Non-enzymic tetramisation of ethyl 3-(4-ethoxycarbonylmethyl-1H-pyrrol-3-yl)propionate with formaldehyde follows a similar course to the non-enzymic tetramisation of porphobilinogen

Article abstract of DOI:10.1016/S0040-4039(01)01160-1

The octaethyl esters of uroporphyrins were formed directly from ethyl 3-(4-ethoxy-carbonylmethyl-1H-pyrrol-3-yl)propionate and formalin in a yield of ca. 30% with the isomers I, II, III and IV being formed in the ratio 1:1:4:2. Under anaerobic conditions, the colourless uroporphyrinogen esters were formed in a similar ratio. These observations parallel the non-enzymic formation of uroporphyrinogens from the naturally occurring tetrapyrrole precursor, porphobilinogen, highlighting the similarity in both tetramisation and isomerisation reactions.

Full text of DOI:10.1016/S0040-4039(01)01160-1

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 5973–5976
Non-enzymic tetramisation of ethyl
3-(4-ethoxycarbonylmethyl-1H-pyrrol-3-yl)propionate with
formaldehyde follows a similar course to the non-enzymic
tetramisation of porphobilinogen
Kwai-Ming Cheung and Peter M. Shoolingin-Jordan*
Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton,
Biomedical Sciences Building, Bassett Crescent East, Southampton SO16 7PX, UK
Received 29 May 2001; accepted 28 June 2001
Abstract—The octaethyl esters of uroporphyrins were formed directly from ethyl 3-(4-ethoxy-carbonylmethyl-1H-pyrrol-3-
yl)propionate and formalin in a yield of ca. 30% with the isomers I, II, III and IV being formed in the ratio 1:1:4:2. Under
anaerobic conditions, the colourless uroporphyrinogen esters were formed in a similar ratio. These observations parallel the
non-enzymic formation of uroporphyrinogens from the naturally occurring tetrapyrrole precursor, porphobilinogen, highlighting
the similarity in both tetramisation and isomerisation reactions. © 2001 Elsevier Science Ltd. All rights reserved.
Porphobilinogen (3-(5-aminomethyl-4-carboxymethyl-
1H-pyrrol-3-yl)-propionic acid) is the monopyrrolic
building unit from which all tetrapyrroles are formed in
biological systems.1 Four molecules of porphobilinogen
are transformed into uroporphyrinogen III by the
action of two enzymes (Scheme 1); porphobilinogen
deaminase2 that forms a linear 1-hydroxymethylbilane
called preuroporphyrinogen from four molecules of
porphobilinogen and uroporphyrinogen III synthase2
that catalyses the cyclisation and rearrangement of
preuroporphyrinogen specifically to the uroporphyrino-
gen III isomer. The latter reaction has attracted much
interest over several decades since it involves the iso-
merism of the terminal pyrrole ring (ring d) of
preuroporphyrinogen.
Experiments carried out some 40 years ago demon-
strated that any single isomer of uroporphyrinogen,
when heated in dilute acid under anaerobic conditions,3
generated the same ratio of uroporphyrinogen I, II, III
and IV isomers in the ratio 1:1:4:2. In basic or neutral
conditions, the interconversion of the isomers did not
occur significantly. In the presence of dimedone,4
a
formaldehyde scavenger, the yields of uroporphyrino-
gens were reduced substantially suggesting that the
aminomethyl group of porphobilinogen, or its hydroxy-
methyl equivalent, is freely in equilibrium with formal-
dehyde. This was also suggested by carrying out the
non-enzymic polymerisation of porphobilinogen in the
presence of formaldehyde, which again lowered the
yield of uroporphyrinogens.
Scheme 1.
* Corresponding author.
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)01160-1

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K.-M. Cheung, P. M. Shoolingin-Jordan / Tetrahedron Letters 42 (2001) 5973–5976
In a related study, it was demonstrated that the natural
tetrapyrrole precursor, porphobilinogen, may be trans-
formed non-enzymically into uroporphyrinogens in
high yield by heating in dilute acid under anaerobic
conditions. A mixture of all four possible isomers of
uroporphyrinogen (I, II, III and IV) was formed in the
same ratio (1:1:4:2) as in the isomerisation of
uroporphyrinogens.
The meso-protons at ca. l 10.25 and NH protons at l
−3.60 can be clearly observed. The broadness of the
meso-proton peaks arises since all four isomeric uro-
porphyrins are present. The 13C NMR (not shown)
revealed the structure of the octaethyl-uroporphyrins,
in which the meso-carbons are at l 97.8 and the
pyrrolic carbons are at l 144.6, 144.0, 140.9 and 132.9.
A small amount of the octaethyl-uroporphyrin mixture
was hydrolysed in 2 M NaOH/EtOH at room tempera-
ture overnight and the products were purified using an
Amberlite IR 120 (H+ form) column. After evaporation
of the solvent, dark reddish-violet crystals were
obtained.
These observations suggested that the bond lability at the
pyrrole a-positions was likely to be exploited in the
enzyme-catalysed rearrangement of preuroporphyrino-
gen into uroporphyrinogen III. Any mechanism must
take into account the observation that all the enzymic
reactions occur with strict steric control with no evidence
of randomisation of the paired meso-hydrogen atoms.5
In this paper we report the formation of octaethyl-uro-
porphyrinogens and octaethyl-uroporphyrins by reac-
tion of ethyl 3-(4-ethoxy-carbonylmethyl-1H-pyrrol-
3-yl)propionate 2 (opsopyrrole dicarboxylic ester) with
formaldehyde to compare the reaction with the equiva-
lent tetramisation of porphobilinogen.
These isomers of uroporphyrin free acids were sepa-
rated by reverse phase HPLC using a 150 mm×3.9 mm
Nova-pak C18 column connected to a Waters 626 pump
and a Waters 600s controller, eluting with 13% v/v
acetonitrile and 87% 1 M ammonium acetate buffer at
pH 5.16.9 The uroporphyrin free acid isomers I, II, III
and IV were eluted in a ratio of ca. 1:1:4:2.
The preparation of the octaethyl-uroporphyrins is
shown in Scheme 2. Compound 1 was prepared accord-
ing to the literature.6 Compound 27 was then obtained
in 60% yield by hydrolysis of 1 in dilute alcoholic KOH
for 3–4 hours. This was then refluxed with formalin
(37%) and a catalytic amount of p-TsOH in benzene, in
the dark, under nitrogen and then open to the air. After
work-up, crude octaethyl-uroporphyrins 3,8 were
obtained in ca. 30% yield. Recrystallisation from
CHCl3/EtOH, yielded dark brownish-red amorphous
crystals.
The chemical formation of uroporphyrinogen esters
under anaerobic conditions was also investigated by in
situ 1H NMR. Ethyl 3-(4-ethoxycarbonylmethyl-1H-
pyrrol-3-yl)propanoate 2, formalin (37%) and a p-TsOH
crystal in deuterated chloroform were introduced into a
NMR tube and placed in a long Dewar flask containing
liquid nitrogen. The NMR tube was connected to a
three-way tap to allow the contents to be evacuated and
filled with nitrogen gas several times. The NMR tube
was then sealed and warmed to 4°C in the spectrome-
Scheme 2.

K.-M. Cheung, P. M. Shoolingin-Jordan / Tetrahedron Letters 42 (2001) 5973–5976
5975
enzymatically.10 The above results show that a mixture
of uroporphyrins can be prepared by a relatively simple
procedure in yields approaching 30%. By use of HPLC
techniques for the separation of uroporphyrin esters
and free acids, it is possible to obtain pure isomers on
1
a large scale. The in situ H NMR studies clearly show
the formation of a mixture of uroporphyrinogen esters
from ethyl 3-(4-ethoxycarbonylmethyl-1H-pyrrol-3-yl)-
propionate 2 and formalin under anaerobic conditions.
The findings indicate that, whether the staring material
is porphobilinogen alone or opsopyrrole dicarboxylic
ester and formaldehyde, the ratio of isomers is the same
and that therefore the mechanism is likely to be the
same in both processes, with 1-hydroxymethylbilanes,
such as preuroporphyrinogen, as intermediates.
The above study not only provides further insight into
the novel reactivity of reduced porphyrinogen macro-
cyclic systems but, in addition, offers a simple alterna-
tive method for the synthesis of all four
octaethyl-uroporphyrinogen isomers and their oxidised
equivalents.
Acknowledgements
This work was supported by the EPSRC, the BBSRC
and by the Wellcome Trust. We also wish to thank the
EPSRC at Daresbury11 for the Chemical Database
Service provided.
1
Figure 1. The in situ H NMR spectra illustrating the forma-
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100 minutes at 4°C; (c) additional 20 minutes at 25°C.
* indicates the impurities from formalin, partly erased for
clarity.
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at 4°C until after a period of 100 minutes (Fig. 1b).
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(both having an upfield chemical shift of ca. 0.06 ppm),
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sis of 1 in dilute KOH/EtOH. lH (250 MHz, CDCl3):
8.15 (s, 1H), 6.70 (s, 1H), 6.55 (s, 1H), 4.15 (m, 4H), 3.49
(s, 2H), 2.78 (t, 2H, J=7.0 Hz), 2.56 (t, 2H, J=7.0 Hz),
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(37%, 0.12 ml) and a catalytic amount of p-TsOH in
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hours under N2 in the dark. After this, the solution was
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1
ple tube immediately after the in situ H NMR experi-
ment. From the spectrum (not shown), the structure of
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meso-carbons appear at l 21.8 and the pyrrolic carbons
at l 125.6, 124.0, 117.0 and 110.0. The 13C NMR
results of the chemically synthesised uroporphyrinogens
reported here were similar to the 13C NMR data of the
uroporphyrinogens I and III, which were prepared

5976
K.-M. Cheung, P. M. Shoolingin-Jordan / Tetrahedron Letters 42 (2001) 5973–5976
amorphous crystals (40 mg, 12%). Rf=0.65 (EtO-
H−2Et−Me] (18).
Ac:toluene 1:1). UV–vis (CHCl3): umax (m)=405.2 (1.65×
105) [lit.12 umax (m)=405 (2.15×105)], 501.2 (1.39×104),
534.8 (0.83×104), 570.8 (0.6×104) and 624.8 (0.36×104). lH
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8H), 4.20 (m, 16H), 3.40 (m, 8H), 1.20 (m, 24H) and
−3.60 (s, 2H). lC: 173.21, 172.78, 171.11, 144.6, 144.0,
140.93, 132.93, 97.80, 61.09, 60.33, 37.11, 32.69, 21.60,
14.99, 13.89. FAB-MS: m/z (%): 1055 [M+H] (82), 1041
[M+2H−Me] (100), 1027 [M+2H−Et] (70) and 982 [M+
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.