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Biosynthesis of Natural Porphyrins Studies with Isomeric Hydroxymethylbilanes on the Specificity and Action of Cosynthetase.

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symmetry of the compounds. In disubstituted compounds it
is not so extremeL4].
The 'H-NMR signals'51for the cyclopropene protons, like those of the IR bandsf61,correspond to the
data of well-known compounds (Table 1).
Table 1. 'H-NMR data of (I), (21, (4). and (7) (CDCI,, 400 MHz, TMS as internal standard). ',C-NMR data of (I) and (7) (In CDCI,), and further physical
data of ( I ) , (2) and (4)
'H-NMR ( 6 values) [a]
(11
(2)
5.39 ddd
2.01 m
1.95 m
1.41 m
5.35 d (br)
2.2 m
1.95 m
1-H
2a-H
213-H
3-H 1
4-H J
5-H
6a-H
6P-H
7-H
8a-H
8P-H
9a-H
9P-H
13-H
14-H
1.56 dd
2.34ddd
3.05 ddddd
1.70 dddd
2.20 ddddd
2.07 d (br)
2.38 ddddd
8.46 d
0.88 s
15-H
089 s
1
i
-
1.65-1
5 m
1.58 dd
2.10 ddd
2.98 dddd
1.41 dddd
1.79 d (br)
2.33 d (br)
2.2 m
8.39 s
0.95 s
o.84
1
\
(71
165-1.35
2.79 dddd
1.98 m
1 73 m
2.1-1.9
m
rn
I
1.65-1.35
1
j
m
2.34 d (br)
1.98 m
1.73 m
1.62 m
J
\
141
-
2.78 dddd
1.62 m
2.0 m
1.62 m
8.40 s
0.78 s
4.76 ddd
4.44ddd
J
8.38 s
C-7
C-8
C-9
C-I0
C-ll
C-12
C-13
C-14
C-15
122.0
29.1
26.6
340
37.7
41.1
41.1
29.2
25.5
144.5
C-1
C-2,6
C-3.5
C-4
36.7
29.3
24.8
25.6
C-7
C-8
C-9
173.0
159.0
146.7
174.9
158.4
147.4
154
19.5
( I ) , IR (CCI,). Y= 1840. 1588 crn ' (cyclopropenone); MS: m/e=216 151 ( M + ,
14%). 201 (M-CH,.
13). 188 (M-CO, 13). 173 (201-CO. 45). 131
-78 (1=589 nrn),
(173-CHKH - C H > ,69. RDA [bl). 91 (C,H:. 100); [.I2"=
-81 (578). -93 (546). - 141 (436) (c=0.65. CHCI,)
(2). IR (CCI,): Y= 1830. 1585 cm ' (cyclopropenone); M S m/e=216.151 ( M +,
12%)
(4). IR (CCL): v= 1835. 1600cm ': MS: m/e=216.151 ( M i , 38). 201 (11). 188
(21). 173 (45). 91 (100); [.Iz4=
+ 2 2 (h=589 nm), +24 (578). +25 (546). +34
(434) ( c = O . l . CHCI,)
la] Coupling constants J [Hzl: (I): 1.2a=1.9=2; 1,2@=5; 2,9=2; 4,15=6.2,
6a,6P = 14; 6a.7 =6.68,7 = 2.5; 6&8@= 2.5; 7,8a =4.5; 7.8P =8P,9a =8P,9P = 2.4;
7,13=1.5; 8a,9a=4.5; 8a,9P=8a,8P=13; 9a,98=14; (2): 1.2=5: 4,15=6.5;
6a.7 = 1 I;
6p,7 = 3.5;
6p,Sp = 2;
7.8a = 13;
7,8P = 1;
6a,6P = 13 5.
8a,8P = 8a.9P = 13; 8a.9a =4; 9a,98= 15; (4): 3,15 = 5,15 = 15,15' = 1.5;
5,6p= 12.5;
6a.7 = 7,Ra = 4
6P.7 =7,8P = 12.5:
(7):
1.2a = 1.6a =3.5:
1.26 = 1.66 = 9.5. [b] RDA = Retro-Diels-Alder cleavage.
According to the 'H-NMR data (Table 1) the second component is undoubtedly the eudesmane derivative (4). Although, only few signals are interpretable by 1st order rules
the spectrum is, nevertheless, very similar to that of the corresponding eudesmane. The allyic coupling for 13-H is missing; however, the chemical shift of 13-H is almost the same
as that in the case of (1).
We have also isolated (1) from Lychnophora passerina
Gardn. (Compositae, Tribus Vernonieae), as well as an isomeric cyclopropenone which may have the structure (2). The
altered couplings for 7-H show that the cyclopropenone
moiety in (2) must be arranged equatorially. The 'H-NMR
data of (2) (Table 1) are similar to those of (1). Only the
In1
Ed Engl. 20 (1981) No. 3
The overground parts of the plants were extracted with
ether/petroleum ether and the extracts worked up by column
chromatography and by TLC (SO2). 1.5 kg of overground
parts of Telekia speciosa gave 25 mg of (1) and 3 mg of (4)
[TLC:ether/petroleum ether (1 :l)], 650 g of overground
parts of Lychnophorapasserina gave 10 mg of (1) and 6 mg of
(2), while 8 mg of (1) and 6 mg of (2) were isolated from 100
g of roots. (1) and (2) are colorless oils.
(7): To a solution of 100 mg of (5) (prepared by reaction of
l,l-dibromo-2-cyclohexylethylene with n-butyllithium at
-78 "C and subsequent treatment with chlorotrimethylsilane) in 5 cm3 CHC13 is added 15 mg of benzyl(triethy1)ammonium chloride and the stirred mixture treated dropwise at
20°C with 5 cm3 50% NaOH solution. After 2 h the mixture
is poured into 100 cm3 of H20, taken up in ether, and the residue obtained on evaporation purified by TLC (ether);
yield: 60 mg (5) and 8 mg (7).
Received. November 28. 1980 [ Z 710 IE]
German version Angew Chem Y3, 280 (1981)
Miscellaneous data
Angew Chem
Experimental
-
"C-NMR ( 6values)
(71
c-l
C-2
C-3
C-4
c-5
C-6
chemical shifts of 6-H and 8-H are somewhat different from
those of ( l ) ,since the deshielding effects operate differently
owing to the changed stereochemistry, as can be deduced
from Dreiding models. ( I ) , (2) and (4), are the first naturally
occurring cyclopropenone derivatives.
F. Bohlmann, J. Jakupovic, A. Schusrer, Phytochemistry 20 (1981). in press.
G. Hofle, W. Sfeglirh. Synthesis IY72. 619.
F. Bohlmann, C. Zdero, M. Silva, Phytochemistry 16, 1302 (1977).
E. V. Dehmlow, R. Zersberg, S. S. Dehmlow, Org. Magn. Reson. 7. 418
(1975).
151 P. Crabbe. H. Carpio, E. Velarde. J. H. Fried, J . Org. Chem. 38, 1478
(1973).
16) R. Breslow, L. J. Allman, J. Am. Chem. SOC.XX, 504 (1966)
[1]
121
[31
[41
Biosynthesis of Natural Porphyrins:
Studies with Isomeric Hydroxymethylbilanes
on the Specificity and Action of Cosynthetasel'.]
By Alan R. Battersby, Christopher J. R. Fookes,
George W. J. Matcham and Pramod S. Pandey'']
Dedicated to Professor Hans Herloff Inhoffen
on the occasion of his 75th birthday
Uroporphyrinogen-111 (4) is the precursor of the natural
porphyrins, chlorins and corrins and its biosynthesis from
porphobilinogen (1) requires the enzymes deaminase and cosynthetase"). The biosynthesis involves the building of an
(2), folunrearranged tetrapyrrole, the bilane derivativeL3.*]
lowed by a single intramolecular
An enzymic replacement of the amino function of (2), X=NH3
and of (1) by another nucleophile [shown as X in (2)] before
the final cyclization with rearrangement is discussed e. g.
in r31. With deaminase acting alone on porphobilinogen ( l ) ,
the product released is the unrearranged hydroxymethylbilane15.6.71 (3). Natural and syntheticr51(3) were identical substrates for cosynthetase and the product was uroporphyrinogen-111 (4).
The foregoing knowledge and synthetic methodology
made it possible to probe the action of cosynthetase by syn['I Prof. Dr. A. R. Battersby, Dr. c.J . R Fookes, Dr. G. W. J. Matcham, Dr P.
S. Pandey
University Chemical Laboratory
Lensfield Road, Cambridge, CB2 1 EW (England)
[**I
This work was supported by the Science Research Council and by Roche
Products Ltd.
0 Verlag Chemie GmbH. 6940 Weinheim. 19x1
0570-0833/8l/0303-0293
S 02.50/0
293
thesizing hydroxymethylbilanes, which are isomeric with the
natural substrate. Four bilanes were constructed: the reversed ring-B (6), reversed ring-C (7),reversed ring-D (8) and
J
Mamuare
A
P
A
P
synthetic routes were used for all four and the approach for
the reversed ring-D bilane (8) is illustrated. The five formyl
bilane esters as (lo), were shown to be isomerically pure by
HPLC and were characterized by mass spectrometry and
NMR.
The freshly prepared, derived bilanes (3) and (6)-(9)
were then incubated separately (pH= 8.25) with cosynthetase, free from deaminase, which had been isolated from Euglena gracitis; non-enzymic (“chemical”) ring-closure of each
bilane was carried out at the same pH. The unrearranged hydroxymethylbilane (3) was included in the set of experiments
as a standard.
It was found that the natural unrearranged hydroxymethylbilane (3) was by far the best substrate for cosynthetase but
C HO
/
Chemically
PMe
AM‘
>
p
+
’
Me
NH
NH
MeA+<+
HN-
C HO
NH
/
P
P
14)
P
(5)
Uroporphyrinogen-I
P = CHzCHzCOzH
CHZCOzH,
A
(8J
(9), in which rings A, B and C have been reversed relative to
the natural, unrearranged hydroxymethylbilane (3). Similar
A
- OH
P
A
P
A
P
A
/C\
ID\
H
N
H
N
H
N
CHzCOzH
AMe = CHzCOzCH3
P
= CHzCHzCOzH
P M e= CHzCHzCOzCHs
P
A
/B\
H
H N T
\
A
Uroporphyrinogen- 111
CHo
L
n”
,-OH
- O H L
HN
P
(3)
A
(11)
U roporphyrinogen- IV
P
P
P
A
A
A
P
A
P
P _
A
_
A _
P
_
(7)
A
P
A
P
A
P
A
(8)
A
P
(9)
P
A
16)
A
_
P _
A _
P _
A _
P _
A
P
interestingly, the cyclizations of reversed-C (7)and reversedD (8) bilanes were also enzymically accelerated. In contrast,
the reversed-B (6) and reversed A, B, C (9) bilanes did not
Table 1 Ring-closure of hydroxymethylbilane (3) and isomers (6)-(9) chemically and by cosynthetase. Products: uroporphy
rinogen-I [I (5)j; uroporphynnogen-III [ = (4)I;uroporphyrinogen-IV [=(If)].
Substrate
V,,,
@el.)
KJwM
I
Unrearranged bilane (3) [ 5 ]
Reversed ring-B (6)
Reversed nng-C (7)
Reversed ring-D (8)
Reversed rings A B C (9)
100
~ 0 . 5
5
13
<0.5
11.3
-
105
11.4
-
loo
-
-
Uroporphyrinogen isomers yields I%] [a]
Chemically
Enzymically
111
IV
I
I11
IV
-
98
99
97
98
-
8
35
-
92
96
49
64
98
-
51
-
-
[a] Yields of uroporphyrinogen-I1 and of isomers marked with a dash amounted to <3%.
294
0 Verlag Chemie CmbH, 6940 Weinheim,1981
0570-0833/81/0303-0294
$ 02.50/0
Angew. Chem. In?. Ed. Engl. 20 (1981) No. 3
act as substrates (see Table 1). The products from the action
of cosynthetase on bilanes (31, (7) and (S), and also from
chemical ring-closure at pH 8.25, were analyzed by HPLCtS1.
The results (see Table 1) showed in each case that chemical
ring-closure occurred with essentially no rearrangement. Importantly, ca. one third of the enzymic product from the reversed ring-D bilane (8) was uroporphyrinogen-I (5) arising
from enzymic inversion of the terminal ring-D. Knowing the
isomeric composition of the product and also the chemical
and enzymic rates of ring-closure, it can be calculated that
ca. 45% of bilane (8), ring-closed by cosynthetase, undergoes
ring-D reversal to produce uroporphyrinogen-I (5). Although the reversed ring-C bilane (7) is a relatively poor substrate for cosynthetase, similar calculations show that in this
case, the ring-closure promoted by cosynthetase, nevertheless, occurs with efficient formation (>95%) of uroporphyrinogen-IV ( l l ) ,presumably from inversion of the terminal
ring“].
These results interlock with those obtained earlier[’’, in
which deaminase and cosynthetase acted together on mymbers of a set of isomeric aminomethylbilanes (2, X=NH,
and isomers). Interpretation of these early experiments was
complicated by the fact that two enzymic steps were involved; thus, conversion of any of the aminomethyl into hydroxymethyl bilanes by deaminaset4’would enhance the rate
of uroporphyrinogen formation, even when cosynthetase is
not involved, as is the case for the reversed ring-B bilane (6).
The present work focuses attention on cosynthetase alone
and shows the following: (a) Cosynthetase is not an absolutely specific enzyme, but all the studied modifications of pyrrole rings produced a substantial adverse effect on affinity,
rate of reaction and/or efficiency of the inversion process.
(b) Cosynthetase has evolved to invert the terminal ring during ring-closure of the natural .hydroxymethylbilane (3) and
will do a similar inversion when an isomeric hydroxymethylbilane acts as a substrate e. g . it does so efficiently (but slowly) with bilane (7) as substrate. (c) Cosynthetase will even
partially turn back the terminal ring of bilane (8) in which
ring-D had already been inverted by synthesis. This result
excludes the possibility that formation of uroporphyrinogenI11 (4) from the regular hydroxymethylbilane (3) involves
rearrangement of (3) into (8) as a first step.
Received: December I , 1980 [Z ?OX IE]
German version: Angew Chem. 93, 290 (1981)
[ I ] Review: A . R. Baifersby. E. McDonald, in K. M . Smith: Porphyrins and Metalloporphyrins, Elsevier, Amsterdam, 1975, p. 61.
121 A. R. Batrersby, G. L. Hodgson, E. Hunt, E. McDonald & J. Saunders, J
Chem. Soc. Perkin Trans. I, 1976, 273.
131 A. R. Battersbv, E. McDonald, D. C. Williams, H . K. W. Wurnger, J. Chem.
SOC.Chem. Commun 1977. 113; A. R. Batrersby, C. J. R. Fookes, E. McDonald, M . J. Meegan, ibid. 1978. 185; H -0. Dauner, G. Gunzer, I. Heger, G.
Muller, Z . Physiol. Chem., 357. 147 (1976).
141 A . R. Battersby. C. J. R. Fookes, G. W J. Matcham, E. McDonald, J. Chem.
SOC.Chem. Commun. 1979, 539; P. Jordan & J. S. Seehra, FEBS Lett. 104,
364 (1979).
I51 A . R. Barrersbj, C. J. R. Fookes, G. W. J. Marcham, E. McDonald & K. E.
Gusiafson-Pofrer,J. Chem. Soc.Chem. Commun. 1979, 316; A. R. Baitersby,
C. J . R . Fookes, K. E. Gusrafson-Potter, G. W. J. Matcham, E. McDonald,
rbid. IY79. 1155.
161 A . R. Bartersby, R G. Brereron, C J. R. Fookes, E. McDonald, G. W. J. Matcham, J. Chem. SOC.Chem. Commun. 1980, t 124.
17) The intermediate released by deaminase was also observed independently in
Texas and the natural material was shown to be a substrate for cosynthetase
(see G Burton, P. E. Fagerness, S. Hosozawa, P. M . Jordan, A. I . Scott, J.
Chem. SOC.Chem. Commun. 1979, 202) but it was wrongly concluded that
its structure is an N-alkylpyrrole macrocyclic system (see G. Burion, H. NordIOU.S. Hosozawa, H. Malsumoto, P. M . Jordan, P. E. Fagerness, L. M. Pryde,
A. I . Scott, J. Am. Chem. SOC.101, 3114 (1979).
(81 In principle. uroporphyrinogen-IV could also be formed from bilane (7) by
inversion of ring-B but the sum of evidence points to this being highly unlike1Y.
191 A. R. Bariersby. C. J. R. Fookes. G. W. J. Matcham, E. McDonald, J. Chem.
Soc. Chem. Commun. 1978. 1064.
Angew. Chem. Inr. Ed. Engl. 20 (1981) No. 3
“Chemical Mutation” by Amino Acid Exchange
in the Reactive Site of a Proteinase Inhibitor and
Alteration of Its Inhibitor Specifity‘’.”
By Herbert R. Wenzel and Harald Tscheschel’]
Dedicated to Professor Gerhard Pfreiderer on the occasion
of his 60th birthday
Low-molecular weight protein proteinase inhibitors inhibit serine-proteinases by reversible, substrate-analogous association at the active center of the enzyme1’]. The specificity
of an inhibitor is determined by the geometry of the amino
acid residues at the contact surface and in most cases dominated by the amino acid Pl[3a,3bl
in its reactive center. This is
supported by comparison of the sequences of homologous inh i b i t o r ~ ‘and
~ ” ~in two cases by semisynthetic amino acid replacement~‘~~]:
thus, by enzymatic reactions P, = Arg could
be replaced by Lys in the soybean inhibitor (Kunitz) and
Pi = Lys by Arg in the inhibitor from bovine organs. The
anti-tryptic activity of both proteins was preserved. Replacement of the basic amino acids by Trp converted the two inhibitors with primary specificity against trypsin into inhibitors
with primary specificity against chymotrypsin. The “enzymatic mutation” has so far only been used with success in the
case of basic and aromatic amino acids, since no suitable enzyme system could be found for other amino acids.
We describe here a method which enables insertion of almost every amino acid in the position PI of the inhibitor
(Kunitz) from bovine organs by a peptide-chemical method.
The natural inhibitor (PI = Lys”) inhibits trypsin extremely
strongly, chymotrypsin strongly, leukocytic elastase scarcely,
and pancreatic elastase not at all. (A projection of the a-carbon atoms of the inhibitor is shown in‘”.)
The Lys1’-Ala16 peptide bond in the reactive site of the
native inhibitor is hydrolyzed according to previously described methods; with carboxypeptidase B one then obtains
the inactive modified des-Lys” inhibitort6! Water-soluble
carbodiimides enable coupling of Val-OMe with the free
carboxyl group of CysL4[’1(Scheme 1).
The product is an excellent inhibitor for elastase from human leukocytes. The maximum inhibition is only reached
after several minutes; obviously the complex between elastase
and Val15-inhibitor is formed relatively slowly. If this complex is dissociated by drastic lowering of the pH (kinetically
controlled dissociation), an inhibitor is obtained which immediately inhibits elastase to the maximum. This finding
suggests resynthesis of the peptide bond Vaf‘S-Ala’6 by the
proteinase during the complex formation‘”.
The semisynthesis has so far also been carried out according to the same procedure with the methyl esters of Arg, Phe,
Met, Leu, Ala and Gly. According to the amino-acid analysis
of the crude material about 2.2 to 4.1 amino acids were inserted per inhibitor molecule, up to 0.3 thereof in the decisive
position P1,i. e. 30% yield. The other part was coupled to the
five carboxy groups (Asp3, Glu7, Glu4’, AspSo,Ala5’) already
present in the unmodified inhibitor. These are so far removed from the reactive site, that their derivatization has hardly
any influence on the inhibitor activity.
[‘I
Prof. Dr. H. Tschesche, Dr. H. R. Wenzel
Fakultat fur Chemie, Lehrstuhl Biochemie der Universitat
Postfach 8640, 4800 Bielefeld 1 (Germany)
I“] Delivered in part in a lecture at the Spring Conference of the Gesellschaft
f i r Biologische Chemie, Miinster, March 1980 [I]. This work was supported
by the Deutsche Forschungsgemeinschaft, the Stiftung Volkswagenwerk.
and the Fonds der Chemischen Industrie We thank Bayer AG for supplying us with Trasylola [Trypsin-Kallikreina inhibitor (Kunitz) from bovine
lung].
0 Verlag Chemie GmbH, 6940 Weinheim, 1981
0570-0833/81/0.703-0295
$
02.50/0
295
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cosynthetase, natural, porphyrio, action, specificity, studies, isomeric, biosynthesis, hydroxymethylbilane
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