Vanampally Chandrashaker

Abiotic formation of uroporphyrinogen and coproporphyrinogen from acyclic reactants

Tetrapyrrole macrocycles (e.g., porphyrins) have long been proposed as key ingredients in the emergence of life, yet plausible routes for forming their essential pyrrole precursor have previously not been identified. Here, the anaerobic reaction of δ-aminolevulinic acid (ALA, 5–240 mM) with 5-methoxy-3-(methoxyacetyl)levulinic acid (1-AcOH, 5–240 mM) in water (pH 5–7) at 25–85 °C for a few hours to a few days affords uroporphyrinogen, which upon chemical oxidation gives uroporphyrin in overall yield of up to 10%. The key intermediate is the α-methoxymethyl-substituted analogue of the pyrrole porphobilinogen (PBG). Reaction of ALA and the decarboxy analogue of 1-AcOH (1-Me) gave coproporphyrinogen (without its biosynthetic precursor uroporphyrinogen as an intermediate); oxidation gave the corresponding coproporphyrin in yields comparable to those for uroporphyrin. In each case a mixture of porphyrin isomers was obtained, consistent with reversible oligopyrromethane formation. The route investigated here differs from the universal extant biosynthetic pathway to tetrapyrrole macrocycles, where uroporphyrinogen (isomer III) – natures last common precursor to corrins, heme, and chlorophylls – is derived from eight molecules of ALA (via four molecules of PBG). The demonstration of the spontaneous self-organization of eight acyclic molecules to form the porphyrinogen under simple conditions may open the door to the development of a chemical model for the prebiogenesis of tetrapyrrole macrocycles.

A tandem combinatorial model for the prebiogenesis of diverse tetrapyrrole macrocycles

The extant biosynthesis of tetrapyrrole macrocycles has been considered a paradigm for the prebiotic formation of such molecules, yet only a few analogous non-enzymic reactions along the overall process have been demonstrated. In a prior study, the aqueous non-enzymic reaction of a dione and an aminoketone (δ-aminolevulinic acid) afforded uroporphyrinogen, Natures last universal precursor to all extant tetrapyrroles. Here, in one flask the non-enzymic combinatorial reaction of two diones (substituents = acetic acid and methyl) and two aminoketones (substituents = propionic acid and ethyl) yields four pyrroles, which upon subsequent combinatorial reaction afford a distribution of porphyrinogens. A software program for virtual library generation predicts 538 porphyrinogens from this [2 × 2] reaction (owing to combinations and permutations) of which there are 25 sets of isomers based on condensed formulas of substituents. The collection spans the entire range of polarity enabled by the biosynthesis including uro- (number of carboxylic acids = 8), copro- (4), meso- (2), and etio-porphyrinogen (0). The first two are successive intermediates in the extant biosynthesis, the latter two resemble in polarity the advanced biosynthetic products protoporphyrin and chlorophyll. Upon consideration of substituent patterns, the porphyrins (obtained by oxidation of the porphyrinogens) can be grouped into one of four polarity categories (predicted percentage): hydrophilic (0.4%), uncertain (83.6%), amphiphilic (15.6%), and hydrophobic (0.4%). HPLC and mass spectrometry data are consistent with expectations to the limit of analytical capabilities. Thus, in terms of the polarity of the tetrapyrrole macrocycles formed, an all-at-once non-enzymic combinatorial process recapitulates features of the stepwise biosynthetic pathway.

Self-organization of tetrapyrrole constituents to give a photoactive protocell

A tandem combinatorial reaction of four acyclic, colorless compounds (two α-aminoketones and two diones; one polar and one nonpolar for each) in aqueous solution (30 mM each, 60 °C, pH 7, 24 h) containing lipid vesicles and with or without quinones and solar illumination affords a distribution of up to 538 porphyrins in 1.6–3.9% overall yield. The reactions leading to the porphyrins can occur in either or both the aqueous phase and the hydrophobic membrane of the vesicles. The porphyrins encompass a broad range of polarity and partition in the aqueous-lipid medium. Two fractions obtained by size-exclusion chromatography include porphyrins associated with the lipid vesicles and porphyrins in the aqueous phase. The porphyrins in both phases are photoactive as demonstrated by fluorescence quantum yield measurements (Φf ∼ 0.07–0.08) and by the Krasnovsky reaction (a photosynthetic-like process). The constituents of the Krasnovsky reaction employed here are methyl red, ascorbic acid, and 2,6-dichlorophenolindophenol. Illumination of the two porphyrin-containing samples (aqueous phase or vesicles) in the presence of the Krasnovsky constituents results in the reduction of methyl red and oxidation of ascorbic acid. Thus, the overall process transforms a colorless aqueous suspension via four stages (two combinatorial reactions, oxidation, physical partitioning) to photoactive porphyrins in distinct venues. The process may provide a model for the origin of pigments that enable proto-photosynthesis.

Primordial Oil Slick and the Formation of Hydrophobic Tetrapyrrole Macrocycles

The functional end products of the extant biosynthesis of tetrapyrrole macrocycles in photosynthetic organisms are hydrophobic: chlorophylls and bacteriochlorophylls. A model for the possible prebiogenesis of hydrophobic analogues of natures photosynthetic pigments was investigated by reaction of acyclic reactants in five media: aqueous solution (pH 7, 60°C, 24 h); aqueous solution containing 0.1 M decanoic acid (which forms a turbid suspension of vesicles); or aqueous solution accompanied by dodecane, mesitylene, or a five-component organic mixture (each of which forms a phase-separated organic layer). The organic mixture was composed of equimolar quantities of decanoic acid, dodecane, mesitylene, naphthalene, and pentyl acetate. The reaction of 1,5-dimethoxy-3-methylpentan-2,4-dione and 1-aminobutan-2-one to give etioporphyrinogens was enhanced in the presence of decanoic acid, affording (following chemical oxidation) etioporphyrins (tetraethyltetramethylporphyrins) in yields of 1.4-10.8% across the concentration range of 3.75-120 mM. The yield of etioporphyrins was greater in the presence of the five-component organic mixture (6.6% at 120 mM) versus that with dodecane or mesitylene (2.1% or 2.9%, respectively). The reaction in aqueous solution with no added oil-slick constituents resulted in phase separation-where the organic reactants themselves form an upper organic layer-and the yield of etioporphyrins was 0.5-2.6%. Analogous reactions leading to uroporphyrins (hydrophilic, eight carboxylic acids) or coproporphyrins (four carboxylic acids) were unaffected by the presence of decanoic acid or dodecane, and all yields were at most ∼2% or ∼8%, respectively. Taken together, the results indicate a facile means for the formation of highly hydrophobic constituents of potential value for prebiotic photosynthesis.

Expanded combinatorial formation of porphyrin macrocycles in aqueous solution containing vesicles. A prebiotic model

The role of combinatorial processes in the origin of life remains relatively unexplored. In a chemical model for the possible prebiogenesis of tetrapyrrole macrocycles reported previously, a tandem combinatorial reaction of two diones (substituents = methyl, acetic acid) and two aminoketones (substituents = ethyl, propanoic acid) afforded up to 538 porphyrins (upon oxidation of the corresponding porphyrinogens). The reaction was performed at a 1 : 1 ratio of hydrophobic and hydrophilic substituents in each pool of reactants, and the resulting porphyrins partitioned in ∼1 : 1 ratio between aqueous solution and phosphatidylcholine vesicle membranes. Here, a change in the ratio of hydrophobic and hydrophilic substituents of the [2 × 2] reaction gave corresponding changes in the polarity profile of the resulting porphyrins (3.5–9.0% yield). Reaction of four diones and four aminoketones (bearing hydrophilic or hydrophobic substituents) in the presence of lipid vesicles followed by photooxidation afforded porphyrins in 8.7% yield. The resulting porphyrins partitioned in ∼1 : 1 ratio between phosphatidylcholine vesicles and aqueous solution, as observed previously for the [2 × 2] reaction. Both the aqueous fraction and the vesicles fraction were photochemically active as evidenced by the fluorescence quantum yield (Φf ∼ 0.1). Software (PorphyrinViLiGe) for virtual library generation indicates that the [4 × 4] reaction affords up to 131 464 porphyrins. The relative insensitivity of physicochemical properties (partitioning, photoactivity) toward combinatorial expansion may be a valuable yet unappreciated attribute for prebiotic functionality.

Catalytic diversification upon metal scavenging in a prebiotic model for formation of tetrapyrrole macrocycles

A prebiotic model for the formation of tetrapyrrole macrocycles was examined in aqueous solution containing representative Earth-available metals [Mg(II), Mn(II), Fe(II), Co(II), Ni(II), Cu(II), Zn(II) and Pd(II)]. First, a hydrophilic porphyrin (uroporphyrin I) was found to undergo metalation with all metals examined except Mg(II). Second, a competition experiment among the eight metals with uroporphyrin in limiting quantity afforded preferential metalation with Mn(II), Co(II), Cu(II) and Ni(II). A multicomponent analysis method enabled absorption spectrophotometric detection of 8 distinct uroporphyrins (7 metallo-, 1 free base) in a single mixture. Third, a dione–aminoketone reaction was performed in aqueous solution containing the metals followed by photooxidation in the presence of a quinone. The reaction proceeds through multiple stages: (1) dione–aminoketone condensation to give a pyrrole equipped for self-condensation, (2) tetramerization of the pyrrole and cyclization to give uroporphyrinogens, (3) 6e−/6H+ dehydrogenation (e.g., photooxidation) to give the uroporphyrins, and (4) metalation of the uroporphyrins. The presence versus absence of metals resulted in lower yields, yet Mn(II), Fe(II), Co(II), Cu(II) and Zn(II) each individually gave the corresponding metallouroporphyrin [with trivalent metals observed in three cases: Mn(III), Fe(III), and Co(III)]. Analogous reaction in the presence of all eight metals together gave the free base, Mn(III), and Zn(II) chelates whereas other metal chelates could not be reliably detected by absorption spectroscopy or mass spectrometry. Such metalloporphyrins greatly broaden the accessible redox levels, catalytic avenues, and photochemical features versus those of the free base porphyrins. Taken together, scavenging of metals is expected to increase the functional diversity of tetrapyrroles on early Earth.

Bioconjugatable, PEGylated hydroporphyrins for photochemistry and photomedicine. Narrow-band, red-emitting chlorins

Chromophores that absorb and emit in the red spectral region (600-700 nm), are water soluble, and bear a bioconjugatable tether are relatively rare yet would fulfill many applications in photochemistry and photomedicine. Here, three molecular designs have been developed wherein stable synthetic chlorins - analogues of chlorophylls - have been tailored with PEG groups for use in aqueous solution. The designs differ with regard to order of the installation (pre/post-formation of the chlorin macrocycle) and position of the PEG groups. Six PEGylated synthetic chlorins (three free bases, three zinc chelates) have been prepared, of which four are equipped with a bioconjugatable (carboxylic acid) tether. The most effective design for aqueous solubilization entails facial encumbrance where PEG groups project above and below the plane of the hydrophobic disk-like chlorin macrocycle. The chlorins possess strong absorption at ~400 nm (B band) and in the red region (Qy band); regardless of wavelength of excitation, emission occurs in the red region. Excitation in the ~400 nm region thus provides an effective Stokes shift of >200 nm. The four bioconjugatable water-soluble chlorins exhibit Qy absorption/emission in water at 613/614, 636/638, 698/700 and 706/710 nm. The spectral properties are essentially unchanged in DMF and water for the facially encumbered chlorins, which also exhibit narrow Qy absorption and emission bands (full-width-at-half maximum of each <25 nm). The water-solubility was assessed by absorption spectroscopy over the concentration range ~0.4 μM - 0.4 mM. One chlorin was conjugated to a mouse polyclonal IgG antibody for use in flow cytometry with compensation beads for proof-of-principle. The conjugate displayed a sharp signal when excited by a violet laser (405 nm) with emission in the 620-660 nm range. Taken together, the designs described herein augur well for development of a set of spectrally distinct chlorins with relatively sharp bands in the red region.

Complexity in structure-directed prebiotic chemistry. Effect of a defective competing reactant in tetrapyrrole formation

Chemical reactions in prebiotic environments likely entailed complex combinatorial processes with mixtures of reactants. As a case study, the effect of a defective reactant has been examined in a chemical model for the prebiogenesis of tetrapyrrole macrocycles (uroporphyrins). The latter are formed by a multistep process that includes (1) condensation of a β-diketone and an α-aminoketone to form a pyrrole, (2) self-condensation of the pyrrole to form a bilane (tetrapyrromethane), (3) cyclization of the bilane to form a porphyrinogen, and (4) oxidation of the porphyrinogen to form the porphyrin. A defective β-diketone (acetylacetone) affords a pyrrole that can react with nascent oligopyrromethane chains and in so doing terminate chain growth. Quantitative studies show that the porphyrin yield decreases with the fourth power of the mole fraction of the non-defective reactant. Mass spectral examination of the reaction mixture revealed the presence of the pyrrole and of end-capped oligomers derived from the defective diketone. Taken together, the results show the poisoning effect of a small amount of defective reactant in repetitive, irreversible structure-directed reactions, which have been a mainstay in thinking about chemistry leading to the origin of life.

The Porphobilinogen Conundrum in Prebiotic Routes to Tetrapyrrole Macrocycles

Attempts to develop a credible prebiotic route to tetrapyrroles have relied on enzyme-free recapitulation of the extant biosynthesis, but this process has foundered from the inability to form the pyrrole porphobilinogen (PBG) in good yield by self-condensation of the precursor δ-aminolevulinic acid (ALA). PBG undergoes robust oligomerization in aqueous solution to give uroporphyrinogen (4 isomers) in good yield. ALA, PBG, and uroporphyrinogen III are universal precursors to all known tetrapyrrole macrocycles. The enzymic formation of PBG entails carbon-carbon bond formation between the less stable enolate/enamine of one ALA molecule (3-position) and the carbonyl/imine (4-position) of the second ALA molecule; without enzymes, the first ALA reacts at the more stable enolate/enamine (5-position) and gives the pyrrole pseudo-PBG. pseudo-PBG cannot self-condense, yet has one open α-pyrrole position and is proposed to be a terminator of oligopyrromethane chain-growth from PBG. Here, 23 analogues of ALA have been subjected to density functional theoretical (DFT) calculations, but no motif has been identified that directs reaction at the 3-position. Deuteriation experiments suggested 5-(phosphonooxy)levulinic acid would react preferentially at the 3- versus 5-position, but a hybrid condensation with ALA gave no observable uroporphyrin. The results suggest efforts toward a biomimetic, enzyme-free route to tetrapyrroles from ALA should turn away from structure-directed reactions and focus on catalysts that orient the two aminoketones to form PBG in a kinetically controlled process, thereby avoiding formation of pseudo-PBG.

Scope and limitations of two model prebiotic routes to tetrapyrrole macrocycles

Two routes have been proposed as chemical models for the prebiogenesis of tetrapyrrole macrocycles. In each case, the formation of a pyrrole equipped for self-condensation has proved to be the key step, given that the pyrrole self-condensation readily affords porphyrinogen macrocycles. The scope of the two routes is investigated herein. In the first route, a β-ketoester bearing a 4-phosphonooxy substituent was found to react with δ-aminolevulinic acid (ALA) at lower temperature (35 °C) than that of the 4-methoxy substituent (90 °C) to give the corresponding tetraalkyl-tetraester porphyrin, but eight other 4-substituents (Cl-, HO-, AcO-, AcS-, NCS-, MeS-, chloropyridinium-, and morpholino-) failed at 60 or 90 °C. In the second route, reaction of 1,5-dimethoxypentane-2,4-dione (bearing an acetic acid substituent at the 3-position) with 1-aminoacetone gave the corresponding octaalkylporphyrin in a yield comparable to that of the α-aminoketone ALA. Each pyrrole in the resulting porphyrin bears one methyl group and one acetic acid group, thereby constituting a des-methyl homologue of coproporphyrin. The results delineate the features of structure-directed reactions and highlight the limitations of such reactions where no explicit enzyme-like catalyst is present to guide the outcome.