Synthesis of compact water-soluble bioconjugatable porphyrins for life sciences applications

Abstract

Porphyrins substituted in a trans-AB pattern bearing a single bioconjugatable group (A) and a single water-solubilizing group (B) with limited molecular weight were synthesized for potential use in aqueous solution. The synthetic approach employed the established reaction of a dipyrromethane and a 1,9-bis(N,N-dimethylaminomethyl)dipyrromethane bearing A and B substituents, respectively, which afforded in low yield the corresponding trans-AB-porphyrin for direct use or subsequent tailoring. Altogether, 30 porphyrins were prepared. The synthetic intermediates entailed the following (given as number of new/known): PEG alkylating agent (1/0), aldehydes (3/6), dipyrromethanes (7/4), and Eschenmoser dipyrromethanes (5/2); totaling 46 new compounds. The candidate bioconjugatable linkers included carboxaldehyde, ethylphenol, phenylisothiocyanate, ethylisothiocyanate, NHS ester of benzoic acid, and NHS ester of phenylpropanoic acid. The water-solubilization motifs included polar groups appended to the two ortho (2,6-) positions of a meso-aryl group. Water solubility was assessed by spectral-band integrity in absorption spectroscopy with a 1000-fold reciprocal change of concentration (0.1–100 μM) and cuvette pathlength (100–0.1 mm). Selected porphyrins were examined for efficacy in bioconjugation. Among all, the most suitable porphyrins were those equipped with (A) a benzoic acid or phenylpropanoic acid group and (B) a meso-aryl group bearing (OCH₂CH₂)₇-CO₂H groups at the 2,6-positions. The studies taken together have pruned candidate designs for bioconjugations in dilute aqueous media.

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Introduction

Practitioners of porphyrin chemistry are well familiar with the diverse repertoire of functional properties presented by this extraordinarily versatile class of compounds. Yet the realization of those rich properties in applications in the life sciences may be impeded by the hydrophobic nature of the disk-like macrocycles. A large literature is available concerning the synthesis of porphyrins destined for use in biological or biotechnological systems where at least some degree of solubility in aqueous solution is desired.^1–5

Our particular interest concerns the design and synthesis of porphyrins that bear only two substituents: a water-solubilizing motif (A) and a bioconjugatable group (B). We have prepared a number of trans-AB-porphyrins, where A and B are not identical and are located at opposite positions across the macrocycle (i.e., 5,15-disubstitution).^6–21 The presence of only two substituents provides a compact architecture. In a recent example, a generic trans-AB-porphyrin chassis (I) was prepared for derivatization via click chemistry with oligo(ethyleneoxy) groups (Chart 1).²⁰ [The oligoethylene glycol groups that were employed were monodisperse (i.e., uniform length), yet are generally referred to under the umbrella term polyethylene glycol (PEG).] Two propargyloxy groups project over the face of the porphyrin. The attachment of polar groups at these sites results in facial encumbrance and enhances solubility in aqueous media. Studies carried out to identify water-solubilizing groups revealed (1) substantial differences between carboxy-terminated and methyl-terminated PEG units, and (2) the importance of a PEG₆versus a PEG₂ group.²² The chassis was derivatized to create a porphyrin–folic acid conjugate (II, Chart 1) that was subsequently metalated with the radionuclide ⁶⁴Cu for studies related to ovarian cancer, in cells and in mice.²⁰

Chart 1 trans-AB-porphyrin chassis and a porphyrin–folate conjugate.

One objective of the present work has been to extend beyond the use of click chemistry for attachment of water-solubilization and bioconjugation groups. While click chemistry^23,24 enables a “snap-on” approach well-suited for use with a generic chassis, and is growing in importance in tetrapyrrole chemistry,^2,25 the method as typically practiced now to form simple triazoles requires blocking the porphyrin core (e.g., with zinc metalation) to preclude copper insertion. A second objective was to explore a larger collection of bioconjugatable groups. The bioconjugatable linkers²⁶ include those that enable joining via bonds such as amide,^27,28 thiourea,^29,30 and carbamate.^31,32 A design feature that was considered essential was facial encumbrance^33–35 engendered by the 2,6-disubstitution pattern of a meso-aryl group to impart water solubility.

The present work reports the synthesis of trans-AB-porphyrins each chiefly equipped with a 2,6-bis(PEG)phenyl group for solubilization and one of a variety of bioconjugatable groups. The general synthetic approach to trans-AB-porphyrins relies on the reaction of a dipyrromethane and a 1,9-bis(N,N-dimethylaminomethyl)dipyrromethane wherein each bears a single substituent, the A or B group, at the meso-position.⁶ The availability of diverse meso-substituted dipyrromethanes^36–38 facilitates work in this area.

The paper begins with considerations that have influenced the choice of bioconjugatable tethers and PEG groups. The synthesis of 12 target porphyrins is presented next; the candidate bioconjugatable tethers include carboxaldehyde, ethylphenol, phenylisothiocyanate, ethylisothiocyanate, N-hydroxysuccinimidyl (NHS) ester of benzoic acid, and NHS ester of phenylpropanoic acid. Selected porphyrins were examined for water solubility by absorption spectroscopy over a 1000-fold range of concentration (0.1–100 μM). The preferred candidates that emerged from this broad survey were porphyrins equipped with NHS esters and a meso-aryl group bearing –(OCH₂CH₂)₇-CO₂H (or –(OCH₂CH₂)₄-OMe) groups at the 2,6-positions. The companion paper³⁹ focuses on quantitative evaluation of the kinetics and yields of conjugation of porphyrin-NHS esters with amines. Taken together, the studies provide insights into molecular design as well as quantitative data for bioconjugation of porphyrins with amines in aqueous solution under mild conditions.

Results and discussion

Bioconjugatable linkers – reconnaissance

The literature on bioconjugation is vast.⁴⁰ Application of new linkers in tetrapyrrole science is an ongoing area of research.⁴¹ Only a few comments are warranted here bearing on factors that have influenced our choice of bioconjugation motifs. Treatment of an aniline with succinic anhydride affords the corresponding amido-succinimic acid, which has been used with water-soluble chlorins (e.g., III → III-NHS, Scheme 1, panel 1).^42,43 Analysis of the reaction mixture upon attempted bioconjugation of the NHS ester (III-NHS) revealed the presence of a succinimide reaction product (IV) due to intramolecular cyclization,⁴³ a process also noted by others.^44,45 Thus, the simplicity of the installation of the amido-succinimic acid group presents a beguiling trap, which is best avoided by elision of the amido moiety; in other words, by use of the analogous phenylpropanoic acid^43,46 or the benzoic acid unit itself.

Scheme 1 Bioconjugation design considerations.

Earlier we had employed the phenoxyacetic acid unit as a water-solubilization motif^14,15 and as a linker (via an NHS ester).^{8,15,16,42,47} While attractive from the viewpoint of convenient synthetic installation and versatile conjugation with amides, it has come to our attention that the linkage is not stable. Data concerning the hydrolysis half-lives of diverse NHS esters are published on the website of a bioconjugation company,⁴⁸ and while not peer-reviewed, the results, if credible, point to the instability of the oxyacetic acid motif: at pH 8 and 25 °C, the half-life of PEG–OCH₂CO₂–NHS (V-NHS, Scheme 1, panel 2) is 0.75 min, to be compared with PEG–OCH₂CH₂CO₂–NHS (16.5 min) and PEG–OCH₂CH₂CH₂CO₂–NHS (23.3 min).⁴⁸ Studies carried out in the mid-20th century – and seemingly little noticed since then – by the estimable Campaigne and Boucher flagged the instability of the p-aminophenoxyacetate systems denoted by VI.⁴⁹ The electron-rich amino and oxygen atoms in a 1,4-arrangement are susceptible to oxidation leading to a quinonoid moiety, from which subsequent bond cleavage occurs as demonstrated by studies of p-anisidine (VII). Indeed, in the presence of a mild oxidizing agent,⁴⁹ “Aniline, methanol and anisole were isolated from the reaction mixtures even after the reactions [of p-anisidine (VII)] had been run only a few minutes” as shown in Scheme 1, panel 2, reaction iii. The tetrapyrrole macrocycle typically is electron-rich and likely could serve an analogous function as the amino group in VI. All told, the p-substituted phenoxyacetate systems that we employed previously (VIII, IX)^{8,14–16,42,47} are undesired and henceforth shunned either as a bioconjugatable linker (VIII) or as a solubilization motif (IX) (Scheme 1, panel 3). Again, a stable analogue is the phenylpropanoic acid group, where the ether oxygen atom of the phenoxyacetate is replaced with a methylene group, or simply the benzoic acid itself.

The above considerations led to the design of trans-AB-porphyrins (labeled P1–P9 with various modifiers to designate functional group changes) that are equipped with a variety of candidate bioconjugatable linkers (Chart 2). The ethyl groups in selected designs were proposed with the notion to shift the reaction site some distance from the porphyrin ring. By contrast with the choice of bioconjugatable linkers, the designer faced with choice of PEG groups encounters an almost unlimited range of candidates. PEG groups are widely used for aqueous solubilization across the molecular sciences,^50,51 in the pharmaceutical industry to increase systemic circulation,^52–54 and in tetrapyrrole chemistry.^2,5,25,55 The special properties of PEG groups have been the topic of extensive studies.^56–59 The PEG groups here differ in length (although in all cases short: 4–7 oligoethylene units), position of substitution on the aryl ring, and the presence of carboxylic acid or methyl terminal groups. The synthesis is reported next.

Chart 2 Target porphyrins.

Syntheses of building blocks

Heterotelechelic monodisperse PEG reagents are widely available,^53,60–62 but often additional tailoring is required for specific applications. Here, m-PEG₄-OH (PEG-A) was reacted with 4-nitrobenzenesulfonyl chloride (NsCl) under basic condition⁶³ to give 2,5,8,11-tetraoxatridecan-13-yl 4-nitrobenzenesulfonate (PEG-A-Ns) in 84% yield (Scheme 2).

Scheme 2 Activation of a PEG moiety.

Two aldehydes used for the ultimate preparation of NHS esters include 4-formylbenzoic acid (A1) and 3-(4-formylphenyl)propanoic acid (A2). The coupling reaction⁶⁴ of aldehyde A2 and 2-(trimethylsilyl)ethanol with N,N′-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) gave aldehyde A3 in 78% yield (Scheme 3).

Scheme 3 New aldehyde building blocks.

For facial encumbrance of the porphyrin, 2,6-dihydroxybenzaldehyde (A4) was used as a scaffold onto which two types of PEG groups were grafted. The reaction⁶⁵ of PEG-A-Ns with A4 in N,N-dimethylformamide (DMF) containing K₂CO₃ gave the diPEGylated benzaldehyde A5 as an oil in 60% yield (Scheme 3). Similarly, the reaction of A4 with PEG-B gave the diPEGylated benzaldehyde A6 in 65% yield.

The aldehydes used here for preparing dipyrromethanes (Scheme 4) include those that are new (A3, A5, A6), known (A7,²⁰A8,⁶A9,⁶⁴A10,⁶⁶A11,⁶⁷A14⁶⁸), or commercially available (A1, A2, A4, A12, A13). Known aldehyde A14⁶⁸ was prepared herein from p-hydroxybenzaldehyde and bromo PEG-C (see Scheme S1, ESI†), which proceeded in higher yield and larger scale (94%, 1.51 g) than with the corresponding iodo-PEG reagent⁶⁸ (67%, 0.12 g). Each aldehyde was subjected to a standard dipyrromethane-formation strategy,^69,70 taking neat pyrrole as the solvent and reactant under acidic condition following by treatment of base (typically triethylamine or NaOH) to give the desired product. In this manner, aldehydes A3 and A5–A14 were converted to the corresponding dipyrromethanes D3 and D5–D14. The dipyrromethanes include those that are new (D3, D5, D6, D10, D11, D13, D14) and those that are known (D7,²⁰D8,⁷¹D9,⁷²D12⁹). The reactions were carried out using trifluoroacetic acid (TFA), a catalyst employed in an early method of synthesis,⁶⁹ with triethylamine for neutralization upon workup. In so doing, the yields varied from 47% to 89%. Examination in one case (aldehyde A5) of InCl₃⁷⁰ as catalyst instead of TFA, and workup with NaOH instead of triethylamine, gave the dipyrromethane D5 in 33% yield. The reaction of aldehyde A13 was worked up with NaOH.

Scheme 4 Synthesis of dipyrromethanes.

Seven of the dipyrromethanes were treated with Eschenmoser's reagent (N,N-dimethylmethyleneiminium iodide)⁶ to afford the desired counterparts D3-Esch and D8-Esch–D13-Esch in 33% to quantitative yield (Scheme 4). The dipyrromethane derivatives include two that are known (D8-Esch,¹⁹D12-Esch⁹) whereas all others are new. The reaction was carried out at 0 °C to room temperature and worked up by aqueous–organic extraction. In general, the crude product so obtained was examined by ¹H NMR spectroscopy to confirm the integrity and then used without further purification in the following reaction. A more expansive description of conditions for use of the Eschenmoser salt with dipyrromethanes is provided in the Experimental section.

Rational syntheses of porphyrins

One rational synthetic approach to access trans-AB-porphyrins entails the condensation of a dipyrromethane and a 1,9-bis(N,N-dimethylaminomethyl)dipyrromethane in the presence of zinc acetate followed by oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ).⁶ The product is the zinc(II) trans-AB-porphyrin (Table 1). Thus, dipyrromethanes (D5–D7, and D14) characterized by distinct R¹ substituents and 1,9-bis(N,N-dimethylaminomethyl)dipyrromethanes (D3-Esch, D8-Esch, D9-Esch, D10-Esch, D11-Esch, D12-Esch, and D13-Esch) with varying R² groups were used to form the corresponding trans-AB-porphyrins. This route yielded nine trans-AB-porphyrins in yields ranging from 5–12% (Table 1). The porphyrin abbreviations denote key protecting groups tert-butyl (Bu), tert-butoxycarbonyl (Boc), 2-(trimethylsilyl)ethyl (ETMS), and 4,4-dimethylisopropylidene acetal (acetal). The dipyrromethanes were used to carry the moieties for aqueous solubilization (R¹) whereas the 1,9-bis(N,N-dimethylaminomethyl)dipyrromethanes carried the bioconjugatable linker (R²). The R¹ substituents in these porphyrins were PEG groups except for the alkyne-functionalized R¹ in ZnP1-acetal, which provides a versatile handle for click chemistry. Conversely, under appropriate deprotection conditions, the R² substituents in all porphyrins (except ZnP5 and ZnP6) function as reactive sites for bioconjugation. In the case of porphyrin ZnP5, the reactive site is directly available without deprotection. The O-acetyl group of D10-Esch was cleaved during the formation of ZnP5. Porphyrin ZnP5, which contains a p-hydroxyphenethyl group, was unstable at room temperature. The attempted reduction of the nitro group of ZnP6 by treatment with SnCl₂ failed to give the p-aminophenethyl free base porphyrin. Both phenethyl designs (ZnP5, ZnP6) were discontinued.

Table 1 Synthesis of trans-AB zinc(II) porphyrins

D + DEsch/porphyrin	R^1	R^2	Yield

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The removal of protecting groups or modification of functional groups is required in some cases to unveil moieties for bioconjugation.^73,74 Here, treatment of ZnP1-acetal to acidic conditions¹⁹ unveiled the formyl group and caused dezincation to give free base porphyrin-aldehyde P1-CHO (Scheme 5). The aldehyde group allows further bioconjugation through the formation of oxime or hydrazone linkages.⁷⁵ For click chemistry,^23–25 to prevent the insertion of copper ion into the porphyrin core,^22,46 zinc was subsequently reintroduced using zinc acetate in a solution of dichloromethane and methanol to give ZnP1-CHO in 87% yield. The protected trimethylsilylethyl ester of ZnP2-ETMS was cleaved with tetrabutylammonium fluoride (TBAF) in tetrahydrofuran (THF)²¹ to give zinc porphyrin–carboxylic acid ZnP2 in 70% yield. Treatment of the latter with TFA caused dezincation, and reaction with N-hydroxysuccinimide (NHS) mediated by DCC⁴² afforded the porphyrin-NHS ester P2-NHS following preparative thin layer chromatography (TLC) in 94% yield (3.5 mg) suitable for bioconjugation (Scheme 5). Additionally, ZnP2-ETMS was treated²² with TFA in CH₂Cl₂ to give deprotected free base porphyrin P2 in 91% yield.

Scheme 5 Elaboration of porphyrins.

Porphyrins ZnP3-Bu/ETMS and ZnP4-Bu/ETMS resemble ZnP2-ETMS but contain a tert-butyl ester at the terminus of each PEG chain. Treatment with TBAF in THF selectively cleaved the ETMS protecting group in the presence of the tert-butyl esters to give porphyrin–carboxylic acid ZnP3-Bu or ZnP4-Bu in 85% or 88% yield, respectively (Scheme 6). Subsequent esterification with NHS and DCC⁴² gave the respective porphyrin-NHS ester ZnP3-Bu/NHS or ZnP4-Bu/NHS in high yield. Finally, treatment of ZnP3-Bu/NHS with TFA in CH₂Cl₂ gave the bis(carboxy-terminated PEG)porphyrin-NHS ester P3-NHS in 90% yield (8.7 mg, by preparative TLC), whereas ZnP4-Bu/NHS gave the analogous free base porphyrin-NHS ester P4-NHS in 85% yield (2.4 mg, by reversed-phase HPLC).

Scheme 6 Synthesis of bioconjugatable porphyrins.

Hydrolyzed products of porphyrin-NHS esters P3-NHS and P4-NHS were sought for use in studies of amidation. Thus, treatment of porphyrin ZnP3-Bu/ETMS or ZnP4-Bu/ETMS with TFA in CH₂Cl₂ (1 : 1) gave global deprotection and dezincation to afford the free base porphyrin–tricarboxylic acid P3 or P4 in 63% yield or 89% yield, respectively (Scheme 7).

Scheme 7 Synthesis of hydrolyzed porphyrin standards.

Porphyrins ZnP7-Bu/Boc and ZnP8-Bu/Boc differ from ZnP3-Bu/ETMS and ZnP4-Bu/ETMS in the nature of the bioconjugatable group – the presence of a protected amino group rather than a protected carboxylic acid group. Treatment of porphyrin ZnP7-Bu/Boc or ZnP8-Bu/Boc with TFA in CH₂Cl₂ gave free base porphyrin-amine P7 or P8, respectively (Scheme 8). Subsequent reaction with 1,1′-thiocarbonyldi-2(1H)-pyridone⁷⁶ in CH₂Cl₂ gave the corresponding bioconjugatable porphyrin-isothiocyanate P7-NCS or P8-NCS upon preparative TLC in 80% (5.6 mg) or 64% (2.6 mg) yield, respectively.

Scheme 8 Synthesis of porphyrins for bioconjugation.

Finally, treatment of zinc porphyrin ZnP9-ETMS with TFA in CH₂Cl₂ caused cleavage of the ETMS group and dezincation to afford free base porphyrin–carboxylic acid P9 in 78% yield (Scheme 9).

Scheme 9 Synthesis of porphyrin–benzoic acid P9.

Statistical syntheses of porphyrins

Studies of bioconjugation showed the value of porphyrins P3-NHS and P4-NHS, as reported in the companion paper.³⁹ The syntheses shown in Scheme 6 afforded each porphyrin-NHS ester in small quantities (8.7 mg and 2.4 mg, respectively). A chief focus became to increase the quantities of the target porphyrins. While doing so, we also sought to avoid reliance on time-consuming and scale-limiting specialized chromatography: P3-NHS was prepared with use of two preparative TLC procedures, while P4-NHS was prepared with use of one preparative TLC procedure and with reversed-phase HPLC.

trans-AB-porphyrins can be prepared in a rational manner as described above, or in a statistical process.⁶⁹ The latter, with two aldehydes and one dipyrromethane, affords a mixture of three trans-substituted porphyrins bearing A₂-, B₂-, or AB-substituents. The statistical process is more expedient but relies on the ability to separate the resulting mixture of porphyrins. Thus, the condensation of aldehyde A1, dipyrromethane, and aldehyde A6 with TFA catalysis followed by oxidation with DDQ gave porphyrin P3-Bu in 10% yield (Scheme 10). Similarly, statistical condensation of aldehyde A2, dipyrromethane, and aldehyde A6 gave porphyrin P4-Bu in 11% yield. In each case, three red bands were observed upon chromatography, with the desired porphyrin as the middle band, which was easily isolated by column chromatography. Treatment of porphyrin P3-Bu or P4-Bu with NHS and DCC⁴² afforded P3-Bu/NHS or P4-Bu/NHS in high yield. Finally, P3-Bu/NHS or P4-Bu/NHS was treated with TFA in CH₂Cl₂ to obtain the porphyrin-NHS ester P3-NHS or P4-NHS in 97% or 94% yield, respectively. The two target porphyrins were obtained in quantity of 19.7 or 26.6 mg, respectively, without use of preparative TLC or HPLC procedures for purification.

Scheme 10 Synthesis of porphyrins.

Characterization

The porphyrins were typically characterized by absorption spectroscopy, ¹H NMR and ¹³C{¹H} NMR spectroscopy, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), and electrospray ionization mass spectrometry (ESI-MS). Selected results are described in the following.

The ¹H NMR spectra of the zinc trans-AB-porphyrins ZnP9-ETMS and ZnP2-ETMS are displayed in Fig. 1. The resonances from the PEG unit in the 4-phenyl-substituted ZnP9-ETMS appear in the range δ 4.4–2.8 ppm, while those of 2,6-disubstituted ZnP2-ETMS were observed in the range δ 4.0–1.4 ppm. The lone terminal methyl group of the 4-phenyl-substituted ZnP9-ETMS resonates as a singlet at δ 2.85 ppm, whereas both terminal methyl groups of the 2,6-disubstituted ZnP2-ETMS resonate as a singlet at δ 2.33 ppm. The distinct chemical shift pattern is attributed to the location of the PEG groups with respect to the aromatic ring current of the porphyrin macrocycle: thrust outward in the equatorial plane of ZnP9-ETMSversus projected over the faces of the porphyrin π-system in ZnP2-ETMS.

Fig. 1 Partial upfield region of the ¹H NMR spectra in CDCl₃ at room temperature of two zinc porphyrins. Peaks assigned to the PEG chains and the ethyl moiety of the ETMS linker are indicated.

The absorption spectra of the free base or zinc porphyrins showed the characteristic strong (Soret) band in the near-ultraviolet region and the expected manifold of weaker peaks in the visible region. Representative spectra are shown in methanol for free base porphyrin P3-Bu/NHS (405 nm Soret; 500, 534, 573, 629 nm) and zinc porphyrin ZnP3-Bu/NHS (412 nm Soret; 544, 580 nm) in Fig. S1 (ESI†).

The structure of dipyrromethane D7 (Fig. S2, ESI†) and zinc porphyrin ZnP1-acetal were determined by single-crystal X-ray diffraction (Fig. S3, ESI†). In the porphyrin, the propargyloxy groups project above and below the plane of the porphyrin macrocycle, as observed for analogous porphyrins bearing 2,6-disubstituted aryl groups.^22,77

The ESI-MS spectrum of free base porphyrin–tricarboxylic acid P3 displayed the expected m/z isotopic manifolds for species with z = 1 and z = 2, where the charged species stemmed from protonation. The most intense peak in each respective manifold was as follows: 1211.5260 (z = 1) and 606.2669 (z = 2). The observed and calculated masses were in good agreement for double protonation, Δ[M + 2H]²⁺ = 0.0007 m/z, which corresponds to 1.15 ppm (Fig. S4, ESI†). Multiply charged ions have been observed of analogous PEGylated porphyrin¹⁹ or PEGylated chlorins.⁴⁶

Solubility assessments in aqueous solution

Porphyrins P2, P3, P4, and P9 were selected for self-aggregation studies and log P measurements, where P is the partition coefficient between 1-octanol and an aqueous medium. The rationale for selecting such porphyrins was as follows. Porphyrins P3 and P4 were of highest importance given structural similarity to the porphyrin chassis used to prepare porphyrin–folate II (Chart 1). Porphyrin P2 was chosen as an analogue of P3 but without carboxylate-terminated PEG groups. Porphyrin P9 was chosen as an additional analogue but with a single PEG group disposed at the p-position rather than the o-positions of the meso-aryl group.

Self-aggregation in aqueous solution. Following the general method for aggregation studies,^22,78 the samples were dissolved in phosphate-buffered saline (PBS). Here, PBS has pH 7.4 and contains 137 mM NaCl, 10 mM phosphate, and 2.7 mM KCl. Self-aggregation was assessed by the integrity of the spectral bands in absorption spectroscopy upon examination of the porphyrin in solution over a large concentration range. With a fixed cuvette pathlength, the range of available concentrations is limited, and observed changes may reflect limitations in dynamic range of the absorption spectrophotometer rather than changes in solution of the solubilized/aggregated chromophores. But upon reciprocal change of analyte concentration (0.1–100 μM) and cuvette pathlength (100–0.1 mm), a concentration range of 1000-fold or larger can be examined.^22,78 For homogeneous dispersions of chromophores in solution, the absorbance (A) is expected to remain constant as the product of the concentration (c) and pathlength (l) remains constant, as specified in Beer's law: A = εlc. On the other hand, the occurrence of self-aggregation is expected to give rise to band broadening.

The results over a 1000-fold range of concentration and of cuvette pathlength are shown in Fig. 2. Porphyrin P2 displayed peak broadening at 100 μM, indicative of aggregation. No peak broadening was detected at concentrations less than 10 μM. The absorption spectra of porphyrin P3 were essentially identical with each other over the 1000-fold range of concentration, consistent with a homogeneous solution at each concentration. The absorption spectra of porphyrin P4 showed a very slight change in intensity; on the other hand, spectral broadening, a telltale sign of aggregation, was hardly detected. Porphyrin P9, which bears a neutral PEG group at the p-position, displayed an expected broad Soret band even at 1 μM (not shown). Examination of P9 at higher concentrations was not carried out due to insolubility in aqueous solution. As observed previously,²² PEG groups at the o-aryl positions (P2, P3 and P4) project above and below the plane of the macrocycle, and may suppress π–π stacking, with additional repulsion imparted by the negative charges of the carboxylate termini in porphyrins P3 and P4.

Fig. 2 Absorption spectra of porphyrins in aqueous PBS at room temperature. Spectra were collected as described previously^22,78 with reciprocal change of cuvette pathlength (0.1, 1.0, 2.0, 10, and 100 mm) and sample concentration (100, 10, 5, 1 and 0.1 μM).

Log P measurements. The partitioning of an organic compound between octanol and aqueous solution was assessed for porphyrins P2, P3, P4, and P9 (∼10⁻⁴ M total porphyrin concentration). A sample was removed from each layer and analyzed by absorption spectroscopy in dimethylsulfoxide (DMSO).^22,78 Porphyrin P2, which bears neutral PEG groups at the 2,6-positions of the aryl group, was found entirely in the octanol layer (log P > 2), while porphyrin P4, which bears PEG groups with carboxylic acid termini, was found entirely in the aqueous solution (log P < −2). Porphyrins P3 and P4, which bear identical PEG chains but different functional groups on the distal side (benzoic acid and phenylpropanoic acid, respectively), exhibited slightly different log P values: −1.5 for P3, <−2 for P4. A log P value of P9 could not be determined due to insolubility in aqueous solution, which was as expected given the position of the PEG group relative to the porphyrin macrocycle (Table 2).

Table 2 Aqueous-organic partitioning of porphyrins

Compounds	Projection	PEG number	Terminus	Log P
a Not determined because of insolubility in aqueous solution.
P2	2,6-	4	–OMe	>2
P3	2,6-	6	–CO2H	−1.5
P4	2,6-	6	–CO2H	<−2
P9	4-	6	–OMe	ND^a

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Initial assessments of bioconjugation in aqueous solution

Porphyrins P3-NHS, P4-NHS, P7-NCS, and P8-NCS were subjected to reaction with a short amino-PEG to assess conjugation efficacy. The porphyrin NHS-esters (P3-NHS, P4-NHS) gave excellent results, which are described in the companion paper.³⁹ With P7-NCS, the crude mixture after 1 h showed numerous components upon HPLC analysis (Fig. S5, ESI†). With P8-NCS, almost no reaction occurred after 24 h, but a major unknown product derived from the porphyrin was observed (Fig. S6, ESI†). The isothiocyanate derivatives proved quite inferior to the NHS esters in the conjugation reactions, and were not studied further. More extensive bioconjugation studies are described in the companion paper.³⁹

Outlook

The challenges and solutions for creating water-soluble tetrapyrrole macrocycles have been reviewed by several research groups.^1–5 The evolution of tetrapyrrole-compatible water-soluble designs investigated by our group has been outlined in various treatments.^22,43,46,55 The target porphyrins prepared herein were designed to bear one water-solubilization group and one bioconjugatable linker in the sparsely substituted architecture of a trans-AB-porphyrin. The syntheses of the 12 target porphyrins (Chart 2) have pointed up limitations, particularly in yields, of existing approaches for porphyrin synthesis. The low yield in turn generally gave small quantities of porphyrins, which while workable for studies of solubility properties and bioconjugation, often led to challenges in purification. Porphyrins P2-NHS, P3-NHS, P4-NHS, P7-NCS, and P8-NCS were prepared in a rational fashion in quantities of 2.4–8.7 mg. On the other hand, porphyrins P3-NHS and P4-NHS were prepared in a statistical fashion at larger scale (19.7 or 27.6 mg, respectively).

A comparison of the rational versus statistical methods can be seen upon examination of the syntheses of P3-NHS and P4-NHS. Both afford good yields following macrocycle formation but differ in accessible scale.

• The rational method requires two designer dipyrromethanes (each of which requires a synthetic aldehyde), affords a single zinc porphyrin, and requires use of an ester (here, ETMS) in the constituent that is used to make the Eschenmoser dipyrromethane (Table 1 and Scheme 6). The rational method required one or two chromatography columns to obtain the macrocycle, preparative TLC following TBAF cleavage of the ETMS group, one chromatography column upon NHS installation, and preparative TLC or reversed-phase HPLC upon TFA cleavage of the tert-butyl groups to give the porphyrin-NHS ester.

• The ostensibly more complex statistical method requires one generic dipyrromethane and two aldehydes (one of which is commercially available and is used as the free carboxylic acid rather than the ester) to afford three free base porphyrins (Scheme 10). The statistical method required two chromatography columns to obtain the free base trans-AB-porphyrin, one chromatography column upon NHS installation, and a C18-silica pad upon TFA cleavage of the tert-butyl groups. Given the availability of the two aldehydes and the dipyrromethane, the overall synthesis entails three steps (macrocycle formation, NHS ester formation, and tert-butyl ester cleavage.

• Quantitative comparison is provided here for the syntheses of P3-NHS. The statistical route used 461 mg of A6 (and stoichiometric amounts of 4-formylbenzoic acid (A1) and dipyrromethane) to obtain 65.9 mg (10% yield) of free base porphyrin P3-Bu. One-third thereof was carried forward to obtain P3-Bu/NHS (23.5 mg) and the latter then on to P3-NHS (19.7 mg). The rational route used 208 mg of D6 and 96 mg of DEsch-9 to obtain 20 mg (7% yield) of zinc porphyrin ZnP3-Bu/ETMS, which upon TBAF treatment gave porphyrin ZnP3-Bu (16.2 mg). Subsequent transformations mirrored the statistical route to give ZnP3-Bu/NHS (13.2 mg) and P3-NHS (11.0 mg), with about 85% of each intermediate carried through to the following step. The total quantity of P3-NHS obtained via the two routes was not substantially different, but the rational route entailed a greater number of steps and more extensive chromatography.

The implementation of the C18-silica pad for final purification (following TFA cleavage of the tert-butyl groups) was adopted at a later stage during the course of the research program, and likely could be used in both routes; however, the impediments to scale in the rational route appear to accrete in several stages: (1) the several-step synthesis of bespoke meso-substituted dipyrromethanes, (2) the acutely low yield in the Eschenmoser route to the porphyrin macrocycles, and (3) the difficulties in purification upon cleavage of ETMS esters with these intrinsically polar PEG-containing porphyrins. While both routes install the PEG-containing water-solubilization motifs at early stages of the synthesis (prior to macrocycle formation), the statistical route employs the free carboxylic acid and does not require a deprotection step with TBAF. Higher yields in macrocycle formation would obviously be greatly beneficial, but short of that refined ideal, further studies are required to better understand the issues of synthetic steps, purification challenges, and yields to gain better access to the PEG-containing porphyrin-NHS esters.

Water-soluble synthetic porphyrins have been known for more than one-half century, yet most contain four meso-substituents and are not equipped for bioconjugation.²² Key to the use of a single bioconjugatable motif is a companion design strategy for aqueous solubilization. Here, solubilization in aqueous media was gauged by investigation of the absorption spectrum upon reciprocal change in concentration and cuvette pathlength. Upon doing so with a porphyrin bearing a single benzoic acid, the presence of a 2,6-bis[CH₃O(CH₂CH₂O)₄-]phenyl group (P2) or 2,6-bis[HO₂C(CH₂CH₂O)₇-]phenyl group (P3 or P4) was found to afford solubility up to 10 or 100 μM, respectively. In this design, the ionizable carboxylic acid unit at the terminus of each short PEG unit imparts a significant difference for aqueous solubility at slightly basic pH values. The PEG groups, while relatively short, provide adequate facial encumbrance of the hydrophobic porphyrin π-system. While an effort has been made in recent years to investigate other chromophores, there remains much interest in porphyrins. Indeed, PEGylated porphyrins have been widely used; representative examples include to treat solid tumors with photodynamic therapy (PDT),^79–82 to facilitate imaging-guided disease treatment,^83–86 to detect toxic inorganic species in water via turn-off^87,88 or turn-on^89–91 of generic fluorescent probes, and to achieve oxidative catalysis in aqueous media.^92–96

Porphyrins P3, P4, and III–VIII displayed no band broadening over the accessible concentration regime, which reaches to ∼0.1 mM, and are regarded as highly soluble in aqueous solution. It warrants emphasis that in the presence of success – where no spectral band broadening is observed at the highest concentrations accessible (∼0.1 mM) – little insight can be gleaned; in other words, the reciprocal concentration–cuvette pathlength method is not effective for discrimination among the various architectures to identify superior molecular designs for aqueous solubilization. Regardless, the emergence of P3 and P4 as compact porphyrins that are water-soluble and accessible without use of copper-mediated click chemistry represents a successful outcome. Quantitative studies of bioconjugation with the porphyrin-NHS esters P3-NHS and P4-NHS are reported in the companion paper.³⁹ Taken together, the work reported herein should facilitate the design, synthesis, and bioconjugation of water-soluble porphyrins in diverse applications.

Experimental section

General methods

THF was freshly distilled from sodium/benzophenone ketyl and used immediately. Commercially available compounds were used as received. Silica (SiliaFlash® P60, 40–63 μm, Silicycle, R12030B) and C₁₈-silica (C18 60A, 40–63 μm, Silicycle, R33230B) were used for column chromatography. Preparative reversed-phase HPLC was performed on a Buchi® PrepPure C850 with a Buchi® column (C18 100 Å, 10 μm, LC column 250 × 10 mm).

¹H and ¹³C{¹H} NMR spectra were recorded in CD₃OD, CD₂Cl₂ or CDCl₃ at room temperature. Chemical shifts for ¹H NMR spectra are reported in parts per million (δ) relative to tetramethylsilane or a solvent signal [CD₃OD, δ = 3.31 ppm; CD₂Cl₂, δ = 5.32 ppm; CDCl₃, δ = 7.26 ppm]. Chemical shifts for ¹³C{¹H} NMR spectra are reported in parts per million (δ), and spectra were calibrated by using tetramethylsilane or solvent signals [CD₃OD, δ = 49.00 ppm; CD₂Cl₂, δ = 53.84 ppm; CDCl₃, δ = 77.16 ppm].

MALDI-MS was performed using the matrix α-cyano-4-hydroxycinnamic acid (α-CHCA). Absorption spectra were collected in toluene, MeOH, DMSO, MeCN/H₂O (80 : 20), and MeCN/H₂O (95 : 5) at room temperature. ESI-MS data are reported for the molecular ion, anionized or cationized molecular ion.

Known compounds

Known aldehydes (A7,²⁰A8,⁶A9,⁶⁴A10,⁶⁶A11,⁶⁷A14⁶⁸), dipyrromethanes (D7,²⁰D8,⁷¹D9,⁷²D12⁹), and Eschenmoser dipyrromethanes (D8-Esch,¹⁹D12-Esch⁹) were prepared as described in the literature. Known aldehyde A14⁶⁸ was prepared as described in the ESI.†

SCXRD analysis

The diffraction data of known dipyrromethane D7 and the porphyrin ZnP1-acetal reported here were collected using a Bruker D8 Venture diffractometer at 100 K with MoKα (0.71073 Å). The diffraction data were obtained and integrated by APEX4 software with SAINT and SADABS. The crystal structure was calculated, refined, and visualized using ShelXLe,⁹⁷ OLEX2 with ShelXL,⁹⁸ and Mercury 4.0⁹⁹ software. Hydrogen atoms were placed at calculated positions and refined using a riding model.

Examination of concentration-dependent self-aggregation

A porphyrin sample was dissolved in PBS 1× to prepare a 1 mM stock solution of porphyrin. PBS 1× (pH 7.4) contains 137 mM NaCl, 10 mM phosphate, and 2.7 mM KCl. The porphyrin stock solution was filtered through a 0.2 μm PTFE filter, and the concentration of the filtrate was determined based on the absorbance. The filtrate was diluted with PBS 1× to prepare solutions of porphyrin at 100 μM, 10 μM, 5 μM, 1 μM and 0.1 μM solutions. The absorption spectra of those solutions were recorded at room temperature.

Determination of log P values

Values were determined at room temperature following a reported method.⁷⁸ The quantity of porphyrin employed was ∼0.2–0.4 mg in an octanol–aqueous solution of 2 mL. An aliquot (100 μL) of each phase was removed by microsyringe and was placed in 3.0 mL of DMSO, and the absorption spectrum of each phase was measured. In each case, octanol refers to 1-octanol. The ratio between the intensity of maximum absorption for the two phases (octanol/water) was calculated.

Initial bioconjugation assessments

The initial bioconjugation assessment was carried out with a porphyrin and the amino-PEG reactant NH₂-PEG₆-OH in aqueous solution (pH 9.0) at room temperature. Reaction mixtures were analyzed after 1 h by HPLC with absorption spectral and mass spectral detection. The experimental methods are described in the companion paper.³⁹

Formation of 1,9-bis(N,N-dimethylaminomethyl)dipyrromethanes by use of Eschenmoser's reagent

Key points are as follows:

(1) Eschenmoser's reagent (N,N-dimethylmethyleneiminium iodide) was employed as obtained commercially as a fine powder.

(2) For workup, the reaction mixture typically was diluted with additional CH₂Cl₂ and washed several times with aqueous NaHCO₃ solution to completely remove unreacted remaining reagent and any iodide. The organic layer was collected, washed with brine, dried with Na₂SO₄, and concentrated under reduced pressure to afford a dark (brown or black) amorphous solid that often consisted of a dry powder.

(3) Thorough washing of the crude reaction product is essential to avoid carryover of iodide into the porphyrin-forming reaction, whereupon DDQ oxidation, a mono-iodo-substituted porphyrin can result. Here, the high polarity of the PEG-containing porphyrins rendered difficult the separation of any iodo-porphyrin byproduct from the target porphyrin.

(4) The crude, Eschenmoser dipyrromethane was generally characterized by ¹H NMR and ¹³C{¹H} NMR spectroscopy and then used in the next step without further purification.

(5) The Eschenmoser dipyrromethanes (as dry solids under an inert atmosphere) can be stored at room temperature for a long period.

(6) Use of the chloride salt of Eschenmoser's reagent was not fruitful. The synthesis of D13-Esch was attempted in this manner, but a complex reaction mixture formed, containing tri- or tetra-alkylated products, which were difficult to remove from the desired dialkylated, 1,9-bis(N,N-dimethylaminomethyl)dipyrromethane (the Eschenmoser dipyrromethane).

PEG reagent

2,5,8,11-Tetraoxatridecan-13-yl 4-nitrobenzenesulfonate (PEG-A-Ns). Following a reported method⁶³ with some modifications, a solution of PEG-A (2.1 g, 10 mmol) and 4-nitrobenzenesulfonyl chloride (2.7 g, 12 mmol) in CH₂Cl₂ (20 mL) at 0 °C was treated dropwise with triethylamine (2 mL) for 10 min. The reaction mixture was allowed to warm to room temperature and stirred overnight. After the reaction was completed on the basis of TLC analysis [silica, CH₂Cl₂/ethyl acetate (1 : 1)], the resulting solution was washed with water and brine, dried (Na₂SO₄), and then concentrated to dryness under reduced pressure. The crude product was chromatographed [CH₂Cl₂/ethyl acetate (1 : 1)] to afford a colorless liquid (3.3 g, 84%): ¹H NMR (500 MHz, CDCl₃) δ 8.16 (d, J = 7.8 Hz, 1H), 7.83–7.73 (m, 3H), 4.43–4.41 (m, 2H), 3.79–3.77 (m, 2H), 3.64–3.53 (m, 12H), 3.37 (s, 3H); ¹³C{¹H} NMR (175 MHz, CDCl₃) δ 148.5, 134.8, 132.5, 131.4, 130.0, 124.9, 72.1, 71.4, 70.9, 70.71, 70.70, 70.66, 70.6, 68.7, 59.1; ESI-MS obsd 394.1157, calcd 394.1161 [(M + H)⁺], M = C₁₅H₂₃NO₉S.

Aldehydes

4-(3-(2-(Trimethylsilyl)ethoxy)-3-oxopropyl)benzaldehyde (A3). Following a reported method⁶⁴ with some modifications, a solution of A2 (356 mg, 2.00 mmol) and 2-(trimethylsilyl)ethanol (1.18 g, 10.0 mmol) in CH₂Cl₂ (10.0 mL) was stirred at room temperature. After 10 min, DMAP (36.7 mg, 0.300 mmol) and DCC (454 mg, 2.20 mmol) were added. The reaction mixture was stirred at room temperature for 2.5 h, then filtered to remove the precipitate. The filtrate was concentrated under reduced pressure. The crude residue was chromatographed [silica, hexanes/CH₂Cl₂ (1 : 2)] to afford a colorless oil (436 mg, 78%): ¹H NMR (500 MHz, CDCl₃) δ 9.98 (s, 1H), 7.81 (d, J = 8.2 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H), 4.18–4.14 (m, 2H), 3.03 (t, J = 7.7 Hz, 2H), 2.64 (t, J = 7.7 Hz, 2H), 0.97–0.94 (m, 2H), 0.03 (s, 9H); ¹³C{¹H} NMR (125 MHz, CDCl₃) δ 192.0, 172.7, 148.1, 135.0, 130.2, 129.2, 63.0, 35.5, 31.2, 17.5, −1.4; ESI-MS obsd 301.1221, calcd 301.1230 [(M + Na)⁺], M = C₁₅H₂₂O₃Si.

2,6-Bis((2,5,8,11-tetraoxatridecan-13-yl)oxy)benzaldehyde (A5). Following a reported method⁶⁵ with some modifications, a solution of A4 (31 mg, 0.22 mmol) in DMF (1.0 mL) was treated with K₂CO₃ (125 mg, 0.904 mmol) at 60 °C (oil bath). After 30 min, a sample of PEG-A-Ns (224 mg, 0.570 mmol) was slowly added, and the resulting suspension was heated to 80 °C (oil bath) and stirred overnight at 80 °C (oil bath). The reaction mixture was washed with water (×2) and brine, dried (Na₂SO₄), and then concentrated to dryness under reduced pressure. The crude residue was chromatographed [CH₂Cl₂/MeOH (12 : 1)] to afford a brown liquid (69 mg, 60%): ¹H NMR (700 MHz, CDCl₃) δ 10.51 (s, 1H), 7.39 (t, J = 8.4 Hz, 1H), 6.58 (d, J = 8.5 Hz, 2H), 4.19 (t, J = 5.0 Hz, 4H), 3.90–3.89 (m, 4H), 3.76–3.75 (m, 4H), 3.67–3.63 (m, 16H), 3.55–3.54 (m, 4H), 3.37 (s, 6H); ¹³C{¹H} NMR (175 MHz, CDCl₃) δ 189.1, 161.3, 135.5, 115.1, 105.4, 71.9, 71.0, 70.7, 70.59, 70.58, 70.5, 69.4, 68.8, 59.0; ESI-MS obsd 519.2800, calcd 519.2787 [(M + H)⁺], M = C₂₅H₄₂O₁₁.

2,6-Bis((1-tert-butyloxy-1-oxo-4,7,10,13,16,19-hexaoxahenicosan-21-yl)oxy)benzaldehyde (A6). Following a reported method⁶⁵ with some modifications, a solution of A4 (414 mg, 3.00 mmol) in DMF (7.5 mL) was treated with K₂CO₃ (1.66 g, 0.0120 mol) for 30 min at 65 °C (oil bath) followed by the addition of PEG-B (2.98 g, 6.30 mmol). The mixture was stirred overnight at 80 °C (oil bath). After the reaction was completed as determined by TLC (CH₂Cl₂/MeOH, 15 : 1) analysis, the reaction mixture was concentrated under reduced pressure to remove DMF. The crude product was dissolved in CH₂Cl₂ (50 mL), extracted with water (2 × 50 mL), extracted with brine (2 × 50 mL), and dried (Na₂SO₄). The combined organic extract was dried under reduced pressure. The residue was purified by chromatography [silica, CH₂Cl₂/MeOH (40 : 1)] to give a yellow liquid (1.79 g, 65%): ¹H NMR (700 MHz, CDCl₃) δ 10.25 (s, 1H), 7.15 (t, J = 8.4 Hz, 1H), 6.34 (d, J = 8.4 Hz, 2H), 3.94 (q, J = 4.9, 4.2 Hz, 4H), 3.50–3.32 (m, 48H), 2.24–2.21 (m, 4H), 1.21–1.19 (m, 18H); ¹³C{¹H} NMR (175 MHz, CDCl₃) δ 188.5, 170.5, 161.0, 135.4, 114.8, 105.2, 80.0, 70.7, 70.4, 70.3, 70.2, 70.1, 69.1, 68.6, 66.6, 36.0, 27.9; ESI-MS obsd 923.5191, calcd 923.5210 [(M + H)⁺], M = C₄₅H₇₈O₁₉.

Dipyrromethanes

5-(4-(3-(2-(Trimethylsilyl)ethoxy)-3-oxopropyl)phenyl)dipyrromethane (D3). Following a reported method⁶⁹ with some modifications, a sample of A3 (399 mg, 1.43 mmol) in distilled pyrrole (20.0 mL, 286 mmol) was stirred at 0 °C (ice bath) under argon. After 10 min, TFA (16 μL, 0.210 mmol) was slowly added, and the reaction mixture was stirred at 0 °C (ice bath) for 30 min. Then, triethylamine (200 μL, 1.43 mmol) was added to quench the reaction. The solvent was removed under reduced pressure. The crude residue was chromatographed [silica, CH₂Cl₂] to afford a pale-yellow solid (459 mg, 81%): ¹H NMR (500 MHz, CDCl₃) δ 7.93 (s, 2H), 7.16–7.12 (m, 4H), 6.69 (td, J = 2.7, 1.5 Hz, 2H), 6.15 (q, J = 2.9 Hz, 2H), 5.92–5.90 (m, 2H), 5.45 (s, 1H), 4.19–4.15 (m, 2H), 2.93 (t, J = 7.9 Hz, 2H), 2.61–2.58 (m, 2H), 1.00–0.96 (m, 2H), 0.04 (s, 9H); ¹³C{¹H} NMR (125 MHz, CDCl₃) δ 173.2, 140.1, 139.5, 132.7, 128.7, 128.6, 117.3, 108.6, 107.3, 62.8, 43.8, 36.1, 30.7, 17.5, −1.3; ESI-MS obsd 417.1954, calcd 417.1969 [(M + Na)⁺], M = C₂₃H₃₀N₂O₂Si.

5-[2,6-Bis((2,5,8,11-tetraoxatridecan-13-yl)oxy)phenyl]dipyrromethane (D5). Following a reported method⁷⁰ with some modifications, a solution of A5 (295 mg, 0.569 mmol) in pyrrole (4.0 mL) was stirred at room temperature under argon. After 15 min, InCl₃ (38 mg, 0.17 mmol) was added to the mixture. The reaction mixture was stirred for 1.5 h at room temperature, then NaOH (68 mg, 1.7 mmol) was added, and the mixture was stirred for 45 min. The mixture was filtered (Fisher brand qualitative paper), and the filtrate was concentrated under reduced pressure to remove residual pyrrole. The crude mixture was chromatographed [silica, CH₂Cl₂/MeOH (15 : 1)] to give a brown liquid (118 mg, 33%): ¹H NMR (500 MHz, CDCl₃) δ 9.30 (s, 2H), 7.07 (t, J = 8.4 Hz, 1H), 6.62 (q, J = 2.1 Hz, 2H), 6.50 (s, 2H), 6.22 (s, 1H), 6.03 (q, J = 2.8 Hz, 2H), 5.90–5.88 (m, 2H), 4.04 (s, 4H), 3.72–3.49 (m, 28H), 3.35 (s, 6H); ¹³C{¹H} NMR (125 MHz, CDCl₃) δ 156.9, 132.9, 127.7, 120.6, 116.1, 107.2, 105.8, 71.9, 70.8, 70.58, 70.55, 70.45, 70.43, 69.7, 59.0, 32.9; ESI-MS obsd 635.3521, calcd 635.3538 [(M + H)⁺], M = C₃₃H₅₀N₂O₁₀.

5-[2,6-Bis((1-tert-butyloxy-1-oxo-4,7,10,13,16,19-hexaoxahenicosan-21-yl)oxy)phenyl]dipyrromethane (D6). Following a reported method⁶⁹ with some modifications, a sample of A6 (1.38 g, 1.50 mmol) in distilled pyrrole (20.8 mL, 300 mmol) was stirred at 0 °C (ice bath) under argon. After 10 min, TFA (18 μL, 0.235 mmol) was slowly added, and the reaction mixture was stirred at 0 °C (ice bath) for 30 min. Then, triethylamine (209 μL, 1.50 mmol) was added. The solvent was removed under reduced pressure. The crude residue was chromatographed [silica, CH₂Cl₂/MeOH (40 : 1)] to afford a yellow liquid (1.40 g, 89%): ¹H NMR (500 MHz, CDCl₃) δ 9.25 (s, 2H), 7.06 (t, J = 8.2 Hz, 1H), 6.61–6.60 (m, 2H), 6.49 (s, 2H), 6.21 (s, 1H), 6.03–6.01 (m, 2H), 5.90–5.85 (m, 2H), 4.05 (s, 4H), 3.72–3.53 (m, 48H), 2.49 (t, J = 6.6 Hz, 4H), 1.44 (s, 18H); ¹³C{¹H} NMR (125 MHz, CDCl₃) δ 170.9, 132.9, 127.7, 120.6, 116.1, 107.3, 105.8, 80.5, 77.3, 70.8, 70.59, 70.57, 70.54, 70.52, 70.51, 70.48, 70.43, 70.35, 69.7, 66.9, 36.3, 32.9, 28.1; ESI-MS obsd 1039.5939, calcd 1039.5948 [(M + H)⁺], M = C₅₃H₈₆N₂O₁₈.

5-(2-(4-Acetoxyphenyl)ethyl)dipyrromethane (D10). Following a reported method⁶⁹ with some modifications, a sample of A10 (288 mg, 1.50 mmol) in distilled pyrrole (20.8 mL, 300 mmol) was stirred at 0 °C (ice bath) under argon. After 10 min, TFA (18 μL, 0.235 mmol) was slowly added, and the reaction mixture was stirred at 0 °C (ice bath) for 45 min. Then, triethylamine (209 μL, 1.50 mmol) was added. The solvent was removed under reduced pressure. The crude residue was chromatographed [silica, CH₂Cl₂/ethyl acetate (40 : 1)] to afford a pale-yellow solid (366 mg, 79%): ¹H NMR (500 MHz, CDCl₃) δ 7.76 (s, 2H), 7.16 (d, J = 8.5 Hz, 2H), 6.99 (d, J = 8.5 Hz, 2H), 6.63 (td, J = 2.7, 1.5 Hz, 2H), 6.16 (q, J = 2.9 Hz, 2H), 6.09 (td, J = 2.9, 1.7 Hz, 2H), 3.97 (t, J = 7.6 Hz, 1H), 2.63–2.60 (m, 2H), 2.30–2.24 (m, 5H); ¹³C{¹H} NMR (125 MHz, CDCl₃) δ 169.8, 148.9, 139.5, 133.2, 129.5, 121.5, 121.4, 117.3, 108.3, 105.7, 37.0, 36.1, 33.0, 21.3; ESI-MS obsd 309.1590, calcd 309.1598 [(M + H)⁺], M = C₁₉H₂₀N₂O₂.

5-(2-(4-Nitrophenyl)ethyl)dipyrromethane (D11). Following a reported method⁷⁰ with some modifications, a solution of A11 (964 mg, 5.38 mmol) and pyrrole (70 mL) was stirred at 0 °C (ice bath) under argon for 5 min, then TFA (62 μL) was added slowly. The reaction was stirred at 0 °C (ice bath) for 30 min, then triethylamine (750 μL) was added. The mixture was concentrated under reduced pressure. The crude product was chromatographed [silica, hexanes/CH₂Cl₂ (1 : 4) to CH₂Cl₂] to afford a yellow liquid (1.2 g, 76%): ¹H NMR (600 MHz, CDCl₃) δ 8.14 (d, J = 6.0 Hz, 2H), 7.78 (s, 2H), 7.31 (d, J = 6.0 Hz, 2H), 6.66–6.65 (m, 2H), 6.18–6.15 (m, 2H), 6.11–6.09 (m, 2H), 3.99 (t, J = 7.6 Hz, 1H), 2.74 (t, J = 6Hz, 2H), 2.32–2.28 (m, 2H); ¹³C{¹H} NMR (150 MHz, CDCl₃) δ 149.8, 146.4, 132.5, 129.3, 123.7, 117.5, 108.3, 105.7, 37.0, 35.6, 33.5; ESI-MS obsd 296.1388, calcd 296.1394 [(M + H)⁺], M = C₁₇H₁₇N₃O₂.

5-(4-(tert-Butyloxycarbonylamino)phenyl)dipyrromethane (D12). Following a reported method⁶⁹ with some modifications, a sample of A12 (443 mg, 2.00 mmol) in distilled pyrrole (28.0 mL, 400 mmol) was stirred at 0 °C (ice bath) under argon. After 10 min, TFA (23 μL, 0.30 mmol) was slowly added, and the reaction mixture was stirred at 0 °C (ice bath) for 30 min. Then, triethylamine (280 μL, 2.0 mmol) was added. The mixture was concentrated under reduced pressure. The crude residue was chromatographed [silica, CH₂Cl₂] to afford a yellow solid (580 mg, 86%): ¹H NMR (500 MHz, CDCl₃) δ 7.91 (s, 2H), 7.30 (d, J = 8.1 Hz, 2H), 7.13 (d, J = 8.5 Hz, 2H), 6.68 (td, J = 2.6, 1.5 Hz, 2H), 6.46 (s, 1H), 6.15 (q, J = 2.9 Hz, 2H), 5.92–5.90 (m, 2H), 5.43 (s, 1H), 1.52 (s, 9H); ¹³C{¹H} NMR (125 MHz, CDCl₃) δ 152.9, 137.3, 136.8, 132.7, 129.1, 119.0, 117.3, 108.6, 107.2, 43.5, 28.5; ESI-MS obsd 360.1674, calcd 360.1683 [(M + Na)⁺], M = C₂₀H₂₃N₃O₂.

5-(2-(tert-Butyloxycarbonylamino)ethyl)dipyrromethane (D13). Following a reported method⁷⁰ with some modifications, a solution of A13 (2.3 g, 13 mmol) in pyrrole (50 mL, 720 mmol) was treated dropwise with TFA (99 μL, 1.3 mmol) over 5 min. The mixture at room temperature was stirred under argon for 1 h followed by the addition of NaOH (1.0 g, 25 mmol). The crude mixture was stirred for 1 h, then filtered through a Celite pad (5 cm × 2 cm). The filtrate was concentrated and then chromatographed [silica, CH₂Cl₂/ethyl acetate (20 : 1 to 10 : 1)] to give a brown oil (2.07 g, 55%): ¹H NMR (500 MHz, CDCl₃) δ 8.52 (s, 2H), 6.75–6.57 (m, 2H), 6.14 (q, J = 2.9 Hz, 2H), 5.98 (s, 2H), 4.61 (t, J = 6.6 Hz, 1H), 4.03 (t, J = 7.4 Hz, 1H), 3.18 (q, J = 6.4 Hz, 2H), 2.09 (q, J = 6.7 Hz, 2H), 1.45 (s, 9H); ¹³C{¹H} NMR (125 MHz, CDCl₃) δ 156.9, 133.3, 117.2, 108.2, 105.4, 79.7, 38.6, 36.2, 34.8, 28.5; ESI-MS obsd 290.1860, calcd 290.1863 [(M + H)⁺], M = C₁₆H₂₃N₃O₂.

5-(4-((2,5,8,11,14,17-Hexaoxanonadecan-19-yl)oxy)phenyl)dipyrromethane (D14). Following a procedure⁶⁹ with modification, a sample of TFA (28 μL, 0.36 mmol) was added to a solution of A14 (1.47 g, 3.67 mmol) in freshly distilled pyrrole (26.0 mL, 382 mmol) at 0 °C (ice bath). The reaction mixture was stirred for 1 h at 0 °C (ice bath). Subsequently, triethylamine (0.55 mL) was added, and the resulting mixture was stirred for 15 min at 0 °C (ice bath). The mixture was concentrated under reduced pressure and then chromatographed [silica, CH₂Cl₂/ethyl acetate (2 : 1) followed by CH₂Cl₂/methanol (15 : 1)] to afford a brownish oil (0.89 g, 47%): ¹H NMR (600 MHz, CDCl₃) δ 8.01 (s, 2H), 7.12–7.10 (m, 2H), 6.68–6.84 (m, 2H), 6.69–6.68 (m, 2H), 6.14 (q, J = 3.0 Hz, 2H), 5.90–5.89 (m, 2H), 5.42 (s, 1H), 4.11–4.10 (m, 2H), 3.85–3.83 (m, 2H), 3.72–3.70 (m, 2H), 3.67–3.61 (m, 16H), 3.54–3.52 (m, 2H), 3.36 (s, 3H); ¹³C{¹H} NMR (150 MHz, CDCl₃) δ 157.8, 134.4, 132.9, 129.4, 117.1, 114.7, 108.3, 107.0, 71.9, 70.8, 70.64, 70.59, 70.57, 70.55, 70.5, 69.7, 67.5, 59.0, 43.1; ESI-MS obsd 534.3177, calcd 534.3174 [(M + NH₄)⁺], M = C₂₈H₄₀N₂O₇.

Eschenmoser dipyrromethanes

1,9-Bis(N,N-dimethylaminomethyl)-5-(4-(3-(2-(trimethylsilyl)ethoxy)-3-oxopropyl)phenyl)dipyrromethane (D3-Esch). Following a reported method⁶ with some modifications, a sample of D3 (450 mg, 1.14 mmol) in CH₂Cl₂ (11.4 mL) was stirred at 0 °C (ice bath) under argon. After 15 min, N,N-dimethylmethyleneiminium iodide (442 mg, 2.39 mmol) was added, and the reaction mixture was stirred at 0 °C (ice bath) for 35 min. Then, saturated aqueous NaHCO₃ (7.0 mL) was added. The reaction mixture was diluted with CH₂Cl₂ (40 mL) and washed with saturated aqueous NaHCO₃ (7 × 40 mL). The organic extract was dried (Na₂SO₄) and concentrated under reduced pressure to afford a yellow solid (561 mg, 97%): ¹H NMR (500 MHz, CDCl₃) δ 8.31 (s, 2H), 7.12 (s, 4H), 5.90 (t, J = 2.9 Hz, 2H), 5.75–5.73 (m, 2H), 5.34 (s, 1H), 4.18–4.15 (m, 2H), 3.38–3.29 (m, 4H), 2.91 (dd, J = 8.7, 7.2 Hz, 2H), 2.59–2.58 (m, 2H), 2.17 (s, 12H), 0.99–0.96 (m, 2H), 0.04 (s, 9H); ¹³C{¹H} NMR (125 MHz, CDCl₃) δ 173.2, 140.5, 139.1, 132.8, 128.9, 128.6, 128.5, 107.5, 106.8, 62.8, 56.8, 45.1, 44.0, 36.2, 30.7, 17.4, −1.3; ESI-MS obsd 509.3295, calcd 509.3306 [(M + H)⁺], M = C₂₉H₄₄N₄O₂Si.

1,9-Bis(N,N-dimethylaminomethyl)-5-(4-(2-(trimethylsilyl)ethoxycarbonyl)phenyl)dipyrromethane (D9-Esch). Following a reported method⁶ with some modifications, a solution of D9 (366 mg, 1.00 mmol) in CH₂Cl₂ (10 mL) was treated with N,N-dimethylmethyleneiminium iodide (726 mg, 3.92 mmol) under argon. The mixture was stirred for 1 h at room temperature, then CH₂Cl₂ (40 mL) was added to afford a homogeneous solution. The organic layer was washed (saturated aqueous NaHCO₃ (50 mL) and brine (50 mL)), dried (Na₂SO₄), and concentrated under reduced pressure to give a brown solid (479 mg, 100%): mp 67–69 °C; ¹H NMR (500 MHz, CDCl₃) δ 8.60 (s, 2H), 7.98–7.93 (m, 2H), 7.30–7.26 (m, 2H), 5.93 (t, J = 2.9 Hz, 2H), 5.77–5.73 (t, J = 2.9 Hz, 2H), 5.43 (s, 1H), 4.43–4.37 (m, 2H), 3.50–3.34 (m, 4H), 2.23 (s, 12H), 1.15–1.08 (m, 2H), 0.08 (s, 9H); ¹³C{¹H} NMR (150 MHz, CDCl₃) δ 168.1, 149.0, 133.5, 131.1, 130.5, 129.75, 129.67, 109.4, 108.5, 64.6, 57.9, 46.1, 45.7, 18.8, 0.00; ESI-MS obsd 479.2848, calcd 479.2848 [(M − H)⁻], M = C₂₇H₄₀N₄O₂Si.

1,9-Bis(N,N-dimethylaminomethyl)-5-(2-(4-acetoxyphenyl)ethyl)dipyrromethane (D10-Esch). Following a reported method⁶ with some modifications, a sample of D10 (158 mg, 0.513 mmol) in CH₂Cl₂ (5.1 mL) was stirred at 0 °C (ice bath) under argon. After 10 min, N,N-dimethylmethyleneiminium iodide (199 mg, 1.08 mmol) was added, and the reaction mixture was stirred at 0 °C (ice bath) for 35 min. Then, saturated aqueous NaHCO₃ (5 mL) was added. The reaction mixture was diluted with CH₂Cl₂ (20 mL) and washed with saturated aqueous NaHCO₃ (6 × 25 mL). The organic extract was dried (Na₂SO₄), and concentrated under reduced pressure to afford a yellow solid (183 mg, 85%): ¹H NMR (500 MHz, CDCl₃) δ 8.30 (s, 2H), 7.15 (d, J = 8.5 Hz, 2H), 6.97 (d, J = 8.5 Hz, 2H), 5.90 (d, J = 2.7 Hz, 4H), 3.91 (t, J = 7.6 Hz, 1H), 3.36–3.29 (m, 4H), 2.61–2.58 (m, 2H), 2.28 (s, 3H), 2.26–2.21 (m, 2H), 2.15 (s, 12H); ¹³C{¹H} NMR (125 MHz, CDCl₃) δ 169.8, 148.9, 139.7, 133.5, 129.6, 128.6, 121.4, 107.4, 105.0, 56.8, 45.1, 37.4, 36.1, 33.2, 21.3; ESI-MS obsd 423.2744, calcd 423.2755 [(M + H)⁺], M = C₂₅H₃₄N₄O₂.

1,9-Bis(N,N-dimethylaminomethyl)-5-(2-(4-nitrophenyl)ethyl)dipyrromethane (D11-Esch). Following a reported method⁶ with some modifications, N,N-dimethylmethyleneiminium iodide (740 mg, 4.00 mmol) was added to a solution of D11 (614 mg, 2.08 mmol) in CH₂Cl₂ (20 mL). The mixture was stirred at room temperature for 1 h, then saturated aqueous NaHCO₃ was added. The organic phase was washed saturated aqueous NaHCO₃ (20 mL × 5) and brine), dried (Na₂SO₄), and then concentrated under reduced pressure to afford a yellow solid (268 mg, 33%): ¹H NMR (500 MHz, CDCl₃) δ 8.30 (s, 2H), 8.13 (d, J = 8.7 Hz, 2H), 7.29 (d, J = 8.7 Hz, 2H), 5.90 (s, 4H), 3.90 (t, J = 7.7 Hz, 1H), 3.32 (q, J = 13.4 Hz, 4H), 2.73–2.70 (m, 2H), 2.29–2.24 (m, 2H), 2.15 (s, 12H); ¹³C{¹H} NMR (125 MHz, CDCl₃) δ 150.0, 146.4, 133.0, 129.3, 128.5, 123.6, 107.5, 104.9, 56.6, 44.9, 37.4, 35.6, 33.7.

1,9-Bis(N,N-dimethylaminomethyl)-5-(4-(tert-butyloxycarbonylamino)phenyl)dipyrromethane (D12-Esch). Following a reported method⁶ with some modifications, a sample of D12 (236 mg, 0.700 mmol) in CH₂Cl₂ (7.0 mL) was stirred at 0 °C (ice bath) under argon. After 15 min, N,N-dimethylmethyleneiminium iodide (272 mg, 1.47 mmol) was added, and the reaction mixture was stirred at 0 °C (ice bath) for 35 min. Then, saturated aqueous NaHCO₃ (7 mL) was added. The reaction mixture was diluted with CH₂Cl₂ (25 mL) and washed with saturated aqueous NaHCO₃ (6 × 30 mL). The organic extract was dried (Na₂SO₄) and then concentrated under reduced pressure to afford a yellow solid (285 mg, 90%): ¹H NMR (500 MHz, CDCl₃) δ 8.40 (s, 2H), 7.25 (d, J = 8.0 Hz, 2H), 7.11 (d, J = 8.6 Hz, 2H), 6.56 (s, 1H), 5.90 (t, J = 2.9 Hz, 2H), 5.74 (t, J = 3.0 Hz, 2H), 5.32 (s, 1H), 3.38–3.29 (m, 4H), 2.16 (s, 12H), 1.51 (s, 9H); ¹³C{¹H} NMR (125 MHz, CDCl₃) δ 153.0, 137.2, 137.1, 132.9, 129.0, 128.8, 118.8, 107.6, 106.7, 56.8, 45.1, 43.7, 28.5; ESI-MS obsd 452.3009, calcd 452.3020 [(M + H)⁺], M = C₂₆H₃₇N₅O₂.

1,9-Bis(N,N-dimethylaminomethyl)-5-(2-(tert-butyloxycarbonylamino)ethyl)dipyrromethane (D13-Esch). Following a reported method⁶ with some modifications, a solution of D13 (1.87 g, 6.50 mmol) in CH₂Cl₂ (65 mL) was treated with N,N-dimethylmethyleneiminium iodide (2.6 g, 14 mmol) at room temperature under argon. After 1 h, the crude reaction mixture was washed three times with saturated aqueous NaHCO₃. The organic phase was dried (Na₂SO₄) and concentrated under reduced pressure to afford a black, non-crystalline solid (2.3 g, 88%): ¹H NMR (500 MHz, CDCl₃) δ 9.10 (s, 2H), 5.95 (t, J = 2.9 Hz, 2H), 5.88–5.87 (m, 2H), 4.68 (s, 1H), 3.99 (t, J = 7.5 Hz, 1H), 3.56–3.50 (m, 4H), 3.12 (q, J = 6.2 Hz, 2H), 2.29 (s, 12H), 1.42 (s, 9H); ESI-MS obsd 404.3017, calcd 404.3020 [(M + H)⁺], M = C₂₂H₃₇N₅O₂.

Porphyrin formation

Zn(II)-5-(2,6-bis(propargyloxy)phenyl)-15-(4-(5,5-dimethyl-1,3-dioxan-2-yl)phenyl)porphyrin (ZnP1-acetal). Following a method⁶ with some modifications, a solution of D8-Esch (544 mg, 1.21 mmol) and D7 (396 mg, 1.20 mmol) in EtOH (120 mL) was treated with Zn(OAc)₂·2H₂O (2.63 g, 0.0120 mol). The mixture was heated to reflux. After 2 h, the reaction mixture was allowed to cool to room temperature. The reaction mixture was treated with DDQ (821 mg, 3.62 mmol) and stirred at room temperature for 15 min followed by the addition of triethylamine (850 μL, 6.10 mmol). After 10 min, the crude mixture was concentrated under reduced pressure followed by column chromatography [CH₂Cl₂] and recrystallization (hexane/CHCl₃) to afford a red solid (73 mg, 8%): ¹H NMR (700 MHz, CDCl₃) δ 10.23 (s, 2H), 9.39 (d, J = 4.4 Hz, 2H), 9.35 (d, J = 4.4 Hz, 2H), 9.09 (d, J = 4.4 Hz, 2H), 9.06 (d, J = 4.4 Hz, 2H), 8.25 (d, J = 7.5 Hz, 2H), 7.94 (d, J = 7.5 Hz, 2H), 7.81 (t, J = 8.7 Hz, 1H), 7.28 (d, J = 8.7 Hz, 2H), 5.79 (s, 1H), 4.36 (s, 4H), 4.00 (d, J = 10.3 Hz, 2H), 3.89 (d, J = 10.3 Hz, 2H), 2.31 (s, 2H), 1.51 (s, 3H), 0.94 (s, 3H); ¹³C{¹H} NMR (175 MHz, CDCl₃) δ 158.6, 150.8, 149.71, 149.67, 149.4, 143.6, 137.7, 134.7, 132.4, 132.3, 132.0, 131.5, 129.9, 124.6, 122.0, 119.8, 110.7, 107.2, 106.0, 102.3, 78.8, 78.1, 75.4, 56.4, 53.6, 30.6, 23.4, 22.2; λ_abs (toluene) 414, 539, 573 nm; MALDI-MS obsd 746.43, calcd 746.19 [M⁺]; ESI-MS obsd 747.1930, calcd 747.1944 [(M + H)⁺], M = C₄₄H₃₄N₄O₄Zn. Slow evaporation from THF at 20 °C afforded a crystalline sample that was then examined by SCXRD analysis.

Zn(II)-5-(2,6-bis((2,5,8,11-tetraoxatridecan-13-yl)oxy)phenyl)-15-(4-(2-(trimethylsilyl)ethoxycarbonyl)phenyl)porphyrin (ZnP2-ETMS). Following a reported method⁶ with some modifications, a solution of D5 (64 mg, 0.10 mmol) and D9-Esch (48 mg, 0.10 mmol) in EtOH (10 mL) was treated with Zn(OAc)₂·2H₂O (222 mg, 1.01 mmol) at room temperature. The mixture was heated to reflux for 2 h, then allowed to cool to room temperature. The reaction mixture was treated with DDQ (70 mg, 0.31 mmol). The reaction mixture was stirred for 30 min, and then triethylamine (100 μL) was added. After 15 min, the solution was concentrated and the crude product was chromatographed using a gradient [silica, CH₂Cl₂/ethyl acetate (4 : 1) to (3 : 2) to (1 : 1)] to give a red solid (5.5 mg, 5%): ¹H NMR (700 MHz, CDCl₃) δ 10.19 (s, 2H), 9.37 (d, J = 4.2 Hz, 2H), 9.32 (d, J = 4.2 Hz, 2H), 9.04 (d, J = 4.2, 2H), 9.02 (d, J = 4.2 Hz, 2H), 8.46 (d, J = 7.7 Hz, 2H), 8.32 (d, J = 7.7 Hz, 2H), 7.73 (t, J = 8.7 Hz, 1H), 7.05 (d, J = 8.7 Hz 2H), 4.65 (t, J = 8.4 Hz, 2H), 3.97 (t, J = 4.5 Hz, 4H), 2.90 (t, J = 4.5 Hz, 4H), 2.33 (s, 6H), 2.29–2.24 (m, 4H), 2.14–2.08 (m, 4H), 1.79–1.72 (m, 4H), 1.70–1.61 (m, 8H), 1.43 (t, J = 4.9 Hz, 4H), 1.35–1.30 (m, 2H), 0.21 (s, 9H); ¹³C{¹H} NMR (175 MHz, CDCl₃) δ 167.4, 160.0, 151.1, 149.6, 149.2, 149.1, 148.3, 134.8, 132.1, 131.9, 131.7, 131.6, 130.1, 129.6, 127.8, 121.9, 118.2, 106.1, 105.6, 70.7, 69.6, 68.99, 68.94, 68.8, 68.6, 68.51, 68.49, 63.7, 58.1; 29.9, 17.8, −1.1; λ_abs (toluene) 417, 548, 582 nm; MALDI-MS obsd 1080.26, calcd 1080.39 [M⁺]; ESI-MS obsd 1081.3955, calcd 1081.3967 [(M + H)⁺], M = C₅₆H₆₈N₄O₁₂SiZn.

Zn(II)-15-(2,6-bis((1-tert-butyloxy-1-oxo-4,7,10,13,16,19-hexaoxahenicosan-21-yl)oxy)phenyl)-15-(4-(2-(trimethylsilyl)ethoxycarbonyl)phenyl)porphyrin (ZnP3-Bu/ETMS). Following a reported method⁶ with some modifications, a solution of D6 (208 mg, 0.200 mmol) and D9-Esch (96 mg, 0.20 mmol) in EtOH (20 mL) was treated with Zn(OAc)₂·2H₂O (439 mg, 2.00 mmol) at room temperature. The mixture was heated to reflux for 2 h, then allowed to cool to room temperature followed by treatment with DDQ (136 mg, 0.600 mmol). The reaction mixture was stirred for 30 min, then triethylamine (140 μL) was added. After 15 min, the solution was concentrated under reduced pressure. Chromatography using aluminum oxide (basic, Brockman grade I) with a gradient [CH₂Cl₂/ethyl acetate (4 : 1) to ethyl acetate] gave a red solid (20 mg, 7%): ¹H NMR (700 MHz, CD₃OD) δ 10.26 (s, 2H), 9.44 (d, J = 4.2 Hz, 2H), 9.37 (d, J = 4.2 Hz, 2H), 9.00–8.97 (m, 4H), 8.50 (d, J = 8.4 Hz, 2H), 8.39 (d, J = 8.4 Hz, 2H), 7.79 (t, J = 8.4 Hz, 1H), 7.16 (d, J = 8.4 Hz, 2H), 4.70–4.67 (m, 2H), 4.02 (t, J = 4.2 Hz, 4H), 3.55 (t, J = 6.3 Hz, 4H), 3.33–3.31 (m, 8H), 3.24–3.22 (m, 4H), 3.13–3.10 (m, 4H), 2.92–2.90 (m, 8H), 2.59–2.57 (m, 4H), 2.38 (t, J = 6.3 Hz, 4H), 2.12–2.11 (m, 4H), 1.85–1.82 (m, 8H), 1.58–1.56 (m, 4H), 1.43 (s, 18H), 1.38–1.35 (m, 2H), 0.24 (s, 9H); ¹³C{¹H} NMR (175 MHz, CD₃OD) δ 171.3, 167.2, 166.8, 159.9, 151.0, 149.6, 149.3, 148.9, 148.5, 134.6, 131.5, 131.4, 131.3, 130.9, 130.0, 129.3, 127.3, 121.2, 117.6, 112.1, 105.3, 105.1, 80.3, 76.8, 70.14, 70.13, 70.0, 69.82, 69.78, 69.6, 69.4, 69.1, 68.9, 68.8, 68.5, 68.38, 68.35, 66.5, 66.4, 63.2, 54.8, 53.4, 35.8, 29.3, 27.0, 17.1, −2.7; λ_abs (toluene) 417, 548, 582 nm; MALDI-MS obsd 1484.57, calcd 1484.63 [M⁺]; ESI-MS obsd 1507.6133, calcd 1507.6197 [(M + Na)⁺], M = C₇₆H₁₀₄N₄O₂₀SiZn.

Zn(II)-5-(2,6-bis((1-tert-butyloxy-1-oxo-4,7,10,13,16,19-hexaoxahenicosan-21-yl)oxy)phenyl)-15-(4-(3-(2-(trimethylsilyl)ethoxy)-3-oxopropyl)phenyl)porphyrin (ZnP4-Bu/ETMS). Following a reported method⁶ with some modifications, a solution of D3-Esch (332 mg, 0.653 mmol) and D6 (678 mg, 0.653 mmol) in EtOH (65.3 mL) was treated with Zn(OAc)₂·2H₂O (1.43 g, 6.53 mmol) at room temperature. The reaction mixture was heated to reflux for 2 h, then allowed to cool to room temperature. The reaction mixture was treated with DDQ (445 mg, 1.96 mmol). The resulting mixture was stirred for 30 min, and then triethylamine (910 μL, 6.53 mmol) was added. After 15 min, the solvent was removed under reduced pressure, and the crude product was chromatographed first [silica, CH₂Cl₂/ethyl acetate (1 : 1)] and second [silica, CH₂Cl₂/MeOH (25 : 1)] to afford a red solid (58.6 mg, 6%): ¹H NMR (500 MHz, CDCl₃) δ 10.16 (s, 2H), 9.34 (d, J = 4.4 Hz, 2H), 9.31 (d, J = 4.4 Hz, 2H), 9.06 (d, J = 4.4 Hz, 2H), 9.02 (d, J = 4.3 Hz, 2H), 8.17–8.15 (m, 2H), 7.72 (t, J = 8.5 Hz, 1H), 7.62 (d, J = 7.8 Hz, 2H), 7.05 (d, J = 8.5 Hz, 2H), 4.37–4.33 (m, 2H), 3.96–3.95 (m, 4H), 3.52 (t, J = 6.6 Hz, 4H), 3.35 (t, J = 7.9 Hz, 2H), 3.24 (dd, J = 6.0, 3.9 Hz, 4H), 3.16 (dd, J = 5.9, 3.9 Hz, 4H), 2.98–2.93 (m, 6H), 2.89–2.87 (m, 4H), 2.83–2.81 (m, 4H), 2.63–2.61 (m, 4H), 2.38 (t, J = 6.6 Hz, 4H), 2.29–2.28 (m, 4H), 1.76–1.74 (m, 4H), 1.58–1.54 (m, 8H), 1.41 (s, 18H), 1.36–1.36 (m, 4H), 1.16–1.12 (m, 2H), 0.14 (s, 9H); ¹³C{¹H} NMR (125 MHz, CDCl₃) δ 173.4, 171.0, 160.0, 151.0, 149.6, 149.4, 149.2, 141.3, 139.7, 134.9, 132.1, 132.0, 131.7, 131.2, 130.0, 126.6, 122.1, 119.5, 111.7, 106.2, 105.3, 80.6, 70.7, 70.10, 70.07, 70.0, 69.8, 69.6, 69.5, 69.2, 68.9, 68.8, 68.6, 68.44, 68.39, 66.8, 63.0, 36.5, 36.3, 31.2, 28.2, 17.6, −1.2; λ_abs (toluene) 415, 544, 579 nm; MALDI-MS obsd 1513.44, calcd 1513.67 [(M + H)⁺]; ESI-MS obsd 1535.6470, calcd 1535.6510 [(M + Na)⁺], M = C₇₈H₁₀₈N₄O₂₀SiZn.

Zinc(II)-5-(2,6-bis((1-tert-butyloxy-1-oxo-4,7,10,13,16,19-hexaoxahenicosan-21-yl)oxy)phenyl)-15-(2-(4-acetoxyphenyl)ethyl)porphyrin (ZnP5). Following a reported method⁶ with some modifications, a solution of D10-Esch (183 mg, 0.433 mmol) and D6 (450 mg, 0.433 mmol) in EtOH (43.0 mL) was treated with Zn(OAc)₂·2H₂O (950 mg, 4.33 mmol) at room temperature. The reaction mixture was heated to reflux for 3 h, then allowed to cool to room temperature, and treated with DDQ (295 mg, 1.30 mmol). The resulting mixture was stirred for 30 min, and then triethylamine (600 μL, 4.33 mmol) was added. After 15 min, the solution was concentrated, and the crude product was chromatographed [silica, CH₂Cl₂/ethyl acetate (1 : 1) to (1 : 6)] to give a dark red solid (34.2 mg, 6%): ¹H NMR (700 MHz, CDCl₃) δ 10.07 (s, 2H), 9.59 (d, J = 4.3 Hz, 2H), 9.36 (d, J = 4.3 Hz, 2H), 9.27 (d, J = 4.3 Hz, 2H), 8.98 (d, J = 4.2 Hz, 2H), 7.71 (t, J = 8.7 Hz, 1H), 7.09 (d, J = 8.0 Hz, 2H), 7.04 (d, J = 8.7 Hz, 2H), 6.66–6.65 (m, 2H), 6.14 (s, 1H), 5.35 (t, J = 8.1 Hz, 2H), 3.96 (t, J = 4.4 Hz, 4H), 3.85 (t, J = 8.0 Hz, 2H), 3.54 (t, J = 6.6 Hz, 4H), 3.28 (dd, J = 5.9, 4.0 Hz, 4H), 3.21 (dd, J = 5.8, 4.0 Hz, 4H), 3.01 (t, J = 4.9 Hz, 4H), 2.90–2.87 (m, 8H), 2.62–2.60 (m, 4H), 2.39 (t, J = 6.6 Hz, 4H), 2.25 (dd, J = 5.7, 4.0 Hz, 4H), 1.61 (dd, J = 5.7, 3.9 Hz, 4H), 1.51 (t, J = 5.4 Hz, 4H), 1.42 (s, 18H), 1.34–1.33 (m, 4H), 1.17 (t, J = 5.3 Hz, 4H); ¹³C{¹H} NMR (175 MHz, CDCl₃) δ 171.2, 160.0, 154.7, 151.0, 149.8, 149.2, 148.7, 133.7, 131.8, 131.7, 131.6, 130.0, 129.8, 129.1, 122.2, 118.4, 115.5, 111.2, 106.3, 105.0, 80.7, 70.14, 70.13, 70.0, 69.8, 69.7, 69.5, 69.1, 68.85, 68.78, 68.5, 68.3, 68.2, 66.8, 44.1, 38.0, 36.3, 28.2; λ_abs (toluene) 415, 545, 579 nm; MALDI-MS obsd 1407.87, calcd 1407.58 [(M + Na)⁺]; ESI-MS obsd 1407.5818, calcd 1407.5852 [(M + Na)⁺], M = C₇₂H₉₆N₄O₁₉Zn.

Zinc(II)-5-(2,6-bis((1-tert-butyloxy-1-oxo-4,7,10,13,16,19-hexaoxahenicosan-21-yl)oxy)phenyl)-15-(2-(4-nitrophenyl)ethyl)porphyrin (ZnP6). Following a reported method⁶ with some modifications, a solution of D11-Esch (206 mg, 0.504 mmol) and D6 (523 mg, 0.503 mmol) in EtOH (50 mL) was reacted with Zn(OAc)₂·2H₂O (1.11 g, 5.04 mmol) and then refluxed for 2 h. The reaction mixture was allowed to cool to room temperature. The reaction mixture was treated with DDQ (341 mg, 1.50 mmol), stirred for 15 min, and then treated with triethylamine (700 μL). The crude mixture was dried under high vacuum and then passed through a silica pad (3 cm × 3 cm, CH₂Cl₂) to give elution of black byproducts followed by a fraction containing the desired porphyrin (∼80% pure). The porphyrin fraction was concentrated and chromatographed [silica, CH₂Cl₂/ethyl acetate/methanol (6 : 6 : 1)] to give the desired product as a non-crystalline, red solid (44 mg, 7%): ¹H NMR (700 MHz, CDCl₃) δ 10.07 (s, 2H), 9.61–9.56 (m, 2H), 9.42–9.36 (m, 2H), 9.27 (d, J = 4.3 Hz, 2H), 8.97 (d, J = 4.3 Hz, 2H), 8.27–8.19 (m, 2H), 7.71 (t, J = 8.7 Hz, 1H), 7.65–7.59 (m, 2H), 7.04 (d, J = 8.7 Hz, 2H), 5.46–5.33 (m, 2H), 4.08–3.99 (m, 2H), 3.98–3.88 (m, 4H), 3.50 (t, J = 6.6 Hz, 4H), 3.27–3.21 (m, 4H), 3.20–3.13 (m, 4H), 2.98–2.94 (m, 4H), 2.92–2.87 (m, 4H), 2.87–2.80 (m, 4H), 2.65–2.59 (m, 4H), 2.36 (t, J = 6.6 Hz, 4H), 2.34–2.30 (m, 4H), 1.89–1.82 (m, 4H), 1.71 (t, J = 5.2 Hz, 4H), 1.68–1.63 (m, 4H), 1.52–1.47 (m, 4H), 1.40 (s, 18H); ¹³C{¹H} NMR (175 MHz, CDCl₃) δ 171.0, 159.8, 151.2, 150.1, 149.3, 149.2, 148.7, 146.7, 132.0, 131.9, 130.1, 129.6, 128.4, 127.7, 124.1, 121.9, 116.7, 113.0, 111.4, 106.2, 105.1, 100.3, 80.6, 70.01, 69.99, 69.9, 69.7, 69.4, 69.3, 69.1, 68.9, 68.8, 68.6, 68.4, 66.7, 44.7, 37.2, 36.2, 28.2; λ_abs [MeCN/H₂O (80 : 20)] 412, 546, 579 nm; satisfactory MALDI-MS and ESI-MS data could not be obtained; LC-MS gave obsd m/z 1436.55, calcd 1436.58, [(M + Na)⁺], M = C₇₂H₉₅N₅O₂₀Zn.

Zn(II)-5-(2,6-bis((1-tert-butyloxy-1-oxo-4,7,10,13,16,19-hexaoxahenicosan-21-yl)oxy)phenyl)-15-(4-(tert-butyloxycarbonylamino)phenyl)porphyrin (ZnP7-Bu/Boc). Following a reported method⁶ with some modifications, a solution of D12-Esch (271 mg, 0.600 mmol) and D6 (624 mg, 0.600 mmol) in EtOH (60.0 mL) was treated with Zn(OAc)₂·2H₂O (1.32 g, 6.00 mmol) at room temperature. The reaction mixture was heated to reflux for 1.5 h, then allowed to cool to room temperature. The reaction mixture was treated with DDQ (409 mg, 1.80 mmol). The resulting mixture was stirred for 30 min, and then triethylamine (836 μL, 6.00 mmol) was added. After 15 min, the solvent was removed under reduced pressure, and the crude product was chromatographed first [silica, CH₂Cl₂/MeOH (25 : 1)] and second [silica, CH₂Cl₂/ethyl acetate (1 : 9)] to afford a red solid (42.8 mg, 5%): ¹H NMR (500 MHz, CDCl₃) δ 10.14 (s, 2H), 9.34 (d, J = 4.4 Hz, 2H), 9.30 (d, J = 4.4 Hz, 2H), 9.09 (d, J = 4.4 Hz, 2H), 9.01 (d, J = 4.4 Hz, 2H), 8.17–8.14 (m, 2H), 7.81 (d, J = 7.8 Hz, 2H), 7.72 (t, J = 8.5 Hz, 1H), 7.07–7.03 (m, 3H), 3.96–3.95 (m, 4H), 3.54 (t, J = 6.6 Hz, 4H), 3.24 (dd, J = 5.9, 3.9 Hz, 4H), 3.16 (dd, J = 5.9, 3.9 Hz, 4H), 2.94–2.92 (m, 4H), 2.89–2.87 (m, 4H), 2.82–2.80 (m, 4H), 2.63–2.61 (m, 4H), 2.39 (t, J = 6.6 Hz, 4H), 2.27–2.25 (m, 4H), 1.73–1.71 (m, 4H), 1.67 (s, 9H), 1.55–1.52 (m, 8H), 1.42 (s, 18H), 1.34–1.32 (m, 4H); ¹³C{¹H} NMR (125 MHz, CDCl₃) δ 171.0, 160.0, 153.3, 151.0, 149.7, 149.4, 149.2, 138.1, 137.9, 135.3, 132.1, 131.9, 131.7, 131.2, 130.0, 122.1, 119.2, 116.8, 111.6, 106.1, 105.3, 80.6, 70.1, 70.0, 69.74, 69.68, 69.5, 69.1, 68.9, 68.8, 68.5, 68.4, 68.3, 66.8, 36.3, 28.6, 28.2; λ_abs (toluene) 416, 546, 580 nm; MALDI-MS obsd 1455.92, calcd 1455.63 [M⁺]; ESI-MS obsd 1478.6191, calcd 1478.6224 [(M + Na)⁺], M = C₇₅H₁₀₁N₅O₂₀Zn.

Zn(II)-5-(2,6-bis((1-tert-butyloxy-1-oxo-4,7,10,13,16,19-hexaoxahenicosan-21-yl)oxy)phenyl)-15-(2-(tert-butyloxycarbonylamino)ethyl)porphyrin (ZnP8-Bu/Boc). Following a reported method⁶ with some modifications, a solution of D6 (600 mg, 0.58 mmol) and D13-Esch (278 mg, 0.69 mmol) in ethanol (58 mL) at room temperature was treated with Zn(OAc)₂·2H₂O (1.27 g, 5.8 mmol) and then heated to 80 °C (oil bath). After 2 h, the reaction mixture was allowed to cool to room temperature. The reaction mixture was treated with DDQ (386 mg, 1.7 mmol), stirred at room temperature for 15 min, and then treated with triethylamine (406 μL, 2.9 mmol). The crude mixture was dried under high vacuum and then passed through a silica pad (3 cm × 3 cm; ethyl acetate then CH₂Cl₂/methanol 100 : 1 to 10 : 1) to give elution of black byproducts followed by a fraction containing the desired porphyrin (∼80% pure). The porphyrin fraction was concentrated and chromatographed [silica, CH₂Cl₂/ethyl acetate/methanol (6 : 6 : 1)] to give the desired product as a non-crystalline, red solid (72 mg, 7%): ¹H NMR (500 MHz, CDCl₃) δ 10.12 (s, 2H), 9.75 (d, J = 4.5 Hz, 2H), 9.44 (d, J = 4.5 Hz, 2H), 9.28 (d, J = 4.4 Hz, 2H), 8.99 (d, J = 4.3 Hz, 2H), 7.72 (t, J = 8.5 Hz, 1H), 7.04 (d, J = 8.5 Hz, 2H), 5.34 (t, J = 6.9 Hz, 2H), 5.11–5.05 (m, 1H), 4.28 (q, J = 6.9 Hz, 2H), 3.98–3.93 (m, 4H), 3.54 (t, J = 6.6 Hz, 4H), 3.33–3.21 (m, 8H), 3.06–3.01 (m, 4H), 2.89 (t, J = 4.4 Hz, 8H), 2.61–2.56 (m, 4H), 2.38 (t, 4H), 2.27–2.21 (m, 4H), 1.62–1.60 (m, 8H), 1.56 (s, 9H), 1.41 (s, 18H), 1.34–1.29 (m, 4H), 1.28–1.22 (m, 4H); ¹³C{¹H} NMR (125 MHz, CDCl₃) δ 171.0, 156.0, 151.1, 150.0, 149.2, 148.9, 148.2, 132.0, 131.8, 130.1, 129.4, 112.5, 106.3, 105.1, 80.6, 70.19, 70.18, 70.1, 69.9, 69.6, 69.5, 69.2, 68.9, 68.8, 68.5, 68.30, 68.26, 66.9, 36.3, 28.8, 28.2; λ_abs [MeCN/H₂O (95 : 5)] 411, 546, 578; ESI-MS obsd 1408.6400, calcd 1408.6404 [(M + H)⁺], M = C₇₁H₁₀₁N₅O₂₀Zn.

Zn(II)-5-(4-((2,5,8,11,14,17-hexaoxanonadecan-19-yl)oxy)phenyl-15-(4-(2-(trimethylsilyl)ethoxycarbonyl)phenyl)porphyrin (ZnP9-ETMS). Following a procedure⁶ with modification, a sample of Zn(OAc)₂ (1.10 g, 5.00 mmol) was added to a solution of D9-Esch (240 mg, 0.50 mmol) and D14 (258 mg, 0.50 mmol) in ethanol (50.0 mL). The resulting mixture was stirred and refluxed (oil bath) for 1.5 h. The reaction mixture was allowed to cool to room temperature. A sample of DDQ (340 mg, 1.50 mmol) was added, and the reaction mixture was stirred for 15 min. Triethylamine (700 μL, 5 mmol) was added, and the reaction mixture was stirred for 15 min. The mixture was concentrated under reduced pressure. Purification of the crude mixture by column chromatography [silica, CH₂Cl₂/ethyl acetate (2 : 1)] afforded reddish fractions (λ_abs 415–420 nm) that were set aside for further purification; all black fractions were discarded. Purification of the reddish fractions by column chromatography [silica, CH₂Cl₂/ethyl acetate (2 : 1)] afforded a dark-red solid (55.3 mg, 12%): ¹H NMR (500 MHz, CDCl₃) δ 10.24 (s, 2H), 9.39–9.38 (m, 4H), 9.13 (d, J = 4.4 Hz, 2H), 9.03 (d, J = 4.4 Hz, 2H), 8.43–8.41 (m, 2H), 8.31–8.30 (m, 2H), 8.13–8.11 (m, 2H), 7.27–7.25 (m, 2H, buried in CDCl₃), 4.61–4.57 (m, 2H), 4.32–4.26 (m, 2H), 3.89–3.87 (m, 2H), 3.68–3.64 (m, 2H), 3.57–3.55 (m, 2H), 3.53–3.47 (m, 4H), 3.44–3.39 (m, 4H), 3.37–3.35 (m, 2H), 3.29–3.27 (m, 2H), 3.12–3.10 (m, 2H), 2.98–2.96 (m, 2H), 2.85 (s, 3H), 1.34–1.28 (m, 2H), 0.20 (s, 9H); ¹³C{¹H} NMR (125 MHz, CDCl₃) δ 168.4, 159.8, 151.7, 150.78, 150.76, 150.6, 148.8, 136.9, 136.5, 136.0, 133.9, 133.2, 133.0, 131.0, 129.0, 121.4, 119.6, 114.2, 107.5, 72.6, 72.0, 71.80, 71.76, 71.7, 71.62, 71.59, 71.52, 71.48, 71.2, 71.1, 69.0, 64.9, 59.8, 18.9, 0.00; λ_abs (toluene) 415, 540, 576 nm; MALDI-MS obsd 986.94, calcd 985.32 [(M + Na)⁺]; ESI-MS obsd 963.3321, calcd 963.3337 [(M + H)⁺], M = C₅₁H₅₈N₄O₉SiZn.

Porphyrin elaboration

Zn(II)-5-(2,6-bis(propargyloxy)phenyl)-5-(4-formylphenyl)porphyrin (ZnP1-CHO). Following a method¹⁹ with some modifications, a sample of ZnP1-acetal (24.8 mg, 0.0331 mmol) was added to a solution of TFA (1 mL), 5% H₂SO₄ in water (504 μL), and acetic acid (2 mL). The mixture was heated to 80 °C (oil bath). After 1.5 h, the mixture was allowed to cool to room temperature. Then, CH₂Cl₂ (50 mL) and saturated aqueous NaHCO₃ (50 mL) were added. The organic phase was washed (saturated aqueous NaHCO₃ (50 mL × 3) and brine), dried (Na₂SO₄), and then concentrated to dryness under reduced pressure. The crude was dissolved in the mixed solvent CH₂Cl₂/MeOH (9 : 1) (18 mL), and then Zn(OAc)₂·2H₂O (373 mg, 1.70 mmol) was added. The mixture was stirred overnight at room temperature. The mixture was washed with saturated aqueous NaHCO₃ (50 mL × 3) and extracted with CH₂Cl₂ (50 mL). The organic extract was washed with brine, dried (Na₂SO₄), concentrated under reduced pressure, and recrystallized (hexane/CHCl₃) to give a red solid (19 mg, 87%): ¹H NMR (700 MHz, CD₂Cl₂) δ 10.37 (s, 1H), 10.29 (s, 2H), 9.44–9.43 (m, 4H), 9.07 (d, J = 4.3 Hz, 2H), 9.03 (d, J = 4.3 Hz, 2H), 8.43 (d, J = 7.5 Hz, 2H), 8.30 (d, J = 7.5 Hz, 2H), 7.85 (t, J = 8.8 Hz, 1H), 7.29 (d, J = 8.8 Hz, 2H), 4.43 (d, J = 2.4 Hz, 4H), 2.33 (t, J = 2.4 Hz, 2H); ¹³C{¹H} NMR (175 MHz, CD₂Cl₂) δ 192.4, 158.5, 150.6, 149.6, 149.35, 149.26, 149.0, 135.5, 135.3, 132.1, 131.8, 131.6, 130.0, 127.8, 121.3, 118.3, 111.2, 106.7, 105.9, 78.4, 75.0, 56.3; λ_abs (toluene) 414, 539, 573 nm; MALDI-MS obsd 660.36, calcd 660.11 [M⁺]; ESI-MS obsd 661.1202, calcd 661.1213 [(M + H)⁺], M = C₃₉H₂₄N₄O₃Zn.

Zn(II)-5-(2,6-bis((2,5,8,11-tetraoxatridecan-13-yl)oxy)phenyl)-15-(4-carboxyphenyl)porphyrin (ZnP2). Following a reported procedure,²¹ a solution of ZnP2-ETMS (3.8 mg, 3.5 μmol) in THF (350 μL) was treated with TBAF (1 M in THF, 32 μL, 32 μmol) at 40 °C. The reaction mixture was stirred for 1 h and then concentrated under reduced pressure to remove residual THF. The crude product was dissolved in CH₂Cl₂ (50 mL), washed with water (3 × 50 mL) and brine (50 mL), dried (Na₂SO₄), concentrated under reduced pressure, and finally chromatographed [silica, CH₂Cl₂ to CH₂Cl₂/MeOH (15 : 1)] to afford a red solid (2.4 mg, 70%): ¹H NMR (700 MHz, CDCl₃) δ 10.20 (s, 2H), 9.39 (d, J = 4.2 Hz, 2H), 9.33 (d, J = 4.2 Hz, 2H), 9.04 (d, J = 4.2 Hz, 4H), 8.54 (d, J = 7.4 Hz, 2H), 8.37 (d, J = 7.4 Hz, 2H), 7.74 (t, J = 8.7 Hz, 1H), 7.06 (d, J = 8.7 Hz, 2H), 3.98 (t, J = 4.5 Hz, 4H), 2.92 (t, J = 4.2, 4H), 2.36 (s, 6H), 2.31 (t, J = 4.2, 4H), 2.14 (t, J = 4.2, 4H), 1.80 (dd, J = 4.9, 4.2 Hz, 4H), 1.68–1.64 (m, 8H), 1.45 (t, J = 4.9 Hz, 4H); ¹³C{¹H} NMR (175 MHz, CDCl₃) δ 159.8, 151.0, 149.5, 149.1, 148.9, 134.8, 132.0, 131.8, 131.54, 131.48, 130.0, 128.3, 121.8, 117.9, 112.1, 105.9, 105.5, 70.6, 69.4, 68.9, 68.8, 68.7, 68.5, 68.4, 58.0; λ_abs (DMSO) 417, 548, 582 nm; MALDI-MS obsd 980.30, calcd 980.32 [M⁺]; ESI-MS obsd 981.3253, calcd 981.3259 [(M + H)⁺], M = C₅₁H₅₆N₄O₁₂Zn.

5-(2,6-Bis((2,5,8,11-tetraoxatridecan-13-yl)oxy)phenyl)-15-(4-(N-succinimidyloxycarbonyl)phenyl)porphyrin (P2-NHS). A solution of ZnP2 (3.6 mg, 3.7 μmol) in CH₂Cl₂ (1.0 mL) was treated with TFA (100 μL) at room temperature for 5 min. The resulting solution was diluted with CH₂Cl₂ (10 mL), washed extensively (H₂O (20 mL), aqueous NaHCO₃ (20 mL × 2) and brine (20 mL × 2)), and then dried (Na₂SO₄). The organic layer was collected and dried under vacuum to give a dark red solid. The solid was dissolved in CH₂Cl₂ (290 μL) and then treated with DCC (6.0 mg, 29 μmol) and N-hydroxysuccinimide (3.3 mg, 29 μmol). The mixture was stirred at room temperature for 1 h and then filtered. The filtrate was purified by preparative TLC [silica, 5 cm × 20 cm × 0.5 mm; CH₂Cl₂/ethyl acetate/methanol (5 : 5 : 1)] to give the final product as a non-crystalline red solid (3.5 mg, 94%): ¹H NMR (700 MHz, CDCl₃) δ 10.28 (s, 2H), 9.42 (d, J = 4.4 Hz, 2H), 9.33 (d, J = 4.4 Hz, 2H), 9.02 (d, J = 4.4 Hz, 2H), 9.00 (d, J = 4.5 Hz, 2H), 8.61 (d, J = 7.8 Hz, 2H), 8.45 (d, J = 7.7 Hz, 2H), 7.75 (t, J = 8.7 Hz, 1H), 7.06 (d, J = 8.7 Hz, 2H), 4.02 (t, J = 4.9 Hz, 4H), 3.18–3.16 (m, 10H), 3.08–3.01 (m, 8H), 2.92 (t, J = 4.9 Hz, 4H), 2.66–2.63 (m, 4H), 2.31 (dd, J = 5.7, 4.3 Hz, 4H), 2.06–2.03 (m, 4H), 1.88 (dd, J = 5.6, 3.8 Hz, 4H), −3.13 (s, 2H); ¹³C{¹H} NMR (175 MHz, CDCl₃) δ 170.4, 169.5, 162.3, 159.8, 148.9, 148.4, 145.9, 145.5, 145.2, 135.3, 131.94, 131.85, 131.3, 130.7, 130.3, 129.3, 124.5, 119.5, 116.8, 112.3, 105.9, 105.2, 71.7, 70.0, 69.7, 69.4, 69.3, 69.2, 68.8, 59.0, 26.0, 25.3; λ_abs (MeOH) 404, 500, 534, 574, 629 nm; MALDI-MS obsd 1038.47, calcd 1038.41 [(M + Na)⁺]; ESI-MS obsd 1016.4278, calcd 1016.4288 [(M + H)⁺], M = C₅₅H₆₁N₅O₁₄.

5-(2,6-Bis((2,5,8,11-tetraoxatridecan-13-yl)oxy)phenyl)-15-(4-carboxyphenyl)porphyrin (P2). Following a standard procedure,²² a solution of ZnP2-ETMS (16.0 mg, 14.8 μmol) in CH₂Cl₂ (1.0 mL) was treated with TFA (1.0 mL) at room temperature. The reaction mixture was stirred for 5 h, then diluted with CH₂Cl₂ (20 mL), washed with water (3 × 15 mL), and dried (Na₂SO₄). The crude residue was purified by column chromatography [silica, CH₂Cl₂/MeOH (20 : 1)] to afford a red solid (12.4 mg, 91%): ¹H NMR (700 MHz, CDCl₃) δ 10.27 (s, 2H), 9.39 (d, J = 4.4 Hz, 2H), 9.35 (d, J = 4.4 Hz, 2H), 9.05 (t, J = 3.8 Hz, 4H), 8.58 (d, J = 7.3 Hz, 2H), 8.40 (d, J = 7.4 Hz, 2H), 7.76 (t, J = 8.7 Hz, 1H), 7.08 (d, J = 8.8 Hz, 2H), 4.05 (t, J = 4.8 Hz, 4H), 3.23–3.19 (m, 10H), 3.05 (t, J = 4.7 Hz, 4H), 2.96 (t, J = 4.9 Hz, 4H), 2.68 (t, J = 5.0 Hz, 4H), 2.34 (t, J = 5.0 Hz, 4H), 2.07 (t, J = 4.6 Hz, 4H), 1.90 (t, J = 4.7 Hz, 4H), −3.08 (s, 2H); ¹³C{¹H} NMR (175 MHz, CDCl₃) δ 171.3, 159.9, 148.3, 147.1, 146.2, 145.5, 145.2, 135.0, 131.8, 131.7, 131.2, 130.7, 130.6, 129.4, 128.8, 119.6, 117.6, 112.1, 105.9, 105.0, 71.7, 70.7, 70.0, 69.7, 69.33, 69.27, 69.2, 68.8, 59.0; λ_abs (MeOH) 404, 500, 533, 574, 629 nm; MALDI-MS obsd 919.34, calcd 919.41 [(M + H)⁺]; ESI-MS obsd 919.4095, calcd 919.4124 [(M + H)⁺]; M = C₅₁H₅₈N₄O₁₂.

Zn(II)-5-(2,6-bis((1-tert-butyloxy-1-oxo-4,7,10,13,16,19-hexaoxahenicosan-21-yl)oxy)phenyl)-5-(4-carboxyphenyl)porphyrin (ZnP3-Bu). Following a reported procedure,²¹ a solution of ZnP3-Bu/ETMS (20.4 mg, 13.7 μmol) in THF (1.4 mL) was treated with TBAF (1 M in THF, 28 μL, 28 μmol) at 35 °C (oil bath). The reaction mixture was stirred for 1.5 h, then allowed to cool to room temperature. The solvent was removed under reduced pressure. The crude product was purified by preparative TLC [silica, 1 mm, 20 × 20 cm, CH₂Cl₂/MeOH (20 : 1)] to give a red solid (16.2 mg, 85%): ¹H NMR (700 MHz, CD₃OD) δ 10.27 (s, 2H), 9.45 (d, J = 4.2 Hz, 2H), 9.38 (d, J = 4.2 Hz, 2H), 9.01 (d, J = 4.2 Hz, 2H), 8.98 (d, J = 4.2 Hz, 2H), 8.50 (d, J = 7.7 Hz, 2H), 8.37 (d, J = 7.7 Hz, 2H), 7.81 (t, J = 8.4 Hz, 1H), 7.17 (d, J = 8.4 Hz, 2H), 4.04–4.02 (m, 4H), 3.46 (t, J = 6.3 Hz, 4H), 3.30–3.26 (m, 8H), 3.20–3.19 (m, 4H), 3.09–3.08 (m, 4H), 2.94–2.93 (m, 4H), 2.89–2.88 (m, 4H), 2.58–2.57 (m, 4H), 2.31 (t, J = 6.3 Hz, 4H), 2.13–2.11 (m, 4H), 1.86–1.83 (m, 8H), 1.56–1.55 (m, 4H), 1.40 (s, 18H); ¹³C{¹H} NMR (175 MHz, CD₃OD) δ 171.4, 159.9, 151.0, 149.6, 149.3, 149.0, 148.1, 134.5, 131.5, 131.4, 131.3, 131.0, 130.0, 127.5, 121.3, 117.9, 112.0, 105.4, 105.1, 80.3, 69.7, 69.5, 69.3, 68.94, 68.88, 68.7, 68.38, 68.36, 68.3, 68.2, 66.3, 35.6, 27.0; λ_abs (toluene) 417, 548, 582 nm; MALDI-MS obsd 1385.72, calcd 1385.57 [(M + H)⁺]; ESI-MS obsd 1407.5413, calcd 1407.5489 [(M + Na)⁺], M = C₇₁H₉₂N₄O₂₀Zn. Note: in a prior synthesis, attempted separation by column chromatography (silica) afforded the product and unknown species, hence the reliance here on preparative TLC for purification.

Zn(II)-5-(2,6-bis((1-tert-butyloxy-1-oxo-4,7,10,13,16,19-hexaoxahenicosan-21-yl)oxy)phenyl)-5-(4-(N-succinimidyloxycarbonyl)phenyl)porphyrin (ZnP3-Bu/NHS). Following a reported method⁴² with some modifications, a solution of ZnP3-Bu (14.5 mg, 10.5 μmol) and N-hydroxysuccinimide (12.1 mg, 105 μmol) in CH₂Cl₂ was stirred at room temperature. After 5 min, DCC (8.7 mg, 42 μmol) and DMAP (1.3 mg, 1.1 μmol) were added. The reaction mixture was stirred at room temperature for 1 h, and then filtered to remove a white precipitate. The filtrate was concentrated under reduced pressure and then chromatographed [silica, CH₂Cl₂/ethyl acetate (1 : 2) to (1 : 4)] to afford a red solid (13.2 mg, 85%):¹H NMR (500 MHz, CDCl₃) δ 10.19 (s, 2H), 9.39 (d, J = 4.4 Hz, 2H), 9.31 (d, J = 4.4 Hz, 2H), 9.03 (d, J = 4.4 Hz, 2H), 8.99 (d, J = 4.3 Hz, 2H), 8.59–8.56 (m, 2H), 8.42–8.40 (m, 2H), 7.73 (t, J = 8.5 Hz, 1H), 7.05 (d, J = 8.5 Hz, 2H), 3.98–3.96 (m, 4H), 3.51 (t, J = 6.6 Hz, 4H), 3.22 (dd, J = 6.0, 3.9 Hz, 4H), 3.14 (dd, J = 5.9, 3.9 Hz, 4H), 3.03 (s, 4H), 2.94–2.90 (m, 8H), 2.82 (dd, J = 5.8, 4.0 Hz, 4H), 2.65–2.64 (m, 4H), 2.37 (t, J = 6.6 Hz, 4H), 2.33–2.31 (m, 4H), 1.83–1.81 (m, 4H), 1.66–1.60 (m, 8H), 1.45–1.43 (m, 4H), 1.40 (s, 18H); ¹³C{¹H} NMR (125 MHz, CDCl₃) δ 171.0, 169.6, 162.5, 159.9, 151.1, 150.9, 149.7, 149.2, 148.7, 135.3, 132.2, 131.9, 131.8, 131.4, 128.9, 123.9, 117.2, 112.5, 106.1, 105.7, 80.6, 70.09, 70.05, 70.0, 69.8, 69.6, 69.5, 69.2, 68.9, 68.8, 68.6, 68.52, 68.47, 66.8, 49.3, 36.3, 34.0, 28.2, 26.0, 25.7, 25.0; λ_abs (MeOH) 412, 544, 580 nm; MALDI-MS obsd 1481.96, calcd 1481.58 [M⁺]; ESI-MS obsd 1504.5610, calcd 1504.5652 [(M + Na)⁺], M = C₇₅H₉₅N₅O₂₂Zn.

5-(2,6-Bis((1-hydroxy-1-oxo-4,7,10,13,16,19-hexaoxahenicosan-21-yl)oxy)phenyl)-15-(4-(N-succinimidyloxycarbonyl)phenyl)porphyrin (P3-NHS). Following a reported method²² with some modifications, a sample of ZnP3-Bu/NHS (11.0 mg, 7.4 μmol) in CH₂Cl₂ (370 μL) was treated with TFA (370 μL) at room temperature. The reaction mixture was stirred for 2 h at room temperature. The mixture was concentrated under reduced pressure. Given the high polarity and chromatographic tailing of the product, the mixture was purified by preparative TLC [silica, 0.25 mm, 20 × 20 cm, CH₂Cl₂/MeOH (9 : 1)] to afford a red solid (8.7 mg, 90%): ¹H NMR (700 MHz, CDCl₃) δ 10.29 (s, 2H), 9.43 (d, J = 4.5 Hz, 2H), 9.35 (d, J = 4.5 Hz, 2H), 9.02 (d, J = 4.4 Hz, 2H), 9.00 (d, J = 4.4 Hz, 2H), 8.61 (d, J = 7.7 Hz, 2H), 8.45 (d, J = 7.7 Hz, 2H), 7.75 (t, J = 8.7 Hz, 1H), 7.06 (d, J = 8.7 Hz, 2H), 4.02 (t, J = 4.6 Hz, 4H), 3.57 (t, J = 6.0 Hz, 4H), 3.45–3.43 (m, 12H), 3.38 (dd, J = 5.8, 3.6 Hz, 4H), 3.25 (t, J = 4.8 Hz, 4H), 3.05–3.00 (m, 8H), 2.95 (t, J = 4.7 Hz, 4H), 2.60 (t, J = 4.8 Hz, 4H), 2.41 (t, J = 6.0 Hz, 4H), 2.23 (t, J = 4.8 Hz, 4H), 1.99 (t, J = 4.5 Hz, 4H), 1.78 (t, J = 4.5 Hz, 3H), −3.14 (s, 2H); ¹³C{¹H} NMR (175 MHz, CDCl₃) δ 174.3, 169.6, 162.3, 159.8, 148.8, 135.3, 132.03, 131.96, 131.3, 130.8, 130.4, 129.3, 124.6, 119.4, 116.8, 112.3, 105.9, 105.2, 70.7, 70.5, 70.4, 70.3, 70.12, 70.06, 69.9, 69.44, 69.36, 69.2, 69.04, 69.02, 68.8, 66.7, 35.2, 29.9, 26.0; λ_abs (MeOH) 405, 500, 534, 573, 629 nm; λ_abs (DMSO) 410, 503, 538, 575, 630 nm; MALDI-MS obsd 1307.96, calcd 1307.54 [M⁺]; ESI-MS obsd 1308.5418, calcd 1308.5446 [(M + H)⁺], M = C₆₇H₈₁N₅O₂₂.

Zn(II)-5-(2,6-bis((1-tert-butyloxy-1-oxo-4,7,10,13,16,19-hexaoxahenicosan-21-yl)oxy)phenyl)-15-(4-(3-hydroxy-3-oxopropyl)phenyl)porphyrin (ZnP4-Bu). Following a reported procedure,²¹ a solution of ZnP4-Bu/ETMS (29.0 mg, 19.1 μmol) in THF (1.90 mL) was treated with TBAF (1 M in THF, 48 μL, 48 μmol) at 35 °C (oil bath). The reaction mixture was stirred for 1 h, then allowed to cool to room temperature. The solvent was removed under reduced pressure. Given the similarity with the homologue ZnP3-Bu, the crude product was purified by preparative TLC [silica, 1 mm, 20 × 20 cm, CH₂Cl₂/MeOH (20 : 1)] to give a red solid (23.8 mg, 88%): ¹H NMR (700 MHz, CDCl₃) δ 10.15 (s, 2H), 9.34 (d, J = 4.3 Hz, 2H), 9.30 (d, J = 4.3 Hz, 2H), 9.08 (d, J = 4.3 Hz, 2H), 9.01 (d, J = 4.3 Hz, 2H), 8.16 (d, J = 7.5 Hz, 2H), 7.72 (t, J = 8.6 Hz, 1H), 7.61 (d, J = 7.2 Hz, 2H), 7.04 (d, J = 8.7 Hz, 2H), 3.96–3.95 (m, 4H), 3.54 (t, J = 6.6 Hz, 4H), 3.28–3.25 (m, 6H), 3.17–3.16 (m, 4H), 2.95 (t, J = 4.9 Hz, 4H), 2.89 (t, J = 4.4 Hz, 4H), 2.83 (t, J = 5.0 Hz, 4H), 2.65 (t, J = 4.8 Hz, 4H), 2.40 (t, J = 6.6 Hz, 4H), 2.31–2.29 (m, 4H), 1.78–1.74 (m, 4H), 1.59–1.51 (m, 8H), 1.42 (s, 18H), 1.34 (q, J = 5.2 Hz, 4H); ¹³C{¹H} NMR (175 MHz, CDCl₃) δ 171.1, 159.9, 150.9, 149.6, 149.4, 149.2, 141.4, 139.5, 135.0, 132.1, 131.9, 131.7, 131.2, 130.0, 126.6, 122.0, 119.5, 111.6, 106.1, 105.3, 80.7, 70.0, 69.9, 69.7, 69.6, 69.5, 69.1, 68.83, 68.77, 68.5, 68.4, 68.3, 66.8, 36.2, 28.2; λ_abs (MeOH) 411, 544, 575 nm; MALDI-MS obsd 1413.03, calcd 1413.60 [M⁺]; ESI-MS obsd 1435.5778, calcd 1435.5802 [(M + Na)⁺], M = C₇₃H₉₆N₄O₂₀Zn.

Zn(II)-5-(2,6-bis((1-tert-butyloxy-1-oxo-4,7,10,13,16,19-hexaoxahenicosan-21-yl)oxy)phenyl)-15-(4-(3-(N-succinimidyloxy)-3-oxopropyl)phenyl)porphyrin (ZnP4-Bu/NHS). Following a reported method⁴² with some modifications, a solution of ZnP4-Bu (6.1 mg, 4.3 μmol) and N-hydroxysuccinimide (4.9 mg, 43 μmol) in CH₂Cl₂ was stirred at room temperature. After 5 min, DCC (8.9 mg, 43 μmol) was added. The reaction mixture was stirred at room temperature for 1 h, and then the resulting mixture was filtered to remove a white precipitate. The filtrate was concentrated under reduced pressure and then chromatographed [silica, CH₂Cl₂/ethyl acetate (1 : 2) to (1 : 4)] to afford a red solid (5.2 mg, 80%): ¹H NMR (700 MHz, CDCl₃) δ 10.15 (s, 2H), 9.34 (d, J = 4.3 Hz, 2H), 9.30 (d, J = 4.3 Hz, 2H), 9.07 (d, J = 4.3 Hz, 2H), 9.02 (d, J = 4.2 Hz, 2H), 8.18–8.17 (m, 2H), 7.73 (t, J = 8.6 Hz, 1H), 7.64 (d, J = 7.7 Hz, 2H), 7.05 (d, J = 8.6 Hz, 2H), 3.97 (dd, J = 5.2, 3.6 Hz, 4H), 3.51 (t, J = 6.6 Hz, 4H), 3.46 (t, J = 7.9 Hz, 2H), 3.30–3.28 (m, 2H), 3.22 (t, J = 4.9 Hz, 4H), 3.14 (dd, J = 5.9, 4.1 Hz, 4H), 2.99–2.86 (m, 12H), 2.80 (t, J = 5.0 Hz, 4H), 2.63–2.60 (m, 4H), 2.37 (t, J = 6.6 Hz, 4H), 2.28 (t, J = 4.9 Hz, 4H), 1.76 (dd, J = 5.7, 3.8 Hz, 4H), 1.59–1.57 (m, 8H), 1.40 (s, 18H), 1.38 (t, J = 5.4 Hz, 4H); ¹³C{¹H} NMR (175 MHz, CDCl₃) δ 171.0, 169.3, 168.3, 160.0, 151.0, 149.6, 149.5, 149.2, 141.9, 138.2, 135.1, 132.1, 132.0, 131.7, 131.3, 130.1, 126.7, 122.1, 119.3, 111.8, 106.1, 105.4, 80.6, 70.08, 70.06, 69.9, 69.74, 69.67, 69.5, 69.2, 68.9, 68.8, 68.6, 68.5, 68.4, 66.8, 56.1, 36.3, 33.1, 30.6, 29.9, 28.2, 25.9; λ_abs (MeOH) 411, 545, 580 nm; MALDI-MS obsd 1509.12, calcd 1509.61 [M⁺]; ESI-MS obsd 1532.5932, calcd 1532.5965 [(M + Na)⁺], M = C₇₇H₉₉N₅O₂₂Zn.

5-(2,6-Bis((1-hydroxy-1-oxo-4,7,10,13,16,19-hexaoxahenicosan-21-yl)oxy)phenyl)-15-(4-(3-(N-succinimidyloxy)-3-oxopropyl)phenyl)porphyrin (P4-NHS). Following a reported method²² with some modifications, a sample of ZnP4-Bu/NHS (3.2 mg, 2.1 μmol) in CH₂Cl₂ (150 μL) was treated with TFA (150 μL) at room temperature. The reaction mixture was stirred for 2 h at room temperature. The mixture was concentrated under reduced pressure and then purified by reversed-phase HPLC [C18-silica, H₂O (0.1% TFA)/CH₃CN (0.1% TFA) (1 : 1 to 1 : 9)] to afford a red solid (2.4 mg, 85%): ¹H NMR (700 MHz, CD₂Cl₂) δ 10.30 (s, 2H), 9.43 (d, J = 4.5 Hz, 2H), 9.38 (d, J = 4.4 Hz, 2H), 9.10 (d, J = 4.5 Hz, 2H), 9.02 (d, J = 4.4 Hz, 2H), 7.77 (t, J = 8.7 Hz, 1H), 7.72 (d, J = 7.8 Hz, 2H), 7.11 (d, J = 8.7 Hz, 2H), 4.04–4.01 (m, 4H), 3.60 (t, J = 6.1 Hz, 4H), 3.49–3.43 (m, 14H), 3.38–3.36 (m, 4H), 3.30–3.27 (m, 2H), 3.25–3.23 (m, 4H), 3.02–2.99 (m, 4H), 2.96–2.94 (m, 4H), 2.93–2.85 (m, 4H), 2.60–2.58 (m, 4H), 2.45 (t, J = 6.1 Hz, 4H), 2.27–2.24 (m, 4H), 2.15–2.12 (m, 4H), 1.92–1.90 (m, 4H), −3.14 (s, 2H); ¹³C{¹H} NMR (175 MHz, CD₂Cl₂) δ 174.5, 169.9, 168.9, 160.3, 148.6, 147.3, 145.8, 140.3, 139.5, 135.6, 132.2, 131.9, 131.4, 131.3, 131.1, 127.6, 119.9, 119.5, 112.0, 106.4, 105.2, 70.9, 70.84, 70.81, 70.58, 70.56, 70.4, 69.98, 69.96, 69.7, 69.6, 69.5, 69.2, 66.9, 35.3, 33.4, 31.0, 26.3; λ_abs (MeOH) 403, 500, 533, 573, 628 nm; λ_abs (DMSO) 408, 503, 536, 574, 630 nm; MALDI-MS obsd 1336.21, calcd 1336.58 [(M + H)⁺]; ESI-MS obsd 1336.5739, calcd 1336.5759 [(M + H)⁺], M = C₆₉H₈₅N₅O₂₂.

5-(2,6-Bis((1-hydroxy-1-oxo-4,7,10,13,16,19-hexaoxahenicosan-21-yl)oxy)phenyl)-15-(4-carboxyphenyl)porphyrin (P3). A solution of ZnP3-Bu/ETMS (15 mg, 10 μmol) in CH₂Cl₂ (1.0 mL) was treated with TFA (1.0 mL) at room temperature. The reaction mixture was stirred for 2 h and then diluted with CH₂Cl₂ (10 mL). Saturated aqueous NaHCO₃ was added to the reaction mixture until pH = 9. The aqueous phase was washed once with CH₂Cl₂. The aqueous phase was acidified with aqueous 1 M HCl until pH = 2, and then extracted with CH₂Cl₂ (2 × 10 mL). The combined organic extract was washed with brine (10 mL), dried (Na₂SO₄), and concentrated under reduced pressure to give a red solid (7.6 mg, 63%): ¹H NMR (700 MHz, CD₂Cl₂) δ 10.29 (s, 2H), 9.41–9.36 (m, 4H), 9.05–9.01 (m, 4H), 8.48–8.46 (m, 2H), 8.35 (d, J = 7.3 Hz, 2H), 7.77 (t, J = 8.7 Hz, 1H), 7.10 (d, J = 8.7 Hz, 2H), 4.04 (t, J = 4.7 Hz, 4H), 3.59 (t, J = 6.0 Hz, 5H), 3.50–3.40 (m, 12H), 3.34 (dd, J = 5.8, 3.5 Hz, 4H), 3.20 (t, J = 4.7 Hz, 4H), 2.96–2.92 (m, 8H), 2.51 (t, J = 4.7 Hz, 4H), 2.45 (t, J = 6.2 Hz, 4H), 2.15 (t, J = 4.7 Hz, 4H), 2.07 (dd, J = 5.6, 3.7 Hz, 4H), 1.80 (dd, J = 5.7, 3.7 Hz, 4H), −3.14 (s, 2H); ¹³C{¹H} NMR (175 MHz, CD₂Cl₂) δ 174.6, 170.1, 159.7, 148.1, 146.7, 146.1, 145.4, 145.1, 134.9, 131.8, 131.7, 130.9, 130.6, 130.4, 129.8, 129.2, 128.6, 119.2, 117.5, 111.9, 105.8, 104.9, 70.23, 70.18, 70.1, 70.0, 69.9, 69.7, 69.3, 69.2, 69.0, 68.9, 68.8, 68.6; λ_abs (DMSO) 409, 503, 538, 575, 630 nm; MALDI-MS obsd 1211.42, calcd 1211.52 [(M + H)⁺]; ESI-MS obsd 606.2670, calcd 606.2678 [(M + 2H)²⁺]; M = C₆₃H₇₈N₄O₂₀.

5-(2,6-Bis((1-hydroxy-1-oxo-4,7,10,13,16,19-hexaoxahenicosan-21-yl)oxy)phenyl)-15-(4-(3-hydroxy-3-oxopropyl)phenyl)porphyrin (P4). A solution of ZnP4-Bu/ETMS (15.2 mg, 10.0 μmol) in CH₂Cl₂ (1.0 mL) was treated with TFA (1.0 mL) at room temperature. The reaction mixture was stirred for 6 h and then diluted with CH₂Cl₂ (10 mL). Saturated aqueous NaHCO₃ was added to the reaction mixture until pH = 9. The aqueous phase was washed once with CH₂Cl₂. The aqueous phase was acidified with aqueous 2 M HCl until pH = 2, and then extracted with CH₂Cl₂ (2 × 10 mL). The combined organic extract was washed with brine (10 mL), dried (Na₂SO₄), and concentrated under reduced pressure to give a red solid (11.0 mg, 89%): ¹H NMR (700 MHz, CD₂Cl₂) δ 10.19 (s, 2H), 9.32 (d, J = 4.4 Hz, 2H), 9.28 (d, J = 4.5 Hz, 2H), 8.99 (d, J = 4.4 Hz, 2H), 8.92 (d, J = 4.4 Hz, 2H), 8.12–8.09 (m, 2H), 7.68 (t, J = 8.7 Hz, 1H), 7.60–7.57 (m, 2H), 7.00 (d, J = 8.7 Hz, 2H), 3.95–3.92 (m, 4H), 3.53 (t, J = 6.0 Hz, 4H), 3.40–3.38 (m, 4H), 3.36–3.34 (m, 4H), 3.33–3.30 (m, 4H), 3.26–3.21 (m, 6H), 3.06–3.02 (m, 4H), 2.91 (t, J = 7.1 Hz, 2H), 2.87–2.84 (m, 4H), 2.76–2.73 (m, 4H), 2.39 (t, J = 6.0 Hz, 4H), 2.24–2.21 (m, 4H), 1.90–1.88 (m, 4H), 1.86–1.83 (m, 4H), 1.56–1.53 (m, 4H), –3.23 (s, 2H); ¹³C{¹H} NMR (175 MHz, CD₂Cl₂) δ 178.5, 175.7, 160.3, 148.6, 147.3, 145.7, 140.6, 139.9, 135.5, 132.2, 131.9, 131.4, 131.3, 131.1, 127.6, 119.9, 119.7, 112.0, 106.3, 105.2, 70.89, 70.87, 70.7, 70.6, 70.5, 70.2, 69.8, 69.68, 69.66, 69.34, 69.27, 69.1, 66.8, 35.4, 30.3; λ_abs (MeOH) 404, 500, 533, 573, 628 nm; λ_abs (DMSO) 408, 503, 536, 574, 631 mm; MALDI-MS obsd 1239.31, calcd 1239.56 [(M + H)⁺]; ESI-MS obsd 1238.5513, calcd 1238.5522 [M⁺]; M = C₆₅H₈₂N₄O₂₀.

5-(2,6-Bis((1-hydroxy-1-oxo-4,7,10,13,16,19-hexaoxahenicosan-21-yl)oxy)phenyl)-15-(4-aminophenyl)porphyrin (P7). A solution of ZnP7-Bu/Boc (12.6 mg, 8.64 μmol) in CH₂Cl₂ (580 μL) was treated with TFA (290 μL) at room temperature for 2 h. The reaction was monitored by LC-MS. The reaction mixture was concentrated under reduced pressure and then passed through a C18-silica pad (1 cm × 1 cm) by eluting with H₂O (to remove the remaining TFA) followed by acetonitrile. The collected solution was dried under vacuum to give a red solid (7.6 mg, 75%): ¹H NMR (700 MHz, CDCl₃) δ 10.22 (s, 2H), 9.36 (d, J = 4.4 Hz, 2H), 9.32 (d, J = 4.4 Hz, 2H), 9.13 (d, J = 4.4 Hz, 2H), 8.98 (d, J = 4.4 Hz, 2H), 8.01 (d, J = 7.5 Hz, 2H), 7.72 (t, J = 8.7 Hz, 1H), 7.06–7.02 (m, 4H), 4.01–3.99 (m, 4H), 3.54 (t, J = 6.0 Hz, 4H), 3.39–3.36 (m, 12H), 3.29 (dd, J = 5.8, 3.7 Hz, 4H), 3.12 (dd, J = 5.7, 3.8 Hz, 4H), 2.93 (t, J = 4.6 Hz, 4H), 2.85 (dd, J = 5.7, 3.8 Hz, 4H), −3.07 (s, 2H); ¹³C{¹H} NMR (175 MHz, CDCl₃) δ 159.8, 148.2, 147.3, 146.3, 145.1, 136.0, 131.8, 131.6, 131.22, 131.20, 130.8, 130.6, 120.0, 119.7, 114.0, 111.1, 105.8, 104.7, 70.3, 70.1, 69.9, 69.6, 69.35, 69.27, 69.2, 69.1, 68.9, 68.8, 68.7, 67.2, 35.8; λ_abs (DMSO) 410, 505, 543, 577, 634; MALDI-MS obsd 1204.22, calcd 1204.53 [(M + Na)⁺]; ESI-MS obsd 1204.5290, calcd 1204.5312 [(M + Na)⁺], M = C₆₂H₇₉N₅O₁₈.

5-(2,6-Bis((1-hydroxy-1-oxo-4,7,10,13,16,19-hexaoxahenicosan-21-yl)oxy)phenyl)-15-(4-isothiocyanatophenyl)porphyrin (P7-NCS). Following a reported method⁷⁶ with some modifications, a sample of P7 (6.8 mg, 5.8 μmol) in CH₂Cl₂ (1.5 mL) was treated with 1,1′-thiocarbonyldi-2(1H)-pyridone (2.7 mg, 12 μmol) at room temperature for 2 h. The mixture was concentrated under reduced pressure. Given the high polarity and chromatographic tailing of the product, the mixture was purified by preparative TLC [silica, 0.25 mm, 20 × 20 cm, CH₂Cl₂/MeOH (9 : 1)] to give a red solid (5.6 mg, 80%): ¹H NMR (700 MHz, CDCl₃) δ 10.28 (s, 2H), 9.39 (d, J = 4.5 Hz, 2H), 9.36 (d, J = 4.5 Hz, 2H), 9.01 (d, J = 4.4 Hz, 2H), 8.99 (d, J = 4.5 Hz, 2H), 7.73 (t, J = 8.7 Hz, 1H), 7.68–7.65 (m, 2H), 7.05 (d, J = 8.7 Hz, 2H), 4.01 (t, J = 4.5 Hz, 4H), 3.45 (t, J = 6.1 Hz, 4H), 3.38 (dd, J = 5.9, 3.5 Hz, 4H), 3.36–3.30 (m, 12H), 3.16 (dd, J = 5.8, 3.6 Hz, 4H), 2.95 (t, J = 4.6 Hz, 4H), 2.91 (dd, J = 5.7, 3.7 Hz, 4H), 2.56–2.48 (m, 4H), 2.27 (t, J = 6.2 Hz, 4H), 2.18–2.12 (m, 4H), 1.96–1.90 (m, 4H), 1.75–1.69 (m, 4H), −3.17 (s, 2H); ¹³C{¹H} NMR (175 MHz, CDCl₃) δ 177.4, 159.8, 148.3, 146.3, 145.5, 145.2, 140.9, 136.6, 135.8, 132.0, 131.9, 131.3, 131.2, 130.8, 130.5, 124.5, 119.3, 117.2, 112.1, 105.8, 105.1, 70.7, 69.9, 69.85, 69.78, 69.73, 69.68, 69.4, 69.3, 69.23, 69.20, 69.0, 68.85, 68.82, 68.6, 68.1, 37.0, 29.8; λ_abs (DMSO) 411, 504, 539, 576, 632; MALDI-MS obsd 1245.84, calcd 1246.49 [(M + Na)⁺]; ESI-MS obsd 1246.4856, calcd 1246.4877 [(M + Na)⁺], M = C₆₃H₇₇N₅O₁₈S.

5-(2,6-Bis((1-hydroxy-1-oxo-4,7,10,13,16,19-hexaoxahenicosan-21-yl)oxy)phenyl)-15-(2-aminoethyl)porphyrin (P8). A solution of ZnP8-Bu/Boc (5.4 mg, 3.8 μmol) in CH₂Cl₂ (225 μL) was treated with TFA (25 μL) at room temperature for 3 h. The reaction was monitored by LC-MS. The reaction mixture was concentrated and then passed through a C18-silica pad (1 cm × 1 cm) by eluting with H₂O (to remove the remaining TFA) followed by acetonitrile. The collected solution was dried under vacuum to give the desired product as a non-crystalline red solid (3.9 mg, 90%): ¹H NMR (500 MHz, CDCl₃) δ 10.13 (s, 2H), 9.85 (d, J = 4.6 Hz, 2H), 9.40 (d, J = 4.6 Hz, 2H), 9.27 (d, J = 4.6 Hz, 2H), 8.96 (d, J = 4.5 Hz, 2H), 7.72 (t, J = 8.5 Hz, 1H), 7.01 (d, J = 8.6 Hz, 2H), 5.55 (t, J = 8.3 Hz, 2H), 4.08–4.02 (m, 2H), 4.00 (t, J = 4.3 Hz, 4H), 3.88 (t, J = 6.0 Hz, 4H), 3.62–3.56 (m, 4H), 3.51–3.45 (m, 4H), 3.35–3.27 (m, 4H), 3.18–3.10 (m, 4H), 2.93 (t, J = 4.3 Hz, 4H), 2.82–2.70 (m, 8H), 2.20 (t, J = 4.8 Hz, 4H), 1.57–1.39 (m, 8H), 0.95–0.82 (m, 4H), 0.81–0.63 (m, 4H), −3.16 (s, 2H). This crude sample was used in the next step without further purification.

5-(2,6-Bis((1-hydroxy-1-oxo-4,7,10,13,16,19-hexaoxahenicosan-21-yl)oxy)phenyl)-15-(2-isothiocyanatoethyl)porphyrin (P8-NCS). Following a reported method⁷⁶ with some modifications, a solution of P8 (3.9 mg, 3.4 μmol) in CH₂Cl₂ (500 μL) was treated overnight with 1,1′-thiocarbonyldi-2(1H)-pyridone (2.3 mg, 10 μmol) at room temperature. The resulting reaction mixture was purified by preparative thin layer chromatography [silica, 20 cm × 20 cm × 0.5 mm; CH₂Cl₂/methanol (9 : 1, R_f = 0.45)] to give the desired product as a non-crystalline, dark-red solid (2.6 mg, 64%): ¹H NMR (500 MHz, CDCl₃) δ 10.26 (s, 2H), 9.58 (d, J = 4.6 Hz, 2H), 9.50 (d, J = 4.6 Hz, 2H), 9.35 (d, J = 4.6 Hz, 2H), 8.99 (d, J = 4.6 Hz, 2H), 7.74 (t, J = 8.5 Hz, 1H), 7.05 (d, J = 8.6 Hz, 2H), 5.43 (t, J = 7.3 Hz, 2H), 4.64 (t, J = 7.3 Hz, 2H), 4.09–3.96 (m, 4H), 3.52–3.31 (m, 16H), 3.18–3.11 (m, 4H), 3.02–2.93 (m, 4H), 2.90–2.82 (m, 4H), 2.50–2.42 (m, 4H), 2.35–2.25 (m, 4H), 2.11–2.03 (m, 8H), 1.95–1.87 (m, 4H), 1.67–1.58 (m, 4H), −3.15 (d, J = 21.3 Hz, 2H); λ_abs [MeCN/H₂O (95 : 5)] 402, 500, 532, 574, 627; MALDI-MS obsd 1199.7, calcd 1198.5 [(M + Na)⁺]; ESI-MS obsd 1176.5075, calcd 1176.5057 [(M + H)⁺], M = C₅₉H₇₇N₅O₁₈S.

Zn(II)-5-(4-((2,5,8,11,14,17-hexaoxanonadecan-19-yl)oxy)phenyl-15-(4-carboxyphenyl)porphyrin (P9). A solution of ZnP9-ETMS (17.0 mg, 17.6 μmol) in CH₂Cl₂ (880 μL) was treated dropwise with TFA (880 μL) and then stirred overnight at room temperature. The resulting dark-green reaction mixture was diluted with CH₂Cl₂ (20.0 mL). The organic solution was washed with 5% NaHSO₄ (20.0 mL) and saturated aqueous NH₄Cl (20.0 mL) to give a reddish solution. Each aqueous solution was extracted with CH₂Cl₂ (2 × 15.0 mL). The combined organic extract was dried (Na₂SO₄) and then concentrated under reduced pressure. Column chromatography [silica, CH₂Cl₂/MeOH (9 : 1)] afforded a dark-red solid (11.0 mg, 78%): ¹H NMR (500 MHz, CDCl₃) δ 10.21 (s, 2H), 9.33–9.32 (m, 4H), 9.09 (d, J = 4.4 Hz, 2H), 9.02 (d, J = 4.4 Hz, 2H), 8.62–8.60 (m, 2H), 8.41–8.40 (m, 2H), 8.17–8.14 (m, 2H), 7.37–7.35 (m, 2H), 4.46–4.45 (m, 2H), 4.10–4.08 (m, 2H), 3.92–3.89 (m, 2H), 3.84–3.82 (m, 2H), 3.80–3.75 (m, 4H), 3.74–3.72 (m, 4H), 3.71–3.70 (m, 2H), 3.69–3.67 (m, 2H), 3.66–3.65 (m, 2H), 3.56–3.55 (m, 2H), 3.38 (s, 3H), −3.15 (s, 2H), –CO₂H signal is not observed; ¹³C{¹H} NMR (125 MHz, CDCl₃) δ 170.9, 158.8, 147.5, 147.0, 146.5, 145.2, 145.1, 135.9, 135.0, 133.8, 131.9, 131.6, 131.2, 130.4, 128.9, 128.8, 119.5, 117.1, 113.3, 105.4, 71.9, 71.0, 70.8, 70.71, 70.66, 70.62, 70.60, 70.58, 70.5, 70.4, 69.9, 67.8, 59.0; λ_abs (toluene) 411, 504, 539, 578, 634 nm; MALDI-MS obsd 801.43, calcd 801.35 [(M + H)⁺]; ESI-MS obsd 801.3478, calcd 801.3494 [(M + H)⁺], M = C₄₆H₄₈N₄O₉.

Statistical syntheses of porphyrins

5-(2,6-Bis((1-tert-butyloxy-1-oxo-4,7,10,13,16,19-hexaoxahenicosan-21-yl)oxy)phenyl)-5-(4-carboxyphenyl)porphyrin (P3-Bu). Following a reported method⁶⁹ for statistical condensations with some modifications, a solution of A1 (75.1 mg, 0.500 mmol), dipyrromethane (146 mg, 1.00 mmol), and A6 (461 mg, 0.500 mmol) in CH₂Cl₂ (50.0 mL) was stirred at room temperature. After 5 min, TFA (134 μL, 1.75 mmol) was added, and the reaction mixture was stirred at room temperature for 30 min. Then, DDQ (341 mg, 1.50 mmol) was added. The resulting mixture was stirred at room temperature for 15 min, and then triethylamine was added (350 μL, 2.50 mmol). After stirring for 15 min, the solution was concentrated under reduced pressure, and the crude product was chromatographed [silica, CH₂Cl₂/MeOH (15 : 1)]. Three porphyrin bands were observed, whereupon the most mobile band and second-most mobile band were collected together. A second column [silica, CH₂Cl₂/MeOH (25 : 1)] was used to obtain in pure form the second band, which upon concentration afforded a red solid (65.9 mg, 10%): ¹H NMR (700 MHz, CDCl₃) δ 10.18 (s, 2H), 9.36–9.19 (m, 4H), 9.03 (d, J = 4.4 Hz, 2H), 9.00 (d, J = 4.4 Hz, 2H), 8.58–8.50 (m, 2H), 8.31 (d, J = 7.3 Hz, 2H), 7.74 (t, J = 8.7 Hz, 1H), 7.08 (d, J = 8.7 Hz, 2H), 4.04 (t, J = 4.8 Hz, 4H), 3.67–3.65 (m, 4H), 3.55–3.50 (m, 12H), 3.44 (dd, J = 5.8, 4.0 Hz, 4H), 3.31 (t, J = 4.9 Hz, 4H), 3.09 (t, J = 4.9 Hz, 4H), 2.95 (t, J = 4.9 Hz, 4H), 2.70 (t, J = 4.9 Hz, 4H), 2.47 (t, J = 6.5 Hz, 4H), 2.36 (t, J = 4.9 Hz, 4H), 2.08 (t, J = 4.6 Hz, 4H), 1.91 (t, J = 4.6 Hz, 4H), 1.42 (s, 18H), −3.10 (s, 2H); ¹³C{¹H} NMR (175 MHz, CDCl₃) δ 171.1, 159.9, 148.3, 146.3, 145.6, 145.4, 145.1, 134.8, 131.8, 131.6, 131.1, 130.7, 128.7, 119.6, 118.2, 111.9, 105.9, 104.9, 80.7, 70.7, 70.50, 70.47, 70.41, 70.38, 70.2, 70.0, 69.62, 69.58, 69.3, 69.23, 69.16, 68.8, 67.0, 36.3, 28.2; λ_abs (MeOH) 404, 500, 533, 574, 629 nm; MALDI-MS obsd 1323.39, calcd 1323.65 [(M + H)⁺]; ESI-MS obsd 1345.6317, calcd 1345.6354 [(M + Na)⁺], M = C₇₁H₉₄N₄O₂₀.

5-(2,6-Bis((1-tert-butyloxy-1-oxo-4,7,10,13,16,19-hexaoxahenicosan-21-yl)oxy)phenyl)-5-(4-(N-succinimidyloxycarbonyl)phenyl)porphyrin (P3-Bu/NHS). Following a reported method⁴² with some modifications, a solution of P3-Bu (24.3 mg, 18.4 μmol) and N-hydroxysuccinimide (21.2 mg, 184 μmol) in CH₂Cl₂ was stirred at room temperature. After 5 min, DCC (38.0 mg, 184 μmol) was added. The reaction mixture was stirred at room temperature for 1 h, and then the resulting mixture was filtered to remove a white precipitate. The filtrate was concentrated under reduced pressure and then chromatographed [silica, CH₂Cl₂/ethyl acetate (1 : 1) to (1 : 3)] to afford a red solid (23.5 mg, 90%): ¹H NMR (700 MHz, CDCl₃) δ 10.29 (s, 2H), 9.43 (d, J = 4.4 Hz, 2H), 9.34 (d, J = 4.5 Hz, 2H), 9.02 (d, J = 4.4 Hz, 2H), 9.00 (d, J = 4.4 Hz, 2H), 8.61 (d, J = 8.1 Hz, 2H), 8.46 (d, J = 8.0 Hz, 2H), 7.75 (t, J = 8.7 Hz, 1H), 7.07 (d, J = 8.7 Hz, 2H), 4.02 (t, J = 4.9 Hz, 4H), 3.66 (t, J = 6.6 Hz, 4H), 3.55–3.48 (m, 12H), 3.43 (dd, J = 5.9, 4.0 Hz, 4H), 3.31–3.28 (m, 4H), 3.10–2.97 (m, 8H), 2.93 (t, J = 4.9 Hz, 4H), 2.70–2.67 (m, 4H), 2.46 (t, J = 6.6 Hz, 4H), 2.35–2.32 (m, 4H), 2.08–2.05 (m, 4H), 1.92–1.89 (m, 4H), 1.42 (s, 18H), −3.13 (s, 2H); ¹³C{¹H} NMR (175 MHz, CDCl₃) δ 171.4, 171.0, 169.5, 162.3, 159.8, 148.8, 148.3, 145.9, 145.5, 145.2, 135.3, 131.95, 131.86, 131.2, 130.7, 130.3, 129.3, 124.5, 119.5, 116.7, 112.3, 105.9, 105.2, 80.6, 70.54, 70.50, 70.46, 70.4, 70.3, 70.1, 69.6, 69.34, 69.26, 69.1, 68.8, 67.0, 36.3, 28.2, 25.9, 25.3; λ_abs (MeOH) 405, 500, 534, 573, 629 nm; MALDI-MS obsd 1420.23, calcd 1420.67 [(M + H)⁺]; ESI-MS obsd 1420.6659, calcd 1420.6698 [(M + H)⁺], M = C₇₅H₉₇N₅O₂₂.

5-(2,6-Bis((1-hydroxy-1-oxo-4,7,10,13,16,19-hexaoxahenicosan-21-yl)oxy)phenyl)-15-(4-(N-succinimidyloxycarbonyl)phenyl)porphyrin (P3-NHS). A solution of P3-Bu/NHS (22.0 mg, 15.5 μmol) in CH₂Cl₂ (1.1 mL) was treated with TFA (550 μL) at room temperature. The reaction mixture was stirred for 2 h at room temperature and then diluted with CH₂Cl₂ (20.0 mL). The organic solution was washed water (3 × 20.0 mL), dried (Na₂SO₄), and concentrated under reduced pressure. The crude product was loaded on a C18-silica pad (2 cm × 1 cm) and washed with water (40 mL) followed by acetonitrile (8 mL) to give an eluant that upon concentration afforded a red solid (19.7 mg, 97%) with identical characterization data (¹H NMR, ¹³C{¹H} NMR, MALDI-MS) as above.

5-(2,6-Bis((1-tert-butyloxy-1-oxo-4,7,10,13,16,19-hexaoxahenicosan-21-yl)oxy)phenyl)-15-(4-(3-hydroxy-3-oxopropyl)phenyl)porphyrin (P4-Bu). Following a reported method⁶⁹ for statistical condensations with some modifications, a solution of A2 (89.1 mg, 0.500 mmol), dipyrromethane (146 mg, 1.00 mmol), and A6 (461 mg, 0.500 mmol) in CH₂Cl₂ (50.0 mL) was stirred at room temperature. After 5 min, TFA (134 μL, 1.75 mmol) was added, and the reaction mixture was stirred at room temperature for 30 min. Then, DDQ (341 mg, 1.50 mmol) was added. The resulting mixture was stirred at room temperature for 15 min, and then triethylamine was added (350 μL, 2.50 mmol). After stirring for 15 min, the solution was concentrated under reduced pressure, and the crude product was chromatographed [silica, CH₂Cl₂/MeOH (20 : 1)]. Three porphyrin bands were observed, whereupon the most mobile band and second-most mobile band were collected together. A second column [silica, CH₂Cl₂/MeOH (25 : 1)] was used to obtain in pure form the second band, which upon concentration afforded a red solid (75.0 mg, 11%): ¹H NMR (700 MHz, CDCl₃) δ 10.20 (s, 2H), 9.32 (d, J = 4.5 Hz, 2H), 9.30 (d, J = 4.5 Hz, 2H), 9.08 (d, J = 4.4 Hz, 2H), 9.00 (d, J = 4.4 Hz, 2H), 7.73 (t, J = 8.7 Hz, 1H), 7.67 (d, J = 7.3 Hz, 2H), 7.06 (d, J = 8.7 Hz, 2H), 4.02 (t, J = 4.8 Hz, 4H), 3.65 (t, J = 6.4 Hz, 4H), 3.53–3.47 (m, 12H), 3.41 (dd, J = 5.9, 3.9 Hz, 4H), 3.33 (t, J = 7.9 Hz, 2H), 3.25 (dd, J = 5.8, 4.2 Hz, 4H), 3.01 (dd, J = 5.7, 4.1 Hz, 4H), 2.94–2.92 (m, 6H), 2.57 (t, J = 5.0 Hz, 4H), 2.47 (t, J = 6.5 Hz, 4H), 2.20 (t, J = 5.0 Hz, 4H), 2.00 (t, J = 4.7 Hz, 4H), 1.76 (t, J = 4.7 Hz, 4H), 1.42 (s, 18H), −3.09 (s, 2H); ¹³C{¹H} NMR (175 MHz, CDCl₃) δ 171.1, 159.8, 148.2, 146.9, 145.2, 145.1, 141.3, 139.2, 135.0, 131.7, 131.3, 131.1, 130.9, 130.6, 127.2, 119.7, 119.4, 111.4, 105.9, 104.7, 80.7, 70.7, 70.44, 70.40, 70.35, 70.33, 70.2, 69.9, 69.5, 69.4, 69.2, 69.10, 69.08, 68.8, 66.9, 37.5, 36.3, 31.7, 28.2; λ_abs (MeOH) 404, 500, 532, 574, 629 nm; MALDI-MS obsd 1351.34, calcd 1351.68 [(M + H)⁺]; ESI-MS obsd 1373.6636, calcd 1373.6667 [(M + Na)⁺], M = C₇₃H₉₈N₄O₂₀.

5-(2,6-Bis((1-tert-butyloxy-1-oxo-4,7,10,13,16,19-hexaoxahenicosan-21-yl)oxy)phenyl)-15-(4-(3-(N-succinimidyloxy)-3-oxopropyl)phenyl)porphyrin (P4-Bu/NHS). Following a reported method⁴² with some modifications, a solution of P4-Bu (24.3 mg, 18.4 μmol) and N-hydroxysuccinimide (21.2 mg, 184 μmol) in CH₂Cl₂ was stirred at room temperature. After 5 min, DCC (38.0 mg, 184 μmol) was added. The reaction mixture was stirred at room temperature for 1 h, and then the resulting mixture was filtered to remove a white precipitate. The filtrate was concentrated under reduced pressure and then chromatographed [silica, CH₂Cl₂/ethyl acetate (1 : 1) to (1 : 3)] to afford a red solid (23.5 mg, 89%): ¹H NMR (700 MHz, CDCl₃) δ 10.25 (s, 2H), 9.38 (d, J = 4.4 Hz, 2H), 9.32 (d, J = 4.4 Hz, 2H), 9.09 (d, J = 4.4 Hz, 2H), 9.00 (d, J = 4.4 Hz, 2H), 8.23 (d, J = 7.7 Hz, 2H), 7.74 (t, J = 8.7 Hz, 1H), 7.69 (d, J = 7.4 Hz, 2H), 7.06 (d, J = 8.7 Hz, 2H), 4.01 (t, J = 4.8 Hz, 4H), 3.65 (t, J = 6.6 Hz, 4H), 3.54–3.44 (m, 14H), 3.41 (dd, J = 5.8, 4.0 Hz, 4H), 3.30–3.26 (m, 6H), 3.05 (dd, J = 5.7, 4.2 Hz, 4H), 2.96–2.91 (m, 8H), 2.65–2.62 (m, 4H), 2.46 (t, J = 6.6 Hz, 4H), 2.29 (dd, J = 5.8, 4.1 Hz, 4H), 2.07–2.04 (m, 4H), 1.88 (dd, J = 5.7, 3.6 Hz, 4H), 1.42 (s, 18H), −3.11 (s, 2H); ¹³C{¹H} NMR (175 MHz, CDCl₃) δ 171.2, 171.0, 169.3, 168.3, 159.7, 148.2, 146.8, 145.3, 145.1, 140.1, 138.7, 135.2, 131.7, 131.4, 131.1, 131.0, 130.6, 127.1, 119.7, 118.9, 111.5, 105.9, 104.8, 80.6, 70.54, 70.50, 70.45, 70.4, 70.3, 70.0, 69.63, 69.61, 69.3, 69.23, 69.18, 68.8, 67.0, 36.4, 33.0, 30.6, 28.2, 25.8, 25.2; λ_abs (MeOH) 404, 500, 533, 573, 628 nm; MALDI-MS obsd 1448.25, calcd 1448.70 [(M + H)⁺]; ESI-MS obsd 1448.6966, calcd 1448.7011 [(M + H)⁺], M = C₇₇H₁₀₁N₅O₂₂.

5-(2,6-Bis((1-hydroxy-1-oxo-4,7,10,13,16,19-hexaoxahenicosan-21-yl)oxy)phenyl)-15-(4-(3-(N-succinimidyloxy)-3-oxopropyl)phenyl)porphyrin (P4-NHS). A solution of P4-Bu/NHS (31.8 mg, 22.0 μmol) in CH₂Cl₂ (1.5 mL) was treated with TFA (750 μL) at room temperature. The reaction mixture was stirred for 2 h at room temperature and then diluted with CH₂Cl₂ (20.0 mL). The organic solution was washed with water (3 × 25.0 mL), dried (Na₂SO₄), and concentrated under reduced pressure. The crude product was loaded on a C18-silica pad (2 cm × 1 cm) and washed with water (40 mL) followed by acetonitrile (8 mL) to give an eluant that upon concentration afforded a red solid (27.6 mg, 94%) with identical characterization data (¹H NMR, ¹³C{¹H} NMR, MALDI-MS) as above.

Conflicts of interest

The authors declare competing financial interests.

Data availability

Crystallographic data for ZnP1-acetal has been deposited at the CCDC under 2321456. Crystallographic data for D7 has been deposited at the CCDC under 2415855. ¹H NMR and ¹³C{¹H} NMR spectra for new compounds; and absorption spectra of selected compounds in solution. All other data are contained in the paper.

Acknowledgements

We thank Ms Zainab Aleem for technical assistance and Dr Bruce Pitner (NIRvana Sciences, Inc.) for stimulating comments. Funding was provided by the National Science Foundation to Oncurie, Inc. (NSF 2136700), and by NC State University. NMR spectroscopy and mass spectrometry measurements were carried out in the Molecular Education, Technology, and Research Innovation Center (METRIC) at NC State University. SCXRD analyses were performed at University of Tennessee at Knoxville.

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Footnote

† Electronic supplementary information (ESI) available: ¹H NMR and ¹³C{¹H} NMR spectra for new compounds; absorption spectra of selected compounds in solution. Single-crystal X-ray diffraction data. CCDC 2321456 (ZnP1-acetal) and 2415855 (D7). For ESI and crystallographic data in CIF or other electronic format see DOI: https://doi.org/10.1039/d5nj02077h

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