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of a complex of water in benzonitrile (17) are listed in the same table. Also included are the .... nuclear magnetic resonance spectroscopy of chlorophyll. In The.

The influence of water on the spectroscopic properties of a liposoluble magnesium porphyrin CAMILLE CHAPADOS' Centre de recherche en photobiophysique. Universite' du Que'bec a Trois-RiviPres, Trois-RiviPres (Que'.), Canada G9A 5 H 7 AND

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DENISGIRARD A N D MICHELRINGUET De'pnrtement de chimie-biologic, Universite' du Que'bec a Trois-Rivieres, Trois-Rivieres (Qut.), Cnnnda G9A 5H7

Received August 7, 1987 CAMILLE CHAPADOS, DENISGIRARD, and MICHELRINGUET. Can. J. Chem. 66, 273 (1988). Complexation of water by chlorophyll molecules in natural photosynthetic processes is a matter of great interest. In the present work the influence of water on a model porphyrin, magnesium 2,7,12,17-tetrahexyl-3,8,13,18-tetramethyl porphin (MgTHTMP) has been studied by infrared spectroscopy. A CC14(spectroscopicgrade) solution of MgTHTMP showed two small bands in the 3600 cm-' infrared region. These bands are assigned to v, and v , vibrations of water coordinated to the Mg atom. For MgTHTMP in the solid state, either deposited on an infrared plate or suspended in a Nujol film, none of the OH bands is observed. An elemental analysis confirmed that the solid porphyrin was anhydrous. This evidence indicates that while no water is complexed with MgTHTMP in the solid state, this porphyrin is hlghly hygroscopic in a CCI, solution and will easily complex with the few water molecules (less than 0.0190) present in the spectro grade solvent by forming a coordinate bond between the central magnesium atom and the oxygen of water. Several other metallic porphyrins in CCI4 solutions have been studied for comparison with the Mg porphyrin. The spectra of these substances dld not show any complexed water. A model of the H20-MgTHTMP interaction is given and the implication of this model on the aggregation states of chlorophyll will be discussed. DENISGIRARD et MICHEL RINGUET. Can. J. Chem. 66, 273 (1988). CAMILLE CHAPADOS, Le complexation de I'eau par des molCcules de chlorophy lle, dans des processus naturels, est une question fondamentale. Dans le present travail, on a Ctudit l'influence de l'eau sur une porphyrine modele, la tttrahexyl-2,7,12,17 tCtramtthy1-3,8,13,18 porphine - magnksium (THTMPMg),en faisant appel i la spectroscopie infrarouge. Une solution de THTMPMg dans du CC14 (qualitt spectroscopique)prCsente deux petites bandes dans la rtgion de 3600 cm-' de l'infrarouge. On a attribut ces bandes aux vibrations v j et v , de l'eau coordonnte a l'atome de Mg. Dans le cas du THTMPMg a 1'Ctat solide, soit dtpost sur une plaque infrarouge soit suspendu dans le Nujol, on n'observe aucune de ces bandes. Une analyse tltmentaire a permis de confirmer que la porphyrine solide est anhydre. Ces donntes indiquent que, m&mes'il n'y a pas d'eau complexCe avec la THTMPMg a l'ttat solide, cette porphyrine en solution dans le CC14est tellement hygroscopique qu'elle se complexe facilement avec les quelques moltcules d'eau prtsentes (moins que 0,01%) dans le solvant de qualit6 spectroscopiqueen formant une liaison de coordinence entre l'atome de magntsium central et l'oxygkne de l'eau. Pour fins de comparaison avec la porphyrine de Mg, on a aussi ttudiC plusieurs autres porphyrines mCtalliques en solution dans le CCl,. Les spectres de ces substances ne prCsentent pas de bandes correspondant a de l'eau complexke. On prtsente un modkle de l'interaction HrO-THTMPMg et on discute des implications de ce modkle sur les ttats d'agrtgation de la chlorophylle. [Traduit par la revue]

1. Introduction In algae and higher plants, water is being oxidized by photosynthetic process to produce oxygen at the reaction center of photosystem 2. Some chlorophyll a (Chl a ) molecules are present in this reaction center as well as in the reaction center of photosystem 1 and in the antenna chlorophyll. These Chl a molecules are situated in the chloroplast where water is abundant. The aggregation state of chlorophyll within these systems is known to vary. The influence of water on these aggregation states is not fully understood because water can make several types of bonding with the chlorophyll at different sites: i.e. with the Mg atom, and with the carbonyl groups. No experimental technique has given a definitive answer for the structures of these organizations. Infrared spectroscopy has been used in several occasions to study the influence of water on the chlorophyll molecules (1-8). Typical ir spectra of some chlorophylls and some Chl derivatives have been obtained by Katz et al. (1). Most of the spectra taken in C C 4 solutions showed a broad Teatureless band in the 3400cm-' ir region. This band has been assigned to the OH stretch band of bonded water. Efforts to remove the water from the chlorophyll samples were mostly unsuccessful. 'TO whom correspondence may be addressed.

An infrared study of chlorophyll-chlorophyll and chlorophyll-water interactions in organic solutions (2) has shown in the OH stretch region a complex set of bands comprising some sharp bands in the 3700 cm-' region and a broad band with unresolved features in the 3300 cm-' region. An effort to dry the solutions did not remove those bands entirely. Pheophytin a in CC14 showed some sharp bands at 3397, 3538, 3622, and 3690 cm-'. On exchange with D 2 0 , the first two bands were displaced to lower frequencies and the last two were replaced by one at 3665 cm-'. The 3538 cm-' band was assigned to enolic chlorophyll a . Solutions of chlorophyll made up in undried solvents showed a broad, medium intensity band centered at 3400 cm-' (3). This absorption maximum appears to include the bands of free water dissolved in the solvent ( v , at 3620 cm-' and v3 ;t 3710 cm-'), of water coordinated to Mg at 3380 and 3665 cm- , and of water hydrogen bonded to the coordinated water molecule. Codistillation with dry solvents removes water to the point that no appreciable absorption can be detected in the OH stretch region although the spectrum is not coincident with the base line. When a solution of Chl a hydrate in dry CC14 was studied by ir spectroscopy (4), the spectrum contained several peaks assigned to the symmetric (3620 cm-') and anti-symmetric

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(3715 cm-I) stretch of free water, to OH stretch of water coordinated to the Mg atom of Chl (3665 cm-I), and to hydrogen bonded water (3380 cm-I). Two shoulders at 3300 and 3440 cm-' are assigned to carbonyl overtones. Dry Chl a in dry CCl, showed three small bands (3280, 3365, and 3450 cm-I) which are assigned to either carbonyl overtones following Ballschmiter and Katz (2) or to residual hydrogen bonded water (less than 3% mol relative to Chl). Solid samples of anhydrous and hydrated chlorophyll species showed a broad, mostly featureless band in the 3350 cm-' region (5). Depending on the hydration conditions, the band is more or less intense and the position of the maxima is slightly different for each aggregation species. Microcrystalline Chl a hydrate deposited on a germanium ATR plate showed in the infrared a large structureless band centered 3 150 cm- indicative of bonded water molecules (unpublished results). To remove water, the sample is placed in a high vacuum of lop7Torr for more than 72 h. This treatment gave an amorphous dried species that showed no bands in the OH region (6). The dryness of the sample was further substantiated by a test with SO2 vapor that showed no reaction (unpublished results), whereas, in the presence of water, Chl a reacts with SO2 to give pheophytin a (7). The influence of water on mono- and multilayers of Chl a obtained by the Langmuir-Blodgett technique have been reported previously (7, 8). A fresh multilayer kept in a moist atmosphere showed in the infrared a broad band centered at 3400 cm-' which disappears rapidly under a vacuum of lop3 Torr (7). A fresh monolayer deposited on a germanium ATR plate and placed in the dry sample compartment of the spectrometer showed no band in the OH stretch region (8). The only study of the interaction of water with another metallic porphyrin is an X-ray diffraction study on a magnesium tetraphenylporphyrin (MgTPP) made by Tirnkovich and Tulinsky (9). The presence of water was confirmed by density measurements carried out by flotation in aqueous silver nitrate solution. The lack of other studies is probably due to the insolubility of most porphyrin molecules in ir organic solvents. In order to understand the interaction of water with the Mg atom in a porphyrin environment, we studied by ir spectroscopy a model molecule, a synthetic liposoluble magnesium porphyrin, MgTHTMP (Fig. I), in which the absence of other polar functional groups would limit the interactions of water to the Mg atom site and to the .rr electron system of the chromophore. This work was intended to verify the assignments reviewed above and to complement our knowledge of H20-Mg interactions in chlorophyll. Several samples were made of MgTHTMP (Fig. 1): a suspension in Nujol, several solutions in CCl, of different dryness conditions, a solution in a mixture of pyridine and CCl,, and a solid sample exposed to different moisture conditions. For comparison, we have obtained the ir spectra of several other metallic porphyrin compounds. The results of these experiments will be given and a model of the interaction between water and MgTHTMP will be presented.

MgTHTMP

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2. Experimental 2 . 1 . Synthesis The synthesis of THTMP was made following the method used by Smith and Bisset for the synthesis of tetrapentyltetramethylporphin (10). The synthesis of ZnTHTMP was made by incorporating zinc in THTMP by the acetate method (1 1). The MgTHTMP compound was obtained by the reaction of propioxymagnesium bromide on THTMP

FIG. 1. Molecular model of MgTHTMP. at low heating temperature (70-75°C) (12). All manipulations of this compound have been done under red light because solutions of MgTHTMP are sensitive to white light. The samples were dried in a vacuum desiccator over CaC12 before recording the spectra. Elemental analyses were made by Guelph Chemical Lab., Guelph, Ont. and showed that these substances were pure and contained no water. Anal. calcd. for C48H68N4Zn(ZnTHTMP): C 75.54, H 9.05, N 7.03; found: C 75.22, H 8.94, N 7.31. Anal. calcd. for C48H68N4Mg (MgTHTMP): C 79.75, H 9.60, N 7.68; found: C 79.48, H 9.4% N 7.72. 2.2 Spectrum recording The CC14 solutions of the porphyrin compounds were made using spectro grade solvent (Anachemia Chemicals Ltd.; water