Fluorescence Assay for Tolyporphins Amidst Abundant Chlorophyll in Crude Cyanobacterial Extracts

Abstract

Tolyporphins are distinctive tetrapyrrole natural products found singularly in a filamentous cyanobacterial‐microbial holobiont (termed HT‐58‐2) from Micronesia. The absorption and fluorescence features of tolyporphins resemble those of chlorophyll a, complicating direct analysis of culture samples. Treatment of the crude (unfractionated) organic extract (CH2Cl2/2‐propanol, 1:1) of HT‐58‐2 cultures with NaBH4 in methanol causes reduction of the peripheral ketone auxochromes, whereupon tolyporphins (predominantly 7,17‐dioxobacteriochlorins) exhibit a bathochromic shift (λabs ˜ 676 → ˜ 700 nm) and chlorophyll a (a 131‐oxochlorin) exhibits a hypsochromic shift (λabs 665 → 634 nm). Fluorescence excitation spectroscopy (at 368 and 491 nm with λem 710 nm) enabled detection of reduced tolyporphins amidst abundant reduced chlorophyll a (1:19 ratio), a detection sensitivity >5 times that without reduction. The resulting assay combines simple sample preparation from non‐axenic cultures at microscale quantities (2 mL, 2 μm), absence of any fractionation procedures, and fluorescence detection. Tolyporphins were readily detected in cultures of HT‐58‐2 at reasonable growth periods in the absence of environmental stressors, which was not possible previously.

[3 + 2]-Cycloadditions with Porphyrin β,β′-Bonds: Theoretical Basis of the Counterintuitive meso-Aryl Group Influence on the Rates of Reaction

“…While chlorins and bacteriochlorins may be extracted from natural sources, synthetic hydroporphyrins are most commonly studied; these can be made through total synthesis from pyrrolic precursors. − An alternative approach is the modification of a fully synthetic porphyrin; many reactions that accomplish the removal of one or two pseudo-olefinic double bonds from conjugation are known. ,, Among them are reduction, oxidation, or cycloaddition reactions (though it should be noted that a formally strict distinction between these three reactions cannot always be made, as the examples below will illustrate). For example, the β,β′-bond of porphyrins and chlorins can be dienophiles in Diels-Alder reactions or they can undergo oxidation/cycloaddition reactions with osmium tetroxide, ruthenium tetroxide, permanganate, or, in the case of octaalkylporphyrins, ozone.…”

Stepwise Reduction of β-Trioxopyrrocorphins: Collapse of the Oxo-Induced Macrocycle Aromaticity

“…The regioselective reduction of one of the two ketone functionalities of the dioxo chromophores 6-O and 7-O to the corresponding alcohols with NaBH 4 was previously shown to be possible . Likewise, we find now that the reduction of the pyrrocorphin trioxoisomers 8-O 2,7,12 and 8-O 2,7,18 with NaBH 4 over the course of several hours generates the monoreduced products 9-O 2,12 (OH) 7 - rac and 9-O 7,18 (OH) 2 - rac , respectively (Scheme ).…”

Synthesis of bacteriochlorins bearing diverse β-substituents

“…We recently developed a sensitive fluorescence assay for the detection of dioxobacteriochlorin-type tolyporphins in the presence of chlorophyll a by reduction of the respective ketones. 81 In so doing, the Q y band of the tolyporphins shifted bathochromically whereas that of chlorophyll a shifted hypsochromically; the latter shift stems from the removal of a potent auxochrome (the 13-ketone) aligned along the y -axis. A model system for the development of the assay was provided by the synthetic dioxobacteriochlorin prepared as shown in Scheme 9, given the known sensitivity of the methylene unit flanking the gem -dimethyl group.…”

“…The resulting shift has been employed in a fluorescence assay for naturally occurring dioxobacteriochlorins. 81…”

“…The resulting shift has been employed in a fluorescence assay for naturally occurring dioxobacteriochlorins. 81 In summary, the ability to tailor the bacteriochlorin by condensation of diverse dihydrodipyrrin-acetals or by derivatization of bacteriochlorins provides flexibility in pursuit of target molecular designs. The present paper concerns pairwise installation of substituents at the b-pyrrole or b-pyrroline sites.…”