low-temperature photcoxidation of chlorophyll-a

Pkorot~kemisr,y undPhorobioloyy, 1972, Vol. 16, pp. 499--509. Pergamon Press. Printed in Great Britain

LOW-TEMPERATURE PHOTCOXIDATION OF CHLOROPHYLL-A”

GWENDOLYN SHERMAN?, T. ,MARC KORNS and HENRY LINSCHITZ Department of Chemistry, Elrandeis University, Waltham, Mass. 02 154, U S A .

(Received 3 November 1971; accepted 30 M a y 1972)

Abstract - Illumination of chlorophyll at low temperature, in solvents containing organic Lewis bases and oxygen, leads to oxidation of the C-2 vinyl group. This oxidation proceeds through a series of thermal and photochemical changes, including initial formation of a new compound (I) absorbing at 620 nm. This undergoes further rapid photochemical (or thermal) conversion back to a chlorin-like substance (14, and much slower phototransformation to a third intermediate (111) absorbing near 500 nim. The reaction requires complexed metal (Mg or Zn), but not an intact cyclopentanone ring. The ‘620’ photoproduct is assigned to a cyclic peroxide in- volving C- 1 of chlorophyll and the vinyl group.

INTRODUCTION

NUMEROUS studies have been conducted on the photochemical transformations of chlorophyll (Chl) in vitro (Seely, 1966a). In connection with Chl’s photocatalytic function, its photo-redox and sensitizer reactions are of particular interest. Illumination of Chl solutions with white light., at room temperature and in contact with air, leads to slow, irreversible fading of the pigment (Livingston and Owens, 1956). The detailed nature of this undoubtedly complicated process has not been established. However, by irradiating at low temperatures and at controlled wavelengths, and by following the changes spectrophotometricdly, we have discovered a complex set of photochemical and thermal reactions leading to a quite specific photooxidation of Chl (Korn, 1955). In this paper we describe varialus stages of this reaction, as well as studies on Chl derivatives, which establish that the reaction requires complexed Mg (or Zn) but not an intact cyclopentanone ring and that the site of reaction involves the vinyl group. More- over, organic bases such as pyridine or alcohol, as well as molecular oxygen, are needed. All these characteristics sharply distinguish the oxygen reaction from the low-temperature reaction between Chl and quinones, which we have reported earlier (Linschitz and Rennert, 1952). Independent work on the low-temperature oxygen reaction has been published by Dilung and Karpitskaya (1 963).

A room-temperature photoreduction of Chl in oxygen-free pyridine-ethanol, containing ascorbic acid and 11,4-diazabicyclo(2.2.2) octane, has been reported by Seely (1 966b). The “hypochlorophyll” resulting from this photoreduction has a spectrum markedly similar to our first unstable intermediate and it also requires the C-2 vinyl group and Mg or Zn for its formation.

*This work was assisted by a grant from the U.S. Atomic Energy Commission to Brandeis University

tPresent address: Research Laboratory, Mt. Auburn Hospital, Cambridge, Mass. 02140. $Present address: Creole Petroleum Corp., 15 West 51 Street, New York, N.Y.

[Contract No. AT(30-1)-2003].

499

500 G . SHERMAN, T. M. KORN and H. LINSCHITZ

MATERIALS A N D METHODS Photoclzemical procedure

For irradiation and spectrophotometry at low temperature, a thermostated cell- holder was used. The absorption cell, made of 1 cm precision-bore tubing, sealed to a long Pyrex tube, was held in a square hole in a cylindrical copper block. Slots were cut into opposite sides of the block for passage of light. The block constituted the base of a copper can, provided with a central tube, and the annular space was filled with an isopentane-cyclopentane bath. This was cooled by liquid nitrogen circulated through a copper coil. The entire assembly was mounted in a tall Pyrex Dewar equipped with plane windows. This arrangement provided a clear optical path free of refrigerant and, with suitable cooling or heating coils and baths, permitted any temperature to be main- tained from liquid nitrogen to +200"C. At - 17O"C, the rate of warming without added nitrogen was about 2"C/hr. Temperatures were monitored by a copper-constantan thermocouple placed in a capillary directly in the solution, just above the optical path. Irradiations were carried out with the Dewar mounted at the center of an optical bench between two 1000-W projection lamps, with the light focused through condenser lenses and water filters onto opposite faces of the cell. Appropriate Corning filters were placed at the Dewar windows as needed. To record spectra, the Dewar was supported on a cork ring held in a suitable adapter in the cell compartment of a Cary Model 14 spectrophotometer.

The usual solvent mixtures were EPA (ether-isopentane-ethanol, 8 : 3 : 5 ) or E-Py (ether-pyridine, 6 : l), prepared from reagent-grade solvents with no further treatment except that pyridine was refluxed with barium oxide and distilled. Concentra- tions were adjusted to give initial optical densities between 0.8 and 2 at the red peak (ca. 5 x 10-6M).

Preparative low-temperature photochemical technique An apparatus was constructed in which appreciable amounts of photoproduct could

be prepared by low-temperature irradiation with filtered light. The apparatus consisted of a chrome-plated can, 60mm 0.d. and 300mm long, fitted inside a flat-bottomed cylindrical Pyrex vessel and centered by metal spacers. The solution to be irradiated was contained in the 6 mm annular space between the metal can and Pyrex tube. The central can was cooled by an isopentane-methylcyclohexane (1 : 1) bath, kept below - 159°C by a copper coil through which liquid nitrogen was blown. Temperatures were measured by a thermocouple directly immersed in the irradiated solution. The assembly of Pyrex vessel and cooling finger was supported in an unsilvered Dewar, surrounded by a Pyrex jacket. An aqueous dye solution (0.0013% Fast Green and Metanil Yellow) was circulated through the jacket and a water cooled coil, and served both as optical f i l - ter and coolant. Six 500-W projection lamps, placed within a cylindrical matte reflector, were spaced around the filter jacket, and were cooled by a fan mounted underneath. With this equipment, 5 mg of chlorophyll in 250 ml of EPA solvent (3 X 10-5M) could be fairly completely converted to Stage I material (see below) in about a 20-min irradiation.

After photoconversion, the solution was exposed to room light (Stage 11), treated while still cold (0°C) with 2 N HCl, washed several times with water and saturated NaCI, dried over sodium sulfate, flash-evaporated at room temperature, and chromatographed.

Low-temperature photooxidation of chlorophyll-a 50 1

Materials Ethyl chlorophyllide a. was isolated from the leaves of Ailanthus altissima by the

method of Holt and Jacobs (1954) and has been previously described (Sherman and Wang, 1966).

Chlorophyll a was isolated from a mixture of chlorophylls a and b obtained from fresh spinach by the method of Jacobs, Vatter and Holt (1 954). Chl a was separated by column chromatography on sucrose, and obtained as a deep blue-black material with a metallic luster by evaporating the ether extract over water. This material has no definite melting point but softens at about 115°C. The extinction coefficient at 428 nm was 109,000 and at 660 nm, 86,100, with a 428/660 nm ratio of 1.27 and 660/505 nm ratio of 72.5. Calc. for C,,H,,MgN,O,. 2H,O: C, 71.06; H, 8.24; N, 6-03. Found: C, 70.83; H, 8.66; N, 5.83.

Zn pheophytin a was prepared by heating a small amount of pheophytin a with zinc acetate in dioxane just below reflux temperature under a nitrogen atmosphere for 2 hr. When a spectrum indicated complexing of zinc, the dioxane solution was added to ether, washed several times with water, dried over anhydrous sodium sulfate, and the ether evaporated. The material was used for irradiation without further purification. The absorption maxima are at 655,607, 564, 520 and 425 nm (sh.) with a 425/655 nm ratio of 1 -41.

Mg-chlorin e,-trimethyl ester. Ethyl chlorophyllide a , 66 mg, was added to 1.0 g of KOH dissolved in 20 ml of methanol, which previously had been boiled to expel dissolved oxygen. After refluxing for 1 hr, and acidification with 1 N HCI while cooling in an ice bath, the mixture was diluted with water and extracted with ether. The extract was washed several times with water and saturated NaC1, and dried over anhydrous sodium sulfate. The visible spectrum agreed with that reported by Fischer (1935) for chlorin e,. The ethereal solution of dye was then treated with diazomethane. After this treatment the material was not extractable with 0.01 N KOH. After evaporation of the ether, the residue was washed with petroleum ether and collected by filtration. Re- crystallization from acetone-methanol produced dull green, stout needles, 32 mg, m.p. 21 1-212°C (soften 105"C), reported (Woodward et al., 1960) m.p. 207.5-208-5°C.

Magnesium was introduced by adding methyl magnesium iodide to a solution of chlorin e, ester in anhydrous ether. A green precipitate immediately formed and was decomposed by adding a saturated solution of ammonium chloride. The ethereal solution was washed, dried, and the ether evaporated. The absorption maxima shifted from 665 and 399 nm to 645 and 418 nm with a 4181645 nm ratio of 2.80 and a 6451502 nm ratio of 24, compared to 2-82 and 4, respectively, for the compound before insertion of Mg.

Meso-ethyl chlorophyllide a. Ethyl chlorophyllide a, 50 mg, was dissolved in 20 ml of acetone with a small amount of 10% palladium on charcoal. Hydrogenation under atmospheric pressure was carried out for 30 min with stirring. The color of the solution changed from greenish blue to deep blue early in the reaction. The reaction mixture was filtered, diluted with water, and extracted with ether, which was washed, dried and evaporated. On washing with petroleum ether, brilliant blue, lustrous plates appeared, which melted at about 170"C, followed by resolidification. The material was used for irradiation without recrystallization. The visible absorption spectrum agreed with that reported by Fischer (1933) with maxima at 650 and 426 nm in ether and a 426/650 nm ratio of 1-14 compared to a 405/657 nm ratio of 2.0 without magnesium.

5 0 2 G . SHERMAN, T. M. KORN and H. LINSCHITZ

10-Hydroxy-chlorophyll a. A solution of 80 mg of chlorophyll a in 80 rnl of 0.1 M ethanolic (anhydrous) ammonium acetate was allowed to stand in the dark at room temperature for 2 hr. After chromatography on sucrose, 66 mg of lustrous blue material was obtained. The visible absorption spectrum agreed with those reported by Holt (1958) Gilman (1956) and Pennington et al. (1967). The alkoxyl content for IO-hydroxy-chlorophyll a was found to be 3.41% (calc., 3.41%).

9-Deoxo-9-hydroxychlorophyllide a was prepared by reducing an ethereal solution of ethyl chlorophyllide with sodium borohydride. The product was purified by sugar chromatography and had absorption maxima at 412 and 635 nm in ether with a 41 21635 nm ratio of 3.42.

The preparation of chlorophyll d and dihydroxy ethyl chlorophyllide a have been previously described (Sherman and Wang, 1966).

Bacteriochlorophyll was kindly given to us from a preparation made by Holt and Jacobs.

RESULTS A N D DISCUSSION Spectra in EPA

Photochemical reactions. In undegassed EPA at low temperature (- 175" & 5°C) chlorophyll a shows three well-defined stages of phototransformation caused by succes- sive irradiations, as shown in Figs. 1-3.

Stage I results from illumination with red light in the main long-wave chlorophyll band, until no further spectral change occurs. At this point, the reaction is marked by the disappearance of the original red band at 669 nm and development of a strong new band at 620 nm (Fig. 1 , spectrum 3). The Soret region is also transformed into a sharp long-wave band at 442 nm, at slightly higher energy than the original Soret position, and a somewhat more intense broad band with two closely spaced peaks at 407 and 4 18

1 0 , I I I I I I I I

0 9

0 8

0 7

-

0 4 0

03

0 2

01

0 350 400 450 500 550 600 650 700 750

Wovelength. nm

Fig. 1. Conversion of chlorophyll a to Stage I ('620') photoproduct, by low-temperature illumination in EPA solvent. Spectrum 1 (solid line)-Chl a at -180°C, before irradiation. Spectrum 2 (dashed line)-after 1.5 min illumination with red light (A > 640nm), at -180°C. Spectrum

3 (dotted line)-after 10 min further illumination with red light; final temperature - 168°C.

Low-temperature photooxidation of chlorophyll-a 503

07 O8 r, 2 0 6

0 05 -

0 4 0

03

0 2

01

0 350 400 450 500 550 600 650 700 750

Wavelength, nm

Fig. 2. Photoconversion of Stage I + Stage I1 in EPA, by low-intensity irradiation at low temperature. Spectrum 1 -Stage I photoproduct (‘620’), prepared as above, at -185°C. Spec- trum 2-after weak white light illuimination for 10 sec. Spectrum 3-after weak white light il-

luminatioa for 25 sec. Spectrum 4-after weak white light illumination for 55 sec.

nm. The almost total disappearance of the original chlorophyll red band establishes that the sharp 442 nm Soret peak cannot be assigned to incomplete transformation.

The formation of Stage I itself :seems to occur in more than one step. In passing from the starting spectrum 1 to the final spectrum 3 (Fig. l), we note that isosbestic points are not formed. Indeed at some wavelengths the direction of absorbance change is re- versed during the irradiation. Thus, the initial rise at 520 or 360 nm (1 + 2) is followed by bleaching at these wavelengths. The possibility that spectrum 3 (Fig. 1) corresponds to at least two absorbing species is supported by the sharp separation between the long-wave Soret band at 442 nm and the remaining absorption in this region, between 400 and 430 nm.

0.7 k I

0 3

0.2

01

0 350 400 450 500 550 600 650 700 750

Wavelength, nm

Fig. 3. Formation of Stage 111 in EIPA. Spectrum 1 (solid line)-Stage I photoproduct (‘620’) at - 175°C. Spectrum 2 (dashed line)-after strong irradiation with white light for IS min; final temperature - - 160°C. Spectrum 2 contains both Stage III (absorbing around 500 nm) and

Stage 11 material.

SO4 G . SHERMAN, T. M. KORN and H. LINSCHITZ

If the Stage I reaction product is irradiated at low temperature with 620 nm or weak white light, the transformation shown in Fig. 2 (spectrum 1 -+ 4) occurs very rapidly. A strong red band develops at 677 nm while the bands at 620 and 570 nm disappear, leav- ing a weaker residue at 6 18 nm. The sharp Soret peak at 442 nm moves up, to become a long-wave shoulder (435 nm) on the broad double-peaked Soret band of Stage I, which is essentially retained in Stage 11. The reaction I -+ I1 is characterized by excellent isosbestic points (Fig. 2), indicating the photoconversion of only a single absorbing species.

Further low-temperature irradiation of Stage I1 with intense white light gives rise to a remarkable multi-peaked absorption in the 450-550 nm region (Stage 111, Figs. 3 and 5). If the cold chlorophyll solution is exposed from the beginning to strong unfiltered white light, Stage I can hardly be observed because of immediate transformation to 11 and 111.

Thermal reactions, Stage I -+ Stage I I . The red peak of Stage I1 shifts reversibly from 677 nm at -170°C to 666 nm at room temperature. This same 666 nm band is formed from Stage I simply by allowing the solution to warm in the dark and stand at room temperature (Fig. 4).

When Stage I11 solutions are warmed to room temperature, a new spectrum similar to that of Stage I1 is formed but with much loss of intensity and irreversible side reactions.

Spectra in E-Py In E-Py (ether-pyridine, 6 : 1) solvent, the general pattern of photochemical and

thermal transformations is similar to that seen in EPA, except that the orange product of Stage I11 is formed much more easily and completely. Thus, a cleaner and more accurate spectrum of this substance can be obtained (Fig. 5). The intermediates of Stages I and I1 in E-Py have low-temperature longwave maxima at 623 and 681 nm,

0 7 O 8 3 0 6

D 0 5 -

0 4 0

0 3

0 2

01

0 350 400 450 500 550 600 650 700 7 5 0

W o v e l e n g i h , nm

Fig. 4. Thermal conversion in EPA; Stage I + Stage I1 and Stage I1 a ‘378’. Spectrum 1 (solid line)- Stage I photoproduct, at - 173°C prepared as in Fig. 1 . Spectrum 2 (dashed line)- above solution, after slow warming to room temperature and standing in the dark for 10 hr ( I 4 11). Spectrum 3 (dash-dot line)-above solution, after exposure to room light for 2 min (completion of formation of 11). Spectrum 4 (dotted line)-solution of 3 after treatment with

2 N HCl (Stage I1 a ‘378’).

Low-temperature photooxidation of chlorophyll-u 505

O 7 t I

350 400 4 5 0 500 550 600 650 700 750

Wavelength, nm

Fig. 5 . Photoconversions in E-Py solvent: Stage I + Stage I1 -+ Stage 111. Spectrum I (solid line)-Stage I photoproduct ('620') at - 162"C, formed from ChI n by low temperature irradiation with red light. Spectrum 2 (dashed line)-after 2min irradiation of above solution with weak white light; temperature -174°C (Stage 1 -+ Stage 11). Spectrum 3 (dotted line)-after further I 2 min irradiation with intense white light; temperature - 174°C (Stage I1 + Stage 111).

compared with 620 and 677 nm, respectively, in EPA. The room-temperature spectrum of the Stage I1 product in E-Py is, also similar but not identical with that in EPA (Figs. 6 and 7). Figure 6 shows that good isosbestics are found for the thermal Stage I -+ Stage I1 conversion as well as the corresponding photochemical process (Fig. 2).

Conditions necessary for reaction Oxygen. The photoreactions described above do not occur in carefully degassed

solvent. While a quantitative study of the effect of oxygen concentration has not been made, qualitative experiments indicate that the rate remains high even when a major fraction of the oxygen has been removed by bubbling with inert gas.

0 6 t 0.5

350 400 4 5 0 500 550 600 6 5 0 700 750

Wavelength. nm

Fig. 6. Thermal conversion of Stage I + Stage 11, in E-Py. Spectrum 1 -Stage I photoproduct ('620') at -17O"C, formed by low-temperature irradiation of Chl u with red light. Spectrum 5 - above solution, after allowing to warm to room temperature over 6.5 hr period (Stage I - I I) .

Intermediate spectra (2-4) were taken at intervals during warm-up period.

506 G. SHERMAN, T. M. KORN and H. LINSCHITZ

350 400 450 500 550 600 650 700 750

Wavelength, n m

Fig. 7. Room temperature spectra of Chl a, Stage I1 and ‘378’ products, in E-Py (from same ex- periment shown in Fig. 6). Spectrum 1 (solid line)-Initial Chl a solution. Spectrum 2 (dashed line)- Final spectrum, after exposing solution of Fig. 6, spectrum 5, to room light (completion of I 4 I1 reaction). Spectrum 3 (dotted line)-after t:eatment with 2 N HCI in absorption cell

(Stage I1 378).

Solvent. No reaction occurs in ether-isopentane solvent. Addition of small quanti- ties of ethanol or pyridine permit the reaction to take place.

Structure of reactant. Chlorophyll derivatives in which the vinyl group unsaturation is removed by hydrogenation (ethyl meso-chlorophyllide a), hydroxylation (ethyl 2- desvinyl-2 ( 172-dihydroxy ethyl) chlorophyllide a) or group replacement ethyl 2- desvinyl-2-formyl chlorophyllide a) do not react.

Removal of magnesium (ethyl pheophorbide a) also prevents the reaction, but sub- stitution of zinc for magnesium (zinc pheophytin) gives a good Stage I intermediate with peak at 6 1 1 nm.

Derivatives in which the cyclopentanone ring is modified (ethyl 9-deoxo-9-hydroxy chlorophyllide a) or destroyed (Mg chlorin e6 trimethyl ester, allomerized chlorophyll) react, not only to Stage I, but also to form the orange intermediate of Stage 111 upon further illumination. In addition, chlorophyll b and bacteriochlorophyll also react. It is noteworthy that, in the case of Zn pheophytin and 9-hydroxy chlorophyllide, warming the ‘Stage I’ reaction product appears to regenerate starting material, rather than material showing new chlorin-like spectra.

Attempted isoiation and characterization of product: acid treatment Among the three photoproducts discussed here, that of Stage 11, ‘677’, is the only

one which can be handled at room temperature. Attempts to chromatograph such material, obtained from preparative low-temperature EPA photooxidation of ethyl chlorophyllide, were not successful, owing to its instability in solution and its high pol- arity. However, treatment with 2 N HC1 gave a magnesium-free product (‘378’) which was more stable and which could be separated on a column. The absorption spectrum of the main chromatographic fraction thus obtained is identical with that of the acid- treated material shown in Figs. 4 and 7. The Soret region is characteristically complex, with a main band (in ether) at 378 nm, and broader, lower peaks at 398 and 419 nm. The main red peak is at 65 1 nm. The constancy of this spectrum under repeated chrom-


atographic treatment, using a variety of columns and developers, convinces us that the ‘378’ substance is pure, despite its, unusual spectrum.

The spectrum of ‘378’ is remarkable in a further respect, in that it exhibits none of the marked green absorption expected for a pheophytin. Indeed, the spectrum in the red and green corresponds to a Mgcontaining chlorin. To be certain of this point, the pigment was dissolved in concentrated HCI, diluted with water, extracted back into ether, and pyridine then added to excess. The anomalous spectrum remained un- changed after this treatment. We #conclude that, despite its spectrum, ‘378’ cannot be a hydrochloride salt containing magnesium.

Solutions of ‘378’ slowly decompose on standing to yield products absorbing at 690 nm and 6 18-620 nm, respectively. Separated chromatographically, both the visible and the i.r. spectra of the 690nm compound are identical with those of authentic ethyl pheophorbide d (Sherman and Wang, 1966). The visible spectrum of pheophorbide d is quite unique and could hardly be assigned reasonably to any other compound.

The ‘378’ compound gives a positive phase test, and its i.r. spectrum and conversion to pheophorbide d indicate further that it contains an intact cyclopentanone ring. Reduc- tion of the ‘378’ compound with zinc-acetic acid gives a product with a typical zinc chlorin spectrum. When the zinc is removed with mineral acid, a normal pheophytin spectrum is obtained, with a marked green peak.

Nature of the reaction Characterization of the reaction intermediates described here evidently requires

determination of the chemical structure of the final stable reaction product, a task still incomplete. However, some conclusions may be drawn from the data thus far obtained. The failure of any compound lacking the vinyl group to undergo reaction, and the formation of pheophorbide d from the final ‘378’ product, require that the vinyl group be the site (or a site), of oxidation. In agreement with this, the i.r. spectrum of ‘378’ shows a hydroxyl and a new carbonyl function.

We suspect, further, that ‘378’ may actually be a dimeric structure, formed from two chlorophyll molecules. The peculiar absorption spectrum, with three peaks in the Soret region and an intense, quite broad, red band suggests two different absorbing cen- ters in the molecule, of which at least one is not a porphyrin. In addition, we note the lack of isosbestic points in the initial photoconversion to the 620 nm spectrum (Stage I), and their presence in the photo or thermal reaction, I + 11. The absence of a normal pheophytin spectrum in ‘378’ may indicate some type of intermolecular interaction at the center of the metal-free ring system, which may arise from a dimeric structure. It is of interest to note that the spectrum of Stage I1 suggests a superposition of chlorophyllide d and pheophorbide d (Sherman and Wang, 1966).

The primary reaction with oxygen presumably leads to a peroxide, either through direct interaction of oxygen and e:xcited chlorophyll, or via singlet oxygen, which may be formed at the reacting molecule or at a neighboring excited chlorophyll (self-sensiti- zation). The low-temperature requirement may indicate the participation of a ground- state chlorophyll-oxygen complex (the reaction rate is not changed by removal of considerable oxygen) as well as stabilization of an initial labile peroxide intermediate (Schenck and Dunlap, 1956; Dufraisse et al., 1967). In this connection, however, it is observed that lowering the temperature to - 190°C (liquid nitrogen) prevents the Stage I reaction. EPA solvent undergoes a marked increase in rigidity between -180” and

508 G. SHERMAN, T. M. KORN and H. LINSCHITZ

- 190°C. This would argue against high concentration of ground-state photoreactive dye-oxygen complex.

The requirement for ring-complexed metal and presence of ‘activators’ (Lewis bases) is also found in other chlorophyll photo-redox reactions (Korn, 1955; Seely, 1966b). It is remarkable, however, that the reaction dealt with here involves a peripheral (vinyl) group and not the main conjugated ring structure.

In connection with the structure of the presumed initial peroxide, we emphasize that the spectrum of our ‘620’ photoproduct is strikingly similar to that of so-called ‘hypochlorophyll’, produced by room-temperature irradiation of chlorophyll in ascorbic acid- amine solutions (Seely, 1966b). Both photoproducts show bands near 620nm and a triple-peaked Soret region with one peak sharply set off on the long wave side. The two reactions resemble each other also in their requirement for Mg (or Zn), the intact vinyl group and basic activators, and their indifference to the presence of the cycfopentanone ring. Seely has given evidence that ‘hypochlorophyll’ is 1,2-dihydromesochlorophyll (although mesochlorophyll does not undergo the hypochlorophyll reaction). This would suggest that the ‘620’ photoproduct should be formulated either as a Chl 1,2 dioxetane or as a cyclic peroxide resulting from 1,4 addition of oxygen between C-1 of chlorophyll and the vinyl group (Korn, 1955). Structures in which the chlorin ring con- jugation is broken (Barrett, 1967; Fuhrhop and Mauzerall, 1971) are quite unlikely in view of the absence of photoproduct absorption beyond 700nm, as well as the appearance of pheophorbide d as an ultimate oxidation product. Moreover, the Stage I absorption lies too high (620nm) to be consistent with dioxetane formation at the vinyl double bond. A Chl 1,2 dioxetane structure also encounters difficulties in explaining the need for intact vinyl and the mode of conversion of Stage I to the chlorin of Stage 11. It is thus most reasonable, and consistent with singlet oxygen chemistry (Gollnick and Schenk, 1%7), to assume 1,4-peroxidation as the initial step (Korn, 1955). A similar initial reaction has been proposed in the photooxidation of protoporphyrin IX dimethyl ester (Inhoffen et al., 1969).

The results of Fig. 1 suggest that the primary 1,4 photoperoxide, A, next undergoes at least one subsequent reaction, which is rapid even at low temperature and of such

O* CH

\ C A

\ \ 0-CH, I I

D


type as to retain the 620 nm absorption band. This could conceivably involve a second chlorophyll molecule, as indicated above, or may be an internal bond rearrangement. Two such possibilities, for example, are peroxide cleavage with p-elimination of hydrogen (Wasserman, 1970), giving B, and diepoxide formation (Wasserman, 1970; Boche and Runquist, 1968), as in C. Conversion of these intermediates to Stage I1 would then have to occur by as yet uncertain transfer reactions, perhaps oxidizing the side chain further (to hydroxyl and carbonyl functions, as observed) and regenerating a chlorin structure. Structure B corresponds to the product of the room temperature photooxidation of protoporphyrin IX dimethyl ester, which has been shown to undergo acid-catalyzed conversion back to a porphyrin (Inhoffen er al., 1969). Another simple possibility for the Stage I -+ Stage I1 reaction would be transformation of the 1,4-peroxide to a vinyl dioxetane (Wassennan, 1970) with chlorin structure, D, whose cleavage would lead either to hydroxy carbonyl compounds or to the 2-formyl group of pheophorbide d (Kopecky and Mumford, 1969).

The orange intermediate of Stage I11 remains to be identified. Its stabilization by basic media (E-Py) and reversion to a chlorin spectrum on warming in the dark suggest that it may possibly be an anion, resulting from loss of a proton from an acidic excited state of the oxidized (Stage 11) chlorin.

REFERENCES Barrett, J. (1967) Nature 215,733. Boche, J. and 0. Runquist (1968)J. Org. Chem. 33,4285. Dilung, I. I. and V. E. Karpitskaya (1963) P'roc. Acad. Sci., USSR, 367. Dufraisse, C., G. Rio and A. Ranjon (1967) Compr. Rendu 264,516. Fischer, H. (1933)Ann. 506, 146. Fischer,H.(1935)Z.physikChem. (Leipzi,q)A 174,81. Fuhrhop, J. H. and D. Mauzerall(1971) Ph,orochem. Phorobiol. 13,453. Gilman, S . (1956) Thesis, Syracuse University, Syracuse, New York. Gollnick, K. and G. 0. Schenck (1967) In 1,l-Cycloaddition Reactions (Edited by J. Hamer). Academic

Holt, A. S. (1958) Can. J . Biochern. Physiol'. 36,439. Holt, A. S. and E. E. Jacobs (1954) Can. J. Bot. 41,710. Inhoffen, H. H., H. Brockmann and K. Bliesener (1969)Ann. 730. Jacobs, E. E., A. E. Vatter and A. S. Holt (1954)Arch. Biochem. Biophys. 53,228. Kopecky, K. and C. Mumford (1969) Canad. J. Chem. 47,709. Kom, T. M. (1955) Ph.D. thesis, Syracuse University, Syracuse, New York. Linschitz, H. and J. Rennert (1952) Nature 169, 193. Livingston, R. L. and K. E. Owens (1956)J. Am. Chem. Soc. 78,3305. Pennington, F. C., H. H. Strain, W. A. Svec and J. J. Katz (1967)J. Am. Chem. Soc. 89,3875. Schenck, G. 0. and D. E. Dunlap (1956)Angew. Chemie 68,248. Seely, G . R. (1966a) In The Chlorophylls (Edited by L. P. Vernon and G. R. Seely) Chapter 17 and

Seely, G. R. (1 966b) J. Am. Chem. Soc. 88,34 17. Sherman, G. and S. F. Wang (1966)J. Org. Chem. 31,1465. Wasserman, H. H. (1970)Ann. N . Y.Acad. Sci. 171, 108. Woodward, R. B., et al. (1960)J.Am. Chenz. Soc. 82,3800.

Press, N.Y.

references therein. Academic Press, New York.

low-temperature photcoxidation of chlorophyll-a

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