Methylcorrinoids methylate radicals - Their second biological mode of action?

Full text of DOI:10.1002/(SICI)1521-3773(20000117)39:2<393::AID-ANIE393>3.0.CO;2-M

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steps (Scheme 1, top).[2, 3] Extensive investigations of the B -
After the reaction with the phosphane the solvent was distilled off at
308C under vacuum, the molecular organic compounds were extracted
with CH Cl
at � 308C, and the solid residue was dissolved in MeCN (main
product: [C Xe][BF ] (Table 1)). The extract of the reaction with the
1
2
�
dependent methionine synthase have indeed shown that the
methyl transfer reactions occur by two nucleophilic substitu-
2
2
6
F
5
4
5
1
9
tion steps and convert homocysteine and N -methyltetrahy-
phosphane was characterized by F NMR spectroscopy: d ˆ 1.32 (dhept.,
1
4
J(F,P) ˆ 694.5, J(F,F) ˆ 16.3, 2F; PF
2
), � 132.73 (6F, o-F), � 146.50 (3F, p-
drofolate (with net retention) into methionine and tetrahy-
‡
F), � 159.48 (6F, m-F); GC-MS (70 eV): m/z (%): 570 (5) [M ], 551 (3)
[4]
drofolate.
‡
‡
[
M � F], 403 (100) [M � C
6 5
F ].
Received: August 26, 1999 [Z13929]
[
[
1] J. H. Holloway, E. G. Hope, Adv. Inorg. Chem. 1999, 46, 51 ± 100, and
references therein.
2] a) D. Naumann, W. Tyrra, J. Chem. Soc. Chem. Commun. 1989, 47 ±
5
6
0; b) H.-J. Frohn, S. Jakobs, J. Chem. Soc. Chem. Commun. 1989,
25 ± 627.
[
3] a) H.-J. Frohn, V. V. Bardin, J. Chem. Soc. Chem. Commun. 1993,
1
9
072 ± 1074; b) H.-J. Frohn, V. V. Bardin, Chem. Commun. 1999, 919 ±
20.
[
[
[
[
[
[
4] V. V. Zhdankin, P. J. Stang, N. S. Zefirov, J. Chem. Soc. Chem.
Commun. 1992, 578 ± 579.
5] H.-J. Frohn, T. Schroer, G. Henkel, Angew. Chem. 1999, 111, 2751 ±
2753; Angew. Chem. Int. Ed. 1999, 38, 2554 ± 2556.
6] D. Naumann, W. Tyrra, R. Gnann, D. Pfolk, J. Chem. Soc. Chem.
Commun. 1994, 2651 ± 2653.
7] H.-J. Frohn, St. Jakobs, G. Henkel, Angew. Chem. 1989, 101, 1534 ±
1536; Angew. Chem. Int. Ed. Engl. 1989, 28, 1506 ± 1507.
8] H.-J. Frohn, H. Franke, P. Fritzen, V. V. Bardin, J. Organomet. Chem.,
in press.
9] H.-J. Frohn, J. Fluorine Chem. 1998, 89, 59 ± 63.
[
[
10] H.-J. Frohn, W. Breuer, J. Fluorine Chem. 1990, 47, 301 ± 315.
11] K. O. Christe, E. C. Curtis, D. A. Dixon, H. P. Mercier, J. C. P. Sanders,
G. J. Schrobilgen, J. Am. Chem. Soc. 1991, 113, 3351 ± 3361.
[
[
12] M. Schmeisser, K. Dahmen, P. Sartori, Chem. Ber. 1970, 103, 307 ± 311.
13] L. M. Yagupolꢁskii, V. V. Lyalin, V. V. Orda, L. A. Alekseeva, Zh. Org.
Khim. 1970, 6, 329 ± 332; J. Org. Chem. (USSR) 1970, 6, 317 ± 319.
14] H.-J. Frohn, J. Helber, J. Fluorine Chem. 1980, 16, 568.
Scheme 1.
[
[
15] M. Schmeisser, K. Dahmen, P. Sartori, Chem. Ber. 1967, 100, 1633 ±
1
637.
Recent biosynthetic investigations on archaebacterial lip-
ids[ and the antibiotics bottromycin, thienamycin, and
thiostrepton[ provided evidence for unprecedented biolog-
ical methylation reactions (and other alkylations) at saturated
and inactivated carbon centers. In these methylation reactions
methyl groups that originate from methionine are incorpo-
rated with net retention into the products.[ Arigoni and co-
workers have considered a radical mechanism involving
methylcorrin cofactors for these methylation reactions.[
Herein we report the first experiments that establish the
5a]
[5b]
[6a]
6b]
5, 6]
Methylcorrinoids Methylate RadicalsÐTheir
Second Biological Mode of Action?**
5a]
Herv e Mosimann and Bernhard Kräutler*
(
very efficient!) methylation of alkyl radicals by 1 (see
Dedicated to Professor Rolf Thauer
on the occasion of his 60th birthday
Scheme 1, bottom).
Heating an oxygen-free aqueous solution of 2'-bis(ethoxy-
carbonyl)propylcobalamin (2)[ and methylcobalamin (1)
7]
[1, 8]
The vitamin B12 derivative methylcobalamin (1), as well as
related methylcorrinoids, are fundamentally important orga-
nometallic cofactors of methylation reactions. The known
(1:2 ˆ 1.6:1) at 708C for about 5 h (at pH7, with protection
from light) completely decomposed the organocobalamin 2.
The organic decomposition products obtained after oxidation
of the reaction mixture with air (in the dark and with the
addition of potassium cyanide) were analyzed after extraction
with deuterochloroform.[ The 200 MHz H NMR spectrum
of the product exhibited signals for a 4.6:1 mixture of 2-ethyl-
2-methylmalonic acid diethyl ester (3) and of 2,2-dimethyl-
malonic acid diethyl ester (4), with a combined yield
amounting to approximately 70% (Scheme 2). In an analo-
[
1]
enzymatic reactions with 1 depend on the heterolytic organo-
III
I
metallic reactivities of methyl-Co -corrins and Co -corrins
and the methylations proceed by nucleophilic substitution
9]
1
[
*] Prof. Dr. B. Kräutler, Dr. H. Mosimann
Institut für Organische Chemie der Universität
Innrain 52a, 6020 Innsbruck (Austria)
Fax : (‡ 43)512-507-2892
E-mail: bernhard.kraeutler@uibk.ac.at
gous experiment using trideuteromethylcobalamin ([D ]1)
3
[
**] We thank Prof. D. Arigoni for discussions that induced the present
project. We are grateful to the Swiss National Science Foundation
instead of 1 a combined yield of approximately 70% of [D ]3
3
and 4 was obtained as a 4.7:1 mixture (degree of deuteration
(H.M.) and to the Austrian National Science Foundation (FWF-
Project No. 12639) for financial support.
of [D ]3: 95 Æ 5% from NMR studies). Storage of a deoxy-
3
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Scheme 2.
genated aqueous solution of 2 and 1 at room temperature and
in the absence of light for about 17 h led to the decomposition
of about 30% of 2 and to the formation of an approximately
3
:1 mixture of 3 and 4.
The organometallic vitamin B12 derivative 2 is thermally
Scheme 3.
labile and decomposes even at room temperature to cob(ii)al-
amin (5) and the 2-bisethoxycarbonylpropyl radical (6).[
The effective half-life of 2 at 278C in an oxygen-saturated,
neutral aqueous solution is about 50 min, which is in
7, 10]
an intermolecular, homolytic substitution reaction by a (short-
lived, organic) radical at a saturated carbon atom directly
bound to a metal center is without precedence,
corresponding reactions of (persistent) metal radicaloids
(such as Co -complexes, etc.) are known.
of related intramolecular mechanisms was suggested to be
responsible for the formation of (substituted) cycloalkanes
[
7]
[17]
qualitative agreement with earlier experiments. However,
remains practically intact in a strictly oxygen-free aqueous
but
2
II
[18, 19]
solution (258C, pH7), even after 20 h (UV/Vis). In a similar
experiment 2 was completely decomposed after 7 h at 708C to
The occurrence
II
Co -corrins (>90%, UV/Vis) and to 2,2-dimethylmalonic
[
11]
[20]
acid diethyl ester (ca. 40%). In contrast, aqueous solutions
of methylcobalamin (1) are stable for a practically unlimited
time at room temperature (in the absence of nucleophiles and
in the dark). In the presence of the radical trap 2,2,6,6,-
tetramethyl-piperidinyl-1-oxyl (TEMPO) 1 is decomposed
with an effective half-life of only about 4 h at 1308C (in
from organometallic, bridged B dimers, as well as from
12
hexenyl cobaloximes.[ Homolytic substitution reactions at
carbon centers, when examined more closely, were found to
take place with stereochemical inversion of configura-
tion.[
18]
17, 18]
As a result of its characteristical low homolytic (Co-C)-
[
12]
[12]
ethylene glycol). Compound 1 thermolytically decomposes
at 2008C with formation of methane and ethane (ca. 1:1).[
Our experimental results can be explained by a decom-
BDE 1 has the reactivity of a ªlatentº (persistent) methyl
13]
[19, 21]
radical towards radicals
and, similar to coenzyme B (8, a
12
[
23]
ªreversibly functioning source of an alkyl radicalº), should
be considered a partner in biological radical reactions. The
enzymatic transfer of a methyl group from 1 to a carbon-
centered radical should proceed with inversion of the config-
uration of the methyl group (and should therefore occur with
net retention of configuration from methionine). In the
known cases of unusual biosynthetic C-methylation reactions
the configuration of the methyl group in the methylation
II
position of the thermolabile 2 in the presence of 1 to the Co -
corrin 5 and to the 2-bisethoxycarbonylpropyl radical (6),
which abstracts the cobalt-bound methyl group of 1, to
produce the methylation product 3 (Scheme 3).
The proposed abstraction of the cobalt-bound methyl group
of methylcobalamin (1) by a primary alkyl radical (6) can be
estimated from homolytic bond dissociation energies (BDE)
to be a very exothermic reaction step (DH ꢀ� 48 kcal
product is the same as the one in methionine, the biosynthesis
precursor.
�
1
[14]
[5, 6]
mol ). Our experiments indicate that the abstraction of
Our investigations thus support the suggested
the oganometallically bound methyl group of 1 by the radical
participation of methylcorrinoids in biosynthetic methylations
of carbon radicals.[
The property of efficient radical methylation agent should,
therefore, be added to the characteristic organometallic
5]
6
competes effectively with the recombination reaction of 6
II
with the Co -corrin 5 (this recombination to 2 is also strongly
exothermic and presumably nearly diffusion-controlled.[
)
16]
We explain the formation of 3 from 1 and 2 by the substitution
of the cobalt ± corrin moiety of 1 by the alkyl radical 6. Such
reactivities of methylcobalamin (1), the known biologically
important methylation agent of nucleophiles.[
2, 4]
Methylcor-
394
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Angew. Chem. Int. Ed. 2000, 39, No. 2

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1
):[25b] d ˆ 4.19 (q, J ˆ 7.1, OEt),
anthracene). 3: H NMR (200 MHz, CDCl
.92 (q, J ˆ 7.5, Et-C(2)), 1.39 (s, CH
J ˆ 7.5, Et-C(2)).
Thermolysis of 2 and trideuteromethylcobalamin ([D
carried out as above, but with [D ]1 instead of 1, and gave 2-(2,2,2-
3
1
3
-C(2)), 1.25 (t, J ˆ 7.1, OEt), 0.88 (t,
3
]1): The reaction was
3
trideuteroethyl)-2-methylmalonic acid diethyl ester ([D
3
]3) and 4 (4.7:1,
1
7
1%). [D
3
]3: H NMR (200 MHz, CDCl
3
): d ˆ 4.19 (q, J ˆ 7.1, OEt), 1.89
(
3
brs, Et-C(2)), 1.39(s, CH -C(2)), 1.25(t, J ˆ 7.1, OEt).
Received: June 25, 1999 [Z13632]
[1] W. Friedrich, Vitamin B12 und verwandte Corrinoide, Vol. III/2 of
Fermente, Hormone und Vitamine (Ed.: R. Ammon, W. Dirscherl),
Georg Thieme, Stuttgart, 1975.
[
[
[
2] J. M. Pratt, Inorganic Chemistry of Vitamin B12, Academic Press, New
York, 1972.
3] B. Kräutler in Vitamin B12 and B12 Proteins (Eds.: B. Kräutler, D.
Arigoni, B. T. Golding), WILEY-VCH, Weinheim, 1998, pp. 3 ± 43.
4] a) C. L. Drennan, S. Huang, J. T. Drummond, R. G. Matthews, M. L.
Ludwig, Science 1994, 266, 1669 ± 1674; b) M. L. Ludwig, R. G.
Matthews, Anun. Rev. Biochem. 1997, 66, 269 ± 313.
rinoids are to be considered also as ªlatent methyl radicalsº
and are cofactors suitable for homolytic biosynthetic methyl-
ation reactions.
[
5] a) P. Galliker, O. Gräther, M. Rümmler, W. Fitz, D. Arigoni in
Vitamin B12 and B12 Proteins (Eds.: B. Kräutler, D. Arigoni, B. T.
Golding), WILEY-VCH, Weinheim, 1998, pp. 447 ± 458; b) L. Kel-
lenberger, D. Holmes, B. Martinoni, D. Arigoni, Chimia 1996, 50, 344.
6] a) D. R. Houck, K. Kobayashi, J. M. Williamson, H. G. Floss, J. Am.
Chem. Soc. 1986, 108, 5365 ± 5366; b) P. Zhou, D. OꢁHagan, U. Mocek,
Z. Zeng, L.-D. Yuen, T. Frenzel, C. J. Unkefer, J. M. Beale, H. G.
Floss, J. Am. Chem. Soc. 1989, 111, 7274 ± 7276.
[
Experimental Section
Trideuteromethylcob(iii)alamin ([D
3
]1): Prepared according to Tollinger
et al.,[ but using trideuteromethyl iodide instead of [ C]methyl iodide.
8]
13
1
[7] J. H. Grate, J. W. Grate, G. N. Schrauzer, J. Am. Chem. Soc. 1982, 104,
588 ± 1594.
8] M. Tollinger, T. D e rer, R. Konrat, B. Kräutler, J. Mol. Catal. A 1997,
16, 147 ± 155.
Degree of deuteration was determined as 98 Æ 5% from H NMR and
1
FAB-MS studies.
[
[
2
'-Bis(ethoxycarbonyl)propylcobalamin (2): An oxygen-free solution of
aquocobalamin chloride (200 mg, 0.145 mmol) in aqueous 0.1m LiClO
5 mL, <10 ppm O ) was reduced (UV/Vis) in a glove box at a mercury
electrode to give cob(i)alamin (� 1.1 V versus a 0.1n calomel reference
1
4
9] The aqueous fraction of a similar experiment contained vitamin B12
(
2
1
(
7) and 1 in the ratio 1.7:1.0 ( H NMR).
‡
[
10] In acidic solutions 2 is protonated at the nucleotide base to give 2-H ,
which tends to decompose more than 100 times less rapidly than 2.
11] The NMR data provided no evidence for the existence of products
derived from a rearrangement of 6 to a methylsuccinyl radical (for
studies with 6 and with related radicals in solution, see S. Wollowitz, J.
Halpern, J. Am. Chem. Soc. 1988, 110, 3112 ± 3120).
�
1
(
0.1NCE), consumption: 2.06 Fmol ). An 0.5m aqueous solution of p-
toluene sulfonic acid (0.520 mL) and 2-bromomethyl-2-methylmalonic acid
[
[
25a]
diethyl ester (0.178 mL)
(0.867 mmol) in methanol (1 mL) were then
added in the dark. The electrolysis was continued at � 1.0 V versus 0.1NCE
for 2.5 h. The solvents were removed at room temperature. The residue was
dissolved in a little methanol and precipitated with acetone. The precipitate
[
[
12] B. D. Martin, R. G. Finke, J. Am. Chem. Soc. 1992, 114, 585 ± 592.
13] G. N. Schrauzer, J. W. Sibert, R. J. Windgassen, J. Am. Chem. Soc.
‡
was washed with acetone and dried to give 2-H (as the tosylate, 231 mg,
94% yield) as an orange coloured solid (still containing 5% aquocobala-
1
968, 90, 6681 ± 6688.
min). UV/Vis (H
2
O, pH2): lmax (l g e) ˆ 451s (3.76), 421 (3.78), 296s (4.17),
� 1 [12]
� 1 [15]
[
[
14] (Co-C)-BDE (1) ꢀ 37 kcalmol
;
(C�C)-BDE (3) ꢀ 85 kcalmol
.
1
285 (4.23), 266 (4.30); H NMR (500 MHz, D
2
O, pD 2): d ˆ 9.10 (s, 1H),
15] A. J. Gordon, R. A. Ford in The Chemistꢁs Companion, Wiley, New
York, 1972.
7.55 (d, 2H), 7.41 (s, 1H), 7.35 (s, 1H), 7.22 (d, 2H), 7.04 (s, 1H), 6.40 (d,
1H), 4.82 (dd, 1H), 4.66 (dd, 1H), 4.62 (m, 1H), 4.58 (d, 1H), 4.21 ± 4.15 (m,
1H), 4.07 (d, 1H), 3.92 ± 3.85 (m, 2H), 3.80 ± 3.72 (m, 4H), 3.64 (dd, 1H),
3.43 (d, 1H), 3.18 (dd, 1H), 3.06 (dd, 1H), 2.77 ± 2.64 (m, 3H), 2.45 ± 2.08
[
[
[
[
[
16] J. F. Endicott, G. J. Ferraudi, J. Am. Chem. Soc. 1977, 99, 243 ± 245.
17] J. C. Walton, Acc. Chem. Res. 1998, 31, 99 ± 107.
18] M. D. Johnson, Acc. Chem. Res. 1983, 16, 343 ± 349.
19] B. Kräutler, Helv. Chim. Acta 1987, 70, 1268 ± 1278.
20] B. Kräutler, T. D e rer, P. Liu, W. Mühlecker, M. Puchberger, K.
Gruber, C. Kratky, Angew. Chem. 1995, 107, 66 ± 69; Angew. Chem.
Int. Ed. Engl. 1995, 34, 84 ± 86.
(
5
1
m, 10H), 2.27 (s, 6H), 2.24 (s, 3H), 2.08 (s, 3H), 2.04 (s, 3H), 2.04 ± 1.89 (m,
H), 1.80 ± 1.60 (m, 4H), 1.75 (s, 3H), 1.53 (s, 3H), 1.40 (s, 3H), 1.30 (s, 3H),
.29 (d, 1H), 1.15 (s, 3H), 1.03 (d, 3H), 0.95 (t, 3H), 0.86 (t, 3H), 0.54 (d,
1
H), 0.50 (s, 3H), � 0.07 (s, 3H); FAB-MS (o-nitrobenzyl alcohol (NOBA)
‡
matrix): m/z (%): 1518.5/1517.5/1516.5 (17/22/28) [M‡H ]; 1331.5/1330.5/
[13]
[
21] The observation of ethane as the main product of the photolysis and
‡
1
329.4 (38/76/100) [M‡H � C
9
H
15
O
4
].
[22]
of the reductive electrolysis of 1 was rationalized by dimerization of
‡
[13, 22]
Thermolysis of 2 in presence of methylcobalamin (1): 2-H (30.0 mg
methyl radicals.
Our results suggest an alternative consideration,
containing 17% water, 0.0135 mmol) was dissolved in hydrochloric acid
namely of a direct reaction of methyl radicals with the intact
(
3 mL, 10 mm). The solvents were removed and the solid residue was dried.
methylcorrinoid (1).
Similarly 1 (29.0 mg, 0.0216 mmol) was dissolved in water (5 mL) and then
dried. Both compounds were then dissolved in phosphate buffer (4 mL,
[22] K. A. Rubinson, E. Itabashi, H. B. Mark, Jr., Inorg. Chem. 1982, 21,
3571 ± 3573.
0
.1m, pH7) in the dark and in the glove box, and the deoxygenated mixture
was heated in the dark at 708C for 5 h. Oxygen-saturated water (2 mL) and
.5m aqueous KCN (0.054 mL) were then added and the mixture was
[23] J. Halpern, Science 1985, 227, 869 ± 875.
[24] Co -corrinoids (such as 5, the biologically important radical traps)
II
[3]
0
then may act, in a complementary way, in homolytic demethylations of
reactive (radicaloid) methylating agents.[8, 19]
extracted with CDCl
through a plug of dried cotton wool. Analysis by 200 MHz H NMR
spectroscopy identified the product as a 4.6:1 mixture of 2-ethyl-2-
methylmalonic acid diethyl ester (3)
diethyl ester (4).[ The total yield (ca. 70%) was estimated by comparison
3
(3 Â 0.6 mL). The extract was dried by passing
1
[25] a) Y. Murakami, Y. Hisaeda, T. Ozaki, T. Tashiro, T. Ohno, Y. Tani, Y.
Matsuda, Bull. Chem. Soc. Jpn. 1987, 60, 311 ± 324; b) F. Björkling, J.
Boutelje, S. Gatenbeck, K. Hult, T Norin, P. Szmulik, Tetrahedron
1985, 41, 1347 ± 1352; c) L. Kiriazis, E. Kalatzis, N. E. Alexandrou, J.
Heterocycl. Chem. 1989, 26, 155 ± 160.
[25b]
and 2,2-dimethyl-malonic acid
25c]
with the signal of the residual CHCl
3
in CDCl
3
(standardized against
Angew. Chem. Int. Ed. 2000, 39, No. 2
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0570-0833/00/3902-0395 $ 17.50+.50/0
395