Preparation Of Porphyrins And Chlorins By Degradation Of Natural Pigments

It is truly fortunate that massive amounts of natural products containing both hemin [11] and chlorophylls a and b [12,13] can be accessed. Fischer's three volumes (20,22,24), Die Chemie des Pyrrols, report an astonishing array of procedures for obtaining tetrapyrrole compounds from natural sources. Thus, large volumes of blood can be processed to provide hemin in kilogram quantities. From hemin, a large number of porphyrins and derivatives can be obtained (see later). Similarly, chlorophyll derivatives in the a and b series can be obtained by extraction of leaves, usually spinach. But if only chlorophyll a derivatives are desired, one can take advantage of the fact that certain algae, such as Spirulina, produce only chlorophyll a; thus, a laborious separation of the chlorophyll a and b series can be avoided. If chlorophyll b derivatives are required, there used to be no option but to extract plant chlorophylls and perform the chromatographic separation, either by preparative scale high-performance liquid chromatography (HPLC) or by gravity column chromatography on sucrose. Some years ago, a chemical deriva-tization approach was developed to make the chromatographic separations more palatable, and that will be discussed later.

3.1. Porphyrins from Hemoglobin

Because of the relative ease with which hemin can be obtained from blood, it can be purchased from a number of chemical companies at costs around a few dollars per gram. The method of choice (19) for preparation of hemin from blood involves addition of heparinized, citrated, or defibrinated blood to hot acetic acid containing sodium chloride. After cooling and removal of coagulated protein (usually with a wooden stick), the hemin separates and can be collected by filtration. Alternatively, the messy protein can be precipitated by addition of a solution of strontium chloride, followed by concentration to give hemin as above

(16,44). Hemes [iron(II) porphyrins] can be obtained from hemins [iron(III) chloride porphyrins] most commonly by reduction with sodium dithionite under nitrogen or argon. Since autoxidation of iron(II) to iron(III) porphyrins is very facile in air, use of nitrogen or (preferably the heavier) argon

is absolutely essential. Chromatographic purification of hemins is best accomplished on the corresponding (usually methyl) esters; but hemins [e.g., 11] bearing car-boxylic acid groups should not be esterified with diazomethane—a side-reaction takes place with the iron atom. For methyl esters (the simplest and best ester to use under normal circumstances), 5% sulfuric acid in methanol is the best mixture to use (CAUTION: take care to gently add the acid to the stirred and cooled alcohol) (66). Hemin esters can be hydrolyzed to the corresponding free carboxylic acids using base (66).

3.1.2. Protoporphyrin IX [1]

Protoporphyrin IX [1] is the product obtained by removal of iron from hemin [11], but acid alone does not accomplish this result because iron(III) is very difficult to eject from a porphyrin. Commercial samples of protoporphyrin IX are usually not very pure because of the sensitivity of protoporphyrin to photo-oxygenation at the vinyl groups (see above). The best method for obtaining protoporphyrin IX is to treat hemin [11] with ferrous sulfate in hydrochloric acid (46,51,52); the hemin is reduced to heme, and the iron(II), in strict contrast to iron (III), is readily removed by the acid. Commercial hematoporphyrin IX [14] is often very pure (unlike protopor-phyrin IX), so a method for the preparation of [1] by double dehydration of hematopor-phyrin IX [14] has been reported (40). This involves brief heating of [14] with toluene ^-sulfonic acid in 1,2-dichlorobenzene. The dimethyl ester [2] of protoporphyrin IX can be obtained by esterification with either dia-zomethane (CAUTION: diazomethane can be explosive under certain circumstances) or with methanol-sulfuric acid (CAUTION) as mentioned above for hemin. The very useful Grinstein method (33) can be used to prepare protoporphyrin IX dimethyl ester [2] in one step from hemin [11].

3.1.3. Mesoporphyrin IX [15]

Mesoporphyrin IX [15] is related to protoporphyrin IX [1] with the important difference that the sensitive 3- and 8-vinyl groups in [1] are replaced with durable ethyl groups—hence, mesoporphyrin IX does not undergo the photo-oxygenation reaction mentioned above for protopor-phyrin. Early biosynthetic investigations of the metabolism of protoporphyrin IX often used the easy to handle mesoporphyrin IX [15], and so incorporated a hydrogenation step to accomplish reduction of the 3- and 8-vinyl groups in protoporphyrin IX (9); the method of choice (22) is catalytic hydrogenation over palladium in formic acid. Either protoporphyrin IX, its ester, or hemin are used, and the iron in [11] is removed concomitantly during the reaction. Mesohemin IX [16], the iron(III) chloride of mesoporphyrin IX, is best obtained by the introduction of iron into [15] rather than by reduction of hemin [11]. Esterification of mesoporphyrin IX can be carried out using either diazo-methane or sulfuric acid acid-alcohol.

3.1.4. Hematoporphyrin IX [14]

Hematoporphyrin IX [14] was the first porphyrin to be isolated (in 1867) (69); it was obtained by treatment of blood with concentrated sulfuric acid. Nominally, hematoporphyrin IX [14] is obtained chemically from protoporphyrin by hydration of both of the 3- and 8-vinyl groups. Since the 31- and 81-carbon atoms are chi-ral in [14], a mixture of four optical isomers (enantiomers and diastereomers) is obtained, and these can be separated by HPLC. Porphyrin [14] can also be purchased from commercial sources.

Using protoporphyrin IX [1] as the starting material, hematoporphyrin IX is best prepared by treatment with hydrogen bromide in acetic acid, followed by hydrol-

ysis of the resulting 3,8-di(1-bromoethyl)-derivative [17] with water (22). If a common alcohol (R1OH) such as methanol (R1 = CH3) is used in this last stage, then the 3,8-di(1-alkoxyethyl) analogue [18] is produced. Alternatively, reduction of 3,8-diacetyldeuteroporphyrin IX dimethyl ester [19] with sodium borohydride affords hematoporphyrin IX dimethyl ester [20]

[e.g., Reference 66]. 3,8-Diacetyldeutero-porphyrin IX [21] can be prepared by oxidation of hematoporphyrin IX (62), or by Friedel-Crafts acetylation of deuterohemin IX [22] using acetic anhydride and pyridine, followed by removal of the iron (66). Use of sulfuric acid and methanol to ester-ify the propionic acids in [14] is not advised because acid-catalyzed dehydra

tion, or ether formation, at the 3,8-(1 -hydroxyethyl) groups is a problem; it is best to use diazomethane in methanol to obtain the dimethyl ester [20] (CAUTION).

3.1.5. Deuteroporphyrin IX [23]

Deuteroporphyrin IX [23] is of significant historical importance because it was the first porphyrin isolated in Fischer's Nobel Prize winning synthesis of hemin

[11] (29). Deuterohemin [22] can be obtained from "proto" hemin by brief heating of [11] in a resorcinol melt (60), via the so-called Schumm reaction in which the vinyl groups are replaced by hydrogen atoms (10,12,17,42). Demetalation, as reported above for the preparation of pro-toporphyrin IX from hemin, then affords deuteroporphyrin IX [23].

Numerous 3,8-disubstitution products (and 3- or 8-monosubstitution analogues)

of deuteroporphyrin IX and its esters can be prepared, usually by way of aromatic electrophilic substitution on the hemin or its copper(II) complex. A typical example is 3,8-diacetyldeuteroporphyrin IX [21] (see above), which was also an intermediate in the Fischer's hemin total synthesis.

3.1.6. Coproporphyrin III [24]

Coproporphyrin III [24] is a biologically significant porphyrin because its hexahydro-derivative, coproporphyrinogen III [25], is a colorless intermediate on the pathway between uroporphyrinogen III [26], proto-porphyrinogen IX [27], and protoporphyrin IX [1] in normal porphyrin metabolism. Under normal circumstances, the amount of [25] present at steady state is small. However, biological oxidation of coproporphyrino-gen III yields the colored coproporphyrin III, which takes it out of the normal metabolic sequence. Hence, certain diseases of porphyrin metabolism can result in a buildup of excess photochemically active porphyrins in tissues; such diseases are known collectively as porphyrias. Chemically, porphyrinogens can be oxidized very efficiently to porphyrins by use of 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ). If biosynthetic work using porphyrinogens is to be carried out, the corresponding porphyrin can usually be reduced to por-phyrinogen using sodium amalgam or catalytic hydrogenation (15). When vinyl groups are present on the porphyrin macrocycle, of course, only the sodium amalgam route is recommended—catalytic hydrogenation will probably reduce the vinyls to ethyls. It must be kept in mind when handling porphyrinogens, that oxygen and light can efficiently oxidize the hexahydro material to the porphyrin level, which will make it inactive in biosynthetic investigations—the first true porphyrin in porphyrin biosynthesis is protoporphyrin IX itself.

3.2 Porphyrins and Chlorins from Plants and Algae

In this section, some simple degradation reactions, which furnish porphyrins and chlorins in useful quantities from plants and algae, will be described.

The traditional source for chlorophylls a [12] and b [13], usually present in a ratio of about 3:1, was leaf tissue, usually spinach (25,68). A very useful chemical adjunct for simplification of the mandatory chromatographic separation of the chlorophyll a and b pigments has been reported (41); it employs the Girard reagent T as a means of dramatically increasing the polarity of the series b component in the mixture. For example, reaction of methyl pheophorbide a [28] and b [29] mixture (see above) with Girard's reagent T gives a mixture consisting of unreacted a series compound, i.e., methyl pheophorbide a [28], and the salt [30] from the b series. Column chromatography then achieves a very simple separation in which [30] remains adsorbed to the top of the column, whereas the relatively nonpolar a series compound [28] is eluted quickly. Use of a polar solvent then elutes the b series salt, which can be hydrolyzed to give pure methyl pheophorbide b [29].

Investigators wishing only to deal with chlorophyll derivatives in the a series were advantaged when it was shown that Spir-ulina maxima (from Mexico) or S. pacifica (from Hawaii) contain only the chlorophyll a series of pigments. On account of the fairly drastic extraction conditions, chlorophyll a itself is usually not obtained directly from the alga, but large quantities of pheophytin a [31] and methyl pheophorbide a [28] (up to 0.4% measured by dry weight) can be obtained (67).

Treatment of the plant chlorophylls (either separately or as a mixture) with mild acid gives the metal-free pheophytins a [31] and b [32]; this, as a dried paste, is usually the form in which the pigments are stored prior to further degradation to useful materials. Hydrolysis of the pheo-phytins gives the corresponding pheophor-bides a [33] and b [34]; (note that the pheophorbides still contain one ester, and that hydrolysis of this ester will cause concomitant decarboxylation on ring E). Alternatively, and preferably (for ease of handling), methanolysis of pheophytin a

c02H c02h

(23) (deutero porphyrin tX)

c02H c02h

(23) (deutero porphyrin tX)

cojh c02H co,H co2h

(24) (coproporphyrin III} (25) (coproporphyrinogen III)

cojh c02H co,H co2h

(24) (coproporphyrin III} (25) (coproporphyrinogen III)

co2h coah co2h co2h

(26) (uroporphyrinogen III) (27) (protoporphyrinogen IX)

co2h coah co2h co2h

(26) (uroporphyrinogen III) (27) (protoporphyrinogen IX)

or b provides the corresponding methyl pheophorbides a or b [28 or 29, respectively]—these contain two methyl esters. Transesterification of the phytyl ester for methyl, without removal of the magnesium atom, can be accomplished to afford the methyl chlorophyllides [35] and [36] (26).

A number of simple to perform but mechanistically complex reactions can be carried out on chlorophyll derivatives. For example, oxidation of pheophytin a [31] under highly alkaline conditions accomplishes cleavage the 131-132 bond in the P-ketoester ring E, with hydrolysis of the of phytyl ester, to give Fischer's "unstable chlorin" [37] (28). Simple evaporation of the solution affords the so-called purpurin

(37) ("unstable chlorin") (38) {purpurin 18)

(39) (purpurin 7 trimethyl ester) (40) R = CH=CH2 (3-vinylrhodoporphyrin XV

dimethyl ester)

(41) R = Et (rhodoporphyrin XV dimethyl ester)

18 [38], which bears a very useful anhydride ring [45]. On the other hand, dia-zomethane esterification (CAUTION) yields purpurin 7 trimethyl ester [39] (2628,45). Heating of [39] in collidine gives a diversely substituted porphyrin, 3-vinylrhodoporphyrin XV dimethyl ester [40] (28). If the so-called "meso" (i.e., 3-ethyl instead of 3-vinyl) series of pigments is used, another porphyrin, rhodoporphyrin XV dimethyl ester [41], is obtained.

The isocyclic ring (E) in chlorophylls and their derivatives contains a P-ketoester function which imparts a high degree of chemical reactivity upon the compounds containing it. Such lability is often a disadvantage in the use of chlorophyll derivatives for specific purposes; the spectrum of chemical reactivity in the ring E portion of the pigments can be dramatically decreased by removal of the 132-CO2Me group. When the 132-CO2Me group is removed, the so-called "pyro" series of chlorophyll derivatives are obtained. Basically, ketones are much less reactive than are conjugated ketoesters. Thus, heating of methyl pheo-phorbide a [28] (or b [29]) in collidine (30) gives methyl pyropheophorbide a [42] (or b [43]) in virtually quantitative yield; use of collidine is a yield-enhancing improvement upon the classical method (28) which uti

lized pyridine. Identical demethoxycar-bonylation reactions take place with the so-called meso- (3-ethyl) series of compounds.

The 5-membered isocyclic ring in the pyro-series of chlorophylls cannot be cleaved, but the ring E in its P-ketoester form can be readily opened since the highly reactive conjugated functionality provides a handle for chemical elaboration of ring E. For example, pheophorbide a [33] and 3-vinylpheoporphyrin aj [44, vide infra] can be treated with alkali to give, after esterification with diazomethane, chlorin eg trimethyl ester [45] and chloro-porphyrin-eg trimethyl ester [46], respectively (24). Methanolysis of pheophorbide a also affords [45]. This reaction can be reversed, and ring E is reformed either by treatment with methoxide (24), with tert-butoxide (65), or best of all using tri-phenylphosphine and bis(trimethylsilyl) amide (31).

Although the chlorophyll b series of pigments is less accessible than those from chlorophyll a (and indeed, as mentioned above, Spirulina algae contains no chlorophyll b) a series of reactions parallel to those described above also occurs in the b series; the analogue of chlorin e6 trimethyl ester in the b series is called rhodin g7 trimethyl ester [47] and of chloropor-phyrin e6 trimethyl ester is rhodinpor-phyrin g7 trimethyl ester [48].

Chlorins can be converted into por-phyrins by using DDQ as a dehydrogena-tion agent. The P-ketoester functionality does not take kindly to oxidative stress, so methyl pheophorbide a [28] gives only a low yield of 3-vinylpheoporphyrin a5 dimethyl ester [44]. Using a "sledgehammer" approach to preparation of porphyrins from chlorophyll derivatives, chlorophyll a under very vigorous basic conditions followed by esterification (methanolysis), affords phylloporphyrin XV methyl ester

[49] and pyrroporphyrin XV methyl ester

❖ Procedure 1. Isolation of Methyl Pheophorbide a [28] from S. maxima (67)

1. About 500 g of dried S. maxima alga is slurried in 2 L of acetone, and then liquid nitrogen is added to form a frozen slush.

2. After transferring to a 5-L 3-necked round-bottom flask, the mixture is heated at reflux with mechanical stirring for 2 hours. The supernatant is filtered through a Whatman filter paper (Whatman, Clifton, NJ, USA) using a Buchner funnel, and more acetone is added to the solid debris.

3. The extraction process, with refluxing, is repeated twice more—note that the debris retains its deep green color, but amounts of additional chlorophyll obtained are marginal.

4. The green filtrate is evaporated and then purified by flash chromatography on Grade V neutral alumina, eluting first with n-hexane to remove a fast running yellow band, with dichlormethane to remove the major blue-gray pheo-phytin a band, and finally with 97:3 dichloromethane:tetrahydrofuran to remove some bright green magnesium-containing pigments.

5. The evaporated pheophytin a fraction is treated with 500 mL of 5% sulfuric acid in methanol (degassed by bubbling with nitrogen gas) for 12.5 hours at room temperature in the dark (aluminum foil) under nitrogen, followed by dilution with dichloromethane, and rinsing with water.

6. The mixture is rinsed with 10% saturated aqueous sodium bicarbonate, the organic layer is dried over anhydrous sodium sulfate, followed by evaporation and crystallization of the residue from dichloromethane:methanol. This gives methyl pheophorbide a [28] (average yield 1.8 g).

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