Anion Recognition by a Mettaloporphyrin Host

Robert Chaz Cardenas 8/1/2008

Mentor: Dr. Stephen Starnes

1

Abstract

The purpose of this project is to synthesize a meso-functionalized porphyrin

receptor that is pre-organized to fit trigonal planar anions. By using a UV-Visible

Spectrophotometer we can then study different geometrical shaped anions and how they

differ in affinity when compared to trigonal planar anions. This project will also be

continued to synthesize a water soluble porphyrin for possible environmental and

biological use. The porphyrin with the sulfonamide sidearm is formed to make a pocket

effect so that the eleven targeted anions can fit inside the structure. These eleven targeted

anions have four different geometrical shapes, spherical, bent, trigonal planar, and

tetrahedral. Our results were conclusive to the speculation that the trigonal planar and the

spherical anions had a greater affinity for the porphyrin, due to the pre-organized binding

sites of the porphyrin.

2

Background Information

Introduction: The last ten years has seen an explosion in research efforts geared

towards selective anion recognition and sensing.[1] Anions are ubiquitous in biological

systems where they play roles in diverse areas such as osmotic pressure regulation, cell

signaling, energy transduction, genetic information control, and the regulation of

metabolism and defining cell status.[2] Receptors have been designed that recognize

peptide surfaces (through carboxylate side chain recognition),[3] and carboxylates

(malate, fumarate, succinate, oxaloacetate, and ketoglutarate are key components of the

citric acid and glyoxylate cycles; asparate and glutamate are excitatory amino acid

neurotransmitters).[4] Research in the area of anion recognition is aiding the drive to

discover bioactive vancomycin[5] and ristocetin[6] (antibiotics) analogs by yielding a

better understanding of protein-substrate (carboxylate) interactions. Anion recognition

has played a role in the development of enzyme mimics that cleave phosphate ester bonds

(through phosphate recognition)[7] and artificial adolases (through enolate recognition).

[8]

Environmental applications of anion recognition; there are many anions present in

nature that carry with them deleterious health effects upon human exposure. For example,

perchlorate salts have recently been identified in lettuce grown in California and Arizona.

This is a biomedical concern because perchlorate inhibits the uptake of iodine by the

thyroid gland, which in turn inhibits thyroid hormone production[9]. Recent reports

indicated perchlorate above current EPA accepted standards in store-bought milk.[10]

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Biological Anions of Interest Environmental Anions of Interest

2/3 of all enzyme substrates are anionic in nature

• Carboxylates (RCO2- , (amino acids))

• Nitrate (NO3-) - levels in drinking water lower than accepted EPA standards (10 ppm) have been linked to bladder cancer. Has also been linked to “blue baby syndrome”

• Dicarboxylates - oxalate, malonate, malate, fumarate succinate, oxaloacetate, ketoglutarate(intermediates in the citric acidand Glyoxylate cycles), aspartate, glutamate (excitatory amino acid neurotransmitters)

• Nucleotides, oligonucleotides (DNA, RNA pyrophosphate, inositol triphosphate) Phosphorylated species play essential roles inosmotic pressureregulation, cell signaling, energy transduction, genetic information control, regulating metabolism...

• Chloride (Cl- (cystic fibrosis))

• Nitrate and Phosphate (PO43-) induceeutrophication in lakes, streams, ponds…

• Pertechnetate (TcO4-) - by-product of the nuclear fuel cycle

• Nitrate present in huge quantities in these nuclear waste tanks. They present a problem in the vitrification of the spent fuel

• Perchlorate (ClO4-) - rocket fuel, fertilizer, explosives industry - health hazard if concentration is greater than 1 ppb in drinking water

4

Porphyrin Sensors

NNN

N

NH

NOH

Zn

NNN

N

NH

NOH

NNH

NS

SO

O

O H

O

Zn

1 2 3

76

NNN

N

NH

NOH

NO

NOctyl

Zn

HH

NNN

N

NH

NOH

NO

NOctyl

Zn

HH

NNN

N

NH

NOH

CN

N

O

N

ON

Zn

H HH

HH

NNN

N

NH

NOH

N

Zn

SO

O

H

8

Receptors 1-6 were synthesized. 1 showed very nice UV/Vis spectral data (sharp

isosbestic points). 2-6 did not. Brad Smith, Professor of Chemistry at The University at

Notre Dame, suggested we make receptor 7 since, in his experience, he found that

sulfonamide were better behaved, easier to work with than ureas, and showed strong

binding to anions. This was subsequently confirmed by Professor Franz Smitdchen,

University of in Germany, who pointed out at the recent International Symposium of

Macrocyclic and Supramolecular Chemistry that in his experience ureas formed

aggregates in solution. This is what we believe to be true of receptors 2-6 as their UV

NNN

N

NH

NOH

N

Zn

4 5

NNN

N

NH

NOH

NN

N

O

N

ON

Zn

HH

HH

NNN

N Zn

Zn-TPP9

NNN

N

NH

NOH

NH

Zn

SO

O NNN

N H

TPP

H

5

titrations with anions show complex UV/Vis spectral data (few anions show sharp

isosbestic points-which will be illustrated later in the talk). In response to Brad Smith’s

suggestion, receptor 7 was synthesized. It exhibited ideal UV/Vis spectral data when

titrated with anion solutions.

This is where Hikma’s (my lab partner) and my piece of the puzzle fits in. Hikma

and I turned our attention to the synthesis of receptors 8 and 9 when we joined the group.

Compounds 8 and 9 are sulfonamide versions of 2 and 3, analogous to receptor 7 which

is a sulfonamide version of 5.

CC003 HJ005

6

Methodology

The porphyrin that is synthesized in this experiment has basically two parts, a

porphyrin ring, and a meso-functionalized arm that hangs over the top of the molecule.

So to start we had to synthesize the functionalized arm called a sulfonamide:

A B C (CC003)

NNN

N

NH

NOH

NH

Zn

Ts

Chemical Formula: C58H41N7O3SZnExact Mass: 979.23

Molecular Weight: 981.45m/z: 979.23 (100.0%), 980.23 (66.7%), 981.23 (60.1%), 982.23 (48.3%),

983.22 (41.5%), 984.23 (27.1%), 981.24 (19.7%), 983.23 (19.2%), 985.23 (9.0%), 981.22 (4.6%), 982.24 (4.4%), 985.22 (3.7%), 984.22 (3.4%), 986.23 (2.9%), 984.24 (2.4%), 982.22 (2.1%), 986.22 (1.2%)

Elemental Analysis: C, 70.98; H, 4.21; N, 9.99; O, 4.89; S, 3.27; Zn, 6.66

Compound: C58 H41 N7 O3 S Zn

Rel. Intensity [%]100

50

0 m/z

980 985

NH2

NH2

S

O

O

Cl

NH2

NHS

O

O

THF

D

Pyridine (E)

This reaction is usually done with one of the free amines protected by a boc-

anhydried, but for this reaction the materials are relatively cheap so we used a large

excess of the phenylene diamine with the tosyl chloride.

Procedure: Dissolved B and E in a round bottom flask, then in a separate flask

dissolve A into D. While stirring add A and D to the round bottom flask containing B and

E. The color changed from clear to a dark orange. Stir for one hour, add 150ml of water,

7

heat to boiling point, cool to room temperature, filter off grey precipitation, re-crystallize

using methanol, then filter off orange crystals (5.11g of product).

After the product was thought to be completed we used the proton NMR (Nuclear

Magnetic Resonance) to confirm that our product was pure in form.

The small peak at about 5.0 ppm is the single free amine that we were initially

after, because it will be needed for the next chemical reaction where we attach the

sulfonamide to the porphyrin.

8

When we discovered that we have made the sulfonamide successfully we then set

up for the next reaction which is to attach the sulfonamide to the porphyrin:

NHNNH

N

H2N

NHNNH

N

O2NO

H

O

H

NO2

HN

Boiling

OH

O

1. SnCl2 HCl

2. NH4OH

triphosgene

CH2Cl2, pyridineNH

NNHN

NC

O

CH2Cl2

NHNNH

N

NH

NOH

Zn(OAc)2

1:1 CHCl3:CH3OH

NH2

NHS

O

O

NH

NNN

N

NH

NOH

NH

Zn

S

O

O

Ts

CC003

We took the porphyrin and 300ml of DCM (dichloromethane), then added

triphosgene then triethylamine, finally we put a nitrogen balloon on it and then added

CC001 then let it stir overnight.

After the reaction was completed we purified it by using Column

Chromatography. When the mettaloporphyrin was extracted we then turn our attention to

how well the anions are going to bind. This was achieved by using an UV

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spectrophotometer. By titrating anions into a DCM/Porphyrin solution we were able to

see absorbance peaks shift and cross at a particular point called the isosbestic point.

As one can see here CC003 has three point binding (left), and here (right) is the

UV spectrum of the porphyrin plus sixteen additions of nitrate. This data is derived from

the Bensi-Hildebrand equation:

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K= [Porphyrin.Anion] /[ Porphyrin][Anion]

By using this excel template we are able to take the raw data from the UV

spectrophotometer and crunch the numbers to see the correlation and the binding curve

which can tell us also which concentration of the guest anion is liked best by the

porphyrin. This template shows us sixteen scans using 10ul of 100 equivalences of

nitrate.

11

Conclusion

We have concluded that out of the eleven anions that were introduced only the

spherical and trigonal planar geometrical shaped anions had very high affinities for the

host/guest concentrations, but the tetrahedral and bent anions had very low binding

constants, which was hypothesized in the beginning due to the pre-organized shape of the

porphyrin.

Acknowledgments

I would like to first off thank the NSF and the REU program for giving the

opportunity to be a successful and knowledgeable person. Second I would like to thank

my mentor Dr. Stephen Starnes for showing me and sharing his knowledge, also my

wonderful research group: Joey Ramos, Kyle Fort, Anna Vladmirova, Hikma Jemal.

Final thanks to my family, friends and any other supporters.

12

Reference:

[1]. For recent reviews of anion recognition see: (a) Schmidtchen, F. P.; Berger, M.

“Artificial organic host molecules for anions,” Chem. Rev. 1997, 97, 1609-1646. (b)

Gale, F. P. “Anion Coordination and anion-directed assembly: highlights from 1997 and

1998.” Coord. Chem. Rev. 2000, 199, 181-233.

[2]. Sessler, J. L.; Tvermoes, N. A.; Davis, J.; Anzenbacher, P. J.; Jursikov á, K.; Sato,

W.; Seidel, D.; Lynch, V. M.; Black, C. B.; Try, A.; Andrioletti, B.; Hemmi, G.; Mody,

T. D.; Magda, D. J.; Král, V. "Expanded porphyrins. Synthetic materials with potential

medicinal utility."Pure Appl. Chem. 1999, 71, 2009-2018 and references therein.

[3]. Salvatella, X.; Peczuh, M. W.; GairÍ, M.; Jain, R. K.; Sánchez-Quesada, J.; de

Mendoza, J.; Hamilton, A. D.; Giralt, E. "Side chain elongation causes a change from

enthalpy driven to entropy driven binding in the molecular recognition of tetraanionic

peptides."Chem. Commun. 2000, 1399-1400.

[4]. Sessler, J. L.; Andrievsky, A.; Král, V.; Lynch, V. M. "Chiral recognition of

dicarboxylate anions by sapphyrin-based receptors."J. Am. Chem. Soc. 1997, 119, 9385-

9392 and references therein.

[5]. Pieters, R. "Synthesis and binding studies of carboxylate binding pocket analogs of

vancomycin."Tetrahedron 2000, 41, 7541-7545[6]. Albert, J. S.; Hamilton, A. D.

"Synthetic analogs of the ristocetin binding site: Neutral, multidentate receptors for

carboxylate recognition."Tetrahedron Lett. 1993, 34, 7363-7366.

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[7]. Zepik, H. H.; Benner, S. A. "Catalysts, anticatalysts, and receptors for unactivated

phosphate diesters in water."J. Org. Chem. 1999, 64, 8080-8083.

[8]. Kimura, E.; Gotoh, T.; Koike, T.; Shiro, M. "Dynamic enolate recognition in

aqueous solution by Zn(II) in a phenacyl-pendant cyclen complex: Implications for the

role of Zn(II) in class II aldolases."J. Am. Chem. Soc. 1999, 121, 1267-1274.

[9]. Logan, B. E. "Assessing the outlook for perchlorate remediation."Environ. Sci.

Technol. 2001, 483A-487A.

[10]. Kirk, A. B.; Smith, E. E.; Tian, K.; Anderson, T. A.; Dasgupta, P. K. "Perchlorate

in milk."Environ. Sci. Technol. 2003, 37, 4979-4981.