Protoporphyrin IX

Biosynthesis, Functionalization Chemistry, and Applications

Protoporphyrin IX Plays a Vital Role in Biological Systems

Life on earth is critically dependent on the existence of certain key molecules; among these critical molecules are a group of compounds containing tetra-pyrrole macrocycles as an important part of them.

Heme (Iron protoporphyrin IX), chlorophyll, and cobalamin (vitamin B12) are some of the tetra-pyrrole compounds vital to the survival of life — all of which require protoporphyrin IX to exist. Iron protoporphyrin IX plays a pivotal role in oxygen transportation when in the form of hemoglobin and is an active component of biologically essential molecules such as cytochrome p450, myoglobin, and numerous other metalloenzymes.1

Table of Contents

1. Mense, S., Zhang, L. Heme: a versatile signaling molecule controlling the activities of diverse regulators ranging from transcription factors to MAP kinases. Cell Res 16, 681–692 (2006). https://doi.org/10.1038/sj.cr.7310086

What is Protoporphyrin IX?

Protoporphyrin IX (also referred to as Protoporphyrin or PPIX) is the macrocyclic ring system of heme without the iron chelated into the center of the molecule. The enzyme ferrochelatase is responsible for facilitating the chelation of iron into the center of protoporphyrin to form heme. Interestingly, the aromatic porphyrin ring system in protoporphyrin IX strongly absorbs light in specific regions of the visible spectrum of light giving blood its characteristic red color. Of its many important roles, perhaps most notable is that protoporphyrin IX is a critical intermediate in the biosynthesis of heme, chlorophyll, and cobalamin.

Did you know? 

Protoporphyrin IX’s chemical structure was first proposed by Kuster in 1912 and was confirmed when Fischer reported the chemical synthesis of heme in 1929.

Protoporphyrin IX contains functional groups (carboxylic acids and vinyl groups) that have been modified to generate many unique compounds of interest. The free base form of protoporphyrin IX has been metalated with a variety of metals other than iron, imparting unique properties that can be used to research biological mechanisms and may impact the activity of enzymes in various biological pathways. The introduction of metalo-protoporphyrins using metals that don’t occur in nature have been used to explore a multitude of applications such as photosensitizers used in photodynamic therapy (PDT) to treat cancer, catalysts in organic transformations, and as a unique antimicrobial agent. 

It is protoporphyrin IX’s extensive customizability that gives it such a unique and impactful role in various fields of research and application. You can see how vastly it can be customized by checking out our exhaustive (but not comprehensive!) list of custom porphyrins that we offer.

The Biosynthesis of Protoporphyrin IX for Hemoglobin, Chlorophyll, and Cobalamin (Vitamin B12)

The biosynthetic pathway to produce protoporphyrin IX represents one of the most essential metabolic pathways in living organisms, serving as a critical juncture in cellular metabolism. Protoporphyrin IX is the key precursor for the biosynthesis of several vital biomolecules, including hemoglobin, chlorophyll, and cobalamin (vitamin B12).

Hemoglobin, an essential protein found in red blood cells, is responsible for oxygen transport throughout the body. The incorporation of iron into protoporphyrin IX forms heme, the functional component of hemoglobin, enabling efficient oxygen binding and release.

In plants and certain algae, protoporphyrin IX is the precursor for chlorophyll, the green pigment crucial for photosynthesis. Chlorophyll’s role in capturing light energy and converting it into chemical energy through the photosynthetic process is fundamental to the energy balance of our planet.

Additionally, protoporphyrin IX serves as a precursor in the synthesis of cobalamin, commonly known as vitamin B12. Cobalamin is an enzymatic cofactor essential for both mammals and bacteria. This vitamin is vital for various metabolic functions, including DNA synthesis, fatty acid metabolism, and neurological function.

Thus, the pathway leading to the production of protoporphyrin IX is indispensable for sustaining life, influencing a wide array of physiological processes across different biological kingdoms.

Protoporphyrin IX is synthesized in 7 steps

The first step of protoporphyrin IX biosynthesis starts with condensation of glycine and succinyl CoA to form d-aminolevulinic acid (ALA) by d-aminolevulinic acid synthase (ALAS)2.

Chemical reaction showing the condensation of glycine and succinyl CoA to form delta-aminolevulinic acid (ALA) by delta -aminolevulinic acid synthase (ALAS).
Buy delta-aminolevulinic acid (ALA)

For the second step, ALA dehydratase (ALAD)3 combines two molecules of ALA in a condensation to form porphobilinogen (PBG).

Chemical reaction shown where delta-aminolevulinic acid dehydratase (ALAD) combines two molecules of delta-aminolevulinic acid (ALA) in a condensation to form porphobilinogen (PBG).
Buy porphobilinogen

In the third step, four molecules of porphobilinogen are subsequently used to form the unstable hydroxymethylbilane (HMB) by porphobilinogen deaminase (PBGD)4.

Chemical reaction showing how four molecules of porphobilinogen are subsequently used to form the unstable hydroxymethylbilane (HMB) by porphobilinogen deaminase (PBGD)

The fourth step uses hydroxymethylbilane as the substrate for uroporphyrinogen III synthase (UROS)5.  Interestingly, uroporphyrinogen III synthase changes the orientation of one of the pyrrole subunits before catalyzing the macrocyclization to form uroporphyrinogen III. In the absence of uroporphyrinogen III synthase, hydroxymethylbilane will spontaneously cyclize to uroporphyrinogen I6.

Chemical reaction showing how hydroxymethylbilane is the substrate for uroporphyinogen III synthase (UROS).

The fifth step of protoporphyrin IX synthesis continues by decarboxylation all of the four acetic side chains to methyl groups by uroporphyrinogen III decarboxylase (UROD)7 to form coproporphyrinogen III.

Chemical reaction showing the decarboxylation all of the four acetic side chains to methyl groups by uroporphyrinogen III decarboxylase (UROD) to form coproporphyrinogen III.

The sixth step of the synthesis is catalyzed by coproporphyrinogen III oxidase (CPO)8, 9 that changes two of the propionic acid side chains to vinyl groups resulting in the formation of protoporphyrinogen IX.

Chemical reaction showing the catalyzation of coproporphyrinogen III oxidase (CPO) that changes two of the propionic acid side chains to vinyl groups resulting in the formation of protoporphyrinogen IX

In the seventh step, protoporphyrinogen oxidase (PPO)10, 11 mediates a six-electron oxidation forming protoporphyrin IX (PPIX).

Chemical reaction showing how protoporphyrinogen oxidase (PPO) mediates a six-electron oxidation forming protoporphyrin IX (PPIX). This is the final unifying step in the biosynthesis of heme, chlorophyll, and cobalamin.

At last, the formation of protoporphyrin IX marks the final unifying phase in the biosynthesis of heme, chlorophyll, and cobalamin; from this point onward, the synthesis pathways diverge, each leading to the formation of these distinct but equally crucial biomolecules.

How is protoporphyrin IX used in further biosynthetic pathways?

The biosynthesis of heme only requires 1 additional step…

Read more about heme biosynthesis

The biosynthesis of chlorophyll continues with 7 more steps…

Coming soon: Chlorophyll Biosynthesis

The biosynthesis of cobalamin requires 17 steps of additional transformations…

Coming soon: Cobalamin Biosynthesis
  1. Ferreira, G.C., Gong, J. 5-Aminolevulinate synthase and the first step of heme biosynthesis. J Bioenerg Biomembr 27, 151–159 (1995). https://doi.org/10.1007/BF02110030
  2. Jaffe, E.K. Porphobilinogen synthase, the first source of Heme’s asymmetry. J Bioenerg Biomembr 27, 169–179 (1995). https://doi.org/10.1007/BF02110032
  3. Grandchamp, B., De Verneuil, H., Beaumont, C., Chretien, S., Walter, O. and Nordmann, Y. (1987), Tissue‐specific expression of porphobilinogen deaminase. European Journal of Biochemistry, 162: 105-110. doi:1111/j.1432-1033.1987.tb10548.x
  4. Battersby, A., Fookes, C., Matcham, G. et al. Biosynthesis of the pigments of life: formation of the macrocycle. Nature 285, 17–21 (1980). https://doi.org/10.1038/285017a0
  5. Paul R. Ortiz de Montellano (2008). “Hemes in Biology”. Wiley Encyclopedia of Chemical Biology. John Wiley & Sons. doi:1002/9780470048672.wecb221
  6. Straka, J.G., Kushner, J.P Purification and characterization of bovine hepatic uroporphyrinogen decarboxylase. Biochemistry 1983, 22, 20, 4664-4672. https://doi.org/10.1021/bi00289a009
  7. T. Yoshinaga, S. Sano Coproporphyrinogen oxidase: I. Purification, properties, and activation by phospholipids. Biol. Chem., 255 (10) (1980), pp. 4722-4726.
  8. Yoshinaga, S. Sano Coproporphyrinogen oxidase: II. Reaction mechanism and role of tyrosine residues on the activity. J. Biol. Chem., 255 (10) (1980), pp. 4727-4731.
  9. Dailey HA. 1990. Conversion of coproporphyrinogen to protoheme in higher eukaryotes and bacteria: Terminal three enzymes. In Biosynthesis of heme and chlorophylls (ed. Dailey HA), pp. 123–161. McGraw-Hill, New York.
  10. Dailey, T. A. & Dailey, H. A. Human protoporphyrinogen oxidase: Expression, purification, and characterization of the cloned enzyme. Protein Sci. 5, 98–105 (1996). https://doi.org/10.1002/pro.5560050112

Functionalization Chemistry of Protoporphyrin IX

Protoporphyrin IX is a versatile intermediate in the laboratory synthesis of many unique derivatives of protoporphyrin. The functionalization of protoporphyrin has allowed access to hundreds of new molecules with potential applications in medicine, materials science, and catalysis.12,13

Incorporation of Various Metals

Metallated protoporphyrins are extensively used in biological studies because they are competitive inhibitors of heme oxygenase, thus they’re often used to explore processes to breakdown and eliminate heme.15
 
Some of the most potent heme inhibitors are:15
There are other notable heme inhibitors, such as:
The following compounds have little effect on heme oxygenase, so they’re often used as controls:17
Additionally, a considerable amount of research has been conducted regarding the use of metalloporphyrins as preventative agents for neonatal hyperbilirubinemia . This work is comprehensively covered in a review by Peixoto, et al16.
Example illustration of protoporphyrin IX metalation to become a metalloporphyrin.

Modification of Carboxylic Acids

Protoporphyrin IX dimethyl ester is useful for further derivatization since it is much more soluble in organic solvents than protoporphyrin IX itself. In addition to ester forms, peptide coupling conditions can be used to produce bis-amides from PPIX for use in the field of photodynamic therapy research14,15.

Molecular structure formation showing esterification and amide formation using protoporphyrin IX.

Need protoporphyrin IX dimethyl ester? 

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Modification of Vinyl Groups

Functionalization of the bis-vinyl groups can be accomplished by several different synthetic transformations. Bromination of the vinyl groups followed by the addition of alcohols gives access to mesoporphyrin derivatives that contain ether groups. Reduction of the vinyl groups affords mesoporphyrin IX as the product.

Hydroformylation of the vinyl groups with a rhodium catalyst produces the corresponding homo-elongated bis-aldehyde16.
Regioselective bromination of the vinyl groups followed by conversion of the resulting bromide to a boronic acid and cross coupling produces aryl and alkynyl functionalized protoporphyrin derivatives18.

A multistep procedure has been reported for the conversion of both vinyl groups into the bis-aldehyde species17.

Illustration of molecular structures starting with protoporphyrin IX and then showing the various ways the vinyl groups can be modified through bromination, suzuki coupling, carbonylation, bromination, ether formation, hydrogenation, diels alder, esterification, and oxidation.
Addition of dienophiles to protoporphyrin IX leads to ring annulated chlorin derivatives, one of which is commercially used as a photosensitizer in photodynamic therapy under the name of Verteporfin (trade name Visudyne™). The addition of maleic anhydride to protoporphyrin followed by nucleophilic attack produced a new series of photosensitizers19. Research into synthetic modification of protoporphyrin continues to be active and is allowing access to unique molecules with specifically designed properties and functionalities.    
  1. Sitte, Elisabeth., Senge, Mathias O., The Red Color of Life Transformed – Synthetic Advances and Emerging Application of Protoporphyrin IX in Chemical Biology. Eur. J. Org. Chem. 2020, 3171-3191. 
  2. Pavlov, V. Yu., Modern Aspects of the Chemistry of Protoporphyrin IX. Russian Journal of Organic Chemistry, 2007, 43, 1-34. 
  3. Maximova, Ksenia., Pisarek, Sabina, Gryko, Dorota., A Practical Protocol for the Conjugation of various Amino Acids to Protoporphyrin IX. Synthesis 2013; 45, 1099-1105. 
  4. Uchoa, Adjaci, F., Oliveira, Carla, S., Baptista, Mauricio, S. Relationship between structure and photoactivity of porphyrins derived from protoporphyrin IX. Journal of Porphyrins and Phthalocyanines, 2010, 14, 832-845. 
  5. Peixoto, Andreia., Pareira, Mariette M., Neves, Graca M., Silva, Artur, M.S., Cavaleiro, Jose, A.S., Hydroformylation: a versatile tool for the synthesis of new B-formyl-metalloporphyrins. Tetrahedron Letters, 2003, 44, 5593-5595. 
  6. Miyata, Kota., Yasuda, S., Masuya, Takuto., Ito, Satoshi., Kinoshita, Yusuke., Tamiaki, Hitoshi., Oba, Toru., Facile iodination of the vinyl groups in protoporphyrin IX dimethyl ester and subsequent transformation of the iodinated moieties. Tetrahedron, 2018, 74, 3707-3711. 
  7. O’Brien, Jessica, M., Sitte, Elisabeth., Flanagan, Keith, J., Kuhner, Hannes., Hallen, Lukas j., Gibbons, Daire., Senge, Mathias O., Functionalization of Deutero- and Protoporphyrin IX Dimethyl Esters via Palladium-Catalyzed Coupling Reactions. J. Org. Chem. 2019, 84, 6158-6173. 
  8. 19. de Oliveira, Kleber T., Silva, Artur M.S., Tome, Augusto C., Neves, Maria G.P.M.S., Neri, Claudio, R., Garcia, Vinicius S., Serra, Osvaldo, A., Iamamoto, Yassuko., Cavaleiro, Jose A.S., Synthesis of new amphiphilic chlorin derivatives from protoporphyrin-IX dimethyl ester. Tetrahedron, 2008, 64, 8709-8715. 

Applications of Protoporphyrin IX

Protoporphyrin IX and its metalated derivatives have a myriad of applications due to unique biological activity20,21,22,23, acid-base and pi-pi stacking and optical properties.

Heme oxygenase research & heme oxygenase inhibitors

Heme oxygenase is an enzyme that catalyzes the degradation of heme resulting in the opening of the porphyrin ring releasing ferrous iron, biliverdin IX alpha, and carbon monoxide. Biliverdin IX alpha is first converted to bilirubin IX alpha by a NADPH dependent reductase then gluconorated and finally excreted24,25,26 primarily through the digestive tract.
Molecular reaction illustrating how heme becomes biliverdin via heme oxygenase

G-quadruplex-Protoporphyrin IX-based sensors:

Guanine rich sequences can form guanine-quartets (G-quartets) by Hoogsteen base pairing, resulting in the formation of G-quadruplexes31. Aromatic molecules known to complex with G-quadruplexes through interactions of the aromatic ring systems (pi-pi interactions). Protoporphyrin IX and similar compounds have been reported to form complexes with G-quadruplexes with unique chemical sensing properties. For example, Protoporphyrin IX and Zn(II) protoporphyrin IX have been used in the detection of nucleic acids via the interaction with G-quadraplexes32. Telomerase activity in cancer cells was quantified by the enhancement of Zn(II) protoporphyrin IX fluorescence by binding to G-quadruplexes33. Since G-quadruplexes are also known to bind to metals, protoporphyrin IX has been used as a fluorescence signal reporter to detect metal binding by G-quadruplexes34. N-methyl mesoporphyrin is also widely used as a fluorescence probe for G-quadruplex DNA34.

Ferrochelatase Inhibitors:

The terminal step in the heme biosynthesis pathway is the insertion of Fe(II) into protoporphyrin IX by protoporphyrin IX ferrochelatase27. N-methyl protoporphyrin is a strong inhibitor of ferrochelatase with a Ki of 10 nM28. This compound is commonly used to induce heme deficiency in cell cultures29. N-methyl mesoporphyrin is also a ferrochelatase inhibitor30.

Antimicrobial activity:

Ga(III) protoporphyrin IX, In(III) protoporphyrin IX, Co(III) protoporphyrin IX, Mn(III) protoporphyrin IX, and Cu(II) protoporphyrin IX exhibit antimicrobial activity against several pathogenic microorganisms such as Staphylococcus aureus, Mycobacterium abscessus, and Haemophilus influenzae. This activity is attributed to reduced uptake of heme or adverse properties of the non-native metalloporphyrins38,39,40.

Reconstitution of Metalloproteins:

Metalated protoporphyrin IX derivatives such as Mn(III) protoporphyrin IX, Co(III) protoporphyrin IX, and Cu(II) Protoporphyrin IX have been used in experiments to reconstitute hemoproteins in place of Fe(II) protoporphyrin IX for mechanistic studies and to catalyze reactions that are impossible with heme. While challenges exist in scale and durability, advances have been made in the area of engineered proteins designed to have optimum catalytic activity. Other proteins with metalated protoporphyrin derivatives have been used as Redox catalysts35, to catalyze carbene insertion reactions36, and as water splitting catalysts37.
  1. Vreman,J., Ekstrand, B.C., Stevenson, D.K. Selection of metalloporphyrin heme oxygenase inhibitors based on potency and photoreactivity. Pediatr Res. 1993, 33, 195-200. DOI: 10.1203/00006450-199302000-00021
  2. Schulz, S., Wong, R.J., Vreman, H.J., Stevenson, D.K. Metalloporphyrins – an update. Front. Pharmacol., 2012. https://doi.org/10.3389/fphar.2012.00068
  3. Drummond, G.S., Kappas, A. Prevention of neonatal hyperbilirubinemia by tin protoporphyrin IX, a potent competitive inhibitor of heme oxidation. Proc. Natl. Acad. Sci. USA., 1981, 78, 6466-6470. https://doi.org/10.1073/pnas.78.10.6466
  4. Morioka, I., Wong, R., Abate, A. et al. Systemic Effects of Orally-Administered Zinc and Tin (IV) Metalloporphyrins on Heme Oxygenase Expression in Mice. Pediatr Res 59, 667–672 (2006). https://doi.org/10.1203/01.pdr.0000215088.71481.a6
  5. Kikuchi G, Yoshida T, Noguchi M (2005). “Heme oxygenase and heme degradation”. Biochem. Biophys. Res. Commun. 338 (1): 558–567. doi:1016/j.bbrc.2005.08.020
  6. Ahmad Z, Salim M, Maines MD (Mar 2002). “Human biliverdin reductase is a leucine zipper-like DNA-binding protein and functions in transcriptional activation of heme oxygenase-1 by oxidative stress”. The Journal of Biological Chemistry. 277 (11): 9226–32. doi:1074/jbc.M10823920
  7. Jansen, P.L.M., Mulder, G.J., Burchell, B. and Bock, K.W. (1992), New developments in glucuronidation research: Report of a workshop on “Glucuronidation, its role in health and disease”. Hepatology, 15: 532-544. doi:1002/hep.1840150328
  8. Ferreira GC, Franco R, Lloyd SG, Moura I, Moura JJ, Huynh BH. Structure and function of ferrochelatase. J Bioenerg Biomembr. 1995 Apr;27(2):221-9. doi: 10.1007/BF02110037
  9. Cole, S.P.C., Marks, G.S. Ferrochelatase and N-alkylated porphyrins. Mol Cell Biochem 64, 127–137 (1984). https://doi.org/10.1007/BF00224769
  10. Zhen Shi, Gloria C. Ferreira; Modulation of inhibition of ferrochelatase by N-methylprotoporphyrin. Biochem J 1 October 2006; 399 (1): 21–28. doi: https://doi.org/10.1042/BJ20060753
  11. F De Matteis, A H Gibbs, A G Smith; Inhibition of protohaem ferro-lyase by N-substituted porphyrins. Structural requirements for the inhibitory effect. Biochem J 1 September 1980; 189 (3): 645–648. doi: https://doi.org/10.1042/bj1890645
  12. Ida, J.; Chan, S.K.; Glökler, J.; Lim, Y.Y.; Choong, Y.S.; Lim, T.S. G-Quadruplexes as An Alternative Recognition Element in Disease-Related Target Sensing. Molecules 2019, 24, 1079. https://doi.org/10.3390/molecules24061079
  13. Z.-Z. Yang, Z.-B. Wen, X. Peng, Y.-Q. Chai, W.-B. Liang, R. Yuan, A novel fluorescent assay for the ultrasensitive detection of miRNA-21 with the use of G-quadruplex structures as an immobilization material for a signal indicator. Chem. Commun. 2019, 55, 6453-6456.
  14. Z. Zhang, E. Sharon, R. Freeman, X. Liu, I. Willner, Fluorescence Detection of DNA, Adenosine-5’-Triphosphate (ATP), and Telomerase Activity by Zinc(II)-Protoporphyrin IX/G-Quadruplex Labels. Anal. Chem. 2012, 84, 4789–4797;
  15. Yett, A., Lin, L.Y., Beseiso, D., Miao, J., Yatsunyk, L.A. N-methyl mesoporphyrin IX as a highly selective light-up probe for G-quadruplex DNA. Journal of Porphyrins and Phthalocyanines Vol. 23, No. 11n12, pp. 1195-1215 (2019) https://doi.org/10.1142/S1088424619300179
  16. Stone, K.L.; Hua, J.; Choudhry, H. Manganese-Substituted Myoglobin: Characterization and Reactivity of an Oxidizing Intermediate towards a Weak C-H Bond. Inorganics 2015, 3, 219-229. https://doi.org/10.3390/inorganics3020219
  17. Key, H., Dydio, P., Clark, D. et al. Abiological catalysis by artificial haem proteins containing noble metals in place of iron. Nature 534, 534–537 (2016). https://doi.org/10.1038/nature17968
  18. Teruyuki Komatsu, Rong-Min Wang, Patricia A. Zunszain, Stephen Curry, and Eishun Tsuchida Journal of the American Chemical Society 2006 128 (50), 16297-16301 DOI: 10.1021/ja0656806
  19. Stojiljkovic I, Kumar V, Srinivasan N. Non-iron metalloporphyrins: potent antibacterial compounds that exploit haem/Hb uptake systems of pathogenic bacteria. Mol Microbiol. 1999 Jan;31(2):429-42. doi: 10.1046/j.1365-2958.1999.01175.x. PMID: 10027961.
  20. Morales-de-Echegaray, A.V., Maltais, T.R., Lin, L., Younis, W., Kadasala, N.R., Seleem, M.N., Wei, A. ACS Infect. Dis. 2018, 4, 11, 1564-1573. https://doi.org/10.1021/acsinfecdis.8b00125
  21. Visaggio, D., Frangipani, E., Hijazi, S., Pirolo, M., Leoni, L., Rampioni, G., Imperi, F., Bernstein, L., Sorrentino, R., Ungaro, F., Visca, P. ACS Infect. Dis. 2022, 8, 1, 78-85. 10.1021/acsinfecdis.1c00409 
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