Observation of the pathway from lysine to the isoprenoidal lipid of halophilic archaea, Halobacterium halobium and Natrinema pallidum, using regiospecifically deuterated lysine

Article abstract of DOI:10.1246/bcsj.74.2199

We examined the incorporation of lysine into archaeal isoprenoidal lipids of halophilic archaea, Natrinema pallidum and Halobacterium halobium using two regiospecifically deuterium-labeled derivatives, [3,3-²H₂] and [6,6-²H₂]lysines. The two deuterated lysines were synthesized, and the incorporation of deuterium to the lipid core was defined by ²H NMR. The results revealed that lysine is degraded to crotonoyl-CoA by the decarboxylation of carboxylate in the metabolism of halophilic archaea, much like the metabolism of lysine in aerobic bacteria; the process converts lysine to isoprenoidal lipids via the mevalonate pathway through glutaryl-CoA, crotonoyl-CoA, and acetoacetyl-CoA.

Full text of DOI:10.1246/bcsj.74.2199

c. Jpn.
© 2001 The Chemical Society of Japan
Bull. Chem. Soc. Jpn., 74, 2199–2205 (2001) 2199
e Chemical
ciety of Ja-
n
e Chemical
ciety of Ja-
n
Observation of the Pathway from Lysine to the Isoprenoidal Lipid of
Halophilic Archaea, Halobacterium halobium and Natrinema pallidum,
Using Regiospecifically Deuterated Lysine
The Pathway from Lysine to Isoprenoidal Lipid
N.Yamauchi et al.
01
99
Noriaki Yamauchi,*,# Satoshi Endoh, Keiko Kato,† and Tatsushi Murae
05
Department of Earth and Planetary Sciences, Graduate School of Science, Kyushu University,
Hakozaki, Fukuoka 812-8581
SJA8
09-2673
170
†Laboratory of Chemistry, Faculty of Education, Yamagata University, Yamagata 990-8560
(Received May 21, 2001)
01
01
We examined the incorporation of lysine into archaeal isoprenoidal lipids of halophilic archaea, Natrinema palli-
dum and Halobacterium halobium using two regiospecifically deuterium-labeled derivatives, [3,3-2H2] and [6,6-
2H2]lysines. The two deuterated lysines were synthesized, and the incorporation of deuterium to the lipid core was de-
fined by 2H NMR. The results revealed that lysine is degraded to crotonoyl-CoA by the decarboxylation of carboxylate
in the metabolism of halophilic archaea, much like the metabolism of lysine in aerobic bacteria; the process converts
lysine to isoprenoidal lipids via the mevalonate pathway through glutaryl-CoA, crotonoyl-CoA, and acetoacetyl-CoA.
.4
4
Archaea (archaebacteria) live in relatively hostile environ-
ments, such as those characterized by low pH (2–3), high tem-
perature (~110 °C), completely anaerobic conditions, or high
salt concentration. They can now be clearly distinguished
from eucarya and eubacteria from the 16S ribosomal RNA se-
quences, the cell-wall structure, and the membrane lipid pat-
terns.1,2 Many investigators regard the archaea as the evolu-
tionarily oldest among living organisms based on their molecu-
lar biological characteristics, while others insists archaea
evolved from existing microorganisms as a way of adapting to
their living environments. Among archaea, halophilic archaea
live in an environment such as a medium with nearly saturated
NaCl concentration. The existence of an unusual ether lipid in
archaea is one of the clearest distinctions between archaea and
other organisms. Distinct from the lipids of eucarya or eubac-
teria, the lipids of halophilic archaea characteristically consist
Fig. 1. Structure of liquid core and related hydrocation frag-
of saturated C20 (and C25) isoprenoids bonded with glycerol by
ment in halophilic archaea.
an ether-linkage (1 and 2), as shown in Fig. 1.3
The entire outer sphere of archaea consists of isoprenoid-
tabolites and plant secondary metabolites (included in terpe-
noids and pigments, respectively) have been shown to be bio-
synthesized, was not involved in these archaea. However,
Smith et al. pointed out that 13C-labeled acetate was not incor-
porated in the branched methyl and methine carbon of the iso-
prenoidal lipid in Halobacterium salinarium NRC34001.7 Our
recent incorporation experiments for the halophilic archaea
Natrinema pallidum, using [3-(2H3)methyl]-mevalonolactone
and [13C]labeled acetate,8 suggested that some metabolic path-
ways from amino acid to mevalonate, in keeping with Smith’s
proposal,7 cooperate with the usual pathway from acetate to
mevalonate.
based biomembrane lipids. The isoprenoidal diether lipid core
is completely absent in membrane lipids of eubacteria and eu-
carya. Therefore, the biosynthesis of isoprenoidal lipid in ar-
chaea has intrigued many scientists, who have investigated its
structural analysis in biosynthetic studies.3 Kates et al. have
shown the incorporation of radiolabeled mevalonate in diether
lipid,4 and Kakinuma et al. recently reported the incorporation
of [2H9]mevalonate in halophilic archaea.5 These results sug-
gest that isoprenoid was biosynthesized from acetate to meva-
lonate, a ubiquitous biosynthetic precursor of various types of
isoprenoid. They also suggest that a mevalonate-independent
pathway,6 from which many parts of bacterial secondary me-
Smith’s result and our result show a strong correlation of
lysine metabolism and the mevalonate pathway. In typical mi-
croorganisms, it has been shown that lysine is degraded to cro-
# Graduate School of Sciences, Kyusyu University, and Faculty of
Education, Yamagata University.

2200 Bull. Chem. Soc. Jpn., 74, No. 11 (2001)
The Pathway from Lysine to Isoprenoidal Lipid
Scheme 1. The two metabolic pathway of lysine in microorganisms.
Scheme 2. Synthesis of [3,3-2H2] and [6,6-2H2]Lysines.
tonoyl-CoA by a rearrangement of the amino group with lysine
cally deuterium-labeled lysine. We herein report on our re-
sults.
2,3-aminomutase (Clostridium),9 or by the decarboxylation of
carboxylate (Pseudomonas), as shown in Scheme 1.10 In ar-
chaea, however, it is unclear which pathway exists, or if both
exist, which is predominant. In order to eradicate this ambigu-
ity, we investigated the metabolic pathways using regiospecifi-
Synthesis of [3,3-2H2] and [6,6-2H2]Lysines
The fatty acid content in halophilic archaea is very low;
however, researchers have observed the existence of fatty acid

N.Yamauchi et al.
Bull. Chem. Soc. Jpn., 74, No. 11 (2001) 2201
synthase (FAS) activity and the inhibition of its activity in high
salt concentration.3,11 This may indicate that a part of the inter-
mediates in fatty acid biosynthesis would be converted to iso-
prenoid biosynthesis in archaea. Initially, we thought that our
observation and Smith’s result could be explained by the link
of the known pathway for a microorganism in lysine metabo-
lism to the intermediate of fatty acid biosynthesis. Thus, one
of the pathways between the above-mentioned two paths of
lysine degradation would be relatively active in halophilic ar-
chaea. The intermediate in the lysine-degradation pathway,
crotonoyl-CoA, is converted to mevalonate and isoprenoid
through the mevalonate pathway, which is the main pathway in
the biosynthesis of the isoprenoidal lipid core in halophilic ar-
chaea.
C4 unit for synthesizing the two-labeled amino acid, the same
C4 component 4, which was easily prepared from an inexpen-
sive starting material, 1,4-butanediol (3), was selected and pre-
pared. In the synthesis of [3,3-2H2]lysine, 4 was converted to
bromide 6 and coupled with diethyl t-butoxycarbonylamino-
malonate to yield amide 7. The terminal protection group at 7
was deprotected and converted to azide 8. Finally, catalytic
hydrogenation and acid hydrolysis of 8 gave [3,3-2H2]lysine 9.
Next, in the synthesis of [6,6-2H2]lysine 14, 4 was converted to
phtalimide 10, and the benzyl-protecting group of 10 was
deprotected to give 11 and further converted to bromide 12.
Then, 12 was coupled with diethyl acetamidomalonate to give
13, which was finally deprotected to give 14. The isotopic en-
richment of the product was > 95%, as determined from its 1H
NMR spectrum.
If the predominant catabolism of lysine in the archaea was
via lysine 2,3-aminomutase, the hydrogen at C-3 of lysine was
finally lost at the crotonoyl-CoA, and C-6 of lysine was con-
verted to the terminal methyl group. If, on the other hand, the
predominant catabolism was via decarboxylation at C-1 of
lysine, the hydrogen at C-6 of lysine was finally lost at the cro-
tonoyl-CoA, and C-3 of lysine was converted to the terminal
methyl group and C-2 of crotonoyl-CoA. To discriminate be-
tween the two, [3,3-2H2]lysine and [6,6-2H2]lysine were syn-
thesized. Several reports on the synthesis of 3- and 6-labeled
lysine have appeared in the literature recently.12 Among them,
Aberhart and Gould et al. synthesized [3,3-2H2]lysine and ste-
reoselectively 3-deuterated lysine, and used them as a probe in
work concerning the mechanistic implication of the stereo-
chemistry of lysine 2,3-aminomutase from Clostridium and the
biosynthesis of streptothricin.13 Their method appears to be
convenient for large-scale preparations. According to their
concept concerning the preparation, the C4 units for [3,3-
2H2]lysine and [6,6-2H2]lysine were synthesized and coupled
with protected aminomalonate,14 as shown in Scheme 2. The
Incorporation of [3,3-2H2] and [6,6-2H2]Lysines
Incorporation experiments of Halobacterium halobium
(which contains 1 as a lipid-core component) and Natrinema
pallidum15 (which contains 1 and the C20–C25 diether 2) were
performed as reported previously.8 In brief, Halobacterium
halobium JCM 9120 and Natrinema pallidum IAM 13147
were incubated for 6 days at 37 °C in 300 mL of a medium
containing 250 mg of 9 or 14. Lipids were extracted, then hy-
drolyzed with methanolic HCl. A mixture of diethers 1 and 2
was separated from the remaining mixture of the other hydro-
lyzate.
1
The resulting lipid core 1 and 2 were identified by H and
13C NMR, respectively. Because of the low incorporation of
deuterium, no significant difference was caused by deuterium
incorporation on the 1H and 13C NMR spectra. The determina-
2
tion of the position of deuterium was examined by H NMR
(61.4 MHz, CDCl3 standard (7.26 ppm)). The 2H NMR spec-
tra of each lipid obtained from a feeding experiment along
Fig. 2. 1H NMR spectrum and 2H NMR spectra of 1 and 2 from Natrinema pallidum: (A) (1H) non-labeled standard; (B) (2H) the
sample derived from [6,6-2H2]-lysine-incubation experiment; (C) (2H) the sample derived from [3,3-2H2]lysine-incubasion experi-
ment. Similar result was obtained from 1 of Halobacterium halobium.

2202 Bull. Chem. Soc. Jpn., 74, No. 11 (2001)
The Pathway from Lysine to Isoprenoidal Lipid
Scheme 3. Proposed pathway from lysine to mevalonate in halophilic archaea.
1
with the H NMR of non-labeled lipid diether are shown in
would be retained via crotonoyl-CoA through lysine metabo-
lism, as shown in Scheme 2.
Fig. 2. No significant 2H was incorporated in the lipids afford-
ed by these bacteria cultivated in a 14-containing medium (Fig.
2, B). On the other hand, a significant H signal at the non-
Our observation and Smith’s proposed pathway can be ex-
plained as a known lysine metabolic pathway in aerobic bacte-
ria that has not been shown to exist in archaea, however, as
shown in Scheme 3. In these archaea, lysine would be convert-
ed to “free” glutaric acid, like in the catabolism of lysine in
Pseudomonas by the action of glutarate semialdehyde dehy-
drogenase.16 Then, because of the C2 symmetry of the mole-
cule, two labeling patterns of glutaric acid must be obtained
from [3,3-2H2]lysine 9. [2,2-2H2]glutaric acid, which is pre-
sumably formed from 9, was converted to crotonoyl-CoA, ace-
toacetyl-CoA, and mevalonate, and deuterium was incorporat-
ed to the C-3 methyl hydrogens of mevalonate. [4,4-2H2]glu-
taric acid, which is also formed from 9, was converted similar-
ly, and deuterium was incorporated to the C-2 methylene hy-
drogens of mevalonate. If these conversions were to occur, the
deuterium at C-3 of lysine would be converted to hydrogens
connected with the branched methyl carbon via the C-3 methyl
of mevalonate, and C-2, C-6, C-10, and C-14 (methylene) car-
bon via the C-2 of mevalonate. However, a part of the deuteri-
um at the C-2 of mevalonate would be lost because of the low
pKa of the methylene hydrogens in acetoacetyl-CoA. Further-
more, the C-2 hydrogen in mevalonate may be lost during
isomerization at IPP and DMAPP, regardless of the stereo-
2
substituted methyl region (δ 0.81) was observed in the NMR
spectrum of the lipids obtained by incubation using a medium
containing 9 (Fig. 2, C). The signal corresponded to hydro-
gens at branched methyl in isoprenoidal lipids. No other deu-
terium signal was observed in the 2H NMR spectrum. Further,
the lipid core was degraded by HI and reduced to hydrocar-
bons 15 and 16; then, GC-MS analysis was performed. The
mass spectra of 15 and 16 showed a slight incorporation of
deuterium from a sample in the feeding experiment of 9 (ca.
3% deuterium incorporation). No incorporation of deuterium
was observed in the hydrocarbon fraction from a sample in a
feeding experiment of 14. The result was that the incorpora-
tion of deuterium occurred in the isoprenoidal portion.
These results indicated that the C-3 carbon (and hydrogen)
in lysine was converted to a branched methyl carbon in iso-
prenoidal lipid, showing the presence of an efficient route, as
suggested by Smith et al.,7 for the conversion of lysine to the
acetate unit at the branched methyl and methine carbon. This
conclusion is based on the logic that if lysine was degraded to
crotonoyl-CoA by rearranging an amino group, then the C-3
hydrogens in lysine would be lost and the C-6 hydrogens

N.Yamauchi et al.
Bull. Chem. Soc. Jpn., 74, No. 11 (2001) 2203
MHz, CDCl3) δ 1.71 (4H, m), 2.31 (1H, broad, -OH), 3.50 (2H, t,
J = 7.2 Hz), 3.61 (2H, m), 4.59 and 4.61 (2H, d, J = 11.0 Hz),
7.30 (5H, aromatic). IR (CHCl3) 3619, 3422, 3011, 2940, 2866,
1602, .1496, 1464, 1362, 1234 cm−1. HRMS Calcd for C11H16O2:
180.1150. Found: 180.1138.
chemical ambiguity at the methylene hydrogen at this posi-
tion.17 Thus, because of the low level of incorporation of deu-
terium, the latter could not be detected and the former deuteri-
um was only observed by 2H NMR. Therefore, our result sug-
gests that lysine is efficiently converted to glutarate, crotonoyl-
CoA, acetoacetyl-CoA, and mevalonate in halophilic archaea.
The results also suggest that the lysine metabolism in halo-
philic archaea resembles that in Pseudomonas.
Because of the low incorporation level of lipid diether from
9, all of Smith’s proposed pathway, the C2 unit of amino acid is
efficiently converted to the isoprenoidal lipid core at the
branched methyl and methyne region, could not explain our
lysine-incorporation experiments. A search for other carbon
sources (such as the degradation of leucine or valine) is still
needed to clarify the unique path for the efficient biosynthesis
of isoprenoidal lipid in halophilic archaea.
Recently, the complete genome sequence of one halophilic
archea was published; however, only one gene with enzyme
coding related to lysine metabolism could be observed from
the sequence similarity.18 Additional experiments are needed to
clarify the metabolic pathway of the unique microorganism,
and further investigations of other species of archaea (e.g.,
thermophilic archaea) and differences in the biosynthetic path-
way are currently underway.
[1,1-2H2]-4-Benzyloxy-butan-1-ol (5). To a solution of 4
(2.8 g, 15.6 mmol) in acetone (25 mL) was added Jones Reagent
(0.3 mL, 0.81 mmol) in a dropwise manner, and the solution was
stirred for 30 min. Excess reagent was quenched by the addition
of iPrOH, and the mixture was stirred for several minutes. The
mixture was evaporated to ca. 10 mL, and then ether (50 mL) was
added. The carboxylate was extracted with 1 M NaOH (50 mL),
and the water layer was acidified with conc. HCl. The acidified
water layer was extracted with ether (50 mL × 2). The combined
organic phase was washed with brine and dried over anhydrous
Na2SO4. After filtration, the organic solution was evaporated to
dryness. Thirty milliliters of anhydrous methanol and 3 drops of
conc. H2SO4 were added to the residue (2.9 g), and the mixture
was refluxed for 3 h. The mixture was evaporated to ca. 30 mL
and diluted with ether (30 mL), and then Na2CO3 solution was
added to it. The organic layer was separated, and the water layer
was extracted with ether (30 mL × 2). The combined organic lay-
er was dried over Na2SO4, then filtrated, and evaporated to dry-
ness. The residue was purified by silica-gel column chromatogra-
phy (100 g, hexane : EtOAc = 4 : 1) to give 2.56 g of ester. To a
mixture of ester in 60 mL of THF was added 600 mg of LiAl2H4
with three portions and the mixture was stirred for 1 h. The mix-
ture was diluted with ether (60 mL), and a small amount of water
was added to quench any excess reagent and to precipitate
Al(OH)3; then Na2SO4 was added. The mixture was filtrated,
evaporated, and the residue was purified by silica-gel column
chromatography (200 g, hexane : EtOAc = 2 : 1) to give 2.05 g of
5 (72.5%). 1H NMR (90 MHz, CDCl3) δ 1.69 (2H, m), 2.31 (1H,
broad, -OH), 3.50 (2H, t, J = 7.2 Hz), 4.59 and 4.61 (2H, d, J =
11.0 Hz), 7.30 (5H, aromatic). IR (CHCl3) 3619, 3422, 3011,
2940, 2866, 1602, 1496, 1464, 1362, 1234 cm−1. HRMS Calcd
Experimental
Infrared spectra were obtained with a Perkin Elmer 1600 FT-IR
1
spectrometer. H NMR spectra were recorded with a JEOL EX-90
spectrometer. 13C NMR spectra were recorded with a JEOL EX-
90 spectrometer. 2H NMR spectra were recorded with a JEOL
GX-400 spectrometer. For the 1H spectra and 13C NMR, tetrame-
thylsilane (0 ppm) was used as an internal standard at CDCl3. For
the 2H NMR spectra, the natural abundance of the deuterium sig-
nal of CDCl3 solvent (7.26 ppm) was used as an internal standard.
HR-EI-MS spectra were recorded with a JEOL-D300 spectrome-
ter. GC-MS were recorded with a Shimadzu QP-5000 spectrome-
ter. Chromatographic separations were carried out with Merck
Kieselgel 60, 70–230 mesh columns.
2
for C11H14O2 H2: 182.1274. Found: 182.1314.
[4,4-2H2]-4-Benzyloxy-1-bromobutane (6). To a solution of
5 (3.38 g, 18.5 mmol) in CH2Cl2 (140 mL) was added triphe-
nylphosphine (10.99 g, 41.7 mmol) and CBr4 (13.89 g, 41.8
mmol); the mixture was stirred at rt for 3 h, and then evaporated.
The residue was purified by silica-gel column chromatography (90
Synthesis of [3,3-2H2]Lysine. 4-Benzyloxybutan-1-ol (4).
1,4-Butanediol 3 (12.0 g, 110 mmol) and freshly distilled benzal-
dehyde (10.0 g, 110 mmol) in 70 mL of benzene was added to 100
mg of p TsOH and refluxed while removing water that evolved
during the reaction procedure. The reflux was continued for 12 h,
the mixture was cooled, and 50 mL of aq Na2CO3 was added. The
organic layer was separated and the water layer was extracted with
50 mL of ether. The combined organic layer was dried over
NaSO4. After filtration, the organic solution was evaporated.
Then, 18.7 g of the crude 7-membered ring ketal was obtained. A
part of the product (12.5 g) was dissolved into the mixture of
CH2Cl2–ether (60 mL–60 mL). After three portions of LiAlH4
(3.0 g) were added to the mixture with stirring, it was slowly heat-
ed to 40 °C (oil bath). AlCl3 (9.0 g) in ether (60 mL) was added
during 30 min, and the boiling was continued for 2 h. The mixture
was cooled, the excess of LiAlH4 was decomposed with ethyl ace-
tate (30 mL), and Al(OH)3 was precipitated by the addition of wa-
ter. After dilution with ether (100 mL), the organic layer was fil-
tered and the residue was washed with ether. The combined or-
ganic layer was washed with water, dried, and concentrated. The
residue was purified by silica-gel column chromatography (300 g,
1
g, hexane : EtOAc = 10 : 1) to give 4.41 g of 6 (97.3%). H NMR
(90 MHz, CDCl3) δ 1.90 (4H, m), 3.50 (2H, t, J = 7.0 Hz), 4.49
(2H, s), 7.30 (5H, aromatic). IR (CHCl3) 3011, 2940, 2866, 1602,
1496, 1454, 1362, 1234 cm−1. Anal. Calcd for C11H13BrO2H2: C,
53.89; H + 2H, 6.16%. Found: C, 53.57; H + 2H, 6.54%.
Diethyl
[1,1-2H2]-4-Benzyloxybutyl-N-t-butoxycarbonyl-
aminomalonate (7). To a solution of sodium ethoxide (0.87
mmol/L, 7.4 mL, 6.44 mmol) in 10 mL of EtOH was added dieth-
yl t-butoxycarbonylaminomalonate (1.70 g, 5.86 mmol); and the
solution was stirred at rt for 30 min. Then, bromide 6 (1.43 g,
5.82 mmol) in 10 mL of EtOH was added, and the mixture was re-
fluxed for 3 h. After removing the precipitate, the mixture was di-
luted with 20 mL of ethyl acetate and water was added. The or-
ganic layer was separated, and the water layer was reextracted
with ethyl acetate (20 mL). The combined organic layer was
washed, dried, filtered, and evaporated. The residue was purified
by silica-gel column chromatography (50 g, hexane : EtOAc = 10
: 1 to 2 : 1) to give 2.18 g of 7 (83.2%). 1H NMR (90 MHz,
CDCl3) δ 1.22 (6H, t, J = 6.8 Hz), 1.42 (6H, s), 3.46 (2H, t, J =
6.3 Hz), 4.21 (4H, q, J = 6.8 Hz), 4.46 (2H, s), 5.90 (1H, broad,
1
hexane : EtOAc = 2 : 1) to give 12.0 g of 4 (90.7%). H NMR (90

2204 Bull. Chem. Soc. Jpn., 74, No. 11 (2001)
The Pathway from Lysine to Isoprenoidal Lipid
2
-NH), 7.30 (5H, aromatic). IR (CHCl3) 3011, 2940, 2866, 1602,
C12H11NO3 H2: 221.1019. Found: 221.0968.
2
1496, 1454, 1362, 1234 cm−1. Anal. Calcd for C23H33NO7 H2: C,
N-(4-Bromo-[1,1-2H2]butyl)phthalimide (12). To
a solu-
62.85; H + 2H, 8.03; N, 3.19%. Found: C, 62.91; H + 2H, 7.95;
N, 3.60%.
tion of 11 (2.88 g, 13.1 mmol) in CH2Cl2 (130 mL) were added
triphenylphosphine (9.64 g, 36.6 mmol) and CBr4 (12.17 g, 36.6
mmol); the mixture was stirred at rt for 2.5 h, and then evaporated.
The residue was purified by silica-gel column chromatography (80
g, hexane : EtOAc = 10 : 1) to give 2.98 g of 12 (80.1%). 1H
NMR (90 MHz, CDCl3) δ 1.90 (4H, m), 3.45 (2H, m), 7.77 (4H,
aromatic), 7.30 (5H, aromatic). IR (CHCl3) 3028, 2940, 2866,
Diethyl [1,1-2H2]-4-Azidobutyl-N-t-butoxycarbonylamino-
malonate (8). To a solution of 7 (4.58 g, 10.4 mmol) in 50 mL
of AcOH was added 100 mg of Pd–C, and the mixture was stirred
vigorously under a H2 atmosphere for 4 h. The mixture was fil-
tered and evaporated to give 3.58 g of the alcohol (98.4%). The
product was converted to azide with no further purification. To a
solution of the above product (3.58 g, 10.3 mmol) in 30 mL of py-
ridine was added TsCl (2.5 g, 13.0 mmol); and the mixture was
stirred at rt for 1 h, and then worked up and purified by silica-gel
column chromatography (50 g, hexane : EtOAc = 10 : 1 to 2 : 1)
to give 2.86 g of tosylate. Then, the tosylate was dissolved into 20
mL of DMF, and sodium azide (715 mg, 11 mmol) was added to
the mixture. The mixture was stirred at 100 °C (bath temp) for 2 h
and cooled to rt. The usual work-up and chromatography (50 g,
hexane : EtOAc, 4 : 1 to 2 : 1) were done, to give 1.96 g of 8
(50.2%). 1H NMR (90 MHz, CDCl3) δ 1.25 (6H, t, J = 6.8 Hz),
1.44 (6H, s), 3.29 (2H, t, J = 6.7 Hz), 4.21 (4H, q, J = 6.8 Hz),
4.46 (2H, s), 5.90 (1H, broad, -NH). IR (CHCl3) 3454, 3011,
2940, 2866, 1744, 1725, 1496, 1362, 1055 cm−1. Anal. Calcd for
1770, 1711, 1602, 1468, 1454, 1394 cm−1
C12H11BrNO2 H2: 283.0175. Found: 283.0200.
. HRMS Calcd for
2
Diethyl N-Acetamido-([4,4-2H2]-4-phthalimidobutyl)malo-
nate (13). To a solution of sodium ethoxide (0.87 mmol/L, 5.3
mL, 6.0 mmol) in 10 mL of EtOH was added diethyl acetami-
domalonate (0.62 g, 5.7 mmol); the solution was warmed to 70 °C
in an oil bath and stirred for 30 min. Then, 12 (1.01 g, 3.55 mmol)
in 10 mL of hot EtOH was added, and the mixture was refluxed
for 12 h. After removing the precipitate, the mixture was diluted
with 20 mL of ethyl acetate and water was added. The organic
layer was separated, and the water layer was reextracted with ethyl
acetate (20 mL). The combined organic layer was washed, dried,
filtered, and evaporated. The residue was purified by silica-gel
column chromatography (50 g, hexane : EtOAc = 10 : 1 to 2 : 1)
to give 0.77 g of 13 (51.6%). 1H NMR (90 MHz, CDCl3) δ 1.25
(6H, t, J = 6.8 Hz), 1.71 (2H, m), 2.03 (3H, s), 2.37 (2H, m), 4.21
(4H, q, J = 6.8 Hz), 6.75 (1H, broad, -NH), 7.77 (4H, aromatic).
2
2
C16H26N4O6 H2 C, 51.32; H + H, 7.54; N, 14.96%. Found: C,
51.82; H + 2H, 8.01; N, 15.06%.
[3,3-2H2]Lysine (9). To a solution of 8 (1.96 g, 5.23 mmol) in
20 mL of AcOH was added 100 mg of Pd–C, and the mixture was
stirred vigorously for 12 h under a H2 atmosphere, and then fil-
tered, evaporated, and dissolved into a THF-1M HCl mixture (20
mL–5 mL). The mixture was stirred for 2 h at 100 °C (bath temp)
and evaporated. It was then dissolved into 10 mL of 6 M HCl and
stirred at 100 °C for 2 h. The filtrate was evaporated to ca. 10 mL
and applied to IR120B (H+) ion-exchange column chromatogra-
phy (r 15 mm × 70 mm, 35 mL). The column was washed with 3
volumes of water, and the product was eluted with 1 M aq NH3.
The eluent was evaporated and dried under reduced pressure to
give 653 mg of the product (84.4%).
IR (CHCl3) 3414, 3028, 2940, 2866, 1736, 1706, 1678, 1602,
2
1468, 1454, 1394 cm−1
.
Anal. Calcd for C21H24N2O7 H2: C,
59.99; H + 2H, 6.23; N, 6.66%. Found: C, 59.91; H + 2H, 6.25;
N, 6.55%.
[6,6-2H2]Lysine (14). 13 (1.50 g, 3.57 mmol) was suspended
in 15 mL of 6 M HCl, and the mixture was refluxed for 12 h. The
mixture was diluted with 30 mL of water, and precipitated phthal-
imide was filtered under reduced pressure. The filtrate was evapo-
rated to ca. 10 mL and applied to an IR-120B (H+) ion-exchange
column chromatography (r 15 mm × 70 mm, 35 mL). The col-
umn was washed with 3 volumes of water, and the product was
eluted with 1 M aq. NH3. The eluent was evaporated and dried un-
der reduced pressure to give 450 mg of the product (85.1%). IR
Synthesis of [6,6-2H2]Lysine. N-(4-Benzyloxy-[1,1-2H2]bu-
tyl)phthalimide (10). To a solution of 6 (4.41 g, 18.0 mmol) in
N,N-dimethylformamide (50 mL) was added potassium N-phthal-
imide (5.40 g, 0.81 mmol); and the resulting solution was refluxed
at 120 °C (bath temp) for 2 h. The mixture was diluted with ether
(50 mL), and sat. NaCl (30 mL) was added. The organic phase
was separated, the water phase was reextracted with ether (50 mL
× 3), and the combined organic phase was washed with brine, and
then dried over anhydrous Na2SO4. The residue was purified by
silica-gel column chromatography (90 g, hexane : EtOAc = 7 : 1)
to give 4.12 g of 10 (73.3%). 1H NMR (90 MHz, CDCl3) δ 1.71
(4H, m), 3.70 (2H, t, J = 7.0 Hz), 4.49 (2H, s), 7.30 (5H, aromat-
ic) 7.77 (4H, aromatic). 13C NMR (22 MHz, CDCl3) δ 40.24,
67.81, 73.09, 73.42, 112.20, 127.75, 128.43, 144.49. IR (CHCl3)
(KBr) 3500–2500, 1627, 1586, 1418 cm−1
.
Bacterial Culture and Lipid Extraction. Natrinema palli-
dum (formerly Halobacterium halobium) IAM 13147 was ob-
tained from the Institute of Applied Microbiology, University of
Tokyo, and Halobacterium halobium JCM 9120 was obtained
from Riken. The cultivation and isolation of lipid diether were
carried out according to our previous report. Three hundred milli-
liters of the medium were incubated for each experiment, and 250
mg of deuterium-labeled lysine was applied to the medium. Cells
were harvested by centrifugation to yield typically 2 g of wet
cells. Lipids were extracted, and the non-polar contaminants were
separated by acetone-precipitation. After methanolysis of the po-
lar lipid fraction, the residue was chromatographed over silica gel
with hexane–dietheyl ether (4 : 1) to give, typically, about 4 to 6
mg of lipid (8 and 9, as a mixture in the case of Natrinema palli-
dum; 8, as a single compound in the case of Halobacterium halo-
bium).
3028, 2940, 2866, 1770, 1711, 1602,1496,1454, 1394,1234 cm−1
.
2
HRMS Calcd for C12H10O3 H2 ((M−C7H7 (Bn)+): 220.0941.
Found: 220.0916.
N-(4-Hydorxy-[1,1-2H2]butyl)phthalimide (11). To a solu-
tion of 10 (4.12 g, 13.2 mmol) in 50 mL of ethanol was added 200
mg of 10% Pd–C. The mixture was stirred for 30 min in a H2 at-
mosphere for 20 h. The mixture was filtered, and the filtrate was
evaporated to give 2.89 g of 11 (98.5%). 1H NMR (90 MHz,
CDCl3) δ 1.71 (4H, m), 3.70 (2H, t, J = 7.0 Hz), 4.49 (2H, s), 7.77
(4H, aromatic). IR (CHCl3) 3624, 3465, 3028, 2940, 2866, 1770,
Degradation of Lipid-Core and Conversion to Hydrocarbon
by HI Cleavage and LiAlH4 Reduction. HI (1 mL) and Lipid
(1 mg) were refluxed for 12 h at 100 °C (bath temp). The mixture
was cooled to rt, and 5 mL of water was added to it. The iodide
was extracted with hexane (10 mL × 2), and the hexane layer was
dried with Na2SO4. After filtration, the hexane layer was evapo-
1711, 1602, 1468, 1454, 1394 cm−1
.
HRMS Calcd for

N.Yamauchi et al.
Bull. Chem. Soc. Jpn., 74, No. 11 (2001) 2205
rated, and then 1 mL of THF and 20 mg of LiAlH4 were added.
After the mixture was stirred for 30 min, a small amount of water
was added and the mixture was diluted with hexane. The mixture
was filtrated, and hexane was removed under a stream of nitrogen.
The products were analyzed by GC-MS.
Jpn., 73, 2513 (2000).
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We are grateful to Ms. Kayoko Ogi of Kyushu University
for 2H NMR measurements. This work was supported in part
by a Grant-in-Aid for Scientific Research from the Ministry of
Education, Science, Sports and Culture.
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