TOWARDS NOVEL NAPHTHALENE BASED NEAR INFRARED DYES FOR BIOIMAGING APPLICATIONS GOUTAM PRAMANIK (M.Sc), NUS A THESIS SUBMITTED FOR THE DEGREE OF MASTERS BY RESEARCH Under the supervision of ASSOCIATE PROFESSOR TANJA WEIL DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2010 1 Acknowledgement: I offer my sincerest gratitude to my supervisor, Associate Professor Tanja Weil, who has supported me throughout my thesis with her patience and knowledge whilst allowing me the room to work in my own way. I attribute the level of my Masters degree to her encouragement and effort and without her, this thesis, too, would not have been completed or written. One simply could not wish for a better or friendlier supervisor. 2 Table of Content:  SUMMARY......................................................................... 4  ABBREVIATIONS AND DEFINITIONS........................ 5  INTRODUCTION .............................................................9  THE AIM OF THE THESIS ............................................25  RESULTS AND DISCUSSION .......................................25  CONCLUSION.................................................................36  EXPERIMENTAL DETAILS ........................................37  OVERVIEW OF RELEVANT SPECTRA………........42  BIBLIOGRAPHY ............................................................47 3 Summary: Fluorescent dyes based on small organic molecules that emit light in the near infrared (NIR) region are of great current interest in material science as well as in bio-imaging and chemical biology. They allow imaging of biological samples with minimal autofluorescence, reduced light scattering, and high tissue penetration. In the present work, reaction schemes towards blue coloured NIR-dyes based on the naphthalene diimide (NDI) scaffold have been designed starting from 2, 6-dibromonaphthalene dianhydride as the central building block. Different substituents have been attached to the NDI scaffold via condensation and nucleophilic substitution of the bromo-substituents with derivatives carrying primary or secondary amino groups. In this way, symmetrically substituted naphthalene diimide (NDI) derivatives displaying high quantum yields and large stokes’ shifts have been achieved. 4 Abbreviations and definitions DCM Dichloromethane DMF Dimethylformamide NMR Nuclear magnetic resonance TLC Thin layer chromatography MW Microwave DBI Dibromoisocyanuric acid Et3N Triethylamine UV Ultraviolet NIR Near Infrared NIRF Near Infrared Fluorophore nm Nanometer NDI Naphthalenediimide CT Charge transfer Conc concentrated 5 List of figure: Figure 1- Jablonski diagram.............................................10 Figure 2- Stokes’ shift.......................................................12 Figure 3- Near Infrared (NIR) Window……………………19 Figure 4 -HOMO, LUMO, and transition density............23 Figure 5- LCMS analysis of crude N,N´-Bis(2-hydroxyethyl)-2, 6-di (n-2-hydroxyethyl)-1, 4, 5, 8Naphthalene tetracarboxylic Acid Diimide (NDI-1).......35 6 List of scheme: Scheme 1: General scheme for synthesis of symmetric and unsymmetric core-substituted naphthalenediimide chromophores...........................26. Scheme 2: Synthesis and reaction mechanism of the preparation of dibromoisocyanuric acid...................................................................27. Scheme 3: Synthesis and reaction mechanism of 2, 6dibromonaphthalene-1,4,5,8-dianhydride........................................30. Scheme 4: Synthesis of NDI-1 & NDI-2..........................................32. Scheme 5: Discussion of the challenges focussing on the low reaction yields of NDI-1 & NDI-2.................................................................34. Scheme 6: Synthesis of N, N´-Bis-(ethyl)-1, 4, 5, 8naphthalenetetracarboxylic acid diimide (3)..................................36. 7 List of spectra: 1 1. The H NMR (300 MHz) spectrum dibromoisocyanuric acid (1) in DMSO-d6.....................................................................42. 1 2. The H NMR (300 MHz) spectrum monobromanaphthalene dianhydride in DMSO-d6........................................................43. 3. The 1H NMR (300 MHz) spectrum of NDI-1 in D2O..........44. 1 4. The H NMR (300 MHz) spectrum of NDI-2 in D2O….....45. 1 5. The H NMR (300 MHz) spectrum of 3 in CDCl3................46. 6. Optical spectra of NDI-1……………………………............46. 7. Optical spectra of NDI-2…....................................................47. 8 Introduction: Introduction to fluorescence: In 2008, the Nobel Prize in chemistry was given to Osamu Shimomura, Martin Chalfie and Roger Y. Tsien for their discovery and development of green fluorescent protein (GFP). GFP represents a fluorescent protein that can be genetically encoded to be attached to a large variety of different proteins that become fluorescent after labeling. In this context, 2008 can be considered an auspicious year for fluorescence-based bio-imaging. This innovation has revolutionized the way cellular processes; protein interactions and biological process are visualized and largely improved our understanding of fundamental cellular processes. To date, the detection of emitted light is an indispensable tool to detect and visualize all different kinds of processes and it is successfully applied in many different disciplines. 9 The term 'fluorescence' was coined by George Gabriel Stokes in his 1852 paper titled "On the Change of Refrangibility of Light".1 Fluorescence is an optical process, by which a molecule is promoted to an excited state by absorption of photons and then emits a photon as it relaxes to its ground state. This possible process of interaction between light and molecules can be explained by using the Jablonoski Diagram (Figure 1). Figure 1. The Jablonski diagram. It illustrates the electronic &vibrational states of a molecule and the transitions between them. 10 Absorption of photons excites the molecule from the ground state (S0) to an excited stated state (typically S1 or S2). From the excited state, the molecule can relax back to the ground state by several pathways. Non radiative transition between two states of same multiplicity (S2→S1) or different vibration levels of the same electronic state is termed as internal conversion (IC). In this process, the molecule looses vibrational and rotational energy. Relaxation of a molecule from S1→S0 with emission is called fluorescence. Absorption from S0 can proceed to a higher vibrational level of the S1 state and decay from the S1 to S0 state might not proceed to the lowest vibrational level. In this case, some energy is lost during this internal conversion process. In this case, the emission spectrum reveals bands of lower energy and consequently at longer wavelength. Stokes shift is the difference between positions of the band maxima of the absorption and emission spectra of the same electronic transition (Figure 2). It can be expressed in frequency unit (cm-1). 11 Figure 2. Stokes shift. When a molecule absorbs a photon, it gains energy and enters an excited state. The molecule losses some energy in non-radiative pathway. Thus the emitted photon has less energy than the absorbed photon, this energy difference is the Stokes shift. Another possibility of deactivating the excited state is called intersystem crossing (ISC) and refers to a process in which the S1 state transition proceeds first to the triplet transition state (T1). From the T1 state, the S0 state could be reached by a slow radiative process called phosphorescence. Transition from T1 to S0 is forbidden and therefore, timescales for phosphorescence are usually much slower than those for fluorescence. Since T1 has lower energy than S1, emission of 12 phosphorescence is usually more bathochromically shifted than fluorescence emission. There are some other important characteristics which are related to fluorescence and which are important to characterize dye molecules and allow a comparison of their relative performance such as the fluorescence quantum yield (фF) and the excited state lifetime (τs). фF refers to the ratio of the number of photon emitted versus the number of photon absorbed. It can also be defined as the rate of radiative decay ( Ksr) from S1 to S0 to the sum of the rate of the radiative decay ( Ksr) from S1 to S0 and the rate of the nonradiative decay ( Ksnr) from S1 to S0. Equation 1: The excited state lifetime is defined in equation 23 13 Equation2: Thus, the fluorescence quantum yield is proportional to the fluorescence lifetime. Equation 3: There are several mechanism by which fluorophores can act. The most common is staining, where the fluorophore gets accumulated at a particular organelle of a cell, which can be visualised by fluorescence techniques. In some cases, probes undergo a physical change which alters their optical properties during excitation. An analyte can covalently interact with the dye which leads to spectra change of the dye. Cleavage of certain functional group might 14 quench the fluorescence of the dye and can ‘turn on’ an optical response.2 Fluorescence Resonance Energy Transfer (FRET) is a very important concept that relies on the distance-dependent transfer of energy from a donor molecule to an acceptor molecule. Due to its sensitivity to the distance between the chromophores, FRET has been used to investigate and characterize molecular interactions. FRET refers to the radiationless transmission of energy from a donor molecule to an acceptor molecule. The donor molecule represents the dye or chromophore that initially absorbs the energy and the acceptor represents the chromophore to which the energy is subsequently transferred. This resonance interaction occurs over greater than interatomic distances, with neglectable conversion to thermal energy and usually without any molecular collision. The transfer of energy leads to a reduction in the donor’s emission intensity and the excited state lifetime, and an increase in the acceptor’s emission intensity. A pair of molecules that interact in such a manner that FRET occurs is often referred to as a donor/acceptor pair. Cleavage of one entity of the FRET pair generally affects the absorption and emission maxima. 4 15 The polarity of the environment also affects the photophysical properties of the dye molecule. Solvatochromic dyes change their colour according to the polarity of the liquid in which they are dissolved due to a significant difference in the dipole moment between the ground state and the first excited state.5 For example; the long-wavelength absorption of pyridinium betaine dyes is shifted towards shorter wavelengths by changing from a nonpolar to a polar solvent.6 One of the common electronic interactions is photoinduced electron transfer (PET) between organic fluorophores and suitable electron donating moieties. PET-quenching has been used as reporter for monitoring conformational dynamics in polypeptides, proteins, and Oligonucleotides.7 In PET, electrons from the HOMO of the donor are transferred to the LUMO of the fluorophore at the excited state thus quenching the fluorescence. Upon binding, homo of the donor is lowered, PET is disrupted and fluorescence is recovered.8 The self‐association of dye molecules in solution can occur due to intermolecular van der Waals like attractive forces between the 16 molecules. The aggregates in solution exhibit distinct changes in the absorption band as compared to the monomeric species. From the spectral shifts, various aggregation patterns of the dyes in different media can be proposed. Near Infrared Dyes ( NIR ) for Bioimaging: In recent years, fluorescence imaging using Near Infrared dyes has attracted much attention as it affords the opportunity for non-invasive in vivo imaging.9 Researchers are also encouraged by the continuous developments of imaging equipment, reconstruction algorithms, and more importantly the availability of imaging reporter molecules. These reporter molecules encompass exogenously administered probes detectable by fluorescence and/or bioluminescence imaging. One particularly enticing aspect of optical imaging is the ability to design reactive probes with inherent amplification.10 Optical imaging, which uses light at various wavelengths (UV to Near Infrared) for image generation, includes many 17 different acquisition techniques. Optical image contrast can be based on absorption, fluorescence, fluorescence lifetime and polarization.11 For fluorescence-based bioimaging, the optimum wavelength for excitation and emission ranges from 650–900 nm.12 This range of wavelength is called Near Infrared (NIR) Window (Figure-3). The interfering background signal of cells in the UV and visible region is due to autofluorescence of biological targets, which occurs when tissues, proteins or other biomarkers fluoresce naturally. Thus, a high background signal usually appears in the detection of biological samples when visible light used for excitation and collected after emission. The major advantage of fluorescence spectroscopy lies in a high signal to noise ratio and thereby achieving low detection limits. The distinct features of NIRF over UV and visible region fluorescence include a lower background signal from biological samples enabling higher signal to - noise ratios (SNR). 18 Figure 3. Near Infrared (NIR) Window. The NIR window is ideally suited for in vivo imaging because of minimal light absorption by hemoglobin (900 nm). Characteristics of suitable NIR dyes for bioimaging applicatons: The ideal NIRF fluorophore for in vivo bio-imaging should reveal the following characteristics: 1. A peak fluorescence close to 700–900 nm. 2. High quantum yield. 3. Narrow excitation/emission spectrum. 19 4. High chemical and photo-stability. 5. Non-toxicity. 6. Excellent cell permeability, biocompatibility, biodegradability, or excretability. 7. Availability of monofunctional derivatives for conjugations 8. Commercial viability and production scalability for large quantities ultimately required for human use. 9. Large stokes shift. 10. Easily tunable optical property. Despite the multitude of available dyes there is still considerable interest in new chromophore systems that satisfy the special demands of emerging technologies different disciplines such as e.g. biological and physical sciences. For example, new interest in fluorophores with NIR emission has arisen in conjunction with singlemolecule spectroscopy of biomolecules12 where most traditional NIR dyes lack the required fluorescence quantum yield and photostability or whose performance is hampered by aggregation of their extendedconjugated cores. The second point holds especially true for rylene 20 dye.13 Rylene dyes are ideally suited for single molecule spectroscopy (SMS) owing to their high fluorescence quantum yields and photostability.14 The rylene dyes mostly synthesized by the groups of Muellen, Wuerthner and Langhals opened up new possibilities on organic field effect transistors (OFETs), bioimaging. However, as a significant drawback, such dyes are difficult to solubilize sometimes even in organic solvents and exhibit a high tendency for the formation of dye aggregates due to their extended aromatic scaffolds which quenches fluorescence.15 On the other hand, the smallest representative of the rylene diimides, naphthalene diimide (NDI), is a colorless compound that emits below 400 nm and is considered nonfluorescent. It has been extensively applied as an extended aromatic building block in supramolecular chemistry. In recent years, core-unsubstituted NDIs were tailored for applications in numerous research fields such as light harvesting, design of supramolecular architectures, DNA intercalation. Due to their n-type semiconducting properties, core-unsubstituted NDIs bearing alkyl or fluorinated alkyl groups in the imide positions have been of interest as active layer in organic field effect transistors 21 (OFETs).16 Naphthalene diimide has also been used extensively by other groups as an electron acceptor in molecular arrays for photoinduced electron transfer owing to its low reduction potential, its high-lying excited-state and the intense and well-defined spectroscopic signature of the radical anion.17 NDIs can form large supramolecular structures through hydrogen bonding, leading to helical organic nanotubes of defined chirality.18 Also, supramolecular arrangement by ð-ð interactions were achieved resulting in rigid-rod ð-helical architectures, whose architectures are untwisted into open cation channels by intercalation of dialkoxynaphthalene ligands. Naphthalene diimide organogels were built by noncovalent interactions such as ð-ð stacking, hydrogen bonding, and van der Waals forces which serve as supramolecular hosts and sensors for different types of electron-rich naphthalene derivatives.19 Unlike the colourless NDI without substituents within the bay region of the NDI core, NDIs bearing two electron-donating substituents, reveal highly brilliant colours and strong fluorescence. Functionalization of NDIs by core substitution in the bay region triggered an eminent progress in controlling the optical and redox 22 properties of this class of dyes and thus extended the scope of their application. Their interesting electronic properties arise from a new CT transition in the visible wavelength range, which is strongly influenced by the electron-donating strength of the core substituents. It has been described that there are nodes on the HOMO and LUMO orbitals at the imide nitrogen atoms (Figure 4).20 Figure 4. HOMO, LUMO, and transition density for a) N,N - dimethyl naphthalene 1,4,5,8-tetracarboxylic acid bisimide and its b) 2-chloro-6-dimethylamino- and c) 2,6-dimethylamino-substituted derivatives according to CNDO/S calculations of AM1 optimized molecules.20 23 Accordingly, the electronic properties of naphthalene diimide are weakly affected by the substituents at the diimide region. Introduction of substituents onto these positions usually requires tedious, multi-step transformations. Very recently, Wuerthner’s group has simplified the synthetic procedure of core substituted NDI dyes.21 They have used 2,6dibromonaphthalene dianhydride as precursor molecule for achieving core-disubstituted NDIs. Two bromine substituents were introduced into the naphthalene core of 1,4,5,8-naphthalenetetracarboxylic acid dianhydride by electrophilic aromatic substitution using stoichiometric amounts of dibromoisocyanuric acid (DBI) in oleum (20% SO3) at room temperature. Alternatively, bromine has been used as bromination agent in the presence of catalytic amounts of iodine using oleum as a solvent resulting mainly in the desired dibrominated product as well as byproducts.22 Two bromo-substituents of 2,6-dibromonaphthalene dianhydride can substituted by nucleophiles such as alcohols, amines as well as thiol-derivatives yielding NDIs with electron-donating coresubstituents. 24 The aim of the thesis: This thesis aims at synthesizing core-substituted water soluble NDI chromophores by varying the amino-substituents at the imide and at the bay position and to study their optical and electrochemical properties. At a later stage these NDI chromophores will be applied for bioimaging applications. RESULT AND DISCUSSION: The synthesis of substituted NDI chromophores as reported by the group of Wuerthner is summarized in Scheme 1.20 The differences from the original scheme are 1) Conc sulphuric acid is used as a solvent for the preparation of 2, 6dibromonaphthalene dianhydride (2) from 1,4,5,8-Naphthalene dicarboxylic dianhydride (SM), instead of oleum (20 % SO3) because the oleum is not allowed to use in Singapore due to environmental reason. (2) Imidisation of 2, 6-dibromonaphthalene dianhydride (2) with amine I decided to take the advantage of microwave heating instead of normal heating. 25 2 SM M Scheme 1: General scheme for synthesis of symmetric and unsymmetric core substituted naphthalenediimide chromophore. Synthesis of dibromoisocyanuric acid (1) Since dibromoisocyanuric acid was not available for us, this important building block needed to be prepared on larger scale. Dibromoisocyanuric acid can be prepared from the commercially 26 available cyanuric acid by following the reported procedures (Scheme2).23 In order to stop the reaction at the required dibromo- product and to prevent cleavage of the urea ring, the reaction was performed at 4oC. The white, crystalline powder of dibromoisocyanuric acid gives a strong bromine odour and it needed to be stored in the refrigerator and wrapped with aluminium foil to protect it from light and moisture to avoid decomposition. Dibromoisocyanuric acid (1) was characterised by NMR and mass spectroscopy. The yield of is 37 %. 1 Mechanism 1 27 Scheme 2: Synthesis and reaction mechanism of dibromoisocyanuric acid Cyanuric acid tautomerise to ketone form in water solution. LiOH removes the acidic hydrogen from imide. Then the negative charge on the imide attacks the bromine to give N-bromo compound. Repeat of the same process give dibromoisocyanuric acid (1). Synthesis of 2,6-dibromonaphthalene-1,4,5,8-dianhydride (2): Prepartion of 2,6-dibromonaphthalene-1,4,5,8-dianhydride (2) from commercially available naphthalene dianhydride (SM) is very important step for this whole project. It can be either achieved via aromatic electrophilic substitution of aromatic protons or via aromatic nucleophilic substitution of hydride anions of the naphthalene dianhydride (SM) (scheme 3). Aromatic electrophilic substitution: 28 SM 2 Mechanism: SM 2 Aromatic nucleophilic substitution: 29 2 SM Mechanism: SM 2 Scheme 3: Synthesis and Mechanism of 2,6-dibromonaphthalene1,4,5,8-dianhydride (2). 30 Introduction of two bromine-groups at the core of the naphthalene dianhydride (SM) requires harsh condition since the naphthalene ring is highly deactivated for aromatic electrophilic substitution due to the presence of the two anhydride moieties. 2, 6-dichloro naphthalene dianhydride has successfully been prepared starting from pyrene24 but the yield is very low and this approach also requires the use of chlorine gas which is considered a safety risk if no dedicated equipment is available. Finally, 2, 6-dibromonaphthalene-1,4,5,8-dianhydride was obtained starting from naphthalene dianhydride (SM) by aromatic electrophilic substitution (Scheme-3). It has been reported before that the application of stoichiometric amounts of DBI and oleum (20% SO3) and naphthalene dianhydride yields dianhydride (2) as major product. 25 2,6-dibromonaphthalene-1,4,5,8Alternatively, elemental bromine can be used in the presence of catalytic amounts of iodine in oleum to give dibromonaphthalene dianhydride (2).26 Another approach to prepare 2,6-dibromonaphthalene dianhydride (2) includes aromatic nucleophilic substitution of aromatic H- by Br- in oleum (Scheme-3).27 All these procedures require oleum in which could not be purchased in 31 Singapore since it is prohibited by environmental law. Therefore, as an alternative strategy, bromination with DBI was selected and performed in readily available 98% concentrated H2SO4. After dibromination, no purification was done. Because 2,6-dibromonaphthalene dianhydride (2) has two anhydride group so the compound get stick in silica gel it did not come out of the column. The crude product was used for the next step condensation reaction with the primary or secondary aminoderivatives. Symmetrically substituted NDI-1 and NDI-2 chromophores were prepared from 2,6-dibromonaphthalene anhydride after heating in a microwave oven for 30 min and at 140oC with ethanol amine and 3amino propanol respectively in DMF and in the presence of triethylamine (Scheme 4). 32 Scheme 4: Synthesis of NDI-1 & NDI-2 But the yields of both NDI-1 and NDI-2 with respect to Naphthalene dianhydride (SM) were less than 1%. To find out the reason behind such a low yield LCMS analysis was done. For NDI-1 LCMS analysis data is shown in Figure-5. From LCMS data it is very clear that the major product was mono substituted naphthalene diimide 33 (4) and very little amount of disubstitued naphthalene diimide (NDI-1) was obtained as shown in scheme 5. From LCMS peak area analysis the ratio of mono substituted and bisubstitued product was approximately 100:3.26. Optical property of NDI-1 (Figure 6) and NDI-2 (Figure 7) were studied. NDI-1 (ethanolamine), NDI-2 (3-aminopropanol) show absorbtion maxima at 600 nm and 610 nm respectively in water. NDI-1 (ethanolamine), NDI-2 (3-aminopropanol) give emission maxima at 690 nm and 680 nm respectively. NDI-2 has stokes shift of 70 nm, where`as NDI-1 has 90 nm. The emission quantum yield of NDI-1 and NDI-2 were measured using N,N-di(2,6-diisopropylphenyl)-1,6,7,12- tetraphenoxyperylene-3,4:9,10-tetracarboxylic acid bisimide (0.96, CHCl3) as a reference and were found to be 0.57 and 0.46 in water respectively. 34 4 NDI-1 NDI-2 5 Scheme 5: Discussion of the challenges focussing on the low reaction yields of NDI-1 & NDI-2 LCMS analysis of crude NDI-1 [MS Spectrum] Base Peak m/z 413.2321 (Inten : 578,693) m/z Rel. Inten. 158.9468 12.15 391.2563 5.82 413.2321 100.00 414.2401 17.72 35 473.132 3.26 Inten.(x100,000) 6.0 413.232 5.0 4.0 3.0 2.0 1.0 158.947 0.0 250 500 750 1000 1250 m/z Figure 5- LCMS analysis of crude N,N´-Bis-(2-hydroxyethyl)-2,6di(N-2-hydroxy ethyl)-1,4,5,8-naphthalenetetracarboxylic Acid Diimide (NDI-1). 36 To verify that imidisation of naphthalene derivatives works well, the following reported reaction scheme was explored.28 Condensation of ethylamine with 1,4,5,8 tetracarboxylic naphthalene dianhydride works very well with 64% of yield in my hand. The product was characterised by NMR and Mass spectroscopy. SM 3 Scheme 6: Synthesis of N,N´-Bis-(ethyl)-1,4,5,8naphthalenetetracarboxylic Acid Diimide (3) Conclusion: Two new symmetrical core substituted naphthalene diimide blue coloured dye NDI-1 and NDI-2, which emits at near infrared region were synthesized with very low yield. The reason behind the low yield is the use of conc H2SO4 instead of oleum during bromination of naphthalene anhydride (SM). Conc H2SO4 is less oxidising than oleum. 37 Conc H2SO4 leads to mono bromination (major product) of naphthalene anhydride (SM) and subsequently mono substituted naphthalene diimide (4, 5) was formed as major product. Mono substituted naphthalene diimide (4, 5) dye emits bellow 600 nm.27 But mono substituted naphthalene diimide dye cannot take the advantage of near infrared region in bioimaging application. So to explore core substituted naphthalene diimide dye in more details alternative route to the core substitution of naphthalene dianhydride (SM) by using easily available and environment friendly chemicals need to be investigated. EXPERIMENTAL DETAILS: All reactions were performed under argon atmosphere and stirred magnetically in oven-dried glassware fitted with a rubber septum. Commercial anhydrous solvents were used in every reaction step. Inorganic salts and acids were used in aqueous solution and are reported in % w/v. Unless otherwise stated, all reagents were obtained from Aldrich, Alfa Aesar, or TCI America and used without further purification. Flash chromatography was performed using 38 silica gel (25–40 mm particle size), respectively. Thin layer chromatography analyses were performed using pre-coated Merck Silica Gel 60 F254 and visualized with ultraviolet light. Rf values were obtained by elution in the stated solvent ratios (v/v). All solvent mixtures are reported as (v/v) unless noted otherwise. The microwaveassisted reactions were performed using the Biotage Initiator microwave synthesizer at 300 W. 1H NMR spectra were measured at 298 K on a Bruker ACF 300 or AMX 500 Fourier Transform spectrometer. Chemical shifts were reported in δ (ppm), relative to the residual undeuterated solvent which was used with an internal reference. The signals observed were described as s (singlet), d (doublet), t (triplet), and m (multiplet). The number of protons (n) for a given resonance were indicated as nH. Mass spectral analyses were recorded on a Finnigan MAT 95/XL-T spectrometer under electron impact (EI) or electrospray ionization (ESI) techniques. 39 Preparation of Dibromocyanuric acid (1): To brominate naphthalene dianhydride, dibromoisocyanuric (DBI) acid is used is precursor. DBI is prepared from cyanuric acid using the following method. Procedure: 645 mg (5 mmol) of cyanuric acid and 23.95 mg (10 mmol) LiOH were dissolved in 50 ml of water. Bromine (1ml) was added and the mixture was kept in a 40C freezer for 24 h. After 24 h, the white precipitate was filtered and washed with cold water and used for next step. 1H NMR (300 MHz, DMSO): δ 8.71 (s, 1H ) ppm. Preparation of 2,6-dibromonaphthalene diianhydride (2) from 1,4,5,8- Naphthalenetetracarboxylic acid anhydrides (SM): 50 mg (0.186mmol) of 1,4,5,8- naphthalenetetracarboxylic acid anhydride and 159 mg (0.556 mmol) of dibromoisocyanuric acid were dissolved in 5 ml conc. H2SO4 and stirred at room temperature for 5 h. Then, the orange solution was poured into ice and a solid yellow product was filtered and dried in high vacuum and the crude product was used for next step. 1H NMR (300 MHz, DMSO): δ 8.78 (s, 2H) ppm. 40 Synthesis of N,N´-bis-(2-hydroxyethyl)-2,6-di(N-2-hydroxy ethyl)1,4,5,8-naphthalene diimide (NDI-1): 200 mg (0.047mmol) of 2,6dibromo-1,4,5,8- naphthalenetetracarboxylic acid anhydrides and 102.4 µL (1.88 mmol) of ethanolamine were dissolved in 5 ml DMF and 148 µL triethylamine was added to it and irradiated with Microwave irradiation for 30 min at 145oC. Then the reaction mixture was cooled to room temperature and DMF was evaporated. Flash column chromatography (silica gel, DCM) yielded a blue solid NDI-1 (1 mg, 4.65 µmol, 1% yield). Rf = 0.86 (silica gel, DCM: MeOH 4:1); 1H NMR (300 MHz, D2O): δ 8.06 (s, 2H ), 3.83 (m, 8H), 3.38 (m, 8H) ppm. Synthesis of N,N´-Bis-(3-hydroxypropyl)-2,6-di(N-3-hydroxy propyl)-1,4,5,8-naphthalenetetracarboxylic Acid Diimide (NDI-2): 200 mg (0.047mmol) of 2,6-dibromo-1,4,5,8- Naphthalenetetracarboxylic acid anhydrides and 141.2 µL (1.88 mmol) of 3-aminopropanol was dissolved in 5 ml DMF and 148 µL triethylamine was added to it and irradiated with Microwave irradiation 41 for 30 min at 1450C. Then the reaction mixture was cooled to room temperature and DMF was evaporated. Flash column chromatography (silica gel, DCM) yielded a blue solid NDI-2 (2 mg, 4.65 µmol, 1% yield). Rf = 0.8 (silica gel, DCM: MeOH 4:1); 1H NMR (300 MHz, D2O): δ 7.98 (s, 2H ), 2.97 (t, 4H), 2.81 (t, 4H), 1.8 (t, 4H), 1.38 (t, 4H), 0.83 (m, 8H) ppm. Synthesis of N,N´-Bis-(ethyl)-1,4,5,8-naphthalenetetracarboxylic Acid Diimide (3): An aqueous solution of 200 mg (0.74 mmol) of 1,4,5,8naphthalenetetracarboxylic acid anhydrides and 120.73 mg (1.48 mmol) ethylaminehydrochloride was refluxed for 6 h. Then the reaction mixture was cooled and the product was extracted in chloroform. Then the product was purified by flash column chromatography. Total 153.9 mg (64%) product was obtained. Rf = 0.7 (silica gel, hexane: EtOAc 4:1); 1H NMR (300 MHz, D2O): δ 8.74 (s, 4H ), 4.28-4.23 (m, 4H), 1.56-1.33 (t, 6H) ppm. 42 Overview of relevant spectra: 1. The 1H NMR (300 MHz) spectrum of dibromoisocyanuric acid (1) in DMSO-d6. 43 2. The 1H NMR (300 MHz) spectrum of monobromanaphthalene dianhydride in DMSO-d6. 44 3. The 1H NMR (300 MHz) spectrum of NDI-1 in D2O. 45 4. The 1H NMR (400 MHz) spectrum of NDI-2 in D2O. 46 5. The 1H NMR (300 MHz) spectrum of 3 in CDCl3. 6. Optical spectra of NDI-1. 0.7 1 Fluorescence Intensity (6a) Absorbance 0.6 0.5 0.4 0.3 0.2 0.1 0 (6b) 0.75 0.5 0.25 0 350 450 550 650 750 Wavelength (nm) 640 690 740 790 840 Wavelength (nm) 47 (6a). UV-vis-NIR absorption spectra of NDI 1 (0.1 mg/mL) in water. Absorbtion maxima 600 nm. (6b) Emission spectra of NDI1 in water. The excitation wavelength was 600 nm. The emission maxima is 690 nm. 7. Optical spectra of NDI-2 0.08 1.2 Fluorescence Intensity (7a) Absorbance 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0 (7b) 1 0.8 0.6 0.4 0.2 0 350 450 550 650 750 Wavelength (nm) 640 690 740 790 Wavelength (nm) (7a). UV-vis-NIR absorption spectra of NDI 2 (0.1 mg/mL) in water. Absorbtion maxima 610 nm. (7b) Emission spectra of NDI2 in water. The excitation wavelength was 610 nm. The emission maxima is 680 nm. Bibliography: 1. Stokes, G. G. Philosophical Transactions of the Royal Society of London. 1852, 142, 463–56. 2. Li, J.; Yao, S. Q. Org. Lett. 2009, 11, 1671. 3. Lakowicz, J. R. 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Zhurnal Organicheskoi Khimii. 1977, 13, 1262. 52 [...]... attractive forces between the 16 molecules The aggregates in solution exhibit distinct changes in the absorption band as compared to the monomeric species From the spectral shifts, various aggregation patterns of the dyes in different media can be proposed Near Infrared Dyes ( NIR ) for Bioimaging: In recent years, fluorescence imaging using Near Infrared dyes has attracted much attention as it affords... various wavelengths (UV to Near Infrared) for image generation, includes many 17 different acquisition techniques Optical image contrast can be based on absorption, fluorescence, fluorescence lifetime and polarization.11 For fluorescence -based bioimaging, the optimum wavelength for excitation and emission ranges from 650–900 nm.12 This range of wavelength is called Near Infrared (NIR) Window (Figure-3)... positions usually requires tedious, multi-step transformations Very recently, Wuerthner’s group has simplified the synthetic procedure of core substituted NDI dyes. 21 They have used 2,6dibromonaphthalene dianhydride as precursor molecule for achieving core-disubstituted NDIs Two bromine substituents were introduced into the naphthalene core of 1,4,5,8-naphthalenetetracarboxylic acid dianhydride by electrophilic... these NDI chromophores will be applied for bioimaging applications RESULT AND DISCUSSION: The synthesis of substituted NDI chromophores as reported by the group of Wuerthner is summarized in Scheme 1.20 The differences from the original scheme are 1) Conc sulphuric acid is used as a solvent for the preparation of 2, 6dibromonaphthalene dianhydride (2) from 1,4,5,8 -Naphthalene dicarboxylic dianhydride... architectures are untwisted into open cation channels by intercalation of dialkoxynaphthalene ligands Naphthalene diimide organogels were built by noncovalent interactions such as ð-ð stacking, hydrogen bonding, and van der Waals forces which serve as supramolecular hosts and sensors for different types of electron-rich naphthalene derivatives.19 Unlike the colourless NDI without substituents within... higher signal to - noise ratios (SNR) 18 Figure 3 Near Infrared (NIR) Window The NIR window is ideally suited for in vivo imaging because of minimal light absorption by hemoglobin (900 nm) Characteristics of suitable NIR dyes for bioimaging applicatons: The ideal NIRF fluorophore for in vivo bio-imaging should reveal the following characteristics: 1 A peak fluorescence close to 700–900... sciences For example, new interest in fluorophores with NIR emission has arisen in conjunction with singlemolecule spectroscopy of biomolecules12 where most traditional NIR dyes lack the required fluorescence quantum yield and photostability or whose performance is hampered by aggregation of their extendedconjugated cores The second point holds especially true for rylene 20 dye.13 Rylene dyes are ideally... equipment is available Finally, 2, 6-dibromonaphthalene-1,4,5,8-dianhydride was obtained starting from naphthalene dianhydride (SM) by aromatic electrophilic substitution (Scheme-3) It has been reported before that the application of stoichiometric amounts of DBI and oleum (20% SO3) and naphthalene dianhydride yields dianhydride (2) as major product 25 2,6-dibromonaphthalene-1,4,5,8Alternatively, elemental... triplet transition state (T1) From the T1 state, the S0 state could be reached by a slow radiative process called phosphorescence Transition from T1 to S0 is forbidden and therefore, timescales for phosphorescence are usually much slower than those for fluorescence Since T1 has lower energy than S1, emission of 12 phosphorescence is usually more bathochromically shifted than fluorescence emission There... biocompatibility, biodegradability, or excretability 7 Availability of monofunctional derivatives for conjugations 8 Commercial viability and production scalability for large quantities ultimately required for human use 9 Large stokes shift 10 Easily tunable optical property Despite the multitude of available dyes there is still considerable interest in new chromophore systems that satisfy the special ... the dyes in different media can be proposed Near Infrared Dyes ( NIR ) for Bioimaging: In recent years, fluorescence imaging using Near Infrared dyes has attracted much attention as it affords... and polarization.11 For fluorescence -based bioimaging, the optimum wavelength for excitation and emission ranges from 650–900 nm.12 This range of wavelength is called Near Infrared (NIR) Window... the present work, reaction schemes towards blue coloured NIR -dyes based on the naphthalene diimide (NDI) scaffold have been designed starting from 2, 6-dibromonaphthalene dianhydride as the central