Synthesis of 13C-labeled γ-hydroxybutyrates for EPR studies with 4-hydroxybutyryl-CoA dehydratase

Article abstract of DOI:10.1016/j.bioorg.2004.09.001

4-Hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum catalyses the reversible dehydration of its substrate 4-hydroxybutyryl-CoA (4-HB-CoA) to crotonyl CoA. The enzyme contains one [4Fe-4S]²⁺ cluster and one flavin adenine dinucleotide (FAD) molecule per homotetramer. Incubation of the enzyme with its substrate under equilibrium conditions followed by freezing at 77 K induced the EPR-spectrum of a neutral flavin semiquinone (g = 2.005, linewidth 2.1 mT), while at 10 K additional signals were detected. In an attempt to characterize these signals, 4-HB-CoA molecules specifically labeled with ¹³C have been synthesized. This was achieved via ¹³C- labeled γ-butyrolactones, which were obtained from ¹³C-labeled bromoacetic acids by efficient synthetic routes. Incubation of the ¹³C-labeled 4-hydroxybutyrate-CoA molecules with 4-hydroxybutyryl-CoA dehydratase did not lead to marked broadening of the signals.

Full text of DOI:10.1016/j.bioorg.2004.09.001

Bioorganic Chemistry 33 (2005) 53–66
www.elsevier.com/locate/bioorg
13
Synthesis of C-labeled c-hydroxybutyrates
for EPR studies with 4-hydroxybutyryl-CoA
dehydratase
Ulrike Nasera, Antonio J. Pierikb, Richard Scotta,
¨
Irfan C¸ inkayab, Wolfgang Buckelb, Bernard T. Goldinga,*
a
School of Natural Sciences—Chemistry, University of Newcastle upon Tyne, Bedson Building,
Newcastle upon Tyne, NE1 7RU, United Kingdom
Laboratorium fu¨r Mikrobiologie, Fachbereich Biologie, Phillips-Universita¨t, Marburg, D-35032, Germany
b
Received 11 August 2004
Available online 13 October 2004
Abstract
4-Hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum catalyses the revers-
ible dehydration of its substrate 4-hydroxybutyryl-CoA (4-HB-CoA) to crotonyl CoA. The
enzyme contains one [4Fe–4S]2+ cluster and one flavin adenine dinucleotide (FAD) molecule
per homotetramer. Incubation of the enzyme with its substrate under equilibrium conditions
followed by freezing at 77 K induced the EPR-spectrum of a neutral flavin semiquinone
(g = 2.005, linewidth 2.1 mT), while at 10 K additional signals were detected. In an attempt
to characterize these signals, 4-HB-CoA molecules specifically labeled with 13C have been
synthesized. This was achieved via 13C-labeled c-butyrolactones, which were obtained from
13C-labeled bromoacetic acids by efficient synthetic routes. Incubation of the 13C-labeled
4-hydroxybutyrate-CoA molecules with 4-hydroxybutyryl-CoA dehydratase did not lead to
marked broadening of the signals.
ꢀ 2004 Elsevier Inc. All rights reserved.
Keywords: Isotopic labeling; c-Butyrolactone; 4-Hydroxybutyryl-CoA dehydratase; Flavin adenine
dinucleotide semiquinone; EPR spectroscopy
*
Corresponding author. Fax: +44 191 222 6929.
E-mail address: B.T.Golding@ncl.ac.uk (B.T. Golding).
0045-2068/$ - see front matter ꢀ 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2004.09.001

54
U. Na¨ser et al. / Bioorganic Chemistry 33 (2005) 53–66
1. Introduction
Clostridium aminobutyricum ferments c-aminobutyrate (GABA, 4-aminobutyrate)
to ammonia, acetate, and butyrate [1]. c-Aminobutyrate is converted into 4-hydrox-
ybutyrate via amino transfer to 2-oxoglutarate giving glutamate and succinate sem-
ialdehyde, which is reduced by NADH [2,3]. Reaction of 4-hydroxybutyrate with
acetyl-CoA mediated by 4-hydroxybutyrate CoA-transferase yields 4-hydroxybuty-
ryl-CoA [4], which is reversibly dehydrated to crotonyl-CoA [3,5]. In the final step,
disproportionation of crotonyl-CoA leads to acetate and butyrate. 4-Hydroxybuty-
ryl-CoA dehydratase has been characterized as a homotetramer (260 kDa) contain-
ing one FAD and one [4Fe–4S]2+ cluster per subunit [5,6]. The crystal structure has
recently been solved and shown to be related to that of medium chain acyl-CoA
dehydrogenase. Modeling of 4-hydroxybutyryl-CoA into the putative active site re-
vealed an interaction of the hydroxyl group with the [4Fe–4S] cluster, which is coor-
dinated by three cysteine and one histidine side chains [7]. The dehydratase catalyses
a mechanistically most intriguing transformation, because the C-3 hydrogen that
needs to be eliminated is not activated (pKa = 40) [8]. The proposed catalytic mech-
anism starts with the abstraction of the C-2 proton from 4-hydroxybutyryl-CoA, fol-
lowed by one-electron transfer to FAD [9,10]. The resulting semiquinone radical
anion removes the pro-S hydrogen from C-3 of the accompanying enoxy radical,
[11] the pKa of which is estimated to be ca. 14 [12]. This process yields a ketyl radical
anion and a neutral FAD semiquinone radical. The ketyl radical anion eliminates the
hydroxyl group to give a dienoxy radical, which is reduced by the FAD semiquinone
radical to a dienolate. Finally, this species is protonated at the C-4 position to afford
the product crotonyl-CoA (Scheme 1).
When isolated under strictly anaerobic conditions, about 20% of the FAD of 4-
hydroxybutyryl-CoA dehydratase is in the neutral semiquinone form, indicating that
the enzyme is able to stabilize this radical. Full enzymatic activity is achieved after
oxidation of the FAD semiquinone to FAD quinone with ferricyanide [13].
We have found that upon addition of substrate, part of the FAD is reduced to the
semiquinone as revealed by EPR spectroscopy at 10 and 77K. At the lower temper-
ature, additional signals were obtained which could be due to substrate-derived rad-
icals interacting with the [4Fe–4S]2+ cluster. To characterize these signals we have
synthesized specifically 13C-labeled c-butyrolactones (Fig. 1), which were hydrolyzed
with aq. sodium hydroxide to 4-hydroxybutyrates and converted enzymatically into
4-hydroxybutyryl-CoA species using 4-hydroxybutyrate CoA-transferase [4].
2. Results and discussion
2.1. Chemistry
All the syntheses were optimized on unlabeled starting material. For 13C-labeled
c-butyrolactone, a synthesis was devised starting from commercially available 13C-
labeled bromoacetic acid. The acid was esterified with diazomethane, and then either

U. Na¨ser et al. / Bioorganic Chemistry 33 (2005) 53–66
55
Scheme 1. Proposed mechanism for the dehydration of 4-hydroxybutyryl-CoA mediated by 4-hydroxy-
butyryl-CoA dehydratase.
Fig. 1. 13C-labeled c-butyrolactones synthesized and used in the present study: 11a c-[2-13C]butyrolactone,
11b c-[3-13C]butyrolactone, 11c c-[4-13C]butyrolactone, 11d c-[5-13C]butyrolactone, 11e c-[2,3,4,
5-13C]butyrolactone.
converted to a Wittig salt or an aldehyde. Combining the Wittig salt with the alde-
hyde yielded an alkene which upon reduction and deprotection afforded the appro-
priate c-butyrolactone (Scheme 2).
2.2. Labeling
Starting from [1-13C]-, [2-13C]-, or [1,2-13C]-labeled bromoacetic acid and employ-
ing either labeled or unlabeled starting material in each branch of the synthesis,
enabled the introduction of 13C into all individual positions of the target c-butyro-
lactone (Scheme 3). 13C4-labeled c-butyrolactone was also synthesized.
Bromoacetic acid (1) was converted to the corresponding methyl ester (2) using
diazomethane, as the method is clean and quantitative [14]. The synthesis of

56
U. Na¨ser et al. / Bioorganic Chemistry 33 (2005) 53–66
Scheme 2. Synthetic route to c-butyrolactone.
Scheme 3. Syntheses of the labeled c-butyrolactones.
unlabeled (4-methoxybenzyloxy)acetaldehyde (6) was achieved in three steps starting
from rac.-1,2-di-O-isopropylideneglycerol, Solketal (3), to give the desired unlabeled
aldehyde in an overall yield of 61% [15]. The synthesis of labeled (4-methoxybenzyl-
oxy)acetaldehyde (6) was achieved in three steps: reaction of (2) with the sodium
salt of 4-methoxybenzyl alcohol using the method of Lal et al. [16] followed by a

U. Na¨ser et al. / Bioorganic Chemistry 33 (2005) 53–66
57
reduction of (4-methoxybenzyloxy)acetic acid methyl ester (7) with lithium alumin-
ium hydride to give 2-(4-methoxybenzyloxy)ethanol (8). Oxidation of alcohol (8) to
the corresponding aldehyde (6) was performed using Dess–Martin periodinane
(1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-3(1H)) [17,18]. The key step in the
synthetic pathway is the Wittig reaction of (6) with labeled or unlabeled bromoacetic
acid methyl ester (2) under mild conditions [19]. The alkene 4-(4-methoxybenzyl-
oxy)but-2-enoic acid methyl ester (9) was reduced to 4-(4-methoxybenzyloxy)buta-
noic acid methyl ester (10) and finally deprotection, using 2,3-dichloro-5,
6-dicyano-1,4-benzoquinone (DDQ), gave c-butyrolactone (11) [20]. The crude
13C-labeled c-butyrolactones (11a–e) were purified by preparative gas chromatogra-
phy using dichloromethane as the trapping solvent. The dichloromethane was removed
in vacuo to give a c-butyrolactone suitable for conversion into 4-hydroxybutyrate.
2.3. Biochemistry and EPR-spectroscopy
The c-butyrolactones (11a–e) were each hydrolyzed with one equivalent of
sodium hydroxide to yield the corresponding 4-hydroxybutyrate, which was subse-
quently converted to its CoA-ester by incubation with acetyl-CoA and 4-hydroxybu-
tyrate CoA-transferase [4]. The integrity and 13C-labeling of the CoA-esters was
confirmed by MALDI-TOF mass spectrometry. The 4-hydroxybutyryl-CoA dehy-
dratase was incubated anoxically with ferricyanide and desalted by gel filtration.
The oxidized enzyme and the CoA-thiol esters were combined and frozen at 77 K.
Fig. 2. EPR spectra of the neutral FAD semiquinone radical (A) and neutral FAD semiquinone radical
with superimposed additional signals (B) of 9.9 lM tetrameric 4-hydroxybutyryl-CoA dehydratase in
100 mM potassium phosphate (pH 7.4) incubated in the presence of (a) 2 mM CoA, (b) unlabeled and
[1-13C]-, [2-13C]-, [3-13C]-, [4-13C]-, [1,2,3,4-13C]-labeled 4-hydroxybutyryl-CoA (trace c–g, respectively). 4-
Hydroxybutyryl-CoA dehydratase was oxidized with potassium ferricyanide and desalted, as described in
materials and methods. EPR conditions: microwave frequency 9460 1 MHz; microwave power 2 lW (A)
or 20 mW (B); modulation amplitude 0.55 mT; modulation frequency 100 kHz; temperature 77 K (A),
10 K (B).

58
U. Na¨ser et al. / Bioorganic Chemistry 33 (2005) 53–66
The EPR signal of the oxidized enzyme markedly increased upon addition of sub-
strate, regardless of whether unlabeled substrate or the five differently 13C-labeled 4-
hydroxybutyryl-CoA species were used. The EPR spectra at 77 K and at 10 K (Fig.
2) clearly revealed an intense isotropic signal of the neutral FAD semiquinone rad-
ical (line width 2.1 0.1 mT g = 2.005, cf. [13]). At the temperature of 10 K addi-
tional signals above and below g = 2.005 were detected (g values 2.033, 1.969, and
1.911), and probably arise from a partner radical of the FAD semiquinone radical.
All spectra with the unlabeled and labeled substrates were standardized to equal
intensity at the sharpest signal at g = 2.033 (linewidth 1.2 mT). The small variations
observed at the broader signals (g = 1.969 and 1.911, with linewidths of 2.8 and
3.2 mT, respectively) can be attributed to unavoidable variations of the experimental
conditions. However, the interaction of the substrate-derived radicals with excited
states of the [4Fe–4S]2+ cluster could have caused broadening of the signals in such
a way that 13C-induced coupling could no longer be observed. The low temperature
(10 K) required for visualization of the additional signals is consistent with such an
interaction.
3. Conclusion
We have shown that the synthesis of 13C-labeled c-butyrolactones can be readily
achieved from commercially available starting materials. Using the methods described,
it is possible to insert the 13C-label in any one or more of the carbon positions. All of the
steps proceeded in good to excellent yield, therefore avoiding significant loss of 13C-la-
bel. The use of diazomethane and Dess–Martin periodinane, although potentially haz-
ardous, posed no problem in our hands, and were milder and cleaner than the
alternatives. Alkaline hydrolysis of the c-butyrolactones obtained, followed by enzy-
matic thioesterification, afforded the corresponding labeled 4-hydroxybutyryl-CoA.
Incubation of 4-hydroxybutyryl-CoA dehydratase with 4-hydroxybutyryl-CoA
led to the formation of radicals. Whereas one radical could be identified as the neu-
tral flavin semiquinone radical, the identity of the other species remains elusive. Ow-
ing to a possible interaction with excited states of the [4Fe–4S]2+ cluster, the
additional signals observed at 10 K could have arisen from substrate-derived radi-
cals, despite the lack of 13C-induced line broadening. This is the first indication of
the involvement of substrate-derived radicals in the reversible dehydration of 4-
hydroxybutyryl-CoA to crotonyl-CoA and is consistent with the proposed mecha-
nism (Scheme 1).
4. Experimental
4.1. Caution
Dess–Martin periodinane [17,18] must be handled with caution as it can hydro-
lyze to iodoxybenzoic acid, which is shock sensitive. Diazomethane is a potent

U. Na¨ser et al. / Bioorganic Chemistry 33 (2005) 53–66
59
carcinogen and is also shock sensitive, and therefore must be handled in an efficient
hood behind a safety screen.
4.2. General
All chemicals were obtained from either Aldrich (Gillingham, Dorset, UK) or
Lancaster (Newgate, Lancashire, UK) and were used without further purification.
1
Solvents were dried by standard techniques. H NMR spectra were run at 200,
300, or 500 MHz and 13C NMR at 50.3, 75.45, or 125.75 MHz, using residual signals
from the deuterated solvent as references; D2O (4.81 ppm), d6-DMSO (2.50 ppm),
chloroform (7.25 ppm) for 1H spectra; d6-DMSO (39.7 ppm), chloroform
(77.0 ppm) for 13C spectra. All coupling constants were measured in Hertz. Sep-
Pak C18-cartridges were supplied by Whatman International (Maidstone, Kent,
UK). Other chromatographic materials were obtained from VWR International
(Poole, Dorset, UK).
4.3. Enzyme experiments and EPR spectroscopy
c-Butyrolactone was converted into the corresponding 4-hydroxybutyrate using
1.1 eq. of 1 M NaOH solution. The formation of the sodium salt was quantitative.
The 1 M solution was neutralized to pH 7 using 0.5 M HCl. The synthesis of 4-
hydroxybutyryl-CoA esters was performed using 1 mM labeled 4-hydroxybutyrate
(50 ll) in 1 M potassium phosphate (50 ll) pH 7.4; 32 mM acetyl-CoA (16 ll), trans-
ferase (15 ll, 53 U/ml), total sample volume of 0.5 ml. After incubation at rt for
10 min, a DTNB-assay [5,50-dithiobis(2-nitrobenzoate)] [4] was carried out to deter-
mine whether the conversion of 4-hydroxybutyrate into its corresponding CoA-ester
was complete. The synthesized 4-hydroxybutyryl-CoA esters were purified using a
C18-cartridge. The column was initially washed with 1 column volume (cv) of meth-
anol and with 10 cvÕs of 0.1% TFA to equilibrate. The CoA-ester was loaded and
washed with 10 cvÕs of 0.1% TFA, which were discarded. The column was dried
by purging with pressurized air, eluted with 1 cv of 0.1% TFA/50% MeCN, and fur-
ther rinsed with 5 cvÕs of 0.1% TFA/50% MeCN. The fractions containing the CoA
ester were collected, the solutions were concentrated (speed vac) and the aqueous
residue was freeze dried. MALDI-TOF mass spectrometry of the CoA-esters was
performed with an Applied Biosystems Voyager System 2048 using a-cyano-4-
hydroxycinnamic acid as the matrix [21].
The 4-hydroxybutyryl-CoA dehydratase was treated in an anaerobic chamber
with 10 mM ferricyanide at pH 7.0 for 30 min. Low molecular mass compounds were
separated from the enzyme by gel filtration on Sephadex G25. Labeled or unlabeled
6 mM hydroxybutyryl-CoA ester (59 ll) in 500 mM potassium phosphate (35 ll, pH
7.4), 396 lM 4-hydroxybutyryl-CoA dehydratase (17.5 ll, 21.4 mg mlꢀ1) and water
(63.5 ll) were mixed and transferred to an EPR tube, which was immediately cooled
in liquid nitrogen. The EPR spectra of these samples were recorded using an X-band
Bruker EPR spectrometer equipped with an Oxford Instruments flow cryostat using
liquid nitrogen or helium as coolant.

60
U. Na¨ser et al. / Bioorganic Chemistry 33 (2005) 53–66
4.4. Synthesis
4.4.1. Conversion of the 13C-labeled bromoacetic acid (1) to 13C-labeled methyl
bromoacetate (2)
4.4.1.1. This reaction must be performed in an efficient hood and behind a safety
screen. To a labeled bromoacetic acid (1) (1.0 g, 7.1 mmol) in diethyl ether (20 mL)
was added a 0.3–0.4 M solution of diazomethane in diethyl ether [14] until the reaction
mixture remained yellow. The excess of diazomethane was removed by passing N2
through the ethereal mixture and into a trap containing dilute acetic acid. The ether
was removed to yield labeled methyl bromoacetate (2) as a pale yellow liquid (1.1 g,
1
7.2 mmol, 100%). H NMR (200 MHz, CDCl3) methyl bromo[1-13C]acetate (2a): d
3.78 (2H, d, J 4.7 Hz, CH2), 3.73 (3H, d, J 3.9 Hz, OCH3); methyl bromo[2-13C]acetate
(2b): d 3.78 (2H, d, J 153.4 Hz, CH2), 3.73 (3H, s, OCH3); methyl bromo[1,2-13C]ace-
tate (2c): d 3.79 (2H, dd, J 4.7 and 153.3Hz, CH2), 3.74 (3H, d, J 3.9 Hz, OCH3).
4.5. Syntheses of (4-methoxybenzyloxy)acetaldehyde (6)
4.5.1. Method A
4.5.1.1. Rac.-3-O-40-methoxybenzyl-1,2-di-O-isopropylideneglycerol (4). Rac.-1,2-
di-O-isopropylideneglycerol (3) (8.9 g, 67 mmol) in THF (30 mL) was added dropwise
to a stirred suspension of sodium hydride (60% oil dispersion, 3.0 g, 80 mmol, pre-
washed with petrol) in dry THF (30 mL) under N2. After stirring for 15 min, p-meth-
oxybenzyl chloride (10.6 g, 67 mmol) in dry THF (30 mL) was added dropwise. The
mixture was heated at reflux overnight and the progress of reaction was monitored
by TLC (petrol/ethyl acetate, 4:1, starting material Rf = 0.12 and product
Rf = 0.56). After 20 h, the mixture was cooled in an ice-bath and water (25 mL) was
added. The volatile solvents were removed and the orange residue was partitioned be-
tween water (30 mL) and ethyl acetate (30 mL). The aqueous phase was further ex-
tracted with ethyl acetate (4 · 20 mL). The combined organic fractions were washed
with water (3 · 20 mL), sat. NaHCO3 (2 · 20 mL), dried (MgSO4), filtered, and con-
centrated to give a yellow liquid (15.1 g). The crude product was purified by medium
pressure chromatography (MPC) on silica eluting with petrol/ethyl acetate (4:1) to af-
ford (4) as a colorless liquid (11.6 g, 68%). 1H NMR (200 MHz, CDCl3) d 7.20 (2H, d, J
8.5 Hz, H-20 and H-60), 6.82 (2H, d, J 8.6 Hz, H-30 and H-50), 4.44 (2H, s, pmb-OCH2),
4.21 (1H, m, H-2), 3.97 (2H, m, 2· H-1), 3.73 (3H, s, OCH3), 3.39 (2H, m, 2· H-3), 1.34
(3H, s, CH3), 1.29 (3H, s, CH3). 13C NMR (50.3 MHz, CDCl3) d 159.66, 130.41, 129.79,
114.18, 109.77, 75.14, 73.56, 71.16, 67.32, 55.66, 27.17, and 25.77.
4.5.1.2. 3-O-40-methoxybenzylglycerol (5). Aqueous hydrochloric acid (1 M,
100 mL) was added to a solution of (4) (11.5 g, 45.6 mmol) in THF (100 mL) at room
temperature. The reaction was monitored by TLC (ether, starting material Rf = 0.95
and product Rf = 0.21) until the starting material had disappeared. After 6h the
solution was neutralized with sat. NaHCO3 and the volatiles were removed under
reduced pressure. The resulting solution was extracted with ethyl acetate
(4 · 50 mL), the combined organic layers were dried (MgSO4), filtered, and the

U. Na¨ser et al. / Bioorganic Chemistry 33 (2005) 53–66
61
solvent was removed. The residue was taken up in ether and treated dropwise with
petrol until precipitation persisted and a two-phase system was formed. The mixture
was stirred vigorously and cooled in an ice bath to give a white solid that was dried in
1
vacuo to give crystalline (5) (9.54 g, 98%). H NMR (200 MHz, CDCl3) d 7.28 (2H,
d, J 8.6 Hz, H-20 and H-60), 6.91 (2H, d, J 8.7Hz, H-30 and H-50), 4.51 (2H, s, pmb-
OCH2), 3.89 (1H, m, H-2), 3.84 (3H, s, OCH3), 3.67 (2H, m, 2· H-1), 3.56 (2H, m,
H-3), 2.96 (1H, s, HOCH), 2.54 (1H, s, HOCH2). 13C NMR (50.3 MHz, CDCl3) d
159.54 (C-40), 129.92 (C-10), 129.63 (C-20 and C-60), 114.05 (C-30 and C-50), 73.39
(pmb-OCH2), 71.65 (C-2), 70.78 (C-1), 64.24 (OCH3), 55.45 (C-3).
4.5.1.3. (4-Methoxybenzyloxy)acetaldehyde (6). Sodium(meta)periodate (2.1 g,
9.9 mmol) and water (20 lL) were added to (5) (2.0 g, 9.0 mmol) in dichloromethane
(20 mL) and the solution was vigorously stirred overnight. After the starting material
had disappeared (TLC ethyl acetate, starting material Rf = 0.32 and product
Rf = 0.78), the mixture was filtered and concentrated, and the residue was purified
by medium pressure chromatography (MPC) on silica with petrol/ethyl acetate (2:3)
1
as eluent to give (6) as a colorless oil (1.47 g, 91%). H NMR (200 MHz, CDCl3) d
9.78 (1H, s, CHO), 7.23 (2H, d, J 8.6 Hz, H-20 and H-60), 6.83 (2H, d, J 8.7 Hz, H-30
and H-50), 4.49 (2H, s, pmb-OCH2), 4.00 (2H, s, 2· H-2), 3.74 (3H, s, OCH3). 13C
NMR (50.3 MHz, CDCl3) d 200.72 (CHO), 159.73 (C-40), 129.85 (C-20 and C-60),
128.93 (C-10), 114.08 (C-30 and C-50), 75.08 (pmb-OC H2 or C-2), 73.43 (pmb-OC
H2 or C-2), 55.38 (OCH3).
4.5.2. Method B
4.5.2.1. (4-Methoxybenzyloxy)acetic acid methyl ester (7). Sodium hydride (60%
oil dispersion, 0.60 g, 16 mmol, pre-washed with petrol) was suspended in dry
THF (10 mL) under N2. 4-Methoxybenzyl alcohol (1.9 g, 14 mmol) was added por-
tionwise (1 h) with stirring at room temperature until the evolution of H2 ceased.
Methyl bromoacetate (2) (2.1 g, 14 mmol) was added dropwise at 0 ꢁC (1 h). The
reaction mixture was slowly brought to room temperature and stirring was contin-
ued for 20 h. After the starting material had disappeared (TLC petrol/ethyl acetate,
3:2, starting material Rf = 0.51 and product Rf = 0.74) the reaction mixture was di-
luted with ethyl acetate (42 mL) and slowly poured over crushed ice (56 g) containing
ammonium chloride (1.4 g). The ethyl acetate layer was separated, washed with sat.
brine (100 mL) and dried (MgSO4). The solvent was removed and the residual oil
was purified by MPC on silica with petrol/ethyl acetate (2:3) as eluent to yield (7)
1
as a pale yellow liquid (1.60 g, 56%). H NMR (200 MHz, CDCl3) d 7.19 (2H, d,
J 8.6 Hz, H-20 and H-60), 6.81 (2H, d, J 8.7 Hz, H-30 and H-50), 4.50 (2H, s, pmb-
OCH2), 4.01 (2H, s, 2· H-2), 3.74 (3H, s, OCH3), 3.69 (3H, s, COOCH3). 13C
NMR (50.3 MHz, CDCl3) d 171.52 (C OOCH3), 159.82 (C-40), 129.94 (C-20 and
C-60), 129.22 (C-10), 114.04 (C-30 and C-50), 73.13 (pmb-OCH2), 66.92 (CH2),
55.43 (OCH3), 51.98 (COOCH3).
4.5.2.2. 2-(4-Methoxybenzyloxy)ethanol (8). Compound (7) (0.50 g, 2.4 mmol)
was added to a solution of LiAlH4 (0.10 g, 2.6 mmol) in ether (20 mL) under N2

62
U. Na¨ser et al. / Bioorganic Chemistry 33 (2005) 53–66
in a way that the reaction mixture gently boiled at reflux. After the addition was
complete, the mixture was further heated at reflux for 2 h until the starting material
had disappeared (TLC petrol/ethyl acetate, 3:2, starting material Rf = 0.74 and
product Rf = 0.21). Ethyl acetate (500 lL) and water (250 lL) were added and the
mixture was filtered. The solvent was removed and the residual oil was purified by
MPC on silica with petrol/ethyl acetate (3:2) as eluent to yield (8) as a colorless liquid
(0.32 g, 70%). 1H NMR (200 MHz, CDCl3) d 7.20 (2H, d, J 8.6 Hz, H-20 and H-60),
6.82 (2H, d, J 8.7 Hz, H-30 and H-50), 4.42 (2H, s, pmb-OCH2), 3.74 (3H, s, OCH3),
3.67 (2H, m, 2· H-1), 3.49 (2H, t, J 4.5 Hz, 2· H-2), 2.03 (1H, br s, OH). 13C NMR
(50.3MHz, CDCl3) d 159.85 (C-40), 130.36 (C-10), 129.74 (C-20 and C-60), 114.19 (C-
30 and C-50), 73.36 (pmb-OCH2), 71.37 (C-1), 62.29 (C-2), 55.60 (OCH3).
4.5.2.3. (4-Methoxybenzyloxy)acetaldehyde (6). Compound (8) (0.30 g, 1.6 mmol)
was added to a solution of Dess–Martin periodinane (0.80 g, 1.9 mmol) in dry
dichloromethane (20 mL) in the dark at room temperature. The reaction was vigor-
ously stirred and monitored by TLC (petrol/ethyl acetate, 3:2, starting material
Rf = 0.22 and product Rf = 0.5) until no starting material remained. The reaction
was poured onto a silica column and eluted with petrol/ethyl acetate (2:3) to give
(6) as a colorless oil (0.27 g, 91%). NMR data as quoted before.
4.6. Syntheses leading to c-butyrolactone (11)
4.6.1. 4-(4-Methoxybenzyloxy)but-2-enoic acid methyl ester (9)
A solution of (4-methoxybenzyloxy)acetaldehyde (0.50 g, 2.8 mmol), triphenyl-
phosphine (1.9 g, 7.3 mmol) and methyl bromoacetate (0.65 g, 4.3 mmol) in dry
DCM (20 mL) was cooled to 0 ꢁC. Propylene oxide (2.44 g, 42.1 mmol) was added
under N2 and the resulting mixture was stirred at room temperature for 4 days (mon-
itored by TLC, petrol/ethyl acetate, 3:2, starting material Rf = 0.45 and product
Rf = 0.72). Removal of the solvent gave an oil, which was triturated with petrol
(3 · 10 mL). After the last addition the mixture was left overnight. Filtration and re-
moval of the solvent gave a crude product that was purified by MPC on silica, elut-
ing with petrol/ethyl acetate (5:1) to give (9) as a colorless liquid (0.39 g, 60%), which
1
comprised of a mixture of two alkene isomers. H NMR (500 MHz, CDCl3) d 7.21
(2H, d, J 8.6 Hz, H-20 and H-60), 6.92 (1H, dt, J 4.2 and 15.5 Hz, H-2trans), 6.82 (2H,
d, J 8.7 Hz, H-30 and H-50), 6.36 (1H, dt, J 4.9 and 11.9 Hz, H-2cis), 6.05 (1H, dt, J
2.1 and 15.9 Hz, H-3trans), 5.75 (1H, dt, J 2.1 and 11.6 Hz, H-3cis), 4.42 (2H, s, pmb-
OCH2), 4.08 (2H, dd, J 2.2 and 4.6 Hz, CH2), 3.74 (3H, s, OCH3), 3.68 (3H, s,
13
COOCH3). C NMR (50.3 MHz, CDCl3) d 166.78 (COOCH3), 159.36 (C-40),
148.73 (CH = CHcis), 144.72 (CH = CHtrans), 129.77 (C-10), 129.32 (C-20 and C-
60), 120.91 (CH = CHtrans), 119.01 (CH = CHcis), 113.87 (C-30 and C-50), 72.44
(pmb-OCH2), 68.18 (CH2), 55.38 (OCH3), 51.31 (COOCH3).
4.6.2. 4-(4-Methoxybenzyloxy)butanoic acid methyl ester (10)
Compound (9) (0.10 g, 0.42 mmol) and palladium catalyst (10wt. % on activated
carbon, 0.08 g) were stirred in dry methanol (25 mL) under a hydrogen atmosphere.

U. Na¨ser et al. / Bioorganic Chemistry 33 (2005) 53–66
63
The reaction was monitored by TLC (petrol/ethyl acetate, 3:2, starting material
Rf = 0.74 and product Rf = 0.65). After 30 h the catalyst was removed by filtration
(Celite) and the filtrate was concentrated to yield (10) as a colorless liquid (0.90 g,
1
96%). H NMR (500 MHz, CDCl3) d 7.19 (2H, d, J 8.6 Hz, H-20 and H-60), 6.80
(2H, d, J 8.7 Hz, H-30 and H-50), 4.37 (2H, s, pmb-OCH2), 3.73 (3H, s, OCH3),
3.58 (3H, s, COOCH3), 3.41 (2H, t, J 6.1 Hz, 2· H-4), 2.36 (2H, t, J 7.3 Hz, 2·
H-2), 1.87 (2H, quint, J 6.4 Hz, 2· H-3). 13C NMR (50.3MHz, CDCl3) d 166.58
(COOCH3), 159.26 (C-40), 129.41 (C-10), 129.15 (C-20 and C-60), 113.97 (C-30 and
C-50), 72.34 (pmb-OCH2), 69.03 (C-4), 55.47 (OCH3), 51.41 (COOC H3), 31.08
(C-2), 25.26 (C-3).
4.6.3. c-Butyrolactone (11)
2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (70 mg, 0.34 mmol) was
added to a solution of (10) (70 mg, 0.29 mmol) in dichloromethane (10 mL) and
water (0.5 mL) at 0 ꢁC. The mixture was stirred until TLC showed disappearance
of the starting material (petrol/ethyl acetate, 3:2, starting material Rf = 0.68 and
product Rf = 0.45). The resulting solution was stirred with aq. NaOH (1 M,
10 mL) overnight. The aqueous layer was washed with DCM (4 · 20 mL) and the
washings discarded. After acidification of the aqueous layer with aq. HCl (3 M,
20 mL), DCM (20 mL) was added and the mixture was stirred for 2 h. The layers
were separated and the aqueous phase was further extracted with DCM
(4 · 20 mL). The organic layers were combined, dried (MgSO4) and the solvent
was removed to give crude c-butyrolactone (11) (30 mg, 81%). Further purification
was achieved by preparative GC (23 mg, 62%). 1H NMR (500 MHz, CDCl3) d
4.32 (2H, t, J 7.1 Hz, 2· H-5), 2.48 (2H, t, J 7.6 Hz, 2· H-3), 2.24 (2H, quint, J
2.4 Hz, 2· H-4). 13C NMR (125.75 MHz, CDCl3) d 177.76 (C-2), 68.59 (C-5),
27.89 (C-3), 22.29 (C-4).
4.7. NMR-data of the labeled compounds
4.7.1. 4-(4-Methoxybenzyloxy)but-2-enoic[1-13C]acid methyl ester (9a)
1H NMR (200 MHz, CDCl3) d 7.21 (2H, d, J 8.6 Hz, H-20 and H-60), 6.97 (1H, m,
H-2trans), 6.82 (2H, d, J 8.7 Hz, H-30 and H-50), 6.37 (1H, m, H-2cis), 6.02 (1H, m, H-
3
trans), 5.77 (1H, m, H-3cis), 4.41 (2H, s, pmb-OCH2), 4.05 (2H, dd, J 2.4 and 4.8 Hz,
CH2), 3.74 (3H, s, OCH3), 3.64 (3H, d, J 3.9 Hz, 13COOCH3).
4.7.2. c-[2-13C]Butyrolactone (11a)
1H NMR (500 MHz, CDCl3) d 4.29 (2H, dt, J 3.1 and 7.0 Hz, 2· H-5), 2.43 (2H,
dt, J 5.8 and 8.3 Hz, 2· H-3), 2.20 (2H, m, 2· H-4). 13C NMR (125.75 MHz, CDCl3)
d 177.67 (s, 13C-2, enhanced), 68.45 (d, J 3.0 Hz, C-5), 27.74 (d, J 49.0 Hz, C-3),
22.14 (s, C-4).
4.7.3. 4-(4-Methoxybenzyloxy)but-2-enoic[2-13C]acid methyl ester (9b)
1H NMR (200 MHz, CDCl3) d 7.19 (2H, d, J 8.6 Hz, H-20 and H-60), 6.96 (1H, m,
H-2trans), 6.82 (2H, d, J 8.7Hz, H-30 and H-50), 6.39 (1H, m, H-2cis), 6.19 (1H, m,

64
U. Na¨ser et al. / Bioorganic Chemistry 33 (2005) 53–66
H-3trans), 5.67 (1H, m, H-3cis), 4.42 (2H, s, pmb-OCH2), 4.54 (2H, m, CH2), 3.74 (3H,
s, OCH3), 3.67 (3H, s, COOCH3).
4.7.4. c-[3-13C]Butyrolactone (11b)
1H NMR (500 MHz, CDCl3) d 4.29 (2H, dt, J 2.5 and 7.0 Hz, 2· H-5), 2.43 (2H,
dm, J 81.0 Hz, 3-13CH2), 2.20 (2H, m, 2· H-4). 13C NMR (125.75 MHz, CDCl3) d
177.75 (d, J 50.0 Hz, C-2), 68.47 (d, J 2.0 Hz, C-5), 27.96 (s, 13C-3, enhanced),
22.19 (d, J, 34.0 Hz, C-4).
4.7.5. (4-Methoxybenzyloxy)acetic[1-13C]acid methyl ester (7a)
1H NMR (500 MHz, CDCl3) d 7.21 (2H, d, J 8.6 Hz, H-20 and H-60), 6.81 (2H, d,
J 8.7 Hz, H-30 and H-50), 4.50 (2H, s, pmb-OCH2), 4.05 (2H, dd, J 7.1 and 14.4 Hz,
2· H-2), 3.83 (3H, s, OCH3), 3.75 (3H, d, J 1.3 Hz, 13COOCH3).
4.7.6. 2-(4-Methoxybenzyloxy)[1-13C]ethanol (8a)
1H NMR (500 MHz, CDCl3) d 7.23 (2H, d, J 8.6 Hz, H-20 and H-60), 6.83 (2H, d,
J 8.7 Hz, H-30 and H-50), 4.41 (2H, s, pmb-OCH2), 3.75 (3H, s, OCH3), 3.69 (2H, m,
2· H-1), 3.29 (2H, m, 2· H-2), 2.01 (1H, br s, OH).
4.7.7. (4-Methoxybenzyloxy)[1-13C]acetaldehyde (6a)
1H NMR (500 MHz, CDCl3) d 9.68 (1H, d, J 175.5 Hz, 13CHO), 7.29 (2H, d, J
8.6 Hz, H-20 and H-60), 6.88 (2H, d, J 8.7 Hz, H-30 and H-50), 4.55 (2H, s, pmb-
OCH2), 4.05 (2H, dd, J 0.6 and 4.0 Hz, CH2), 3.79 (3H, s, OCH3).
4.7.8. 4-(4-Methoxybenzyloxy)but-2-enoic[3-13C]acid methyl ester (9c)
1H NMR (500 MHz, CDCl3) d 7.19 (2H, d, J 8.6 Hz, H-20 and H-60), 6.92 (1H, dt,
J 4.3 and 15.9 Hz, H-2trans), 6.83 (2H, d, J 8.7 Hz, H-30 and H-50), 6.75 (1H, dt, J 4.3
and 15.6 Hz, H-2cis), 6.05 (1H, m, H-3trans), 5.75 (1H, m, H-3cis), 4.43 (2H, s, pmb-
OCH2), 4.09 (2H, m, CH2), 3.75 (3H, s, OCH3), 3.68 (3H, s, COOCH3).
4.7.9. c-[4-13C]Butyrolactone (11c)
1H NMR (500 MHz, CDCl3) d 4.27 (2H, dt, J 2.2 and 7.0 Hz, 2· H-5), 2.43 (2H,
dt, J 2.7 and 8.3 Hz, 2· H-3), 2.20 (2H, dm, J 81.0 Hz, 4-13CH2). 13C NMR
(125.75 MHz, CDCl3) d 177.43 (s, C-2), 68.26 (d, J 31.0 Hz, C-5), 27.53 (d, J
33.0 Hz, C-3), 22.19 (s, 13C-4, enhanced).
4.7.10. (4-Methoxybenzyloxy)acetic[2-13C]acid methyl ester (7b)
1H NMR (500 MHz, CDCl3) d 7.21 (2H, d, J 8.6 Hz, H-20 and H-60), 6.83 (2H, d,
J 8.7 Hz, H-30 and H-50), 4.50 (2H, d, J 4.0 Hz, pmb-OCH2), 4.05 (2H, dd, J 7.1 and
14.4 Hz, 2· H-2), 3.82 (3H, s, OCH3), 3.75 (3H, s, COOCH3).
4.7.11. 2-(4-Methoxybenzyloxy)[2-13C]ethanol (8b)
1H NMR (500 MHz, CDCl3) d 7.24 (2H, d, J 8.6 Hz, H-20 and H-60), 6.84 (2H, d,
J 8.7 Hz, H-30 and H-50), 4.39 (2H, d, J 4.0 Hz, pmb-OCH2), 3.75 (3H, s, OCH3),
3.69 (2H, m, 2· H-1), 3.45 (2H, m, 2· H-2), 2.01 (1H, br s, OH).

U. Na¨ser et al. / Bioorganic Chemistry 33 (2005) 53–66
65
4.7.12. (4-Methoxybenzyloxy)[2-13C]acetaldehyde (6b)
1H NMR (500 MHz, CDCl3) d 9.68 (1H, d, J 26.0 Hz, CHO), 7.28 (2H, d, J 8.6 Hz,
H-20 and H-60), 6.87 (2H, d, J 8.7 Hz, H-30 and H-50), 4.55 (2H, d, J 4.0 Hz, pmb-
OCH2), 4.05 (2H, d, J 141.0, CH2), 3.79 (3H, s, OCH3).
4.7.13. 4-(4-Methoxybenzyloxy)but-2-enoic[4-13C]acid methyl ester (9d)
1H NMR (500 MHz, CDCl3) d 7.21 (2H, d, J 8.6 Hz, H-20 and H-60), 6.93 (1H,
dt, J 4.3 and 15.9 Hz, H-2trans), 6.83 (2H, d, J 8.7 Hz, H-30 and H-50), 6.75 (1H, dt,
J 4.3 and 15.6 Hz, H-2cis), 6.05 (1H, m, H-3trans), 5.75 (1H, m, H-3cis), 4.23 (2H, d,
J 4.0 Hz, pmb-OCH2), 3.94 (2H, m, CH2), 3.72 (3H, s, OCH3), 3.68 (3H, s,
COOCH3).
4.7.14. c-[5-13C]butyrolactone (11d)
1H NMR (300 MHz, CDCl3) d 4.28 (2H, dt, J 151.9 and 7.0 Hz, 5-13CH2),
2.43 (2H, m, 2· H-3), 2.21 (2H, m, 2· H-4). 13C NMR (125.75 MHz, CDCl3)
d 177.42 (s, C-2), 68.88 (s, 13C-5, enhanced), 28.19 (s, C-3), 22.16 (d, J 31.0
Hz, C-4).
4.7.15. (4-Methoxybenzyloxy)acetic[1,2-13C]acid methyl ester (7c)
1H NMR (200 MHz, CDCl3) d 7.19 (2H, d, J 8.6 Hz, H-20 and H-60), 6.81 (2H, d,
J 8.7 Hz, H-30 and H-50), 4.50 (2H, d, J 4.0 Hz, pmb-OCH2), 4.02 (2H, m, 2· H-2),
3.76 (3H, s, OCH3), 3.69 (3H, d, J 4.0 Hz, COOCH3).
4.7.16. 2-(4-Methoxybenzyloxy)[1,2-13C]ethanol (8c)
1H NMR (500 MHz, CDCl3) d 7.25 (2H, d, J 8.6 Hz, H-20 and H-60), 6.84 (2H, d,
J 8.7 Hz, H-30 and H-50), 4.47 (2H, d, J 4.0 Hz, pmb-OCH2), 3.79 (3H, s, OCH3),
3.69 (2H, m, 2· H-2), 3.44 (2H, m, 2· H-1), 2.03 (1H, br s, OH).
4.7.17. (4-Methoxybenzyloxy)[1,2-13C]acetaldehyde (6c)
1H NMR (500 MHz, CDCl3) d 9.68 (1H, dd, J 26.0 and 175.5 Hz, CHO), 7.28
(2H, d, J 8.6 Hz, H-20 and H-60), 6.87 (2H, d, J 8.7 Hz, H-30 and H-50), 4.51 (2H,
d, J 4.0 Hz, pmb-OCH2), 4.05 (2H, dd, J 4.0 and 141.0, CH2), 3.79 (3H, s,
OCH3).
4.7.18. 4-(4-Methoxybenzyloxy)[1,2,3,4-13C]but-2-enoic acid methyl ester (9e)
1H NMR (500 MHz, CDCl3) d 7.19 (2H, d, J 8.6 Hz, H-20 and H-60), 7.05 (1H, m,
H-2trans), 6.83 (2H, d, J 8.7Hz, H-30 and H-50), 6.72 (1H, m, H-2cis), 6.22 (1H, m, H-
3
trans), 5.88 (1H, m, H-3cis), 4.43 (2H, d, J 4.3 Hz, pmb-OCH2), 4.21 (2H, m, CH2),
3.74 (3H, s, OCH3), 3.69 (3H, d, J 4.0 Hz, COOCH3).
4.7.19. 4-(4-Methoxybenzyloxy)butanoic-[1,2,3,4-13C] acid methyl ester (10e)
1H NMR (500 MHz, CDCl3) d 7.18 (2H, d, J 8.6 Hz, H-20 and H-60), 6.81 (2H, d,
J 8.7 Hz, H-30 and H-50), 4.37 (2H, d, J 4.0, pmb-OCH2), 3.76 (3H, s, OCH3), 3.59
(3H, dd, J 4.0 and 10.1 Hz, COOCH3), 3.41 (2H, m, 2· H-2), 2.47 (2H, t, J 7.3 Hz,
2· H-4), 1.72 (2H, m, 2· H-3).

66
U. Na¨ser et al. / Bioorganic Chemistry 33 (2005) 53–66
4.7.20. c-[2,3,4,513C]Butyrolactone (11e)
1H NMR (500 MHz, CDCl3) d 4.28 (2H, dm, J 150.0 Hz, 2· H-5), 2.41 (2H, m, 2·
H-3), 2.23 (2H, dm, J 255.0 Hz, 2· H-4). 13C NMR (125.75 MHz, CDCl3) d 177.77
(dd, J 4.0 and 50.0 Hz, 13C-2), 68.47 (dt, J 4.0 and 31 Hz, 13C-5), 28.02 (ddd, J 2.0,
33.0 and 50.0 Hz 13C-3), 22.49 (t, J 31.0 Hz, 13C-4).
Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft and the Euro-
pean Commission (Contract No HPRN-CT-2002-00195). We thank Professor Rolf
K Thauer for access to the EPR spectrometer in the Max-Planck-Institut fur terres-
¨
trische Mikrobiologie, Karl-von-Frisch-Straße, Marburg, Germany.
References
[1] J.K. Hardman, T.C. Stadtman, J. Bacteriol. 79 (1960) 544.
[2] J.K. Hardman, T.C. Stadtman, J. Biol. Chem. 238 (1963) 2081.
[3] P. Willadsen, W. Buckel, FEMS Microbiol. Lett. 70 (1990) 187.
[4] U. Scherf, W. Buckel, Appl. Environ. Microbiol. 57 (1991) 2699.
[5] U. Scherf, W. Buckel, Eur. J. Biochem. 215 (1993) 421.
[6] U. Muh, I. C¸ inkaya, S.P.J. Albracht, W. Buckel, Biochemistry 35 (1996) 11710.
¨
[7] B.M. Martins, H. Dobbek, I. C¸ inkaya, W. Buckel, A. Messerschmidt, submitted to Proc. Natl. Acad.
Sci. USA 2004 (in press).
[8] T.L. Amyes, J.P. Richard, J. Am. Chem. Soc. 114 (1992) 10297.
[9] W. Buckel, FEBS Lett. 389 (1996) 20.
[10] W. Buckel, B.T. Golding, FEMS Microbiol. Rev. 22 (1999) 523.
[11] R. Scott, U. Na¨ser, P. Friedrich, T. Selmer, W. Buckel, B.T. Golding, Chem. Commun. 10 (2004)
1210.
[12] D.M. Smith, W. Buckel, H. Zipse, Angew. Chem. Int. Edn. Engl. 42 (2003) 1867.
[13] I. C¸ inkaya, W. Buckel, M. Medina, C. Gomez-Moreno, R. Cammack, Biol. Chem. 378 (1997) 843.
[14] B.S. Furniss, A.J. Hannaford, V. Rogers, P.W.G. Smith, A.R. Tatchell, VogelÕs Textbook of
Practical Organic Chemistry, fourth ed., Longman, London and New York, 1978, 291.
[15] B.T. Golding, L. Cottrell, D. Mackay, D. Zhang, W.P. Watson, Chem. Res. Toxicol. 16 (2003) 933.
[16] B. Lal, A.K. Gangopadhyay, R. Rajagopalan, A.V. Ghate, Bioorg. Med. Chem. 6 (1998) 2061.
[17] D.B. Dess, J.C. Martin, J. Org. Chem. 48 (1983) 4155.
[18] D.B. Dess, J.C. Martin, J. Am. Chem. Soc. 113 (1991) 7277.
[19] M. Bennet, G.B. Gill, G. Pattenden, A.J. Shuker, A. Stapleton, J. Chem. Perkin Trans. 1 (1991) 929–
937.
[20] T.W. Green, P.G.M. Wuts, Protective Groups in Organic Synthesis, John Wiley, New York, 1999.
[21] R.C. Beavis, T. Chaughary, B.T. Chait, Org. Mass. Spectrom. 27 (1992) 156.