Abiotic formation of uroporphyrinogen and coproporphyrinogen from acyclic reactants

Article abstract of DOI:10.1039/c0nj00716a

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) - nature's 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. The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2011.

Full text of DOI:10.1039/c0nj00716a

New Journal of Chemistry
An international journal of the chemical sciences
www.rsc.org/njc
Volume 35 | Number 1 | January 2011 | Pages 1–244
ISSN 1144-0546

Dynamic Article Links
NJC
Cite this: New J. Chem., 2011, 35, 65–75
www.rsc.org/njc
PAPER
Abiotic formation of uroporphyrinogen and coproporphyrinogen from
acyclic reactantsw
Jonathan S. Lindsey,* Vanampally Chandrashaker, Masahiko Taniguchi and
Marcin Ptaszek
Received (in Montpellier, France) 18th September 2010, Accepted 14th October 2010
DOI: 10.1039/c0nj00716a
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 d-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 1C
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 a-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) – nature’s
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.
upon decarboxylation and dehydrogenation of two of the four
Introduction
propionic acid side chains affords protoporphyrinogen IX.
Dehydrogenation (À6eÀ, À6H+) of the protoporphyrinogen
Tetrapyrrole macrocycles play a central role in most present
living organisms yet the prebiotic origin of such molecules
IX macrocycle yields protoporphyrin IX, the ligand for heme
remains obscure.1 The dearth of understanding exists despite a
and a precursor to all chlorophylls and bacteriochlorophylls.
recurring view since the 1950s that such molecules were likely
Granick and Mauzerall put forth the view that the biosynthesis
to be valuable if not essential for the origin of life.2–18 Such
provides a ‘‘window on evolution’’ where each molecule
views stem from the unique attributes of tetrapyrroles for
served a function in its time and then was replaced with
diverse bioenergetic processes (including photosynthesis) and
molecules formed later in the biosynthetic pathway as trans-
formations were appended over time.3,4,8,18,20–22 The rationale
have been further encouraged by the universality, concision,
and molecular logic of the tetrapyrrole biosynthetic pathway.
for this view stems from the multifaceted change in features of
The early steps of the biosynthesis19 are shown in Fig. 1. Two
the molecules in proceeding from the early to later stages
molecules of ALA dimerize to give porphobilinogen (PBG),
of the biosynthesis: (i) from water-soluble compounds to
which undergoes tetramerization and macrocyclization
hydrophobic macrocycles, (ii) from saturated to unsaturated
(accompanied by inversion of the orientation of one pyrrole)
molecules, and (iii) from simple to complex molecular
to give uroporphyrinogen III. Uroporphyrinogen III is the
architectures. The evolutionary perspective that ‘‘biosynthesis
last common progenitor of the corrinoid, porphyrin, and
recapitulates biogenesis’’20 has prompted consideration as
(bacterio)chlorin macrocycles known as the pigments of
to whether the earliest steps might have been operative in
life. Decarboxylation of the four acetic acid side chains of
the prebiotic era – via purely structure-directed processes
uroporphyrinogen III affords coproporphyrinogen III, which
(i.e., without enzymes or other catalysts) – thereby yielding
tetrapyrole macrocycles that underpinned early catalysis and
Department of Chemistry, North Carolina State University, Raleigh,
NC 27695, USA. E-mail: jlindsey@ncsu.edu
proto-photosynthesis.
A proposed prebiotic pathway that closely mirrors the
w Electronic supplementary information (ESI) available: Mass
extant biosynthesis is outlined in Fig. 2. Examples of trans-
spectral data for the porphyrinogens, porphyrins, and various pyrroles.
See DOI: 10.1039/c0nj00716a
formations and products in each of the steps include the (1)
c
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2011
New J. Chem., 2011, 35, 65–75 65

Fig. 2 Conceptual steps in a pre-biosynthetic route to tetrapyrroles.
to form macrocycles such as metalated uroporphyrin,
coproporphyrin, and hydroporphyrin (e.g., chlorin, bacterio-
chlorin) analogues. Uroporphyrin (and uroporphyrinogen)
have been proposed as prebiotic photosensitizers given their
water solubility, characteristic reactions, and potential for
prebiotic formation.9–11,18,22,23 Studies to address the occurrence
of the individual steps include the following:
ꢀ Diverse modifications (step 5): Uroporphyrin under-
goes smooth metalation [e.g., with zinc(II) or copper(II)]
in neutral aqueous solution at room temperature;24,25 facile
photoreduction to give a variety of porphomethenes;26,27 and,
under exceptionally forcing conditions, decarboxylation of the
acetic acid side chains.28
ꢀ Dehydrogenation (step 4): The dehydrogenation of a
porphyrinogen to give the porphyrin can be achieved with
oxidants under a variety of conditions.29 The dehydrogenation
of uroporphyrinogen can be achieved in aqueous solution
Fig. 1 Contemporary biosynthesis of tetrapyrrole macrocycles.
conversion of early-Earth available substances to ALA, (2)
dimerization of ALA to give PBG, (3) tetramerization of PBG
and macrocyclization to give uroporphyrinogen (four isomers),
(4) dehydrogenation to give uroporphyrin (four isomers), and
(5) subsequent metalation, decarboxylation, and/or reduction
Fig. 3 Step 4: photochemical oxidation of uroporphyrinogen affords
uroporphyrin in an autocatalytic manner.
c
66 New J. Chem., 2011, 35, 65–75
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2011

by chemical or photochemical oxidation, and moreover,
uroporphyrin can catalyze the dehydrogenation in an auto-
catalytic manner as shown in Fig. 3.30 The autocatalytic
process has been demonstrated with oxygen but is expected
to proceed with diverse (prebiotic) oxidants.
ꢀ Tetramerization and macrocyclization (step 3): PBG
undergoes condensation in the absence of enzymes to give
the estimated statistical mixture of four uroporphyrinogen
isomers as shown in Fig. 4. Examination of the distribution
of uroporphyrinogens (inferred upon oxidation to give the
corresponding uroporphyrin isomers) indicates that isomer III
is the most abundant, as expected on statistical considerations.31
Although strong acid was predominantly employed for the
condensation, a few reactions have been reported under neutral
conditions. At pH 7.6 and 60 1C for 21 h under anaerobic
conditions, PBG (10 mM) affords the uroporphyrinogen
isomers in 70% yield.31 Similarly, PBG (0.26 mM) at pH 7.4
and 70 1C for 2 h under anaerobic conditions affords the
uroporphyrinogen isomers in B65% yield.32 Treatment of a
given uroporphyrinogen isomer to the acidic macrocyclization
conditions results in the statistical distribution of isomers,
providing evidence for the thermodynamic stability of the
macrocycles.33 These data indicate the robustness of the
macrocyclization process under plausible prebiotic conditions.
ꢀ Pyrrole formation (step 2): ALA in solution affords a
dihydropyrazine and pseudo-porphobilinogen rather than
PBG, the latter outcome owing to the greater reactivity of the
d versus b methylene unit (Fig. 5).34 Despite extensive studies,
prebiotically plausible conditions have not been identified for
the solution dimerization of ALA to give PBG.35
Fig. 5 Attempted step 2: solution self-condensation of ALA affords
pseudo-porphobilinogen and a dihydropyrazine rather than PBG.
The lack of a simple route to PBG, while incongruous given
the facile occurrence of the seemingly more complex steps
3 and 4 of the pathway, effectively crimps the entire process.
The failure to find a viable structure-directed pathway to PBG
has been referred to as the ‘‘Achilles’ heel’’ of the prebiogenesis
of tetrapyrrole macrocycles.13,18 The resulting impasse leads to
Fig. 4 Step 3: solution condensation of porphobilinogen affords four uroporphyrinogen isomers.
c
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2011
New J. Chem., 2011, 35, 65–75 67

two opposing views concerning the origin of uroporphyrinogen
in the prebiotic era: (i) uroporphyrinogen formed via structure-
directed pathways through use of precursors other than
PBG,13,18 or (ii) uroporphyrinogen did not form until a later
era when appropriate catalysts (e.g., enzymes) were present to
direct conversion of ALA to PBG. A parallel possibility is that
diverse abiotic porphyrinogens – distinct from (and prebiotic
predecessors to) uroporphyrinogen – formed in a structure-
directed manner prior to the emergence of catalysts.
are not found in contemporary living organisms; however,
the success of this route provided a conceptual foundation for
addressing the uroporphyrinogen problem. Here we describe a
structure-directed route to uroporphyrinogen that entails
reaction of acyclic species in anaerobic aqueous solution under
mild conditions of temperature and pH. The same route
with a different precursor affords coproporphyrinogen without
the intermediacy of uroporphyrinogen, its contemporary bio-
synthetic precursor.
An investigation of the chemical foundation for the latter
possibility revealed a new process that encompasses two
of the distinct reaction steps (2 and 3) shown in Fig. 2: ALA
and a 4-methoxy-substituted b-ketoester (2) react to yield a
porphobilinogen analogue (3) that self-condenses to give the
corresponding porphyrinogen (4, Fig. 6). The reaction proceeds
under plausible prebiotic conditions [anaerobic reaction
of ALA (1–5 mM) with 2 (2–40 mM) in water (pH 5–7) at
70–100 1C for >6 h]; upon oxidation the porphyrin is
obtained in up to 10% yield.36 The 1,3-dicarbonyl motif
directs enamine formation to the meso-carbon (C2), whereas
the 4-methoxymethyl unit is untouched and becomes the
a-methoxymethyl unit that equips the pyrrole for self-
condensation. The resulting porphyrinogen and porphyrin
Results and discussion
Uroporphyrinogen
The conceptual approach we investigated to form a PBG
analogue from acyclic materials is shown in Fig. 7. A
2,4-diketone (1-AcOH) was chosen for reaction with ALA.
Compound 1-AcOH is a hybrid of 5-methoxylevulinic acid
and the simple 2,4-diketone acetylacetone. As a 5-methoxyl-
evulinic acid species, 1-AcOH bears a methoxyacetyl unit at
Fig. 7 Compound 1-AcOH is a hybrid of the 2,4-diketone acetyl-
acetone and the monoketone levulinic acid. Reaction with ALA is
expected to preferentially yield the enaminone whereupon 3-g bond
formation closes the ring. Loss of the sacrificial methoxyacetyl unit
(upon attack of HOÀ, H2O, or other nucleophile) followed by
tautomerization affords the porphobilinogen analogue MeOPBG-U.
Other pathways and products are possible (vide infra).
Fig. 6 Integration of steps 2 and 3: hybrid condensation of ALA and
a 4-methoxy-b-ketoester (2) affords pyrrole 3 equipped for self-
condensation to give the non-biological porphyrinogen 4.
c
68 New J. Chem., 2011, 35, 65–75
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2011

the 3-position. As an acetylacetone species, 1-AcOH bears
methoxy groups at the two terminal methyl groups and an
acetic acid substituent at the meso-carbon (C3). Thus, 1-AcOH
is an ALA analogue yet differs in that 1-AcOH (i) does not
self-condense; (ii) is expected to form the enamine wherein the
carbon–carbon double bond encompasses the meso carbon
(3-position), and thereby direct carbon–carbon bond forma-
tion at the 3-g positions; (iii) contains an a-methoxyacetyl unit
that is lost upon pyrrole formation; and (iv) contains a
methoxy substituent destined to be the leaving group at the
a-methylpyrrole site upon reaction with ALA. The resulting
pyrrole (MeOPBG-U) is an analogue of PBG that is equipped
for self-condensation, bears the same b-substituents as PBG,
and differs only in the presence of an a-methoxymethyl group
rather than an a-aminomethyl unit.
The anaerobic reaction of ALA and 1-AcOH (120 mM each)
at 60 1C and pH 5 for 24 h yielded the uroporphyrinogen
[ESI-MS analysis gave m/z = 837.2873, (M + H)+, Fig. S1w]
as shown in Fig. 8. The uroporphyrinogen was accompanied
by a small amount of uroporphyrin (owing to background
oxidation). Also present upon ESI-MS examination was a
peak attributed to the formation of a pyrrole via the Fischer-Fink
pathway, termed MeO-FF-U [m/z = 314.1234, (M + H)+;
Table S1w]; vide infra. Treatment of an aliquot of the reac-
tion mixture with iodine in aqueous acid, a standard
technique developed for conversion of uroporphyrinogen to
uroporphyrin,31,33 enabled spectrophotometric quantitation
on the basis of the characteristic Soret absorption band
(Fig. 9A). Extraction of the acidic oxidized mixture with
cyclohexanone gave uroporphyrin in crude form, which upon
dissolution in neutral aqueous solution gave Soret and visible
absorption bands (Fig. 9B) as well as fluorescence emission
and excitation spectra (Fig. 9C), all of which were almost
identical with that of an authentic sample of uroporphyrin
(isomer III). ESI-MS analysis gave the uroporphyrin molecular
ion [m/z = 831.2350, (M + H)+, Fig. S1w]. HPLC analysis
with mass or absorption detection of the crude uroporphyrin
gave three peaks, two of which exhibited retention times
identical with those of commercially available samples of
two uroporphyrin isomers (I and III) (Fig. 9D). Note that
chromatographic conditions have only been developed for
separation of three of the four uroporphyrin isomers.37 The
ratio of uroporphyrin isomers observed from reaction of ALA
and 1-AcOH resembled the equilibrium distribution obtained
upon non-enzymic condensation of PBG (Fig. 9D).31
The reaction of ALA and 1-AcOH was examined under a
range of conditions spanning concentration (5–240 mM), time
(6–96 h), temperature (25–85 1C), and pH (5–7). Illustrative
examples are as follows.
The reaction at 60 1C for 24 h afforded yields that increased
from o0.5% to 7.9% (pH 5) or 10.7% (pH 7) upon going
from dilute (5 mM) to concentrated (240 mM) reactions, with
yields peaked in the 120–170 mM region (Fig. 10).
The reaction for 72 h at 60 1C gave uroporphyrin in 10.6%
(120 mM, pH 7) or 4.3% yield (120 mM, pH 5).
The reaction at 25 1C (120 mM, pH 7) afforded uroporphyrin
in 0.9% and 4.1% yield after 1 and 4 days, respectively,
whereas at 85 1C for 6 h the yield was 2.2%.
In summary, while the interplay of concentration, pH,
temperature and time are not yet fully delineated, a broad range
of plausible early-Earth conditions38 was found to give successful
condensation with yields of up to 10% of uroporphyrin. These
Fig. 8 Condensation of ALA and 1-AcOH affords a porphobilinogen analogue (MeOPBG-U) that self-condenses to give uroporphyrinogen
isomers (all four are shown). The competing Fischer-Fink pathway affords a fully substituted pyrrole (MeO-FF-U).
c
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2011
New J. Chem., 2011, 35, 65–75 69

results constitute the first laboratory demonstration of the
structure-directed formation of uroporphyrinogen from acyclic
reactants.
Coproporphyrinogen, a biosynthetically advanced tetrapyrrole
macrocycle
The scope was examined by reaction of a substrate other
than 1-AcOH under conditions identical to those to form
uroporphyrinogen (Fig. 11). Thus, the reaction of the decarboxy
analogue of 1-AcOH (1-Me) gave a molecular ion [m/z =
661.3236, (M + H)+] consistent with coproporphyrinogen
(Fig. S2w). Use of LC-ESI-MS revealed the presence of four
peaks as expected for the four possible coproporphyrinogen
isomers (Fig. 12). Examination of the crude reaction mixture also
revealed the presence of the Fischer-Fink pyrrole MeO-FF-C
[m/z = 292.1161, (M + Na)+; Table S1w]; vide infra.
Fig. 10 The yield of uroporphyrin as a function of the concentration
of 1-AcOH and ALA (equimolar) upon reaction for 24 h at 60 1C at
pH 5 or pH 7 determined upon oxidation of reaction aliquots. The
data points are positioned at factors of 2À1/2 from the highest
concentration (240 mM) examined.
Oxidation of the reaction mixture obtained from 1-Me and
ALA gave coproporphyrin in 10.6% overall yield (upon reaction
at 120 mM, pH 7, 60 1C, 24 h). The absorption spectrum,
fluorescence excitation spectrum, and fluorescence emission
spectrum were nearly identical with those from an authentic
sample of coproporphyrin III (Fig. 13A,B). Characterization
of the coproporphyrin sample by mass spectrometry gave the
expected molecular ion, and LC-ESI-MS showed three
coproporphyrin isomers (Fig. S3w). HPLC with absorption
detection also showed three coproporphyrin isomers
(Fig. 13C). The same reaction at 25 1C for 4 days afforded
coproporphyrin in 3.1% yield, whereas at 85 1C for 6 h the
yield was 6.4%.
Fig. 9 Characterization data for the uroporphyrin product obtained
from reaction of 1-AcOH and ALA (120 mM each, pH 5, 60 1C, 24 h).
(A) Absorption spectra in 0.1 M HCl. An aliquot from the reaction
mixture diluted 850-fold into a 2.6 mL cuvette (solid blue line) shows a
trace of porphyrin (Soret band at 405 nm) and dipyrrin species
(490 nm) owing to background oxidation. Upon addition of excess
I2 to the cuvette (followed by discharging with Na2S2O3) the colorless
porphyrinogen30 is converted to the porphyrin (solid black line). The
spectrum of an authentic sample of uroporphyrin III is included for
comparison (dotted red line, normalized at the Soret band of the crude
sample). (B) Absorption spectra in 0.1 M sodium phosphate (pH 7).
The crude uroporphyrin extracted from acidic brine (solid black line)
is compared with an authentic sample of uroporphyrin III (dotted red
line). (C) Fluorescence excitation (lem 678 nm) and emission spectra
(lexc 398 nm) in 0.1 M sodium phosphate (pH 7) of the crude
uroporphyrin (solid lines) are compared with those of an authentic
sample of uroporphyrin III (dashed lines, normalized at the peak band
of the crude sample). (D) HPLC with mass spectral detection
[m/z = 831 Æ 2, (M + H)+] of the crude uroporphyrin (showing
three putative isomers) is compared with data from authentic samples
of uroporphyrin I and uroporphyrin III. The chromatogram from the
acidic condensation (90 1C, 1 h, air oxidation) of PBG is shown in
overlay. Slight variation in retention times (e.g., 6 s at 4.4 min)
occurred for a given sample on multiple runs. See Materials and
methods for HPLC conditions.
c
70 New J. Chem., 2011, 35, 65–75
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2011

Fig. 11 Condensation of 1-Me and ALA provides direct access to coproporphyrinogen isomers. The competing Fischer-Fink pathway affords a
fully substituted pyrrole (MeO-FF-C).
proceed beneficially via a lactone intermediate remains to be
determined.
Competing pathways to pyrroles
The reaction of an a-aminoketone and a b-diketone can
proceed via multiple pathways, including here the desired
Knorr and undesired Fischer-Fink routes (Fig. 15). Indeed,
examination of a variety of reactions of ALA and 1-AcOH
(or 1-Me) by ESI-MS revealed the presence of a peak due to
the porphyrinogen (derived from the porphobilinogen-like
Knorr pyrrole) as well as a peak due to the Fischer-Fink
pyrrole (Table S1w). The Knorr and Fischer-Fink routes have
their branch point at the step where the initial enaminone
undergoes cyclization.
Fig. 12 LC-ESI-MS analysis of a sample from the reaction of 1-Me
The Fischer-Fink route stems from condensation at the
and ALA with detection at the mass channel m/z 659.4–663.1 shows
four putative coproporphyrinogen isomers [(M + H)+]. See Materials
d-methylene of ALA with the 2-carbonyl of 1-AcOH (or 1-Me)
followed by dehydration. The Fischer-Fink pyrrole (MeO-FF-U
or MeO-FF-C) is expected to be relatively unreactive owing to
the deactivating effect of the a-acyl substituent. The Knorr
pathway entails condensation of the enaminone C3-carbon
with the ALA g-ketone to yield the pyrroline intermediate
(see Fig. 7). Because the enaminone nucleophile is a tertiary
carbon, cleavage of the methoxyacetyl unit at the resulting
quaternary carbon is required (followed by loss of hydroxide
and tautomerization) to give the pyrrole equipped for self-
condensation (MeOPBG-U or MeOPBG-C). Thus, one
methoxyacetyl unit becomes an integral part of the structure
(of MeOPBG-U or MeOPBG-C) while the second serves only
as a temporary aide to direct enamine formation at the C3 site
and methods for HPLC conditions.
It is noteworthy that the pyrrole (MeOPBG-C) obtained in
the reaction of 1-Me and ALA is not equipped to form a
lactone with the b-alkylcarboxylic acid, as can occur with the
pyrrole (MeOPBG-U) derived from 1-AcOH and ALA; with
PBG;31 and perhaps with an a-(methoxymethyl)pyrrole used
in a chemical synthesis of coproporphyrin39 (Fig. 14). Thus,
the formation of such a lactone intermediate and/or intra-
molecular catalysis by the b-acetic acid unit is not essential to
promote the condensation in step 3 (tetramerization and
macrocyclization). Whether there are reaction conditions that
c
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2011
New J. Chem., 2011, 35, 65–75 71

Fig. 14 Possible lactone formation in step 3 (tetramerization and
macrocyclization).
Butler and George carried out the reaction of ALA and
3-methylpentan-2,4-dione (5-Me), a substrate closely resembling
the core structure of 1-AcOH or 1-Me.40 The Fischer-Fink
pyrrole (6-Me) was the only solid product isolated, albeit in
16.7% yield.40 Were the Fischer-Fink pathway the only
pathway available, of course, no porphyrinogen would result
upon reaction of ALA and 1-AcOH or 1-Me. Because the
result of Butler and George taken on face value indicated the
reaction of ALA and 1-AcOH or 1-Me would not lead
to porphyrinogen, we repeated the reaction of ALA and
3-substituted pentan-2,4-diones to assess the products under
the exact conditions employed here that led to porphyrinogens.
The 3-substituted pentan-2,4-diones lack the 1,5-dimethoxy
substituents of 1-AcOH or 1-Me and hence are arrested
with regards to subsequent condensations (Fig. 16). To assess
the products unbiased by chemical separation methods,
we employed ESI-MS analysis of the crude reaction mixtures
(a measurement method not commonly available at the time
when Butler and George performed their study).
Fig. 13 Data for coproporphyrin obtained upon oxidation of a
sample from the reaction of 1-Me and ALA (pH 7, 60 mM ALA
and 120 mM 1-Me, 24 h, 60 1C). (A) Absorption spectra in n-propanol
of the crude porphyrin extract (solid line) and comparison with the
spectrum of an authentic sample of coproporphyrin III (dashed line);
the spectra are normalized at the Soret band. (B) Fluorescence
excitation (lem 678 nm) and emission spectra (lexc 398 nm) in
n-propanol of the crude coproporphyrin (solid lines) obtained upon
oxidation and extraction are compared with an authentic sample of
coproporphyrin III (dashed lines, normalized at the peak bands). (C)
HPLC with absorption spectral detection of the crude porphyrin
extract compared with authentic samples of coproporphyrin I and
coproporphyrin III. Three putative coproporphyrin isomers are
observed in the crude sample. See Materials and methods for HPLC
conditions.
The reaction of ALA and 3-methylpentan-2,4-dione
(5-Me, 60 mM) at 60 1C and pH 5 for 24 h upon ESI-MS
analysis showed peaks for both the Fischer-Fink pyrrole 6-Me
and the Knorr pyrrole 7-Me (Table S2w). Identical reaction of
ALA and 3-acetyllevulinic acid (5-AcOH) showed peaks for
both the Fischer-Fink pyrrole 6-AcOH and the Knorr pyrrole
7-AcOH. Whether the isolation of pyrrole 6-Me and not also
7-Me by Butler and George reflects relative yield or relative
stability of the two pyrroles remains to be determined. In
summary, the competing formation of the Fischer-Fink
and Knorr pyrroles appears to be intrinsic to the reaction of
a 3-substituted 2,4-diketone with ALA. Quantitation of the
two pathways and examination of simple catalysts or con-
ditions that might suppress the Fischer-Fink shunt require
further study.
(Fig. 7). Other pyrroles are possible (e.g., by cleavage of the
succinyl moiety along the Fischer-Fink route) but were not
observed upon ESI-MS analysis of the reaction mixtures.
Isomers of the Fischer-Fink pyrroles formed by 5 - g closure
(rather than d - 2 closure as shown in Fig. 15) cannot be
discriminated by nominal mass analysis, and while considered
to be less likely, cannot at present be ruled out.
c
72 New J. Chem., 2011, 35, 65–75
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2011

Fig. 16 Competing Fischer-Fink and Knorr routes with 3-substituted
pentan-2,4-diones.
molecules of ALA. The enzyme porphobilinogen synthase is
believed to direct an aldol condensation (to set the critical
b–g bond, Fig. 5) between two molecules of ALA followed by
imine formation to give the 5-membered ring.46 The dimerization
of ALA in solution, which presumably entails initial formation
of the imine, is thwarted by the greater reactivity of the d versus b
site for aldol condensation.34 The ALA-dimerization problem
has been sidestepped here through a hybrid reaction of ALA
with a 2,4-diketone bearing a 3-substituent and 1,5-dimethoxy
units; one keto moiety functions sacrificially to direct reaction
at the 3-position (corresponding to the b-site in ALA, which is
comparatively unreactive in solution) and then is lost upon
pyrrole formation. Other sacrificial directing groups (e.g.,
cyano) at the 3-position might serve equally well. The
simplicity, structure-directed transformations, mild conditions,
and non-negligible yields for the multistep process identified
herein would appear to meet many criteria for a plausible
prebiotic process.
Fig. 15 Competing Fischer-Fink and Knorr routes on the pathway to
porphyrinogens.
On the other hand, one criterion that has not yet been met is a
prebiotic route to 1-AcOH (or 1-Me), or for that matter to ALA.
Ultimately, a claim to prebiotic relevance for the proposed route
is contingent on substantiation of routes to such precursors from
early Earth-available substances. With regards to the b-diketones
(1-AcOH, 1-Me), b-dicarbonyl compounds are central to fatty
acid biosynthesis/degradation and microbial polyketide
synthesis.47 In addition, the a-methoxyacetyl unit is employed
in polyketide synthesis.48 While it may never be possible to
prove the occurrence of a prebiotic process, at a minimum, the
study herein may reveal ‘‘indispensable chemical requirements
that restrict the range of boundary conditions compatible with
a chemistry of biogenesis.’’49 In this context, the process highlights
a number of issues central to studies of the origin of life.
A key issue concerns proto-metabolism versus extant
metabolism, which de Duve has emphasized may be broadly
parallel yet have distinct features.14 The following points
deserve comment in this regard.
Outlook
The prior impasse concerning plausible prebiotic routes to
tetrapyrrole macrocycles stemmed from inability to form the
pyrrole PBG or equivalent thereof. Numerous routes are
known for the chemical synthesis of pyrroles,41 including
PBG;42–45 however, none employs relatively simple substances
that spontaneously give rise to a pyrrole that contains all of
the essential features of PBG. PBG (i) is water-soluble owing
to the two carboxylic acid side chains and the aminomethyl
unit, (ii) reacts as a 11 electrophile (or azafulvene equivalent) at
the a-methyl site, and (iii) undergoes electrophilic substitution
at the open a-site, a process potentiated by the two activating
b-alkyl groups. Together, such features enable self-condensation
of PBG in aqueous solution under mild conditions.
The reaction pathway identified herein to form PBG analogues
is fundamentally different from both the (efficient) enzymatic
transformation and the (failed) solution dimerization of two
c
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2011
New J. Chem., 2011, 35, 65–75 73

ꢀ The macrocycles uroporphyrinogen and coproporphyrinogen
are ‘generationally simple yet architecturally complex’49 and
form spontaneously from acyclic reactants; however,
structure-directed reactions in general are far slower
than enzymatically catalyzed processes.50 Here, the obvious
limitations of the structure-directed processes include the
competing formation of the undesired Fischer-Fink pyrrole,
the formation of four porphyrinogen isomers, the requirement
for somewhat high concentrations, and the comparatively low
yields. It remains unknown how/when processes dictated
solely by the intrinsic molecular structure of reactants gave
way to more efficient and selective catalyzed reaction
processes.
Materials and methods
Reactants
ALA is commercially available. The syntheses of compounds
1-AcOH and 1-Me (by alkylation of the known 1,5-dimethoxy-
pentan-2,4-dione53) and 5-AcOH will be reported elsewhere.
Reactions
All experiments were carried out at 0.5 mL scale under
anaerobic conditions following techniques that have been
described in detail for the reaction of ALA and 2.36 Reactions
were performed in 0.5 M sodium acetate (pH 5) or 0.5 M
3-(N-morpholinyl)propylsulfonic acid (pH 7), respectively,
with pH values determined at 25 1C.
ꢀ Uroporphyrin, derived oxidatively from uroporphyrinogen,
has been proposed as an ideal prebiotic photosensitizer,9–11,18,22,23
as has coproporphyrin.10 Both can now be readily formed
from acyclic reactants yet neither is a constituent of extant
biochemistry.4,19
Porphyrin yields
The yield of porphyrin was determined by addition of a
reaction aliquot (50 mL of a 30 mM reaction) to 0.1 M HCl
(2.6 mL) in a cuvette under aerobic conditions, to which was
then added 56 mL of I2 solution (50 mM in ethanol); sub-
sequent treatment with 112 mL of Na2S2O3 (100 mM in water)
discharged excess iodine. The absorption spectrum was
ꢀ Coproporphyrinogen is a more advanced intermediate than
uroporphyrinogen along the extant biosynthetic path to hemes
and chlorophylls. The facile formation of coproporphyrinogen
without uroporphyrinogen as a precursor raises questions
concerning how/why the extant biosynthesis originated with
reliance on ALA as the sole precursor followed by decarboxyl-
ation of the acetic acid side chains of uroporphyrinogen,
versus employing two distinct substrates to directly form
coproporphyrinogen.
quantitated using eSoret = 505 000 MÀ1 cmÀ1 33 The oxidation
.
process was scaled linearly for reactions at other concentra-
tions, and for oxidations at larger scale. Treatment of the
oxidized reaction mixture with acidic brine (3.6 M NaOAc,
pH 3.2), extraction into cyclohexanone,54 and concentration
of the organic extract afforded the crude porphyrin mixture.
ꢀ The yields to date are not high, but tetrapyrrole macro-
cycles persist for hundreds of millions of years in anaerobic
environments,51 and hence would be expected to accumulate
under prebiotic conditions.
HPLC conditions
HPLC of the uroporphyrin sample with mass spectral detec-
tion was carried out at 50 1C on a C18 column (50 Â 2.1 mm,
2.7 mm particles) with the following solvents [A = aqueous
NH4OAc (1.0 M, pH 5.15); B = 98% CH3CN, 2% water, and
0.3% formic acid; the CH3CN itself contained 2% water] and
flow rate 0.35 mL minÀ1. The elution regimen consisted of 7%
B for 4 min followed by transition to 10% B over 6 min. See
Fig. 9D.
The demonstration of a route from acyclic materials
to porphyrinogens opens the door to the development
of a chemical model for the prebiogenesis of tetrapyrrole
macrocycles. Ultimately, such a model must begin with simple,
early Earth-available precursors to ALA and 1-AcOH
(or analogues) and integrate multiple successive transfor-
mations (steps 1-5, Fig. 2). Concerning step 1, we note that
both ALA and 1-AcOH are of structural complexity inter-
mediate between that of accepted prebiotic species such
as amino acids and nucleosides. Steps 2 and 3 have been
demonstrated herein, and precedent exists for step 4 (albeit as
a stand-alone process).30 Toward greater molecular complexity,
the reaction of diverse analogues of ALA and of 1-AcOH
alone or in combinatorial processes, as well as in situ
modification processes (step 5: side-chain alteration, reduc-
tion, metalation), are expected to yield diverse biotic and
abiotic macrocycles encompassing a range of polarity and
functionality.
HPLC of the coproporphyrinogen sample with mass spectral
detection was carried out at 50 1C on a C18 column (50 Â 2.1 mm,
2.7 mm particles) with the following solvents [A = 98% water
and 2% CH3CN; B = 98% CH3CN, 2% water, and 0.3%
formic acid; the CH3CN itself contained 2% water] and flow
rate 0.4 mL minÀ1. The elution regimen consisted of 25% B
for 2 min, transition to 75% B over 8 min, then transition to
25% B over 3 min. See Fig. 12.
HPLC of the coproporphyrin sample with absorption
spectral detection (ldet = 405 nm) was carried out at 25 1C
on a C18 column (100 Â 2.1 mm, 5 mm particles) in isocratic
mode (flow rate 0.8 mL minÀ1) using 26% of CH3CN and
74% of aqueous NH4OAc (1.0 M, pH 5.15). See Fig. 13C.
The availability of tetrapyrrole macrocycles via model
prebiotic processes enables examination of a variety of catalytic
and energy-utilization processes central to origin-of-life
problems. For example, the question of whether the earliest
cells52 were chemotrophic or phototrophic has animated
the life sciences since the time of Oparin and Haldane. The
facile formation of diverse tetrapyrrole macrocycles from
simple precursors may provide an opportunity to explore
systematically the de novo emergence of proto-photosynthetic
processes.
Fluorescence spectroscopy
The fluorescence spectra were obtained at room temperature
with excitation and emission slit widths of 1.5 nm, and a sweep
rate of 1 nm/2 s. The spectra were corrected for excitation
intensity and instrument response on emission. The fluores-
cence emission spectra were obtained upon excitation at the
c
74 New J. Chem., 2011, 35, 65–75
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2011

peak of the Soret band with samples having absorbance values
o0.06 in 1 cm pathlength cuvettes. Fluorescence excitation
spectra were obtained upon emission detection at the Q(0,1)
peak of the same samples.
16 M. T. Madigan, J. M. Martinko and J. Parker, in Brock Biology of
Microorganisms, Prentice Hall, Upper Saddle River, NJ, USA,
Eighth edn, 1997, p. 612.
17 D. W. Deamer, Microbiol. Mol. Biol. Rev., 1997, 61, 239–261.
18 D. C. Mauzerall, Clin. Dermatol., 1998, 16, 195–201.
19 R. J. Porra, Photochem. Photobiol., 1997, 65, 492–516.
20 S. Granick, Harvey Lectures, 1950, 44, 220–245.
21 D. Mauzerall, in The Photosynthetic Bacteria, ed. R. K. Clayton
and W. R. Sistrom, Plenum Press, New York, USA, 1978,
pp. 223–231.
22 D. Mauzerall, in Bioorganic Chemistry, ed. E. E. van Tamelen,
Academic Press, New York, USA, 1978, vol. IV, pp. 303–314.
23 S. Granick, in Biochemistry of Chloroplasts, ed. T.W.
Goodwin, Academic Press, New York, USA, 1967, vol. 2,
pp. 373–410.
24 P. A. Carapellucci and D. Mauzerall, Ann. N. Y. Acad. Sci., 1975,
244, 214–238.
25 J. Feitelson and D. Mauzerall, J. Phys. Chem., 1996, 100,
7698–7703.
26 D. Mauzerall, J. Phys. Chem., 1962, 66, 2531–2533.
27 D. Mauzerall, J. Am. Chem. Soc., 1962, 84, 2437–2445.
28 T. K. With, Biochem. J., 1975, 147, 249–251.
29 J. S. Lindsey, Acc. Chem. Res., 2010, 43, 300–311.
30 D. Mauzerall and S. Granick, J. Biol. Chem., 1958, 232,
1141–1162.
Mass spectrometry
High resolution exact mass measurements of the porphyrins
and porphyrinogens were carried out using electrospray ioni-
zation (ESI) time-of-flight on an Agilent Technologies 6224
LC-TOF mass spectrometer operated in positive-ion mode
(Duke University, with assistance of Dr George Dubay). High
resolution exact mass measurements of the pyrroles were
carried out using electrospray ionization (ESI) time-of-flight
on an Agilent Technologies 6210 LC-TOF mass spectrometer
operated in positive-ion mode (NC State University, with
assistance of Ms. Danielle Lehman).
Acknowledgements
This work was supported by a grant from the NSF Chemistry
of Life Processes Program (NSF CHE-0953010). JSL wishes to
thank Dr David C. Mauzerall for critical encouragement over
many years concerning this ‘‘Achilles’ heel’’ of porphyrin
evolution.13,18 The journal cover art accompanying this article
was prepared by Ms. Margot Geist (www.geistlight.com) using
a seascape image obtained at Cape Perpetua, Oregon.
31 D. Mauzerall, J. Am. Chem. Soc., 1960, 82, 2605–2609.
32 R. B. Frydman, S. Reil and B. Frydman, Biochemistry, 1971, 10,
1154–1160.
33 D. Mauzerall, J. Am. Chem. Soc., 1960, 82, 2601–2605.
34 A. R. Butler and S. George, Tetrahedron, 1992, 48, 7879–7886.
35 A. R. Chaperon, H. Bertschy, A.-L. Franz-Schrumpf, B. Hugelet,
A. Neels, H. Stoeckli-Evans and R. Neier, Chimia, 2003, 57,
601–606.
36 J. S. Lindsey, M. Ptaszek and M. Taniguchi, Origins Life Evol.
Biosphere, 2009, 39, 495–515.
37 J. M. Rideout, D. J. Wright and C. K. Lim, J. Liq. Chromatogr.
Relat. Technol., 1983, 6, 383–394.
References
1 S. L. Miller, in The Molecular Origins of Life, ed. A. Brack,
Cambridge University Press, Cambridge, UK, 1998, pp. 59–85.
2 H. Gaffron, in Rhythmic and Synthetic Processes in Growth, ed.
D. Rudnick, Princeton University Press, Princeton, NJ, USA,
1957, pp. 127–154.
3 S. Granick, Ann. N. Y. Acad. Sci., 1957, 69, 292–308.
4 S. Granick, in Evolving Genes and Proteins, ed. V. Bryson and
H. J. Vogel, Academic Press, New York, USA, 1965, pp. 67–88.
5 J. D. Bernal, The Origin of Life, The World Publishing Company,
Cleveland, OH, USA, 1967.
6 A. A. Krasnovsky, Origins Life, 1974, 5, 397–404.
7 A. A. Krasnovsky, Origins Life, 1976, 7, 133–143.
8 D. Mauzerall, Philos. Trans. R. Soc. London, Ser. B, 1976, 273,
287–294.
9 J. A. Mercer-Smith and D. Mauzerall, Photochem. Photobiol.,
1981, 34, 407–410.
38 J. F. Kasting and M. T. Howard, Philos. Trans. R. Soc. London,
Ser. B, 2006, 361, 1733–1742.
39 I. T. Kay, Proc. Natl. Acad. Sci. U. S. A., 1962, 48, 901–905.
40 A. R. Butler and S. D. George, Tetrahedron, 1993, 49, 7017–7026.
41 G. P. Bean, in Pyrroles, Part 1, ed. R. A. Jones, John Wiley &
Sons, Inc., New York, USA, 1990, pp. 105–294.
42 P. Bobal and R. Neier, Trends Org. Chem., 1997, 6, 125–144.
43 A. R. Chaperon, T. M. Engeloch and R. Neier, Angew. Chem., Int.
Ed., 1998, 37, 358–360.
44 R. Neier, J. Heterocycl. Chem., 2000, 37, 487–508.
45 C. P. Soldermann, R. Vallinayagam, M. Tzouros and R. Neier,
J. Org. Chem., 2008, 73, 764–767.
46 E. K. Jaffe, Bioorg. Chem., 2004, 32, 316–325.
47 S. Smith and S.-C. Tsai, Nat. Prod. Rep., 2007, 24, 1041–1072.
48 Y. A. Chan, A. M. Podevels, B. M. Kevany and M. G. Thomas,
Nat. Prod. Rep., 2009, 26, 90–114.
10 J. A. Mercer-Smith and D. C. Mauzerall, Photochem. Photobiol.,
1984, 39, 397–405.
11 J. A. Mercer-Smith, A. Raudino and D. C. Mauzerall, Photochem.
Photobiol., 1985, 42, 239–244.
12 J. M. Olson and B. K. Pierson, Origins Life Evol. Biosphere, 1987,
17, 419–430.
49 A. Eschenmoser, Tetrahedron, 2007, 63, 12821–12844.
50 R. Wolfenden and M. J. Snider, Acc. Chem. Res., 2001, 34,
938–945.
51 H. J. Callot and R. Ocampo, in The Porphyrin Handbook,
ed. K. M. Kadish, K. M. Smith and R. Guilard, Academic Press,
San Diego, USA, 2000, vol. 1, pp. 349–398.
13 D. C. Mauzerall, Origins Life Evol. Biosphere, 1990, 20, 293–302.
14 C. de Duve, in Blueprint for a Cell: The Nature and Origin of Life,
Neil Patterson Publishers, Carolina Biological Supply Company,
Burlington, NC, USA, 1991.
52 T. Cavalier-Smith, Philos. Trans. R. Soc. London, Ser. B, 2006,
361, 969–1006.
53 A. H. Alberts and D. J. Cram, J. Am. Chem. Soc., 1979, 101,
3545–3553.
54 C. Rimington and A. Benson, Biochem. J., 1967, 105, 1085–1090.
15 D. Mauzerall, Photosynth. Res., 1992, 33, 163–170.
c
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2011
New J. Chem., 2011, 35, 65–75 75