrophilic compounds are advantageous as they possess polar (ionic) groups that provide high solubility in aqueous buffers (enabling to increase the values of DOL), prevent aggregation and self-quenching of the dye residues, reduce unspecific binding, and increase the fluorescence quantum yields.[26] Therefore, as a test substance, we chose the phosphorylated coumarin 9,CH=CH-2-
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py,H (Table 1 and Figure 4) obtained from the nonpolar precursor 7 c,CH=CH-2-py (Scheme 2) and evaluated the possibility of using it in two-color STED microscopy together with the Star635 dye as reference (for spectra and structures, see Figures 2 and 4). The results are illustrated in Figure 5 and Figure S1 in the Supporting Information, which present the image of the Golgi apparatus in a HeLa cell. A ribbon-like structure localized in the perinuclear region is typical of Golgi staining. Golgi cisternae are impossible to image in sufficient detail by using a conventional confocal microscope with a lateral resolution of around 250 nm, which is much larger than the thickness of a single Golgi cisterna (around 100 nm). The transmembrane protein GPP130 is localized to the cisGolgi,[27] whereas p230 is associated with the trans-Golgi network.[28] These two Golgi markers were imaged by using indirect immunofluorescence with secondary antibodies labeled with Star470SX + and Star635 dyes (with a Stokes shift of 128 and 20 nm, respectively). For this purpose, one
FULL PAPER mentary dye with a large Stokes shift that can be used in multicolor confocal and STED microscopy without appreciable cross-talk. In this respect, it is a valuable addition to the family of red-emitting fluorescent dyes with long Stokes shifts (e.g., ChromeoTM 494)[29] intended for use in STED microscopy. The absence of cross-talk is particularly important because it provides an additional “degree of freedom” and enables two-color imaging and co-localization of biological objects without using any linear unmixing schemes, which require supplementary experiments and complicate the acquisition and procession of data.
Conclusion
Coumarins with a CF3 group attached at C-4 and 3-(hetero)aryl or 3-[2-(hetero)arylethenyl] groups have been synthesized. Aminophenols 4 a–c were used as starting materials and various coumarins with large Stokes shifts were prepared by the synthetic routes given in Scheme 1. This approach enACHTUNGREabled us to modify the positions of the absorption and emission bands and, if necessary, shift them to the red spectral region in which the autofluorescence of cells and their organelles is negligible. Dyes with diverse polarities were synthesized by fusion of the 2,2,4-trimethyl-1,2-dihydroquinoline fragment to the coumarin fluorophore (providing the basic nonpolar scaffold), subsequent decoration with a primary hydroxy group (giving a dye of medium polarity), and phosFigure 5. STED (upper panel) and confocal (lower panel) two-color imaging of the Golgi apparatus. Localizaphorylation (providing a highly tion of Gpp130 (cis-Golgi) and p230 (trans-Golgi) in HeLa cells by indirect immunofluorescence using antiGpp130 and anti-p230 antibodies. Cells were then stained with Star470SX + and Star635 conjugated secondary polar dye). This approach alantibodies as indicated in the figure. Scale bars: 1 mm. lowed the synthesis of 9,CH= CH-2-py,H (Star470SX +), which performed well in twotunable STED depletion laser (750–770 nm) and two excitacolor confocal and STED microscopy when used together tion sources (532 and 640 nm pulsed diode lasers) were with the reference red-emitting rhodamine dye Star635, used. Under these conditions, both dyes provide an optical which has a small Stokes shift. Other interesting dyes were lateral resolution of 61–66 nm (see Figure S2), which is near identified, for example, compound 7 c,4-py-B (see Tables 1 the spatial resolution of the Leica TCS STED microscope. and 2) also emits in the red region, possesses a huge Stokes The imaging results obtained with the Star470SX + dye are shift of 213 nm (free dye as ethyl ester in ethanol) and 163 nm (in antibody conjugates), as well as acceptable fluocomparable to the imaging performance of Star635: Both rescence quantum yields (e.g., 12 % in protein conjugates dyes are bright and provide an excellent signal-to-noise with DOL = 0.9). The properties of this dye may be further ratio with almost no detectable background (unspecific improved by introducing a polar sulfonic or phosphoric acid binding is very low). Taking into account the fact that residue into the 2,2,4-trimethyl-1,2-dihydroquinoline moiety. Star635 was shown to be a reference dye (with a small Other interesting dyes emitting orange or green light, comStokes shift) in immunofluorescence assays,[25] we may conpounds 7 b,2-py and 7 c,H, respectively, also deserve further clude that the new coumarin dye Star470SX + is a comple-
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improvement by “hydrophilization”, which is expected to increase their solubility in aqueous solutions, fluorescence quantum yields, and brightness in optical microscopy.
Acknowledgements The authors are grateful to Nina Ohm, Marianne Pulst, and Maxim Sednev for recording optical spectra and calculating the DOL values and fluorescence quantum yields. We appreciate the assistance of E. Rothermel in the cell culture. We thank Dr. Haisen Ta for measurements of the fluorescence lifetimes of the Star470SX + dye and its bioconjugates. We are indebted to Bundesministerium fr Bildung und Forschung (BMBF 513) for financial support within the program Optische Technologien fr Biowissenschaften und Gesundheit (FKZ 13N11066). F.B. was supported by a Post-Doctoral Research Fellowship from the American-Italian Cancer Foundation. We are thankful to J. Jethwa for his critical reading of the manuscript.
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