Normal and abnormal heme biosynthesis. 2.1 Synthesis and metabolism of type-III pentacarboxylic porphyrinogens: Further experimental evidence for the enzymic clockwise decarboxylation of uroporphyrinogen-III

Article abstract of DOI:10.1021/jo9814748

Uroporphyrinogen decarboxylase catalyses the sequential decarboxylation of uroporphyrinogen-III (1) to give coproporphyrinogen-III (2), a precursor to the hemes and chlorophylls. This involves the decarboxylation of four nonequivalent acetate side chains to produce methyl units and in principle could take place by 24 different pathways involving up to 14 intermediary porphyrinogens. In the past, seemingly contradictory data have been presented that either support an ordered 'clockwise' decarboxylation pathway or a random decarboxylation process. Four pentacarboxylate porphyrinogens might be involved immediately before the formation of 2, and these compounds have been synthesized as the corresponding porphyrin pentamethyl esters via tripyrrene and a,c-biladiene intermediates. Hydrolysis of the methyl esters and reduction with 3% sodium amalgam gave the required porphyrinogens, and these were incubated with crude enzyme preparations derived from chicken red cell hemolysates. One of these pentacarboxylate porphyrinogens. (5dab) consistently proved to be a much better substrate than the other three, providing new support for the 'clockwise' pathway for coproporphyrinogen-III formation.

Full text of DOI:10.1021/jo9814748

478
J . Org. Chem. 1999, 64, 478-487
Nor m a l a n d Abn or m a l Hem e Biosyn th esis. 2.1 Syn th esis a n d
Meta bolism of Typ e-III P en ta ca r boxylic P or p h yr in ogen s: F u r th er
Exp er im en ta l Evid en ce for th e En zym ic Clock w ise
Deca r boxyla tion of Ur op or p h yr in ogen -III
Timothy D. Lash,* Ukti N. Mani, Elizabeth A. Lyons, Pornlert Thientanavanich, and
Marjorie A. J ones
Department of Chemistry, Illinois State University, Normal, Illinois 61790-4160
Received J uly 27, 1998
Uroporphyrinogen decarboxylase catalyses the sequential decarboxylation of uroporphyrinogen-
III (1) to give coproporphyrinogen-III (2), a precursor to the hemes and chlorophylls. This involves
the decarboxylation of four nonequivalent acetate side chains to produce methyl units and in
principle could take place by 24 different pathways involving up to 14 intermediary porphyrinogens.
In the past, seemingly contradictory data have been presented that either support an ordered
“clockwise” decarboxylation pathway or a random decarboxylation process. Four pentacarboxylate
porphyrinogens might be involved immediately before the formation of 2, and these compounds
have been synthesized as the corresponding porphyrin pentamethyl esters via tripyrrene and a,c-
biladiene intermediates. Hydrolysis of the methyl esters and reduction with 3% sodium amalgam
gave the required porphyrinogens, and these were incubated with crude enzyme preparations
derived from chicken red cell hemolysates. One of these pentacarboxylate porphyrinogens (5d a b)
consistently proved to be a much better substrate than the other three, providing new support for
the “clockwise” pathway for coproporphyrinogen-III formation.
Ch a r t 1. Ur o’gen - a n d Cop r o’gen -Typ e Isom er s
In tr od u ction
Uroporphyrinogen decarboxylase (UPD; EC 4.1.1.37)
catalyses the conversion of uroporphyrinogen-III (uro’gen-
III; 1) to coproporphyrinogen-III (copro’gen-III; 2), whereby
four acetate side chains are decarboxylated to give methyl
units (Chart 1).2 This is the first enzymic step in the
heme and chlorophyll biosynthetic pathways to occur
solely at the tetrapyrrolic macrocycle level and represents
the initial branch point from corrin/vitamin B12 biosyn-
thesis.3 UPD has a low substrate specificity and also
converts the type isomers uro’gens I, II, and IV (3a -c)
to the corresponding copro’gens 4a -c (Chart 1).4 The
mechanism for this reaction is believed to involve initial
protonation onto the pyrrole nucleus, which facilitates a
deprotonation-decarboxylation (Scheme 1). Subsequent
protonation onto the resulting exocyclic methylene, fol-
lowed by a tautomerization, affords the methyl-substi-
tuted pyrrole.5 This process has been shown to occur with
retention of configuration using stereospecifically doubly
labeled 2H/3H succinate,6 implying that the chemistry
occurs in a highly ordered “micro-environment”.5 UPD
has been isolated and fully or partially purified from
several sources, including human7,8 and chicken eryth-
rocytes,9 bovine liver,10 tobacco leaves,11 the yeast Sac-
charomyces cerevisiae,12 and the bacteria Rhodobacter
palustrus13 and Rhodobacter sphaeroides.14 The enzyme
is sensitive to polychlorionated hydrocarbons15 and is
inhibited by heavy metals16 and sulfhydryl reagents,7
indicating the presence of an essential cysteine residue
* To whom correspondence should be addressed. E-mail: tdlash@
ilstu.edu.
(1) Part 1: Lash, T. D.; Mani, U. N.; Drinan, M. A.; Zhen, C.; Hall,
T.; J ones, M. A. J . Org. Chem. 1999, 64, 464-477.
(2) Biosynthesis of Tetrapyrroles; J ordan, P. M., Ed.; Elsevier:
London, 1991.
(3) Scott, A. I. Angew. Chem., Int. Ed. Engl. 1993, 32, 1223.
Battersby, A. R. Science 1994, 264, 1551. Battersby, A. R.; Leeper, F.
J . In Topics in Current Chemistry 195. Biosynthesis: Polyketides and
Vitamins; Leeper, F. J ., Vederas, J . C., Eds.; Springer-Verlag: Berlin,
1998; pp 143-193.
(4) Smith, A. G.; Francis, J . E. Biochem. J . 1979, 183, 455.
(5) Akhtar, M. In The Biosynthesis of Tetrapyrrolic Pigments; Wiley:
Ciba Foundation Symposium 180; Chichester, 1994; pp 130-155.
(6) Barnard, G.; Akhtar, M. J . Chem. Soc., Chem. Commun. 1975,
494.
(7) De Verneuil, H.; Sassa, S.; Kappas, A. J . Biol. Chem. 1983, 258,
2454. Elder, G. H.; Tovey, J . A.; Sheppard, D. M. Biochem. J . 1983,
215, 45.
(8) Mukerji, S. K.; Pimstone, N. R. Int. J . Biochem. 1992, 24, 105.
(9) Kawanishi, S.; Seki, Y.; Sano, S. J . Biol. Chem. 1983, 258, 4285.
(10) Straka, J . G.; Kushner, J . P. Biochemistry 1983, 22, 4664.
(11) Chen, T. C.; Miller, G. W. Plant Cell Physiol. 1974, 15, 993.
(12) Felix, F.; Brouillet, N. Eur. J . Biochem. 1990, 188, 393.
(13) Koopman, G. E.; J uknat de Gweralnik, A. A.; Battle, A. M. del
C. Int. J . Biochem. 1986, 18, 935.
(14) J ones, R. M.; J ordan, P. M. Biochem. J . 1993, 293, 703.
10.1021/jo9814748 CCC: $18.00 © 1999 American Chemical Society
Published on Web 12/29/1998

Normal and Abnormal Heme Biosynthesis. 2
J . Org. Chem., Vol. 64, No. 2, 1999 479
Sch em e 1. P r op osed Mech a n ism for th e En zym ic
Deca r boxyla tion of th e Aceta te Sid e Ch a in s of
Ur op or p h yr in ogen -III
Ch a r t 2. Typ e-III a n d Typ e-I In ter m ed ia tes a n d
Abn or m a l Meta bolites
at the active site. In humans, defects in hepatic UPD give
rise to the disease porphyria cutanea tarda (PCT),17,18
which is associated with skin photosensitivity and liver
damage. Hepatic porphyrias may be induced by polyha-
logenated aromatic hydrocarbons,19,20 including 2,3,7,8-
tetrachlorodibenzo-p-dioxin,21 and can also result from
heavy metal poisoning22 and acute alcoholism.23 An
underlying association of PCT with human immunode-
ficiency viral (HIV) infection has also been reported.24
from several natural sources and in enzyme incubation
studies.25-27 In addition, relatively large amounts of these
porphyrins are excreted in the urine and feces of patients
suffering from PCT26,28 and rats poisoned with hexachlo-
robenzene (HCB) (the latter represents an animal model
for PCT).26 Due to the asymmetry of uro’gen-III, the first
decarboxylation can occur, in principle, at four different
sites to produce four heptacarboxylate porphyrinogens
7a -d (Scheme 2; Chart 2).26 Each “hepta” could be
decarboxylated in three ways to give a total of 6 hexa-
carboxylate porphyrinogens 6a b, 6a c, 6d a , 6bc, 6bd , and
6cd , and these “hexas” can each be converted in two ways
to produce four different pentacarboxylate porphyrino-
gens 5d a b, 5a bc, 5a cd , and 5bcd , which in turn would
all produce copro’gen-III.26 Hence, there are 4! or 24
possible pathways between 1 and 2 (Scheme 2) that could
involve a total of 14 different intermediates (Chart 2).
Enzyme-catalyzed reactions are nearly always highly
regio- and stereoselective, and only one of these pathways
might be expected to be favored under physiological
conditions. In a monumental and groundbreaking en-
deavor, J ackson et al. isolated the hepta-, hexa-, and
pentacarboxylate porphyrins from the feces and urine of
HCB poisoned rats and compared these materials by
europium shift reagent proton NMR spectroscopy to
synthetic samples of all 14 of the possible intermediates
in the form of the corresponding porphyrin methyl
esters.26,28,29 These results clearly demonstrated that
Clock w ise Deca r boxyla tion Hyp oth esis
UPD is known to sequentially decarboxylate the four
acetate residues of uro’gen-III via hepta-, hexa-, and
pentacarboxylate intermediates 5-7 (Chart 2).25,26 These
species, usually observed after conversion to the corre-
sponding porphyrins, have been isolated and identified
(15) De Verneuil, H.; Sassa, S.; Kappas, A. Biochem. J . 1983, 214,
145. Kawanishi, S.; Seki, Y.; Sano, S. J . Biol. Chem. 1983, 258, 4285.
(16) Kushner, J . P.; Lee, G. R.; Nacht, S. J . Clin. Invest. 1972, 51,
3044.
(17) Elder, G. H. Semin. Liver Dis. 1998, 18, 67.
(18) Moore, M. R. Int. J . Biochem. 1993, 25, 1353. McDonagh, A.
F.; Bissell, D. M. Semin. Liver Dis. 1998, 18, 3.
(19) Strik, J . J . W. T. A.; Koeman, J . H. Chemical Porphyria in Man;
Elsevier: Amsterdam, 1979.
(20) Elder, G. H. In Heme and Hemoproteins; De Matteis, F.,
Aldridge, W. N., Eds.; Springer-Verlag: Berlin, 1978; pp 157-200.
Sinclair, P. R.; Elder, G. H.; Bement, W. J .; Smith, S. G.; Bonkowsky,
H. L.; Sinclair, J . F. FEBS Lett. 1983, 152, 217. Swain, M. G.; Follows,
S. B.; Marks, G. S. Can. J . Physiol. Pharmacol. 1983, 61, 105.
(21) Goldstein, J . A.; Links, P.; Bergman, H. Biochem. Pharmacol.
1982, 31, 1607.
(22) Woods, J . S.; Kardish, R.; Fowler, B. A. Biochem. Biophys. Res.
Commun. 1981, 103, 264.
(23) McColl, K. E. L.; Thompson, G. G.; Moore, M. R.; Goldberg, A.
Eur. J . Clin. Invest. 1980, 10, 107.
(24) Wissel, P. S.; Sordillo, P.; Anderson, K.; Sassa, S.; Savillo, R.
L.; Kappas, A. Am. J . Hemat. 1987, 25, 107. Cohen, P. R. J . Acq.
Immune Def. Synth. 1991, 4, 1112. Conlan, M. G.; Hoots, W. K. J . Am.
Acad. Dermat. 1992, 26, 857.
(25) Mauzerall, D.; Granick, S. J . Biol. Chem. 1958, 232, 1141.
Battle, A. M. del C.; Grinstein, M. Biochim. Biophys. Acta 1964, 82, 1.
San Martin de Viale, L. C.; Grinstein, M. Biochim. Biophys. Acta 1968,
158, 79. Frydman, R. B.; Tomaro, M. L.; Wanschelbaum, A.; Frydman,
B. FEBS Lett. 1972, 26, 203. Smith, S. G. Br. J . Dermat. 1975, 93,
291.
(26) J ackson, A. H.; Sancovich, H. A.; Ferramola, A. M.; Evans, E.;
Games, D. E.; Matlin, S. A.; Elder, G. E.; Smith, S. G. Philos. Trans.
R. Soc. London B 1976, 273, 191. The number/letter codes for 5-7,
which were proposed in this paper, indicate the total number carboxy-
late side chains and which acetate units have been decarboxylated
(relative to uro’gen-III). For instance, 5d a b is a pentacarboxylate
porphyrinogen that has lost the acetate moieties on rings D, A, and B
but retains this unit on ring C.
(27) Battersby, A. R.; Hunt, E.; McDonald, E.; Paine, J . B., III;
Saunders, J . J . Chem. Soc., Perkin Trans. 1 1976, 1008.

480 J . Org. Chem., Vol. 64, No. 2, 1999
Lash et al.
give copro’gen-I (4a ).38 Only one hepta- (7-I) and one
pentacarboxylate (5-I) intermediate are possible in this
case, although two isomeric hexacarboxylate species
6-Ia b and 6-Ia c (Chart 2) might arise (i.e., there are two
possible metabolic pathways).39 HPLC analyses and total
synthesis were used to conclusively demonstrate the
presence of both type-I hexacarboxylate isomers in
samples derived from human and bovine porphyrics as
well as for biochemical studies with chicken red cell
hemolysates, and thus, it was concluded that the decar-
boxylation of uro’gen-I occurs by “a nonspecific route”.39
However, the data for the type-III series are far from
clear-cut. Analysis of the porphyrins present in normal
human urine showed the presence of mixed type-III
pentacarboxylate isomers,40 but these were assumed to
be minor metabolites that resulted from leakage from this
relatively “promiscuous” enzyme.5,40 However, more de-
tailed chromatographic investigations by Lim and co-
workers showed41 that mixed hepta-, hexa-, and penta-
carboxylate fractions were present in normal and PCT
urine, although the “hepta” fraction from PCT did indeed
appear to be primarily 7d ,42 and this group concluded
that the enzymic decarboxylation of uro’gen-III does not
always proceed in a clockwise manner.41,42 More damning
evidence came from enzymic studies, initially using
chicken red cell hemolysates (CRH).32,43 Incubations of
uro’gen-III with CRH were shown to produce at least two
heptacarboxylate isomers, while 7d gave at least two
hexacarboxylate porphyrins (three are possible) and three
different pentacarboxylate porphyrins.32,43 6d a , 6bd , and
6cd , the hexacarboxylate porphyrinogens that might
arise from 7d , all gave mixtures of two pentacarboxylate
metabolites on incubation with CRH.43 Subsequently, Luo
and Lim demonstrated that incubations of uro’gen-III
with erythocyte and hepatic human UPD gave all 14 of
the possible intermediates (Chart 2),42 while J ones and
J ordan showed that UPD derived from Rh. sphaeroides
also produced random mixtures of heptacarboxylate
metabolites.14 Luo and Lim concluded that “the normal
decarboxylation pathway is random in nature” and
suggested that 7d only predominated under abnormal
circumstances.42 While these data seem to support such
a statement, the observations of single isomers in the
feces and urine from HCB poisoned rats and PCT,26 as
well as from incubations of PBG with highly saline
preparations of CRH,27 were difficult to reconcile with
this hypothesis. It is not really plausible that abnormal
conditions would result in a preferred pathway while
normal metabolism of uro’gen-III by UPD is random. The
putative intermediates 7d , 6d a , and 5d a b are all excel-
lent substrates for UPD,26,28,43,44 and while all 14 possible
intermediates are metabolized by UPD not all of them
are good substrates, so this phenomenom cannot be
ascribed to the accumulation of the poorer substrates
Sch em e 2. Ra n d om Deca r boxyla tion of
Ur o’gen -III
single hepta-, hexa-, and pentacarboxylate isomers pre-
dominate and these corresponded to 7d , 6d a , and 5d a b.
A sample of a heptacarboxylate porphyrin was also
isolated from a PCT patient and shown to be indistin-
guishable from 7d .28,31 Furthermore, in concurrent in-
vestigations, Battersby and co-workers isolated a hep-
tacarboxylate porphyrin from incubations of porphobili-
nogen (PBG, a precursor to uro’gen-III1) with avian red
cells containing high concentrations of NaCl and again
demonstrated that this metabolic product corresponded
to 7d .27 These results suggested that the decarboxylation
of uro’gen-III occurs via a preferred pathway starting at
ring D and proceeds in a clockwise fashion to ring A, then
B, and finally ring C (Scheme 3).26,32 Further evidence
for this pathway comes from the isolation of a group of
abnormal metabolites known as the isocoproporphyrins
(Chart 2; 8a -e), which have been isolated from porphyric
patients and HCB poisoned rats.33,34 These are believed
to arise from 5d a b being acted upon out of sequence by
the next enzyme in the heme pathway, coproporphyrino-
gen oxidase,1 after it has escaped from UPD.35,36 Porphy-
rin 8b (dehydroisocoproporphyrin) would be formed from
oxidation of the resulting porphyrinogen, while 8a and
8c-e presumably result from bacterial degradation in
the intestinal tract. Under abnormal conditions dehydro-
isocopro’gen may be involved in an alternative, albeit less
efficient, route for heme biosynthesis.35-37 Uro’gen-I (3a )
is generated in substantial quantities in congenital
erythropoietic porphyria and is metabolized by UPD to
(28) J ackson, A. H.; Ferramola, A. M.; Sancovich, H. A.; Evans, E.;
Matlin, S. A.; Ryder, D. J .; Smith, S. G. Ann. Clin. Res. 1976, 8, Suppl.
17, 64.
(29) J ackson, A. H.; Sancovich, H. A.; Ferramola de Sancovich, A.
M. Bioorg. Chem. 1980, 9, 71. See also: Chakrabarty, M.; Ali, S. A.;
Philip, G.; J ackson, A. H. Heterocycles 1981, 15, 1199.
(30) J ackson, A. H. Seminar Hematol. 1977, 14, 193.
(31) Ryder, D. J . Ph.D. Dissertation, University of Wales, College
of Cardiff, 1977.
(37) Kurlandzka, A.; Zoladek, T.; Rytka, J .; Labbe-Bois, R. Biochem.
J . 1991, 273, 246.
(32) Lash, T. D. Biochem. J . 1991, 278, 901.
(38) Desnick, R. J .; Glass, I. A.; Xu, W.; Solis, C.; Astrin, K. H. Semin.
Liver Dis. 1998, 18, 77.
(39) J ackson, A. H.; Rao, K. R. N.; Supphayen, D. M.; Smith, S. G.
J . Chem. Soc., Chem. Commun. 1977, 696. J ackson, A. H.; Supphayen,
D. M. J . Chem. Soc., Perkin Trans. 1 1987, 277.
(40) Smith, S. G.; Rao, K. R. N.; J ackson, A. H. Int. J . Biochem.
1980, 12, 1081.
(33) Elder, G. H. Biochem. J . 1972, 126, 877. Elder, G. H. J . Clin.
Pathol. 1975, 28, 601. Stoll, M. S.; Elder, G. H.; Games, D. E.;
O’Hanlon, P.; Millington, D. S.; J ackson, A. H. Biochem. J . 1973, 131,
429. Elder, G. H. Seminar Hematol. 1977, 14, 227. Smith, S. G.
Biochem. Soc. Trans. 1977, 5, 1472. Luo, J .; Lim, C. K. Biomed.
Chromatogr. 1995, 9, 113.
(34) (a) J ackson, A. H.; Lash, T. D.; Ryder, D. J . J . Chem. Soc.,
Perkin Trans. 1 1987, 287. (b) Clezy, P. S.; Hai, T. T. Aust. J . Chem.
1976, 29, 1561.
(35) Elder, G. H.; Evans, J . O. Biochem. J . 1978, 169, 205.
(36) J ackson, A. H.; Lash, T. D.; Ryder, D. J .; Smith, S. G. Int. J .
Biochem. 1980, 12, 775.
(41) Lim, C. K.; Rideout, J . M. J . Liquid Chromatogr. 1983, 6, 1969.
Lim, C. K.; Rideout, J . M.; Wright, D. J . J . Chromatogr. 1983, 282,
629. Li, F.; Lim, C. K.; Peters, T. J . Biochem. J . 1987, 243, 621. Lim,
C. K.; Li, F.; Peters, T. J . Biochem. J . 1987, 247, 229.
(42) (a) Luo, J .; Lim, C. K. Biochem. J . 1990, 268, 513; (b) J .
Chromatogr. 1991, 566, 409.

Normal and Abnormal Heme Biosynthesis. 2
J . Org. Chem., Vol. 64, No. 2, 1999 481
Sch em e 3. “Clock w ise” Deca r boxyla tion of Ur op or p h yr in ogen -III
under abnormal or pathological conditions. One possibil-
ity is that the previous enzyme in the heme pathway,
uro’gen-III synthetase, hands on the substrate to UPD
in a specific orientation, and under these circumstances
selective “clockwise” decarboxylation takes place.32 On
the other hand, when UPD is presented with uro’gen-III
or a later intermediate, the substrate may associate with
the active site(s)45 in a random orientation, and this leads
to isomeric mixtures of metabolites. The ability of the
enzyme to accept porphyrinogens randomly may reflect
the need to process and excrete type-I isomers that may
arise even in normal metabolism. This proposal implies
that uro’gen-III undergoes a selective clockwise decar-
boxylation under physiological conditions and the prod-
ucts observed in urine are minor abnormal metabolites
that have escaped from the binding pocket. Further, bio-
synthetic precursors to uro’gen-III, such as PBG, would
be expected to produce single type-III hepta-, hexa-, and
pentacarboxylate porphyrinogens in biochemical studies
using mixed enzyme systems.32 To test this postulate, Luo
and Lim recently carried out incubations of PBG with
crude enzyme preparations from human erythrocytes and
analyzed the resulting products by reversed phase HPLC.46
These data showed that 7d , 6d a , and 5d a b were virtually
the only detectable products, lending support to the
proposal. However, incubations of very low concentra-
tions of radiolabeled uro’gen-III with UPD also appeared
to show an increased selectivity for the “clockwise”
pathway, and this may suggest that the enzyme selectiv-
ity is in actual fact concentration dependent.46
possible intermediates between 1 and 2 (Chart 2). Due
to the relative ease of synthesis, and their potential
involvement in alternative biosynthetic pathways,35-37
the four type-III pentacarboxylate porphyrinogens were
selected for further study. Although all four of these
isomers have been previously shown to be metabolized
by CRH, the kinetic studies were based on minimal data
(in some cases only two data points were used), and it is
difficult to draw conclusions from these results.28,44 While
5bcd was reported to be a poor substrate, the other three
isomers were apparently easily metabolized by UPD.44
We anticipated that more subtle distinctions could be
discerned by analysis of more detailed kinetic studies.47
Syn th esis of Typ e-III P en ta ca r boxyla te
P or p h yr in s
The kinetic studies required synthetic samples of
5d a b, 5a bc, 5a cd , and 5bcd , and these were prepared
as the corresponding porphyrin pentamethyl esters.
These compounds were previously synthesized by J ack-
son et al.29 and Clezy et al.48 using a combination of the
MacDonald49 and b-oxobilane routes.50 In addition, the
porphyrin corresponding to 5d a b was prepared by the
copper(II) chloride mediated cyclization of an a,c-biladi-
ene.51 In this work, we wanted to minimize the number
(45) Evidence has been presented for the presence of more than one
active site in UPD: Cornford, P. Biochem. J . 1964, 91, 64. Tomio, J .;
Garcia, R.; San Martin de Viale, L.; Grinstein, M. Biochim. Biophys.
Acta 1970, 198, 353. Garcia, R. C.; San Martin de Viale, L. C.; Tomio,
J . M.; Grinstein, M. Biochim. Biophys. Acta 1973, 309, 203. Rasmussen,
G. L.; Kusner, J . P. J . Lab. Clin. Med. 1979, 93, 54. Smith, A. G.;
Francis, J . E. Biochem. J . 1979, 183, 455. De Verneuill, H.; Grand-
champ, G.; Nordmann, Y. Biochim. Biophys. Acta 1980, 611, 174.
Straka, J . G.; Kushner, J . P.; Burnham, B. F. Fed. Proc. Fed. Am. Soc.
Exp. Biol. 1980, 39, 2005.
An orderly “clockwise” decarboxylation pathway is
likely to be prevalent under normal physiological condi-
tions, and this should result in UPD having a preference
for porphyrinogens 7d , 6d a , and 5d a b over the other 11
(46) Luo, J .; Lim, C. K. Biochem. J . 1993, 289, 529.
(43) Lash, T. D. Ph.D. Dissertation, University of Wales, College of
Cardiff, 1979.
(44) Smith, S. G.; Ferramola, A. M.; Sancovich, H. A.; Evans, E.;
Matlin, S. A.; Ryder, D. J .; J ackson, A. H. Ann. Clin. Res. 1976, 8,
Suppl. 17, 89.
(47) Preliminary kinetic results for type-III pentacarboxylate por-
phyrinogens were presented at the 209th National ACS Meeting,
Anaheim, CA, April 1995 (Abstract: Lash, T. D.; Mani, U. N.; Lyons,
E. A.; Thientanavanich, P.; Hall, T.; J ones, M. A. Book of Abstracts,
ORGN 344).

482 J . Org. Chem., Vol. 64, No. 2, 1999
Lash et al.
Sch em e 4
Sch em e 5
of dibenzyl ester 9 over 10% palladium on activated car-
bon gave the related dicarboxylic acid 17. Subsequent
decarboxylation by treatment with TFA, followed by
condensation with 2 equiv of pyrrole aldehyde 18a or 18b
in the presence of HBr and precipitation with ether, gave
the tetrapyrrolic a,c-biladiene dihydrobromide salts 19
as red powders (Scheme 5). Cyclization with CuCl2 in
DMF at room temperature,59 followed by demetalation
with 15% sulfuric acid-TFA and reesterification with 5%
H2SO4-methanol, gave the porphyrin pentamethyl esters
20 (precursor to 5a cd ) and 21 (precursor to 5d a b) in 42%
and 61% yields, respectively (Scheme 5).
The remaining pentacarboxylate porphyrin isomers
were prepared from dipyrrole 10, but this required the
use of a more stepwise approach using tripyrrene inter-
mediates.51 Initially, 10 was hydrogenolyzed over Pd/C
to give carboxylic acid 22 (Scheme 4), and attempts were
made to condense the monodeprotected dipyrrylmethane
with pyrrole aldehydes in the presence of p-toluene-
sulfonic acid,51 but tripyrrolic products could not be
isolated in worthwhile yields. To overcome problems of
this type, an alternative tripyrrene-a,c-biladiene route
was developed59 where the tert-butyl ester is cleaved prior
to removal of the benzyl ester (Scheme 6). Treatment of
10 with TFA, followed by condensation with formylpyr-
roles 18 and HBr, gave the tripyrrene benzyl ester
hydrobromide salts 23 as orange-red powders in 43-60%
yield. Subsequent treatment with HBr-TFA for 6 h,
followed by condensation with the complementary pyrrole
aldehyde (i.e., the second aldehyde is different from the
one used to prepare the tripyrrene), gave the a,c-biladiene
dihydrobromide salts 24. Cyclization with CuCl2, followed
by demetalation and reesterification, afforded the pen-
tamethyl esters 25 and 26 in excellent yields. These are
of synthetic intermediates used to prepare all four
isomers and selected the dipyrrylmethanes 9 and 10, both
of which can be generated from a common pyrrole 11
bearing an acetate side chain, as the key precursors
(Scheme 4). Condensation of dione 12 with ethyl bro-
moacetate in the presence of potassium carbonate in
refluxing acetone gave the diester 13 in moderate yield,
and subsequent Knorr-Kleinspehn condensation52,53 with
dibenzyl oximinomalonate (14) in the presence of zinc
dust and sodium acetate in acetic acid gave the mixed
triester 15 in 39% yield. The required (acetoxymethyl)-
pyrrole 11 was obtained by the regioselective oxidation
of 15 with lead tetraacetate in acetic acid. Montmorillo-
nite clay catalyzed condensation54 of 11 with R-unsub-
stituted pyrroles 16a 55 or 16b56 in dichloromethane gave
the pivotal intermediates 9 and 10 as white powders in
excellent yields.
Although dipyrroles 9 and 10 could be taken on to the
targeted pentacarboxylate porphyrin by a number of
established methods, the a,c-biladiene route51,57 was
selected on the basis of past experience58 and the avail-
ability of the other pyrrolic precursors. Hydrogenolysis
(48) Clezy, P. S.; Hai, T. T.; Gupta, P. C. Austr. J . Chem. 1976, 29,
393.
(49) Arsenault, G. P.; Bullock, E.; MacDonald, S. F. J . Am. Chem.
Soc. 1960, 82, 4384. Cavaleiro, J . A. S.; Rocha Gonsalves, A. M. d’A.;
Kenner, G. W.; Smith, K. M. J . Chem. Soc., Perkin Trans. 1 1974,
1771.
(50) J ackson, A. H.; Kenner, G. W.; McGillivray, G.; Smith, K. M.
J . Chem. Soc. C 1968, 294. J ackson, A. H.; Kenner, G. W. Nature 1967,
215, 1126.
(51) Baptista de Almeida, J . A. P.; Kenner, G. W.; Rimmer, R.;
Smith, K. M. Tetrahedron 1976, 32, 1793.
(52) Kleinspehn, G. G. J . Am. Chem. Soc. 1955, 77, 1546.
(53) Plieninger, H.; Hess, P.; Ruppert, J . Chem. Ber. 1968, 101, 240.
(54) J ackson, A. H.; Pandey, R. K.; Rao, K. R. N.; Roberts, E.
Tetrahedron Lett. 1985, 26, 793.
(55) Crook, P. J .; J ackson, A. H.; Kenner, G. W. J . Chem. Soc. C
1971, 474. Battersby, A. R.; Hunt, E.; McDonald, E.; Paine, J . B., III;
Saunders: J . J . Chem. Soc., Perkin Trans. 1 1976, 1008. Lash, T. D.;
Bellettini, J . R.; Bastian, J . A.; Couch, K. B. Synthesis 1994, 170.
(56) Drinan, M. A.; Lash, T. D. J . Heterocycl. Chem. 1994, 31, 255.
(57) Grigg, R.; J ohnson, A. W.; Kenyon, R.; Math, V. B.; Richardson,
K. J . Chem. Soc. C 1969, 176.
(58) Lash, T. D.; Balasubramaniam, R. P. Tetrahedron Lett. 1990,
31, 7545. May, D. A., J r.; Lash, T. D. J . Org. Chem. 1992, 57, 4820.
Lash, T. D. Tetrahedron 1998, 54, 359.
(59) Smith, K. M.; Minnetian, O. M. J . Chem. Soc., Perkin Trans. 1
1986, 277.

Normal and Abnormal Heme Biosynthesis. 2
J . Org. Chem., Vol. 64, No. 2, 1999 483
Sch em e 6
F igu r e 1. Time course study for incubations of pentacarboxy-
late porphyrinogens 5d a b, 5a bc, 5a cd , and 5bcd with chicken
red cell hemolysates showing percentage of product formation
vs time. Product is defined as copro’gen-III (isolated as
coproporphyrin-III) and its metabolites (harderoporphyrin and
protoporphyrin-IX).
Ta ble 1. In itia l Velocities a n d Ma xim u m P r od u ct
F or m a tion for th e F ou r Syn th etic P en ta ca r boxyla te
P or p h yr in ogen Su bstr a tes (m ea n ( SDa )
the precursors to porphyrinogens 5a bc and 5bcd , re-
spectively.
initial velocity
(% product/min)
substrates
% product formedb
5dab
5abc
5bcd
5acd
2.0
2.0
0.4
0.4
90 ( 12
57 ( 11
6 ( 3
Meta bolism of Typ e-III P en ta ca r boxyla te
P or p h yr in ogen s
6 ( 3
Due to their inherent instability, porphyrinogens are
generated immediately prior to use in a given biochemical
experiment. Hence, porphyrin pentamethyl esters 20, 21,
25, and 26 were treated with 25% hydrochloric acid for
16 h at room temperature and reduced under buffered
conditions with 3% sodium-amalgam to generate the
porphyrinogen pentacarboxylic acids. These products
were incubated with CRH for different time periods and
the products analyzed by TLC and normal-phase HPLC
(analyses of the metabolic products were carried out on
the esterified porphyrins derived from these materials).
The kinetic profiles for these studies (Figure 1) consis-
tently showed that 5d a b was by far the best substrate
in these incubations in terms of the final amount of
product produced, followed by 5a bc, while the remaining
two pentacarboxylate porphyrinogens, 5a cd and 5bcd ,
appeared to be equally poor substrates for UPD. The
initial velocities for 5d a b and 5a bc were indistinguish-
able, although they were far greater for these substrates
than for 5bcd or 5a cd , but the maximum product for-
mation was significantly greater for 5d a b (Table 1). Not
only were these results highly reproducible, but similar
data were also obtained for hemolysates derived from
turkey and duck blood. It is interesting that 5a bc should
be the second best substrate as this is the only penta-
carboxylate porphyrinogen that could not be derived from
7d , the first intermediate in the “clockwise” decarboxy-
lation pathway. The striking differences in these kinetic
profiles provide strong support for 5d a b being an inter-
mediate in the preferred decarboxylation pathway lead-
ing to copro’gen-III.
a
b
For three replicate experiments. After a 60 min incubation
with CRH.
Ta ble 2. Effect of 5d a b Con cen tr a tion on th e % P r od u ct
F or m ed by UP Da
concentration of 5d a b (µM)
% productb
2
5
8
13
19
66 ( 2.6
46 ( 3.5
26 ( 2.5
20 ( 3.5
12 ( 1.7
a
b
20 min incubations with CRH at 37 °C. For three replicate
experiments.
strate. Using a range from 2 to 19 µM, 5d a b was incu-
bated for 10 min with CRH. The results (Table 2) clearly
show that above 2 µM the substrate appears to be
inhibitory for the enzyme. Others have also reported that
high substrate concentrations were inhibitory for UPD.9,14
Using uro’gen-III as substrate with preparations of UPD
derived from Rh. sphaeroides, J ones and J ordan14 re-
ported inhibition at concentrations greater than 12 µM,
and similar observations have been reported for chicken
erythrocyte UPD by Kawanishi et al.9 Taken together,
these data are suggestive that during porphyria high
levels of substrate can inhibit the activity of UPD and
this may result in the processing of uro’gen-III via the
nonpreferred (abnormal) hepta-, hexa-, and pentacar-
boxylate porphyrinogens.
Con clu sion s
In addition, the effect of varying substrate concentra-
tion on enzyme activity was assessed using 5d a b as sub-
Although it was proposed over 20 years ago,26 the
“clockwise” decarboxylation hypothesis for the enzymic

484 J . Org. Chem., Vol. 64, No. 2, 1999
Lash et al.
bromoacetate (16.7 mL) was added dropwise to a stirred
solution of dione 12 (35.00 g), potassium carbonate (23.40 g),
and reagent-grade acetone (150 mL) while a gentle reflux was
sustained. After the addition was complete, the solution was
refluxed for 5 h and then filtered while hot. The solid was
washed with acetone and the solvent evaporated under
reduced pressure. The residue was dissolved in chloroform,
washed thoroughly with water to remove all inorganic salts,
dried over magnesium sulfate, and filtered and the solvent
evaporated under reduced pressure. Vacuum distillation gave
methyl 5-[(ethoxycarbonyl)methyl]-4,6-dioxoheptanoate (13) as
a pale yellow oil (20.88 g; 40%): bp 125-138 °C/0.25 Torr.
conversion of uro’gen-III to copro’gen-III has remained
controversial. We have synthesized the four possible
pentacarboxylate intermediates and demonstrated that
one isomer, 5d a b, is a significantly better substrate for
uro’gen decarboxylase than the other three. These kinetic
studies provide further support for the preferred decar-
boxylation pathway proposed by J ackson et al. Further
investigations at the hexa- and heptacarboxylate levels
should also give insights into the selectivity of this
important metabolic process.
A solution of dibenzyl oximinomalonate (40.47 g) in glacial
acetic acid (80 mL) was added to a stirred solution of the
foregoing dione (20.88 g) in glacial acetic acid (100 mL) at 70
°C, while a mixture of zinc dust (32.8 g) and sodium acetate
(16.4 g) was slowly added and the reaction temperature was
maintained near 90 °C. After the addition was complete, the
mixture was heated on a boiling water bath for 1 h, cooled to
70 °C, and poured into 4 L of ice-water. The yellow oil was
extracted into ether, washed with water, dried over magne-
sium sulfate, and filtered and the solvent evaporated under
reduced pressure to give a yellow oil. The pyrrole was purified
by chromatography on silica using a gradient elution of
petroleum ether (60-90 °C), dichloromethane, and methanol
(4:1:0 to 0:1:0.05). Fractions containing the product were
combined, recrystallized from 95% methanol-water, and dried
in vacuo to give the title pyrrole (12.3 g; 39%) as white
crystals: mp 75-76 °C; IR (Nujol mull) ν 3316 (st, sh, NH),
1756, 1719, 1674 cm-1 (st, sh, 3 × CO); 1H NMR (CDCl3) δ
1.25 (3H, t, J ) 7.3 Hz), 2.23 (3H, s), 2.55 (2H, t, J ) 8.1 Hz),
3.02 (2H, t, J ) 8.1 Hz), 3.42 (2H, s), 3.63 (3H, s), 4.12 (2H, q,
J ) 7.3 Hz), 5.29 (2H, s), 7.37 (5H, m), 9.00 (1H, br s); 13C
NMR (CDCl3) δ 11.70, 14.23, 20.70, 29.99, 34.85, 51.45, 60.80,
65.88, 96.17, 114.55, 116.71, 128.22, 128.36, 128.60, 130.86,
Exp er im en ta l Section 61
Enzyme incubations and analyses of metabolic products
were carried out as described in the prequel to this paper,1
except that HPLC analyses were carried out using 50% ethyl
acetate/cyclohexane as the mobile phase.
Diben zyl Ma lon a te. A stirred mixture of malonic acid (104
g), benzyl alcohol (207 mL), p-toluenesulfonic acid (0.50 g), and
toluene (300 mL) was heated under reflux in a round-bottom
flask fitted with a Dean-Stark apparatus until 35.5 mL of
water had been collected (theoretical 36 mL). The solvent was
evaporated under reduced pressure, and subsequent vacuum
distillation gave dibenzyl malonate (249.3 g; 87%) as a colorless
oil: bp 180-185 °C/0.2 Torr (lit.62 bp 188 °C/0.2 mmHg); H
1
NMR (CDCl3) δ 3.22 (2H, s), 4.98 (4H, s), 7.2 (10H, s).
Dib en zyl Oxim in om a lon a t e (14).29 Sodium hydroxide
(20.0 g) was added with stirring to glacial acetic acid (160 mL).
After the pellets were completely dissolved and the mixture
remained hot, dibenzyl malonate (242.2 g) was added, and the
dropwise addition of a solution of sodium nitrite (110.0 g) in
water (150 mL) was immediately initiated. Once the addition
was complete, the mixture was stirred overnight to ensure
complete evolution of nitrogen dioxide. Water and ether were
added so that all the precipitated material dissolved. The
ethereal layer was separated, washed with water and 5%
aqueous sodium bicarbonate solution, and dried over sodium
sulfate and the solvent evaporated under reduced pressure to
give oxime 14 (245.2 g; 92%) as a pale yellow oil that solidified
on standing: 1H NMR (CDCl3) δ 5.20 (2H, s), 5.30 (2H, s), 7.3-
7.6 (10H, m), 10.0 (1H, br).
131.54, 136.22, 160.73, 171.75, 173.66. Anal. Calcd for C21H25
-
NO6: C, 65.10; H, 6.50; N, 3.61. Found: C, 65.00; H, 6.35; N,
3.46.
Ben zyl 3-[2-(Meth oxyca r bon yl)eth yl]-4,5-d im eth ylp yr -
r ole-2-ca r boxyla te. Iodomethane (20.1 g) was added to a
stirred mixture of 12 (27.19 g), potassium carbonate (15.2 g),
and reagent-grade acetone (50 mL) in a round-bottom flask
equipped with two condensers in tandem. The solution was
stirred vigorously at 80 °C (oil bath) overnight. The hot
mixture was filtered and washed with acetone and the solvent
evaporated under reduced pressure. The residue was dissolved
in chloroform, washed thoroughly with water to remove all
the inorganic materials, dried over magnesium sulfate and
filtered and the solvent evaporated under reduced pressure
to give a yellow oil. Vacuum distillation gave methyl 5-methyl-
4,6-dioxoheptanoate (21.70 g; 74%) as a pale yellow oil: bp
85-95 °C/0.025 Torr.
Meth yl 4,6-Dioxoh ep ta n oa te (12). A mixture of magne-
sium turnings (26.7 g), absolute ethanol (135 mL), and carbon
tetrachloride (5 mL) was stirred until the evolution of hydrogen
gas subsided (2 h). Dry benzene (200 mL) was added, and the
mixture was heated under gentle reflux for 4 h or until
hydrogen evolution was complete. The mixture was cooled to
room temperature, and dry diethyl ether (500 mL) was added.
tert-Butyl acetoacetate (170 mL) was then added dropwise at
a rate sufficient to maintain gentle reflux. After the addition
was complete, the solution was further refluxed for 30 min.
The solution was cooled to room temperature, and 3-methoxy-
carbonylpropanoyl chloride63 (207.9 g) was added dropwise to
the stirred mixture. After the initial reaction had subsided,
the solution was refluxed for 1 h and then stirred overnight
at room temperature. The solution was cooled using an ice-
salt bath and treated with 5 M sulfuric acid (200 mL) and
water to dissolve any precipitates that had formed. The organic
layer was separated, and the aqueous layer was extracted with
diethyl ether (3 × 100 mL). The combined organic layers were
washed with brine and dried over magnesium sulfate, and the
solvent was evaporated under reduced pressure. The residual
oil was stirred with p-toluenesulfonic acid (1.20 g) at 140-
150 °C until gas evolution was complete (approximately 2 h).
Vacuum distillation gave the diketone (87.0 g; 50%) as a pale
yellow oil: bp 75-80 °C/0.025 Torr.
Dibenzyl oximinomalonate (30.1 g) in glacial acetic acid (50
mL) was added to the foregoing dione (18.4 g) in glacial acetic
acid (60 mL) while a mixture of zinc dust (26.0 g) and sodium
acetate (12.1 g) was added as described in the previous
procedure. The organic layer was diluted with ether, washed
with water, dried over magnesium sulfate, and filtered and
the solvent evaporated under reduced pressure to give a yellow
solid. Recrystallization from 95% methanol-water gave the
title pyrrole as a white powder (10.1 g; 33%): mp 97-98 °C
(lit.64a mp 93-94 °C; lit.64b mp 90-92 °C); IR (Nujol mull) ν
1
3304 (st, sh, NH), 1735, 1664 cm-1 (st, sh, 2 × CO); H NMR
(CDCl3) δ 1.92 (3H, s), 2.15 (3H, s), 2.48 (2H, t, J ) 8.2 Hz),
3.01 (2H, t, J ) 8.2 Hz), 3.61 (3H, s), 5.27 (2H, s), 7.35 (5H,
m), 8.83 (1H, br s); 13C NMR (CDCl3) δ 8.30, 11.13, 20.60,
34.58, 51.13, 65.37, 115.81, 116.60, 127.80, 127.92, 128.25,
130.01, 130.22, 136.11, 160.66, 173.40.
Ben zyl 4-[(Eth oxyca r bon yl)m eth yl]-3-[2-(m eth oxyca r -
b on yl)et h yl]-5-m et h ylp yr r ole-2-ca r b oxyla t e (15). Ethyl
Ben zyl 5-(Acetoxym eth yl)-4-[(eth oxycar bon yl)m eth yl]-
3-[2-(m eth oxyca r bon yl)eth yl]p yr r ole-2-ca r boxyla te (11).
Lead tetraacetate (9.33 g) was added to a stirred solution of
pyrrole 15 (8.00 g) in glacial acetic acid (200 mL) and acetic
(60) Smith, K. M.; Craig G. W. J . Org. Chem. 1983, 48, 4302.
(61) For general experimental procedures, see ref 1.
(62) Baker, B. R.; Schraub, R. E.; Querry, M. V.; Williams, J . H. J .
Org. Chem. 1952, 17, 77.
(63) Cason, J . J . Am. Chem. Soc. 1942, 64, 1106.

Normal and Abnormal Heme Biosynthesis. 2
J . Org. Chem., Vol. 64, No. 2, 1999 485
anhydride (10 mL) and the solution stirred at room temper-
ature for 2 days. The resulting mixture was poured into ice-
water (1 L) and the precipitate filtered, dissolved in chloroform,
and washed with water. The chloroform solution was filtered
through Celite and dried over magnesium sulfate and the
solvent evaporated under reduced pressure. Crystallization
from 95% methanol-water gave the (acetoxymethyl)pyrrole
(8.65 g; 94%) as a white powder: mp 108-108.5 °C; IR (Nujol
mull) ν 3292 (st, br, NH), 1732, 1670 cm-1 (st, sh, 4 × CO); 1H
NMR (CDCl3) δ 1.24 (3H, t, J ) 7.1 Hz), 2.06 (3H, s), 2.54
(2H, t, J ) 8.4 Hz), 3.01 (2H, t, J ) 8.4 Hz), 3.52 (2H, s), 3.62
(3H, s), 4.12 (2H, q, J ) 7.1 Hz), 5.05 (2H, s), 5.30 (2H, s),
7.36 (5H, s), 9.23 (1H, br s); 13C NMR (CDCl3) δ 14.19, 20.54,
20.90, 29.68, 34.71, 34.90, 51.53, 56.92, 61.04, 66.21, 117.14,
119.09, 128.33, 128.42, 128.64, 128.92, 130.00, 135.91, 160.56,
171.48, 173.59. Anal. Calcd for C23H27NO8: C, 62.01; H, 6.11;
N, 3.14. Found: C, 61.71; H, 5.96; N, 3.19.
4-[2-(Met h oxyca r b on yl)et h yl]-3,5-d im et h ylp yr r ole-2-
ca r boxa ld eh yd e (18a ). Benzyl 4-[2-(methoxycarbonyl)ethyl]-
3,5-dimethylpyrrole-2-carboxylate65 (4.626 g), triethylamine
(20 drops), and absolute ethanol (200 mL) were placed in a
hydrogenation vessel. After the air had been displaced with a
stream of nitrogen gas, 10% palladium charcoal (200 mg) was
added. The mixture was shaken overnight at room tempera-
ture under an atmosphere of hydrogen at 40 psi. The catalyst
was filtered off, and the solvent was evaporated under reduced
pressure. The residue was taken up in dilute ammonia (5%
w/v) and cooled to 0 °C and the solution neutralized with dilute
HCl (5% w/v). The resulting precipitate was filtered, washed
thoroughly with water to remove all traces of acid, and dried
in vacuo to give 4-[2-(methoxycarbonyl)ethyl]-3,5-dimethylpyr-
role-2-carboxylic acid (2.52 g; 76%) as a pale pink powder: mp
131-132 °C dec (lit.66 mp 131 °C dec).
A solution of the foregoing pyrrolecarboxylic acid (1.40 g)
was treated with trifluoroacetic acid (5 mL) for 10 min.
Trimethyl orthoformate (3 mL) was added dropwise to the
reaction mixture and the resulting mixture stirred for 10 min
at 40 °C. The mixture was then cooled to room temperature,
diluted with water (50 mL), and extracted with chloroform (3
× 50 mL). The combined organic layers were washed with
dilute aqueous ammonia (5% v/v; 50 mL) and then again with
water (50 mL). The organic layer was dried over magnesium
sulfate and filtered and the solvent evaporated under reduced
pressure. The residual oil was recrystallized from aqueous
methanol to give the formylpyrrole (0.978 g; 75%) as green
crystals: mp 125-127 °C (lit.67a mp 128-129 °C; lit.67b 123-
125 °C); IR (Nujol mull) ν 3230 (st, sh, NH); 1732, 1634 cm-1
(st, sh, 2 × CO); 1H NMR (CDCl3) δ 2.26 (3H, s), 2.27 (3H, s),
2.45 (2H, t, J ) 7.3 Hz), 2.72 (2H, t, J ) 7.3 Hz), 3.67 (3H, s),
9.38 (1H, br s), 9.46 (1H, s); 13C NMR (CDCl3) δ 8.46, 11.20,
18.97, 34.30, 51.29, 120.67, 127.65, 132.22, 136.26, 173.05,
175.45.
3-[2-(Met h oxyca r b on yl)et h yl]-4,5-d im et h ylp yr r ole-2-
ca r boxa ld eh yd e (18b). Benzyl 3-[2-(methoxycarbonyl)ethyl]-
4,5-dimethylpyrrole-2-carboxylate (10.00 g) was hydrogeno-
lyzed over 10% palladium-charcoal by the procedure described
above. After the mixture was dried overnight in vacuo, 3-[2-
(methoxycarbonyl)ethyl]-4,5-dimethylpyrrole-2-carboxylic acid
(4.81 g; 77%) was obtained as a pale pink powder: 1H NMR
(CDCl3) δ 1.93 (3H, s), 2.18 (3H, s), 2.56 (2H, t, J ) 7.4 Hz),
3.03 (2H, t, J ) 7.4 Hz), 3.66 (3H, s), 8.92 (1H, br s); 13C NMR
(CDCl3) δ 8.84, 11.75, 21.00, 34.90, 35.13, 51.76, 115.81, 117.71,
131.95, 132.45, 166.17, 174.21.
Recrystallization from methanol gave the formylpyrrole (0.888
g; 68%) as brown needles: mp 91-93 °C (lit.68 mp 93-94 °C;
lit.39 mp 92-92.5 °C); IR (Nujol mull) ν 3230 (st, sh, NH), 1732,
1
1634 cm-1 (st, sh, 2 × CO); H NMR (CDCl3) δ 1.96 (3H, s),
2.25 (3H, s), 2.56 (2H, t, J ) 7.4 Hz), 3.02 (2H, t, J ) 7.4 Hz),
3.68 (3H, s), 9.47 (1H, s), 10.0 (1H, br s); 13C NMR (CDCl3) δ
9.39, 14.40, 20.09, 35.51, 51.60, 115.44, 123.53, 136.00, 136.43,
151.81, 173.34.
Ben zyl 5′-(ter t-Bu toxyca r bon yl)-3-[(eth oxyca r bon yl)-
m et h yl]-4,4′-b is[2-(m et h oxyca r b on yl)et h yl]-3′-m et h yl-
2,2′-d ip yr r ylm eth a n e-5-ca r boxyla te (10). Montmorillonite
clay (K10; 2.70 g) was added to a stirred solution of (acetoxy-
methyl)pyrrole 11 (0.501 g) and tert-butyl 3-[2-(methoxycar-
bonyl)ethyl]-4-methylpyrrole-2-carboxylate (16b; 0.295 g) in
dichloromethane (40 mL) and the mixture vigorously stirred
at room temperature overnight. The clay catalyst was filtered
off and the solvent evaporated under reduced pressure to give
a pale pink solid. Recrystallization from methanol gave the
title dipyrrylmethane (0.612 g; 83%) as a white solid: mp 150-
151 °C (softens at 144 °C); 1H NMR (CDCl3) δ 1.25 (3H, t, J )
7.2 Hz), 1.52 (9H, s), 1.99 (3H, s), 2.47-2.54 (4H, m), 2.99 (4H,
t, J ) 7.6 Hz), 3.45 (2H, s), 3.61 (3H, s), 3.66 (3H, s), 3.83 (2H,
s), 4.15 (2H, q, J ) 7.1 Hz), 5.25 (2H, s), 7.25-7.40 (5H, m),
8.74 (1H, br s), 9.23 (1H, br s); 13C NMR (CDCl3) δ 8.80, 14.24,
20.61, 20.92, 23.01, 28.52, 29.72, 34.91, 35.08, 51.63, 61.43,
66.28, 80.73, 115.06, 116.98, 118.21, 119.69, 128.32, 128.54,
128.62, 128.87, 129.04, 130.68, 132.09, 136.23, 160.99, 161.05,
172.71, 173.90, 174.18. Anal. Calcd for C35H44N2O10: C, 64.40;
H, 6.79; N, 4.29. Found: C, 64.40; H, 6.78; N, 4.25.
Diben zyl 3-[(Eth oxyca r bon yl)m eth yl]-3′,4-bis[2-(m eth -
oxyca r bon yl)eth yl]-4′-m eth yl-2,2′-d ip yr r ylm eth a n e-5,5-
d ica r boxyla te (9). Prepared by the previous procedure from
(acetoxymethyl)pyrrole 11 (2.50 g), benzyl 4-[2-(methoxycar-
bonyl)ethyl]-3-methylpyrrole-2-carboxylate (16a ; 1.66 g) and
Montmorillonite clay (K10; 13.50 g). Recrystallization from
methanol gave the title dipyrrylmethane (3.21 g; 83%) as a
white solid: mp 98-99 °C; 1H NMR (CDCl3) δ 1.21 (3H, t, J )
7.2 Hz), 2.28 (3H, s), 2.48 (2H, t), 2.57 (2H, t), 2.76 (2H, t),
3.01 (2H, t), 3.53 (2H, s), 3.51 (3H, s), 3.60 (3H, s), 3.92 (2H,
s), 4.05 (2H, q, J ) 7.2 Hz), 5.24 (2H, s), 7.25-7.41 (5H, m),
9.87 (1H, br s), 10.02 (1H, br s); 13C NMR (CDCl3) δ 7.49, 10.82,
15.53, 17.36, 18.99, 26.40, 30.63, 31.62, 48.23, 48.70, 58.27,
62.06, 62.54, 111.57, 115.00, 115.19, 116.29, 123.84, 124.74,
125.04, 125.07, 125.15, 125.27, 125.44, 126.89, 127.97, 129.28,
133.17, 133.68, 157.61, 158.06, 170.56, 170.61, 171.77. Anal.
Calcd for C38H42N2O10: C, 66.46; H, 6.16; N, 4.08. Found: C,
66.26; H, 6.18; N, 3.92.
5′-(ter t-Bu toxyca r bon yl)-3-[(eth oxyca r bon yl)m eth yl]-
4,4′-bis[2-(m eth oxyca r bon yl)eth yl]-3′-m eth yl-2,2′-d ip yr -
r ylm eth a n e-5-ca r boxylic Acid (22). A mixture of dipyrryl-
methane 10 (0.445 g), triethylamine (20 drops), and ethanol
(100 mL) was shaken with 10% palladium-charcoal (50 mg)
under a hydrogen atmosphere at 40 psi and room temperature
for 16 h. The catalyst was filtered off and the solvent
evaporated under reduced pressure. The residue was taken
up in aqueous ammonia (5%) and cooled to 0 °C using an ice-
salt bath. The solution was neutralized with glacial acetic acid,
maintaining the temperature at 0 °C, and the resulting
precipitate filtered off, washed thoroughly with water, and
dried in vacuo overnight. The title dicarboxylic acid (428 mg;
quantitative) was obtained as an off-white powder: mp 71-
1
72 °C dec; H NMR (CDCl3) δ 1.25 (3H, t), 1.55 (9H, s), 2.01
(3H, s), 2.47 (2H, t), 2.61 (2H, t), 2.97 (2H, t), 3.06 (2H, t),
3.48 (2H, s), 3.65 (3H, s), 3.68 (3H, s), 3.91 (2H, s), 4.14 (2H,
q), 10.79 (1H, br s), 11.33 (1H, br s). Anal. Calcd for
The foregoing carboxylic acid (1.40 g) was reacted with TFA
(5 mL) and trimethyl orthoformate (3 mL) as described above.
The crude product was purified by column chromatography
on silica by elution with cyclohexane-ethyl acetate (1:1).
C
28H38N2O10: C, 59.77; H, 6.81; N, 4.98. Found: C, 59.69; H,
6.71; N, 4.76.
3-[(Eth oxyca r bon yl)m eth yl]-3′,4-bis[2-(m eth oxyca r bo-
n yl)eth yl]-4′-m eth yl-2,2′-d ip yr r ylm eth a n e-5,5′-d ica r box-
ylic Acid (17). Dipyrrylmethane dibenzyl ester 9 (0.382 g),
(64) (a) Crook, P. J .; J ackson, A. H.; Kenner, G. W. Liebigs Ann.
Chem. 1971, 748, 26. (b) Clezy, P. S.; Diakiw, V. Aust. J . Chem. 1975,
28, 2703.
(65) J ohnson, A. W.; Markham, E.; Price, R.; Shaw, K. B. J . Chem.
Soc. 1958, 4254.
(66) J ackson, A. H.; Kenner, G. W.; Wass, J . J . Chem. Soc., Perkin
Trans. 1 1972, 1475.
(67) (a) Davies, J . L. J . Chem. Soc. C 1968, 1392. (b) Al-Hazimi, H.
M. G.; J ackson, A. H.; Knight, D. W.; Lash, T. D. J . Chem. Soc., Perkin
Trans. 1 1987, 265.
(68) Smith, K. M.; Pandey, R. K. J . Heterocycl. Chem. 1985, 22, 1041.

486 J . Org. Chem., Vol. 64, No. 2, 1999
Lash et al.
13.40 (1H, br s), 13.43 (1H, br s). Anal. Calcd for C44H58N4O10
Br2‚H2O: C, 53.88, H, 6.16; N, 5.71. Found: C, 53.02; H, 5.84;
N, 5.77.
triethylamine (20 drops), acetone (200 mL), and 10% pal-
ladium-charcoal (50 mg) were placed in a hydrogenation vessel
and hydrogenolyzed following the previous procedure. After
being dried in vacuo overnight, the title pyrrole (0.257 g; 90%)
was obtained as an off-white powder: mp 158.5-160 °C dec;
1H NMR (DMSO-d6): δ 1.14 (3H, t, J ) 7.2 Hz), 2.12 (3H, s),
2.13 (2H, t), 2.43 (2H, t), 2.53 (2H, t), 2.79 (2H, t), 3.40 (2H,
s), 3.55 (3H, s), 3.56 (3H, s), 3.80 (2H, s), 4.15 (2H, q, J ) 7
Hz), 11.02 (1H, br s), 11.31 (1H, br s), 12.1 (2H, v br). Anal.
Calcd for C24H30N2O10: C, 56.91, H, 5.97; N, 5.53. Found: C,
57.02; H, 6.10; N, 5.34.
Ben zyl 3-[(Eth oxycar bon yl)m eth yl]-2,8,13-tr is[2-(m eth -
oxyca r bon yl)eth yl]-7,12,14-tr im eth yl-5,16-d ih yd r otr ip yr -
r in -1-ca r boxyla te Hyd r obr om id e (23a ). Benzyl 5′-(tert-
butoxycarbonyl)-3-[(ethoxycarbonyl)methyl]-4,4′-bis[2-(meth-
oxycarbonyl)ethyl]-3′-methyl-2,2′-dipyrrylmethane-5-carboxy-
late (10; 0.511 g) was treated with trifluoroacetic acid (3.75
mL) with stirring for 5 min at ambient temperature. A solution
of carboxaldehyde 18a (0.187 g) in methanol (20 mL) was
added all at once and the mixture stirred at room temperature
for 90 min. A mixture of aqueous HBr (48%) and acetic acid
(1:2.33 v/v, 0.1 mL) and ether (30 mL) was added, and the
mixture was stirred for a further 1 h at room temperature and
then placed in a freezer overnight. The precipitate was filtered,
washed thoroughly with ether, and dried in vacuo overnight
to give the title tripyrrene hydrobromide (0.403 g; 60.5%) as
an orange-brown powder: mp 77-78 °C; UV-vis (CHCl3) λmax
(log ꢀ) 493 nm (4.93); 1H NMR (CDCl3) δ 1.23 (3H, t), 2.02 (3H,
s), 2.35 (3H, s), 2.69 (3H, s), 2.51 (6H, m), 2.77 (2H, t), 2.9-
3.0 (4H, m), 3.52 (2H, s), 3.59 (3H, s), 3.61 (3H, s), 3.67 (3H,
s), 4.15 (2H, q), 4.35 (2H, s), 5.30 (2H, s), 7.31 (4H, m), 7.48
(2H, d), 10.82 (1H, br s), 13.13 (2H, br s). Anal. Calcd for
-
12-[(Eth oxycar bon yl)m eth yl]-3,7,13,18-tetr akis[2-(m eth -
oxyca r b on yl)et h yl]-1,2,8,17,19-p en t a m et h yl-10,23-d ih y-
d r obilin Dih yd r obr om id e (24b). Tripyrrene hydrobromide
23b (300 mg) was treated with a mixture of 30% HBr-acetic
acid (1.0 mL) and trifluoroacetic acid (5.0 mL) and stirred at
room temperature for 6 h. A solution of pyrrole aldehyde 18a
(80 mg) in methanol (18 mL) was added all at once and the
mixture stirred for an additional 40 min. Ether (50 mL) was
added dropwise over a short period of time, and stirring was
continued for 1 h. The ether was evaporated and the residue
taken up in methanol-ether and allowed to crystallize in the
freezer overnight. The resulting crystals were filtered and
dried in vacuo overnight to give the a,c-biladiene (186 mg; 53%)
as a red-brown powder: mp 166-167 °C; UV-vis (CHCl3) λmax
(log ꢀ) 455 (4.37), 524 nm (5.11); 1H NMR (CDCl3) δ 0.94 (3H,
t), 1.93 (3H, s), 2.03 (3H, s), 2.38 (3H, s), 2.4-2.6 (8H, m), 2.69
(3H, s), 2.75 (3H, s), 2.78 (2H, t), 2.94 (4H, m), 3.02 (2H, t),
3.62 (6H, s), 3.64 (3H, s), 3.68 (3H, s), 3.65-3.75 (4H, m), 5.20
(2H, s), 7.38 (1H, s), 7.51 (1H, s), 13.18 (1H, br s), 13.30 (1H,
br s), 13.40 (1H, br s), 13.45 (1H, br s). Anal. Calcd for
C
44H58N4O10Br2‚H2O: C, 53.88, H, 6.16; N, 5.71. Found: C,
53.39; H, 5.75; N, 5.54.
8-[(Eth oxycar bon yl)m eth yl]-2,7,12,18-tetr akis[2-(m eth -
oxyca r bon yl)eth yl]-1,3,13,17,19-p en ta m eth yl-10,23-d ih y-
d r obilin Dih yd r obr om id e (19a ). Dipyrrole 17 (250 mg) was
dissolved in trifluoroacetic acid (1.0 mL) and stirred at room
temperature for 10 min. A solution of pyrrole aldehyde 18a
(207 mg) in methanol (2.0 mL) was added, and the residual
material was washed into the reaction flask with methanol
(2.0 mL). HBr in acetic acid (30%; 0.8 mL) was immediately
added, and the mixture was stirred for 30 min at room
temperature. Anhydrous ether (17 mL) was added dropwise,
after which the mixture was stirred at room temperature for
a further 2 h. The resulting precipitate was filtered and rinsed
with anhydrous ether to give the a,c-biladiene dihydrobromide
(325 mg; 68%) as a brick red solid (325 mg; 68%): mp 156-
157 °C dec; UV-vis (CHCl3) λmax (log ꢀ) 451 (4.65), 522 (5.15);
1H NMR (CDCl3) δ 0.94 (3H, t), 2.05 (3H, s), 2.02 (2H, t), 2.09
(3H, s), 2.16 (3H, s), 2.45-2.55 (8H, m), 2.70-2.81 (6H, m),
2.86 (2H, t), 2.70 (3H, s), 2.73 (3H, s), 3.40 (3H, s), 3.61 (3H,
s), 3.67 (6H, s), 3.74 (2H, q), 3.78 (2H, s), 5.24 (2H, s), 7.08
(1H, s), 7.41 (1H, s), 13.24 (1H, s) 13.37 (1H, s) 13.45 (1H, s),
13.54 (1H, s). Anal. Calcd for C44H58N4O10Br2‚H2O: C, 53.88,
H, 6.16; N, 5.71. Found: C, 53.31; H, 6.02; N, 5.89.
C
41H50N3O10Br‚0.6H2O: C, 59.06; H, 6.04, N, 5.04. Found: C,
58.64; H, 5.97; N, 5.03.
Ben zyl 3-[(Eth oxycar bon yl)m eth yl]-2,8,12-tr is[2-(m eth -
oxyca r bon yl)eth yl]-7,13,14-tr im eth yl-5,16-d ih yd r otr ip yr -
r in -1-ca r boxyla te Hyd r obr om id e (23b). Dipyrrylmethane
10 (0.275 g) was treated with trifluoroacetic acid (2.0 mL) with
stirring at ambient temperature for 5 min. A solution of
pyrrole-2-carboxaldehyde 18b (0.101 g) in methanol (5.0 mL)
was added all at once, the dark greenish-orange solution
stirred for an additional 90 min, and a mixture of aqueous
HBr (48%) and acetic acid (1:2.3 v/v, 2 drops) added dropwise.
Rapid but dropwise addition of ether (20 mL) and continuous
stirring of the mixture did not result in precipitation of the
tripyrrene product, so the mixture was left in the freezer
overnight. The resulting precipitate was filtered, washed
thoroughly with ether, and dried in vacuo overnight to give
the desired tripyrrene hydrobromide (0.154 g; 43%) as bright
orange-red crystals, mp 93.5-94.5 °C; UV-vis (CHCl3) λmax
(log ꢀ) 493 nm (4.92); 1H NMR (CDCl3) δ 1.23 (3H, t), 2.02 (6H,
s), 2.64 (3H, s), 2.5-2.6 (6H, m), 2.95-3.08 (6H, m), 3.52 (2H,
s), 3.60 (3H, s), 3.61 (3H, s), 3.63 (3H, s), 4.10 (2H, q), 4.35
(2H, s), 5.30 (2H, s), 7.2-7.3 (3H, m), 7.49 (3H, m), 10.84 (1H,
br s), 13.11 (2H, br). Anal. Calcd for C41H50N3O10Br‚0.5H2O:
C, 59.06; H, 6.04, N, 5.04. Found: C, 58.73; H, 5.90; N, 5.04.
12-[(Eth oxycar bon yl)m eth yl]-2,7,13,17-tetr akis[2-(m eth -
oxyca r bon yl)et h yl]-1,3,8,18,19-p en t a m et h yl-10,23-d ih y-
d r obilin d ih yd r obr om id e (24a ). Benzyl 3-[(ethoxycarbonyl)-
methyl]-2,8,13-tris[2-(methoxycarbonyl)ethyl]-7,12,14-trimethyl-
5,16-dihydrotripyrrin-1-carboxylate hydrobromide (23a ; 0.310
g) was treated with a mixture of 30% HBr-acetic acid (1.0
mL) and trifluoroacetic acid (5.0 mL) with stirring at ambient
temperature for 6 h. A solution of aldehyde 18b (80 mg) in
methanol (18 mL) was added all at once and the mixture
stirred for 40 min. Ether (50 mL) was added dropwise but
rapidly and stirred for 30 min in an ice bath. The precipitate
was filtered, washed thoroughly with ether, and dried in vacuo
overnight to yield the a,c-biladiene (0.221 g; 61%) as red
crystals: mp 165-166 °C; UV-vis (CHCl3) λmax (log ꢀ) 455
(4.38), 524 nm (5.17); 1H NMR (CDCl3) δ 0.95 (3H, t), 1.94 (3H,
s), 2.04 (3H, s), 2.36 (3H, s), 2.4-2.6 (8H, m), 2.71 (3H, s), 2.73
(3H, s), 2.78 (2H, t), 2.90 (2H, t), 2.98 (2H, t), 3.05 (2H, t),
3.62 (6H, s), 3.65 (3H, s), 3.68 (3H, s), 3.72 (4H, m), 5.20 (2H,
s), 7.33 (1H, s), 7.56 (1H, s), 13.19 (1H, br s), 13.33 (1H, br s),
8-[(Eth oxycar bon yl)m eth yl]-3,7,12,17-tetr akis[2-(m eth -
oxyca r bon yl)eth yl]-1,2,13,18,19-p en ta m eth yl-10,23-d ih y-
d r obilin Dih yd r obr om id e (19b). This was prepared by the
procedure detailed above from dipyrrole 17 (500 mg) and
formylpyrrole 18b (414 mg). The title a,c-biladiene dihydro-
bromide salt (347 mg; 73%) was obtained as a brick-red solid:
mp > 260 °C (darkens at 170 °C); UV-vis (CHCl3) λmax (log ꢀ)
453 (4.60), 524 (5.13); 1H NMR (CDCl3) δ 0.93 (3H, t), 2.04
(3H, s), 2.10 (3H, s), 2.29 (3H, s), 2.5-2.65 (8H, m), 2.70 (3H,
s), 2.73 (3H, s), 2.86 (2H, t), 2.95-3.1 (6H, m), 3.40 (3H, s),
3.61 (3H, s), 3.64 (3H, s), 3.65 (3H, s), 3.72 (2H, q), 3.81 (2H,
s), 5.24 (2H, s), 7.28 (1H, s), 7.59 (1H, s), 13.21 (1H, s) 13.35
(1H, s) 13.43 (1H, s), 13.53 (1H, s). Anal. Calcd for C44H58N4O10-
Br2‚H2O: C, 53.88, H, 6.16; N, 5.71. Found: C, 53.76; H, 5.70;
N, 5.58.
2,8,12,17-Tetr akis[2-(m eth oxycar bon yl)eth yl]-7-[(m eth -
oxyca r bon yl)m eth yl]-3,13,18-tr im eth ylp or p h yr in . (25).
a,c-Biladiene dihydrobromide 24a (374 mg) was added to a
stirred solution of copper(II) chloride (1.13 g) in N,N-dimeth-
ylformamide (135 mL), and the resulting mixture was stirred
in the dark for 2 h. The mixture was diluted with dichlo-
romethane (200 mL) and washed with water (2 × 250 mL).
The aqueous layers were back-extracted with dichloromethane,
and the combined organic layers were dried over sodium
sulfate and filtered. The solvent was evaporated on a rotary
evaporator under aspirator pressure and then the residual
DMF removed using a vacuum pump. The solid residue was
taken up in 15% v/v sulfuric acid-trifluoroacetic acid (46 mL)

Normal and Abnormal Heme Biosynthesis. 2
J . Org. Chem., Vol. 64, No. 2, 1999 487
and stirred in the dark at room temperature for 45 min. The
reaction mixture was diluted with dichloromethane (185 mL)
and washed with water (2 × 185 mL) and 5% aqueous sodium
bicarbonate solution (185 mL). The organic layer was dried
over sodium sulfate, and the solvent was evaporated under
reduced pressure. The residue was then dissolved in 5%
sulfuric acid-methanol (50 mL) and stirred in the dark
overnight for reesterification. The reaction mixture was diluted
with dichloromethane and washed with water and then with
5% aqueous sodium bicarbonate solution. The organic layer
was dried over sodium sulfate, and the solvent was evaporated
under reduced pressure. The residue was chromatographed
on grade-III alumina column (2.2 cm × 16 cm), eluting with
dichloromethane. The violet fraction was evaporated under
reduced pressure and the residue recrystallized from chloro-
form-methanol to give the title porphyrin (189 mg; 63%) as
purple crystals, mp 213.5-214.5 °C (lit.29 mp 211-212 °C; lit.48
mp 218-219 °C48); UV-vis (CHCl3) λmax (log ꢀ) 402 (5.23), 500
(4.14), 535 (3.97), 567 (3.79), 623 (3.56); 1H NMR (CDCl3) δ
-3.79 (2H, br s), 3.24-3.32 (6H, m), 3.33 (2H, t), 3.62 (3H, s),
3.66 (3H, s), 3.67 (3H, s), 3.68 (9H, s), 3.69 (3H, s), 3.80 (3H,
s), 4.35-4.48 (8H, m), 5.11 (2H, s), 10.07 (2H, s), 10.16 (1H,
s), 10.17 (1H, s); HRFABMS calcd for C42H48N4O10 + H
769.3449, found, 769.3454.
2,8,13,18-Tetr akis[2-(m eth oxycar bon yl)eth yl]-7-[(m eth -
oxycar bon yl)m eth yl]-3,12,17-tr im eth ylpor ph yr in (26). This
porphyrin was prepared from a,c-biladiene 24b (230 mg) by
the procedure described above. Recrystallization from chloro-
form-methanol gave the title porphyrin (77 mg; 42%) as violet
crystals: mp 154.5-155.5 °C (lit.48 mp 154-155 °C); UV-vis
(CHCl3) λmax (log ꢀ) 402 (5.26), 500 (4.14), 535 (3.98), 567 (3.81),
621 nm (3.59); 1H NMR (CDCl3) δ -3.80 (2H, br s), 3.25-3.36
(8H, m), 3.62 (3H, s), 3.65 (3H, s), 3.67 (9H, s), 3.68 (3H, s),
3.71 (3H, s), 3.77 (3H, s), 4.35-4.5 (8H, m), 5.09 (2H, s), 10.07
(1H, s), 10.08 (1H, s), 10.10 (1H, s), 10.16 (1H, s); HRFABMS:
calcd for C42H48N4O10 + H 769.3449, found: 769.3461.
3,8,13,17-Tetr akis[2-(m eth oxycar bon yl)eth yl]-7-[(m eth -
oxycar bon yl)m eth yl]-2,12,18-tr im eth ylpor ph yr in (20). The
title porphyrin was prepared from a,c-biladiene 19a (250 mg)
by the procedure described above. Recrystallization from
chloroform-methanol gave 20 as a purple solid (85 mg; 42%):
mp 217-218 °C (lit.29 mp 219-220 °C; lit.48 mp 218-219 °C);
UV-vis (CHCl3) λmax 402, 500, 536, 568, 622 nm; 1H NMR
(CDCl3) δ -3.81 (2H, br s), 3.24 (8H, m), 3.55 (3H, s), 3.58
(3H, s), 3.59 (6H, s), 3.60 (3H, s), 3.62 (3H, s), 3.65 (3H, s),
3.71 (3H, s), 4.3-4.5 (8H, m), 5.05 (2H, s), 10.02 (1H, s), 10.03
(1H, s), 10.06 (1H, s), 10.10 (1H, s).
3,8,13,17-Tetr akis[2-(m eth oxycar bon yl)eth yl]-7-[(m eth -
oxycar bon yl)m eth yl]-2,12,18-tr im eth ylpor ph yr in (21). a,c-
Biladiene dihydrobromide 19b (400 mg) was cyclized by the
procedure detailed above. Recrystallization from chloroform-
methanol gave the title porphyrin as a purple solid (196 mg;
61%): mp 209.5-211 °C (lit.29 mp 205-206 °C; lit.48 mp 210-
211 °C; lit.51 mp 214-215.5 °C); UV-vis (CHCl3) λmax 402,
1
500, 536, 568, 622 nm; H NMR (CDCl3) δ -3.81 (2H, br s),
3.25 (8H, m), 3.55 (3H, s), 3.57 (3H, s), 3.60 (12H, s), 3.62 (3H,
s), 3.70 (3H, s), 4.3-4.5 (8H, m), 5.06 (2H, s), 9.99 (1H, s), 10.02
(1H, s), 10.09 (1H, s), 10.11 (1H, s); HRMS calcd for C42H48N4O10
768.3370, found 768.3367.
Ack n ow led gm en t. This material is based upon
work supported by the National Institutes of Health
under Grant Nos. 1 R15 DK45206-01 and 1 R15 GM/
OD52687-01A1 and the National Science Foundation
under Grant No. CHE-9500630.
Su p p or tin g In for m a tion Ava ila ble: Copies of proton
NMR and selected carbon-13 NMR spectra for compounds
9-11 and 15-26 are provided (28 pages). This material is
contained in libraries on microfiche, immediately follows this
article in the microfilm version of the journal, and can be
ordered from the ACS; see any current masthead page for
ordering information.
J O9814748