A synthetic model of the active site of the coenzyme BIZ dependent methylmalonyl-Coh mutase SummaryThe synthesis of a bridged cobaloxime with a built-in methylmalonic ester moiety is described. 2-Brommethyl-2-methylmalonic acid dichloride (5) afforded upon reaction with 5-heptin-1-01 (4) the corresponding diester 6 . Subsequent treatment of 6 with ozone, dimethylsulfide and hydroxylamine hydrochloride led to the pentadentate ligand: 10-brommethy1-10-methy1-9,1 l-dioxo-8,12-dioxa-nonadecane-2,3,17,18-tetraone tetraoxime (8). Reaction of 8 with cobalt (11) chloride, pyridine and sodium borohydride furnished in 7% yield the bridged cobaloxime 10, which was spectroscopically characterized. Short term irradiation of 10 in methanol caused the exchange of the axial pyridine ligand by a solvent molecule affording 10a, the structure of which has been determined by X-ray crystallography. Long term irradiation of 10 in methanol or ethanol led to irreversible cleavage of the Co, C-bond. Upon alkaline hydrolysis the degradation product afforded methylsuccinic acid in 82-95% yield. No incorporation of solvent protons into this product could be observed. Implications of these findings for the mechanism of the coenzyme-B 12 catalysed rearrangement of methylmalonyl-CoA are discussed.Wahrend die Wirkungsweise der meisten Coenzyme in den funfziger Jahren aufgeklart werden konnte, blieb diejenige des Coenzym-B 12 trotz intensiver Forschung bis vor kurzem im Dunkeln. Als Hauptursache muss wohl das Fehlen von Analogien in der (meta1L)organischen Chemie angesehen werden. Die ersten erfolgreichen chemischen Modelle bestanden aus Kobaltkomplexen, in denen das <

Liganden werden synthetisiert, in denen zwei vicinale Dioximfunktionen durch eine Kohlenwasserstoffkette, die in der Mitte eine Brommethyl-oder Chlormethylgruppe tragt, verbunden sind (20, 22, 25). Umsetzung dieser Liganden mit Cobalt(I1)-chlorid in Gegenwart von Pyridin und Natriumboranat ergibt iiberbriickte, intramolekular alkylierte Cobaloxime. Wenn die Dioximfunktionen durch neun C-Atome getrennt sind, entstehen hauptsachlich dimere Produkte, von denen zwei Diastereomere, 26 und 27, isoliert und sowohl spektroskopisch als auch durch Rontgenstrukturanalyse charakterisiert wurden. Bei elf oder dreizehn C-Atomen zwischen den Dioximfunktionen entstehen hauptsachlich monomere Cobaloxime mit cis-Konfiguration, wie 28. Das trans-Monomere 30 entsteht (neben dem cis-Monomeren) erst, wenn die Anzahl der C-Atome in der Brucke auf funfzehn erhoht wird. Synthesis, Characterization, and Stereochemistry of Bridged Intramolecularly Alkylated Cobaloximes; Monomeric and Dimeric Complexes of Different ConfigurationLigands have been synthesized containing two vicinal dioxime functions joined by a carbon chain to which is attached in the centre a bromomethyl or chloromethyl group (20, 22, 25). These ligands, upon reaction with cobalt(I1) chloride in the presence of pyridine and sodium borohydride afforded bridged intramolecularly alkylated cobaloximes. When the vicinal dioxime functions are separated by a chain of nine carbon atoms mainly dimeric complexes are formed. Two of these, 26 and 27, were isolated and characterized by spectroscopic methods and their structures were established by X-ray crystallography. Ligands containing eleven or thirteen carbon atoms between the dioxirne functions afforded mainly monomeric cobaloximes with cis configuration, e. g. 28. The trans-monomeric complex 30 was produced along with the cis isomer when the number of carbon atoms in the bridge was raised to fifteen.Seit dem Vorschlag'), darj Cobaloxime als Modelle fur das Vitamin BIZ dienen kbnnten, ist das Interesse an dieser Verbindungsklasse stark gestiegen. Wir berichteten verschiedentlich uber die Synthese und das Verhalten von uberbruckten Cobalo~imen~-~'. Hier beschreiben wir im Detail die Komplexbildung von Tetraoximliganden mit Cobalt(I1)-chlorid in Abhtingigkeit von der Ligandenstruktur. Zweikernige uberbruckte Cobaloxime und monomere uberbruckte Cobaloxime mit trans-Konfiguration werden erstmalig charakterisiert. Teilaspekte unserer Arbeit wurden bereits in kurzer Form veroffentlicht 3*5).

Für Reaktionen, die durch Vitamin B12 katalysiert werden, gibt es bis heute keinen chemisch plausiblen Mechanismus, was vor allem durch den Mangel an Modellreaktionen bedingt ist. Die photochemische Zersetzung des Cobalt‐Komplexes (1) könnte eine Modellreaktion für die vom Vitamin B12 katalysierte Umlagerung von Methylmalonyl‐CoA in Succinyl‐CoA sein. .

the tetraketone (10) in 70 % yield. The crystalline tetraoxime (1 1) obtained therefrom was then treated with a stoichiometric amount of cobalt(r1) chloride, twice the amount of pyridine, three equivalents of potassium hydroxide, and an excess of NaBH4. C kH3 CHs(61 In continuation of our attempts['% ' 1 to imitate coenzyme B12-dependent enzymatic rearrangements with model catalysts we have synthesized the bridged alkylcobaloxime ( 1 ). The starting material employed was 5-heptyn-1-01 (2) which is obtainable either by condensation of lithium 1 -propyn-1 -ide with tetrahydropyranyl 4-bromobutyl ether or by fragmentation of 2,3-epo_xy-2-methylcyclohexane to~ylhydrazone[~] and subsequent reduction of the resulting aldehyde with LiAIH4.Compound (2) was extended by two C atoms uia the mesylate (3) in a malonic ester synthesis to form 7-nonyn-1-01 (4), the mesylate of which (5) could be used for double alkylation of dimethyl malonate. Upon hydrolysis, decarboxylation, and reduction, the bisalkylated malonic ester (6) afforded 2-(7-nonynyl)-9-undecyn-I -01 (7), which was converted via the mesylate (8) into the bromomethyl derivative (9). Ozonization of (9) at -18°C in and subsequent treatment of the peroxide with dimethylsulfide gave The product mixture contained six alkylated cobalt complexes (overall yield ca. 20 %) which were separated by thinlayer chromatography. The major product ( 1 ) [& =0.39 Using a Picker diffractometer (Mo,, radiation, graphite monochromator) 4625 symmetrically independent reflections were measured of which 2307 were classified as observed

Dedicated to Professor Vladiniir Prelog on the occasion of' liis 70th hirthdaj It has recently been shown by usi1' and by others ['] that a malonic ester substituted in the 0-position with cobalt can rearrange into the corresponding succinic ester. Although a chemically analogous case for the enzyme-catalyzed isomerization of methylmalonyl-CoA to succinyl-CoA has thus been found, this model is in many respects not in keeping with the conditions at the active site of the enzyme. Soon after homolytic cleavage of the cobalt-carbon bond the malonic ester "substrate" loses contact with the central cobalt atom and, thus deprived of that atom's catalytic effect, is no longer able to rearrangei3]. This could explain the 10w'~J and variable['' yields of rearrangement product. In order to better simulate the situation on the enzyme, we synthesized a cobalt complex in which the "substrate" is anchored covalently by two methylene bridges to a planar part of the complex.was reacted with 2-bromomethyl-2-methylmalonic acid dichloride (2) to give the diester ( 3 ) , which on treatment with ozone at -18"C['.71 yielded the crude tetraketone ( 4 ) . The tetraoxime ( 5 ) obtained from ( 4 ) in 80% yield [referred to ( 3 ) ] was finally allowed to react in dilute ethanolic solution with a stoichiometric amount of cobalt(r1) chloride, twice the amount of pyridine, three equivalents of potassium hydroxide, and excess NaBH,. AIkylated cobalt complexes were isolated from the mixture in (with 1 % pyridine) afforded the major product (6) in pure form in 7 % yield [referred to ( 5 ) ] . &=0.44, chloroform: isopropanol : pyridine = 94 : 5 : 1, Macherey-Nagel plates,Comparison of the 'H-NMRl6' and UV spectra with those of known cobaloximes1'. '1 revealed without doubt that the product was the intramolecularly alkylated cobalt complex ( 6 ) .Irradiation of a methanolic solution of (6) with a mercury high-pressure lamp for 60min in the absence of oxygen led to replacement of the pyridine ligand by methanol. Thin-layer chromatography of the photoproduct (chloroform : isopropanol = 9 5 : 5 , Rl=O.l I ; hexane : isopropanol : methanol = 5 : 2 : 1, Rf=O.l 1 ) afforded the cobaloxime (7).( 7 ) crystallizes from C D 3 0 D in the space group P i with a=11.090, b=13.772, c=8.971 A; c~=99.75, p=119.49, y = 84.83": Z = 2. A total of 3076 symmetrically independent reflections were measured on a Picker diffractometer (MoK, radiation), of which 2269 were classified as observed. The positions of the Co atoms were determined by a Patterson synthesis, the positions of all the other atoms other than hydrogen by difference Fourier syntheses. Refinement of the atomic coordinates and the anisotropic temperature factor coefficients for atoms other than hydrogen converged at R = 0.056. Cobaloxime (7) adopts in the crystal lattice a chiral conformation; the ester carbonyl groups of the bridges point in opposite directions. The methyl group in the bridge center (on C-10) projects away from the plane of the equatorial ligands. The angle CO-C~O-C'IO is 127.5", compa...