PUBLISHED IN ISSUE 1 | FALL 2021
Synthesis and Study of a Porphyrin Host for Perfluoroalkyl Contaminants
Maleeha Ahmad
Texas A&M University - Commerce
Maleeha Ahmed attends Texas A&M University - Commerce. As part of her research internship she synthesizes alpha-alpha-alpha-alpha tetraamino porphyrin hosts in an effort to improve negative environmental impacts and adverse health effects created by an accumulation perfluoroalkyl substances.
ABSTRACT
The aim of this project was to synthesize a porphyrin host that can function as a sensor or to
remove accumulating, harmful perfluoroalkyl substances (PFAS) from the environment and the
human body. PFAS are potential carcinogens that cannot be decomposed easily or metabolized.
Thus, we attempted to create a sensor and extraction agent for these compounds. We utilized
H-NMR, 19F-NMR, UV-Visible spectroscopy, and NMR titration studies to evidence the
success of host production and binding between the synthesized porphyrin host and PFAS guests. The results show that the host binds to compounds with longer perfluoroalkyl carbon chains preferentially to those with shorter perfluoroalkyl carbon chains; further, the host binds perfluorinated carboxylates stronger that perfluorinated sulfonate guests. This research presents an advancement for both environmental and human health.
INTRODUCTION
I. Pentadic Criticism
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Anions are negatively charged molecules that have an immense impact on the world around us.
To provide perspective, DNA is negatively charged and, therefore, anionic. Certain anions,
however, can have detrimental impacts on the environment and the human body. For example,
nitrate ions build up in groundwater after being used in fertilizers and can increase the risk of
gastric cancers. (17) Likewise, the negatively charged fluorinated guests used in this thesis
contaminate the environment and can be harmful to humans. Anion recognition is the process in which a host molecule is able to recognize and bind to a negatively charged guest molecule.
Thus, this process is important in order to study the binding properties of the host and guest. We assessed anion recognition through a series of binding studies, including UV-Visible
spectroscopy and NMR titrations.
II. Porphyrin Properties
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Porphyrin is a unique molecule found in heme, the oxygen transporter of blood.(3) Oxygen exchange in blood is an example of host-guest chemistry. Iron is typically bound in the center pocket and acts as the binding agent that carries the oxygen. The same basic structure of porphyrin was used in the production of the host. Zinc was used instead of iron because it has only one oxidation state, +2. This prevents complications during experimentation from the metal oxidizing. The zinc is bound to the center of the porphyrin structure with coordinate covalent bonds to the four nitrogen atoms. Metallation, the process of adding zinc to the center of the host molecule, can increase the binding affinity for guest molecules because of metal coordinate bonds. Binding studies were conducted both with and without zinc in the host molecule.
III. Applications to Thesis
Perfluoroalkyl and polyfluoroalkyl substances (PFAS) are widely used in the creation of industrial and household items. They are composed of long carbon chains saturated with C-F bonds, compounds of which are stable, hydrophobic, and lipophobic.(4) As such, these compounds are typically incorporated into surfactants, flame retardants, fabric protectors (such as ScotchgardTM), and non-stick coatings (such as TeflonTM).(5) PFAS are extremely hard to decompose naturally, which has led to their classification as Persistent Organic Pollutants. Thus, they can build up in water systems and eventually come into human contact. The current methods to remove PFAS entirely from water systems include incinerating concentrated water waste and photochemical oxidation.(14) However, these processes are very costly, both financially and energetically. This makes the investigation of removing PFAS novel and of forefront importance.
PFAS are not only harmful to the environment; they are also poisonous and carcinogenic to humans. PFAS can be ingested through water systems and the residue of household items, like non-stick pans. In recent years, evidence has shown that the ingestion of PFAS in humans can lead to increased risk of prostate cancer3 and negative impacts on the respiratory and immune system.(6) Chronic exposure to fluorinated compounds can also create a condition called fluorosis. The effects of fluorosis include mottled teeth, skin boils, abnormal skin sensations, polyarthritis, hypercoagulation, and affliction of the parathyroid gland.(10) Further pathologies
include granular masses in the bone marrow, similar to that in severe bone marrow diseases like granulating necrosis.(1)1 Current treatments for acute fluoride exposure include intravenous calcium, but more long-term solutions are yet to be found.(13) As potential carcinogens and pollutants, the investigation of PFAS removal is an important one. In order to advance methods for the detection and removal of these contaminants from the environment and from the human body, we synthesized a host compound that can potentially bind these compounds. The synthetic host functions as a grabbing mechanism, with a pocket for the PFAS guest to bind. This host could, thus, serve as a sensor and extraction agent for PFA-containing organic molecules, which could be beneficial to human health and provide environmental applications.
Perfluorinated compounds that served as guests are shown below in Figure 1. PFOA and PFOS, shown below, are two of the commercially utilized PFAS which are known persistent organic pollutants. They are used in Scotchgard and fire suppressants, respectively.
We also studied a long alkyl chain containing non-fluorinated guest, octanoate, and a guest without the perfluoroalkyl or alkyl chain, acetate, to learn what the fluoro groups and the alkyl chain contribute to guest binding. The proposed host contains a perfluorinated pocket where perfluoroalkyl guests can bind. This is theorized to occur due to the high affinity of fluorinated groups to other fluorinated groups.
Figure 1: Perfluoroalkyl guest compounds
Figure 2: Proposed guest binding
METHODOLOGY
I. Production of Host
The host was synthesized according to known procedures as shown in Figure 4 below. The synthesis began with 2-nitrobenzaldehyde and pyrrole to make the known tetranitro compound.(8) The tetranitro groups were reduced to tetraamino groups using tin (II) chloride according to the literature. All synthetic procedures and reactant measurements were completed according to the literature.(9)
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As Figure 5 shows, the tetraamino porphyrin exists in four stereoisomeric forms. The desired α, α, α, α -isomer is a minor product in the reaction initially only obtainable in 14% yield. It can be separated from the other three isomers by column chromatography. Impurities can also be removed through this same technique by changing the polarity of solvents used during the chromatography. For this porphyrin, we found that using 96:4 dichloromethane:methanol (DCM:MeOH) was a successful solvent to separate the isomers. Literature reports the use of a1:1 benzene:ether solvent system for chromatographic separation of the stereoisomers – our DCM:MeOH solvent system is preferable due to the health hazards of benzene and the
instability of ether solvents due to peroxide formation. Since the α, α, α, α-isomer is the most polar of the four stereoisomers, the other three isomers can be isomerized on silica gel to enrich the mixture in the desired α, α, α, α-isomer, as described in the literature.(9)
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​The presence of the α,α,α,α-isomer was confirmed through NMR spectroscopy, shown in Figure 7 in the Appendix. After the correct porphyrin isomer was acquired, the reaction to complete host production began. 75mg of the α,α,α,α-isomer was placed in a round bottom flask with 62µL triethylamine, 110µL of perfluorooctanoyl chloride, and 10mL of dry DCM. A stir bar was added and the reaction was left on a hot plate overnight with no heat. The solvent was then removed on a rotatory evaporator and the product was isolated by column chromatography using a 1:4 ethyl acetate:hexane solvent. The column chromatography isolated the desired product from any impurities and unreacted starting materials. NMR spectroscopy and mass spectrometry were performed to confirm the synthesis and purification of the host
Figure 3: Synthesis of tetra-amino porphyrin and final host
II. Binding Studies: UV-Visible Spectroscopy (UV-Vis) Titrations
After the host was synthesized, preliminary binding studies were performed with UV-Vis spectroscopy. To prepare the host, a stock solution was created in a vial with 1mg of host and 1mL of spectrophotometric-grade DCM (spec-DCM). 200µL of this stock solution was placed in a 25mL volumetric flask. This amount of stock solution was diluted to the 25mL mark with spec-DCM. The guests were also prepared. The first anion guest attempted was perfluorooctylsulfonate (PFOS). Because this guest was already present as a tetraethylammonium salt form, we did not need to prepare the salt form. 25 mg of this guest was placed in a round bottom flask and diluted with 6mL of spec-DCM. A series of dilutions were created from here. 6mL of spec-DCM was placed in the round bottom flask to create a 100eq solution. 0.6mL of the 100eq solution was placed into a vial with 5.4 ml spec-DCM to create the 10eq solution. Similarly, 0.6 ml of the 10eq solution was placed in a new vial with 5.4 ml spec-DCM to create the 1eq solution. Finally, 0.6 ml of the 1eq solution was placed in a new vial with 5.4 ml spec-DCM to create the 0.1eq solution. To conduct the UV-Vis titration studies, 3mL of diluted host solution from the 25mL volumetric flask was placed in a cuvette. Likewise, 3mL of spec-DCM was placed in another cuvette as a reference sample. Binding studies were performed by scanning the pure host and then making different guest additions (10µL, 20µL, 30µL, 40µL, or 50µL additions) for a total of about 15-20 additions. After the 50µL addition of one guest concentration, the next most concentrated guest solution was used. Absorbance changes indicative of binding were decreases in host λmax of between 0.01 and 0.05 per guest addition.
Because the trial with PFOS and the non-metallated host did not show definitive binding, the host molecule was metallated with zinc in order to increase binding affinity. To metallate, 31mg
of host was placed in a round bottom flask. 20mL of 1:1 DCM:MeOH was added, along with 15mg of zinc acetate dihydrate and a stir bar. The round bottom flask was submerged in a beaker of water, which was heated using a hot plate set at 60℃. The mixture was refluxed for 1.5 hours. The solvent system was then removed on a rotatory evaporator and the final product was purified by column chromatography on silica gel. The host stock solution was prepared after metallation using the same technique as the non-metallated host. Titrations were repeated with PFOS in multiple trials. Once this was complete, the other guests were prepared for titration. Like PFOS, acetate (OAc) was received as a salt from the manufacturer. Thus, only the next four guests were reacted to form tetrabutylammonium salts. The first guest created was the tetrabutylammonium salt of PFOA. To create this, 25mg of pentadecafluorooctanoic acid was placed in a round bottom flask with 2mL of MeOH and 46.3µL of tetrabutylammonium hydroxide (Bu4NOH). This was stirred by swirling for a few seconds and then removing the solvent on a rotatory evaporator. The reaction flask was then placed under high vacuum to dry overnight. This process was repeated similarly for each guest, with respective values of solvent and reagents listed in Table 1.
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​After the salt form of the guest was high vacuumed overnight, 6mL of DCM was added to the respective round bottom flask. A series of dilutions were created from here in the same previous manner. The amounts of guest and µL of Bu4NOH to use were attained through the use of an Excel spreadsheet programmed with stoichiometric calculations. The spectra obtained from these UV-Vis binding studies are shown in the Appendix. The binding constants, which were used to compare binding strength, were determined through a linear regression analysis of the binding curves
Table 1: Tetrabutyl Ammonium Salt Preparation
III. Binding Studies: NMR Titrations
Of the guests tested with UV-Vis spectroscopic titrations, PFOA was found to be the strongest binding fluorinated anion of the host molecule, while OA is its nonfluorinated counterpart. NMR titrations were conducted on these molecules to further evidence PFOA’s binding and compare the effect of fluorination on binding to the guests. To prepare the NMR sample, 2.6mg of host were placed in a round bottom flask, with 37.5µL of trifluorotoluene (as internal reference) and 0.75mL of NMR-grade chloroform (CDCl3). This was pipetted into an NMR tube and kept for study. PFOA and OA were also reprepared for the NMR titrations. Similar to in UV-Vis studies, OA was prepared into a salt by placing 15.4mg of octanoic acid, 2mL MeOH, and 76.2µL of Bu4NOH in a vial, swirling the mixture for a few seconds and then evaporation of the solvent on a rotatory evaporator; the sample was then placed under high vacuum overnight. Likewise for PFOA, 25mg of PFOA, 2mL of MeOH, and 45.2µL of
Bu4NOH were reacted then placed on the high vacuum overnight. After this, a series of dilutions were created with CDCl3, rather than spec-DCM. For OA, only a 1eq and 0.1eq solution were prepared, and, for PFOA, only a 5eq and 0.5eq were prepared. For OA, this was completed by adding 1mL of CDCl3 to the sample in its round bottom flask to create a 1eq solution and adding 100µL of the 1eq solution to 900µL of CDCl3 to make the 0.1eq solution. For PFOA, this was completed by adding 0.5mL of CDCl3 to the sample in its round bottom flask to make the 5eq solution and adding 50µL of the 5eq solution to 450µL of CDCl3 in a new vial. The 1H-NMR and 19F-NMR of the host was recorded, and, then, small amounts of the guests, approximately 10µL, were added to the host NMR sample. The spectra was rerecorded after each guest addition. The respective spectra for each of the two guests are shown in the Appendix.
RESULTS
Table 2: Binding Constant Values
I. UV-Vis Titrations
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The results show that OAc has the highest binding affinity to the host molecule, followed by OA, PFOA, PFBA, and PFOS. The data for PFBS was not able to be fit to the linear regression model. Because of this, the results for this compound are inconclusive. However, this compound visually showed binding as indicated by decreases in host absorbance and formation of a new absorbance peak due to host:guest complex formation. This is shown in the Appendix.
II. NMR Titrations
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For OA, the 19F-NMR titrations showed no significant changes in the fluorine signals of the host compound in forming the complex. This is evidenced by the lack of change in the host fluorine signals at about -64 ppm to -128 ppm. This is expected since OA is not fluorinated. Further, the 1H-NMR shows a peak at 8.7ppm that visibly shifts upfield because of a higher electron density created by the coordinate bonds from the guest and zinc. This shift stops moving after approximately 2.0eq of guest, which evidences strong binding.
​
For the 1H-NMR for PFOA, the peak at 8.7ppm shifts towards 8.6ppm. This upfield shift is expected because of the higher electron density created by the coordinate bonds from the guest and zinc. However, because the shift is still moving upfield after the addition of 16eq of guest, this is evidence of weak binding. This weaker binding is also evidenced on the UV-Vis studies. There is a new peak that forms at 3.0, 1.2, and 0.9ppm. These can be explained because the guest was not fully fluorinated; it was received as 96%. In the 19F-NMR, no shifts are observed in the host signals. However, the host 19F-signals decrease in intensity, while guest 19F-signals increase in intensity – this can be observed at -82ppm, -127ppm and at -117ppm from the guest, for example, where guest versus host signals are clearly resolved.
DISCUSSION
We conclude that this host molecule is effective at binding to fluorinated compounds, in addition to non-fluorinated compounds. For the fluorinated compounds, those with carboxylates showed a higher binding affinity than those with a sulfonates. We theorize that this is due to the highly acidic nature of sulfonic acid, which means it conjugate base is very weak and, thus, would be expected to have weak coordinating ability. Additionally, the long-chain perfluorinated 8 carbon guest bound stronger than the short-chain perfluorinated 4 carbon guest. This could be due to stronger non-covalent attraction between the guest perfluoroalkyl carbon chain and the host long perflourinated carbon chain. We suspect that PFBS was not able to be fit to the linear regression model due to the model’s 1:1 binding assumption. It is possible that since PFBS is a smaller molecule, it could exhibit a 1:2 binding structure or another type
of complex binding not known. However, OAc bound stronger than all other guests. This could be due to its small size, which could make it a better fit for the fluorinated pocket of the host. This binding phenomenon could also be due to the smaller number of carbons compared to the other guests; the presence of carbon could decrease basicity and, thus, binding affinity. Likewise, for OA, the lack of fluorine along the alkyl chain could make the guest have a higher binding affinity due to OA being more basic than its perfluorinated version. Comparing OAc to OA, OAc may bind stronger than OA since the host perfluoroalkyl chain is lipophobic and, thus, the long hydrocarbon chain of OA would not interact favorably with the host perfluoroalkyl chain. This is the opposite of PFOA and PFBA where the longer perfluorinated alkyl chain leads to stronger binding.
CONCLUSION
We successfully synthesized a host molecule with a perfluorinated binding pocket that binds to both non-fluorinated and perfluorinated compounds. For perfluorinated compounds, we found that longer-chain molecules bound stronger to the host than short chain molecules. For non-fluorinated compounds, we found the opposite phenomenon; short-chain non-fluorinated molecules bound stronger than long-chain molecules. Finally, carboxylate compounds bound stronger than sulfonate compounds. These results provide useful information for both the
pharmaceutical and environmental industries. By examining the binding properties of this molecule, it has been demonstrated that the host is capable of binding perfluorinated compounds. Since perfluorinated compounds are known organic pollutants with detrimental health consequences, there is much potential to use the results of this study to improve both the health of the human population and the environment.
APPENDIX
Figure 4: 1H-NMR Spectra of α,α,α,α-Isomer
Figure 5: 1H-NMR Spectra of Non-Metallated Host
Figure 6: Mass Spectrometry Spectra of Non-Metallated Host
Figure 7: Mass Spectrometry Spectra of Metallated Host
Figure 8: UV-Vis Titration Curve for PFOS with Metallated Host
Figure 9: UV-Vis Titration Curve for PFOA with Metallated Host
Figure 10: UV-Vis Titration Curve for PFBS with Metallated Host
Figure 11: UV-Vis Titration Curve for PFBA with Metallated Host
Figure 12: UV-Vis Titration Curve for OA with Metallated Host
Figure 13: UV-Vis Titration Curve for OAc with Metallated Host
Figure 14: 1H-NMR Titration for OA with Metallated Host
Figure 15: 1H-NMR Titration for PFOA with Metallated Host
Figure 16: 19F-NMR Titration for PFOA with Metallated Host
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