Hexaazaanthracenes: New Fluorophores with Potential Material Science and Molecular Probe Applications

Ronald G. Brisbois, Macalester College, St. Paul, Minnesota

• Introduction: Wudl and Houk reported the first hexaazaanthracene (HA) derivative (TPHA+) in 1998 (Scheme 1).1 Their curiosity for HA-derivatives centered on the theoretically interesting 16p electron scaffold. Both zwitterionic (i.e. singlet) and diradical (i.e. triplet) ground state resonance structures are possible for the HA-core. The triplet ground state possibility raised the very compelling hope that HA-derivatives could serve as organic conductive materials. However, NMR spectra, dipole moment measurements, crystallographic data, and (U)B3LYP/6-31G* M. O. calculations combined to support modeling TPHA as a ground state zwitterion, in which the top half incorporates an anionic cyanine moiety and the bottom features a cationic cyanine substructure. Practically speaking, the HA-core remains highly intriguing as a function of the unique photophysical behavior it exhibits. For example, TPHA fluoresces strongly (quantum yield = 0.31) and possess an incredible Stoke's shift (lex = 326 nm; lem = 633 nm; Æl = 307 nm). Additionally, Wudl and Levanon subsequently performed time-resolved EPR on TPHA and concluded that excitation to a diradical excited state occurs via novel intramolecular electron transfer (IET) from the singlet excited state.2 Thus, HA-derivatives are indeed theoretically interesting molecules, and the same physical properties that draw the theoretical eye suggest a potential wealth of utility for this fledgling organic scaffold. We seek to develop the promise of HA-derivatives by: (1) improving general synthetic accessibility to HA-derivatives and (2) working toward HA-derived molecular probes for biochemical and molecular biological application.

• Synthesis of HA-Derivatives: We prepared TPHA, as well as a new analog (bis-hexylTPHA; Scheme 1), according to the procedure developed by Wudl and Houk. While their route (UCLA procedure in Scheme 1) is indeed synthetically straightforward, it requires nearly constant attention over three days, involves multiple reaction pots (from the bis-amide 1 or 2), and precludes convergent incorporation of acid-sensitive functionality due to the highly acidic PCl5 melt step. We sought to complement Wudl's and Houk's method with an alternative better suited to the time-frame of academic year undergraduate research (i.e. small blocks of time), as well as featuring more selective and mild activation of bis-amide precursors towards nucleophilic attack by PhNHNH2 (or other hydrazine derivatives). Ideally, we hoped to convert bis-amide starting materials to their corresponding HA-derivatives in one pot. Our preliminary results (Macalester procedure in Scheme 1) towards this set of goals are illustrated in Scheme 1.3 The combination of Tf2O and DIPEA activates the bis-amide 2-6 under mildly basic conditions. Moreover, only one hour and one reaction pot are required to reach the final oxidative cyclization step (i.e. DBU/MeOH). We recognize the need to more fully optimize our alternative procedure with respect to stoichiometry and reaction times, and this will be a critical component of our overall NSF-REU effort. However, we intend to simultaneously explore its scope and limitations, and, in fact, we have scored a few interesting hits in this regard already (Scheme 1; NOTE: Oxidative polymerization of DTHA could yield a novel polythiophene/HA block copolymer having potential material science applications.) Through our NSF-REU efforts we plan to explore more thoroughly preparation of HA-derivatives featuring a wide range of aryl, haloaryl, heteroaryl, and alkyl substituents carried into the final targets via the initial bis-amide. Also, we want to establish that structural variablity is possible via the hydrazine reactant, specifically testing a variety of substituted arylhydrazines and t-BuNHNH2 (as a representative alkylhydrazine). Finally with respect to scope and limitations, access to a range of unsymmetrically substituted HA-derivatives must be demonstrated. As included in Scheme 1, preparation of TBHPHA indicates our preliminary work toward this goal,3 and we submit that this result is an excellent harbinger of future progress. We anticipate devoting significant portions of the first two (10 week) summer research periods to completing this ongoing work. By the third (10 week) summer research period, we hope/intend to begin leveraging our optimized synthetic methodology in the pursuit of HA-based applications (vide infra).

• HA-Derived Molecular Probes: Molecular probes, in all varieties, take advantage of useful photophysical properties to facilitate studies of biological structures, systems, and processes.4 The large Stoke's shift and red fluorescence of HA-derivatives suggest both complementary and novel utility as molecular probes, especially if covalently conjugated to biomolecules and structures as a positional and/or functional tag. In fact, we prepared bis-hexylTPHA and DTBHA in hopes of laying foundations in building towards working molecular probes. That is, the hexyl side-chain and the t-Bu group of TBHPHA serve as rudimentary model tethers, working analogs of which could be incorporated via contemporary synthetic methodologies (Scheme 2). Tethers terminated by -OH and NH2 moieties will permit incorporation into biomaterials via standard ether, ester, amine, and amide linkages.4 Alkyne terminated tethers dovetail nicely into possibilities for DNA or RNA oligiomer labeling via Sonogashira cross-coupling to halogenated C, G, T, A, or U derivatives.5,6 A severe limitation to HA-derivatives as molecular probes may hinge on poor aqueous solubility. Thus, we anticipate exploring HA derivatization strategies that will render these novel fluorophores water-soluble (Scheme 3). Work towards functional molecular probes will be closely informed by results from our scope and limitations study, especially the efficiency of constructing more complex, unsymmetrically substituted HA-derivatives.

+ Acronym Key: TPHA = Tetraphenylhexaazaanthracene; DTBHA = Di-t-butylhexaazaanthracene; DFHA = Difurylhexaazaanthracene; DTHA = Dithienylhexaazaanthracene; TBHPHA = t-Butylhexylphenylhexaazaanthracene

• References: (1) Hutchison, K.; Srdanov, G.; Hicks, R.; Yu, H.; Wudl, F.; Strassner, T.; Nendel, M.; Houk, K. N. J. Am. Chem. Soc. 1998, 120, 2989. (2) Hutchison, K. A.; Hasharoni, K.; Wudl, F.; Berg, A.; Shuali, Z.; Levanon, H. J. Am. Chem. Soc. 1998, 120, 6362. (3) Brisbois, R. G.; Kourdioumov, A.; Duenes, R. A. unpublished results. (4) Handbook of Fluorescent Probes and Research Chemicals, 6th Edition, Haugland, R. P.; Molecular Probes, Inc.: Eugene, OR, 1996. (5) A wide variety of halogenated nucleic, nitrogenous bases are known/commericially available (e.g. Aldrich # 12,750-7 = 8-bromoadenosine; Aldrich # 85,018-7 = (-)5-bromouridine. (6) Our favorite Sonogashira coupling method: Hundertmark, T.; Littke, A. F.; Buchwald, S. L.; Fu, G. C. Org. Lett. 2000, 2, 1729. (7) Byun, H.-S.; Zhong, N.; Bittman, R. Organic Syntheses 1999, 77, 220. (8) Zhu, Y.-P.; Masuyama, A.; Kirito, Y.; Okahata, M. J. Am. Oil Chem. Soc. 1991, 68, 539.