The Chlorophyll b Biosynthetic Pathway: Novel Metabolic Intermediates - Chlorophyll Biosynthesis and Technological Applications - page 277

12.2.2 Rebuttal of Above Claims
The absorbance properties of the tetrapyrrole synthesized by Schoch et al. ( 1995 ),
were assigned to Pchlide b on the basis of (a) their similarity to the absorbance
properties of Chl c 3 reported by Jeffrey and Wright ( 1987 ), and (b) the structural
similarities of MV Pchlide b and Chl c 3 both of which contain a porphin macrocycle
having a double bond at position 7-8 of the macrocycle and a formyl group at
position 3. In this comparison the authors overlooked the fact that in addition to the
rhodofying formyl group at position 3 (a rhodofying group increases the absorbance
intensity of band III, in porphins and band II in phorbins), Chl c 3 also contains a
second rhodofying group (Propyl acrylic group) at position 7 of the porphin
macrocycle (Jeffrey and Wright 1987 ) which is not present in Pchlide b. Instead
the latter contains an etio propyl group at that position. The authors obviously
overlooked the well-known spectroscopic fact that two rhodofying groups on
diagonally opposite pyrrole rings as occurs in Chl c 3, enhance each other
rhodofying effect (Falk 1964 ) and result in the quasi-oxorhodo type spectrum
observed for Chl c 3 . In an oxorhodo spectrum, the ratio of the absorbance intensities
of bands II:I increases significantly (Falk 1964 ). For example in diethyl ether at
room temperature, the ratio of bands II:I in Pchlide a which lacks a rhodofying
group at position 7, is 0.54, it is 1.17 in Chl c 2 which has a rhodofying group at
position 7, and 3.79 in Chl c 3 which has two rhodofying groups on diagonally
opposite pyrrole rings . The rhodofying effects of the formyl group at position 3 of
the macrocycle is usually insignificant as observed for Chl b in comparison to Chl a.
In our opinion the spectral properties assigned by Schoch et al. to their synthetic
putative Pchlide b correspond very closely to the spectral properties of a
pheoporphyrin, that is to a metal-free Pchlide, also referred to as protopheophytin
(Houssier and Sauer 1969 ). For example the ratio of bands II:I in protopheophytin
a in diethyl ether is 1,67; it is 1.57 in the putative Pchlide b spectrum reported by
Schoch et al. in acetone at room temperature (Schoch et al. 1995 ). Loss of Mg may
have occurred during the preparation of Pchlide b in the presence of excess
2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) at room temperature, instead of
under the milder DDQ oxidation conditions (4 C), recommended by Shedbalkar
et al. ( 1991 ).
As for the mass spectroscopic data reported by Shedbalkar et al., it corresponds
to that of a tetrapyrrole axially coordinated to one molecule of methanol the solvent
in which the tetrapyrrole was dissolved prior to FAB mass spectroscopic analysis.
Axial coordination to lewis bases such as methanol is a very well documented
phenomenon particularly in the b tetrapyrrole series (Belanger and Rebeiz 1984 ;
Rebeiz and Belanger 1984 ). As for the failure to detect the 7-8 double bond by mass
spectroscopy it was due to the liability of that bond in Pchlide b , as discussed in
(Shedbalkar et al. 1991 ). Furthermore, Schoch et al. ( 1995 ) completely ignored the
extensive and detailed absorbance, fluorescence and NMR evidence that led to the
assignment of a Pchlide b structure to the synthetic compound by Shedbalkar
et al. ( 1991 ).