Stereochemistry of the methyl group in (R)-3-methylitaconate derived by rearrangement of 2-methylideneglutarate catalysed by a coenzyme B12-dependent mutase

Article abstract of DOI:10.1002/1522-2675(20000906)83:9<2550::AID-HLCA2550>3.0.CO;2-C

2-Methylideneglutarate mutase is an adenosylcobalamin (coenzyme B₁₂)-dependent enzyme that catalyses the equilibration of 2-methylideneglutarate with (R)-3-methylitaconate. This reaction is believed to occur via protein-bound free radicals derived from substrate and product. The stereochemistry of the formation of the methyl group of 3-methylitaconate has been probed using a 'chiral methyl group'. The methyl group in 3-([²H₁,³H]methyl)itaconate derived from either (R)- or (S)-2-methylidene[3-²H₁,3-³H₁]glutarate was a 50 : 50 mixture of (R)- and (S)-forms. It is concluded that the barrier to rotation about the C-C bond between the methylene radical centre and adjacent C-atom in the product-related radical [CH₂CH(^-O₂CC=CH₂)CO₂^-] is relatively low, and that the interaction of the radical with cob(1I)alamin is minimal. Hence, cob(I1)alamin is a spectator of the molecular rearrangement of the substrate radical to product radical.

Full text of DOI:10.1002/1522-2675(20000906)83:9<2550::AID-HLCA2550>3.0.CO;2-C

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Helvetica Chimica Acta ± Vol. 83 (2000)
Stereochemistry of the Methyl Group in (R)-3-Methylitaconate Derived by
Rearrangement of 2-Methylideneglutarate Catalysed by a Coenzyme
B12-Dependent Mutase
by Daniele Ciceria), Antonio J. Pierikb), Günter Hartrampfb), Gerd Brökerb), Giovanna Speranzaa)c),
Wolfgang Buckelb), Sir John Cornforthd), and Bernard T. Golding*a)1)
a) Department of Chemistry, Bedson Building, University of Newcastle upon Tyne, Newcastle upon Tyne,
NE17RU, U.K.
b) Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-Universität, D-35032 Marburg
c) Universitaꢀ degli Studi di Milano, Dipartimento di Chimica Organica e Industriale, via Venezian, 21,
I-20133 Milano
d) The School of Chemistry, Physics and Environmental Sciences, University of Sussex, Falmer, Brighton,
BN19QJ, U.K.
Dedicated to Professor Albert Eschenmoser on the occasion of his 75th birthday
2-Methylideneglutarate mutase is an adenosylcobalamin (coenzyme B12)-dependent enzyme that catalyses
the equilibration of 2-methylideneglutarate with (R)-3-methylitaconate. This reaction is believed to occur via
protein-bound free radicals derived from substrate and product. The stereochemistry of the formation of the
methyl group of 3-methylitaconate has been probed using a ꢁchiral methyl groupꢀ. The methyl group in 3-
([2H1,3H]methyl)itaconate derived from either (R)- or (S)-2-methylidene[3-2H1,3-3H1]glutarate was a 50 :50
mixture of (R)- and (S)-forms. It is concluded that the barrier to rotation about the CÀC bond between the
.
methylene radical centre and adjacent C-atom in the product-related radical [ CH2CH(ÀO2CCˆCH2)CO2À] is
relatively low, and that the interaction of the radical with cob(II)alamin is minimal. Hence, cob(II)alamin is a
spectator of the molecular rearrangement of the substrate radical to product radical.
1. Introduction. ± The enzyme 2-methylideneglutarate mutase isolated from the
anaerobe Eubacterium (Clostridium) barkeri catalyses the equilibration of 2-methyl-
ideneglutarate 12) with (R)-3-methylitaconate 2 (Keq ˆ 0.06) [1]. This process is a
carbon-skeleton rearrangement dependent upon coenzyme B12 (adenosylcobalamin),
and is believed to occur via protein-bound free radicals structurally related to the
substrate and product [2]. Homolysis of the CoÀC bond of coenzyme B12 generates
cob(II)alamin and the 5'-deoxyadenosyl radical, which abstracts a H-atom from C(4) of
2-methylideneglutarate or the Me group of (R)-3-methylitaconate. The resulting
substrate 2-methylideneglutarate or product 3-methylitaconate radical interconvert,
possibly via a cyclopropylmethyl radical (ꢁaddition-elimination mechanismꢀ; Scheme 1
path a) or via acrylate and the 2-acrylate radical (ꢁfragmentation-recombination
1
)
)
Telephone: ‡44-191-222 6647; fax: ‡44-191-222 6929; e-mail: b.t.golding@ncl.ac.uk
2
Abbreviations and nomenclature: 2-methylideneglutarate: 2-methylidenepentanedioic acid; 2-methyl-
idene[3-2H1,3-3H1]glutarate: 2-methylidenepentanedioic acid labelled at C(3), 3-methylitaconate: 2-
methyl-3-methylidenebutanedioic acid; 3-([2H1,3H1]methyl)itaconate: methyl-labelled 3-methylitaconate;
triflic anhydride (Tf2O): trifluoromethanesulfonic anhydride; isocitrate dehydrogenase: ICDH.

Helvetica Chimica Acta ± Vol. 83 (2000)
2551
Scheme 1. Possible Pathways for 2-Methylideneglutarate Mutase (path a, addition-elimination; path b, frag-
mentation-recombination)
mechanismꢀ; Scheme 1 path b) [3]. The product is formed by transferring a H-atom
from the Me group of 5'-deoxyadenosine to the 3-methylitaconate radical.
An issue of critical importance for the mechanistic interpretation is the degree of
interaction of cob(II)alamin with the putative intermediate radicals, which could range
from essentially no interaction to the formation of organocorrinoid intermediates with
a conventional CoÀC s-bond [4]. EPR and X-ray crystallographic data for several
coenzyme B12-dependent systems indicate that intermediate free radicals are bound
some distance from cob(II)alamin (e.g., 6.5 Š in glutamate mutase [5]). It is therefore
expected that the barrier to rotation about the s-bond between the methylidene C-
atom bearing the radical and the adjacent C-atom in the product-related radical for all
coenzyme B12-dependent C-skeleton rearrangements will be relatively low (n.b., there
is essentially free rotation in the ethyl radical [6]) and hardly perturbed by the Co-atom
of cob(II)alamin.
The degree of interaction of a methylidene group with an adjacent centre can be
investigated by means of a ꢁchiral methylꢀ probe [7][8], i.e., a Me group substituted
with deuterium and tritium. If this group is monochiral ((R) or (S))3), and if there is a
significant primary kinetic isotope effect in any reaction abstracting a H-atom from the
Me group, then the chirality information in the Me group may be transferred to a
product. In the classical studies of chiral Me groups, the methylidene group was a
carbanion centre derived from a chiral Me group of a particular chirality [7][8]. As the
methylidene carbanion could engage in a resonance interaction with an adjacent CˆO
group, the barrier to rotation about the CÀC bond between the two centres was
relatively high. The particular chirality of the Me group and the direction of capture of
the carbanion intermediate therefore determined the stereochemical outcome of the
reaction with respect to the reacting methylidene centre. If the methylidene group is a
3
)
This term, which replaces ꢁhomochiralꢀ, ꢁenantiopureꢀ etc. as the designation of a compound consisting of a
single enantiomer, was proposed by Cornforth [9]. It was also proposed that isochiral should replace the
term ꢁracemicꢀ.

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Helvetica Chimica Acta ± Vol. 83 (2000)
radical centre, and the adjacent centre is sp3-hybridised, then the barrier to rotation
about the CÀC bond connecting the two centres is expected to be relatively low (see
above). Hence, a chiral Me group generated from this centre is likely to be a 50 :50
mixture of (R)- and (S)-forms. This outcome has already been shown for the
conversion of either (R)- or (S)-[2-2H1,2-3H1]ethanolamine to [2-2H1,2-3H1]acetalde-
hyde and ammonia catalysed by the coenzyme B12-dependent enzyme ethanolamine
ammonia lyase [10]. The interpretation of this result was that the intermediate radical
.
( CH2CH(NH2)OH) undergoes rotation around its CÀC bond faster than H-atom
transfer from 5'-deoxyadenosine. The use of chiral Me as a mechanistic probe has been
reviewed [11][12].
In this paper, we describe the use of the chiral Me group to probe the mechanism of
the 2-methylideneglutarate mutase reaction. We have investigated the energy barrier to
rotation about the s-bond between the methylidene C-atom bearing the radical and the
adjacent C-atom in the 3-methylitaconate radical. The results indicate that the barrier is
sufficiently low that 2-methylideneglutarate stereospecifically labelled with deuterium
and tritium at C(3), 1a or 1b, leads to 3-([2H1,3H1]methyl)itaconate containing a 50 :50
mixture of (R)- and (S)-([2H1,3H1]methyl) groups (as in 2a and 2b, resp.). A previous
attempt to determine the chirality of the chiral Me group of 3-([2H1,3H1]methyl)itac-
onate derived from both (R)-1a and (S)-2-methylidene[3-2H1,3-3H1]glutarate 1b was
vitiated by the use of impure 2-methylideneglutarate mutase and problems with the
conversion of the 3-([2H1,3H1]methyl)itaconate (2a/2b) to ꢁchiral acetic acidꢀ (3a or 3b)
[13]. We describe experiments with pure recombinant 2-methylideneglutarate mutase
and a protocol for the conversion of the 3-([2H1,3H1]methyl)itaconate (2a/2b) to chiral
acetic acid (3a/3b), which minimises exchange processes in any of the intermediates
containing a chiral methyl group.
2. Results and Discussion. ± 2.1. Synthesis of (R)-and ( S)-2-Methylidene[3-2H1,3-
3H1]glutarates (1a and 1b, resp.). The synthesis was based on the stereospecific
exchange of the pro-S H-atom at C(3) of 2-oxoglutarate (4) catalysed by isocitrate
dehydrogenase [14]. The complete routes from 2-oxoglutarate (4) are shown in
Schemes 2 and 3, and were initially developed by converting 4 to unlabelled 2-
methylideneglutarate and specifically deuteriated 2-methylideneglutarates 1c ± 1e.
Heating 2-oxoglutarate (4) in D2O gave 2-oxo[3-2H2]glutarate 4a, which was converted
into (S)-2-oxo[3-2H1,3-3H1]glutarate (4b) using tritiated water with isocitrate dehy-
drogenase. It was necessary to trap the isotopic labels immediately, and this was done
by the rapid reduction of the (S)-2-oxo[3-2H1,3-3H1]glutarate 4b with NaBH4. The
resulting 2-hydroxy[3-2H1,3-3H1]glutarate (5a) was converted to diethyl 2-hydroxy[3-
2H1,3-3H1]glutarate 6a using Et3O BFÀ4 in water. The OH group of the diethyl ester 6a
‡
was activated as its trifluoromethanesulfonate, which was reacted with Ph3P.
Condensation of the derived phosphorane 7a with formaldehyde gave diethyl (S)-2-
methylidene[3-2H1,3-3H1]glutarate (8a), which was hydrolysed to (S)-2-methylidene[3-
2H1,3-3H1]glutarate (1b) (Scheme 2).
Initial tritiation of 2-oxoglutarate (4) at C(3) by heating with tritiated water to give
4c, followed by washing out tritium at the pro-S position by treatment with isocitrate
dehydrogenase in D2O, gave (R)-2-oxo[3-2H1,3-3H1]glutarate (4d). This was converted
via intermediates 5b ± 8b to (R)-2-methylidene[3-2H1,3-3H1]glutarate 1a (Scheme 3) by

Helvetica Chimica Acta ± Vol. 83 (2000)
2553
Scheme 2. Synthesis of (3S)-2-Methylidene[3-2H1,3-3H1]glutaric Acid (1b), 2-Methylidene[3,3-2H2]glutaric
Acid (1c), (3S)-2-Methylidene[3-2H1]glutaric Acid (1d), and (3R)-2-Methylidene[3-2H1]glutaric Acid (1e)
ia) D2O. ib) Tritiated water. iia) Tritiated water, isocitrate dehydrogenase (ICDH), NADPH. iib) D2O, ICDH,
‡
NADPH. iic) H2O, ICDH, NADPH, iii) NaBH4. iv) Et3O BFÀ4 , NaHCO3. v) Tf2O, py then Ph3P. vi) CH2O,
NaOEt. vii) NaOH. viii) 14CH2O, NaOEt.
Scheme 3. Synthesis of (3R)-2-Methylidene[3-2H1,3-3H1]glutaric acid (1a) (for reagents, see Scheme 2)
following the route described above for (S)-2-methylidene[3-2H1,3-3H1]glutarate (1b)
(Scheme 2).
The analysis of the tritiated samples was performed by measuring the 3H/14C ratio.
For this purpose 2-([14C]methylidene)glutaric acid (1f) was synthesised from [1,3-
bis(ethoxycarbonyl)propyl]triphenylphosphonium triflate (7f) and [14C]formaldehyde
(Scheme 4), and mixed with 1a and 1b.

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Scheme 4. Synthesis of 2-([14C]Methylidene)glutaric acid (1f) (for reagents, see Scheme 2)
2.2. Control Experiments Pertaining to Syntheses and Degradations. The integrity of
the isotopic labels throughout the synthetic route was confirmed by converting 2-
oxo[3,3-2H2]glutarate (4a) to 2-methylidene[3,3-2H2]glutarate (1c) via 5c ± 8c
(Scheme 2). Syntheses were also performed in which 2-oxoglutarate (4) was subjected
to exchange in D2O catalysed by isocitrate dehydrogenase to give (S)-2-oxo[3-
2H1]glutarate (4e), and 2-oxo[3,3-2H2]glutarate was exchanged in H2O to give (R)-2-
oxo[3-2H1]glutarate (4f). These 2-oxoglutarates were converted to the corresponding
2-methylidene[3-2H1]glutarates ((3S)- and (3R)-2-methylidene[3-2H1]glutarates (1d
and 1e, resp.)) via intermediates 5d ± 8d and 5e ± 8e, respectively (Scheme 2). Analysis
of the three deuteriated 2-methylideneglutarates 1c ± 1e by 1H-NMR showed no loss of
deuterium compared to the starting 2-oxoglutarate (i.e., that which was the immediate
precursor of 2-hydroxyglutarate). The 1H- and 2H-NMR analyses of the intermediate
2-hydroxy[3-2H1]glutarate (5e) from reduction of (R)-2-oxo[3-2H1]glutarate (4f)
indicated that it was a 50 :50 mixture of the (2R,3R)- and (2S,3R)-diastereoisomers,
as two resonances were observed for HÀC(3) and 2HÀC(3). Similarly, the 2-
hydroxy[3-2H1]glutarate (5d) from reduction of (S)-2-oxo[3-2H1]glutarate (4e) was a
50 :50 mixture of the (2R,3S)- and (2S,3S)-diastereoisomers.
The determination of the sense of chirality of the chiral Me group in a sample of 3-
([2H1,3H]methyl)itaconate was accomplished via 2,3-dimethylmaleate derived enzy-
matically from the 3-methylitaconate (see below). Acidification of the 2,3-dimethyl-
maleate brought about a rapid conversion to 2,3-dimethylmaleic anhydride [15]. The
anhydride was immediately extracted with CH2Cl2 and saponified to 2,3-dimethylma-
leate, which was oxidised to chiral acetic acid using alkaline permanganate. Since this
oxidation may proceed at least in part via pyruvate, some loss of tritium due to keto-
enol tautomerism could occur. On the other hand, if some Me groups are oxidised, it is
expected that Me containing only protium atoms will be oxidised faster than a chiral
Me group due to a primary kinetic isotope effect. Since the tritium label and the
internal counting standard 14C are located on different Me groups, the effect of the
processes described would be to cause the 3H/14C ratio to rise relative to that in the 2,3-
dimethylmaleate. The actual yield of acetate in the oxidative degradation, which was
developed with unlabelled 2,3-dimethylmaleate, was ca. 80% after isolation by ion-
exchange chromatography. The missing 20% of acetate could be due to over-oxidation
of Me groups or loss during workup. The almost constant 3H/14C ratios observed before
and after oxidation ((R)-series 2.56 to 2.55, (S)-series 2.70 to 2.69) indicates either that
no loss of tritium occurred, or that the loss was counterbalanced by the preferential
oxidation of the 14C-labelled Me groups containing merely protium. In a control
experiment, the oxidation of unlabelled 2,3-dimethylmaleate was performed in the
presence of tritiated water. A complete exchange would have led to a calculated

Helvetica Chimica Acta ± Vol. 83 (2000)
2555
activity of 3.6 Â 105 dpm in the acetate, whereas only 2 dpm were found. It can
therefore be concluded that, during permanganate oxidation, any chirality of the Me
groups in 2,3-dimethylmaleate was completely recovered in acetate.
2.3. Conversion of 3-([2H1,3H1]Methyl)itaconates (2a and/or 2b) to Chiral Acetates
(3a and/or 3b) via 2,3-Dimethylmaleate (9a and/or 9b). To prevent racemisation at the
chiral Me group of 3-methylitaconates derived from (R)- and (S)-2-methylidene-
[2H1,3H1]glutarate, it was necessary to inhibit the reversion to 2-methylideneglutarate.
3-Methylitaconate was removed as it was formed from 2-methylideneglutarate by
including 3-methylitaconate isomerase in the reaction mixture. This brought about the
thermodynamically favourable conversion of 3-methylitaconate to 2,3-dimethylma-
leate in situ (Keq ˆ 7) [16]. The conversion of a labelled 2-methylideneglurarate (1a or
1b) to labelled 2,3-dimethylmaleate (9a and/or 9b) was allowed to proceed to not more
than 5%. The labelled 2,3-dimethylmaleate was then trapped by acidification of the
reaction mixture, which induced rapid formation of its anhydride (10a and/or 10b) [15].
After hydrolysis of the anhydride back to 2,3-dimethylmaleate (9a and/or 9b),
permanganate oxidation gave chiral acetic acid (3a and/or 3b), which was subjected to
a standard analysis [7] to measure its chirality (see Sect. 2.4). The complete sequence
from (S)-2-methylidene[2H1,3H1]glutarate (1b) to chiral acetic acid (3a or 3b) is shown
in Scheme 5.
Scheme 5. Conversion of (3S)-2-Methylidene[3-2H1,3-3H1]glutaric Acid (1b) to Chiral Acetic Acid
(B12-MGM ˆ coenzyme B12-dependent 2-methylideneglutarate mutase)
2.4. Examination of the Acetates for Chirality. This was achieved with the standard
method based on the use of malate synthase and fumarase [7]. Radiochemical analysis
(Table) showed that upon incubation of the labelled malate derived via labelled acetate
from (R)-2-methylidene[3-2H1,3-3H1]glutarate (1a) with fumarase, (50 Æ 2)% of its
tritium content was lost. Hence, the chiral Me group of the 3-methylitaconate produced
from (R)-2-methylidene[3-2H1,3-3H1]glutarate was a 50 :50 mixture of (R)- and (S)-

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Helvetica Chimica Acta ± Vol. 83 (2000)
Table. Specific Activities of Isotopically Labelled Compounds
Labelled
(R)-Series
(S)-Series
compound
3
3
total H/dpm
3H/14C ratio
[dpm/dpm]
total H/dpm
3H/14C ratio
[dpm/dpm]
2-Methylideneglutarate
Acetate
1.6 Â 106
5.3 Â 104
4271
2558
2089
2.56
2.55
1.7 Â 106
5.9 Â 104
4810
2459
2048
2.70
2.69
Malate
t ˆ 0 min
2.14 (100%)
1.03 (48.1%)
1.12 (52.3%)
1.07 (50.0%)
1.07(50 Æ 2%)
2.23 (100%)
1.08 (48.4%)
1.10 (49.3%)
1.08 (48.4%)
1.09 (49 Æ 2%)
Malate/fumarate
Malate/fumarate
Malate/fumarate
Malate/fumarate
t ˆ 60
t ˆ 90
t ˆ 120
2734
2261
Mean value
60 ± 120 min
forms. The labelled malate derived from (S)-2-methylidene[3-2H1,3-3H1]glutarate (1b)
with fumarase lost (49 Æ 2)% of its tritium. Hence, the chiral Me of the 3-
methylitaconate from (S)-2-methylidene[3-2H1,3-3H1]glutarate was also a 50 :50
mixture of (R)- and (S)-forms.
Arigoni and Mazacek demonstrated that the malate derived from monochiral (R)-
acetate, of much higher specific activity than that used in the present work, retained
82% of its tritium content after exchange catalysed by fumarase, whereas that derived
from monochiral (S)-acetate retained 20% [17]. Therefore, the isochiral [9] (racemic)
mixture should give 51% retention, whereas [3H]acetate leads to a malate specimen,
which upon incubation with fumarase, loses exactly 50% of its tritium content. The
results described here, however, are not accurate enough (see above) to distinguish
between these possibilities.
2.5. Mechanistic Implications. We have found that the chiral Me group of 3-
methylitaconate derived from 2-methylideneglutarate in the isomerisation catalysed by
2-methylideneglutarate mutase is essentially a 50 :50 mixture of opposite chiralities.
This implies that the radical centre in the 3-methylitaconate radical is rotating with a
relatively low energy barrier and does not interact significantly with the Co-atom in
cob(II)alamin. This result is consistent with the notion that the rearrangement of 2-
methylideneglutarate to 3-methylitaconate occurs without the participation of co-
b(II)alamin. The addition-elimination and fragmentation-recombination mechanisms
for this enzyme [3], as well as a route involving ꢁpartial protonationꢀ of the substrate
methylidene group [18], are compatible with this finding.
3. Conclusion. ± This is the first time that a chiral Me experiment has been
performed for a coenzyme B12-dependent enzyme using homogenous protein. The
experiments described generated isochiral [9] acetic acid, which we have interpreted as
.
evidence for the intermediacy of the 3-methylitaconate radical ( CH2CH(ÀO2CCˆ
CH2)COÀ2 ) in the equilibration of 2-methylideneglutarate with (R)-3-methylitaconate
catalysed by coenzyme B12-dependent 2-methylideneglutarate mutase. The results
suggest that a protein-mediated rearrangement of the 2-methylideneglutarate radical
to the 3-methylitaconate radical occurs (cf. Scheme 1), with cob(II)alamin as a
spectator. In our experiments, it was necessary to exclude the possibility that isochiral
acetic acid was formed via reversal of the formation of 3-methylitaconate. We believe

Helvetica Chimica Acta ± Vol. 83 (2000)
2557
that this was avoided by trapping the 3-methylitaconate by its conversion into 2,3-
dimethylmaleate catalysed by methylitaconate isomerase.
The authors thank the European Commission, Deutsche Forschungsgemeinschaft and Fonds der
Chemischen Industrie for support.
Experimental Part
General. Commercial reagents were utilised without further purification. CH2Cl2 and pyridine were
distilled from CaH2. EtOH was distilled from Mg/I2. Petrol was the fraction boiling in the range 40 ± 608.
Deuteriated solvents were from Aldrich Chemical Company, Gillingham, Dorset, UK. Tritiated water (300 mCi/
ml) was a gift from Dr. D. Moltzan (Fachbereich Chemie, Philipps-Universität Marburg). [14C]Formaldehyde
(0.1 mCi/ml) was from American Radiolabeled Chemicals Inc. (Avonmouth, Bristol, UK). Acetyl-CoA synthase
from yeast, l-lactate dehydrogenase from rabbit muscle and fumarase from pig heart were from Roche, D-
Mannheim. Maleate synthase, isocitrate dehydrogenase and coenzyme B12 were from Sigma, Poole, Dorset, UK.
TLC: aluminium sheets pre-coated with silica gel (Kieselgel 60 F254, 0.2 mm, Merck, D-Darmstadt). Silica gel
(ꢁflashꢀ, Kieselgel 60) was used for column chromatography (CC). M.p.: determined in capillaries using a
Gallenkamp apparatus and are uncorrected. 1H-, 2H-, and 13C-NMR: recorded at the frequency stated; chemical
shifts (d) in ppm, with residual protons in the deuteriated solvents as an internal standard.
1. Syntheses of Deuteriated Compounds. ± 1.1. 2-Oxo[3,3-2H2]glutarate Disodium Salt (4a). 2-Oxoglutaric
acid disodium salt (2.0 g, 10.5 mmol) was dissolved in D2O (25 ml) and heated for 3 h at 808. The solvent was
1
removed by freeze-drying to give 4a (2.0 g, 10.5 mmol, 100%). White solid. H-NMR (200 MHz, D2O, pD 7):
2.54 (s, 2 H).13C-NMR (50 MHz, D2O): 207.10; 183.07; 171.41; 36.70 (1J(C,D) ˆ 15); 32.09.
1.2. 2-Hydroxy[3,3-2H2]glutaric Acid (5c). NaBH4 (20 mg, 0.52 mmol) was added to an aq. 0.5m soln. of 2-
oxo[3,3-2H2]glutarate (2.0 ml, 1 mmol, pH 7.5) and allowed to react for 5 min. 2m HCl (0.1 ml) was added and
1
the resulting solution was freeze-dried to give a white powder containing 5c. H-NMR (200 MHz, D2O): 2.39
(d, J ˆ 16.7, 1 H); 2.44 (d, J ˆ 16.7, 1 H); 4.18 (s, 1 H). 2H-NMR (500 MHz, H2O): 1.81 (s, 1 2H); 2.20 (s, 1 2H).
13C-NMR (125 MHz, D2O): 181.9; 180.3; 71.1; 32.5; 29.8 (1J(C,D) ˆ 19.9).
1.3. Diethyl 2-Hydroxy[3,3-2H2]glutarate (6c). Method a. Compound 5c (0.79 g, 5.3 mol) was dissolved in
0.23m NaOH (65 ml) and the soln. was stirred at r.t. overnight. Solid NaHCO3 (22.5 g, 0.27mol) and a soln. of
‡
Et3O BFÀ4 (42 g, 0.22 mol) in MeCN (10 ml) were added simultaneously over a period of 10 min, the pH being
kept between 7and 8. The resulting suspension was allowed to stir at r.t. for 20 min. The mixture was diluted
with H2O (50 ml) and decanted. The aq. soln. was extracted with CH2Cl2, and the white precipitate was washed
with CH2Cl2. The combined org. extracts were washed with brine and dried (MgSO4). The solvent was removed
and the resulting yellow oil was purified by chromatography (silica gel; 20% AcOEt in petrol) to afford 6c
(0.80 g, 3.9 mmol, 73%). Colorless oil.
Method b. Compound 5c (400 mg, 2.7mmol) was dissolved in abs. EtOH (150 ml). TsOH (20 mg,
0.1 mmol) was added and the reaction was boiled at reflux for 48 h. The EtOH was removed and the residue was
partitioned between CH2Cl2 and 10% NaHCO3. The org. phase was washed with brine and dried (Na2SO4). The
solvent was removed and the resulting pale yellow oil was purified by chromatography (silica gel; 20% AcOEt in
petrol) to afford 6c (475 mg, 2.3 mmol, 86%). Colorless oil. 1H-NMR (200 MHz, CDCl3): 1.23 (t, J ˆ 7 , 3 H);
1.28 (t, J ˆ 7, 3 H); 2.36 (d, J ˆ 11, 1 H); 2.42 (d, J ˆ 11, 1 H); 3.01 (d, OH); 4.00 ± 4.23 (m, 5 H). 13C-NMR
(50 MHz, CDCl3): 174.6; 173.1; 69.4; 61.9; 60.5; 29.5; 28.8 (1J(C,D) ˆ 19); 14.1; 14.0.
1.4. [1,3-Bis(ethoxycarbonyl)[2,2-2H2]propyl]triphenylphosphonium Triflate (7c). Triflic anhydride
(0.66 ml, 4.0 mmol) was added over a period of 5 min to a chilled (À 58) soln. of 6c (0.80 g, 3.9 mmol) and
pyridine (0.32 ml, 4.0 mmol) in CH2Cl2 (5.6 ml) under N2. The mixture was stirred for 30 min at À58, Ph3P (2.4 g,
9.1 mmol) in CH2Cl2 (3.2 ml) was added and the resulting mixture stirred overnight at r.t. The solvent was
removed and the residue was purified by chromatography on silica using CH2Cl2 as eluent to remove the excess
Ph3P and then AcOEt to recover 7c (1.74 g, 2.9 mmol 74%). The slightly yellow solid was recrystallized with
AcOEt to give 7c. Colorless crystals. M.p. 120 ± 1238. 1H-NMR (200 MHz, CDCl3): 1.02 (t, J ˆ 7.1, 3 H); 1.23
(t, J ˆ 7.1, 3 H); 2.59 (d, J ˆ 17, 1 H); 2.72 (d, J ˆ 17, 1 H); 3.90 (q, J ˆ 7.1, 2 H); 4.01 (q, J ˆ 7.1, 2 H); 5.2
(d, 2J(P,H) ˆ 14.2, 1 H); 7.60 ± 7.90 (m, 15 H). 13C-NMR (50 MHz, CDCl3): 172.1; 167.3; 135.6; 134.4 (3J(P,C) ˆ
9); 134.1 (2J(P,C) ˆ 13); 115.2 (1J(P,C) ˆ 86); 62.2 (1J(P,C) ˆ 120); 40.5, 31.5, 31.2; 23.9 (1J(C,D) ˆ 19); 14.1;
13.6. CI-MS: 451, 363, 279, 185, 145, 107, 79.
1.5. Diethyl 2-Methylidene[3,3-2H2]glutarate (8c). Paraformaldehyde (57mg, 1.9 mmol) was added to a
soln. of 7c (800 mg, 1.33 mmol) in dry EtOH (3.0 ml). To this mixture was added dropwise 1m NaOEt (2.0 ml,

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2 mmol) and the mixture was allowed to stir overnight. The solvent was removed and the residue was purified by
chromatography (silica gel; 10% AcOEt in petrol) to give 8c (250 mg, 1.23 mmol, 92%). Colorless oil. 1H-NMR
(500 MHz, CDCl3): 1.22 (t, J ˆ 7 , 3 H); 1.26 t(, J ˆ 7, 3 H); 2.47 (s, 2 H); 4.10 (q, J ˆ 7, 2 H); 4.17 (q, J ˆ 7 , 2 H);
5.52 (d, 2J ˆ 0.7, 1 H); 6.12 (d, 2J ˆ 0.7, 1 H). 13C-NMR (125 MHz, CDCl3): 172.7; 166.7; 139.2; 125.5; 60.7; 60.4;
33.1 (1J(C,D) ˆ 18); 27.3; 14.2 (2Â).
1.6. 2-Methylidene[3,3-2H2]glutaric Acid (1c). 5m NaOH (1.0 ml) was added to 8c (250 mg, 1.22 mmol) in
MeOH (1.4 ml) and the mixture was stirred for 16 h at r.t. The MeOH was removed, the residual aq. soln. was
acidified with 2m HCl and the resulting mixture was extracted with Et2O (3Â). The combined Et2O extracts
were washed with H2O and brine, and dried (MgSO4). The solvent was removed to afford a white solid. The
crude product was recrystallized from MeCN to give 1c (117mg, 0.80 mmol, 65%). White crystals. M.p. 128 ±
1308 ([19]: 129 ± 1308). 1H-NMR (500 MHz, D2O): 2.46 (s, 2 H); 5.59 (s, 1 H); 6.08 (s, 1 H). 13C-NMR
(125 MHz, D2O): 174.09; 168.20; 140.52; 126.05; 33.21; 27.62 (1J(C,D) ˆ 19.6). EI-MS: 146, 128, 101, 98, 87, 83,
71, 60, 55.
1.7. (3R)-2-Hydroxy[3-2H1]glutaric Acid (5e). A soln. containing 0.5m 4a (2.0 ml, 1.0 mmol, pH 7.5), 20 mm
Tris ´ HCl pH 7.4, 0.3 mm MgCl2, 1.5 mm NADPH and 200 U isocitrate dehydrogenase was incubated at 378 for
2 h. NaBH4 (20 mg, 0.52 mmol) was added to the mixture and allowed to react for 5 min. 2m HCl (100 ml) was
added and the resulting soln. was freeze-dried to give a white powder containing 5e. 1H-NMR (200 MHz, D2O):
1.85 ± 1.95 (m, 0.5 H); 1.96 ± 2.05 (m, 0.5 H); 2.22 ± 2.44 (m, 2 H); 4.06 (d, J ˆ 3.8, 1 H). 2H-NMR (500 MHz,
H2O): 1.82 (s, 0.5 2H); 2.21 (s, 0.5 2H). 13C-NMR (125 MHz, D2O): 181.90; 180.35; 71.14; 32.48; 29.78 (1J(C,D) ˆ
19.9).
1.8. Diethyl (3R)-2-Hydroxy[3-2H1]glutarate (6e). Compound 5e (149 mg, 1.0 mmol) was reacted with an
‡
excess of Et3O BFÀ4 in the manner described for the preparation of 6c (see 1.3, method a) to afford, after
workup, 6e (132 mg, 0.65 mmol, 65%). Colorless oil. 1H-NMR (200 MHz, CDCl3): 1.23 (t, J ˆ 7.0, 3 H); 1.28
(t, J ˆ 7.0, 3 H); 1.81 ± 2.03 (m, 0.5 H); 2.05 ± 2.24 (m, 0.5 H); 2.32 ± 2.58 (m, 2 H); 2.87(br., OH); 4.02 ± 4.24
(m, 5 H). 13C-NMR (50 MHz, CDCl3): 174.6; 173.2; 69.4; 61.8; 60.5; 29.7; 29.3 (1J(C,D) ˆ 20); 14.1; 14.0.
1.9. (3R)-[1,3-Bis(ethoxycarbonyl)[2-2H1]propyl]phosphonium Triflate (7e). Compound 6e (132 mg,
0.65 mmol) was treated with triflic anhydride in the manner described for 7c (see 1.4), followed by reaction
of the intermediate triflate with PPh3. After workup, 7e (290 mg, 0.48 mmol, 73%) was obtained. Colorless
crystals. M.p. 120 ± 1238. 1H-NMR (200 MHz, CDCl3): 1.02 (t, J ˆ 7.1, 3 H); 1.23 (t, J ˆ 7.1, 3 H); 2.00 ± 2.20
(m, 0.5 H); 2.21 ± 2.40 (m, 0.5 H); 2.62 ± 2.82 (m, 2 H); 3.97( q, J ˆ 7.1, 2 H); 4.12 (q, J ˆ 7.1, 2 H); 5.13 (m, 1 H);
7.60 ± 7.90 (m, 15 H). 13C-NMR (50 MHz, CDCl3): 172.1; 167.3; 135.6; 134.4 (3J(P,C) ˆ 9); 134.1 (2J(P,C) ˆ 13);
115.2 (1J(P,C) ˆ 86); 62.2 (1J(P,C) ˆ 120); 40.5; 31.5; 31.2; 23.9 (1J(C,D) ˆ 19); 14.1; 13.6.
1.10. Diethyl (3R)-2-Methylidene[3-2H1]glutarate (8e). Paraformaldehyde was reacted with 7e (290 mg,
0.48 mmol) in the manner described for the preparation of 8c (see 1.5) to give, after workup, 8e (74 mg,
0.37mmol, 76%). Colorless oil. 1H-NMR (500 MHz): 1.22 (t, J ˆ 7 , 3 H); 1.26 (t, J ˆ 7, 3 H); 2.31 ± 2.42 (m, 2 H);
2.42 ± 2.51 (m, 1 H); 4.10 (q, J ˆ 7, 2 H); 4.17 (q, J ˆ 7, 2 H); 5.52 (s, 1 H); 6.12 (s, 1 H). 13C-NMR (125 MHz,
CD3CN): 173.5; 167.6; 140.7; 125.9; 61.7; 61.2; 33.8; 27.9 (1J(C,D) ˆ 17); 14.7; 14.6.
1.11. (3R)-2-Methylidene[3-2H1]glutaric Acid (1e). NaOH (5m, 0.30 ml) was added to 8e (74 mg,
0.37mmol) in MeOH (0.4 ml) and the mixture was stirred for 16 h at r.t. After removing the MeOH, the
residual aq. soln. was acidified with 2m HCl and extracted with Et2O (3Â). The combined Et2O extracts were
washed with H2O and brine, and dried (MgSO4). The Et2O was removed to afford 1e (51 mg, 0.35 mmol, 94%).
White solid. M.p. 129 ± 1308. 1H-NMR (500 MHz, (D6)acetone): 2.38 ± 2.46 (m, 3 H); 5.59 (s, 1 H); 6.08 (s, 1 H).
13C-NMR (125 MHz, (D6)acetone): 174.09; 168.22; 140.52; 126.06; 33.22; 27.61 (1J(C,D) ˆ 19.6).
1.12. (3S)-2-Hydroxy[3-2H1]glutaric Acid (5d). A soln. containing 0.5m 2-oxoglutarate (4, 2.0 ml, 1.0 mmol,
pD 7.5) in D O, 20 mm Tris ´ HCl pD 7.4 in D O, 0.3 mm MgCl2 in D2O, 1.5 mm NADPH in D2O and 200 U
2
2
isocitrate dehydrogenase was incubated at 378 for 2 h. NaBH4 (10 mg, 0.52 mmol, 2.1 equiv.) was added to the
mixture and allowed to react for 5 min. 2m HCl (100 ml) was added and the resulting soln. was freeze-dried to
give a white powder containing 5d. 1H-NMR (500 MHz, D2O): 1.85 ± 1.95 (m, 0.5 H); 1.96 ± 2.05 (m, 0.5 H);
2.22 ± 2.44 (m, 2 H); 4.06 (d, J ˆ 3.8, 1 H). 2H-NMR (76 MHz, H2O): 1.82 (s, 0.5 2H); 2.21 (s, 0.5 2H). 13C-NMR
(125 MHz, D2O): 181.90; 180.35; 71.14; 32.48; 29.78 (1J(C,D) ˆ 19.9).
1.13. Diethyl (3S)-2-Hydroxy[3-2H1]glutarate (6d). Compound 5d (149 mg, 1.0 mmol) was reacted with an
‡
excess of Et3O BFÀ4 in the manner described for the preparation of 6c (see 1.3, method a) to afford, after
workup, 6e (135 mg, 0.66 mmol, 66%). Colorless oil. 1H-NMR (200 MHz, CDCl3): 1.23 (t, J ˆ 7.0, 3 H); 1.28
(t, J ˆ 7.0, 3 H); 1.81 ± 2.03 (m, 0.5 H); 2.05 ± 2.24 (m, 0.5 H); 2.32 ± 2.58 (m, 2 H); 2.87(br., OH); 4.02 ± 4.24
(m, 5 H). 13C-NMR (50 MHz, CDCl3): 174.6; 173.2; 69.4; 61.8; 60.5; 29.7; 29.3 (1J(C,D) ˆ 20); 14.1; 14.0.

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1.14. (3S)-[1,3-Bis(ethoxycarbonyl)[2-2H1]propyl]triphenylphosphonium Triflate (7d). Compound 6d
(135 mg, 0.66 mmol) was treated with triflic anhydride in the manner described for 7c (see 1.4), followed by
reaction of the intermediate triflate with PPh3. After workup, compound 7e (300 mg, 0.49 mmol, 74%) was
1
obtained. Colorless crystals. M.p. 120 ± 1238. H-NMR (200 MHz, CDCl3): 1.02 (t, J ˆ 7.1, 3 H); 1.23 (t, J ˆ 7.1,
3 H); 2.00 ± 2.20 (m, 0.5 H); 2.21 ± 2.40 (m, 0.5 H); 2.62 ± 2.82 (m, 2 H); 3.97( q, J ˆ 7.1, 2 H); 4.12 (q, J ˆ 7.1,
2 H); 5.13 (m, 1 H); 7.60 ± 7.90 (m, 15 H). 13C-NMR (50 MHz, CDCl3): 172.1; 167.3; 135.6; 134.4 (3J(P,C) ˆ 9);
134.1 (2J(P,C) ˆ 13); 115.2 (1J(P,C) ˆ 86); 62.2 (1J(P,C) ˆ 120); 40.5; 31.5; 31.2; 23.9 (1J(C,D) ˆ 19); 14.1; 13.6.
1.15. Diethyl (3S)-2-Methylidene[3-2H1]glutarate (8d). Paraformaldehyde was reacted with 7d (300 mg,
0.49 mmol) in the manner described for the preparation of 8c (see 1.5) to give, after workup, 8d (72 mg,
0.36 mmol, 74%). Colorless oil. 1H-NMR (200 MHz, CDCl3): 1.22 (t, J ˆ 7 , 3 H); 1.26 t(, J ˆ 7, 3 H); 2.44 ± 2.63
(m, 3 H); 4.10 (q, J ˆ 7, 2 H); 4.17 (q, J ˆ 7, 2 H); 5.52 (s, 1 H); 6.12 (s, 1 H). 13C-NMR (50 MHz, CDCl3): 172.7;
166.7; 139.2; 125.5; 60.7; 60.4; 33.1; 27.3 (1J(C,D) ˆ 18); 14.2 (2Â).
1.16. (3S)-2-Methylidene[3-2H1]glutaric Acid (1d). NaOH (5m, 0.30 ml) was added to 8d (72 mg,
0.36 mmol) in MeOH (0.4 ml) and the mixture was stirred for 16 h at r.t. After removing the MeOH, the
residual aq. soln. was acidified with 2m HCl and extracted with Et2O (3Â). The combined Et2O extracts were
washed with H2O and brine, and dried (MgSO4). The Et2O was removed to afford 1d (49 mg, 0.33 mmol, 93%).
White solid. M.p. 128 ± 1308. 1H-NMR (500 MHz, (D6)acetone): 2.38 ± 2.46 (m, 3 H); 5.59 (s, 1 H); 6.08 (s, 1 H).
13C-NMR (125 MHz, (D6)acetone): 174.09; 168.22; 140.52; 126.06; 33.22; 27.61 (1J(C,D) ˆ 20).
2. Synthesis of Radiolabelled Compounds. ± 2.1 The synthesis of each tritiated compound was performed by
a similar procedure and using the same quantities as described for the corresponding deuteriated compound,
except that tritiated water (25 ml, 300 mCi/ml) was introduced in the exchange step catalysed by isocitrate
dehydrogenase (see 1.7) for the series leading to (3S)-2-methylidene[3-2H1,3-3H1]glutaric acid (1b), and 2-
oxoglutarate (190 mg, 1.0 mmol) heated in 2 ml water containing tritiated water (25 ml, 300 mCi/ml) was
converted to 2-oxo-[3,3-3H2]glutarate disodium salt (4c) for the series leading to (3R)-2-methylidene[3-2H1,3-
3H1]glutaric acid (1a). In this way, the following radiolabelled compounds were obtained from 4a: (3S)-2-
hydroxy[3-2H1,3-3H1]glutaric acid (5a), diethyl (3S)-2-hydroxy[3-2H1,3-3H1]glutarate (6a); (3S)-[1,3-bis(eth-
oxycarbonyl)[3-2H1,3-3H1]propyl]triphenylphosphonium triflate (7a); diethyl (3S)-2-methylidene[3-2H1,3-
3H1]glutarate (8a); (3S)-2-methylidene[3-2H1,3-3H1]glutaric acid (1b). The following radiolabelled compounds
were obtained from 4c: (3R)-2-hydroxy[3-2H1,3-3H1]glutaric acid (5b); diethyl (3R)-2-hydroxy[3-2H1,3-
3H1]glutarate (6b); (3R)-[1,3-bis(ethoxycarbonyl)[3-2H1,3-3H1]propyl]triphenylphosphonium triflate (7b); di-
ethyl (3R)-2-methylidene[3-2H1,3-3H1]glutarate (8b); (3R)-2-methylidene[3-2H1,3-3H1]glutaric acid (1a).
2.2. 2-([14C]Methylidene)glutaric Acid (1f). This compound was derived from diethyl 2-([14C])methyl-
idene)glutarate, which was synthesised from [1,3-bis(ethoxycarbonyl)propyl]triphenylphosphonium triflate and
[14C]formaldehyde (0.1 mCi/ml) in the manner described for compounds 1a and 1b. Hydrolysis of the diethyl 2-
([14C]methylidene)glutarate afforded 1f with an activity of 12.8 mCi/mmol.
3. Control Experiment: Oxidation of Dimethylmaleic Anhydride with Permanganate in Tritiated Water. ±
To dimethylmaleic anhydride (4.0 mg, 31 mmol) in 0.4 ml of 0.25m NaOH was added tritiated water (5 ml,
30 mCi/ml) and 1.5 ml of 0.2m KMnO4. The mixture was left overnight at r.t. and then treated with 3% H2O2
(2.0 ml) to reduce the excess KMnO4. Finally, the mixture was boiled to remove unreacted H2O2. The cooled
mixture was filtered and the filtrate was passed through a 5 ml Dowex 1X8 (200 ± 400 mesh) column, ClÀ form.
The resin was washed with H2O (150 ml) and acetate was recovered by elution with 7.5 ml of 0.5m HCl. The
acidic soln. was neutralised with NaOH and freeze-dried twice to afford 300 mg (mainly NaCl) of a white solid.
Acetate was determined enzymatically with acetyl-CoA synthase [20]. This sample, which contained 38.7 mmol
(62%) acetate, was counted against a blank of AcONa and NaCl. No deviation from the background was found
for either sample. Blank: 26 dpm; sample: 28 dpm (for a complete exchange 3.6 Â 105 dpm would have been
expected).
4. Purification of apo-2-Methylideneglutarate Mutase and 3-Methylitaconate Isomerase. ± Overexpression
of the gene coding for 2-methylideneglutarate mutase (mgm) in Escherichia coli DH5a, transformed with the
plasmid pBB2, was performed essentially as described in [21]. Since E. coli lacks adenosylcobalamin, the apo-
enzyme could be purified aerobically and without particular precautions to exclude light. All manipulations
were carried out at 48. Cells (20 g wet mass) were harvested by centrifugation (6000 Â g, 15 min), resuspended in
buffer A (50 mm K3PO4, 1 mm EDTA, pH 7.4) containing 1 mm methylphenylsulfonyl fluoride, and disrupted by
sonication (Branson sonifier B15, USA). Cell debris and membranes were removed by two consecutive
ultracentrifugation steps (200,000 Â g, 30 min). The enzyme was further purified by chromatography on a
Pharmacia FPLC system equipped with Pharmacia columns (D-Freiburg). The supernatant of the second

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ultracentrifugation step was loaded onto DEAE Sepharose (column type HiLoad 26/10) equilibrated with
buffer A (3 ml/min). After washing with 100 ml buffer A, apo-2-methylideneglutarate mutase was eluted with a
linear gradient of 0 ± 1m NaCl in buffer A. The fractions eluting at ca. 300 mm NaCl contained apo-mutase
activity. (NH4)2SO4 was added to a final concentration of 1m to the pooled fractions. The clear soln. was loaded
onto phenyl-Sepharose HP column (HiLoad 26/10) equilibrated with buffer B (buffer A supplemented with 1m
(NH4)2SO4) operated at a flow rate of 3 ml/min. A gradient of 100 ± 50% buffer B in buffer A (100 ml), 50 ± 0%
buffer B in buffer A (200 ml) and 200 ml buffer A eluted the apo-mutase after ca. 400 ml. Fractions which
exhibited activity were concentrated in an Amicon chamber with PM30 membrane (Amicon, D-Witten) and
loaded on a Superdex 200 prep-grade column (type 35/600) equilibrated with buffer A supplemented with
100 mm NaCl (0.5 ml/min). apo-Mutase fractions eluted at a volume of ca. 250 ml. The purity of the
concentrated enzyme was 95 ± 99% as determined by SDS-PAGE. Purified apo-mutase was stable for several
months when stored at protein concentrations of 1 ± 40 mg/ml at À808. The protein content was determined by
the Bradford method [22].
The gene mii coding for (R)-3-methylitaconate isomerase was cloned by PCR from pBB1 into pBluescript
SK ‡ cosmid yielding the plasmid pGB1. Restriction analysis showed that the 5'-region of the mii gene was
oriented to the T7-promoter of the cloning vector. For the overexpression of the mii gene, E. coli BL21(lDE3)
[23] was transformed with the pGB1 plasmid. The E. coli clone was grown and induced by the procedure
described in [21]. The purification of the overproduced enzyme was essentially the same as described above for
the 2-methylideneglutarate mutase, except that the size-exclusion column (Superdex 200 prep-grade) was
omitted. The enzyme with a purity of 95 ± 99% could be stored at À80% for months at protein concentrations of
1 ± 40 mg/ml without loss of isomerase activity.
The activity of apo-2-methylideneglutarate mutase was measured spectrophotometrically at 258 in a
cuvette, d ˆ 1 cm, total volume of 1.0 ml, containing 100 mm K3PO4, pH 7.4, 1 mm dithiothreitol, ca. 1 U 3-
methylitaconate isomerase, and 5 mm 2-methylideneglutarate. After addition of the apo-enzyme, the reaction
was started with 10 mm adenosylcobalamin (final concentration) and was followed at l ˆ 256 nm (e ˆ 0.66 mmÀ1
cmÀ1). The activity of 3-methylitaconate isomerase was determined with a similar assay in which 2-
methylideneglutarate and adenosylcobalamin were replaced by 5 mm (R,S)-3-methylitaconate. The reaction
was started by addition of the isomerase.
5. Enzymology. ± 5.1. Conversion of (R)-2-Methylidene[3-2H1,3-3H1]glutarate to Chiral Acetate. The
incubation contained in a total volume of 1.4 ml: 50 mm K3PO4, pH 7 .4; 10 mm (R)-2-methylidene[3-2H1,3-
3H1]glutarate (ca. 116000 dpm/mmol); 40 mm 2-([14C]methylidene)glutarate (ca. 12000 dpm/mmol); 1.5 U 2-
methylideneglutarate mutase; 5 U 3-methylitaconate isomerase. A 7-ml sample was withdrawn from a mixture
containing only the two 2-methylideneglutarates (460 ml) and its radioactivity was determined: 3H: 24388 dpm;
3
14C: 9511 dpm; H/14C ˆ 2.56. The reaction was started by addition of 28 ml of 1 mm adenosylcobalamin. The
formation of 2,3-dimethylmaleate was followed by the increase of the UV absorbance at l ˆ 270 nm (e ˆ
0.085 mmÀ1 cmÀ1). After 134 s, an increase in absorbance of 0.106 (determined in a cuvette with 5-mm path
length) had occurred corresponding to a 5% (2.5 mm/50 mm) conversion of 2-methylideneglutarate, the
reaction was quenched with 600 ml of 8m (25%) aq. HCl. The resulting soln. was extracted wtih CH2Cl2 (4 Â
10 ml) and dried (MgSO4). The solvent was removed to afford 2,3-dimethylmaleic anhydride.
The dimethylmaleic anhydride (ca. 0.4 mg) was oxidised with alkaline KMnO4 to acetate as described
above (Sect. 3), but on a 10-fold smaller scale. Radiochemical analysis of the acetate soln. (100 ml, 5.73 mmol, ca.
90% yield) gave: 3H: 7123 dpm; 14C: 2825 dpm; 3H/14C ˆ 2.55. According to analysis by 500-MHz 1H-NMR and
UV/VIS spectroscopy, no residual 2,3-dimethylmaleate was present.
5.2. Analysis of Chiral Acetate from (R)-2-Methylidene[3-2H1,3-3H1]glutarate. The incubation contained in
a total volume of 0.5 ml: 33 mm KHCO3/K2CO3, pH 9; 2.0 mm chiral acetate from above; 4 mm glyoxylate;
4 mm ATP ; 4 mm MgCl2; 1 mm Na2EDTA; 2 U malate synthase; 2 U acetyl-CoA synthase (the malate synthase
and acetyl-CoA synthase both contained less than 0.1 mU fumarase). The reaction was started by addition of
0.3 mm CoASH and monitored for the disappearance of glyoxylate. The assay was based on the reduction of
glyoxylate to hydroxyacetic acid (glycolate) mediated by NADH with a non-specific lactate dehydrogenase
from rabbit muscle. The reaction soln. contained 100 mm K3PO4 buffer, pH 7.4, 20 mm NADH, 15 ml of the
sample to be analysed and 6 ml of lactate dehydrogenase (8760 U/ml). The reaction was followed by monitoring
the decrease of absorption at l ˆ 340 nm (e340 ˆ 6.2 mmÀ1 cmÀ1): 0/0, 20/2.04, 30/2.05 (time [min]/glyoxylate
consumed [mmol] from an initial 3.93 mmol). The reaction mixture was diluted to 20 ml and passed through a
Dowex 8 Â 1 (formate form, 20 ml), which was washed with H2O (50 ml). Malic acid was eluted from the
column with 1m HCO2H, collected in 7fractions (10 ml each) and counted. Radioactivity was present in

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fractions 3 and 4. The combined radioactive fractions were evaporated to dryness under vacuum. The residue
was dissolved in ca. 1 ml of 50 mm phosphate buffer, pH 7.4. A sample (200ml) of this soln. was counted.
Fumarase (10 U, 14 ml) was added to the rest of the mixture and the resulting soln. was incubated at 408. Samples
(200 ml) were taken at different times and briefly boiled in order to inactivate the enzyme. Each sample was
diluted with 30 ml of H2O and brought to dryness (this was done twice). Eventually H2O (200 ml) was added and
the whole sample counted to give the following results: 0/2.14/0, 60/1.03/48.1, 90/1.12/52.3, 120/1.07/50.0 (time
[min]/ratio of 3H to 14C/% change compared to malate).
5.3. Conversion of (S)-2-Methylidene[3-2H1,3-3H1]glutarate to Chiral Acetate. This was performed in the
manner described for (R)-2-methylidene[3-2H1,3-3H1]glutarate. A 7-ml sample was withdrawn from a mixture
containing only the two 2-methylideneglutarates (i.e., before adding the other components) and its radioactivity
was determined: 3H: 25778 dpm; 14C: 9539 dpm; 3H/14C ˆ 2.70. Acetate was determined by the acetyl-CoA
synthase method: 5.43 mmol (ca. 78% yield). Radiochemical analysis of the acetate soln. (100 ml) gave: 3H:
3
7748 dpm; 14C: 2884 dpm; H/14C ˆ 2.71.
5.4. Analysis of Chiral Acetate from (S)-2-Methylidene[3-2H1,3-3H1]glutarate. The final sample was counted
to give the following results: 0/2.23/0, 60/1.08/48.4, 90/1.10/49.3, 120/1.08/48.4 (time [min]/ratio of 3H to 14C/%
change compared to malate).
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Received June 2, 2000