Protoporphyrin IX

Uses

hepatoprotectant

Uses

Protoporphyrin IX has been used:

• as a standard in protoporphyrin assays
• in fluorescence spectra analysis
• to treat cells in cell culture to study heme-mediated ferroportin 1 transcription

Definition

ChEBI: A cyclic tetrapyrrole that consists of porphyrin bearing four methyl substituents at positions 3, 8, 13 and 17, two vinyl substituents at positions 7 and 12 and two 2-carboxyethyl substituents at positions 2 and 18. The parent of the class of protoporphyri s.

General Description

Protoporphyrin IX belongs to the porphyrin family, which are a class of tetrapyrroles. It is the iron-free form of hemin and amphiphilic in nature. It is the precursor of heme in its biosynthetic pathway.

Biochem/physiol Actions

Protoporphyrin IXlevels are elevated intumorcells due tometabolism anomaliescompared to normalcells.

Synthesis

5522-66-7

553-12-8

The general procedure for the synthesis of 3,3'-(3,8,13,17-tetramethyl-7,12-divinylporphyrin-2,18-diyl)dipropionic acid from methyl 3,3'-(3,8,13,17-tetramethyl-8,13-divinyl-2,18-porphyrin) dipropionate is as follows: referring to the method of Smith (1999), dimethyl protoporphyrin IX ( 100 mg, 0.169 mmol) was dissolved in a methanol solution (35 mL) containing potassium hydroxide (3.02 g, 53.8 mmol) and heated to reflux overnight under nitrogen protection from light. After completion of the reaction, it was cooled to room temperature and extracted with ethyl acetate. The ethyl acetate layers were combined and back-extracted with 3M hydrochloric acid solution (2 x 50 mL). The aqueous phase was collected and the pH was adjusted to 4 with 3M sodium hydroxide solution, followed by extraction with ethyl acetate (3 x 100 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to afford pure protoporphyrin IX (72.8 mg, 76.6% yield) in the form of a purple-red solid with a melting point of 205 °C (decomposition temperature >300 °C). The 1H NMR spectrum of the product was consistent with the literature reports. UV-Vis absorption spectrum (CHCl3/TFA) λmax/nm: 413 (logε 5.24), 525 (3.25), 556 (3.99), 578 (3.75), 600 (3.63).1H NMR (200 MHz, CDCl3, TMS as internal standard) δ: -3.33 (2H, s, internal NH). 3.19-3.26 (4H, t, J=6.6 Hz, -CH2CH2CO2H), 3.63 (3H, s, -CH3), 3.67 (3H, s, -CH3), 3.69 (3H, s, -CH3), 3.72 (3H, s, -CH3), 4.48 (4H, t, J=5.1 Hz, -CH2CH2CO2H) ), 6.30 (2H, dd, J=17.8 Hz and 10.0 Hz, -CH=CHAHB), 6.47 (2H, dd, J=11.4 Hz and 3.1 Hz, -CH=CHAHB), 8.09-8.27 (2H, m, -CH=CHAHB), 10.63 (1H, s, γ-H), 10.65 (1H, s, δ -H), 10.90 (1H, s, α-H), 10.92 (1H, s, β-H). Mass spectrum (MALDI-TOF) m/z: 563.4 [M+H]+ (calculated value 563.7).

Enzyme inhibitor

This iiron-free, immediate precusor of heme (FWfree-acid = 562.67 g/mol; CAS 553-12-8; brownish-yellow solid; soluble in a number of organic solvents; disodium and dipotassium salts solublized in the presence of Tween 80) has lmax values in 25% HCl are 602.4, 582.2, and 557.2 nm. Protoporphryrin IX is also an activator of guanylate cyclase. See also Iron Protoporphyrin IX; Heme; Hemin Target(s): aminolevulinate aminotransferase; 5-aminolevulinate synthase; glutamate: glyoxylate aminotransferase; glutathione S-transferase; glyoxalase I, or lactoylglutathione lyase; guanylate cyclase; heme oxygenase; hydroxymethylbilane synthase, or porphobilinogen deaminase; nitric-oxide synthase; porphobilinogen synthase, or 5-aminolevulinate dehydratase; succinyl-CoA synthetase; tryptophan pyrrolase, or tryptophan 2,3-dioxygenase; and uroporphyrinogen decarboxylase.

Purification Methods

Protoporphyrin IX (3,18-divinyl-2,7,13,17-tetramethylporphin-8,12-dipropionic acid, ooporphyrin) [553-12-8] M 562.7, pKEst ~ 4.8. Protoporphyrin IX is purified by dissolving (4g) in 98-100% HCOOH (85mL), diluting with dry Et2O (700mL) and keeping at 0o overnight. The precipitate is collected and washed with Et2O, then H2O, and dried in a vacuum at 50o over P2O5. It crystallises from aqueous pyridine and from Et2O in monoclinic, brownish-yellow prisms. The UV max values in 25% HCl are 557.2, 582.2 and 602.4nm. It is freely soluble in ethanolic HCl, AcOH, CHCl3, and Et2O containing AcOH. It forms sparingly soluble diNa and diK salts. [Ramsey Biochemical Preparations 3 39 1953, UV: Holden Aust J. Exptl Biol and Med Sci 15 412 1937, Garnick J Biol Chem 175 333 1948, IR: Falk & Willis Aust J Sci Res [A] 4 579 1951, Beilstein 26 IV 3042.]

Toxicity evaluation

protoporphyrin IX, is highly toxic in the presence of light and molecular oxygen, killing photosynthetic plants very quickly through the generation of singlet oxygen.

References

[1] Patent: WO2017/197104, 2017, A1. Location in patent: Page/Page column 40; 41
[2] Journal of the American Chemical Society, 1982, vol. 104, # 25, p. 6930 - 6937
[3] Patent: US4996200, 1991, A