Vadim P. Boyarskiy

Multicolor Far-Field Fluorescence Nanoscopy through Isolated Detection of Distinct Molecular Species

By combining the photoswitching and localization of individual fluorophores with spectroscopy on the single molecule level, we demonstrate simultaneous multicolor imaging with low crosstalk and down to 15 nm spatial resolution using only two detection color channels. The applicability of the method to biological specimens is demonstrated on mammalian cells. The combination of far-field fluorescence nanoscopy with the recording of a single switchable molecular species at a time opens up a new class of functional imaging techniques.

Rhodamines NN: A Novel Class of Caged Fluorescent Dyes**

Caged (that is, masked) fluorescent dyes are maintained in their nonfluorescent state by the incorporation of a photochemical labile group. The photosensitive masking group or “molecular cage” can be cleaved-off by irradiation with nearUV light, thereby rendering the dye fluorescent. Caged fluorescent dyes are of enormous interest for biological imaging because they may be used, for example, for the analysis of protein dynamics, multicolor fluorescence microscopy, and far-field optical nanoscopy. o-Nitrobenzyl groups are often used as masking groups; however, the use of these dyes is limited because of their rather complex synthesis and the unwanted by-products liberated by photolysis. Herein we report on the synthesis and characterization of a novel class of caged compounds—rhodamine NN dyes, which have a 2-diazoketone (COCNN) caging group incorporated into a spiro-9H-xanthene fragment (compounds 3 and 9-R in Schemes 1 and 3, respectively). This very simple and small caging group is the core element of a new class of masked rhodamines that have remarkable properties. The rhodamine NN dyes can be easily prepared and conjugated with biomolecules, they undergo rapid uncaging under standard irradiation conditions (with wavelengths 420 nm) with formation of highly fluorescent rhodamine derivatives, and they can be used in aqueous buffers, as well as in various embedding media utilized in imaging applications. In microscopy, these novel rhodamines may be used as labels alone or in combination with conventional fluorescent dyes and switchable rhodamine spiroamides. In the latter case, they enable new imaging protocols based on the stepwise activation and detection of several fluorescent markers. The combination of the new rhodamine NN derivative (9-R) with the photochromic spiroamide of rhodamine S and a normal (uncaged) N,N,N’,N’-tetramethylrhodamine resulted in a monochoromatic multilabel imaging scheme with low cross-talk, despite using three fluorophores with very similar absorption and emission spectra. Rhodamines are very photostable and bright fluorescent dyes which can readily be chemically modified and caged. Coumarines and fluorescein have also been used as caged fluorescent dyes. As a photocleavable unit, most of these caged compounds contain a 2-nitrobenzyl group or a derivative with an alkyl or a carboxy group in the a position to the phenyl ring (at the CH2 group) and/or one or two methoxy groups in the aromatic ring. Compounds with a free carboxy group are required for bioconjugation. However, the synthesis of caged rhodamines with a free (“second”) carboxy group is difficult and their yield is low. The 2-nitrobenzyl group and its substitutes are bulky and generate toxic, colored, and highly reactive 2-nitrosobenzaldehyde or 2-nitrosobenzophenone derivatives upon photolysis. These compounds or their oligomers are expected to be poisonous to living cells, and they are also colored and interfere with optical measurements. Other modern caging groups with the required absorption in the near-UV region are also bulky, rather lipophilic, and the procedures for their synthesis and introduction are often complex. For example, 2-(N,N-dimethylamino)-5-nitrophenol was reported to give photocleavable phenyl esters. 7-Diethylamino-4-(hydroxymethyl)-2H-chromen-2-one is known to form esters which can be cleaved easily by irradiation at 412 nm. Derivatives of 8-bromo-7-hydroxyquinoline and 6-bromo-7-hydroxycoumarines have also been proposed as light-sensitive protecting groups. The photolysis of these caged compounds generates light-absorbing by-products. We set out to prepare masked fluorescent dyes without bulky caging groups. A very small 2-diazoketone fragment would be an ideal caging group, provided that it is still possible to integrate this group into the colorless form of a fluorescent dye and then restore the fluorescent state by photolysis. Rhodamines are ideal for this purpose, because they contain a carboxy group, which is known to form colorless and nonfluorescent lactones or lactams with the spiro-9H-xanthene fragment. Furthermore, this carboxy group may be transformed into a 2-diazoketone residue. For the practical realization of this caging strategy, we used rhodamine B as a model compound and performed the reaction of diazomethane with its acid chloride 1. The yellow crystalline diazoketone 3 was obtained in high yield (Scheme 1). In the course of the facile caging reaction, the positively charged C9 atom of the xanthene fragment attacks the negatively charged carbon atom of the diazomethane residue in the intermediate 2. The simultaneous abstraction of a proton furnishes the stable five-membered ring. [*] Dr. V. N. Belov, Dr. C. A. Wurm, Dr. V. P. Boyarskiy, Dr. S. Jakobs, Prof. Dr. S. W. Hell Department of NanoBiophotonics Max Planck Institute for Biophysical Chemistry Am Fassberg 11, 37077 G ttingen (Germany) Fax: (+49)551-201-2506 E-mail: vbelov@gwdg.de cwurm@gwdg.de shell@gwdg.de Homepage: http://www.mpibpc.gwdg.de/abteilungen/200/

Alkenylation of Arenes and Heteroarenes with Alkynes

This review is focused on the analysis of current data on new methods of alkenylation of arenes and heteroarenes with alkynes by transition metal catalyzed reactions, Bronsted/Lewis acid promoted transformations, and others. The synthetic potential, scope, limitations, and mechanistic problems of the alkenylation reactions are discussed. The insertion of an alkenyl group into aromatic and heteroaromatic rings by inter- or intramolecular ways provides a synthetic route to derivatives of styrene, stilbene, chalcone, cinnamic acid, various fused carbo- and heterocycles, etc.

Difference in Energy between Two Distinct Types of Chalcogen Bonds Drives Regioisomerization of Binuclear (Diaminocarbene)PdII Complexes

The reaction of cis-[PdCl2(CNXyl)2] (Xyl = 2,6-Me2C6H3) with various 1,3-thiazol- and 1,3,4-thiadiazol-2-amines in chloroform gives a mixture of two regioisomeric binuclear diaminocarbene complexes. For 1,3-thiazol-2-amines the isomeric ratio depends on the reaction conditions and kinetically (KRs) or thermodynamically (TRs) controlled regioisomers were obtained at room temperature and on heating, respectively. In CHCl3 solutions, the isomers are subject to reversible isomerization accompanied by the cleavage of Pd-N and C-N bonds in the carbene fragment XylNCN(R)Xyl. Results of DFT calculations followed by the topological analysis of the electron density distribution within the formalism of Baders theory (AIM method) reveal that in CHCl3 solution the relative stability of the regioisomers (ΔGexp = 1.2 kcal/mol; ΔGcalcd = 3.2 kcal/mol) is determined by the energy difference between two types of the intramolecular chalcogen bonds, viz. S···Cl in KRs (2.8-3.0 kcal/mol) and S···N in TRs (4.6-5.3 kcal/mol). In the case of the 1,3,4-thiadiazol-2-amines, the regioisomers are formed in approximately equal amounts and, accordingly, the energy difference between these species is only 0.1 kcal/mol in terms of ΔGexp (ΔGcalcd = 2.1 kcal/mol). The regioisomers were characterized by elemental analyses (C, H, N), HRESI+-MS and FTIR, 1D (1H, 13C{1H}) and 2D (1H,1H-COSY, 1H,1H-NOESY, 1H,13C-HSQC, 1H,13C-HMBC) NMR spectroscopies, and structures of six complexes (three KRs and three TRs) were elucidated by single-crystal X-ray diffraction.

Polar Red-Emitting Rhodamine Dyes with Reactive Groups: Synthesis, Photophysical Properties, and Two-Color STED Nanoscopy Applications†

The synthesis, reactivity, and photophysical properties of new rhodamines with intense red fluorescence, two polar residues (hydroxyls, primary phosphates, or sulfonic acid groups), and improved hydrolytic stability of the amino-reactive sites (NHS esters or mixed N-succinimidyl carbonates) are reported. All fluorophores contain an N-alkyl-1,2-dihydro-2,2,4-trimethylquinoline fragment, and most of them bear a fully substituted tetrafluoro phenyl ring with a secondary carboxamide group. The absorption and emission maxima in water are in the range of 635-639 and 655-659 nm, respectively. A vastly simplified approach to red-emitting rhodamines with two phosphate groups that are compatible with diverse functional linkers was developed. As an example, a phosphorylated dye with an azide residue was prepared and was used in a click reaction with a strained alkyne bearing an N-hydroxysuccinimid (NHS) ester group. This method bypasses the undesired activation of phosphate groups, and gives an amphiphilic amino-reactive dye, the solubility and distribution of which between aqueous and organic phases can be controlled by varying the pH. The presence of two hydroxyl groups and a phenyl ring with two carboxyl residues in the dyes with another substitution pattern is sufficient for providing the hydrophilic properties. Selective formation of a mono-N-hydroxysuccinimidyl ester from 5-carboxy isomer of this rhodamine is reported. The fluorescence quantum yields varied from 58 to 92% for free fluorophores, and amounted to 18-64% for antibody conjugates in aqueous buffers. The brightness and photostability of these fluorophores facilitated two-color stimulated emission depletion (STED) fluorescence nanoscopy of biological samples with high contrast and minimal background. Selecting a pair of fluorophores with absorption/emission bands at 579/609 and 635/655 nm enabled two-color channels with low cross-talk and negligible background at approximately 40 nm resolution.

Facile Gold-Catalyzed Heterocyclization of Terminal Alkynes and Cyanamides Leading to Substituted 2-Amino-1,3-Oxazoles

Facile gold-catalyzed heterocyclization based upon intermolecular trapping of the generated α-oxo gold carbenes with various cyanamides R(2)R(3)NCN (R(2)/R(3) = Alk/Alk, -(CH2)2O(CH2)2-, Ar/Ar, Ar/H) has been developed. In most cases, 2-amino-1,3-oxazoles functionalized at the nitrogen atom as well as at the fifth position of the heterocyclic ring (12 examples) were isolated in good to moderate yields.

Metal-mediated coupling of a coordinated isocyanide and indazoles

A reaction between equimolar amounts of cis-[PdCl2(CNCy)2] (1) and indazole (HInd, 2) or 5-methylindazole (HInd(Me), 3) proceeded in refluxing CHCl3 for ca. 6 h affording the aminocarbene species cis-[PdCl2{C(Ind)=N(H)Cy}(CNCy)] (4) or cis-[PdCl2{C(Ind(Me))=N(H)Cy}(CNCy)] (5) in good (72-83%) isolated yields. Complexes 4 and 5 were characterized by elemental analyses (C, H, N), HR ESI(+)-MS, IR, and 1D ((1)H, (13)C{(1)H}) and 2D ((1)H,(1)H-COSY, (1)H,(13)C-HMQC/(1)H,(13)C-HSQC, (1)H,(13)C-HMBC) NMR spectroscopies, and complex 4 as well by X-ray diffraction. The observed coupling represents the first example of metal-mediated integration between any isocyanide and any aromatic heterocyclic system having an HN center.

Coupling of C-amino aza-substituted heterocycles with an isocyanide ligand in palladium(ii) complex

The reaction of cis-dichlorobis(2,6-xylylisocyanide)palladium(ii) with 2-aminopyrazine affords a binuclear palladium complex, in which one of the metal atoms is involved in the palladacyclic ligand. In the contrast, the coupling of isocyanide ligands in cis-dichlorobis-(2,6-xylylisocyanide)palladium(ii) with another C-amino aza-substituted heterocycle, viz.,4-acetyl-3-amino-5-methylpyrazole, gives a mononuclear cationic palladium diaminocarbene complex. Both compounds were characterized by elemental analysis, IR spectroscopy, 1H, 13C{1H}, DEPT90/DEPT135, and 1H,13C-HSQC/1H,13C-HMBC NMR spectroscopy, high-resolution electrospray ionization mass spectrometry, and X-ray diffraction.

Crystal structure of cis-[PdCl2(CNMes)2]

The interaction between PdCl2(CH3CN)2 and 2,4,6-Me3C6H2NC (MesNC) proceeds with the substitution of acetonitrile ligands and leads to the synthesis of a cis-[PdCl2(MesNC)2] complex. The structure of this compound is determined by single crystal X-ray diffraction (XRD). The complex has a slightly distorted square-planar structure of the metal center with two cis-positioned isocyanide ligands. In both CN isocyanide moieties the triple bonds have lengths similar to the lengths of the respective bonds in other isocyanide complexes. In the structure, the cis-[PdCl2(MesNC)2] complexes are bound by weak С–H∙∙∙Cl hydrogen bonds and π-stacking interactions.

Cis/trans equilibrium as the way to form Pd carbene catalyst from trans-isocyanide complex

The behavior of Pd(II) bis-isocyanide complexes in solution was studied for examples of bis-(cyclohexylisocyanide)palladium(II) dichloride (1) and diiodide (2). The equilibrium between isomeric cis- and trans-forms of 1 was observed by NMR and IR spectroscopy. Complex 1 (which has the cis-configuration in the solid state) gives cis-/trans-isomeric mixture after dissolving in CDCl3 for several days at room temperature. The effect of heating on the equilibration rate was considered. The studied equilibrium leads to a purely cis-structure of Pd carbene complex (3) that was synthesized via the interaction of cis-/trans-isomeric mixture of 1 with benzophenone hydrazone.