Synthesis and spectroscopic characterization of [5-13C]- and [6-13C]ubiquinone-10 for studies of bacterial photosynthetic reaction centers

FULL PAPER

Synthesis and Spectroscopic Characterization of [5-¹³C]- and [6-¹³C]-

Ubiquinone-10 for Studies of Bacterial Photosynthetic Reaction Centers

Rutger B. Boers,^[a]Peter Gast,^[b]Arnold J. Hoff,^[b]Huub J. M. de Groot,^[a]and

Johan Lugtenburg*^[a]

Keywords: Natural products / Photosynthesis / Bioorganic chemistry / Isotopic labeling

This paper presents the synthesis and characterization by

into the Q_A-site of the photosynthetic reaction center of Rho-

mass spectrometry and NMR spectroscopy of [2-¹³C]- and [3- dobacter sphaeroides R-26. Magic angle spinning NMR sub-

¹³C]ubiquinone-0 and of [5-¹³C]- and [6-¹³C]ubiquinone-10.

sequently revealed an unperturbed 6-position, while the sig-

A scheme based on the synthetic approach to [5-¹³C]ubiqui- nal of the 5-position was absent. These results corroborate

none-10 has been worked out for the synthesis of ubiqui- the recently reported detection of an asymmetric binding of

nones ¹³C-labeled at any individual position and at every Q_Awith a dynamic perturbation involving the 4-carbonyl

combination of positions in the quinone ring. The [5-¹³C]-

and [6-¹³C]ubiquinone-10 isotopomers were incorporated

functionality.

Introduction

The ubiquinone in the Q_A-site acts as a one-electron gate

only and is tightly bound to the protein. It takes up an

electron derived from the photoexcited special pair, to form

a Q_A-radical anion species. This electron is subsequently

transferred through the Fe^2ϩion to the ubiquinone at the

Q_B-site. In a second photochemically driven step, the Q_B-

ubiquinone acquires another electron and in the process

takes up two protons from the cytoplasm. It then leaves

the reaction center as ubiquinol and is replaced by a new

ubiquinone from the quinone pool in the membrane.^[1]

Thus, the photon energy is converted into chemical en-

ergy by the reduction of ubiquinone into ubiquinol and, at

the same time, the production of a proton gradient across

the plasma membrane. This proton gradient drives the con-

version of ADP into ATP, which is the universal energy

carrier in living organisms.

Ubiquinone-10 (2-decaprenyl-5,6-dimethoxy-3-methyl-

benzoquinone, see Figure 1; decaprenyl ϭ 3,7,11,15,19,

23,27,31,35,39-decamethyl-2,6,10,14,18,22,26,30,34,38-tetra

contadecaenyl) plays an important role in redox chemistry

and electron transport in the living world. It is an essential

cofactor in photosynthesis and in respiration.

Figure 1. Structure and IUPAC numbering of ubiquinone-10 (left)

and ubiquinone-0 (right); note the difference in the numbering in

the quinone ring

Through the use of 1-, 2-, 3-, 3Ј-, and 4-¹³C-labeled

ubiquinone-10 and (¹³CH₃O)₂-ubiquinone-10, obtained by

total synthesis,^[2]a strongly asymmetric binding of Q_Awas

discovered. The MAS ¹³C NMR signal intensity of the 4-

carbonyl carbon atom showed a pronounced temperature

dependence, which provided the first MAS NMR evidence

for dynamic behavior by Q_A.^[3]In contrast, the NMR re-

sponses for the 1-, 2-, 3-, and 3Ј-¹³C-labeled positions indic-

ated that this part of the ubiquinone is essentially immobile

in the binding pocket.

With the aid of FTIR spectroscopy, a dramatic bond or-

der reduction of ca. 10% was found for the 4-carbonyl

bond,^[4,5]which provides additional evidence for perturba-

tion of this side of the Q_A-quinone by the protein.^[6]In the

charge-separated P^·ϩQ_A^·Ϫstate, in contrast, the reduced Q_A

forms its strongest hydrogen bond at the 4-carbonyl posi-

The photosynthetic reaction center membrane protein

complex of Rhodobacter sphaeroides R-26 makes use of two

ubiquinone-10 cofactors for the conversion of light energy

into chemical energy.^[1]These are located in their respective

Q_A- and Q_B-sites at the cytoplasmic side of the reaction

center complex. They are part of two cofactor branches (A,

B) comprising two bacteriochlorophyll units forming the

special pair, two accessory bacteriochlorophyll units, two

bacteriopheophytin units, the two ubiquinone units, and an

Fe^2ϩion. Despite near twofold symmetry in the A and B

branch, the two ubiquinone units have different functions.

[a]

Leiden Institute of Chemistry, Gorlaeus Laboratory, Leiden

University,

P. O. Box 9502, 2300 RA Leiden, The Netherlands

Department of Biophysics, Huygens Laboratory,

[b]

P. O. Box 9504, 2300 RA Leiden, The Netherlands

Eur. J. Org. Chem. 2002, 189Ϫ202

WILEY-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002

1434Ϫ193X/02/0101Ϫ0189 $ 17.50ϩ.50/0

189

R. B. Boers, P. Gast, A. J. Hoff, H. J. M. de Groot, J. Lugtenburg

The first phase in this synthesis of ¹³C-labeled ubiqui-

FULL PAPER

tion, while the 1-carbonyl group is thought to be weakly

bonded.^[4,5,7]EPR experiments have confirmed the mobility none-10 is the preparation of a labeled ubiquinone-0, start-

detected by MAS NMR. In the reduced form there is libra- ing from a suitably and correctly ¹³C-labeled open-chain

tional motion around the axis through the two carbonyl compound. We chose a ring-closure reaction to transform

carbon atoms.^[8]Evidence for mobility of the Q_A-plastoqui- methyl 2-oxohexanoate into a 1,3-cyclohexadione struc-

none in the analogous photosystem II reaction centers in ture^[20]with all the required functionalities for the synthesis

green plants has also been found.^[9]

of ubiquinone-0.^[21Ϫ23]The second phase was the attach-

These results provide convergent evidence for dynamic ment of a polyisoprenoid tail.^[24Ϫ30]

behavior by the Q_A-ubiquinone, which may be essential for

For the synthesis of [5-¹³C]- and [6-¹³C]ubiquinones-0 we

the special function of this Q_A-quinone as a one-electron started with acid chlorides which can be converted into the

gate in the charge separation process. For instance, the dif- corresponding methyl ketones with [¹³C₂]dimethylcadmium

ferent dynamics in the ground state and the radical anion in toluene (see Schemes 1 and 3). The [¹³C₂]dimethylcad-

species may play a functional role as a molecular switch, mium can be prepared by preliminary preparation of the

slowing down the unwanted recombination of the charge- Grignard reagent from commercially available [¹³C]iodome-

separated state.

thane in ether, followed by treatment with cadmium chlor-

It is therefore essential to examine the functional mech- ide. All synthetic steps were first optimized using unla-

anisms involving the ubiquinone in the Q_A-site and to de- beled materials.

termine the as yet uninvestigated interactions of the 5- and

6-positions in the Q_A-ubiquinone with the surrounding pro-

Synthesis of [5-¹³C]Ubiquinone-10 (1a)

tein by MAS NMR, EPR, and FTIR, in order to establish

The first step in the synthesis of [5-¹³C]ubiquinone-10

whether these two positions, similarly to the 4-carbonyl

(1a) was the introduction of a methyl group by treatment

moiety, also display some special dynamic properties in the

of dimethylcadmium with methyl (4-chloroformyl)butyrate

protein.^[10Ϫ12]This determination first of all requires the

(10) (see Scheme 1). The resulting methyl ketone 9 under-

sufficient availability of specifically labeled [5-¹³C]- and [6-

went a ring-closure reaction with potassium tert-butoxide

¹³C]ubiquinone-10.

as the base^[20]to form the anion of 1,3-cyclohexanedione,

Since it was not possible to prepare these specifically [5-

which was subsequently converted into the methyl enol

¹³C]- and [6-¹³C]-labeled ubiquinones-10 efficiently by the

ether 8 with dimethyl sulfate. A selective monoalkylation to

synthetic schemes reported earlier,^[2,13]novel schemes had

give compound 7, by treatment with LDA and iodome-

to be developed. These novel schemes for the preparation

thane, afforded the complete carbon skeleton, with the label

of [5-¹³C]- and [6-¹³C]ubiquinone-10 are discussed in this

at the desired 2-position.

paper. The new synthetic scheme for [5-¹³C]ubiquinone-10

was extendable to enable the preparation of ubiquinones-10

¹³C-labeled at any single position and in all combinations

of positions in the quinone ring. This should be invaluable

for a full vibration analysis of the quinone ring system in

the near future. Finally, the first results from MAS NMR

spectroscopic investigation of the reaction centers of Rho-

dobacter sphaeroides R-26 incorporated with [5-¹³C]- and

[6-¹³C]ubiquinone-10 into the Q_A-site are presented as the

first application.

Synthesis

Most existing schemes for the synthesis of

unlabeled^[14Ϫ17]and deuterium-labeled^[18,19]ubiquinones

begin with aromatic starting materials. These cannot be

modified for the ¹³C-labeling of the ubiquinone ring at

every single position and in all combinations of positions.

Since there are no suitable commercially available ¹³C-la-

Scheme 1. Synthesis of ubiquinone-0 (a and b indicate the positions

of the ¹³C labels; 17 from Scheme 3)

beled aromatic compounds, the synthetic scheme has to

start with ¹³C-enriched compounds such as potassium cy-

anide, iodomethane, or acetic acid.

Methyl enol ether 7 was aromatized to the corresponding

During the synthesis of ¹³C-labeled compounds it is im- phenol 6 by initial treatment with LDA and chlorotrime-

portant to avoid synthetic steps in which dilution of the thylsilane to form a trimethylsilyl ether, followed by oxida-

isotopic label can take place. In addition, scrambling of the tion of this dihydrobenzene with manganese dioxide. To in-

label should be avoided by use of only fully regioselective troduce a precursor for the methoxy functionality at the

reactions. To achieve this it is necessary that all reaction 2-position, a bromination reaction was carried out with

intermediates be asymmetric.

bromine and a catalytic amount of iron. To oxidize di-

190

Eur. J. Org. Chem. 2002, 189Ϫ202

Synthesis and Characterization of [5-¹³C]- and [6-¹³C]Ubiquinone-10

FULL PAPER

bromophenol 5 to quinone 4, a novel treatment with cerium followed by thionyl chloride^[31](see Scheme 3). After ring-

ammonium nitrate (CAN) was applied, as we had found closure of methyl ketone 13, the chlorine atom was specific-

that p-brominated phenols can be specifically oxidized to p- ally introduced with chloramine-T.^[32]The enol ether 15 was

quinones in yields of around 60% with the mild CAN oxid- then formed with trimethyl orthoformate and a catalytic

ant. Quinone 4 has all the functionalities necessary for the amount of HCl. This provided the complete carbon skel-

synthesis of ubiquinone-0 (2).

eton and all the functionalities to make it a suitable starting

To exchange the bromine of quinone 4 for a methoxy compound for the synthesis of ubiquinone-0 (2b).

group, it was necessary to perform a sequence of reac-

tions.^[2]This started with a DielsϪAlder reaction with

freshly distilled cyclopentadiene, and a subsequent 1,4-addi-

tion/elimination reaction with sodium methoxide to provide

adduct 3. The cyclopentadiene was then removed again by

means of a retro-DielsϪAlder reaction in refluxing toluene,

accelerated by addition of an excess of maleic anhydride

to trap the released cyclopentadiene. Ubiquinone-0 (2) was

subsequently purified on silica.

Tributyl(decaprenyl)tin (28) was prepared from solane-

sol.^[28]This 9-isoprenoid alcohol can be lengthened by one

isoprene unit in a five-step reaction sequence.^[2]The thus

synthesized decaprenol (26) was transformed into the allylic

bromide 27 with phosphorus tribromide (see Scheme 2), fol-

lowed by treatment at low temperature with the anion of

tributyltin hydride.^[29]

Scheme 3. Synthesis towards [6-¹³C]ubiquinone-10 (b indicates the

position of the ¹³C-label)

The aromatization was once more performed in two

steps. After formation of the silyl enol ether, it was now

oxidized with iodine in a one-pot procedure.^[33]Phenol 16

was subsequently oxidized by treatment with Fremy’s salt^[21]

to quinone 17, which was converted into ubiquinone-10 (1)

by the same sequence as described for the [5-¹³C]ubiqui-

none-10 (see Schemes 1 and 2).

The synthesis described above was used to synthesize [2-

¹³C]ubiquinone-0 (2b, 0.43 g), starting with [¹³C₂]dimethyl-

cadmium (made from 10.0 g of [¹³C]iodomethane). This

was subsequently transformed into [6-¹³C]ubiquinone-10

(1b, 0.49 g) in an overall yield of 8% (based on ¹³CH₃I).

After final purification on silica, [2-¹³C]ubiquinone-0 (2b,

0.23 g) was recovered.

Scheme 2. Synthesis of ubiquinone-10 (a and b indicate the posi-

tions of the ¹³C labels)

The conversion of ubiquinone-0 (2) into ubiquinone-10

(1) was carried out by means of a boron trifluorideϪdiethyl

ether catalyzed 1,4-addition between the ubiquinone-0 (2)

and tributyl(decaprenyl)tin (28).^[30]Silver oxide was added

to the reaction mixture to oxidize the initially formed prod-

uct (ubiquinol-10) to ubiquinone-10 (1). Any unchanged la-

beled starting material could easily be recovered. The

ubiquinone-10 was finally purified on silica.

Synthesis of Specifically Labeled Ubiquinones

We realized that 3-methoxy-6-methylcyclohex-2-enone 7

(see Scheme 1) was the key molecule for developing a syn-

The procedure described above was used to synthesize [3- thetic scheme to synthesize ubiquinones ¹³C-labeled in all

¹³C]ubiquinone-0 (2a, 0.21 g), starting with [¹³C₂]dimethyl- single positions and in all combinations of positions in their

cadmium (prepared from 10.0 g of [¹³C]iodomethane). This ring systems. It is nonsymmetric, has the complete carbon

was subsequently transformed into [5-¹³C]ubiquinone-10 skeleton, and has all the needed functionalities present. The

(1a, 0.37 g) in an overall yield of 2% (based on ¹³CH₃I). synthesis of this hexene 7 is based on a 1,4-addition be-

In the final purification on silica, [3-¹³C]ubiquinone-0 (2a, tween ethyl 2-methylacetoacetate (18) and ethyl acrylate

0.12 g) was recovered.

(19) (see Scheme 4).

Synthesis of [6-¹³C]Ubiquinone-10 (1b)

For the preparation of [6-¹³C]ubiquinone-10 (1b) we were

able to use the synthetic scheme developed for the synthesis

of [5-¹³C]ubiquinone-10 (1a), with only minor modifica-

tions. The label was now introduced with [¹³C₂]dimethyl-

cadmium by treatment with (4-chloroformyl)-3-methylbu-

tyrate (12), which could be synthesized from 3-methylglut-

aric anhydride (11) by treatment with 1 equiv. of methanol Scheme 4. Synthesis of 4-methyl-1,3-cyclohexanedione

Eur. J. Org. Chem. 2002, 189Ϫ202

191

R. B. Boers, P. Gast, A. J. Hoff, H. J. M. de Groot, J. Lugtenburg

FULL PAPER

Ester 18 can be prepared from ethyl bromoacetate and corporation). The lower value was probably due to addi-

acetonitrile by a Blaise reaction,^[34]followed by subsequent tional reduction of the sample in the MS apparatus.^[43,44]

monoalkylation with iodomethane^[35]in high yield. Ethyl These high incorporation figures confirm that no dilution

acrylate (19) has been prepared from ethyl bromoacetate of the label had taken place during the synthesis of labeled

and formaldehyde.^[36]Since ethyl bromoacetate, iodome- ubiquinone-10.

thane, and formaldehyde are all commercially available in

Examination of the mass spectroscopic fragmentation

all ¹³C-enriched forms, all single and all combinations of pattern of [2-¹³C]- and [3-¹³C]ubiquinone-0 showed some

¹³C-labeled positions of our two starting materials 18 and new insight in its fragmentation. Together with the specific-

19 are accessible.

ally ¹³C-labeled ubiquinones-0 from our earlier work,^[2,38]

These two starting materials underwent a 1,4-addition the fragmentation to fragments of m/z ϭ 83/84 could be

with a catalytic amount of sodium in methanol. The pro- worked out unequivocally (see Table 2 and Scheme 5).

duced diester 20 was converted into the monoacid in one-

pot fashion by saponification and decarboxylation. After

Table 2. Fragments (m/z) from unlabeled [2-¹³C]- and [3-¹³C]ubi-

quinone-0 and of all other single labeled ubiquinones-0

subsequent reesterification in methanol, by use of a cata-

lytic amount of p-toluenesulfonic acid, methyl 4-methyl-5-

oxohexanoate (21) underwent the ring-closure reaction to

give the cyclohexanedione 22. Subsequent synthesis of the

enol ethers 7 and 25, which could be separated on silica in

relative quantities of 3:2, brought us back to the synthetic

route to [5-¹³C]ubiquinone-10 (see Scheme 1). The overall

yield of enol ether 7 was 13% [from ethyl 2-methylacetoacet-

ate (18)]. To increase this yield, enol ether 25 could be sub-

jected to transetherification to provide enol ether 7.

Label position Fragment number

I

II

III

IV

V

VI

VII

unlabeled

1

2

3

182 167 153 137 111

96

83

83/84

83

83/84

84

183 168 154 138 111/112 97

183 168 154 138 111/112 96

183 168 154 138 111/112 97

183 168 154 138 112

184 168 154 137 112

4

5

6

5Ј

97

96

Spectroscopic Characterization

(¹³CH₃O)₂

Mass Spectrometry

To confirm the identity and the degree of ¹³C enrichment

of [2-¹³C]- and [3-¹³C]ubiquinone-0 and [5-¹³C]- and [6-

¹³C]ubiquinone-10, mass spectrometry was performed. The

exact masses of these four compounds, obtained by double

focus mass spectrometry, were the same (within experi-

mental error) as the calculated values. This confirmed their

molecular formulas (see Table 1).

To calculate the degree of ¹³C incorporation, a compar-

ison was made between the pattern of signals around the

M^ϩsignal of natural abundance with the pattern of the

synthesized labeled compounds, which should be shifted

upwards by one mass unit.^[37]All incorporations found in

this way were close to the expected value of 99% (see

Table 1). The value for [2-¹³C]ubiquinone-0 (2b) was found

to be slightly lower. However, we can safely assume that the

degree of incorporation in 2b was just as high as that in 1b,

since [6-¹³C]ubiquinone-10 (1b; 99.6% incorporation) had

been synthesized from [2-¹³C]ubiquinone-0 (2b; 94.7% in-

Scheme 5. Mass spectroscopic fragmentation of ubiquinone-0

Table 1. Elemental compositions of the ubiquinones, their experimental and calculated exact masses and ¹³C incorporation

Compound

Mol. formula

Exact mass (u)

¹³C-label

incorporation

found

calculated

ubiquinone-0 (2)

¹²C₉H₁₀O₄

182.0575

183.0626

183.0637

862.6863

863.6914

863.6871

182.0579

183.0621

862.6839

863.6881

Ϫ

[2-¹³C]ubiquinone-0 (2b)

[3-¹³C]ubiquinone-0 (2a)

ubiquinone-10 (1)

¹³C₁¹²C₈H₁₀O₄

94.7%

98.5%

Ϫ

99.6%

98.8%

¹³C₁¹²C₈H₁₀O₄

12

C H₉₀O₄

59

[5-¹³C]ubiquinone-10 (1a)

[6-¹³C]ubiquinone-10 (1b)

¹³C₁₁₂C₅₈H₉₀O₄

12

¹³C₁C₅₈H₉₀O₄

192

Eur. J. Org. Chem. 2002, 189Ϫ202

Synthesis and Characterization of [5-¹³C]- and [6-¹³C]Ubiquinone-10

FULL PAPER

The M^ϩsignal common to the two compounds can be Hz) can only be due to coupling between the ¹³C label and

found at m/z ϭ 183 (I), whereas the unlabeled quinone is the protons of the methoxy group directly attached to this

found at 182 (I) (see Table 2). Both [2-¹³C]- and [3-¹³C]- label. This allows the signals from the two methoxy groups

ubiquinone-0 show their first two fragments (see Scheme 5) to be assigned unequivocally. The signals at δ ϭ 4.02 and

at m/z ϭ 168 (II) and 154 (III), still containing the label. 4.00 can be assigned to the methoxy groups attached to C²

These fragments have lost either a methyl group or a formyl and to C³, respectively.

group from one of the methoxy groups. The next fragment

In the proton spectra of the two ¹³C-labeled ubiquinones-

IV at m/z ϭ 138 also contains the ¹³C label for both labeled 10, additional J_CϪHcoupling (of 3.5 Hz) with the closest

ubiquinones-0. It can be formed from fragment II by sub- methoxy group signal is observed (see Figure 3). By the

sequent loss of a formaldehyde molecule from the re- same reasoning, the signals at δ ϭ 3.98 and 3.99 can be

maining methoxy group. For the fragments V of both [2- assigned to the methoxy groups at C⁵and C⁶, respectively.

¹³C]- and [3-¹³C]ubiquinone-0 a labeled and an unlabeled

3

signal are simultaneous found at m/z ϭ 111 and 112, due

to the loss either of the C¹C²carbon atoms or of the C⁴C³

carbon atoms in an OϭCϭCϭOCH₃molecule.^[39]Frag-

ment VI at m/z ϭ 96 no longer contains a label. This frag-

ment can therefore only be attributed to the loss of an

H₃COCCOCH₃molecule, containing the C²C³carbon

atoms. The last fragment VII at m/z ϭ 83 also does not

contain the C²C³carbon atoms. Earlier experiments^[38]had

revealed that it still contains the carbonyl carbon atom and

the methoxy carbon atom from fragment V and so must be

formed by a methyl shift from the methoxy group to the

carbonyl oxygen atom, followed by expulsion of the newly

formed carbonyl group. The result is the cyclopropenyl cat-

ion VII.

The mass spectra of [5-¹³C]- and [6-¹³C]ubiquinones-10

are governed by two relatively stable fragments. A bicyclic

ϩ

oxonium ion ¹³C¹²C₁₂H₁₅O₄at m/z ϭ 236 and a

ϩ

¹³C¹²C₉H₁₃O₄fragment at m/z ϭ 198 are observed (see

Figure 2), both of which still contain the labels. The frag-

mentation pattern of ubiquinone-10 clearly differs from that

of ubiquinone-0.

1

Figure 3. 600 MHz H NMR spectra of the methoxy signals in [5-

¹³C]- (1a), [6-¹³C]- (1b), and natural-abundance (1) ubiquinone-10

¹³C NMR Spectroscopy

To verify the structures of the synthesized ubiquinones

1

and the positions of the ¹³C labels, we recorded H noise-

decoupled 75 MHz ¹³C NMR spectra of [2-¹³C]- and [3-

Figure 2. Structures of two stable fragments occurring in the mass

¹³C]ubiquinone-0 and ¹H noise-decoupled 150 MHz ¹³C

spectrum of ubiquinone-10

NMR spectra of [5-¹³C]- and [6-¹³C]ubiquinone-10. The

spectra of the four labeled ubiquinones all show only one

strong signal, at δ ϭ 144.2 and 144.3 for the [5-¹³C]- and

[6-¹³C]ubiquinone-10, respectively and at δ ϭ 144.7 and

144.9 for the [2-¹³C]- and [3-¹³C]ubiquinone-0, respectively.

¹H NMR Spectroscopy

To confirm the structure of the synthesized ubiquinones

and the position of the ¹³C labels, 300 MHz ¹H NMR spec-

tra of [2-¹³C]- and [3-¹³C]ubiquinone-0 and 600 MHz H

1

Table 3. ¹³C-¹³C coupling constants [Hz] in ubiquinone-0

NMR spectra of [5-¹³C]- and [6-¹³C]ubiquinone-10 were re-

corded. All spectra are identical to the spectra of ubiqui-

none-0 and ubiquinone-10 reported in the literature,^[15]ex-

cept for the additional couplings due to the ¹³C isotopes.

This confirms the structures of our synthesized products.

The proton spectra of the [2-¹³C]- and [3-¹³C]ubiqui-

nones-0, for example, show additional coupling for one of

the methoxy groups. Because it is known that three-bond

coupling constants are larger than four-bond coupling con-

Carbon 2

atom

3

4

5

6

5Ј-CH₃2-OCH₃3-OCH₃

1

2

3

4

5

6

58.4 9.8 Ͻ 1.5 8.2

54.6 6.3

ϫ

9.9 Ͻ 1.5 17.3 Ͻ 1.5 8.0

2.8

7.8

59.7 16.7 3.0 1.7

49.9 9.7 Ͻ 1.5

64.5 44.7

2.6

Ͻ 1.5

3

stants,^[40]the J_CϪHcoupling constants (of 3.4 Hz and 3.5

Eur. J. Org. Chem. 2002, 189Ϫ202

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R. B. Boers, P. Gast, A. J. Hoff, H. J. M. de Groot, J. Lugtenburg

FULL PAPER

Table 4. ¹³C-¹³C coupling constants [Hz] in ubiquinone-10

Carbon atom

1

2

3

4

5

6

3Ј-CH₃

5Ј-OCH₃

6Ј-OCH₃

1

2

3

4

51.9

2.8

73.0

3.9

2.4

51.5

9.9

3.0

16.2

59.1

58.9

15.9

3.0

9.9

ϫ

4.4

Ͻ 1.5

45.5

1.5

5

1.6

2.02

ca. 1.5

4.45

6

Ͻ 1.5

2.5

tail, 1-CH₂

tail, 2-CH

tail, 3-C

Ͻ 1.5

44.7

4.1

3.4

Ͻ 1.5

1.6

This proves that only one position is labeled and that no times were performed at various temperatures down to

scrambling has occurred during the synthesis. The signals 180 K. However, no ¹³C-label signal was detected in the

of the remaining natural-abundance carbon atoms of the MAS NMR spectrum (see Figure 4, a and b).

ubiquinones became visible when spectra with a higher sig-

nal-to-noise ratio were obtained. The additional ¹³C-¹³C

coupling constants observed in these signals were used to

confirm the location of the labels (see Tables 3 and 4). In

the spectrum of [3-¹³C]ubiquinone-0, a typical one-bond

coupling constant of 59.7 Hz can be found, for example,

between the (labeled) carbon-3 and the 4-carbonyl carbon

atom. This confirms that the ¹³C labels are in the desired

positions and provides an unambiguous assignment of

these signals.

In fact, it is now possible for the first time to make an

almost complete J-coupling assessment for ubiquinone-0

(see Table 3)^[2,38]and ubiquinone-10 (see Table 4).^[2,38]Only

the one-bond coupling constants between carbon atoms 2

and 3 in ubiquinone-0 and carbon atoms 5 and 6 in ubiqui-

none-10 are missing, due to the higher-order effects found

for these carbon atoms.

Solid-State ¹³C-NMR Reaction Center Measurements

The [5-¹³C]- and [6-¹³C]ubiquinone-10 were incorporated

into the Q_A-sites of purified Rhodobacter sphaeroides R-26

Figure 4. Solid-state NMR spectra of [5-¹³C]- (a), [6-¹³C]- (c), and

natural-abundance (e) ubiquinone-10 incorporated into the Q_A-site

of Rhodobacter sphaeroides R-26; the difference spectra of the two

labeled spectra (a) and (b) with the natural-abundance spectrum

(e) are depicted in spectra (b) and (d); the signal of the 6-carbon

atom is marked by an asterisk

reaction centers by known procedures.^[3,38,41]Subsequent

charge-recombination measurements^[41]revealed incorpora-

tion of the Q_A-site with the labeled ubiquinones-10 of

around 90%. With these reconstituted samples, 100 MHz

cross-polarization ¹³C MAS NMR spectra were recorded

with a Bruker MSL 400 NMR spectrometer. In addition,

difference spectra between the natural abundance protein

(see Figure 4, e) and these ¹³C-incorporated reaction centers

were measured.

The NMR spectroscopic data collected for the reaction

center preparation with the [6-¹³C]ubiquinone-10 show a

signal at δ ϭ 147.5 for the labeled carbon atom (see Fig-

ure 4, c and d). This is a shift of 3.2 ppm compared to the

value of δ ϭ 144.3 of free [6-¹³C]ubiquinone-10. This is

comparable with the relative shifts found for the other car-

bon signals of the ubiquinone ring (see Figure 5).^[3]The sig-

nal intensity and shift are essentially independent of tem-

Figure 5. The chemical shift difference of ubiquinone-10 in the Q_A-

site, compared with ubiquinone-10, dissolved in deuteriochloro-

form^[3]

The difference spectrum of the [6-¹³C]ubiquinone recon-

perature in the range from 210 K to 250 K. For the reaction stituted reaction center protein confirms that the observed

center preparation with the [5-¹³C]ubiquinone-10, measure- signal does not originate from the protein itself (see Fig-

ments with different cross polarizations and relaxation ure 4, d). Similarly, the difference spectrum of the [5-¹³C]-

194

Eur. J. Org. Chem. 2002, 189Ϫ202

Synthesis and Characterization of [5-¹³C]- and [6-¹³C]Ubiquinone-10

FULL PAPER

ubiquinone-10 reconstituted reaction center confirms that signals. The ¹³C enrichment was therefore calculated by

there is no signal from the [5-¹³C]ubiquinone-10 superim- comparison of labeled and unlabeled spectra taken immedi-

posed under one of the protein signals (see Figure 4, b). ately after insertion of the sample.

1

The signals at δ Ͻ 40 observed in these difference spectra

The comparison of the H NMR spectra of ubiquinone-

originate from the detergents used in purifying the protein. 0 and ubiquinone-10 revealed a negligible influence of the

isoprenoid tail on the chemical shift of the ubiquinone ring

protons. The ring methyl group had only shifted downfield

Discussion

by 0.03 ppm and the methoxy signals had shifted upfield by

0.02 and 0.03 ppm after attachment of the isoprenoid tail.

In the carbon spectra, the differences were more pro-

Synthesis and Characterization

The 13-step syntheses described in this paper gives access nounced. The carbon-2 signal had shifted upfield by

to [5-¹³C]- and [6-¹³C]ubiquinone-10 in relatively high over- 10.4 ppm upon attachment of the isoprenoid tail, whereas

all yields of 2 and 8%, respectively, with levels of ¹³C in- the signals of carbon-3 and the 3Ј-methyl group had shifted

corporation above 98% and without isotope scrambling. downfield by 5.1 and 3.5 ppm, respectively. Both carbonyl

This new convergent scheme also makes it possible to label carbon signals showed small upfield shifts (Ͻ 0.4 ppm), the

each of the two methoxy groups separately, together with carbon signals at the methoxy side showed small downfield

the first five positions in the isoprenoid tail.^[2,42]

shifts (Ͻ 0.7 ppm), and the signals of the two methoxy car-

Our earlier work on labeled ubiquinones had an overall bon atoms had negligible shifts (Ͻ 0.1 ppm). From these

yield of nearly 1%.^[2]Its key synthetic step was a nonre- shifts it can be concluded that most of the electron-donat-

gioselective DielsϪAlder reaction between trichloroethylene ing properties of the decaprenyl tail are found at carbon-3.

and 3-methyl-2,5-bis(trimethylsilyloxy)furan (synthesized

Reaction Center Measurements

from 2-methylglutaric anhydride). This DielsϪAlder reac-

tion is unsuitable to introduce the 5- and 6-label with satis-

factory yields. A synthetic scheme has been reported for

the synthesis of [5-¹³C]- and [6-¹³C]ubiquinone-3, where a

similar reaction with a low yield of 0.01% was used.^[13]

The silyl ether from ketone 15 is even more acid-sensitive

then that prepared from ketone 7, probably due to the lack

of the α-methyl group. Oxidation of this silyl ether therefore

had to be carried out with iodine in a one-pot fashion. This

unfortunately gave a mixture of phenol 16 and 3-methoxy-

5-methylphenol, levels of which could be reduced by in-

creasing the reaction temperature and by exclusion of oxy-

gen, which was found to oxidize only to 3-methoxy-5-

methylphenol.

Treatment of the DielsϪAlder adduct of quinone 4a with

methoxide produced, together with the dimethoxy

DielsϪAlder adduct 3a, a small amount of the DielsϪAlder

adduct of cyclopentadiene and 3-methoxy-6-methyl-para-

benzoquinone. This by-product was not found in the similar

substitution reaction of the chlorinated DielsϪAlder ad-

duct made from quinone 17.

Another improvement to our previously published

work^[2]is the procedure for attaching the isoprenoid tail.

DielsϪAlder adduct 3 was previously deprotonated by pot-

assium tert-butoxide to react with an isoprenyl bromide.

This anion proved to be unstable, giving only low yields

(25Ϫ35%) in small-scale reactions. With the boron

trifluorideϪdiethyl ether catalyzed 1,4-addition of tributyl-

(decaprenyl)tin (28) to ubiquinone-0 (2), no ¹³C-enriched

product was lost. Unreacted starting material could be re-

covered and reused, to double the overall yields.

It is generally accepted that the binding of the Q_A-ubiqui-

none is asymmetric both for the neutral and for the charge-

separated states, and that this asymmetric binding is in-

duced by the protein.^[3Ϫ8,45]In earlier MAS NMR studies,

carbon positions 1, 2, 3, and 3Ј gave clear signals.^[3,38]The

4-carbonyl carbon atom gave a temperature-dependent sig-

nal, the intensity of which decreased with increasing tem-

perature and completely disappeared above 250 K. Now,

after measurement of carbon positions 5 and 6, this picture

can be completed. The 6-position gave a clear signal and

the 5-position was not observed, even down to 180 K.

These results corroborate the detection of asymmetric bind-

ing of the Q_A-quinone with the recently reported dynamic

perturbation involving the 4-carbonyl functionality.^[3]The

Q_A-quinone in the ground state apparently has relatively

stationary carbon-1, -2, -3, -3Ј, and -6 and a relatively mo-

bile carbon-4. The absence of a signal for carbon-5 is con-

sistent with dynamic behavior parallel to that of carbon-4.

The dynamic behavior of carbon-5 may be caused by a flex-

ible methoxy group attached to this carbon atom. In con-

formation analysis studies, several energy-minimized meta-

stable conformations for the two methoxy groups have been

reported,^[6,46,47]while different conformations of the me-

thoxy groups have indeed been found in various reaction

center crystal structure studies.^[1,48Ϫ50]Carbon-6 might be

restricted in its mobility by a strong hydrogen bond at the

adjacent 1-carbonyl position.^[51]This hydrogen bond was

recently confirmed by 2D heteronuclear (¹H-¹³C) dipolar

correlation NMR experiments.^[52]

It is known that mass spectra of quinones often show

strong (M ϩ 2)^ϩsignals.^[43,44]This is due to reduction of

the quinone (Q) to the quinol (QH₂) in the mass spectro-

meter. We found that the (M ϩ 2)^ϩsignal increases over

Conclusion

The syntheses of [5-¹³C]- and [6-¹³C]ubiquinone-10 and

time. By using spectra taken immediately after the sample of [2-¹³C]- and [3-¹³C]ubiquinone-0 reported here have suc-

insertion, we were able to obtain spectra without (M ϩ 2)^ϩceeded on a 100-milligram scale in reasonably high yields

Eur. J. Org. Chem. 2002, 189Ϫ202

195

R. B. Boers, P. Gast, A. J. Hoff, H. J. M. de Groot, J. Lugtenburg

FULL PAPER

flux for 45 min. The ether solvent was then evaporated under a

flow of argon and the obtained gray, sandy substance was dispersed

in toluene (100 mL) at room temperature, after which methyl (4-

chloroformyl)butyrate (10; 9.01 mL, 65.2 mmol) in toluene (10 mL)

was added dropwise. After 2 h of subsequent refluxing, the mixture

was cooled down to room temperature, and a saturated NH₄Cl

solution (30 mL) was added, followed by acidification with 1 

HCl. After separation of the water layer, which was extracted twice

with ether, the combined organic layers were washed with saturated

NaHCO₃, NH₄Cl, and NaCl solutions and dried with MgSO₄.

of 2% and 8%, respectively. In addition, a synthetic scheme

to synthesize ubiquinones ¹³C-labeled in all single positions

and combinations of positions, starting from small, com-

mercially available, ¹³C-enriched materials, has been de-

veloped. NMR spectroscopy and mass spectrometry proved

that the incorporations were effected without scrambling or

dilution of the ¹³C label.

The CP/MAS NMR of [6-¹³C]ubiquinone-10 incorpor-

ated into the Q_A-site of the photosynthetic reaction center

of Rhodobacter sphaeroides R-26 gave a clear signal, the After concentration under vacuum, the yellow liquid (8.86 g) was

distilled under vacuum (0.3 Torr). The fraction collected at 60Ϫ70

°C yielded methyl 5-oxohexanoate (9; 7.77 g) as a colorless liquid,

signal intensity and shift of which were essentially inde-

pendent of temperature. For the 5-labeled position, no sig-

nal could be found at temperatures down to 180 K. These

findings are in line with mobility involving the carbon-4

and -5 side of the ubiquinone ring in the Q_A-site.

1

in a yield of 70% with regard to iodomethane. H NMR: δ ϭ 1.9

3

(m, 2 H, center CH₂), 2.15 (s, 3 H, CH₃), 2.35 (t, J_HϪH7.6 Hz, 2

3

H, 2-CH₂), 2.53 (t, J_HϪH7.2 Hz, 2 H, 4-CH₂), 3.68 (s, 3 H,

OCH₃). ¹³C NMR: δ ϭ 19.8 (3-CH₂), 29.3 (CH₃), 32.6 (2-CH₂),

42.0 (4-CH₂), 51.0 (OCH₃), 173.1 (1-CϭO), 207.3 (5-CϭO).

Experimental Section

Methyl [6-¹³C]-5-Oxohexanoate (9a): The procedure described

above was performed with magnesium turnings (1.58 g,

65.0 mmol), [¹³C]iodomethane (10.00 g, 70.0 mmol), CdCl₂(7.82 g,

42.7 mmol), and methyl (4-chloroformyl)butyrate (10; 9.01 mL,

65.2 mmol) to yield a yellow liquid (8.86 g), which was distilled

under vacuum (0.3 Torr). The fraction collected at 60Ϫ70 °C gave

the target compound (7.27 g) as a colorless liquid, in a yield of 72%

Ether refers to diethyl ether, THF refers to tetrahydrofuran, and

PE refers to low-boiling (40Ϫ60 °C) petroleum ether. Silica gel used

for column chromatography was of 40Ϫ63 µm grade. All reactions

were performed in flame-dried glassware under dry nitrogen or ar-

gon and with dry distilled solvents except when stated otherwise.

Ether and PE were distilled from P₂O₅and stored over sodium

˚

1

with regard to [¹³C]iodomethane. ¹H NMR: δ ϭ 2.15 (d, J_CϪH

wire. THF and diisopropylamine were dried and stored over 4 A

127.4 Hz, 3 H, ¹³CH₃). ¹³C NMR: δ ϭ 29.7 (¹³CH₃).

molecular sieves. Methanol was distilled from magnesium turnings

˚

and stored over 3 A molecular sieves. tert-Butyl alcohol was dis-

˚

3-Methoxy-2-cyclohexenone (8): Potassium (1.50 g, 38.4 mmol) was

added to tert-butyl alcohol (250 mL). After all the potassium had

reacted, methyl 5-oxohexanoate (9; 5.00 g, 34.7 mmol) was added

and the mixture was stirred overnight. The tert-butyl alcohol was

evaporated under vacuum and the obtained brown solid was dis-

solved in dichloromethane (150 mL). After addition of dimethyl

sulfate (5.0 mL, 52.8 mmol), the mixture was refluxed for 6 h. After

the mixture had cooled down to room temperature, water (50 mL)

and concentrated ammonia (25 mL) were added and the mixture

was stirred for 15 min, followed by addition of concentrated HCl

(50 mL), dissolved in water (100 mL). The water layer was separ-

ated and extracted three times with dichloromethane. The com-

bined organic layers were dried with MgSO₄. After concentration

under vacuum, a crude yield of 4.37 g (100%) was obtained. ¹H

NMR: δ ϭ 2.0 (m, 2 H, 5-CH₂), 2.34 (t, ³J_HϪH6.9 Hz, 2 H, CH₂),

tilled from CaH₂and stored over 4-A molecular sieves (for distilla-

tion of tert-butyl alcohol a flow of air was used to cool the distilla-

tion cooler). Chlorotrimethylsilane was always freshly distilled from

CaH₂prior to use. Potassium was cut under in dry PE. Mass spec-

trometry was performed with a Finnigan MAT 900 (electron im-

pact, 70 eV). Most NMR spectra were recorded with a Jeol NM

FX 200. The NMR spectra of ubiquinones-0 and ubiquinone-10

were recorded with a Bruker DPX 300 instrument and a Bruker

DMX 600 instrument, respectively. All samples were dissolved in

deuteriochloroform except when noted otherwise. For ¹H NMR

spectra, either tetramethylsilane (δ ϭ 0.00) or deuteriochloroform

(δ ϭ 7.26) were used as internal standards. Deuteriochloroform

(δ ϭ 77.0) was used as internal standard for the ¹³C NMR spectra.

The chemical shifts (δ values) are given in ppm. In the NMR data

of the ¹³C-labeled compounds, only those proton signals with an

additional ¹³CϪH coupling constant, the carbon signal of the ¹³C-

labeled atom, and those carbon signals with an additional ¹³CϪ¹³C

coupling constant are given. The additional couplings found

around the ¹³C-labeled atom are omitted, since these are the same

as found in the other split carbon signals. For clarity, the num-

bering for the carbon atoms has been chosen in such a way that

every carbon atom keeps the same number throughout the syn-

theses and so often does not follow the IUPAC rules. The num-

bering of ubiquinone-0 and ubiquinone-10 does follow the

IUPAC nomenclature.

3

2.43 (t, J_HϪH5.8 Hz, 2 H, CH₂), 3.72 (s, 3 H, OCH₃), 5.37 (s, 1

H, CH). ¹³C NMR: δ ϭ 20.5 (5-CH₂), 28.1 (4-CH₂), 35.9 (6-CH₂),

54.9 (OCH₃), 101.3 (CH), 178.3 (CO), 198.9 (CϭO).

[2-¹³C]-3-Methoxy-2-cyclohexenone (8a): The procedure described

above was performed with tert-butyl alcohol (360 mL), potassium

(2.18 g, 55.8 mmol), methyl [6-¹³C]-5-oxohexanoate (9a; 7.27 g,

50.1 mmol), dichloromethane (220 mL), and dimethyl sulfate (7.27

mL, 76.8 mmol) for the reactions, water (70 mL) and concentrated

ammonia (35 mL) to destroy the dimethyl sulfate, and concentrated

HCl (75 mL) in water (145 mL) for the workup procedure. After

evaporation under vacuum, a crude yield of 5.76 g (94%) was ob-

Synthesis in the Preparation of [3-¹³C]Ubiquinone-0

1

tained. ¹H NMR: δ ϭ 5.37 (d, J_CϪH160.0 Hz, 1 H, ¹³CH). ¹³C

Methyl 5-Oxohexanoate (9): A solution of iodomethane (10.00 g,

4.39 mL, 70.5 mmol) in ether (20 mL) was added dropwise to mag-

NMR: δ ϭ 102.1 (¹³CH).

nesium turnings (1.60 g, 65.8 mmol) in refluxing ether (40 mL) in 3-Methoxy-6-methyl-2-cyclohexenone (7): At Ϫ20 °C, diisopropyl-

such a way as to maintain the ether at a gentle reflux. To make amine (6.26 mL, 44.7 mmol) was dissolved in THF (65 mL), fol-

sure no magnesium was left over, reflux was continued for about

30 min (residual magnesium seriously reduces the yields of the sub- mL, 45.8 mmol) with a syringe. After 10 min, this mixture was

sequent reactions). The Grignard mixture was cooled down to 0 cooled down to Ϫ80 °C, after which 3-methoxy-2-cyclohexenone

°C and dry CdCl₂(7.79 g, 42.5 mmol) was added, followed by re- (8; 4.37 g, 34.7 mmol) in THF (16 mL) was added dropwise. After

lowed by addition of a butyllithium solution in hexane (1.6 , 28.6

196

Eur. J. Org. Chem. 2002, 189Ϫ202

Synthesis and Characterization of [5-¹³C]- and [6-¹³C]Ubiquinone-10

10 min this was followed by dropwise addition of iodomethane

FULL PAPER

[2-¹³C]-3-methoxy-6-methyl-2-cyclohexenone (8.08 g) in undistilled

(3.06 mL, 49.2 mmol). This mixture was allowed to warm up to (wet!) ether (30 mL) and active MnO₂(10 g) to obtain a crude yield

Ϫ15 °C over about 2 h, after which a saturated NH₄Cl solution (25

of 6.26 g (109%). ¹H NMR: δ ϭ 6.36 (dd, ⁴J_HϪH2.8 Hz and ¹J_CϪH

3

4

mL) was added. The water layer was separated and extracted three 156.5 Hz, 1 H, ¹³CH), 6.44 (ddd, J_HϪH8.2, J_HϪH2.8 Hz and

times with ether. The combined organic layers were dried with

MgSO₄. After concentration under vacuum, a crude yield of 4.16 g

³J_CϪH5.2 Hz, 1 H, 4-CH). ¹³C NMR: δ ϭ 105.8 (¹³CH).

2,4-Dibromo-3-methoxy-6-methylphenol (5): Iron powder (0.22 g,

3.9 mmol) was added to 3-methoxy-6-methylphenol (6; 4.97 g,

36.0 mmol) dissolved in dichloromethane (110 mL), cooled to 0

°C. This was followed by dropwise addition of bromine (4.85 mL,

94.1 mmol) in dichloromethane (40 mL) in the dark. After this had

stirred overnight, some saturated sodium dithionite solution was

added to destroy the excess bromine and the mixture was sub-

sequently acidified with 1  HCl to pH ϭ 2Ϫ3. The water layer

3

(86%) was obtained. ¹H NMR: δ ϭ 1.15 (d, J_HϪH6.9 Hz, 3 H,

CH₃), 1.7Ϫ2.5 (several m, 5 H, 2-CH₂and 6-CH), 3.69 (s, 3 H,

OCH₃), 5.34 (s, 1 H, 2-CH). ¹³C NMR: δ ϭ 15.0 (CH₃), 27.8 (5-

CH₂), 28.9 (4-CH₂), 39.8 (6-CH), 55.3 (OCH₃), 101.2 (2-CH), 177.3

(CO), 201.3 (CϭO).

[2-¹³C]-3-Methoxy-6-methyl-2-cyclohexenone (7a): The procedure

described above was performed with diisopropylamine (8.26 mL,

58.9 mmol) in THF (85 mL), a butyllithium solution in hexane (1.6 was separated and extracted three times with dichloromethane. The

, 37.7 mL, 60.3 mmol), [2-¹³C]-3-methoxy-2-cyclohexenone (8a;

combined organic layers were dried with MgSO₄and the solvents

were evaporated under vacuum. This resulted in a crude yield of

5.76 g, 45.4 mmol) in THF (20 mL), and iodomethane (4.03 mL,

64.7 mmol) for the reactions and saturated NH₄Cl solution (25 mL) 8.06 g [73% based on 3-methoxy-6-methyl-2-cyclohexenone (7)]. ¹H

for the workup procedure. After concentration under vacuum, a NMR: δ ϭ 2.21 (s, 3 H, CH₃), 3.82 (s, 3 H, OCH₃), 5.7 (br. s, 1

1

crude yield of 5.80 g (91%) was obtained. H NMR: δ ϭ 5.34 (d,

H, OH), 7.23 (s, 1 H, CH). ¹³C NMR: δ ϭ 16.0 (CH₃), 60.6

(OCH₃), 106.4 (2-CBr), 106.8 (4-CBr), 131.7 (C), 132.9 (5-CH),

150.7 (COH), 151.8 (CO).

¹J_CϪH159.3 Hz, 1 H, ¹³CH). ¹³C NMR: δ ϭ 101.5 (¹³CH).

Trimethylsilyl Enol Ether of 3-Methoxy-6-methyl-2-cyclohexenone:

At Ϫ15 °C, diisopropylamine (4.66 mL, 33.2 mmol) was dissolved [2-¹³C]-2,4-Dibromo-3-methoxy-6-methylphenol (5a): The procedure

in THF (43 mL), followed by addition (by syringe) of a butylli-

described above was performed with 3-methoxy-6-methylphenol

thium solution in hexane (1.6 , 18.7 mL, 29.9 mmol). After 10 (6a, 6.26 g, 45.0 mmol) in dichloromethane (110 mL), iron powder

min, this was followed by dropwise addition of 3-methoxy-6-

methyl-2-cyclohexenone (7; 4.16 g, 29.7 mmol), dissolved in THF

(17 mL). After 5 min, this was followed by addition of chlorotrime-

(0.28 g, 5.0 mmol), and bromine (4.85 mL, 94.1 mmol) in dichloro-

methane (40 mL) to obtain a crude yield of 8.06 g {66% based on

[2-¹³C]-3-methoxy-6-methyl-2-cyclohexenone (7a)}. ¹³C NMR: δ ϭ

thylsilane (8.31 mL, 65.5 mmol). This mixture was allowed to warm 106.4 (¹³CBr).

to room temperature over 1 h, after which the reaction mixture was

2-Bromo-3-methoxy-6-methylparabenzoquinone (4): 2,4-Dibromo-3-

poured into a quickly stirred saturated Na₂CO₃solution, followed

by fast separation in a separating funnel and immediate drying of

the organic layer with MgSO₄. The water layer was quickly ex-

tracted once with ether and the organic phase was combined with

the first MgSO₄-containing batch to dry. After concentration under

vacuum, a crude yield of 7.22 g (115%) was obtained and immedi-

methoxy-6-methylphenol (5; 6.46 g, 21.8 mmol) and 2,6-pyridined-

icarboxylic acid (20.5 g, 123 mmol) were dissolved in a mixture of

water (205 mL) and acetonitrile (205 mL). This mixture was cooled

to

0 °C, after which ammonium cerium(IV) nitrate (51 g,

93.0 mmol) was added in 5-g portions in 1-min intervals. This mix-

ture was stirred overnight at room temperature. Some ether was

then added and the water layer was separated and extracted three

times with ether. The combined organic layers were washed with a

NaHCO₃solution at pH ϭ 9, a saturated NH₄Cl solution, and a

saturated NaCl solution, followed by drying with MgSO₄. After

concentration under vacuum, a crude yield of 4.79 g was obtained.

This was introduced onto a silica column with the aid of some

1

ately used in the subsequent MnO₂oxidation. H NMR: δ ϭ 0.16

[s, 9 H, Si(CH₃)₃], 1.62 (s, 3 H, CH₃), 2.2 (m, 4 H, 2CH₂), 3.55 (s,

3 H, OCH₃), 4.70 (s, 1 H, CH).

Trimethylsilyl Enol Ether of [2-¹³C]-3-Methoxy-6-methyl-2-cyclo-

hexenone: The procedure described above was performed with di-

isopropylamine (6.50 mL, 46.4 mmol) in THF (60 mL), a butylli-

thium solution in hexane (1.6 , 26.1 mL, 41.8 mmol), [2-¹³C]-3- dichloromethane and was eluted with 15% ether/85% PE. This

methoxy-6-methyl-2-cyclohexenone (7a; 5.80 g, 41.4 mmol) in THF yielded 0.83 g of pure quinone [10% based on methyl 5-oxohexano-

1

4

(24 mL), and chlorotrimethylsilane (11.6 mL, 91.4 mmol). A crude

ate (9)]. H NMR: δ ϭ 2.12 (d, J_HϪH1.4 Hz, 3 H, CH₃), 4.21 (s,

yield of 8.08 g (92%) was obtained and immediately used in the

3 H, OCH₃), 6.51 (q, ⁴J_HϪH1.4 Hz, 1 H, CH). ¹³C NMR: δ ϭ 16.2

1

subsequent MnO₂oxidation. ¹H NMR: δ ϭ 4.70 (d, J_CϪH(CH₃), 61.4 (OCH₃), 117.9 (CBr), 131.1 (CH), 145.5 (C),

122.9 Hz, 1 H, ¹³CH). ¹³C NMR: δ ϭ 94.8 (¹³CH).

156.6 (CO), 180.4 (CϭO), 180.9 (CϭO).

3-Methoxy-6-methylphenol (6): Active MnO₂powder (10 g) was ad-

ded to the crude trimethylsilyl enol ether of 3-methoxy-6-methyl-2-

[2-¹³C]-2-Bromo-3-methoxy-6-methyl-para-benzoquinone (4a): The

procedure described above was performed with [2-¹³C]-2,4-di-

cyclohexenone (7.22 g), dissolved in undistilled (wet!) ether (30 bromo-3-methoxy-6-methylphenol (5a; 8.06 g, 27.3 mmol), 2,6-pyr-

mL). This mixture was stirred overnight and subsequently dried

with MgSO₄and filtered through Hyflo Super Cel. The residue was

idinedicarboxylic acid (25.6 g, 153 mmol), water (260 mL), aceto-

nitrile (260 mL), and ammonium cerium nitrate (64 g, 117 mmol) to

obtain a crude yield of 5.15 g. This was introduced onto a silica

extracted thoroughly with ether. After concentration under va-

1

cuum, a crude yield of 6.26 g (121%) was obtained. H NMR: δ ϭ column with the aid of some dichloromethane and was eluted with

4

2.10 (s, 3 H, CH₃), 3.74 (s, 3 H, OCH₃), 6.36 (d, J_HϪH2.8 Hz, 1

15% ether/85% PE to yield pure quinone [1.28 g; 11% based on

3

4

H, 2-CH), 6.44 (dd, J_HϪH8.2 Hz and J_HϪH2.8 Hz, 1 H, 4-CH), methyl 5-oxohexanoate (9a)]. ¹³C NMR: δ ϭ 118.1 (¹³CBr).

7.01 (d, J_HϪH8.2 Hz, 1 H, 5-CH). ¹³C NMR: δ ϭ 15.2 (CH₃),

3

Diels؊Alder Adduct of Cyclopentadiene and 2-Bromo-3-methoxy-6-

54.7 (OCH₃), 105.3 (4-CH), 105.5 (2-CH), 120.6 (C), 130.3 (5-CH),

153.7 (COH), 157.9 (CO).

methyl-para-benzoquinone.

؊

2-Bromo-3-methoxy-8aα-methyl-

4a,5,8,8a-tetrahydro-5α,8α-methano-1,4-naphthoquinone: 2-Bromo-

3-methoxy-6-methyl-para-benzoquinone (4; 0.83 g, 3.59 mmol) and

freshly distilled cyclopentadiene (5 mL) were dissolved in dichloro-

[2-¹³C]-3-Methoxy-6-methylphenol (6a): The procedure described

above was performed with the crude trimethylsilyl enol ether of

Eur. J. Org. Chem. 2002, 189Ϫ202

197

R. B. Boers, P. Gast, A. J. Hoff, H. J. M. de Groot, J. Lugtenburg

FULL PAPER

3

methane (5 mL). After this mixture had been stirred for 40 h, the

NMR: δ ϭ 2.84 (dd, J_HϪH4.1 Hz and J_CϪH2.1 Hz, 1 H, 8a-

solvent was evaporated under vacuum and replaced by PE (10 mL). CH), 3.95 (d, J_CϪH3.4 Hz, 3 H, ¹³COCH₃). ¹³C NMR: δ ϭ

3

The excess cyclopentadiene was removed in a flash column with

PE as eluent. The product was removed from the flash column by

elution with ether. After evaporation of the solvent under vacuum,

150.5 (¹³CO).

Diels؊Alder Adduct of Cyclopentadiene and Ubiquinone-0 (3):

Freshly distilled cyclopentadiene (10 mL) was added to ubiqui-

none-0 (2; 2.00 g, 11.0 mmol) in dichloromethane (5 mL). After 4

d of stirring, the solvent was evaporated under vacuum and re-

placed by PE (10 mL). The excess cyclopentadiene was removed in

a flash column with PE as solvent. The product was obtained by

flushing with ether and evaporation of the solvent under vacuum.

The yield of pure product was 2.72 g (100%).

1

1.06 g (100%) of pure product was obtained. H NMR: δ ϭ 1.53

2

(s, 3 H, 8aα-CH₃), 1.57 (d, J_HϪH8.9 Hz, 1 H, CH_AH_B), 1.71 (d,

²J_HϪH8.9 Hz, 1 H, CH_AH_B), 2.91 (d, ³J_HϪH3.8 Hz, 1 H, 4a-CH),

3.13 (br. s, 1 H, CH), 3.44 (br. s, 1 H, CH), 4.12 (s, 3 H, OCH₃),

6.02 (dd, ³J_HϪH2.7 Hz and 5.5 Hz, 1 H, CHϭCH), 6.15 (dd, ³J_HϪH

2.7 Hz and 5.8 Hz, 1 H, CHϭCH). ¹³C NMR: δ ϭ 26.9 (8aα-CH₃),

46.3 (CH₂), 48.7 (CH), 53.7 (CH), 57.9 (OCH₃), 60.2 (4a-C),

60.9 (4a-CH), 125.2 (CBr), 134.4 (CHϭCH), 138.3 (CHϭCH),

161.5 (CO), 192.2 (CϭO), 194.5 (CϭO).

Ubiquinone-0 (2)

2,3-Dimethoxy-5-methyl-para-benzoquinone:

(1.05 g, 10.7 mmol) and the DielsϪAlder adduct of cyclopentadiene

and 2,3-dimethoxy-5-methyl-para-benzoquinone (3) (1.75 g,

Maleic

anhydride

Diels؊Alder Adduct of Cyclopentadiene and [2-¹³C]-2-Bromo-3-me-

thoxy-6-methyl-para-benzoquinone: The procedure described above

was performed with [2-¹³C]-2-bromo-3-methoxy-6-methyl-para-

benzoquinone (1.28 g, 4.30 mmol) in dichloromethane (5 mL) and

freshly distilled cyclopentadiene (5 mL) to obtain 1.60 g (97%) of

pure product. ¹³C NMR: δ ϭ 125.3 (¹³CBr).

7.06 mmol) were added to toluene (35 mL) and the mixture was

heated at reflux for 4 h. Water (20 mL) at pH ϭ 9 was then added

at room temperature and the mixture was stirred for 15 min. The

water phase was separated and extracted three times with ether. The

combined organic layers were dried with MgSO₄. Concentration un-

der vacuum yielded 2.62 g of crude product. After purification on

silica with 50% ether/50% PE as eluent, pure ubiquinone-0 (2; 1.23 g,

96%) was obtained. ¹H NMR (300 MHz): δ ϭ 2.04 (d, ⁴J_HϪH1.6 Hz,

3 H, CH₃), 4.00 (s, 3 H, 3Ј-OCH₃), 4.02 (s, 3 H, 2Ј-OCH₃), 6.44 (q,

⁴J_HϪH1.6 Hz, 1 H, CH). ¹³C NMR (75 MHz): δ ϭ 15.4 (CH₃), 61.1

(2Ј-OCH₃), 61.2 (3Ј-OCH₃), 131.2 (CH), 143.9 (C), 144.7 (2-CO),

144.9 (3-CO), 183.8 (1-CϭO), 184.3 (4-CϭO).

Synthesis of Ubiquinone-0 (note the change to ubiquinone-0 num-

bering)

Diels؊Alder Adduct of Cyclopentadiene and Ubiquinone-0 (3). ؊

2,3-Dimethoxy-4aα-methyl-4a,5,8,8a-tetrahydro-5α,8α-methano-

1,4-naphthoquinone: Sodium (1.80 g 78.3 mmol) was added to

methanol (20 mL), with sufficient cooling. From this solution, 6.1

mL was taken and added to a solution (cooled to 0 °C) of the

DielsϪAlder adduct of cyclopentadiene and 2-bromo-3-methoxy-

6-methyl-para-benzoquinone (1.06 g, 3.57 mmol) in toluene (40

mL). After 15 min of stirring at 0 °C, the mixture was allowed to

warm to room temperature and stirred for another 1.75 h, after

which the mixture was poured into saturated NH₄Cl solution (20

mL). The water layer was separated and extracted twice with ether.

The combined organic layers were dried with MgSO₄and the solv-

ents were evaporated under vacuum, to yield 0.63 g (71%) of crude

[3-¹³C]Ubiquinone-0 (2a): The procedure described above was per-

formed with maleic anhydride (0.23 g, 2.35 mmol) in toluene (10

mL), and the DielsϪAlder adduct of cyclopentadiene and [3-¹³C]-

2,3-dimethoxy-5-methyl-para-benzoquinone (3a) (0.38 g, 1.53

mmol) to obtain crude product (0.60 g). After purification on silica,

pure [3-¹³C]ubiquinone-0 (0.21 g, 75%) was obtained. ¹H NMR

4

(300 MHz): δ ϭ 4.00 (d, 3.5 Hz, 3 H, 3Ј-OCH₃) 6.44 (dq, J_HϪH

2

product. ¹H NMR: δ ϭ 1.49 (s, 3 H, 4aα-CH₃), 1.55 (d, J_HϪH

1.6 Hz and ⁴J_CϪH0.6 Hz, 1 H, CH). ¹³C NMR (75 MHz): δ ϭ 15.4

2

3

8.9 Hz, 1 H, CH_AH_B), 1.68 (d, J_HϪH8.9 Hz, 1 H, CH_AH_B), 2.84

(d, J_CϪC1.7 Hz, CH₃), 61.1 (d, J_CϪC2.6 Hz, 2Ј-OCH₃), 61.2 (d,

(d, ³J_HϪH4.1 Hz, 1 H, 8a-CH), 3.09 (br. s, 1 H, CH), 3.43 (br. s, 1

²J_CϪC7.8 Hz, 3Ј-OCH₃), 131.2 (d, J_CϪC3.0 Hz, CH), 143.9 (d,

3

H, CH), 3.93 (s, 3 H, OCH₃), 3.95 (s, 3 H, OCH₃), 6.02 (dd, ³J_HϪH

²J_CϪC16.7 Hz, C), no signal found for 2-CO, 144.9 (3-¹³CO), 184.1

3

2

1

2.8 Hz and 5.5 Hz, 1 H, CHϭCH), 6.16 (dd, J_HϪH2.8 Hz and

(d, J_CϪC9.8 Hz, 1-CϭO), 184.3 (d, J_CϪC59.7 Hz, 4-CϭO).

5.5 Hz, 1 H, CHϭCH). ¹³C NMR: δ ϭ 26.3 (4aα-CH₃), 46.2

(CH₂), 48.6 (CH), 52.4 (8a-C), 53.2 (CH), 56.9 (2OCH₃), 60.4 (8a-

CH), 134.3 (CHϭCH), 137.9 (CHϭCH), 150.3 and 150.4 (CO),

194.6 and 198.2 (CϭO).

[2-¹³C]Ubiquinone-0 (2b): The procedure described above was per-

formed with maleic anhydride (0.61 g, 6.22 mmol) in toluene (25

mL) and the DielsϪAlder adduct of cyclopentadiene and [2-¹³C]-

2,3-dimethoxy-5-methyl-para-benzoquinone

(3b,

1.01 g,

Diels؊Alder Adduct of Cyclopentadiene and [3-¹³C]Ubiquinone-0

(3a): The procedure described above was performed with the so-

dium methoxide solution (9.2 mL) and the DielsϪAlder adduct

of cyclopentadiene and [2-¹³C]-2-bromo-3-methoxy-6-methyl-para-

benzoquinone (1.60 g, 4.30 mmol) in toluene (40 mL), to obtain

the crude product (1.26 g, 94%). Purification on a silica column

with 50% ether/50% PE as eluent yielded 0.38 g (28%) of product.

Unfortunately, 0.24 g (20%) of the DielsϪAlder adduct of cyclo-

pentadiene and [2-¹³C]-3-methoxy-6-methyl-para-benzoquinone

4.06 mmol) to obtain the crude product (1.49 g). After purification

on silica, pure [2-¹³C]ubiquinone-0 (0.43 g, 65% based on the

DielsϪAlder adduct of cyclopentadiene and [2-¹³C]-2-chloro-3-me-

thoxy-5-methyl-para-benzoquinone) was obtained. ¹H NMR

3

(300 MHz): δ ϭ 4.02 (d, J_CϪH3.4 Hz, 3 H, 2Ј-OCH₃), 6.44 (dq,

3

⁴J_HϪH1.6 Hz and J_CϪH6.5 Hz, 1 H, CH). ¹³C NMR (75 MHz):

δ ϭ 15.4 (s, CH₃), 61.1 (d, ²J_CϪC8.0 Hz, 2Ј-OCH₃), 61.1 (d, ²J_CϪC

2

3

2.8 Hz, 3Ј-OCH₃), 131.2 (d, J_CϪC17.3 Hz, CH), 143.9 (d, J_CϪC

3.0 Hz, C), 144.7 (2-¹³CO), no signal found for 3-CO, 184.1 (d,

3

was also obtained. ¹H NMR: δ ϭ 3.93 (d, J_CϪH3.4 Hz, 3 H,

¹J_CϪC58.4 Hz, 1-CϭO), 184.3 (d, J_CϪC9.9 Hz, 4-CϭO).

2

¹³COCH₃). ¹³C NMR: δ ϭ 150.4 (¹³CO).

Synthesis of Ubiquinone-10 (note the change to ubiquinone-10

numbering)

Diels؊Alder Adduct of Cyclopentadiene and [2-¹³C]Ubiquinone-0

(3b): The procedure described above was performed with the so-

dium methoxide solution (8 mL) and the DielsϪAlder adduct of Decaprenyl Bromide (27). ؊ (all-E)-3,7,11,15,19,23,27,31,35,39-De-

cyclopentadiene and [2-¹³C]-2-chloro-3-methoxy-5-methyl-para-

camethyl-2,6,10,14,18,22,26,30,34,38-tetracontadecaenyl Bromide:

benzoquinone (1.34 g, 5.28 mmol) in toluene (25 mL). After 2 h of

Decaprenol (26, 2.76 g, 3.96 mmol) and pyridine (10 drops) were

stirring at 0 °C, 1.01 g (77%) of crude product was obtained. ¹H dissolved in ether (38 mL) and PE (24 mL). This mixture was co-

198

Eur. J. Org. Chem. 2002, 189Ϫ202

Synthesis and Characterization of [5-¹³C]- and [6-¹³C]Ubiquinone-10

oled in an ice bath, after which phosphorus tribromide (0.32 mL, tail 1-CH₂), 3.98 (s, 3 H, 5Ј-OCH₃), 3.99 (s, 3 H, 6Ј-OCH₃), 4.93

FULL PAPER

3

3.37 mmol) was added dropwise. The mixture was stirred at 0 °C

for 1 h, followed by 1 h at room temperature, after which the mix-

ture was carefully poured into a separating funnel partially filled

with water, ice, and PE. The water layer was separated and ex-

(t, J_HϪH7.2 Hz, 1 H, tail 2-CH), 5.06 (t, J_HϪH6.0 Hz, 1 H, tail

6-CH), 5.11 (br. t, J_HϪH6.6 Hz, 8 H, tail 8 CH). ¹³C NMR

(150 MHz): δ ϭ 11.9 (3Ј-CH₃), 16.0 (m, tail 7Ј-, 11Ј-, 15Ј-, 19Ј-,

23Ј-, 27Ј-, 31Ј-, and 35Ј-CH₃), 16.3 (tail 3Ј-CH₃), 17.6 (tail end cis-

3

tracted three times with PE. The combined organic layers were 39Ј-CH₃), 25.3 (tail 1-CH₂), 25.7 (tail end trans-40-CH₃), 26.5 (tail

carefully dried with MgSO₄and the solvent was evaporated under

vacuum. A crude yield of 3.03 g (100%) was obtained. H NMR:

4-CH₂), 26.6Ϫ26.7 (m, tail 8-, 12-, 16-, 20-, 24-, 28-, 32-, and 36-

CH₂), 39.7 (m, tail 5-, 9-, 13-, 17-, 21-, 25-, 29-, 33- and 37-CH₂),

61.1 (5Ј-OCH₃), 61.1 (6Ј-OCH₃), 118.8 (tail 2-CH), 123.8 (tail 6-

1

δ ϭ 1.60 (s, 27 H, 9 CH₃), 1.68 (s, 3 H, 40-CH₃), 1.72 (s, 3 H, 3Ј-

CH₃), 2.01 (m, 36 H, 18 CH₂), 4.02 (d, ³J_HϪH8.2 Hz, 2 H, CH₂Br), CH), 124.1Ϫ124.4 (m, tail 10-, 14-, 18-, 22-, 26-, 30-, 34-, and 38-

3

5.1 (br. t, J_HϪH7 Hz, 9 H, 9 CH), 5.53 (t, J_HϪH8.2 Hz, 1 H, CH), 131.2 (tail 39-C), 134.8Ϫ135.0 (tail 11-, 15-, 19-, 23-, 27-,

2-CH).

31-, 35-C), 135.2 (tail 7-C), 137.6 (tail 3-C), 138.8 (3-C), 141.6 (2-

C), 144.2 (5-CO), 144.3 (6-CO), 183.9 (1-CϭO), 184.7 (4-CϭO).

Tributyl(decaprenyl)tin (28)

[5-¹³C]Ubiquinone-10 (1a): The procedure described above was per-

formed with [3-¹³C]ubiquinone-0 (2a; 0.21 g, 1.15 mmol) in dichlo-

romethane (12 mL), silver oxide (0.64 g, 2.76 mmol), freshly dis-

tilled boron trifluorideϪdiethyl ether (0.85 mL, 6.91 mmol), tribu-

tyl(decaprenyl)tin (28; 1.65 g 1.70 mmol) in dichloromethane (10

mL), and KF (4 g) to obtain a crude yield of 1.71 g. After purifica-

tion on silica, pure [5-¹³C]ubiquinone-10 (1a; 0.37 g, 0.43 mmol,

37%) was obtained together with pure [3-¹³C]ubiquinone-0 (2a;

Tributyl[(all-E)-3,7,11,15,19,23,27,31,35,39-decamethyl-

2,6,10,14,18,22,26,30,34,38-tetracontadecaenyl]tin: A solution of

butyllithium in hexane (1.6 , 4.53 mL, 7.25 mmol) was injected

by syringe into diisopropylamine (1.16 mL, 8.28 mmol) in THF (10

mL) cooled to 0 °C. After 5 min, tributyltin hydride (1.18 mL,

4.39 mmol) was added. This mixture was stirred for 15 min and

subsequently cooled to Ϫ60 °C, after which decaprenyl bromide

(27, 2.78 g, 3.66 mmol) in THF (20 mL) was added dropwise. The

mixture was allowed to warm up to Ϫ40 °C over 2 h, after which

the reaction was quenched by pouring it into some saturated

NH₄Cl. The water layer was separated and extracted twice with

ether. The combined organic layers were washed with saturated

NaCl solution and carefully dried with MgSO₄. After evaporation

of the solvent under vacuum, a crude yield of 3.61 g (102%) was

obtained. ¹H NMR: δ ϭ 0.8 (m, 15 H, 3-butyl-CH₂Sn and 3-butyl-

CH₃), 1.3 (m, 6 H, 3-butyl-CH₂), 1.5 (m, 8 H, 3 butyl-CH₂and 1

CH₂Sn), 1.60 (s, 27 H, 9 CH₃), 1.68 (s, 6 H, 3Ј-CH₃and cis-39Ј-

1

3

0.12 g 0.66 mmol, 57%). H NMR (600 MHz): δ ϭ 3.98 (d, J_CϪH

3

3.5 Hz, 3 H, 5Ј-OCH₃). ¹³C NMR (150 MHz): δ ϭ 11.9 (d, J_CϪC

2

1.6 Hz, 3Ј-CH₃), 61.1 (s, 6Ј-OCH₃), 61.1 (d, J_CϪC2.0 Hz, 5Ј-

2

3

OCH₃), 138.8 (d, J_CϪC16.2 Hz, 3-C), 141.6 (d, J_CϪC3.0 Hz, 2-

C), 144.2 (5-¹³CO), no signal found for 6-CO, 183.9 (d, J_CϪC

9.9 Hz, 1-CϭO), 184.7 (d, J_CϪC59.1 Hz, 4-CϭO).

2

1

[6-¹³C]Ubiquinone-10 (1b): The procedure described above was per-

formed with [2-¹³C]ubiquinone-0 (2b; 0.26 g, 1.42 mmol) in dichlo-

romethane (14 mL), silver oxide (0.73 g, 3.15 mmol), freshly dis-

tilled boron trifluorideϪdiethyl ether (0.97 mL, 7.89 mmol), tribu-

tyl(decaprenyl)tin (28; 1.88 g, 1.94 mmol) in dichloromethane (12

mL), and KF (4 g) to obtain a crude yield of 2.06 g. After purifica-

tion on silica, pure [6-¹³C]ubiquinone-10 (1b; 0.48 g, 0.56 mmol,

39%) was obtained together with pure [2-¹³C]ubiquinone-0 (2b;

3

CH₃), 2.00 (m, 36 H, 18 CH₂), 5.1 (br. t, J_HϪH7 Hz, 9 H, 9 CH),

3

5.32 (t, J_HϪH9.3 Hz, 1 H, 3-CH).

Ubiquinone-10 (1)

2-[(all-E)-3,7,11,15,19,23,27,31,35,39-Decamethyl-2,6,10,14,18,

30,34,38-tetracontadecaenyl]-5,6-dimethoxy-3-methylpara-

22,26,benzoquinone: Silver oxide (0.64 g, 2.76 mmol) was added to

ubiquinone-0 (2; 0.20 g, 1.10 mmol) in dichloromethane (12 mL).

This mixture was kept in the dark and cooled to Ϫ80 °C, after

which boron trifluorideϪdiethyl ether (0.85 mL, 6.91 mmol),

freshly distilled from CaH₂, was injected by syringe. Tributyl(decap-

renyl)tin (28; 1.53 g, 1.58 mmol) in dichloromethane (10 mL) was

then added dropwise. This mixture was allowed to warm up to Ϫ20

°C over 2 h and was then cooled down to Ϫ40 °C, after which

saturated NaCl solution (20 mL) was added and the cooling re-

moved. After addition of potassium fluoride (4 g), the mixture was

stirred for 30 min and then filtered through Hyflo Super Cel. The

water layer was separated and extracted twice with dichlorome-

thane. The combined organic layers were dried with MgSO₄and

the solvents were evaporated under vacuum. A crude yield of 1.71 g

was obtained. Purification was on silica with an eluent of 20%

1

0.13 g, 0.71 mmol, 50%). H NMR (600 MHz): δ ϭ 3.99 (d, ³J_CϪH

3.5 Hz, 3 H, 6Ј-OCH₃). ¹³C NMR (150 MHz): δ ϭ 11.9 (s, 3Ј-CH₃),

25.3 (d, ³J_CϪC1.6 Hz, tail 1-CH₂), 61.1 (s, 5Ј-OCH₃), 61.1 (d, ²J_CϪC

3

2

4.5 Hz, 6Ј-OCH₃), 138.8 (d, J_CϪC3.0 Hz, 3-C), 141.6 (d, J_CϪC

15.9 Hz, 2-C), no signal found for 5-CO, 144.3 (6-¹³CO), 183.9 (d,

¹J_CϪC58.9 Hz, 1-CϭO), 184.7 (d, J_CϪC9.9 Hz, 4-CϭO).

2

Syntheses in the Preparation of [2-¹³C]-Ubiquinone-0

Monomethyl 3-Methylglutarate: Methanol (4.71 mL, 116.3 mmol)

was added to 3-methylglutaric anhydride (11; 12.44 g, 97.1 mmol).

This was stirred for 45 min at 80 °C. After evaporation of the excess

methanol under vacuum, 15.39 g (99%) of product was obtained.

3

¹H NMR: δ ϭ 1.05 (d, J_HϪH6.2 Hz, 3 H, CH₃), 2.4 (m, 5 H,

2CH₂and CH), 3.68 (s, 3 H, OCH₃), 10.8 (br. s, OH). ¹³C NMR:

δ ϭ 19.5 (CH₃), 26.9 (CH), 40.2 (2CH₂), 51.3 (OCH₃), 172.7 (Cϭ

O), 177.8 (CϭO).

ether/80% PE until elution of ubiquinone-10, followed by 50% Methyl (4-Chloroformyl)-3-methylbutyrate (12): Thionyl chloride

ether/50% PE for the ubiquinone-0. This yielded pure ubiquinone- (17.6 mL, 203 mmol) was added to monomethyl 3-methylglutarate

10 (1; 0.21 g, 0.24 mmol, 22%) and pure ubiquinone-0 (2; 0.14 g, (15.39 g, 96.2 mmol). After stirring overnight, the excess thionyl

0.77 mmol, 70%). If the product still contained some ubiquinols, chloride was evaporated under vacuum. Subsequent vacuum distil-

then an oxidation procedure was performed with ammonium lation (bp. 60 °C at 0.3 Torr) yielded pure product (16.3 g, 94%).

3

cerium nitrate as described for the oxidation to 2-bromo-3-me-

thoxy-6-methyl-para-benzoquinone (4) prior to purification on sil-

¹H NMR: δ ϭ 1.08 (d, J_HϪH6.5 Hz, 3 H, CH₃), 2.30 (dd, J_HϪH

7.2 Hz and ²J_HϪH15.4 Hz, 1 H, CH_AH_BCOOMe), 2.40 (dd, ³J_HϪH

ica. ¹H NMR (600 MHz): δ ϭ 1.57 (s, 3 H, tail end trans-40-CH₃), 6.2 Hz and J_HϪH15.4 Hz, 1 H, CH_AH_BCOOMe), 2.5 (m, 1 H,

2

3

2

1.59 (s, 3 H, tail-CH₃), 1.60 (s, 21 H, tail 7CH₃), 1.68 (s, 3 H, tail CH), 2.84 (dd, J_{H Ϫ H}7.2 Hz and J_{H Ϫ H}17.0 Hz, 1 H,

3

2

end cis-39Ј-CH₃), 1.74 (s, 3 H, tail 3Ј-CH₃), 1.98 and 2.06 (m, 36 CH_AH_BCOCl), 3.07 (dd, J_HϪH5.8 Hz and J_HϪH17.0 Hz, 1 H,

H, tail 18 CH₂), 2.01 (s, 3 H, 5Ј-CH₃), 3.18 (d, J_HϪH7.2 Hz, 2 H, CH_AH_BCOCl), 3.69 (s, 3 H, OCH₃). ¹³C NMR: δ ϭ 19.3 (CH₃),

3

Eur. J. Org. Chem. 2002, 189Ϫ202

199

R. B. Boers, P. Gast, A. J. Hoff, H. J. M. de Groot, J. Lugtenburg

27.7 (CH), 39.7 (2-CH₂), 51.6 (4-CH₂), 53.0 (OCH₃), 172.0 (CϭO), assium (2.09 g, 53.5 mmol), and methyl [6-¹³C]-3-methyl-5-oxohex-

FULL PAPER

172.5 (CϭO).

anoate (7.64 g, 48.1 mmol) for the reaction, and THF (200 mL)

and concentrated HCl (4.88 mL), diluted to 40 mL with water, for

the workup procedure, to obtain a crude yield of 6.86 g. Recrystal-

lization in 20 mL warm acetone yielded a first fraction (4.71 g) and

a second fraction (0.18 g) of pure [2-¹³C]-5-methylcyclohexane-1,3-

dione (13b), bringing the total yield to 4.89 g (80%). The product

was obtained mostly in its enol form. ¹H NMR: δ ϭ 3.38 (d, ¹J_CϪH

Methyl 3-Methyl-5-oxohexanoate: A solution of iodomethane

(10.00 g, 4.39 mL, 70.5 mmol) in ether (20 mL) was added dropwise

to magnesium turnings (1.58 g, 65.0 mmol) in refluxing ether (40

mL) in such a way as to maintain the ether at a gentle reflux. To

make sure no magnesium was left, heating at reflux was continued

for about 30 min (residual magnesium seriously reduces the yields

of the subsequent reactions). The Grignard mixture was cooled

down to 0 °C and dry CdCl₂(8.00 g, 43.6 mmol) was added, fol-

lowed by reflux for further 45 min. All the ether solvent was then

evaporated under a flow of argon. The obtained gray, sandy sub-

stance was dispersed in toluene (100 mL) at room temperature,

after which methyl (4-chloroformyl)-3-methylbutyrate (12; 9.01

mL) in toluene (10 mL) was added dropwise. After a further 2 h at

reflux, the mixture was cooled down to room temperature, after

which saturated NH₄Cl solution (30 mL) was added, followed by

some 1  HCl. After separation of the water layer, which was ex-

tracted twice with ether, the combined organic layers were washed

with saturated NaHCO₃, NH₄Cl, and NaCl solutions and dried

with MgSO₄. After evaporation under vacuum, the yellow liquid

(9.65 g) was distilled under vacuum (0.3 Torr). The fraction col-

lected at 60Ϫ70 °C gave methyl 3-methyl-5-oxohexanoate (7.34 g)

as a colorless liquid, in a yield of 66% with regard to iodomethane.

1

130.1 Hz, 2 H, ¹³CH₂keto form), 5.48 (d, J_CϪH160.3 Hz, 1 H,

¹³CH enol form). ¹³C NMR: δ ϭ 57.7 (¹³CH₂keto form), 103.9

(¹³CH enol form).

2-Chloro-5-methylcyclohexane-1,3-dione (14): 5-Methyl-1,3-cyclo-

hexanedione (13; 5.0 g, 39.7 mmol) was dissolved in THF (15 mL)

and water (50 mL), which was subsequently cooled to 0 °C. After

dropwise addition of chloramine-T (11.2 g, 49.2 mmol) in water (75

mL), this mixture was stirred for 30 min and then filtered. The

residue was washed with water (5 mL). The filtrate was acidified

to pH ϭ 2 with concentrated HCl and the acidic mixture was satur-

ated with NaCl. This mixture was extracted four times with THF

and the collected organic phases were dried thoroughly with

MgSO₄. After concentration under vacuum, a yield of 4.52 g (71%)

was obtained. The product was obtained in its enol form. ¹H NMR

3

([D₆]acetone): δ ϭ 1.12 (d, J_HϪH5.5 Hz, 3 H, CH₃), 2.3 (m, 3 H,

5-CH and 4-CH₂), 2.6 (m, 2 H, 6-CH₂).

¹H NMR: δ ϭ 0.99 (d, J_HϪH6.5 Hz, 3 H, 3Ј-CH₃), 2.14 (s, 3 H,

3

[2-¹³C]-2-Chloro-5-methyl-1,3-cyclohexanedione (14b): The proced-

ure described above was performed with [2-¹³C]-5-methyl-1,3-cyclo-

hexanedione (13b; 4.71 g, 37.1 mmol) in THF (7 mL) and water

(35 mL), and chloramine-T (10.55 g, 46.3 mmol) in water (69 mL)

to obtain 4.02 g (67%) of product. ¹³C NMR ([D₆]acetone): δ ϭ

109.2 (¹³CCl).

6-CH₃), 2.2Ϫ2.5 (m, 5 H, 2CH₂and CH), 3.67 (s, 3 H, OCH₃). ¹³

C

NMR: δ ϭ 19.7 (3Ј-CH₃), 27.4 (3-CH), 30.2 (6-CH₃), 40.5 (CH₂),

49.8 (CH₂), 51.4 (OCH₃), 172.3 (1-CϭO), 207.2 (5-CϭO).

Methyl [6-¹³C]-3-Methyl-5-oxohexanoate: The procedure described

above was performed with [¹³C]iodomethane (10.00 g, 70.0 mmol)

in ether (20 mL), magnesium turnings (1.58 g, 65.0 mmol) in re-

fluxing ether (50 mL), CdCl₂(8.16 g, 44.5 mmol), and methyl 4-

chloroformyl-3-methylbutyrate (12, 9.01 mL) in toluene (10 mL) to

obtain 10.42 g of a yellow liquid, which, after vacuum distillation

(60 to 70 °C, 0.3 Torr), yielded 7.64 g as a colorless liquid in a yield

2-Chloro-3-methoxy-5-methyl-2-cyclohexenone (15): Trimethyl or-

thoformate (5 mL, 45.7 mmol) and concentrated HCl (6 drops)

were added to 2-chloro-5-methyl-1,3-cyclohexanedione (14; 4.52 g,

28.2 mmol) in methanol (25 mL). After overnight stirring, the solv-

ent was evaporated under vacuum, and a yield of 4.82 g (98%) was

obtained. ¹H NMR: δ ϭ 1.15 (d, ³J_HϪH5.8 Hz, 3 H, CH₃), 2.3 (m,

3 H, 4-CH₂and 5-CH), 2.7 (m, 2 H, 6-CH₂), 3.96 (s, 3 H, OCH₃).

¹³C NMR: δ ϭ 20.7 (CH₃), 27.8 (5-CH), 34.2 (CH₂), 44.7 (CH₂),

56.3 (OCH₃), 111.1 (CCl), 170.4 (3-CO), 190.7 (CϭO).

of 69% with regard to [¹³C]iodomethane. H NMR: δ ϭ 2.14 (d,

1

¹J_CϪH127.4 Hz, 3 H, ¹³CH₃). ¹³C NMR: δ ϭ 30.3 (¹³CH₃).

5-Methyl-1,3-cyclohexanedione (13): Potassium (2.06 g, 52.7 mmol)

was added to tert-butyl alcohol (345 mL). After all the potassium

had reacted, methyl 3-methyl-5-oxohexanoate (6.88 g, 43.5 mmol)

was added and the mixture was stirred overnight. The tert-butyl

alcohol was evaporated under vacuum and the obtained brown

solid was redissolved in THF (200 mL) and concentrated HCl (4.4

mL), diluted to 40 mL with water. The mixture was saturated with

NaCl, after which the water layer was separated and extracted with

THF (100 mL). The combined organic phases were dried carefully

with MgSO₄. After evaporation of the solvent, a crude yield of

6.20 g was obtained. The crude product was dissolved in warm

acetone (20 mL). After overnight crystallization at Ϫ20 °C, a first

fraction of pure product (2.41 g) was obtained. Additional product

(0.43 g) could be obtained from the residue in a second round of

crystallization, bringing the total yield to 2.84 g (53%). The product

was obtained mostly in its enol form. ¹H NMR: δ ϭ 1.09 (d, ³J_HϪH

5.8 Hz, 3 H, CH₃enol form), 1.12 (d, ³J_HϪH6.2 Hz, 3 H, CH₃keto

form), 2.0Ϫ2.8 (several m, 10 H, 2 CH₂and 5-CH for both enol

and keto forms), 3.38 (s, 2 H, 2-CH₂keto form), 5.47 (s, 1 H, 2-

CH enol form), 9.8 (br. s, OH enol form). ¹³C NMR: keto: δ ϭ

19.7 (CH₃), 25.9 (5-CH), 48.0 (2CH₂), 57.7 (2-CH), 203.8 (2 Cϭ

O); enol: δ ϭ 20.7 (CH₃), 28.8 (5-CH), 40.5 (CH₂), 51.3 (CH₂),

103.8 (2-CH), 172.7 (3-COH), 191.6 (1-CϭO).

[2-¹³C]-2-Chloro-3-methoxy-5-methyl-2-cyclohexenone (15b): The

procedure described above was performed with [2-¹³C]-2-chloro-5-

methyl-1,3-cyclohexanedione (14b; 4.02 g, 24.9 mmol) in methanol

(25 mL), trimethyl orthoformate (3.8 mL, 34.7 mmol), and concen-

trated HCl (5 drops) to obtain a yield of 4.34 g (99%). ¹³C NMR:

δ ϭ 111.1 (¹³CCl).

2-Chloro-3-methoxy-5-methylphenol (16): A butyllithium solution

in hexane (1.6 , 5.1 mL, 8.16 mmol) was added by syringe at Ϫ15

°C to diisopropylamine (1.50 mL, 10.7 mmol), dissolved in THF

(22 mL), followed after 10 min by dropwise addition of 2-chloro-3-

methoxy-5-methyl-2-cyclohexenone (15; 0.87 g, 4.99 mmol) in THF

(14 mL). After 5 min, chlorotrimethylsilane (4.56 mL, 35.9 mmol)

was added and the mixture was allowed to warm to room temper-

ature over 1 h. The reaction mixture was then heated to reflux, and

iodine (1.52 g, 5.99 mmol) in THF (22 mL) was added dropwise

under illumination with a 100-W tungsten lamp, followed by 3 h

at reflux under illumination. After the mixture had cooled to room

temperature, some saturated sodium dithionite solution was added

and the water layer was separated and extracted three times with

ether. The combined organic phases were dried with MgSO₄and

the solvent was evaporated under vacuum. The resulting mixture

(1.20 g) was dissolved in undistilled methanol (15 mL) and KF (2 g)

[2-¹³C]-5-Methyl-1,3-cyclohexanedione (13b): The procedure de-

scribed above was performed with tert-butyl alcohol (400 mL), pot-

200

Eur. J. Org. Chem. 2002, 189Ϫ202

Synthesis and Characterization of [5-¹³C]- and [6-¹³C]Ubiquinone-10

FULL PAPER

4

3

was added, followed by 30 min of stirring. Some water was added 6.67 (dq, J_HϪH1.7 Hz and J_CϪH7.6 Hz, 1 H, CH). ¹³C NMR:

to dissolve the salts and the water layer was separated and extracted

three times with ether. The combined organic phases were washed

with saturated NaCl solution and dried with MgSO₄. After concen-

tration under vacuum, crude product (0.77 g, 90%) was obtained.

¹H NMR: δ ϭ 2.29 (s, 3 H, CH₃), 3.86 (s, 3 H, OCH₃), 5.6 (br. s,

OH), 6.38 (s, 1 H, CH), 6.54 (s, 1 H, CH).

δ ϭ 125.0 (¹³CCl).

Diels؊Alder Adduct of Cyclopentadiene and [2-¹³C]-2-Chloro-3-

methoxy-5-methyl-para-benzoquinone

[2-¹³C]-2-Chloro-3-methoxy-4aα-methyl-4a,5,8,8a-tetrahydro-5α,8α-

methano-1,4-naphthoquinone:

[2-¹³C]-2-Chloro-3-methoxy-5-

methyl-para-benzoquinone (17b; 0.99 g 5.31 mmol) and freshly dis-

tilled cyclopentadiene (5 mL) were dissolved in dichloromethane

(10 mL). After this mixture had been stirred for 40 h, the solvent

was evaporated under vacuum and replaced by PE (10 mL). The

excess cyclopentadiene was removed in a flash column, with PE as

eluent. The product was removed from the flash column by elution

with ether. After evaporation of the solvent under vacuum, pure

[2-¹³C]-2-Chloro-3-methoxy-5-methylphenol (16b): The procedure

described above was performed with diisopropylamine (7.43 mL,

53.0 mmol) in THF (110 mL), a butyllithium solution in hexane

(1.6 , 25.3 mL, 40.5 mmol), [2-¹³C]-2-chloro-3-methoxy-5-methyl-

2-cyclohexenone (15b; 4.31 g, 24.6 mmol) in THF (90 mL), chloro-

trimethylsilane (4.56 mL, 35.9 mmol), and iodine (7.53 g,

29.7 mmol) in THF (110 mL) for the reactions. The obtained prod-

uct (7.33 g) was worked up with KF (6 g) in undistilled methanol

(55 mL) to obtain crude product (4.07 g, 96%). ¹H NMR: δ ϭ 6.38

1

product (1.34 g, 100%) was obtained. H NMR: δ ϭ 1.52 (s, 3 H,

2

4aα-CH₃), 1.59 (d, J_HϪH9.3 Hz, 1 H, CH_AH_B), 1.71 (d, J_HϪH

3

9.3 Hz, 1 H, CH_AH_B), CH₂), 2.97 (dd, J_HϪH3.8 Hz and J_CϪH

2.1 Hz, 1 H, 8a-CH), 3.11 (br. s, 1 H, CH), 3.48 (br. s, 1 H, CH),

4.11 (s, 3 H, OCH₃), 6.04 (dd, ³J_HϪH3.1 Hz and 5.8 Hz, 1 H, CHϭ

3

(d, J_CϪH8.6 Hz, 1 H, CH), 6.54 (d, J_CϪH7.9 Hz, 1 H, CH). ¹³C

NMR: δ ϭ 105.4 (¹³CCl).

CH), 6.16 (dd, J_HϪH3.1 Hz and 5.8 Hz, 1 H, CHϭCH). ¹³C

3

Fremy’s Salt. ؊ Potassium Nitrosodisulfonate [K₂(SO₃)₂NO]:

NaNO₂(10.35 g, 150 mmol) was dissolved in water (30 mL) in a

1000-mL round-bottomed flask, which was cooled in an ice-bath

during the whole procedure. To this solution was added crushed

ice (60 g) and NaHSO₃(10.5 g, 101 mmol). After addition of gla-

cial acetic acid (6 mL, 104 mmol), stirring was continued for 4 min,

during which the solution became yellow. Concentrated ammonia

(7.5 mL, 125 mmol) was then added, followed by addition of some

more crushed ice. Some crushed ice should from now on always be

present in the flask. After dropwise addition of KMnO₄solution

(0.2 , 120 mL, 24.0 mmol), stirring was continued for 20 min. The

MnO₂precipitate was removed by paper filtration. The filtrate was

collected in a 1-L Erlenmeyer flask and kept on ice. From this point

on, no further crushed ice was added. To precipitate the Fremy’s

salt, KCl was added until an orange precipitate formed. The precip-

itate was collected in a fritted glass funnel and subsequently washed

with saturated KCl solution (30 mL) containing 5% concentrated

ammonia, with methanol (30 mL) containing 5% concentrated

ammonia, and finally with pure acetone (40 mL). A slightly moist

orange salt (about 12 g) was obtained, and was used without fur-

ther drying or purification. It was kept at 4 °C in a well-sealed jar

with a drop of concentrated ammonia on the inside of the lid.

NMR: δ ϭ 26.2 (4aα-CH₃), 46.2 (CH₂), 48.8 (CH), 53.1 (CH), 57.1

(OCH₃), 57.5 (4a-C), 61.3 (8a-CH), 131.0 (¹³CCl), 135.0 (CHϭ

CH), 138.0 (CHϭCH).

3-Methoxy-6-methyl-2-cyclohexenone (7)

4-Methyl-5-oxohexanoic Acid: Sodium (0.42 g, 18.3 mmol) was ad-

ded to methanol (100 mL). After all the sodium had reacted, ethyl

2-methylacetoacetate (18; 10.00 mL, 70.7 mmol) was added, fol-

lowed by dropwise addition of methyl acrylate (19; 7.00 mL,

77.7 mmol) in methanol (15 mL). After overnight stirring, KOH

(10 g, 178 mmol) in water (15 mL) was added. After further over-

night stirring, concentrated aqueous HCl (15 mL) in water (5 mL)

was added carefully, followed by 1 h of stirring at 65 °C. Additional

concentrated aqueous HCl (2 mL) in water (5 mL) was then added

and stirring was continued for another 1.5 h at 65 °C, followed by

overnight stirring at room temperature. After evaporation of the

solvent, the residue was dissolved in ether and dried with MgSO₄.

A crude yield of 7.63 g (75%) was obtained. ¹H NMR: δ ϭ 1.14

(d, ³J_HϪH6.9 Hz, 3 H, 4Ј-CH₃), 1.68 (m, 1 H, 3-CH_AH_B), 1.99 (m,

3

1 H, 3-CH_AH_B), 2.18 (s, 3 H, 6-CH₃), 2.36 (t, J_HϪH7.2 Hz, 2 H,

2-CH₂), 2.62 (m, 1 H, 4-CH). ¹³C NMR: δ ϭ 16.1 (4Ј-CH₃), 27.0

(3-CH₂), 28.1 (6-CH₃) 31.3 (2-CH₂), 45.8 (4-CH), 178.5 (COOH),

212.2 (5-CϭO).

2-Chloro-3-methoxy-5-methyl-para-benzoquinone (17): A buffer was

prepared from K₂HPO₄(5.0 g, 28.7 mmol) in water (200 mL) and

brought to pH ϭ 6.5 with HCl. To this buffer was added Fremy’s

salt (7.5 g, prepared as described above) and 3-methoxy-5-methyl-

phenol (16; 1.02 g, 5.91 mmol), dissolved in ether (10 mL). After

this mixture had been stirred overnight, ether (100 mL) was added.

The water layer was separated and extracted four times with ether.

The combined organic phasees were washed with saturated NaCl

solution and dried with MgSO₄. After evaporation of the solvent

under vacuum, crude product (0.60 g) was obtained. After purifica-

tion on silica with 15% ether/85% PE as eluent, a yield of 0.28 g

[26% based on 2-chloro-3-methoxy-5-methyl-2-cyclohexenone (15)]

˚

Methyl 4-Methyl-5-oxohexanoate (21): Molecular sieves (3 A, 5 g)

and p-toluenesulfonic acid (0.1 g, 0.53 mmol) were added to 4-

methyl-5-oxohexanoic acid (7.41 g) in methanol (50 mL). After two

nights of stirring, the mixture was filtered through Hyflo Super

Cel and the solvent was evaporated under vacuum. After vacuum

distillation (55Ϫ70 °C, 0.3 Torr), 6.36 g (78%) of product was ob-

1

3

tained. H NMR: δ ϭ 1.12 (d, J_HϪH7.2 Hz, 3 H, 4Ј-CH₃), 1.72

(m, 1 H, 3-CH_AH_B), 1.99 (m, 1 H, 3-CH_AH_B), 2.16 (s, 3 H, 6-CH₃),

3

2.31 (t, J_HϪH7.2 Hz, 2 H, 2-CH₂), 2.60 (m, 1 H, 4-CH), 3.67 (s,

3 H, OCH₃). ¹³C NMR: δ ϭ 16.1 (4Ј-CH₃), 27.3 (3-CH₂), 28.0 (6-

CH₃), 31.4 (2-CH₂), 45.9 (4-CH), 51.4 (OCH₃), 173.4 (1-CϭO),

211.6 (5-CϭO).

4

was obtained. ¹H NMR: δ ϭ 2.08 (d, J_HϪH1.7 Hz, 3 H, CH₃),

4

4.19 (s, 3 H, OCH₃), 6.67 (q, J_HϪH1.7 Hz, 1 H, CH).

[2-¹³C]-2-Chloro-3-methoxy-5-methyl-para-benzoquinone (17b): The 6-Methyl-1,3-cyclohexanedione (22): Potassium (0.56 g, 14.3 mmol)

procedure described above was performed with K₂HPO₄(20 g,

was added to tert-butyl alcohol (100 mL). After all the potassium

had reacted, methyl 4-methyl-5-oxohexanoate (21; 2.04 g,

115 mmol), water (800 mL), Fremy’s salt (30 g), and [2-¹³C]-3-me-

thoxy-5-methylphenol (16b; 4.07 g, 23.5 mmol) in ether (40 mL) to 12.9 mmol) was added and the mixture was stirred overnight. The

obtain crude product (2.91 g), which yielded pure product 1.61 g tert-butyl alcohol was evaporated under vacuum and the obtained

{1.61 g, 35% based on [2-¹³C]-2-chloro-3-methoxy-5-methyl-2- brown solid was dissolved in THF (54 mL) and water (11 mL),

cyclohexenone (15b)} after purification on silica. ¹H NMR: δ ϭ

acidified with concentrated HCl (1.32 mL). The mixture was satur-

Eur. J. Org. Chem. 2002, 189Ϫ202

201

R. B. Boers, P. Gast, A. J. Hoff, H. J. M. de Groot, J. Lugtenburg

FULL PAPER

[18]

H. Dieks, K. Beyer, J. Labelled Comp. Radiopharm. 1990,

ated with NaCl. The water layer was separated and extracted with

THF (100 mL). The combined organic phasees were dried carefully

with MgSO₄. After evaporation of the solvent, a crude yield of

1.77 g (109%) was obtained. The product was obtained as a mixture

28Ϫ29, 1093Ϫ1107.

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A. Duralski, A. Watts, Tetrahedron Lett. 1992, 33, 4983Ϫ4984.

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Goede, A. U. Baldew, J. Lugtenburg, Recl. Trav. Chim. Pays-

Bas 1990, 109, 287Ϫ297.

1

of a keto form and two enol forms. H NMR: δ ϭ 1.20 (m, 9 H,

3 CH₃keto and enol forms), 1.7Ϫ2.7 (several m, 15 H, 2-CH₂and

5-CH for both enol and keto forms), 3.37 (s, 2 H, 2-CH₂keto

form), 5.74 (s, 2 H, 2-CH enol forms), 10.1 (br. s, OH enol forms).

[21]

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6-methyl-1,3-cyclohexanedione (22; 1.77 g, 14.0 mmol) in methanol

(25 mL). After overnight stirring, the solvent was evaporated under

vacuum and a crude yield of 1.95 g (99%) was obtained. The two

isomers (7 ϩ 25) were separated on silica, and 3-methoxy-6-methyl-

2-cyclohexenone (7; 0.41 g, 21%) and 3-methoxy-4-methyl-2-cyclo-

123Ϫ129.

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hexenone (25; 0.36 g, 18%) were obtained. H NMR: δ ϭ 1.15 (d,

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101.2 (2-CH), 177.3 (CO), 201.3 (CϭO).

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Article Doi

Synthesis and spectroscopic characterization of [5-13C]- and [6-13C]ubiquinone-10 for studies of bacterial photosynthetic reaction centers

DOI: 10.1002/1099-0690(20021)2002:1<189::AID-EJOC189>3.0.CO;2-8

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