Normal and abnormal heme biosynthesis. Part 7. Synthesis and metabolism of coproporphyrinogen-III analogues with acetate or butyrate side chains on rings C and D. Development of a modified model for the active site of coproporphyrinogen oxidase

Article abstract of DOI:10.1016/j.bmc.2010.12.053

Analogues of coproporphyrinogen-III have been prepared with acetate or butyrate groups attached to the C and D pyrrolic subunits. The corresponding porphyrin methyl esters were synthesized by first generating a,c-biladienes by reacting a dipyrrylmethane with pyrrole aldehydes in the presence of HBr. Cyclization with copper(II) chloride in DMF, followed by demetalation with 15% H₂SO₄-TFA and reesterification, gave the required porphyrins in excellent yields. Hydrolysis with 25% hydrochloric acid and reduction with sodium-amalgam gave novel diacetate and dibutyrate porphyrinogens 9. Diacetate 9a was incubated with chicken red cell hemolysates (CRH), but gave complex results due to the combined action of two of the enzymes present in these preparations. Separation of uroporphyrinogen decarboxylase (URO-D) from coproporphyrinogen oxidase (CPO) allowed the effects of both enzymes on the diacetate substrate to be assessed. Porphyrinogen 9a proved to be a relatively poor substrate for CPO compared to the natural substrate coproporphyrinogen-III, and only the A ring propionate moiety was processed to a significant extent. Similar results were obtained for incubations of 9a with purified human recombinant CPO. Diacetate 9a was also a substrate for URO-D and a porphyrinogen monoacetate was the major product in this case; however, some conversion of a second acetate unit was also evident. The dibutyrate porphyrinogen 9b was only recognized by the enzyme CPO, but proved to be a modest substrate for incubations with CRH. However, 9b was an excellent substrate for purified human recombinant CPO. The major product for these incubations was a monovinylporphyrinogen, but some divinyl product was also generated in incubations using purified recombinant human CPO. The incubation products were converted into the corresponding porphyrin methyl esters, and these were characterized by proton NMR spectroscopy and mass spectrometry. The results extend our understanding of substrate recognition and catalysis for this intriguing enzyme and have allowed us to extend the active site model for CPO. In addition, the competitive action of both URO-D and CPO on the same diacetate porphyrinogen substrate provides additional perspectives on the potential existence of abnormal pathways for heme biosynthesis.

Full text of DOI:10.1016/j.bmc.2010.12.053

Bioorganic & Medicinal Chemistry 19 (2011) 1492–1504
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Normal and abnormal heme biosynthesis. Part 7. Synthesis and metabolism
of coproporphyrinogen-III analogues with acetate or butyrate side chains
on rings C and D. Development of a modified model for the active site
of coproporphyrinogen oxidase q
⇑
Timothy D. Lash , Teresa R. Lamm, J. Andy Schaber, Wen-hsiang Chung, Eric K. Johnson, Marjorie A. Jones
Department of Chemistry, Illinois State University, Normal, IL 61790-4160, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Analogues of coproporphyrinogen-III have been prepared with acetate or butyrate groups attached to the
C and D pyrrolic subunits. The corresponding porphyrin methyl esters were synthesized by first generat-
ing a,c-biladienes by reacting a dipyrrylmethane with pyrrole aldehydes in the presence of HBr. Cycliza-
tion with copper(II) chloride in DMF, followed by demetalation with 15% H2SO4–TFA and reesterification,
gave the required porphyrins in excellent yields. Hydrolysis with 25% hydrochloric acid and reduction
with sodium-amalgam gave novel diacetate and dibutyrate porphyrinogens 9. Diacetate 9a was incu-
bated with chicken red cell hemolysates (CRH), but gave complex results due to the combined action
of two of the enzymes present in these preparations. Separation of uroporphyrinogen decarboxylase
(URO-D) from coproporphyrinogen oxidase (CPO) allowed the effects of both enzymes on the diacetate
substrate to be assessed. Porphyrinogen 9a proved to be a relatively poor substrate for CPO compared
to the natural substrate coproporphyrinogen-III, and only the A ring propionate moiety was processed
to a significant extent. Similar results were obtained for incubations of 9a with purified human recombi-
nant CPO. Diacetate 9a was also a substrate for URO-D and a porphyrinogen monoacetate was the major
product in this case; however, some conversion of a second acetate unit was also evident. The dibutyrate
porphyrinogen 9b was only recognized by the enzyme CPO, but proved to be a modest substrate for incu-
bations with CRH. However, 9b was an excellent substrate for purified human recombinant CPO. The
major product for these incubations was a monovinylporphyrinogen, but some divinyl product was also
generated in incubations using purified recombinant human CPO. The incubation products were con-
verted into the corresponding porphyrin methyl esters, and these were characterized by proton NMR
spectroscopy and mass spectrometry. The results extend our understanding of substrate recognition
and catalysis for this intriguing enzyme and have allowed us to extend the active site model for CPO.
In addition, the competitive action of both URO-D and CPO on the same diacetate porphyrinogen sub-
strate provides additional perspectives on the potential existence of abnormal pathways for heme
biosynthesis.
Received 2 November 2010
Revised 20 December 2010
Accepted 23 December 2010
Available online 8 January 2011
Keywords:
Heme biosynthesis
Coproporphyrinogen oxidase
Uroporphyrinogen decarboxylase
Porphyrinogens
Porphyrias
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
regioselectively starting with the acetate group on ring D and then
proceeding to ring A, then B and finally ring C (the ‘clockwise’ decar-
In vertebrates, uroporphyrinogen-III (uro’gen-III, Scheme 1) is
generated in four enzyme mediated steps from glycine and succinyl
CoA.1,2 Uro’gen-III is then acted upon by the cytoplasmic enzyme
uroporphyrinogen decarboxylase (URO-D, EC 4.1.1.37) to produce
the tetracarboxylate species coproporphyrinogen-III (copro’gen-
III).3,4 The four decarboxylations occur sequentially and hepta-,
hexa-, and pentacarboxylate intermediates are formed during this
conversion.3 Under physiological conditions, this process occurs
boxylation pathway).3,5–7 However, there are four possible hepta-,
six hexa- and four pentacarboxylate porphyrinogens that could be
formed from uro’gen-III, and overall there are 24 possible pathways
between uro’gen-III and copro’gen-III.3,5–7 Interestingly, in enzyme
incubation studies using uro’gen-III or specific hepta- or hexacarb-
oxylate porphyrinogens, the process appears to be random and all
of the possible porphyrinogen intermediates can be identified.5–8
Moreover, all 14 possible type III intermediates are metabolized by
URO-D and the type I, type II and type IV isomers of uro’gen are also
substrates for this enzyme.9–11 However, the clockwise decarboxyl-
ation pathway does appear to be dominant at very low substrate
concentrations.5,6
q
For part 6 in the series, see Ref. 33b.
⇑
Corresponding author. Tel.: +1 438 8554; fax: +1 309 438 5538.
E-mail address: tdlash@ilstu.edu (T.D. Lash).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.12.053

T. D. Lash et al. / Bioorg. Med. Chem. 19 (2011) 1492–1504
1493
HO2C
CH3
Me
V
P
Me
P
HO2C
5
Me 2 3
Me
Me
7
8
CO2H
6
4
CH3
P
P
A
5
B
7
3
6
4
N
N
9
N
N
1
N
N
2
1
8
H
H
H
H
H
H
CO2H
CO2H
10
20
HO2C
NH
NH
HN
HN
NH HN
NH HN
9
HH
19
11
N
N
10
11
12
20
19
12Me
C
D
Uroporphyrinogen
Decarboxylase
Me18
Me
16
14
13
17
P
15
CH3
CH3
18
16
14
P
P
13
17
15
HO2C
CO2H
Copro'gen-III
Hardero'gen
CO2H
Coproporphyrinogen-III
CO2H
CO2H
Uroporphyrinogen-III
CO2H
Coproporphyrinogen
Oxidase
V
Me
CH3
CH3
P
Me
CH3
CH3
V
CH3
CH3
Me
Me
V
N
N
N
N
N
N
N
N
NH HN
NH HN
N
N
N
N
H H
H
H
H
H
Fe
H
H
Me
CH3
CH3
CH3
CH3
Me
P
P
P
P
CO2H
CO2H
Protoporphyrinogen-IX
CO2H
CO2H
Proto'gen-IX
Isohardero'gen
Heme b
P = CH2CH2CO2H; V = CH=CH2
Scheme 1. Later stages of heme biosynthesis.
Scheme 2. Conversion of copro’gen-III to proto’gen-IX.
Copro’gen-III is transferred into the mitochondria and undergoes
two oxidative decarboxylations, promoted by coproporphyrinogen
oxidase (CPO, EC 1.3.3.3) to produce proto’gen-IX (Scheme 1).4b,12,13
Subsequent dehydrogenation by protoporphyrinogen oxidase affords
the corresponding porphyrin and iron(II) insertion by ferrochelatase
thengiveshemeb (Scheme 1). Protoporphyrin-IX isalsotheprecursor
to most of the other hemes and chlorophylls.1,2,14 CPO remains one of
the least well understood enzymes in the heme biosynthetic path-
way, in part because the mechanism for the conversion of the A and
B ring propionate groups to vinyl moieties remains controversial.15
CPO is a more selective enzyme than URO-D and only metabolizes
two of the four possible type isomers of coproporphyrinogen.16–20
In addition, the two oxidative decarboxylations are known to take
place sequentially, but only one of the two possible tricarboxylate
intermediates is formed.21–23 The corresponding porphyrin, hard-
eroporphyrin, was first isolated and characterized from the harderian
gland of rodents,24 but this monovinylporphyrin and its metabolites
can be observed in numerous other biological materials, including
urine and feces.25–27 Nevertheless, although isoharderoporphyrino-
gen (Scheme 2) is not formed from copro’gen-III, it is a substrate for
CPO.28 A number of synthetic porphyrinogens have also been shown
to be metabolized by CPO using crude enzyme preparations from
chicken red cell hemolysates (CRH), including mesoporphyrinogen-
VI (1a; meso’gen-VI) where the C and D ring propionate units have
been replaced with ethyl groups (Scheme 3).29,30 Meso’gen-VI and re-
lated porphyrinogens 1b–d with methyl, propyl or butyl groups at
these positions are converted byCPO tothe correspondingmonovinyl
products, but the second oxidative decarboxylation is not ob-
served.13,30,32 However, the specific alkyl residues at positions 13
and 17 do have an impact, as meso’gen-VI is the best substrate, fol-
lowed by 1b and 1c. Porphyrinogen 1d is a very poor substrate and
only gives <10% conversion to 2d. These data demonstrate that larger
nonpolar residues are not easily accommodated by CPO. Two related
tripropionate porphyrinogenswerealsoassessed,31,32 where only one
of the propionate residues was replaced by an ethyl group. Porphyri-
nogen 1e, with a 13-ethyl unit, was a good substrate for CPO and was
again converted into a monvinylic product 2e. However, the 17-eth-
ylporphyrinogen 1f was further metabolized by CPO and afforded
the divinylporphyrinogen product 3f. On the basis of these and re-
lated investigations, it was concluded that a porphyrinogen needs
the sequence of peripheral substituents R Me-P Me-P, where
Me
V
Me
P
Me
P
P
Me
Me
N
N
N
N
N
N
N
H
H
H
H
H
H
H
H
N
Me
Me
Me
R13
R17
R13
R17
2
1
a. R13 = R17 = Et; b. R13 = R17 = Me; c. R13 = R17 = Pr;
d. R13 = R17 = Bu; e. R13 = Et; R17 = P; f. R13 = P; R17 = Et
V
Me
for 2f only
V
Me
Me
N
N
N
H
H
H
H
P = CH2CH2CO2H
V = CH=CH2
N
Me
P
Et
3
Scheme 3. Metabolism of substrate analogues by CPO.
R = Me, vinyl, Et or H and P = CH2CH2CO2H, in order for it to be recog-
nized as a good substrate for CPO.12,13 These requirements were
incorporated into a model for the substrate binding sites in CPO
where three regions were designated (Fig. 1). Region Y corresponds
to the catalytic site where the propionate group is converted into
the vinyl moiety, region X designates a site that requires the presence
of a second propionate side chain for substrate binding, and positionZ
represents a region that can accommodate the presence of small non-
polar groups such as H, Me, vinyl (V) or Et, but not propionate or ace-
tate groupings. However, this model does not account for the
influence of alkyl groups at positions 13 and 17 on the first oxidative
decarboxylation, which is favored when the substituents are Et or Me
(1aand 1b), butlesssofor the dipropyl substrate 1candgreatly inhib-
ited for dibutyl porphyrinogen 1d. The 8-butyrate derivative 4a of co-
pro’gen-III has been shown to be a substrate for CPO preparations
from Euglena gracilis, affording 3-vinylporphyrinogen 5a,34 but does
not appear to be metabolized by enzyme preparations from CRH.35

1494
T. D. Lash et al. / Bioorg. Med. Chem. 19 (2011) 1492–1504
strate for the rat liver enzyme.45 These data show that the acetate
Y
moiety inhibits further conversion by CPO. However, 7 is a sub-
strate for URO-D and can be converted into hardero’gen, which
in turn may be metabolized by CPO to produce proto’gen-IX
(Scheme 5).43 It has been proposed that this route (Scheme 5)
may provide an alternative pathway for heme biosynthesis under
abnormal conditions,43,44 although evidence has recently been pre-
sented that suggests that the uncoupling of CPO within the mito-
chondrial intermembrane space may also be a factor.37d Evidence
for this abnormal pathway has also been reported for mutants of
Saccharomyces cerevisiae.46
X
CO2H
CO2H
CH3
N
H
H
N
CH3
H
N
H
CH3
N
CO2H
CH3
Z
CO2H
In aerobic organisms, CPO is an oxygen-dependent enzyme,
although a structurally unrelated CPO is found in anaerobic
bacteria.47 Oxygen-dependent CPO catalyzes the oxidative decar-
boxylation of two propionate groups to vinyl moieties, but the
presence of transition metal ions are not required and no cofactors
are needed.48 Although several mechanisms for this reaction have
been proposed,4 the most plausible explanation involves a base
catalyzed addition of O2 to generate a peroxide anion, followed
by intramolecular deprotonation and elimination of H2O2 and
CO2.15,49 Recently, a crystal structure for the oxygen-dependent
form of CPO from S. cerevisiae was reported,50 and a similar struc-
ture was also published for human recombinant CPO.51 CPO was
characterized as a homodimer52 that generates a nonpolar cleft
that has been tentatively assigned as the active site.50 Subsequent
site-directed mutagenesis studies demonstrated that the invariant
amino acids aspartate 400, arginine 262 and arginine 401 were
essential for significant catalytic activity,53 and these residues
may be involved in substrate binding and catalysis.53 However,
the details of substrate binding interactions and catalytic oxidative
decarboxylation by CPO remains far from complete.
Considerable progress has been made over the last ten years on
the structure and activity of URO-D7,54 and CPO,13,15,31,49–51,53 but a
detailed understanding of the latter enzyme remains elusive. A full
understanding of the later stages of heme biosynthesis is necessary
to fully appreciate nature’s primary pathways for metabolism,
although this goal also has medicinal implications with regard to
the treatment of porphyrias.55 Defects in URO-D and CPO can result
in porphyria cutanea tarda (PCT) and coproporphyria, respectively,
and these diseases are associated with many pathological symp-
toms, including skin photosensitivity, liver damage and neurologi-
cal problems.55 In order to gain a better understanding of substrate
binding and catalysis for CPO, two analogues of copro’gen-III with
acetate or butyrate groups at rings C and D, 9a and 9b (Fig. 3), were
targeted for study. As porphyrinogens are highly unstable, and
prone to oxidations and acid catalyzed rearrangements, it was nec-
essary to synthesize the corresponding porphyrins 10a and 10b.
Full details on the synthesis of porphyrins 10a and 10b, and the ac-
tion of CPO on porphyrinogens 9a and 9b, are described below.
Figure 1. Active site model for substrate binding by coproporphyrinogen oxidase. Y
is the catalytic site, X recognizes and binds a second propionate residue, and Z can
accommodate only small nonpolar groups like Me, Et, V or H.
However, the branched substituents in porphyrinogens 4b and 4c did
allow these species to be metabolized to a limited extent to give pri-
marily the monovinyl products 5b and 5c, respectively (Scheme 4).35
These data show that extended or branched carboxylate units can be
accommodated at site X in the binding model,12,35 but the modified
porphyrinogens are far less effective substrates for CPO.36
Further support for the substrate binding model can be dis-
cerned from the presence of abnormal isocoproporphyrin-type
metabolites 6 (Fig. 2) in the urine and feces of humans suffering
from porphyria cutanea tarda (PCT) and from rats that have been
poisoned with hexachlorobenzene.37 These metabolites were des-
ignated as isocoproporphyrins because the most common member
of the series, 3-ethylporphyrin 6 (R = Et), has the same molecular
weight as coproporphyrin-III.38 The identity of these compounds
was confirmed by total synthesis39–41 and detailed spectroscopic
investigations.42 It was hypothesized that the usual pentacarboxy-
late intermediate, 5dab, formed by the previous enzyme URO-D,
was metabolized out of sequence by CPO to generate dehy-
droisocoproporphyrinogen (7; Scheme 5),43,44 and that this species
gives rise to the observed porphyrins 6. Pentacarboxylate por-
phyrinogen 5dab has been shown to be a substrate for rat liver
CPO and highly purified human CPO, but only the A ring propionate
residue undergoes oxidative decarboxylation to afford 7 and the
hypothetical divinyl metabolite 8 is not seen (Scheme 5).43,44 Syn-
thetic isocoproporphyrinogen has also been shown to not be a sub-
Me
V
Me
P
Me
Me
R
R
Me
Me
N
N
N
N
N
N
N
H
H
H
H
H
H
H
H
N
Me
Me
P
P
P
P
4
5
a. R = B; b. R = CH(CH3)CH2CO2H; c. R = CH2CH(CH3)CO2H
2. Results and discussion
B = (CH2)3CO2H; P = CH2CH2CO2H; V = CH=CH2
Scheme 4. Metabolism of porphyrinogens with modified side chains on ring B.
2.1. Synthesis of substrates as porphyrin methyl esters
Although porphyrin diacetate 10a has not been reported previ-
ously, dibutyrate 10b was synthesized some years ago.56,57 This
earlier work indicated that 9b could be used to probe the enzyme
CPO, but no further information was provided.56 We elected to car-
ry out the synthesis of 9a and 9b using the a,c-biladiene strat-
egy.58,59 A retrosynthetic analysis (Scheme 6) shows that the
syntheses can be accomplished using dipyrrylmethane 11 and dial-
dehydes 12a and 12b. We have made extensive use of dipyrrylme-
thane 11 in our earlier studies,13,31 so it only remained for us to
prepare pyrrole aldehydes 12. These were prepared from pyrrole
esters that were in turn generated using Knorr-type chemistry.60
R
Me
P
Me
Me
N
N
N
N
A = CH2CO2H
P = CH2CH2CO2H
V = CH=CH2
H
H
A
P
P
6
R = V, Et, H, CH(OH)CH3 or COCH3
Figure 2. Isocoproporphyrin series.

T. D. Lash et al. / Bioorg. Med. Chem. 19 (2011) 1492–1504
1495
V
Me
V
Me
Me
P
Me
Me
P
A
P
V
Me
Me
Me
Me
N
N
N
N
N
N
N
N
N
N
N
H
H
H
H
H
H
H
H
H
H
H
CPO
H
N
A
A
P
P
P
P
P
V
P
V
5dab
7
8
URO-D
Me
URO-D
Me
Me
P
Me
Me
P
P
V
Me
Me
Me
Me
N
N
N
N
H
N
N
N
N
H
H
H
H
H
H
CPO
CPO
H
H
H
H
H
N
N
N
N
Me
Me
Me
P
P
P
P
P
P
Proto'gen-IX
Copro'gen-III
Hardero'gen
A = CH2CO2H; P = CH2CH2CO2H; V = CH=CH2
Scheme 5. Normal and abnormal pathways between 5dab and hardero’gen.
PMe
Me
CO2Et
O
Me
P
PMe
Me
Me
Me
P
BrCH2CO2Et
N
N
N
N
N
N
N
H
H
H
H
O
O
K2CO3/Acetone
O
H
H
14
Me
N
Zn/NaOAc
AcOH
Me
Me Me
O
N
CO2Et
R
R
R
R
Me
b. R = BMe
Me
9
a. R = A b. R = B
A = CH2CO2H
P = CH2CH2CO2H
B = (CH2)3CO2H
10
a. R = A
CO2Bn
A
Me = CH2CO2Me
PMe = CH2CH2CO2Me
BMe = (CH2)3CO2Me
CO2Bn
13a
Me
HO
N
H
15
Scheme 7. Synthetic of a pyrrole with an acetate side chain.
Figure 3. New substrate analogues for CPO and the related porphyrin tetramethyl
esters.
butyrate side chain was prepared by a longer but more efficient
route, again starting with 2,4-pentanedione (Scheme 8). Reaction
of 2,4-pentanedione with ethyl 4-bromobutanoate, sodium iodide
PMe
Me
PMe
Me
PMe
Me
Me
Me
PMe
N
N
H
H
NH
N
N
CO2t-Bu
CHO
HO2C
OHC
CO2Et
11
HN
NH
HN
Me
Br(CH2)3CO2Et
Me
R
Me
Me
12
Me
O
O
K2CO3/NaI
Butanone
R
R
R
10
Δ
O
O
16
a. R = AEt; b. R = B Me
AEt =CH2CO2Et
PMe = CH2CH2CO2Me
BMe = (CH2)3CO2Me
AcOH
CO2Et
CO2Et
Δ
H2N CH
CO2Et
Me
CO2Et
17
NaOBn
Scheme 6. Retrosynthetic analysis of porphyrins 10a and 10b.
Me
CO2Bn
N
BnOH
H
Pyrrole 13a with an acetate side chain was prepared in two
steps from 2,4-pentanedione (Scheme 7). Alkylation of the dike-
tone with ethyl bromoacetate and potassium carbonate in reflux-
ing acetone gave ester 14 in 69% yield and this was further
reacted with oxime 15 and zinc dust in acetic acid to give 13a in
33% yield. This relatively low yield is typical of classical Knorr
chemistry,60 but nevertheless still enables significant amounts of
the intermediate to be prepared from reasonably inexpensive
starting materials. The pyrrole is also obtained with a benzyl ester
unit that can be selectively deprotected prior to the formation of
the required pyrrole aldehyde. The corresponding pyrrole with a
18
Δ
Me
CO2Bn
CO2Me
Me
N
H
19
Me
H2SO4
MeOH
Me
CO2Bn
13b
N
H
Scheme 8. Synthesis of pyrroles with butyrate side chains.

1496
T. D. Lash et al. / Bioorg. Med. Chem. 19 (2011) 1492–1504
and potassium carbonate in refluxing methyl ethyl ketone gave the
diketo ester 16 in 76% yield. This was reacted with diethyl amino-
malonate (17)61,62 under modified Kleinspehn conditions61–63 to
give the pyrrole diester 18 in 66% yield. It was then necessary to
the final product. Porphyrins 10a and 10b were characterized by
UV–vis, proton NMR and carbon-13 NMR spectroscopy, and mass
spectrometry. The UV–vis absorption spectra for the diacetate 10a
showed minor bathochromic shifts (0–2 nm) compared to 10b. For
the free base forms in 1% triethylamine-chloroform, 10a gave a kmax
value for the Soret band at 401 nm, and Q bands at 499, 533, 569 and
623 nm; these values were 400, 499, 533, 567 and 621 nm, respec-
tively for dibutyrate 10b. In 1% TFA–chloroform, the corresponding
diprotonated porphyrin dications 10H22þ were formed and these
gave sharper and more intense Soret bands at 400 and 401 nm for
10aH22þ and 10bH22þ, respectively.
The macrocycles were conveniently prepared as porphyrin
methyl esters 10, but needed to be hydrolyzed with 25% hydro-
chloric acid and reduced with 3% sodium amalgam to give the re-
quired porphyrinogen substrates 9.13,65 These were generated
immediately prior to the biochemical studies. The products of
metabolism were isolated as the fully conjugated porphyrins and
converted back into the methyl ester derivatives prior to analysis.
selectively introduce a benzyl ester at the
a-position. Reaction of
18 with sodium benzyloxide in benzyl alcohol gave dibenzyl ester
19, and this was treated with 5% sulfuric acid in methanol to give
the required mixed ester 13b in good overall yields (Scheme 8).
Pyrrole benzyl esters 13a and 13b were deprotected by hydrog-
enolysis over 10% Pd/C to give the related carboxylic acids 20
(Scheme 9). Treatment with TFA decarboxylated these pyrroles,
and subsequent reaction with triethyl orthoformate introduced
the required formyl units to give pyrroles 12 in >70% yield. Dipyrrole
11 (Scheme 10) was treated with TFA for 10 min to cleave the tert-
butyl ester protective group and decarboxylate both terminal
carboxylate units. Two equiv of pyrrole aldehyde 12a or 12b were
then added, followed immediately by HBr in acetic acid, and the
corresponding a,c-biladienes were subsequently precipitated with
ether as the dihydrobromide salts 21 (Scheme 10). Cyclization with
copper(II) chloride in DMF at room temperature64 gave the porphy-
rin products as copper(II) complexes, but these could easily be
demetalated with 15% H2SO4 in TFA and reesterified with 5%
H2SO4 in methanol, and the tetramethyl esters 10 were isolated in
excellent yields. It is worth noting that the acetate side chains were
introduced in a,c-biladiene 21a as the ethyl esters, but following the
final treatment with H2SO4–MeOH the tetramethyl ester 10a was
2.2. Action of CPO and URO-D on the diacetate substrate 9a
Initial biochemical studies were conducted using chicken red
cell hemolysates (CRH). CRH is a convenient source of active CPO
and was used in many of our earlier investigations.13,31–33,35 How-
ever, incubations of 9a with CRH gave complex results that were
not consistent with straightforward metabolism by CPO. CRH con-
tains endogenous protoporphyrin-IX (proto-IX), but HPLC analysis
of the incubation products for 9a showed the formation of several
additional products. CRH also contains URO-D and we speculated
that porphyrinogen 9a might also be a substrate for this enzyme.
Indeed, this type of conversion would parallel the processes that
are believed to lead to the generation of isocoproporphyrins (see
earlier).44 Although a peak could be identified as the expected
monovinyl product 22a, a larger slightly less polar peak was al-
ways present in these extracts. If URO-D can act on 9a, two mono-
acetate porphyrinogens, 23a and 23b, could be formed (Scheme
11). These in turn could be further processed by CPO to give 24a
and 24b, although the substrate binding model suggests that only
23a should be a substrate, while further action by URO-D would
give the hexamethylporphyrinogen 25. Further action by either en-
zyme could lead to a number of other products (Scheme 11). The
predominant porphyrin product was separated by flash chroma-
tography. Although NMR data could not be obtained, the sample
was successfully analyzed by FAB MS and gave a quasimolecular
ion at m/z 625. FAB MS for porphyrins always gives the M+H peak,
and this means that the sample has a molecular weight of 624.
High resolution MS data gave a value for m/z of 625.3029, which
is an excellent match for the formula C36H40N4O6+H (calcd m/z
625.3026), and corresponds to the expected porphyrin trimethyl
esters derived from porphyrinogens 23a and 23b. Although the
precise identity of this product cannot be discerned (23a, 23b, or
a mixture of both), these data strongly imply that URO-D is acting
on this substrate. A second decarboxylation by URO-D to 25, or the
further metabolism of 23a or 23b by CPO, may be occurring, but
these and related processes appear to be slow in comparison to
the formation of the initial metabolites. In order to gain further in-
sights, incubations were carried out in the presence of N-ethylma-
leimide (NEM). NEM is known to be an inhibitor of URO-D66 and it
is notable that the incubations showed an increase in the peak for
the monovinyl product compared to the monoacetate peak corre-
sponding to 22a and/or 22b. Further support for this analysis
was made by carrying out incubations with mitochondrial prepara-
tions derived from CRH. As URO-D is associated with the cyto-
plasm, and CPO is found in the mitochondria, these preparations
would be expected to give 22a but not 23a or 23b. Incubations
of 9a with these mitochondrial preparations did indeed primarily
R
Me
Me
R
H2
Pd/C
Me
CO2H
20
Me
CO2Bn
13
N
H
N
H
R
Me
CHO
a. R = AEt; b. R = B Me
TFA
CH(OEt)3
Me
N
H
12
AEt =CH2CO2Et; BMe = (CH2)3CO2Me
Scheme 9. Synthesis of pyrrole aldehydes.
PMe
Me
Me
OHC
PMe
R
Me
HO2C
+ 2
N
N
N
H
H
H
CO2t-Bu
Me
12
11
a. R = AEt; b. R = B Me
1. TFA
2. HBr
PMe
Me
PMe
2 Br
Me
Me
N
N
H
H
1. CuCl2-DMF
H
H
N
N
2. 15% H2SO4-TFA
3. 5% H2SO4-MeOH
Me
Me
Me
R
R
21 a. R = AEt
PMe
Me
b. R = BMe
PMe
Me
NH
N
N
AEt = CH2CO2Et
AMe =CH2CO2Me
HN
PMe = CH2CH2CO2Me
Me
Me
B
Me = (CH2)3CO2Me
R
R
10 a. R = AMe; b. R = B Me
Scheme 10. Synthesis of porphyrins via a,c-biladienes.

T. D. Lash et al. / Bioorg. Med. Chem. 19 (2011) 1492–1504
1497
V
Me
Me
V
Me
P
Me
Me
P
Me
Me
Me
Me
V
P
N
N
N
N
N
N
N
N
N
N
N
N
H
H
H
H
H
H
H
H
CPO
CPO
CPO
CPO
H
H
H
H
Me
Me
Me
A
Me
V
Me
A
Me
V
A
24a
URO-D
23a
URO-D
URO-D
Me
Me
P
Me
P
Me
N
V
Me
Me
Me
Me
P
P
Me
Me
Me
Me
P
N
N
N
N
N
N
N
N
N
N
N
N
H
H
H
H
H
H
H
HH
H
H
H
H
H
H
CPO
H
N
N
N
Me
Me
Me
Me
A
A
A
A
V
A
9a
Me
A
V
Me
22a
26a
25
URO-D
URO-D
Me
URO-D
P
Me
Me
N
Me
Me
P
Me
Me
P
Me
Me
V
N
N
N
N
N
N
N
H
H
N
H
H
H
H
H
H
H
H
H
N
H
N
N
Me
Me
Me
A
Me
Me
A
Me
A
23b
24b
A = CH2CO2H; P = CH2CH2CO2H; V = CH=CH2
Scheme 11. Multiple pathways for the metabolism of porphyrinogen 9a due to the action of Uro-D and CPO.
show a product peak corresponding to the monovinyl porphyrin
and the results were essentially unchanged when the experiments
were repeated in the presence of NEM. However, the mitochondrial
preparations retained some contamination with URO-D and the re-
sults were difficult to reproduce.
In order to obtain cleaner data and deconvolute the results for
the two enzymes, CRH derived URO-D and CPO were separated
using porphyrin affinity chromatography.67 In addition, purified
human recombinant CPO from Escherichia coli was isolated using
the procedure reported by Medlock and Daily.48,68 For kinetic stud-
ies, the incubation products were extracted using a microassay69
and analyzed by HPLC. Incubations of 9a with the URO-D contain-
ing fraction showed monoacetate 23 as the major product, but a
small peak corresponding to the porphyrin derived from 25 can
also be seen (Fig. 4). Incubations of 6a with CRH-derived CPO or
human recombinant CPO gave rise to a different product peak
corresponding to the monovinyl porphyrin (Fig. 4), confirming
our interpretations of the earlier data. Porphyrinogen 9a is a rea-
sonable substrate for URO-D but appears to be a comparatively
poor substrate for CPO. This is surprising because meso’gen-VI
(1a), and two related porphyrinogens 1b and 1c (Scheme 3), are
much better substrates for CPO even though 9a is structurally
more closely related to the natural substrate copro’gen-III. Trace
amounts (<1%) of a nonpolar porphyrin corresponding to divinyl
product 26 appear to be generated in longer time incubations with
human recombinant CPO (detected by TLC and HPLC), and the data
indicate that the second oxidative decarboxylation step is virtually
shut down for this substrate. This demonstrates that an acetate
group is poorly tolerated at binding site X in the active site model
(Fig. 1).
Figure 4. HPLC traces showing the porphyrin methyl ester products for a 30 min
incubation of 9a with purified recombinant human CPO (red line) and 2 h
incubation with URO-D (blue line). The URO-D fraction was isolated from CRH using
porphyrin affinity chromatography.67 The HPLC analysis was performed on
normal phase 5 L injection loop, with
a
a
l
partisil column (250 ꢀ 4.6 mm), using a 20 l
a mobile phase of 35/65 v/v ethyl acetate–cyclohexane. The UV–vis detector was set
for a wavelength of 404 nm.
150 min (Fig. 5) while CPO produced a maximum product of
35.7 1.2 after 30 min (Fig. 6). The metabolic conversions occur-
ring for 9a with the purified enzymes are summarized in Scheme
12. In both cases, two different side chains might be metabolized,
but only the first conversion is favored. URO-D does appear to rec-
ognize and convert the first formed products 23, but the second
decarboxylation only occurs to a limited extent to form 25.
Kinetic studies were conducted in triplicate using URO-D and
purified human CPO (Figs. 5 and 6). Incubations of URO-D with
9a gave
a maximum product formation of 49.5 1.9% after

1498
T. D. Lash et al. / Bioorg. Med. Chem. 19 (2011) 1492–1504
a. R = AMe, R1 = PMe
b. R = BMe, R1 = PMe
c. R = BMe, R1 = V
V
Me
R1
Me
N
N
N
H
AMe = CH2CO2Me
H
N
P
Me = CH2CH2CO2Me
Me
Me
BMe = (CH2)3CO2Me
V = CH=CH2
R
R
27
Figure 7. Porphyrin products isolated from incubations of porphyrinogens 9a and
9b with CPO.
Figure 5. Kinetic study for incubations of 9a with the CRH URO-D fraction showing
% product formed with time in min.
6.4
8.4
ppm
ppm
10.0
ppm
Figure 8. Partial 400 MHz proton NMR spectrum of the monovinylporphyrin
product 27a isolated from incubations of 9a with purified recombinant human CPO.
The figure shows the downfield region with four singlets for the meso-protons
between 10.0 and 10.2 ppm and resonances for the vinyl moiety at 6.2, 6.4 and
8.3 ppm.
MS results gave a value of m/z 623.2870, which closely matches
the expected value for C36H38N4O6+H.
2.3. Action of CPO on the dibutyrate substrate 9b
Figure 6. Kinetic study for incubations of 9a with purified human CPO showing %
product formed with time in min.
The metabolism of the dibutyrate porphyrinogen 9b was also
investigated using CRH and purified recombinant human CPO.
However, these studies were greatly simplified because 9b is not
affected by URO-D and even crude preparations of CRH only re-
sulted in products due to the catalytic action of CPO. In the initial
studies using CRH, it became clear that 9b is also a mediocre sub-
strate for CPO, although it gave better results than 9a. However,
porphyrinogen 9b proved to be an excellent substrate for purified
human CPO producing 22b as the major metabolic product
(Scheme 13). In the initial studies using CRH, a quasimolecular
ion for the major porphyrin product was obtained using FAB MS
However, as noted above, virtually no conversion of 22a to the
divinyl product 26 by CPO is seen in these studies. Finally, a sample
of the monovinylporphyrin 27a (Fig. 7) derived from 22a was iso-
lated from incubations of 9a with purified human CPO. Poor con-
versions were obtained in these preparative experiments but it
was possible to obtain a low quality 400 MHz proton NMR spec-
trum for this product, and this allowed the presence of a vinyl moi-
ety to be identified ( Fig. 8). Electrospray ionization mass
spectrometry gave an M+H peak at m/z 623. The high resolution
V
Me
V
Me
P
Me
V
Me
Me
Me
Me
P
Me
Me
P
N
N
N
N
N
N
N
N
N
N
N
N
H
H
H
CPO
H
H
H
CPO
<1%
H
H
H
H
HH
Me
Me
Me
A
9a
A
A
A
A
A
26a
22a
URO-D
Me
Me
P
P
Me
Me
P
Me
Me
P
N
N
N
N
N
N
H
H
H
H
H
H
H
URO-D
H
N
N
Me
Me
R2
R1
R2
R1
23
24
A = CH2CO2H; P = CH2CH2CO2H; V = CH=CH2
Scheme 12. Metabolism of porphyrinogen 9a with partially purified CPO and Uro-D.

T. D. Lash et al. / Bioorg. Med. Chem. 19 (2011) 1492–1504
Table 1
1499
V
B
Me
Me
P
Kinetic values obtained with human recombinant CPO using three different
substrates under initial velocity conditions
P
Me
Me
Me
Me
P
N
N
N
N
N
N
N
H
H
H
H
H
Substrate
Km (mM)
Kcat
Kcat/Km
H
H
H
Copro’gen-III68a
9b
9a
0.68
0.76
6.8
3.6
0.90
0.004
5.3
1.2
0.00059
N
Me
Me
B
N
B
B
22b
9b
V
B
Me
CPO and porphyrinogen substrates at concentrations ranging from
0.1 to 10 M obtained under initial velocity conditions. The Km val-
V
Me
Me
N
l
B = (CH2)3CO2H
P = CH2CH2CO2H
V = CH=CH2
H
H
H
ues using copro’gen-III and 9b were essentially the same, while the
value for 9a was about 10 fold higher indicating that the diacetate
substituents fit less well into the active site. The catalytic efficien-
cies (Kcat/Km values) using the three substrates indicate that the
authentic substrate is processed most efficiently and that 9b is al-
most as well processed for the first catalytic step. However, 9a
showed a substantially increased Km value and a reduced Kcat/Km
value, results that strongly suggest that the substrate fit of 9a is
compromised as well as the enzyme’s ability to perform the oxida-
tive decarboxylation reaction.
A preparative experiment was carried out by incubating 9b with
purified recombinant human CPO and the porphyrin products 27b
and 27c (Fig. 8) were separated by flash chromatography. The ma-
jor porphyrin product 27b was characterized by 400 MHz proton
NMR spectroscopy (Fig. 11). The spectrum showed four 1H singlets
for the meso-protons between 10.1 and 10.3 ppm, and three 1H
resonances for the vinyl group at 6.18, 6.38 and 8.32 ppm. The four
porphyrin methyl groups and the three ester methoxy units gave
rise to five 3H singlets and one 6H singlet between 3.6 and
3.8 ppm, and the remaining peaks were also consistent with the
proposed structure 27b. High resolution electron impact mass
spectrometry gave a value for the molecular ion of m/z 678.3415,
closely matching the expected value for C40H46N4O6 (m/z calcd
678.3417). A minor fraction corresponding to the divinylporphyrin
27c was also isolated. It was possible to obtain a low quality proton
NMR spectrum for 27c in CDCl3 that showed the presence of two
vinyl moieties. In addition, high resolution electrospray ionization
mass spectrometry gave a value for the M+H species of m/z
619.3303. This was a good match for the expected molecular for-
mula C38H42N4O6+H (m/z calcd 619.3284).
H
N
N
Me
B
26b
Scheme 13. Metabolism of porphyrinogen 9b with CPO.
at m/z 679. This gave a high resolution peak at m/z 679.3499, which
closely matches the calculated value for C40H46N4O6+H (m/z calcd
679.3496). A time course study for incubations of 9b with CRH
gave only ca. 20% metabolism after 30 min (Fig. 9), but incubations
with purified recombinant human CPO showed much better con-
versions ( Fig. 10). This shows the rapid generation of 22b and
the slow formation of the divinyl product 26b (Scheme 13). The
maximum% product formation (22b + 26b) exceeded 80%, although
about 5% conversion to 26b was observed after 30 min. These re-
sults indicate that unlike the acetate units in 9a, the butyrate side
chains in 9b are a good fit for the first oxidative decarboxylation by
CPO. However, butyrate groups are not very effective in replacing
the propionate residues in binding to site X, and this limits metab-
olism of the second propionate moiety.
Table 1 shows the kinetic constants calculated for 9a, 9b and
the natural substrate copro’gen-III using human recombinant
8.4
10.5
10.0
ppm
6.4
ppm
ppm
ppm
Figure 9. Kinetic study for incubations of 9b with CRH showing % product 22b
(monovinyl) formed with time in min. No more than a trace of 26b (divinyl) was
formed in these experiments.
-3.5
4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8
ppm
Figure 10. Kinetic study for incubations of 9b with purified human CPO showing %
Figure 11. 400 MHz proton NMR spectrum of the monovinylporphyrin product
isolated from incubations of 9b with purified human CPO in CDCl3.
products 22b (monovinyl
) and 26b (divinyl d–d–d) formed with time.

1500
T. D. Lash et al. / Bioorg. Med. Chem. 19 (2011) 1492–1504
These results further extend our understanding of the substrate
Y
recognition requirements for CPO and may help to solve the issue
of how these substrates associate with this enzyme. In addition,
the observation that diacetate 9a can act as a substrate for URO-
D, as well as for CPO, provides additional insights into the viability
of abnormal pathways in heme biosynthesis.44
X
CO2H
CO2H
CH3
B
N
H
A
N
CH3
H
N
H
CH3
C
H
N
CO2H
D
4. Experimental section
4.1. General methods
CH3
N
Z
CO2H
M
Ethyl bromoacetate, ethyl 4-bromobutanoate, 2,4-pentanedi-
one, benzyl acetoacetate, diethyl malonate, benzyl alcohol, 30%
HBr-acetic acid, copper(II) chloride, triethylamine, N,N-dimethyl-
formamide and 10% palladium/charcoal were purchased from
Aldrich or Acros, and were used without further purification. Tri-
ethyl orthoformate was distilled immediately prior to use. Chroma-
tography was performed using grade 3 neutral alumina or 70–230
mesh silica gel. Melting points were determined in open capillary
tubes using a Mel-Temp apparatus and are uncorrected. UV–Vis
absorption spectra were run on a Varian Cary Spectrophotometer.
Proton and carbon-13 NMR data were obtained on a Varian Gemini
400 MHz FT NMR spectrometer or a 500 MHz Bruker NMR Avance III
spectrometer. Mass spectral determinations were conducted at the
Mass Spectral Laboratory, School of Chemical Sciences, University of
Illinois at Urbana-Champaign, and elemental analyses were
obtained from the School of Chemical Sciences Microanalysis Labo-
ratory at the University of Illinois.
Figure 12. Modified model for the active site of CPO showing two new regions (M
and N). These sites accommodate propionate, butyrate, methyl, ethyl and to a lesser
extent propyl groups, but show less tolerance for acetate units.
3. Conclusions
Replacement of the C and D ring propionate residues of copro’-
gen-III with acetate groups greatly reduces substrate recognition
and catalysis by either chicken or human CPO, and gives a com-
paratively low rate of conversion to a monovinylporphyrinogen.
Although copro’gen-III undergoes two oxidative decarboxylations,
the diacetate porphyrinogen 9a gives no more than trace amounts
of the divinyl product. A related dibutyrate 9b was a much better
substrate and was processed at a similar rate to the natural sub-
strate copro’gen-III by human recombinant CPO. Incubations with
CRH gave poorer conversions, possibly due in part to minor differ-
ences in the binding requirements for the avian and human en-
zymes. Porphyrinogen 9b again gave mostly the monovinyl
product, but in this case a more significant amount of divinyl
product was generated, albeit with a maximum product conver-
sion of less than 10%. Previously, porphyrinogens with alkyl
groups (Me, Et, Pr or Bu) at positions 13 and 17 were shown to
be substrates, affording the corresponding monovinyl porphyrin-
ogens.13,32 In those studies, the rates of conversions decreased
as the alkyl group size increased from ethyl to propyl to butyl.
This is probably not due to steric factors, as butyl groups are no
larger than the propionate groups found in copro’gen-III, but this
indicates that larger nonpolar groups cannot be tolerated at these
positions. Figure 12 shows a modified model for the active site,
designating M and N as the substrate binding sites that would
accommodate the 13 and 17-substituents. The new data demon-
strate that the larger butyrate group still fits into these sites very
easily but the smaller acetate groups in 6a do not. This suggests
that these sites favor nonpolar chains close to the macrocycle,
but a polar carboxylate species is needed for longer chains to
be a good fit. This would make sense if the environment immedi-
ately next to the porphyrinogen was lipophilic while a more polar
environment is present further into the pockets described by M
and N. The second oxidative decarboxylation requires the sub-
strate to be rotated through 90° so that the B ring propionate unit
can be placed at the catalytic site Y.13 In copro’gen-III, the C ring
propionate binds to site X and facilitates further metabolism.13
The results in this paper show that butyrate groups can still fit
into site X, in accord with earlier results for E. gracilis,34 but the
unit considerably reduces conversion to the divinyl product. An
acetate group at this position has an even larger negative effect
and allows at best only trace amounts of divinyl porphyrinogen
to be formed. These data demonstrate the substrate binding at
position X shows the following preferences, although propionate
groups are highly preferred.
4.2. Synthetic procedures
4.2.1. Benzyl 4-ethoxycarbonylmethyl-3,5-dimethylpyrrole-2-
carboxylate (13a)
Ethyl bromoacetate (183.4 g, 1.09 mol) was added to a stirred
mixture of 2,4-pentanedione (150.0 g, 1.50 mol), anhydrous potas-
sium carbonate (200 g) and acetone (200 mL) in a 1 L three necked
round bottom flask equipped with a reflux condenser and a
mechanical stirrer. The resulting mixture was stirred under reflux
for 1.5 h. The inorganic salts were removed by suction filtration
and the solvent evaporated under reduced pressure. The residue
was distilled to give ethyl 3-acetyl-4-oxopentanoate (14, 140.7 g,
0.756 mol, 69%) as a pale yellow oil, bp 158–160 °C at 0.05 torr.
A solution of sodium nitrite (106 g) in water (250 mL) was
added dropwise to a solution of benzyl acetoacetate (172 g,
0.896 mol) in acetic acid (300 mL), maintaining the temperature
of the reaction mixture below 15 °C with the aid of a salt-ice bath.
The resulting solution was stirred at room temperature overnight
affording a solution of the corresponding oxime. Ethyl ester 11
(140.7 g, 0.756 mmol) in acetic acid (300 mL) was heated to
70 °C, and the oxime solution was added dropwise while simulta-
neously adding a mixture of zinc dust (200 g) and sodium acetate
(96 g), adjusting the rate of addition so that the reaction tempera-
ture remained between 80 and 90 °C. After the addition was com-
plete, the mixture was heated for 1 h on a boiling water bath. The
mixture was cooled to 70 °C and poured into 4 L of ice-water. The
precipitate was collected by suction filtration, and then taken up in
hot ethanol and filtered to remove zinc and other inorganic mate-
rials. The solvent was removed under reduced pressure and the
residue recrystallized twice from ethanol to give the title pyrrole
(78.10 g, 0.248 mol, 33%) as off-white crystals, mp 81.5–83 °C (lit.
mp70 79–80 °C); IR (Nujol mull):
m 3322 (s, NH str.), 1734 (s, ace-
tate C@O str.), 1668 (s, pyrrole-C@O str.); 1H NMR (400 MHz,
CDCl3): d 1.24 (3H, t, J = 7.2 Hz), 2.22 (3H, s), 2.30 (3H, s), 3.36
(2H, s), 4.11 (2H, q, J = 7.2 Hz), 7.30–7.42 (5H, m), 8.80 (1H, br s);
CH2CH2CO2H ꢁ ðCH2Þ3CO2H ꢁ CH2CO2H or alkyl groups

T. D. Lash et al. / Bioorg. Med. Chem. 19 (2011) 1492–1504
1501
13C NMR (CDCl3): d 10.9, 11.8, 14.4, 30.5, 60.8, 65.7, 115.1, 117.1,
128.26, 128.30, 128.4, 128.7, 131.3, 136.9, 161.5, 171.8.
tion, the solvent evaporated, and the oily residue crystallized from
90% ethanol–water to give the mixed ester pyrrole (36.56 g,
0.111 mol, 90%) as white crystals, mp 66.5–67.5 °C (lit. mp34
4.2.2. Ethyl 4-ethoxycarbonylpropyl-3,5-dimethylpyrrole-2-
carboxylate (18)
66.5–67.5 °C); IR (Nujol mull): m 3319 (s, NH str.), 1729 (s, butyrate
C@O str.), 1666 cmꢂ1 (s, pyrrole-C@O str.); 1H NMR (500 MHz,
CDCl3): d 176 (2H, quintet, J = 7.5 Hz), 2.18 (3H, s), 2.28 (3H, s),
2.30 (2H, t, J = 7.4 Hz), 2.40 (2H, t, J = 7.5 Hz), 3.66 (3H, s), 5.30
(2H, s), 7.30–7.43 (5H, m), 8.84 (1H, br s); 13C NMR (CDCl3): d
10.9, 11.6, 23.4, 25.9, 33.5, 51.6, 65.6, 116.7, 121.2, 127.8, 128.18,
128.20, 128.7, 130.4, 136.8, 161.5, 174.2. Anal. Calcd for
Ethyl 4-bromobutanoate (25.00 g, 0.128 mol) was added to 2,4-
pentanedione (12.8 g, 0.128 mol), anhydrous potassium carbonate
(17.8 g), sodium iodide (19.2 g) and butanone (400 mL), and the
resulting mixture was heated under reflux for 16 h while stirring
vigorously with the aid of a mechanical stirrer. The inorganic salts
were removed by suction filtration and the solvent evaporated un-
der reduced pressure. The residue was distilled to give ethyl 5-
acetyl-6-oxoheptanoate (16, 20.85 g, 97.4 mmol, 76%) as a pale yel-
low liquid, bp 97–105 °C at 0.5 torr.
C19H23NO4: C, 69.28; H, 7.04; N, 4.25. Found: C, 69.17; H, 6.55;
N, 4.18.
4.2.5. 4-Ethoxycarbonylmethyl-3,5-dimethylpyrrole-2-carboxylic
acid (20a)
A mixture of the foregoing diketone 16 (20.85 g, 97.4 mmol)
and diethyl aminomalonate62 (17.0 g, 97 mmol) were added in a
steady stream to gently refluxing acetic acid (55 mL). The resulting
solution was stirred under reflux for 2.5 h. The mixture was cooled
to 70 °C and poured into ice-water (500 mL). The precipitate was
allowed to stand overnight, and then was suction filtered and
recrystallized from ethanol-water to give pyrrole 18 (18.09 g,
64.4 mmol, 66%) as off-white crystals, mp 63–64 °C; IR (Nujol
Benzyl ester 13a (10.00 g, 31.7 mmol) was placed in a hydroge-
nation vessel and dissolved in methanol (200 mL). Triethylamine
(20 drops) was then added, the air was displaced by a stream of
nitrogen, and 250 mg of 10% palladium-charcoal was added. The
mixture was shaken at room temperature under an atmosphere
of nitrogen at 30 psi for 16 h. The catalyst was removed by suction
filtration, and the solvent removed under reduced pressure. The
residue was taken up in 5% aqueous ammonia, cooled in a salt
ice bath, and then neutralized with dilute hydrochloric acid while
maintaining the temperature <10 °C. The resulting precipitate was
suction filtered, washed well with water, and dried in a vacuum
desicator overnight. The pyrrole carboxylic acid (6.35 g, 28.2 mmol,
89%) was isolated as a pink powder, mp 137 °C, dec; 1H NMR
(400 MHz, DMSO-d6): d 1.14 (3H, t, J = 7.2 Hz), 2.09 (3H, s), 2.13
(3H, s), 3.30 (2H, s), 4.01 (2H, q, J = 7.2 Hz), 11.05 (1H, br s),
11.84 (1H, br s); 13C NMR (DMSO-d6): d 11.0, 11.5, 14.8, 30.4,
60.6, 114.4, 117.4, 126.7, 131.5, 162.9, 172.0. Anal. Calcd for
mull):
m
3304 (s, NH str.), 1729 (s, butyrate C@O str.), 1650 cmꢂ1
(s, pyrrole-C@O str.); 1H NMR (500 MHz, CDCl3): d 1.24 (3H, t,
J = 7.1 Hz), 1.34 (3H, t, J = 7.1 Hz), 1.75 (2H, quintet, J = 7.5 Hz),
2.19 (3H, s), 2.26 (3H, s), 2.28 (2H, t, J = 7.4 Hz), 2.40 (2H, t,
J = 7.6 Hz), 4.11 (2H, q, J = 7.1 Hz), 4.29 (2H, q, J = 7.1 Hz), 8.80
(1H, br s); 13C NMR (CDCl3): d 10.8, 11.6, 14.4, 14.8, 23.5, 26.0,
33.8, 59.8, 60.4, 117.1, 121.1, 127.3, 130.0, 162.0, 173.8. Anal. Calcd
for C15H23NO4: C, 64.04; H, 8.24; N, 4.98. Found: C, 64.27; H, 7.91;
N, 5.01.
4.2.3. Benzyl 4-benzyloxycarbonylpropyl-3,5-dimethylpyrrole-
2-carboxylate (19)
C11H15NO4: C, 58.66; H, 6.71; N, 6.22. Found: C, 58.89; H, 6.94;
N, 6.12.
A solution of sodium benzyloxide was prepared by reacting
0.20 g of sodium with 20 mL of benzyl alcohol. Diethyl ester 18
(42.15 g, 0.150 mol) and benzyl alcohol (90 mL) were placed in a
500 mL Erlenmeyer flask clamped in an oil bath. A thermometer
was positioned ca. 5 cm above the surface of the solution and the
temperature was gradually raised from room temperature to
240 °C over a period of 90 min while intermittently adding small
portions of the sodium benzyloxide solution. When the vapor tem-
perature reached 200 °C, a final portion of sodium benzyloxide was
added and the mixture was stirred for an additional 5 min. The hot
solution was poured into a stirred ice cooled mixture of methanol
(240 mL), water (165 mL) and acetic acid (3 mL). The resulting pre-
cipitate was suction filtered and recrystallized from ethanol to give
the dibenzyl ester (52.75 g, 0.130 mol, 87%) as off-white crystals,
4.2.6. 4-Methoxycarbonylpropyl-3,5-dimethylpyrrole-2-carb-
oxylic acid (20b)
Pyrrole benzyl ester 10b (10.00 g, 30.4 mmol) was hydrogeno-
lyzed using the foregoing procedure. The pyrrole carboxylic acid
(6.93 g, 29.0 mmol, 95%) was isolated as a light red powder, mp
124–125 °C, dec; 1H NMR (500 MHz, DMSO-d6): d 1.59 (2H, quin-
tet, J = 7.5 Hz), 2.07 (3H, s), 2.13 (3H, s), 2.23–2.29 (4H, two over-
lapping triplets), 3.56 (3H, s), 10.91 (1H, s), 11.73 (1H, br s); 13C
NMR (DMSO-d6): d 10.5, 10.9, 22.9, 25.7, 32.8, 51.3, 116.6, 119.9,
125.5, 129.9, 162.5, 173.5. Anal. Calcd for C12H17NO4: C, 60.24; H,
7.16; N, 5.85. Found: C, 60.42; H, 6.67; N, 5.88.
4.2.7. 4-Ethoxycarbonylmethyl-3,5-dimethylpyrrole-2-carbal-
dehyde (12a)
mp 64–65 °C; IR (Nujol mull):
m 3302 (s, NH str.), 1722 (s, butyrate
C@O str.), 1658 cmꢂ1 (s, pyrrole-C@O str.); 1H NMR (500 MHz,
CDCl3): d 1.78 (2H, quintet, J = 7.5 Hz), 2.15 (3H, s), 2.26 (3H, s),
2.35 (2H, t, J = 7.4 Hz), 2.40 (2H, t, J = 7.5 Hz), 5.10 (2H, s), 5.29
(2H, s), 7.31–7.43 (10H, m), 8.64 (1H, br s); 13C NMR (CDCl3): d
10.7, 11.5, 23.2, 25.7, 33.6, 65.4, 66.2, 116.5, 121.1, 128.0, 128.1,
128.2, 128.3, 128.5, 128.6, 130.1, 136.0, 136.6, 161.2, 173.4. Anal.
Pyrrole carboxylic acid 20a (1.40 g, 6.22 mmol) was stirred with
TFA (5 mL) at room temperature for 10 min. The solution was
cooled in an ice bath, freshly distilled triethyl orthoformate
(3 mL) was added dropwise, and the mixture was stirred at 40 °C
for a further 10 min. The mixture was then poured into water
(50 mL) and extracted with chloroform (3 x 50 mL). The combined
organic solutions were washed with 5% aqueous ammonia (50 mL)
and water (50 mL), and dried over magnesium sulfate. The drying
agent was removed by suction filtration, the solvent evaporated,
and the residue recrystallized from ethanol to give the pyrrole
aldehyde (0.920 g, 4.40 mmol, 71%) as pale green crystals, mp
114–115 °C (lit. mp71 115–117 °C); 1H NMR (400 MHz, CDCl3): d
1.25 (3H, t, J = 7.2 Hz), 2.279 (3H, s), 2.284 (3H, s), 3.37 (2H, s),
4.13 (2H, q, J = 7.2 Hz), 9.48 (1H, s), 9.67 (1H, br s); 13C NMR
(CDCl3): d 9.0, 11.9, 14.4, 30.2, 61.0, 116.0, 128.3, 132.6, 136.3,
171.5, 176.3.
1
Calcd for C25H27NO4ꢃ /4H2O: C, 73.24; H, 6.76; N, 3.42. Found: C,
73.17; H, 6.57; N, 3.38.
4.2.4. Benzyl 4-methoxycarbonylpropyl-3,5-dimethylpyrrole-2-
carboxylate (13b)
Conc. sulfuric acid (20 mL) was cautiously added to methanol
(400 mL), dibenzyl ester 19 (50.0 g, 0.123 mol) was added, and
the resulting mixture was stirred for 16 h at room temperature.
The solution was diluted with chloroform, washed with water,
5% aqueous sodium bicarbonate, and water, and then dried over
sodium sulfate. The drying agent was removed by suction filtra-

1502
T. D. Lash et al. / Bioorg. Med. Chem. 19 (2011) 1492–1504
4.2.8. 4-Methoxycarbonylpropyl-3,5-dimethylpyrrole-2-carbal-
dehyde (12b)
4.2.11. 3,8-Bis(2-methoxycarbonylethyl)-13,17-bis(methoxycar-
bonylmethyl)-2,7,12,18-tetramethylporphyrin (10a)
Pyrrole carboxylic acid 20b (2.00 g, 8.37 mmol) was reacted un-
der the foregoing conditions. Recrystallization from chloroform–
hexanes gave the aldehyde72 (1.44 g, 6.46 mmol, 77%) as pale green
needles, mp 64–65 °C. Further recrystallization from chloroform–
hexanes gave an analytical sample as small fluffy off-white nee-
dles, mp 64–64.5 °C; 1H NMR (500 MHz, CDCl3): d 1.77 (2H, quin-
tet, J = 7.5 Hz), 2.24 (3H, s), 2.26 (3H, s), 2.31 (2H, t, J = 7.4 Hz),
2.41 (2H, t, J = 7.5 Hz), 3.66 (3H, s), 9.27 (1H, br s), 9.47 (1H, s);
13C NMR (CDCl3): d 8.9, 11.8, 23.3, 25.7, 33.5, 51.6, 122.1, 128.3,
132.3, 135.6, 174.0, 176.0. Anal. Calcd for C12H17NO3: C, 64.55; H,
7.67; N, 6.27. Found: C, 64.35; H, 7.88; N, 6.33.
a,c-Biladiene dihydrobromide 21a (250 mg, 0.281 mmol) was
added to a stirred solution of copper(II) chloride (0.74 g) in DMF
(95 mL), and the resulting mixture was stirred in the dark for 2 h.
The dark red solution was diluted with dichloromethane (100 mL)
and washed with water (3 x 100 mL). The aqueous layers were back
extracted with dichloromethane and the combined organic layers
were dried over sodium sulfate and filtered. The solvent was evapo-
rated on a rotary evaporator under aspirator pressure and then using
a vacuum pump to remove any remaining DMF. The solid residue
was taken up in 15% v/v sulfuric acid–TFA (35 mL) and stirred in
the dark at room temperature for 45 min. The reaction mixture
was diluted with dichloromethane (150 mL), and then washed with
water (2 x 100 mL) and 5% aqueous sodium bicarbonate solution
(100 mL). The aqueous layers were back extracted with dichloro-
methane and the combined organic layers were dried over sodium
sulfate and the solvent was evaporated under reduced pressure.
The porphyrin was then reesterified by dissolving the residue in
5% sulfuric acid–methanol (35 mL) and stirring the mixture in the
dark overnight. The mixture was diluted with dichloromethane,
washed with water and then with 5% aqueous sodium bicarbonate
solution. The aqueous layers were back extracted with dichloro-
methane at each stage, and the combined organic layers were dried
over sodium sulfate and the solvent was evaporated under reduced
pressure. The residue was chromatographed on a grade 3 alumina
column, eluting with dichloromethane. A dark violet product frac-
tion was collected and the solvent removed under reduced pressure.
The residue was recrystallized from chloroform–methanol to give
the title porphyrin (121 mg, 0.177 mmol, 63%) as a maroon solid,
4.2.9. 2,18-Bis(ethoxycarbonylmethyl)-8,13-bis(2-methoxycar-
bonylethyl)-1,3,7,12,17,19-hexamethyl-20,23-dihydrobilin di-
hydrobromide (21a)
Dipyrrylmethane carboxylic acid 1113 (200.0 mg, 0.408 mmol)
was stirred under nitrogen with trifluoroacetic acid (1 mL) for
10 min. A solution of pyrrole aldehyde 12a (170.6 mg, 0.816 mmol)
in methanol (3.6 mL) was added, followed immediately by the addi-
tion of 30% HBr-acetic acid (0.7 mL), and the mixture was stirred for
30 min. Ether (15 mL) was added dropwise and the mixture was
stirred for a further 2 h. The precipitate was filtered off, washed
thoroughly with ether and dried in vacuo overnight to give the title
a,c-biladiene dihydrobromide (269 mg, 0.302 mmol, 74%) as an or-
ange-red powder, mp 185–186 °C; UV–vis (CHCl3): kmax (log10 e)
368 (4.22), 457 (4.43), 523 nm (5.34); 1H NMR (400 MHz, CDCl3): d
1.26 (6H, t, J = 7.1 Hz), 1.92 (2H, t, J = 7.9 Hz), 1.96 (3H, s), 2.26 (3H,
s), 2.33 (3H, s), 2.38 (3H, s), 2.50 (2H, t, J = 7.0 Hz), 2.72 (6H, s),
2.79 (2H, t, J = 7.9 Hz), 2.95 (2H, t, J = 7.1 Hz), 3.41 (3H, s), 3.44 (2H,
s), 3.45 (2H, s), 3.59 (3H, s), 4.14 (4H, q, J = 7.1 Hz), 5.25 (2H, s),
7.16 (1H, s), 7.40 (1H, s), 13.35 (1H, br s), 13.36 (1H, br s), 13.48
(1H, br s), 13.52 (1H, br s); 13C NMR (CDCl3): d 9.5, 10.2, 10.58,
10.63, 13.3, 14.4, 19.8, 20.0, 26.0, 30.3, 33.9, 34.6, 51.5, 52.0, 61.5,
120.6, 121.7, 122.5, 122.6, 125.4, 125.9, 126.1, 127.1, 127.5, 128.6,
143.3, 144.56, 144.64, 145.4, 149.2, 149.9, 156.7, 157.2, 170.0,
172.78, 178.84. Anal. Calcd for C41H54N4O8Br2: C, 55.29; H, 6.11; N,
6.29. Found: C, 55.56; H, 6.25; N, 6.22.
mp 209–210 °C; UV–vis (1% Et3N–CHCl3): kmax (log10
499 (4.20), 533 (4.02), 569 (3.88), 623 nm (3.73); UV–vis (1% TFA–
CHCl3): kmax (log10
) 408 (5.57), 552 (4.19), 594 nm (3.82); 1H
e) 401 (5.27),
e
NMR (400 MHz, CDCl3): d ꢂ3.75 (2H, br s), 3.27 (4H, t, J = 7.8 Hz),
3.63 (3H, s), 3.65 (3H, s), 3.66 (3H, s), 3.682 (3H, s), 3.685 (3H, s),
3.691 (3H, s), 3.769 (3H, s), 3.774 (3H, s), 4.37–4.43 (4H, m), 5.07
(2H, s), 5.08 (2H, s), 10.05 (1H, s), 10.09 (2H, s), 10.14 (1H, s); 1H
NMR (400 MHz, TFA–CDCl3): d ꢂ3.42 (4H, br s), 3.14–3.18 (4H, 2
overlapping triplets), 3.67 (3H, s), 3.68 (6H, s), 3.69 (6H, s), 3.696
(3H, s), 3.698 (3H, s), 3.71 (3H, s), 4.47 (4H, t, J = 7.6 Hz), 5.19 (4H,
s), 10.70 (1H, s), 10.83 (1H, s), 10.85 (2H, s); 13C NMR (TFA–CDCl3):
d 12.10, 12.13, 12.32, 12.34, 21.9, 32.9, 35.67, 35.70, 52.9, 53.5,
99.6, 99.68, 99.75, 99.9, 133.6, 133.7, 139.4, 139.8, 140.5, 140.8,
141.9, 142.1, 142.5, 142.7, 171.7, 174.9. HRMS (EI), m/z Calcd for
4.2.10. 8,13-Bis(2-methoxycarbonylethyl)-2,18-bis(3-methoxy-
carbonylpropyl)-1,3,7,12,17,19-hexamethyl-20,23-dihydrobilin
dihydrobromide (21b)
Dipyrrylmethane 1113 (200.0 mg, 0.408 mmol) was stirred un-
der nitrogen with trifluoroacetic acid (1 mL) for 10 min. A solution
of pyrrole aldehyde 12b (182 mg, 0.816 mmol) in methanol
(3.6 mL) was added, followed immediately by the addition of 30%
HBr-acetic acid (0.7 mL), and the mixture was stirred for 30 min.
Ether (15 mL) was added dropwise and the mixture was stirred
for a further 2 h. The precipitate was filtered off, washed thor-
oughly with ether and dried in vacuo overnight to give the title
a,c-biladiene dihydrobromide (295 mg, 0.321 mmol, 78%) as a red
C
C
8.09.
38H42N4O8: 682.3002. Found: 682.3013. Anal. Calcd for
38H42N4O8: C, 66.85; H, 6.20; N, 8.20. Found: C, 66.61; H, 6.12; N,
4.2.12. 3,8-Bis(2-methoxycarbonylethyl)-13,17-bis(methoxycar-
bonylpropyl)-2,7,12,18-tetramethylporphyrin (10b)
Using the previous procedure, a,c-biladiene 21b (258 mg,
0.281 mmol) was cyclized with CuCl2 (0.74 g), demetalated and
reesterified. Chromatography on a grade 3 alumina column, eluting
with dichloromethane, gave a dark violet band. Recrystallization
from chloroform–methanol gave the dibutyrate (142 mg,
0.192 mmol, 68%) as a maroon solid, mp 140.5–141.5 °C (lit.
powder, mp 197–198 °C; UV–vis (CHCl3): kmax (log10 e) 368
(4.19), 458 (4.44), 525 nm (5.32); 1H NMR (400 MHz, CDCl3): d
1.78 (4H, quintet, J = 7.4 Hz), 1.92 (2H, t, J = 7.9 Hz), 1.96 (3H, s),
2.24 (3H, s), 2.30 (3H, s), 2.35 (3H, s), 2.32–2.37 (4H, m), 2.46–
2.51 (6H, m), 2.71 (6H, s), 2.78 (2H, t, J = 7.9 Hz), 2.94 (2H, t,
J = 7.1 Hz), 3.42 (3H, s), 3.60 (3H, s), 3.683 (3H, s), 3.686 (3H, s),
5.22 (2H, s), 7.10 (1H, s), 7.33 (1H, s), 13.27 (2H, br s), 13.36 (1H,
br s), 13.40 (1H, br s); 13C NMR (CDCl3): d 9.5, 10.2, 10.38, 10.40,
13.3, 19.3, 20.0, 23.5, 25.0, 26.0, 33.4, 33.9, 34.7, 51.5, 51.8, 52.0,
120.0, 121.0, 125.1, 125.5, 125.8, 127.4, 127.9, 128.3, 129.2,
129.3, 142.6, 143.6, 144.0, 144.4, 148.5, 149.1, 157.0, 157.5,
172.9, 173.0, 173.6. Anal. Calcd for C43H58N4O8Br2ꢃH2O: C, 55.10;
H, 6.52; N, 5.98. Found: C, 55.04; H, 6.48; N, 5.95.
mp56 138–140 °C); UV–vis (1% Et3N-CHCl3): kmax (log10
e
) 400
(5.25), 499 (4.14), 533 (3.98), 567 (3.81), 621 nm (3.68); UV-vis
(1% TFA–CHCl3): kmax (log10 ) 407 (5.56), 551 (4.21), 592 nm
e
(3.88); 1H NMR (400 MHz, CDCl3): d ꢂ3.76 (2H, br s), 2.64 (4H,
quintet, J = 7.0 Hz), 2.72–2.77 (H, m), 3.25–3.30 (4H, 2 overlapping
triplets), 3.63 (3H, s), 3.645 (3H, s), 3.649 (3H, s), 3.66 (3H, s), 3.68
(3H, s), 3.70 (3H, s), 3.71 (3H, s), 3.72 (3H, s), 4.15 (2H, t, J = 7.6 Hz),
4.39–4.45 (4H, 2 overlapping triplets), 10.08 (2H, s), 10.09 (1H, s),
10.22 (1H, s); 1H NMR (400 MHz, TFA–CDCl3): d ꢂ3.93 (4H, br s),
2.40–2.46 (4H, m), 2.70–2.76 (4H, 2 overlapping triplets), 3.18

T. D. Lash et al. / Bioorg. Med. Chem. 19 (2011) 1492–1504
1503
(4H, t, J = 7.6 Hz), 3.67 (3H, s), 3.688 (3H, s), 3.694 (3H, s), 3.70 (3H,
s), 3.72 (3H, s), 3.73 (3H, s), 3.78 (3H, s), 3.79 (3H, s), 4.21 (4H, t,
J = 8.0 Hz), 4.50 (4H, t, J = 8.0 Hz), 10.72 (1H, s), 10.83 (1H, s),
10.84 (1H, s), 10.95 (1H, s); 13C NMR (TFA–CDCl3): d 11.89, 11.94,
11.98, 12.04, 21.9, 26.0, 27.2, 33.97, 34.00, 35.77, 52.9, 53.2, 99.2,
99.47, 99.52, 139.4, 139.58, 139.64, 140.0, 140.3, 140.4, 141.4,
141.8, 142.2, 142.3, 142.4, 142.8, 175.9, 176.9. HRMS (EI), m/z Calcd
for C42H50N4O8: 738.3628. Found: 738.3626.
10.17 (1H, s), 10.19 (1H, s), 10.24 (1H, s). HRMS (ESI), m/z Calcd
for C40H46N4O6+H: 679.3496. Found: 679.3486. HRMS (EI), m/z
Calcd for C40H46N4O6: 678.3417. Found: 678.3415.
4.4.3. 13,17-Bis(3-methoxycarbonylpropyl)-2,7,12,18-tetra-
methyl-3,8-divinylporphyrin (27c)
1H NMR (400 MHz, CDCl3; downfield region only): d 6.16–6.22
(2H, m), 6.35–6.42 (2H, m), 8.26–8.36 (2H, m), 10.14 (1H, s),
10.20 (1H, s), 10.22 (1H, s), 10.29 (1H, s). HRMS (ESI), m/z Calcd
for C38H42N4O6+H: 619.3284. Found: 619.3303.
4.3. Enzyme assays
4.3.1. Enzyme incubation studies using chicken red cell
hemolysates (CRH)
Acknowledgements
Enzyme incubations and analyses of metabolic products were
carried out as described elsewhere. For kinetic studies, the incuba-
tion products were isolated using a previously developed microas-
say technique69 and analyzed by TLC and/or HPLC. Incubations of
the diacetate porphyrinogen were carried out in the presence or
absence of N-ethylmaleimide, which is reported to be an inhibitor
for Uro-D.66 The HPLC analyses were performed using normal
phase columns (5 m partisil silica, Alltech) eluting with appropri-
ate ratios of ethyl acetate and cyclohexane. Kinetic data are
reported as mean standard deviation for three replicate experi-
ments and compared statistically using analysis of variance (ANO-
VA) following Fisher’s LSD post Test. Values are considered
different at p < 0.05.
This work was supported by National Institutes of Health under
Grant No. 1 R15 GM/OD52687-01A1 and by the National Science
Foundation under Grant No. CHE-0616555. Funding for
a
500 MHz NMR spectrometer was provided by the National Science
Foundation under grant no. CHE-0722385. The cDNA encoding 6 x
histidine tagged human recombinant CPO was a generous gift from
Dr. H. A. Dailey of the University of Georgia (Athens, Georgia).
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These metabolites were obtained by reducing the tetracarboxy-
lic acids derived from 10a and 10b, respectively, with 3% sodium
amalgam and incubating the resulting porphyrinogens with hu-
man recombinant CPO. The products were purified as their methyl
esters by flash chromatography, eluting with 10% ethyl acetate–
toluene, and characterized by EI and/or ESI MS, and 400 MHz pro-
ton NMR spectroscopy. The diacetate porphyrinogen 9a gave
monovinylporphyrin 27a and only trace amounts of a divinyl spe-
cies. Dibutyrate 6b also gave mostly the monovinylporphyrin 27b
but sufficient divinyl derivative 27c could be isolated in this case
to obtain a low quality proton NMR spectrum as well as MS data.
4.4.1. 8-(2-Methoxycarbonylethyl)-13,17-bis(methoxycarbonyl-
methyl)-2,7,12,18-tetramethyl-3-vinylporphyrin (27a)
1H NMR (400 MHz, CDCl3): d ꢂ3.60 (2H, br s), 3.28 (2H, t,
J = 8.1 Hz), 3.66 (3H, s), 3.678 (3H, s), 3.684 (3H, s), 3.71 (3H, s),
3.74 (3H, s), 3.768 (3H, s), 3.775 (3H, s), 4.1–4.2 (2H, obscured by
impurity peaks), 5.08 (2H, s), 5.12 (2H, s), 6.19 (1H, d,
J = 11.7 Hz), 6.38 (1H, d, J = 16.9 Hz), 8.26–8.34 (1H, m), 10.11
(1H, s), 10.17 (1H, s), 10.19 (1H, s), 10.24 (1H, s). HRMS (ESI), m/z
Calcd for C36H38N4O6+H: 623.2870. Found: 623.2870.
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