Conformational Re‐engineering of Porphyrins as Receptors with Switchable N−H⋅⋅⋅X‐Type Binding Modes

Abstract The selectivity and functional variability of porphyrin cofactors are typically based on substrate binding of metalloporphyrins wherein the pyrrole nitrogen units only serve to chelate the metal ions. Yet, using the porphyrin inner core system for other functions is possible through conformational engineering. As a first step towards porphyrin “enzyme‐like” active centers, a structural and spectroscopic study of substrate binding to the inner core porphyrin system shows that a highly saddle‐distorted porphyrin with peripheral amino receptor groups (1, 2,3,7,8,12,13,17,18‐octaethyl‐5,10,15,20‐tetrakis(2‐aminophenyl)porphyrin) coordinates analytes in a switchable manner dependent on the acidity of the solution. The supramolecular ensemble exhibits exceptionally high affinity to and selectivity for the pyrophosphate anion (2.26±0.021)×109  m −1. 1H NMR spectroscopic studies provided insight into the likely mode of binding and the characterization of atropisomers, all four of which were also studied by X‐ray crystallography.

Abstract: The selectivity and functional variability of porphyrin cofactors are typically based on substrate binding of metalloporphyrins wherein the pyrrole nitrogen units only serve to chelate the metal ions.Y et, using the porphyrin inner core system for other functions is possible through conformational engineering.Asafirst step towardsporphyrin "enzymelike" active centers,astructural and spectroscopic study of substrate binding to the inner core porphyrin system shows that ah ighly saddle-distorted porphyrin with peripheral amino receptor groups (1,2 , 3,7,8,12,13,17,18-octaethyl-5,10,15,20-tetrakis(2-aminophenyl)porphyrin) coordinates analytes in as witchable manner dependent on the acidity of the solution. The supramolecular ensemble exhibits exceptionally high affinity to and selectivity for the pyrophosphate anion (2.26 AE 0.021) 10 9 m À1 . 1 HNMR spectroscopic studies provided insight into the likely mode of binding and the characterization of atropisomers,a ll four of whichw ere also studied by X-ray crystallography.
For the past few decades,the inner core system of conformationally designed nonplanar porphyrins has attracted scientists as an excellent host for various metals [1] with highly tunable basicity. [2] Such porphyrins recently found their first application as organocatalysts [2a,b] and promoters for dioxygen/hydrogen peroxide interconversion. [3] This indicates that specific core NÀH···substrate interactions can be achieved via macrocycle engineering. [4] Since the beginning of porphyrin structural chemistry, [5a] weak interactions have been observed in crystals. [5] Densely packed free base porphyrin systems that encapsulate substrates in their lattices are well known. [4, 5b,c,6] However,alimitation of X-ray crystallography is the structural determination of monocrystalline solids,a chieved by recrystallization from saturated solutions. [7] Alternative spectroscopic detection of NÀH···X-type binding in porphyrin solutions is almost impossible without specially designing the binding pocket, as solvation and dilution drastically affect weak interactions by dispersing the binding agent to its surroundings.Planar porphyrins are more difficult to protonate than their nonplanar counterparts due to the penalty paid for extra distortion required upon protonation. [8] Therefore, the respective symmetric porphyrin dications have yet to find use as either functional or selective anion receptors under normal laboratory conditions. [9] Moreover,t he porphyrin inner core imine and amine units of planar analogues are usually not involved in intermolecular interactions due to the "shielding" properties of the macrocycle system. [10] Distortion can cause an increase in the degree of outwards orientation of the inner pyrrolic entities,making these positions more basic and accessible to substrates,t hereby creating an "active center". [2a,4] To activate the imine and amine moieties,w ec hose ring puckering by steric strain. Since sterically overcrowded porphyrins can have significantly saddle-distorted 3D conformations, [1, 3, 5c, 7, 11] acombination with additional coordinating sites on the periphery of porphyrins can yield porphyrinoid receptors with multiple binding sites to produce as ituation comparable to the enzyme lock-and-key model [4] at am olecular level. Therein, the interplay between all peripheral substituents (i.e., peri interactions and peripheral control via functional groups) would result in aw ell-defined active site directing to the inner core system for substrate/ analyte binding and recognition (Scheme 1).
Many cofactors,enzyme substrates,and DNAare anionic in nature. [13] Among anions of current interest, [14] the pyrophosphate anion has attracted particular attention due to its biological relevance, [15] and significant efforts have been made to develop more potent pyrophosphate sensors. [16] To study the binding capabilities of the isolated tetraaminoporphyrin a 4 -1,U V/Vis titrations were carried out with tris(tetrabutylammonium) hydrogen pyrophosphate (PP i )inthe presence of TFA. TheSoret band of the hexaprotonated porphyrin a 4 -P1 (488 nm) was found to be redshifted by 27 nm compared to the neutral porphyrin a 4 -1 (461 nm), while the new Soret band of a 4 -P1-PP i complex exhibited abathochromic shift of 10 nm (472 nm) compared to a 4 -1 ( Figure 1). Complex formation was accompanied by as trong reduction of the main Q-band of the protonated porphyrin a 4 -P1 (at 696 nm) while two Q-bands arose at new positions (616 and 673 nm), similar to what is observed for the metalloporphyrin complexes [17] (higher symmetry in comparison to the porphyrins). Tw oisosbestic points were identified at 483 and 684 nm. This was followed by ar apid color change from yellow/brown to green. Most often, the variation of the porphyrin color indicates electronic or geometrical changes to the macrocyclic system [9b, 18] which, in this instance,refers to the substrate-core interaction.
To confirm and further investigate NÀH···X-type complexation, the inner core system was blocked from any potential pyrrole-substrate interactions by Ni II insertion. The isolated a 4 -Ni II OET am PP (a 4 -2)( metalated analogue of a 4 -1) showed no observable spectroscopic changes in UV/Vis titration studies (Figure 1). Furthermore,t itration of a 4 -1 with TFAi nt he presence of PP i revealed the formation of core diprotonated porphyrin a 4 -CP1 before transitioning to the a 4 -P1-PP i system ( Figure S46). This,t herefore,i ndicates that:1 )the inner core system plays an essential role in complex formation;2 )protonation of the core imine nitrogens takes place first, followed by the peripheral amines; 3) the charge-carrying peripheral ammonium groups are necessary for the complex formation in order to stabilize the corresponding substrate via the combination of electrostatic and hydrogen-bonding interactions. [19] It should be noted that the nonplanar analogue without any peripheral coordinating groups (H 4 OETPP 2+ )w as incapable of forming any subsequent adducts ( Figure S47).
Given that the protonation of the peripheral amines forms an active probe for PP i detection, amolecular switch between a 4 -P1-PP i and the substrate-free a 4 -CP1 form was developed. Following the use of 14 equiv.o fT FA,t he formation of the hexaprotonated porphyrin a 4 -P1 was observed. Addition of 8equiv.o fPP i promptly formed a 4 -P1-PP i ,w hile another 8equiv.immediately regenerated the substrate-free form (a 4 -CP1), ar esult of the basicity introduced with the PP i salt. Therefore,alack of peripheral charge via deprotonation with excess PP i leads to adestabilized complex. In order to reform the a 4 -P1-PP i ,a cidity must be restored with an additional 14 equiv.ofTFA.These cycles could be repeated at least eight times (in an 8:14 equiv.( PP i :TFA) ratio) (Scheme 1). [20] The molecular switch between active and inactive forms highlights the reversibility and reusability of the current system.
Theanion-recognition properties of a 4 -P1 were studied in CHCl 3 with various anions in the form of TBAs alts (Figures S48 and S49) and different acids ( Figure S50), using UV/Vis spectroscopy.I na ddition to PP i ,f our substratestetrabutylammonium bisulfate (BS), tetrabutylammonium phosphate monobasic (MP), methanesulfonic acid (MSA), and benzenesulfonic acid (BSA)-were pinpointed as complexing substrates.S imilar UV/Vis spectral profiles were observed for all complexes;h owever, the main Soret bands (depending on its position) can be assigned to the anionic moiety present within the analyte (sulfonic at % 464 nm and phosphonic at % 471 nm) (Figure 2). This finding-while preliminary-suggests that the geometrical and electronic prop-  erties of phosphoric and sulfonic moieties influence the porphyrin-analyte complex formation, similar to the proposed "lock-and-key" concept. [4] In operational terms,t he affinities for anions were found to decrease according to the following sequence: PP i > BS > MP as observed from UV/Vis displacement studies ( Figures  S51 and S54). Thea ffinities of MSA and BSA were not investigated due to their poor solubility in CHCl 3 .S toichiometry for a 4 -P1 was determined using Jobsp lots (Figure S52). BS was found to interact with a 4 -P1 in a1 :2 (host:guest) ratio and MP and PP i in a1:1 ratio ( Figure S54). Theb inding constants were calculated using ReactLab software [21] and the corresponding binding parameters are presented in Table 1. UV/Vis titration plots with MP show aw ell-fitted pattern [sum of squares (ssq) = 0.11 and virtual displacement (d r ) = 2.54 10 À3 ]correlating to the 1:1binding mode.Presumably, a 4 -P1 cannot accommodate two MP units, thus leading to only one entity binding in the system. Complexation with BS displayed ab est fitting pattern fixed into the 1:2b inding mode (ssq = 0.11 and d r = 2.33 10 À3) . Interestingly, PP i showed arelatively poor fit (ssq = 3.05 and d r = 0.0132) when fixed into the 1:1binding mode with a 4 -P1. It can be postulated that PP i can act as ab identate-type substrate in complex formation with a 4 -P1.T hereby,c omplexation with PP i had to be treated as a1 :2 binding mode during calculations despite the 1:1r atiometric binding suggested by the Jobsplot (Figure S54). After the new fitting (K 1 = 0a nd 1:2b inding mode), the K 2 value was calculated with as ixfold lower ssq value (0.51) and improved d r value (5.4 10 À3 ), leading to amuch better fit (Table 1). In terms of association constants,the previously performed displacement studies K MP < K BS < K PPi corresponded well with calculated values [(1.04 AE 0.014) 10 5 < (3.44 AE 0.389) 10 6 < (2.26 AE 0.021) 10 9 ,r espectively].N ot only does this show av ery high affinity towards pyrophosphate in contrast to other tetrapyrrole systems, [16b,f,g,k] retaining all the porphyrin functionality as ac hromophore (aromaticity of the macrocycle), but also ar emarkably high selectivity and tolerance in the presence of the most common interfering anions ( Figure S55).
To further investigate the N À H···X-type interactions in a 4 -P1 complex systems,w ec arried out 1 HNMR studies in CD 3 CN.Itshould be noted that aggregation and precipitation in ac oncentrated solution of CHCl 3 prevented any further 1 HNMR analysis in CDCl 3 .M oreover,t he low sensitivity of a 4 -P1 as ar eceptor for analytes in CH 3 CN was detected; [22] thus,excess amounts of substrates were used in the following studies. 1 HNMR spectra in CD 3 CN were recorded with isomerically pure a 4 -1 in the presence of TFA. In contrast to the a 4 -P1 spectra, the addition of MSA, BSA,and BS resulted in new resonances in the aromatic and aliphatic regions ( Figure S56). Complex formation of a 4 -P1-BS, a 4 -P1-MSA, and a 4 -P1-BSA was suggested by the emergence of two sharp inner core proton signals.P resumably,s ubstrate interactions with the inner core system restrict rapid dynamic exchange by blocking the macrocyclic cavity,resulting in the emergence of two differently shifted proton signals.T he ability of the substrates to accept hydrogen bonds from the donor cavity determines the blocking properties by affecting the exchange rates ( Figure 3).
As mentioned above,t he isolation of all individual atropisomers was not yet accomplished for the free-base porphyrin 1 form. However,t he introduction of Ni II to the macrocycle system eliminated any possible inner N À H tautomerism, [23] leading to an increase in structural symmetry. [18] This was achieved by stirring the atropisomeric mixture 1 in boiling toluene (120 8 8C) and in the presence of 5equiv.of nickel(II) acetylacetonate for 4hours,y ielding the nickel(II) porphyrin 2 in 93 %yield as amixture of atropisomers.T hinlayer chromatography (SiO 2 )s howed excellent separation in DCM and, subsequently,a llowed isolation of the individual atropisomers.The relative amounts of individual components (a,b,a,b-2 13 %, a 2 ,b 2 -2 23 %, a 3 ,b-2 49 %, and a 4 -2 15 %) correlated well with previously reported planar free-base analogues (four atropisomers obtained in 1:2:4:1s tatistical abundance ratio) with great stability towards isomerization. [19,24] All of the isolated atropisomers were recrystallized using liquid-liquid diffusion (CHCl 3 /MeOH) and confirmed via X-ray crystallography ( Figures S42  and S44). [25] To our knowledge,t his is the first example of porphyrin atropisomerism where all four isomers have been structurally characterized.
To investigate NÀH···X-type interactions within the different atropisomers of 2, 1 HNMR studies were carried out in CD 3 CN with the addition of MSA (as ad emetalating and complexing agent) ( Figure S57). Thed istribution of the inner NÀH signals of the individual atropisomers was very different ( Figure 4)  Anion [a] Binding mode [b] K showed only one NÀHsignal due to the symmetrical binding motif on both sides of the plane.I nt he case of the a 2 ,b 2 -P1-MSA configuration (Figure 4b), there are potentially three substrates which interact with the N À Hg roups:t wo identically,and one on the other side of the plane leaving one N À H proton "inactive". In a,b 3 -P1-MSA (Figure 4c), four different proton signals were observed due to the highly unsymmetrical system. As previously described, a 4 -P1-MSA (Figure 4d) complexation is followed on one side of the plane making the "blocked" and "open" cavities,r esulting in two differently shifted proton signals.O verall, 1 HNMR spectroscopy was successfully exploited as an instrument for the detection of porphyrin-analyte complexes and could be employed as at ool for the determination of the corresponding conformations.
In this study,w eh ave detailed our insights into the acidactivated nonplanar porphyrin a 4 -P1 N À H···X-type binding motifs observed in solution. High selectivity towards substrates containing phosphonic or sulfonic moieties was spectrophotometrically detected. This was accompanied by ad istinct color transition unlocking the potential of superstructured free-base porphyrins for colorimetric anion recognition. Theh ighest affinity identified for pyrophosphate is rationalized in terms of its ability to form as tronger supramolecular complex with a 4 -P1 compared to other anions tested. Presumably,t his reflects the combined benefit of several favorable interactions,i ncluding electrostatic and hydrogen bonding. 1 HNMR analyses of various complexes revealed highly different inner core proton signals,suggesting ac ombination of "blocked" and "open" cavities due to the binding event. Theproper tuning of various weak interactions combined with a" turned-on" approach, as delivered by the systemsp rotonation, may provide ag eneral strategy for the development of metal-free porphyrin-based probes for awide range of analytes and hints at ap otential path towards artificial porphyrin-based enzyme-like catalysts.