Ewald Daltrozzo

Noninvasive Photoacoustic and Fluorescence Sentinel Lymph Node Identification using Dye-Loaded Perfluorocarbon Nanoparticles

The contrast mechanisms used for photoacoustic tomography (PAT) and fluorescence imaging differ in subtle, but significant, ways. The design of contrast agents for each or both modalities requires an understanding of the spectral characteristics as well as intra- and intermolecular interactions that occur during formulation. We found that fluorescence quenching that occurs in the formulation of near-infrared (NIR) fluorescent dyes in nanoparticles results in enhanced contrast for PAT. The ability of the new PAT method to utilize strongly absorbing chromophores for signal generation allowed us to convert a highly fluorescent dye into an exceptionally high PA contrast material. Spectroscopic characterization of the developed NIR dye-loaded perfluorocarbon-based nanoparticles for combined fluorescence and PA imaging revealed distinct dye-dependent photophysical behavior. We demonstrate that the enhanced contrast allows detection of regional lymph nodes of rats in vivo with time-domain optical and photoacoustic imaging methods. The results further show that the use of fluorescence lifetime imaging, which is less dependent on fluorescence intensity, provides a strategic approach to bridge the disparate contrast reporting mechanisms of fluorescence and PA imaging methods.

Selective NIR chromophores : Bis(Pyrrolopyrrole) Cyanines

New applications have recently led to an increased interest in near infrared (NIR) dyes. Both, strong NIR absorption and fluorescence emission are important phenomena. While fluorescence is exploited especially for labelling purposes in microscopy, NIR absorption has a broad range of applications mainly in material science. Examples of growing fields of interest are NIR light emitting diodes and the use of NIR-photosensitizers in dye-sensitized solar cells. In the latter cases, the aim is to utilize the NIR part of the solar spectrum in order to generate photocurrents. In many applications, especially selective NIR absorbers which absorb strongly in the NIR while exhibiting negligible absorption in the visible spectral range are required. Dyes with these properties find use as NIR absorbers in paints and windows to block off heat, in laser protecting glasses, as dyes for laser polymer welding of transparent components, or as anti-forgery markers. In all these examples it is required that the dye does not influence the spectral properties of the components in the visible. Thus, photostable dyes with strong, narrowbandwidth NIR absorptions and negligible absorption in the visible spectral range are needed. Common strategies to achieve bathochromic shifts of the absorption maximum of a dye are the extension of the chromophoric system or the introduction of donor and acceptor groups into the chromophore. Cyanine dyes and the rylene dyes are the most popular classes showing significant bathochromic shifts upon extension of their π-system. In cyanine dyes, however, the chemical stability decreases with increasing extension. By contrast, rylene dyes are chemically very stable, but, due to comparably large bondlength changes accompanying the S0→S1 transition, they show rather intense 01 and 02 vibronic bands (Franck-Condon principle). Thus, their S0→S1 absorption is extended into the visible range. Other important classes of NIR dyes, showing strong and narrowband NIR absorption are squarine dyes, bodipys and some aza[18]annulene derivatives such as porphyrins phthalocyanines and especially naphtolocyanines. We recently described pyrrolopyrrole cyanine (PPCy) dyes as a new class of NIR dyes and fluorophores. PPCys are synthesized by a condensation reaction of diketopyrrolopyrrole (DPP) 1 and heteroaromatic acetonitriles (HAA) 3. Complexation with either BF2 or BPh2 yields fluorophores which exhibit narrowband absorption between 650 and 900 nm and fluorescence emission with high quantum yields. By isolation of the monoactivated DPP 2, selectivly one of the carbonyl groups of the DPP can be reacted with a HAA 3 to the half converted products 4. Further reaction with another acetonitrile derivative 3 leads to PPCys with an asymmetric substitution pattern. This stepwise reaction scheme allows for the introduction of just one functional group. Here, we describe the synthesis of a new class of NIR chromophores with unprecedented spectral properties. They have very strong and narrowband NIR absorption while featuring negligible visible absorption. The absorption coefficients of these dyes in the NIR belong to the strongest ever reported for organic fluorophores, being twice as high as those of recently reported rylene dyes. Our previous approaches to shift the absorption of the PPCys bathochromically concerned the modification of the heteroaromatic endgroup (A) and/or the complexation agent. The selective synthesis of the halfconverted DPPs 4 opens a strategy to enlarge the chromophore of the PPCys by reacting two equivalents of 4 with a bifunctional bridging heteroaromatic acetonitrile such as 5 (Scheme 1.) The resulting condensation products contain two pyrrolopyrrole moieties and can therefore be called bis-pyrrolopyrrole cyanine dyes.

Pyrrolopyrrole cyanine dyes: a new class of near-infrared dyes and fluorophores.

Pyrrolopyrrole cyanine (PPCy) dyes are presented as a novel class of near-infrared (NIR) chromophores, which are synthesized in a condensation reaction of diketopyrrolopyrrole with heteroarylacetonitrile compounds. Their optical properties are marked by strong and narrow-band NIR absorptions. Complexation products with BF(2) and BPh(2) show strong NIR fluorescence and hardly any absorption in the visible range. We synthesized a series of new PPCys that differ only in the heterocyclic peripheral groups of the chromophore. With this strategy, the absorption spectra can be tuned between 684 and 864 nm, while high fluorescence quantum yields are maintained. The influence of the heterocycle on the optical properties of the dyes is discussed.

Long Fluorescence Lifetime Molecular Probes Based on Near Infrared Pyrrolopyrrole Cyanine Fluorophores for In Vivo Imaging

Fluorescence lifetime (FLT) properties of organic molecules provide a new reporting strategy for molecular imaging in the near infrared (NIR) spectral region. Unfortunately, most of the NIR fluorescent dyes have short FLT typically clustered below 1.5 ns. In this study, we demonstrate that a new class of NIR fluorescent dyes, pyrrolopyrrole cyanine dyes, have exceptionally long FLTs ranging from 3 to 4 ns, both in vitro (dimethyl sulfoxide and albumin/water solutions) and in vivo (mice). These results provide a new window for imaging molecular processes, rejecting backscattered light and autofluorescence, and multiplexing imaging information with conventional NIR fluorescent dyes that absorb and emit light at similar wavelengths.

Azacyanines of the Pyrrolopyrrole Series

The reaction of POCl3-activated, readily soluble diketopyrrolopyrrole (DPP) with 2-aminoheteroaromatics to yield 1:1 and 1:2 hydrogen chelates is described. Complexation of these hydrogen chelates with boron reagents results in thermally and photochemically stable fluorescent dyes (PP-azacyanines). The 1:2 complexes in particular absorb at long wavelengths and are brightly fluorescing. The rich photophysics of the new compounds are presented. Both the pronounced vibrational fine structure of the S0 → S1 transitions and the observed fluorescence phenomena allow detailed conclusions to be made on the correlation between molecular structure and optical properties.

Water-soluble pyrrolopyrrole cyanine (PPCy) near-infrared fluorescent pH indicators for strong acidity

Water-soluble, fluorescence-switchable derivatives of pyrrolopyrrole cyanines (PPCys) which serve as near-infrared pH indicators have been synthesized. The dyes reported have pKa values of 2.4–3.4 and thus are suitable for sensing in highly acidic environments. The new pH probes were evaluated by fluorescence imaging experiments in a cellular environment.

Biological applications of fluorescence lifetime imaging beyond microscopy

Fluorescence lifetime is a relatively new contrast mechanism for optical imaging in living subjects that relies on intrinsic properties of fluorophores rather than concentration dependent intensity. Drawing upon the success of fluorescence lifetime imaging microscopy (FLIM) for investigation of protein-protein interactions and intracellular physiology, in vivo fluorescence lifetime imaging (FLI) promises to dramatically increase the utility of fluorescencebased imaging in preclinical and clinical applications. Intrinsic fluorescence lifetime measurements in living tissues can distinguish pathologies such as cancer from healthy tissue. Unfortunately, intrinsic FLT contrast is limited to superficial measurements. Conventional intensity-based agents have been reported for measuring these phenomena in vitro, but translation into living animals is difficult due to optical properties of tissues. For this reason, contrast agents that can be detected in the near infrared (NIR) wavelengths are being developed by our lab and others to enhance the capabilities of this modality. FLT is less affected by concentration and thus is better for detecting small changes in physiology, as long as sufficient fluorescence signal can be measured. FLT can also improve localization of signals for improved deep tissue imaging. Examples of the utility of exogenous contrast agents will be discussed, including applications in monitoring physiologic functions, controlled drug release and cancer biology. Instrumentation for FLI will also be discussed, including planar and diffuse optical imaging in time and frequency domains. Future applications will also be discussed that are being developed in this exciting field that complement other optical modalities.

‘Green’ Synthesis of 2-Substituted 6-Hydroxy-[3H]-pyrimidin-4-ones and 4,6-Dichloropyrimidines : Improved Strategies and Mechanistic Study

An improved route to 2-substituted 6-hydroxy-[3H]-pyrimidin-4-ones 4 and to 2-substituted 4,6-dichloropyrimidines 5 is reported. Without using highly toxic reactants, compounds 4 can be prepared conveniently in a one pot synthesis on a one mol scale with average yields up to 80 %. 4,6-Dichloropyrimidines 5, which are usually prepared in small quantities, are synthesized with average yields of 80 %, using up to 80 g of starting material. The mechanism of the chlorination of 4 is investigated computationally for the first time. The results suggest that the chlorination with phosphoryl chloride occurs in an alternating phosphorylation–chlorination manner (pathway 1) which is preferred over a sequence which starts with two phosphorylations. The investigated 4,6-dichloropyrimidines described herein form strong complexes with dichlorophosphoric acid but weak complexes with hydrochloric acid (generated during workup). These latter complexes explain the necessity of using aqueous sodium carbonate during the working up. In order to prevent possible formation of pyrimidinium salts between intermediates or the final dichloropyrimidines and unreacted hydroxypyrimidone, the latter could be deactivated with a strong acid such as dichlorophosphoric acid, thus allowing chlorination but prohibiting salt formation. Because of its general applicability to all nitrogen heterocycle chlorinations with phosphoryl chloride, the proposed route to dichloropyrimidines without solvent or side products, using less toxic reactants, is of general synthetic interest.

Pyrrolopyrrole Cyanines: Effect of Substituents on Optical Properties (Eur. J. Org. Chem. 19/2011)

The cover picture shows a series of selected absorption spectra (solid lines) from different substituted pyrrolopyrrole cyanine (PPCy) dyes. This is a new class of chromophores recently developed at the University of Konstanz, located at Lake Constance (upper left). PPCys feature preciously unattainable high fluorescence quantum yields in the near infrared region, which besides other applications makes them attractive as fluorescence labels. One emission spectrum is shown as a dotted line. The core structure of the PPCys is depicted in the upper right corner. Proper substitutions of the core structure allow the generation of dyes absorbing over a broad wavelength range. Details are discussed in the article by A. Zumbusch et al. on p. 3421 ff.