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Heme Biosynthesis from [14C]- and [3H]- Labeled Tripyrroles.

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[l] Tetrapyrrole Biosynthesis, Part 6. This work was supported by the Deut-
[2]
[3]
[4]
[S]
[6]
[7]
sche Forschungsgemeinschaft and the Fonds der Chemischen 1ndustrie.Part 5 : B. Franck, A. Rowold, Ch. Wegner, H.-G. Eckert, Phil. Trans.
R. SOC.London B273, 181 (1976).
B . Franck, D. Gantz, F.-P. MontJorts, F. Schmidtchen, Angew. Chem.
84, 433 (1972); Angew. Chem. Int. Ed. En@. 1 1 , 421 (1972).
A. I. Scott, C . A. Townsend, K . Okada, M . Kajiwara, P . J . Whitman,
R. J . Cushley, J. Am. Chem. SOC.94, 8267 (1972).
0. 7: G. Jones, Phil. Trans. R. SOC. London B 273, 207 (1976); see
also the bibliography given in D. Ronganathon, S. Ranganathan: Art
in Biosynthesis. Vol. I, Academic Press, New York 1976, p. 40.
B. Frydman, R. B. Frydman, Acc. Chem. Res. 8,201 (1975); R. B. Frydman,
A . Valasinas, S . Levy, B. Frydman, FEBS Lett. 38, 134 (1974).
S. n r n e r : The Design of Organic Synthesis. Elsevier, Oxford 1976,
p. 70; L . Velfuz, J . Volfs, J. Mathieu, Angew. Chem. 79, 774 (1967);
Angew. Chem. lnt. Ed. Engl. 6, 778 (1967).
B. Franck, D. Gantz, F. Hiiper, Angew. Chem. 84, 432 (1972); Angew.
Chem. Int. Ed. Engl. 1 1 , 420 (1972). -
Heme Biosynthesis from [‘“CI- and C3H]-Labeled Tripyrroled’]
By Burchard Franck, Gregor Fels, and Giinter Uferpl
The blood pigment heme [ ( S ) , T=H] is biosynthesized
via uroporphyrinogen 111 (2)C31 from four molecules of porphobilinogen (1)121 under inversion of one of the pyrrole
nuclei (in the dotted circle)f41.The course of the condensation
reaction yielding (2) from (1) and the point at which the
“pyrrole inversion” takes place are still uncertain16,71.
Tripyrroles have often been discussed as intermediates in heme
biosynthesis. We have now been able to obtain informative
experimental findings from biosynthetic studies with the [’“Cland [3H]-labeled tripyrroles, ( 3 a ) and (4 a ) [ ’ ] ,respectively.
( 5 a ) , ,\ , . ~‘ e <instead of
\
‘
,,F
e;
CI
In 17 separate experiments various quantities (0.5-4 mg)
of mixtures of the tripyrroles ( 3 a ) and ( 4 a ) and of their
carboxy derivatives ( 3 6 ) and ( 4 b ) were incubated for 5 or
24 h at 32 “C with 25 ml of hemolyzed duck blood[3.91. The
subsequently isolated hemin ( 5 a ) (90-1 17 mg) was purified
until its level of radioactivity was constant.
Table 1. Competitive incorporation of the 14C- and ’H-labeled tripyrroles
( 3 a ) and ( 4 a ) and of ( 3 b ) and ( 4 6 ) into hemin after 5 h.
Previous investigations of heme biosynthesis from isomeric
dipyrroles and oligopyrroles gave inconsistent results16**I. This
can be attributed to the small quantities (nmol) of substances
used, to variation in the enzyme activity in comparison experiments and a tendency for the reactive oligopyrroles to take
part in non-enzymatic condensation (“chemical blank)[’’. In
order to avoid such drawbacks, the multi-step syntheses of
( 3 a ) and ( 4 a ) were carried out in the mmol range. The
two pyrroles were labeled with different isotopes so that they
could compete for the heme-synthesizing enzyme in one experiment. A reliable determination of the “chemical blank[*]
was made possible by the observation that the carboxytripyrroles ( 3 b ) and ( 4 b ) which are also synthesized do not react
with the enzyme system but behave as the tripyrroles (3a)
and (4 a) respectively in the chemical condensation.
p] Prof. Dr. B. Franck, Dip].-Chem. G. Fels, Dip].-Chem. G. Ufer
Organisch-Chemisches Institut der Universitat
Orleans-Ring 23, D-4400 Munster (Germany)
652
Radioactivity of the
tripyrroles [ p a ]
Radioactivity of the
hemin [ E i ]
Incorporation of the
radioactivity of the tripyrroles into hemin
( 3 a ) 14C: 2.37
( 4 a ) ’H: 5.07
‘4C: 0.0122
’H: 0.0118
‘‘C: 0.51 %
’H: 0.23 %
( 3 6 ) 14C: 4.09
( 4 b ) ’H: 23.8
14C: 0.00034
3H: 0.00191
‘4C: 0.008%
3H: 0.008 %
The experimental results shown in Table 1 demonstrate
that the two tripyrroles (3a) and ( 4 a ) are converted into
heme significantly-(3 a), however, 2.2-fold faster. It follows
from the low incorporation values of the “unnatural” carboxytripyrroles ( 3 b ) and ( 4 b ) that the “chemical blanks” can be
neglected under the experimental conditions used.
The competitive incorporation of ( 3 a ) and (4 a ) has thus
shown that, in addition to the preferred biosynthetic precursor,
the enzyme system of duck blood can also use isomeric oligopyrroles. This could result in a partial diffusion of the 14Cand 3H-labelingof the tripyrroles (3 a ) and ( 4 a ) in the heme.
It was confirmed by the chemical degradation of the hemin
( 5 a ) . The phyllopyrrole (6) formed after methylating cleavage
(HI/CH,O) contained, in addition to 14C, 31 % of the 3Hradioactivity of the hemin ( 5 a ) and the phyllopyrrolecarboxylic acid ( 7 ) contained, in addition to 3H, 35% of the I4Cradioactivity.
Angew. Chem. Int. Ed. Engl. 16 ( 1 9 7 7 ) N o . 9
As is the case with other natural products, the enzyme
can use not only one but several intermediates for heme
biosynthesis although these intermediates may be utilized with
different efficiencies. This fact explains the apparently contradictory findings with other precursorsL6-*].
0
hv
- so2
Received: June 23, 1977 [Z 770bl
German version: Angew. Chem. 89. 677 (1977)
CAS Registry numbers:
( 3 a ) , 63448-77-1 ; ( 3 b ) , 63448-85-1; ( 4 a ) , 63448-78-2; (4bJ 63448-86-2;
( 5 a ) , 63527-98-0; (6), 63448-87-3; (7), 63448-884
____
[l] Tetrapyrrole Biosynthesis. Part 7. This work was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen 1ndustrie.Part 6: B. Franck, G. Fels, G. Ufer, A. Rowold, Angew. Chem. 89,
676 (1977); Angew. Chem. Int. Ed. Engl. 16, 651 (1977).
[2] E. 1 . B. Dresel, J . E . Falk, Biochem. J. 63, 80 (1956).
131 B. Franck, D. Gantz, F.-P. Montforts, F . Schmidtchen, Angew. Chern.
84,433 (1972); Angew. Chem. Int. Ed. Engl. 11,421 (1972).
[4] A. R. Battersby, G. L. Hodgson, E. Hunt, E . McDonald, J . Saunders,
J. Chem. SOC.Perkin 11976, 273.
[S] Failure of "pyrrole inversion" to occur results in the formation of uroporphyrinogen I which cannot be converted to heme ( 5 ) and is the cause
of an inborn, lethal blood disease (erythropoetic porpbyria).
[6] B. Frydman, R. B. Frydman, A . Valasinas, E. S . Levy, G . Feinstein, Phil.
Trans. R. SOC.London B273, 137 (1976).
[7] A . R. Battersby, E. McDonaldin K . M . Smith: Porphyrins and Metalloporphyrins. Elsevier, Oxford 1975, p. 61.
[8] A. R. Battersby, E. McDonald, D. C . Williams, H . K . W W r z i g e r , Chem.
SOC.Chem. Commun. 1977, 113.
[9] S . Granick, J. Biol. Chem. 232, 1101 (1958).
A Trimeric Cyclopentadienone Derivative by Photolysis
of 3,5-Diphenyl-4H-thiopyran-4-one 1,l-Dioxide with
Daylight
By Walter Ried and Hubert Bopp['l
Dedicated to Professor Friedrich Asinger on the occasion of
his 70th birthday
Depending upon the nature of substitution, cyclopentadienone derivatives are known either as monomers or, through
Diels-Alder reaction, as dimers1'1. The action of daylight on
3,5-diphenyl-4H-thiopyran-4-one
1,l-dioxide (I ), both in the
solid state as well as in solution, led uia the previously unknown
2,5-diphenylcyclopentadienoneto formation of a trimer (2)
by a double Diels-Alder reaction. Formation of a dimeric
Diels-Alder product, as is usual for monomeric unstable cyclopentadienone derivatives, could not be observed. A photochemical synthetic route to monomeric substituted cyclopentadienones has been described by Ishibe et al.[']: Irradiation
with a
of 2,6-bis(alkylthio)-3,5-diphenyl-4H-thiopyran-4-ones
mercury lamp leads to loss of sulfur and formation of stable
monomers.
The pentacyclic compound ( 2 j is present in the trans formr3]
and can be decarbonylated by briefly heating it to 190°C.
The bicyclic product (3)["],which can be regarded as normal
decarbonylated dimeric cyclopentadienone, is formed. Confirmation of this retro-Diels-Alder reaction is provided by the
decomposition of ( 2j in the mass spectrometer, since occurrence of the fragment m/e=435 as base peak in the spectrum
is consistent with the stability of (31, while no fragment is
observed that would correspond to the tricyclic decarbonylation product.
p] Prof. Dr. W. Ried, Dip].-Chem. H. Bopp
[nstitut fur Organische Chemie der Universitat, Laboratorium Niederrad
Theodor-Stern-Kai 7, D-6000 Frankfurt am Main 70 (Germany)
Angew. Chem. Int. Ed. Engl. 16 (1977) No. 9
Ph
131
The ease of SOz-extrusion from 4H-thiopyran-4-one 1,ldioxides depends decisively upon the choice of substituents.
Whereas, e. g. the 2,6-diphenyl['] and the 3,5-dimethyl derivative16J are completely stable in daylight, in the case of 3,5diphenyl derivatives extrusion of SOz already begins after
ca. 10min. The 2,5-diphenylcyclopentadienonethat is formed
can be trapped with suitable dienophiles. It reacts with
dimethyl acetylenedicarboxylate with loss of CO to give
dimethyl 3,6-diphenylbenzene-1,2-dicarboxylate
(4)121.
t - 1 , t-4, c-5, c-8, t-8a, c-9a-Hexaphenyloctahydro-i.-1,4
: t-5,8dimethanofluorene-9,10,11-trione (2): A solution of ( I )I7]
(2.96g, 0.01 mol) in CHC13 (10ml) is exposed to daylight for
10d at room temperature. The solvent is removed in a rotary
evaporator and the residue recrystallized from CH30H/acetone. 1.7g(73 %) of colorless crystals, m.p. 143°C (red coloration and decomposition).-IR (KBr): C=O 1770 (s), 1690
(s)C=C 1590(s)cm-'. UV(CHzCl,):h,,,(~)=232(3.5 x lo4),
260 (sh) nm ( 1 . 6 ~lo4). 'H-NMR (CDCl3): 6=4.32d (2),
5 = 3 H z ; 6.5d (2), J = 6 H z ; 6.82d (2), J = 6 H z ; 7.2-7.7m
(30). MS: m/e=696 (2 %), M'; 435 (100%).
2,4,7,7a-Tetrapheny1-3~,7a-dihydroinden-l
-one ( 3 ) : ( 2 ) (1 g,
1.4mmol) is heated in an oil-bath at 190°C for 15 min. After
cooling, the residue is recrystallized from acetone. 400mg
(64%) pale yellow crystals, m. p. 186"C.-IR (KBr): C=O
1705 (s), C==C 1590 (s) cm-'. UV (CHZClz):Lax(&)
230
(2.5 x lo4), 335nm (1 x lo"). 'H-NMR (CDC13):6=4.08d (l),
J = 2 H z ; 6.65 and 6.82 AB-dd (2), J = 7 H z ; 7.1-7.7m (21).
Dimethyl 3,6-diphenylbenzene-l,2-dicarboxylate
(4): A sohtion of ( I ) (296 mg, 1 mmol) and dimethyl acetylenedicarboxylate (284 mg, 2 mmol) in CHC13 (10 ml) is exposed to daylight
at room temperature. Separation of the reaction solution by
column chromatography on silica gel (eluent benzene/ethyl
acetate 10: 1) affords-together with small amounts of (2)160mg(46 %)(4jascolorlesscrystalsm.p. 192"C(after recrystallization from methanol).
Received: June 27, 1977 [Z 773 IE]
German version: Angew. Chern. 89.659 (1977)
CAS Registry numbers:
(1), 63448-89-5; (Z), 63448-90-8; (3). 63448-91-9; ( 4 j , 1169-58-0; dimethyl
acetylenedicarboxylate, 762-42-5
[l] M. A. Ogliaruso, M . Romanelli, E. S . Becker, Chem. Rev. 65, 261 (1965).
[2] N. Ishibe, M . Odani, R. Tanuma, J. Chem. SOC.Perkin Trans. 1 1972.
1203.
[3] The exact stereochemistry has not yet been determined. In investigations
on dimeric cyclopentadienone derivatives (B. Fuchs, B. Pazhenchevsky,
M . Pasternak, Tetrahedron Lett. 1972, 3051 and references cited therein)
the endo form has always been confirmed, so most likely the endo-transendo form is favored over the exo-trans-exo form in (2). The endo-transexo form can be ruled out on the basis of spectroscopic data.
653
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14c, tripyrroles, labeled, heme, biosynthesis
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