Panchromatic Absorbers Tethered for Bioconjugation or Surface Attachment

The syntheses of two triads are reported. Each triad is composed of two perylene-monoimides linked to a porphyrin via an ethyne unit, which bridges the perylene 9-position and a porphyrin 5- or 15-position. Each triad also contains a single tether composed of an alkynoic acid or an isophthalate unit. Each triad provides panchromatic absorption (350–700 nm) with fluorescence emission in the near-infrared region (733 or 743 nm; fluorescence quantum yield ~0.2). The syntheses rely on the preparation of trans-AB-porphyrins bearing one site for tether attachment (A), an aryl group (B), and two open meso-positions. The AB-porphyrins were prepared by the condensation of a 1,9-diformyldipyrromethane and a dipyrromethane. The installation of the two perylene-monoimide groups was achieved upon the 5,15-dibromination of the porphyrin and the subsequent copper-free Sonogashira coupling, which was accomplished before or after the attachment of the tether. The syntheses provide relatively straightforward access to a panchromatic absorber for use in bioconjugation or surface-attachment processes.


Introduction
Chromophores that absorb across the visible spectrum (400-700 nm) are of essential importance for diverse studies in the photosciences. The chlorophylls of plant photosynthesis exhibit strong absorption in the violet and red regions, with relatively weak absorption across the rest of the visible region [1]. Natural photosynthetic systems use carotenoids and/or other accessory chromophores in complementary fashion to fill the blue-orange region of the solar spectrum [2]. In this approach, the distinct absorption of a given accessory pigment is followed by excited-state energy transfer among a set of pigments, thereby increasing the wavelength expanse of absorption beyond that of chlorophyll alone. Work that began some 45 years ago [3] led to the realization in the early 1990s that the absorption spectrum of porphyrins could be substantially broadened upon the conjugation of ethynyl groups to the macrocycle [4][5][6][7]. Penetrating studies thereafter by the groups of Anderson [8] and Therien [9][10][11][12][13][14][15][16][17][18][19][20][21] chiefly focused on joining porphyrins via butadiyne and ethyne linkers, respectively, eliciting fascinating spectroscopic features of the resulting arrays. The joining of porphyrins and other chromophores via ethynyl linkers quickly became a prominent molecular motif [22][23][24][25], a topic that has been reviewed [26,27].
For exacting comparison, we then prepared an analogous perylene-ethynyl-porphyrin dyad (1) [42], which differed from the initial dyad (0) in the nature of the non-linking substituents (p-tolyl versus mesityl groups) on the porphyrin, and also the corresponding ethynyl-porphyrin (2) [43,44] and ethynyl-perylene (3) [40] benchmark compounds bearing nearly identical substituents. The change from mesityl to p-tolyl substituents was inconsequential, as shown in the overlay of the spectra of dyads 0 and 1. While the absorption spectrum of each dyad resembles that of a strongly potentiated perylene and diminished porphyrin (Figure 1), the fluorescence emission spectrum resembles that of a porphyrin, albeit one shifted bathochromically (~40 nm) and with substantially increased Figure 1. Absorption spectra of a perylene-ethynyl-porphyrin dyad (1, solid red line), to be com pared with phenylethynyl-porphyrin (2, solid blue line) and perylene-ethyne (3, solid orange lin constituents (in toluene at room temperature). The fluorescence spectrum of 1 also is shown (dash red line; em 695 nm) [42]. The absorption spectrum of the perylene-ethynyl-porphyrin dyad 0 (do ted black trace [41]) is essentially identical with that of analogous dyad 1 (solid red line).
In this paper, we describe the extension of the panchromatic triad for studies in lightharvesting. One design incorporates a single carboxylic acid for bioconjugation, such as in biohybrid assemblies wherein additional solar light capture is advantageous. A second design incorporates an isophthalic acid terminal unit for attachment to surfaces, such as metal oxides for studies of photoinduced charge injection. The syntheses rely on established rational methods and provide relatively direct access to lipophilic panchromatic triads each bearing a single attachment handle.

Synthesis of a Bioconjugatable Panchromatic Triad
We sought to place a hexanoic acid chain on the central porphyrin for bioconjugation purposes. A meso-dibromo-substituted porphyrin building block was prepared for the construction of the bioconjugatable panchromatic triad. The core porphyrin contains an AB pattern of substituents with two open meso-positions and was prepared in a standard manner [51] from two dipyrromethanes (Scheme 1). The dibutyltin-complexed 1,9-diformyl-5-phenyldipyrromethane 5 [52] was treated with propylamine [51] and then reacted with 1,9-diunsubstituted 5-(4-bromophenyl)dipyrromethane 6 [53] in the presence of Zn(OAc) 2 to afford the trans-AB zinc porphyrin 7, which bears a bromine atom at a site for the introduction of a bioconjugatable tether. The bromo-porphyrin 7 thereafter reacted with ethynyl tether 8 [54] via a Sonogashira coupling reaction to form the bioconjugatable zinc porphyrin 9. The Pd-mediated Sonogashira reaction [55] was carried out under copperfree conditions [56,57] using a solvent mixture of toluene and triethylamine (TEA). The bromination [58] of porphyrin 9 using N-bromosuccinimide (NBS) at 0 • C yielded the dibromoporphyrin building block 10. The next step entailed attachment of two peryleneethyne units.
Several points warrant comment concerning the choice of ethynyl-perylene for coupling with the dibromoporphyrin. The chosen perylene (11) bears two aryloxy groups (one in each bay region) and a 2,6-diisopropylphenyl group at the N-imide site, which together impart structural features that enable good solubility of the perylene in hydrocarbon solvents [46]. The ethyne is located at the perylene 9-position, a site of considerable electron density in the frontier molecular orbitals and a site known to afford substantial electronic communication upon covalent attachment to the porphyrin [42,[45][46][47][48][49][50]. The ethynyl-perylene-monoimide 11 is an advanced functional dye that has emerged from extensive studies over several decades beginning with the pioneering work of Langhals [59][60][61][62][63][64][65].
The absorption spectra of ethynyl-perylene-monoimides bearing the 2,6-diisopropylphenyl group (11) or the 2,5-di-tert-butylphenyl (11-tBu [70,71]) are nearly identical, as shown in Figure 3. The similarity of spectra upon use of either solubilization motif is consistent with observation that the 2,6-diisopropyl versus 2,5-di-tert-butyl group has insignificant effects on perylene photophysics [40]. The similar spectra and photophysics are attributed to the presence of a node at the perylene-imide nitrogen atom in both the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) [61]. Thus, the changes in porphyrin substituents from mesityl to p-tolyl groups, and perylene N-imide from a 2,5-di-tert-butylphenyl to a 2,6-diisopropylphenyl group, facilitate synthesis and chemical characterization but have hardly any effects on spectral and photophysical properties. Several points warrant comment concerning the choice of ethynyl-perylene for coupling with the dibromoporphyrin. The chosen perylene (11) bears two aryloxy groups (one in each bay region) and a 2,6-diisopropylphenyl group at the N-imide site, which together impart structural features that enable good solubility of the perylene in insignificant effects on perylene photophysics [40]. The similar spectra and photophysics are attributed to the presence of a node at the perylene-imide nitrogen atom in both the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) [61]. Thus, the changes in porphyrin substituents from mesityl to p-tolyl groups, and perylene N-imide from a 2,5-di-tert-butylphenyl to a 2,6-diisopropylphenyl group, facilitate synthesis and chemical characterization but have hardly any effects on spectral and photophysical properties. The Sonogashira coupling [55] reaction of ethynyl-perylene 11 and dibromoporphyrin 10 was carried out under copper-free [56,57] Pd-mediated conditions (as in the reaction of 7 and 8). Such a reaction of an ethynylphenyl-chromophore and a meso-bromoporphyrin is facile and has been carried out under nearly identical conditions to form porphyrinphenylethynyl-porphyrin dyads [44]. The rationale for copper-free conditions in the synthesis was to avoid any transmetalation of the zinc porphyrin given (1) the avidity of porphyrins for Cu(II); (2) the very short-lived singdoublet excited-state lifetime of copper porphyrins (marked by the absence of fluorescence); and (3) the subsequent difficulty of removing copper from porphyrins, unlike the facile removal of zinc achieved upon treatment with mild acid. Thus, the subsequent acid-mediated demetalation of zinc [42] with The Sonogashira coupling [55] reaction of ethynyl-perylene 11 and dibromoporphyrin 10 was carried out under copper-free [56,57] Pd-mediated conditions (as in the reaction of 7 and 8). Such a reaction of an ethynylphenyl-chromophore and a meso-bromoporphyrin is facile and has been carried out under nearly identical conditions to form porphyrinphenylethynyl-porphyrin dyads [44]. The rationale for copper-free conditions in the synthesis was to avoid any transmetalation of the zinc porphyrin given (1) the avidity of porphyrins for Cu(II); (2) the very short-lived singdoublet excited-state lifetime of copper porphyrins (marked by the absence of fluorescence); and (3) the subsequent difficulty of removing copper from porphyrins, unlike the facile removal of zinc achieved upon treatment with mild acid. Thus, the subsequent acid-mediated demetalation of zinc [42] with trifluoroacetic acid (TFA) afforded the target panchromatic triad 12. Purification was achieved via a three-chromatography sequence that includes use of size-exclusion chromatography (SEC) [42,72]. Removal of the tert-butyl protecting group using 40% TFA [73] in CH 2 Cl 2 gave the carboxy-triad 13 (Scheme 1). For bioconjugation purposes, 13 was further transformed to an N-hydroxysuccinimide ester 14 via reaction [74] with N-hydroxysuccinimide in the presence of N,N-dicyclohexylcarbodiimide (DCC).
The synthesis of the core porphyrin follows that shown for the bioconjugatable triad. Thus, the 1,9-formyldipyrromethane 18 was reacted with propylamine [51] to form the bis(imine), which was then treated with the complementary dipyrromethane 19 [77] to afford the zinc porphyrin 20 in 38% yield (Scheme 2). The bromination [58] of zinc porphyrin 20 afforded the corresponding dibromo zinc porphyrin 21 in 75% yield. Sonogashira coupling [55] of the dibromo zinc porphyrin 21 and ethynyl-perylene 11 under copper-free conditions [56,57] afforded the triad 22 bearing two perylenes and one zinc porphyrin in 90% yield (~200 mg). The reaction progress was monitored by analytical SEC [72], as has been done previously with other panchromatic arrays [42,46]. The analytical SEC traces with absorption spectral determination show the starting materials ( Figure 4, panel a), crude mixture after reaction for several hours (panel b), and the purified triad 22 (panel c) following preparative purification using the three-column chromatography process. The cleavage of the trimethylsilyl (TMS) group [48] of 22 afforded triad 23 bearing a free ethynyl group in 93% yield. ther transformed to an N-hydroxysuccinimide ester 14 via reaction [74] with N-hydroxysuccinimide in the presence of N,N-dicyclohexylcarbodiimide (DCC).

Chart 2. Tetrapyrrole dyes bearing an isophthalate tether.
The synthesis of the core porphyrin follows that shown for the bioconjugatable triad. Thus, the 1,9-formyldipyrromethane 18 was reacted with propylamine [51] to form the bis(imine), which was then treated with the complementary dipyrromethane 19 [77] to afford the zinc porphyrin 20 in 38% yield (Scheme 2). The bromination [58] of zinc porphyrin 20 afforded the corresponding dibromo zinc porphyrin 21 in 75% yield. Sonogashira coupling [55] of the dibromo zinc porphyrin 21 and ethynyl-perylene 11 under copper-free conditions [56,57] afforded the triad 22 bearing two perylenes and one zinc porphyrin in 90% yield (~200 mg). The reaction progress was monitored by analytical SEC [72], as has been done previously with other panchromatic arrays [42,46]. The analytical SEC traces with absorption spectral determination show the starting materials ( Figure 4, panel a), crude mixture after reaction for several hours (panel b), and the purified triad 22 (panel c) following preparative purification using the three-column chromatography process. The cleavage of the trimethylsilyl (TMS) group [48] of 22 afforded triad 23 bearing a free ethynyl group in 93% yield.  The surface-attachment motif was prepared through the DCC-mediated condensation of 5-bromoisophthalic acid (24) and 2-(trimethylsilyl)ethanol (25) in N,N-dimethylformamide (DMF) to give the protected 5-bromo-isophthalate 26 in 23% yield (Scheme 2). The Sonogashira coupling [55] of ethynyl-triad 23 and isophthalate 26 under copper-free conditions [56,57] afforded the protected tethered triad 27 in 69% yield. The removal of the 2-trimethylsilylethyl group [76] of 27 upon treatment with tetrabutylammonium fluoride (TBAF) in tetrahydrofuran (THF) afforded isophthalate-tethered triad 28 in 59% yield. While protected triad 27 was fully characterized, all efforts to characterize 28 by NMR spectroscopy and mass spectrometry were unsuccessful. For confirmation purposes, a small portion of 28 was treated with methanol in the presence of DCC and and 4- The surface-attachment motif was prepared through the DCC-mediated condensation of 5-bromoisophthalic acid (24) and 2-(trimethylsilyl)ethanol (25) in N,N-dimethylformamide (DMF) to give the protected 5-bromo-isophthalate 26 in 23% yield (Scheme 2). The Sonogashira coupling [55] of ethynyl-triad 23 and isophthalate 26 under copper-free conditions [56,57] afforded the protected tethered triad 27 in 69% yield. The removal of the 2-trimethylsilylethyl group [76] of 27 upon treatment with tetrabutylammonium fluoride (TBAF) in tetrahydrofuran (THF) afforded isophthalate-tethered triad 28 in 59% yield. While protected triad 27 was fully characterized, all efforts to characterize 28 by NMR spectroscopy and mass spectrometry were unsuccessful. For confirmation purposes, a small portion of 28 was treated with methanol in the presence of DCC and and 4-dimethylaminopyridine (DMAP) to form the corresponding dimethyl ester 29, which gave the expected mass peak upon matrix-assisted laser-desorption ionization mass spectrometry (MALDI-MS) analysis.

Chemical Characterization
The triads were generally characterized by 1 H NMR spectroscopy, absorption spectroscopy, and MALDI-MS analysis. Limited solubility precluded the collection of 13 C NMR spectra for a number of the triads as well as other compounds. Accurate mass data were obtained by electrospray ionization mass spectrometry (ESI-MS) where possible. The absorption and fluorescence spectra of two tethered triads are shown in Figure 5. The carboxy-triad 13 contains a free base porphyrin, whereas the isophthalate-triad 27 contains a zinc porphyrin. The absorption spectrum of carboxy-triad 13 (panel a) is nearly identical to that of the untethered triad 4 shown in Figure 2. The absorption spectrum of isophthalate-triad 27 (panel b) is nearly identical to that of the untethered triad Zn4 shown in Figure 2. For comparison purposes, the absorption and fluorescence spectra of a benchmark perylene (33) [46] and the trans-AB zinc porphyrin 31 are shown in panels c and d, respectively. Perylene 33 includes a phenyl group at the terminus of the ethyne and a 2,6diisopropylphenyl substituent at the N-imide position (Chart 3) and is displayed correctly in the original report of synthesis and characterization [46], but is shown incorrectly with the 2,5-di-tert-butylphenyl substituent at the N-imide position in a subsequent report [50]. Molar absorption coefficient values are reported for triads 13, 23, 27, and 28, as well as for benchmark ethynyl-perylene-monoimide 11. The f values measured in toluene for triad 13, triad 27, perylene 33, and porphyrin 31 are 0.21, 0.24, 0.94 [50], and 0.011, respectively. For comparison, the f values for free base porphyrin triad 4 and zinc porphyrin triad Zn4 are 0.26 and 0.30, respectively [45]. The fluorescence emission of each triad exhibits (as expected) the general spectral features of a porphyrin but with an enhanced quantum yield. 1 H and 13 C{ 1 H} NMR spectra, mass spectra, and absorption spectra (where availa-Scheme 3. Synthesis of tethered porphyrin 32 lacking perylenes.

Chemical Characterization
The triads were generally characterized by 1 H NMR spectroscopy, absorption spectroscopy, and MALDI-MS analysis. Limited solubility precluded the collection of 13 C NMR spectra for a number of the triads as well as other compounds. Accurate mass data were obtained by electrospray ionization mass spectrometry (ESI-MS) where possible. The absorption and fluorescence spectra of two tethered triads are shown in Figure 5. The carboxy-triad 13 contains a free base porphyrin, whereas the isophthalate-triad 27 contains a zinc porphyrin. The absorption spectrum of carboxy-triad 13 (panel a) is nearly identical to that of the untethered triad 4 shown in Figure 2. The absorption spectrum of isophthalate-triad 27 (panel b) is nearly identical to that of the untethered triad Zn4 shown in Figure 2. For comparison purposes, the absorption and fluorescence spectra of a benchmark perylene (33) [46] and the trans-AB zinc porphyrin 31 are shown in panels c and d, respectively. Perylene 33 includes a phenyl group at the terminus of the ethyne and a 2,6diisopropylphenyl substituent at the N-imide position (Chart 3) and is displayed correctly in the original report of synthesis and characterization [46], but is shown incorrectly with the 2,5-di-tert-butylphenyl substituent at the N-imide position in a subsequent report [50]. Molar absorption coefficient values are reported for triads 13, 23, 27, and 28, as well as for benchmark ethynyl-perylene-monoimide 11. The Φ f values measured in toluene for triad 13, triad 27, perylene 33, and porphyrin 31 are 0.21, 0.24, 0.94 [50], and 0.011, respectively. For comparison, the Φ f values for free base porphyrin triad 4 and zinc porphyrin triad Zn4 are 0.26 and 0.30, respectively [45]. The fluorescence emission of each triad exhibits (as expected) the general spectral features of a porphyrin but with an enhanced quantum yield. 1 H and 13 C{ 1 H} NMR spectra, mass spectra, and absorption spectra (where available) for new compounds are provided in the Supplementary Materials.

General Methods
All chemicals obtained commercially were used as received unless noted otherwise. Reagent-grade solvents (CH2Cl2, hexanes, methanol, toluene, ethyl acetate) and HPLCgrade solvents (toluene, CH2Cl2, hexanes) were used as received. THF was freshly distilled

Purification Following Sonogashira Coupling Reactions
Following a three-chromatography procedure [42,70], reaction mixtures of arrays were first chromatographed with adsorption column chromatography (flash silica, Baker) to remove catalysts and ligands from the coupling reaction. Then, preparative-scale size exclusion chromatography (SEC) was performed using BioRad Bio-Beads S-X1. A preparativescale glass column (4.3 × 53 cm) was packed using BioRad Bio-Beads S-X1 in HPLC grade toluene. The chromatography was performed with gravity flow (~0.2 mL/min). Thereafter, a subsequent adsorption column chromatography (flash silica, Baker) procedure was performed (with HPLC-grade CH 2 Cl 2 and hexanes unless noted otherwise) to remove material that may have leached from the SEC resin.
The preparative purification procedure is generally most effective when the reaction affords a change in size; e.g., in instances where the product is substantially larger than the starting materials. Such is the case of forming triads via Sonogashira coupling procedures as described herein. The purification protocol is applicable to both zinc and free base porphyrins. Here, all Sonogashira coupling reactions were carried out under anaerobic, copper-free conditions [56,57] using zinc porphyrins in relatively dilute solution, as required for homogeneous solubilization of each porphyrin reactant.
Analytical-scale SEC was performed to monitor the purification of arrays [42,56]. Analytical SEC columns (styrene-divinylbenzene copolymer) were purchased from Polymer Laboratories. Analytical SEC was performed with a Hewlett-Packard 1100 HPLC using PLgel 100 Å, Plgel 500 Å, and PLgel 1000 Å columns (each~30-cm in length) in series, eluting with toluene (flow rate = 1.0 mL/min). Sample detection was achieved by absorption spectroscopy using a diode array detector with quantitation at 422, 521, 638, and 726 nm (±8 nm band width), which best captures the peaks of the arrays. In other cases, analytical SEC was performed using PLgel 50 Å, PLgel 100 Å × 2, and PLgel 500 Å columns (each 30-cm in length) in series, eluting with THF (flow rate = 0.8 mL/min) at room temperature. Sample detection was achieved by absorption spectroscopy using a diode array detector with quantitation at 440, 488, 515, 550, and 710 nm.

Outlook
Building block routes have been established for the preparation of triads comprised of two perylene-monoimides, one porphyrin, and a single tether. All of this work has emanated from the unexpected observation a decade ago that a perylene-ethynyl-porphyrin (Dyad 0) exhibits an absorption spectrum essentially lacking a characteristic porphyrin Soret band [41], as shown in Figure 1 [45][46][47]49,50]. The panchromatic absorption provided by the perylene-ethynyl-porphyrin construct differs profoundly from that of the constituent parts. Among all other chromophore-tetrapyrrole constructs subsequently examined [42,[45][46][47][48][49][50], including the exploration of the type and number of chromophores, the attachment site on the tetrapyrrole, and the composition of the tetrapyrrole, the linear (i.e., trans) arrangement of perylene-ethynyl-porphyrin-ethynyl-perylene with attachment at the porphyrin mesopositions has proven superior for panchromaticity and photophysical features. The triads described herein provide absorption across the 350-750 nm region and fluorescence in the near-infrared region. Such spectral features closely resemble those of triads prepared previously that lack tethers. The triads described herein are hydrophobic and may be best suited for use in membraneous assemblies and other lipophilic environments. For perspective, a phenazinyl-ethynyl-porphyrin that bears a benzoic acid tether has been prepared [80]. Triads 13 and 27 provide broader spectral coverage but also are substantially larger. The building block chemistry described herein should enable the preparation of a variety of porphyrin constructs with a range of tethers for studies of panchromatic absorbers in diverse applications.