Improved isotopic deuterium labeling at the diastereotopic methyl group of leucine: A synthetic route to (4S)- and (4R)-[5-2H 1]leucine

Article abstract of DOI:10.1271/bbb.70.276

Two isotopomers of deuterium-labeled leucine in the diastereotopic methyl group were synthesized from inexpensively available (R)- and (S)-citronellol in a more convenient way than by previous methods.

Full text of DOI:10.1271/bbb.70.276

Biosci. Biotechnol. Biochem., 70 (1), 276–278, 2006
Note
Improved Isotopic Deuterium Labeling at the Diastereotopic Methyl Group
of Leucine: a Synthetic Route to (4S)- and (4R)-[5-2H1]Leucine
y
Noriaki YAMAUCHI and Satoshi ENDOH
Department of Earth and Planetary Sciences, Graduate School of Sciences, Kyushu University,
6-10-1 Hakozaki, Fukuoka 812-8581, Japan
Received August 10, 2005; Accepted October 9, 2005
Two isotopomers of deuterium-labeled leucine in the
H
H
H
diastereotopic methyl group were synthesized from
inexpensively available (R)- and (S)-citronellol in a
more convenient way than by previous methods.
(i)
(ii)
(iii)
OH
OMOM
OMOM
OH
1
2
Key words: leucine; diastereotopic methyl group; deu-
terium labeling
3
H
H
H
I
CH22H
(v)
(vi)
(vii)
Deuterium-labeled amino acids are important in
bioorganic chemistry for trace experiments in metabolic
studies of amino acids and related compounds. Thanks
to NMR spectroscopy, the importance of these com-
pounds has recently increased to facilitate the elucida-
tion of the 3-D structures of peptides and proteins.1)
Among these, the leucine residue is important in the
three-dimensional structure of proteins with hydropho-
bic interaction. Assignment of the diastereotopic methyl
groups of leucine by NMR could allow the protein
structures and substrate-enzyme interaction in the
hydrophobic region to be ascertained more precisely.
Several methods have been reported for the specific
chemical and chemo-enzymatic synthesis of deuterium-
labeled leucine in the diastereotopic methyl group.
Young et al. have reported the first synthesis of
(2S,4R)-[5,5,5-2H3]leucine from pyroglutamate.2) Oba
and Nishiyama disclosed the synthesis of stereo- and
regiospecifically deuterium-labeled leucine by a similar
approach.3) Hill et al. described the synthesis of (2S,4S)-
and (2R,4S)-[5,5,5-2H3]leucine, starting from (R)-pule-
gone as the chiral source.4) Willis et al. described the
chemo-enzymatic synthesis of the labeled leucine at the
diastereotopic methyl group.5,6) Their stereogenic center
at C-4 was established by using Evans’s chiral auxil-
iaries and the camphor sultam chiral auxiliary. However,
their method had several problems for scaled-up syn-
thesis of the product or of another isotopomer of the
labeled compound by their use of an unnatural pyroglu-
tamate isomer for one isotopomer, a quantitative amount
of the expensive ruthenium-containing Wilkinson cata-
lyst for decarbonylation of the intermediate, and the
requirement for purifying the labeled isopropanol and
isopropyl bromide.
R
OBn
OBn
OBn
7
4: R=CH2OH
5: R=COOH
6
(iv)
H
H
H
H
CH22H
CH22H
COOH
11
CH22H
COOH
OH
H2N
COOH
H2N
8
9
10
Scheme 1. Reagents and conditions: (i) MOMCl, iPr2NEt, CH2Cl2
(90%); (ii) O3, EtOH, then NaBH4 (89%); (iii) (a) BnCl, NaOH,
DMSO; (b) HCl, MeOH (70% for 2 steps); (iv) Jones reagent,
acetone (80%); (v) I2, PhI(OAc)2, CCl4, W-lamp irr. (100 W ꢁ2)
(87%); (vi) LiAl2H4, THF (80%); (vii) (a) H2/Pd-C, iPrOH; (b)
Jones reagent, acetone (46% for 2 steps).
In our biosynthetic studies of archaeal characteristic
isoprenoidal lipid, we planned to determine the role of
the metabolism of leucine after publication of the results
of lysine metabolism and isoprenoidal lipid biosyn-
thesis.7) We first improved the synthesis for the
diastereotopic labeling of leucine at the specific position
of the methyl group.
The synthetic procedure is shown in Scheme 1.8) The
starting material was citronellol; either the (R) or (S)
isomer is inexpensively available commercially as a
chiral source. The hydroxyl group of (R)-citronellol 1,
25
(½ꢀꢀD ¼ þ3:89 (neat)) was protected with MOM-ether
and double-bond oxidative fission by ozonolysis, and the
resulting aldehyde was reduced with NaBH4 in situ to
give alcohol 3. The newly-formed hydroxyl group was
protected with a benzyl ether, while deprotection of
y
To whom correspondence shoud be addressed. Fax: +81-92-642-4174; E-mail: nyama@geo.kyushu-u.ac.jp

Synthesis of (4S)-[5-2H1]Leucine
277
reflux temperature. The reaction mixture was cooled to
room temperature, and then washed successively with
a dilute sodium thiosulfate solution and water. The
organic phase was dried, evaporated and chromato-
graphed by silica gel (hexane:CHCl3 = 2:1) to give
5.86 g (18.4 mmol, 87.2%) of product 6. IR ꢁmax
(CHCl3) cmꢂ1: 3032, 2859, 1454, 1097. 1H-NMR
(90 MHz) ꢂH (CDCl3): 0.98 (3H, d, J ¼ 6:2 Hz), 1.30–
1.82 (5H, m), 3.25 (1H, dd, J ¼ 5:9, 9.1 Hz), 3.29 (1H,
dd, J ¼ 5:9, 9.1 Hz), 3.57 (2H, t, J ¼ 7:5 Hz), 4.52
(2H, s), 7.32 (5H, aromatic). 13C-NMR (22 MHz) ꢂC
(CDCl3): 17.54, 20.49, 27.15, 32.96, 34.54, 70.23,
72.88, 127.48, 127.57 (ꢁ2), 128.32 (ꢁ2), 138.49.
HRMS m=z (Mþ): calcd. for C13H19IO, 318.0481 found,
318.0495.
H
H
H
H
I
OBz
(iii)
OH
OH
(i)
OBn
OBn
12: R=Bz
OH
14
[α]D = + 9.6
OH
lit. [α]D = - 10.0
6
(ii)
13: R=H
Scheme 2. Reagents and conditions: (i) NaOBz, DMF (99%); (ii)
NaOH, EtOH (61%); (iii) H2/Pd-C (73%).
MOM-ether gave alcohol 4. Jones oxidation of 4 then
yielded carboxylic acid 5. The key step in the synthetic
scheme is the decarboxylation-iodide formation of
carboxylic acid with iodosobenzene diacetate-iodine as
developed by Suarez et al.9) This reaction avoids some
of the difficulties of the modified Hunsdiecker reaction
that needs the use of a quantitative amount of a heavy
metal (e.g. Pb(OAc)4 or Cu(OAc)2). The reaction
proceeded cleanly, and iodide 6 was obtained in good
yield.9) The experimental details for this key step are
shown in the experimental section.
Synthesis of (S)-[5-2H1]-1-O-benzyl-4-methylhexane-
1-ol (7). A solution of 6 (2.42 g, 7.6 mmol) in THF
(40 ml) containing LiAl2H4 (340 mg, 8.1 mmol) was
refluxed for 2 h. The reaction mixture was cooled to
room temperature, and then the excess reagent was
quenched by adding a small amount of water. The
solution was dried by adding anhydrous Na2SO4,
filtered, evaporated and chromatographed by silica gel
(hexane:CHCl3 = 2:1) to give 1.18 g (6.1 mmol, 80.3%)
of product 7. Deuterium incorporation was >96% from
the 1H-NMR and MS data (m=z 101 and 102, (Mþ–
OBn)). IR ꢁmax (CHCl3) cmꢂ1: 3032, 2951, 2867, 1454,
Deuterium was then introduced with the reduction of
6 by LiAl2H4 to give 7. Deprotection and Jones
oxidation of the resulting alcohol yielded labeled 4-
2
methylvaleric acid 8 which is the CH2 H1 derivative of
Hill’s intermediate;4) the product was converted to a
mixture of labeled (2S,4S)- and (2R,4S)-[5-2H1]leucine 9
by their procedure. Another isotopomeric mixture 11
could also be synthesized from (S)-citronellol 10
1
1094 cmꢂ1. H-NMR (90 MHz) ꢂH (CDCl3): 0.87 (2H,
2
m, CH2 H), 0.88 (3H, d, J ¼ 6:3 Hz), 1.10–1.80 (5H,
m), 3.45 (2H, t, J ¼ 6:6 Hz), 4.50 (2H, s), 7.32 (5H,
aromatic). 13C-NMR (22 MHz) ꢂC (CDCl3): 22.25 (t,
J ¼ 19 Hz), 22.52, 27.65, 27.83, 35.29, 70.85, 72.85,
127.42, 127.60 (ꢁ2), 127.60 (ꢁ2), 138.72. EIMS m=z:
193 (Mþ), 102 (Mþ–OBn), 91. Anal. Found: C, 80.59; H
(+2H), 10.38. Calcd. for C13H192H1O: C, 80.78; H
(+2H), 10.42.
25
(½ꢀꢀD ¼ ꢂ3:39 (neat)) in the same manner.
Possible stereochemical ambiguity was then elucidat-
ed. There was the possibility of racemization in the
decarboxylation-iodide formation step; 6 was converted
23
to (R)-2-methylpentane-1,5-diol 14 (½ꢀꢀD ¼ þ9:6
(c ¼ 1:2, ether)) in the three steps shown in Scheme 2,
and the optical rotation of 14 was compared with
25
known (ꢂ)-(S)-2-methylpentane-1,5-diol (½ꢀꢀD ꢂ10:0
Acknowledgments
(c ¼ 0:025, ether),10) or ꢂ8:5 (c ¼ 2:0, ether).11) Thus,
racemization was determined not to have occurred in
this step. This method would provide a pathway for
tritium labeling of leucine in the diastereotopic methyl
group. Utilizing the enzymatic hydrolysis of racemic
leucine acetate, a D or L (2R or 2S) amino acid could
also be obtained.4)
We thank Prof. Katsuki and Assoc. Prof. Irie (Kyushu
University) for measuring the optical rotation. This
study was supported in part by grant-aid from the Kurita
Water and Environmental Foundation.
References and Notes
Experimental
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Syntheses of amino acids incorporationg stable isotopes.
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Infrared spectra were obtained with a Perkin Elmer
1600 FT-IR spectrometer, 1H-NMR spectra were re-
corded with a Jeol EX-90 spectrometer, and HR-MS was
recorded with a Jeol D-300 mass spectrometer. Chro-
matographic separation was carried out with Merck
Kieselgel 60, 70–230 mesh columns.
Synthesis of (R)-1-O-benzyl-5-iodo-4-methylpentane-
1-ol (6). A solution of 5 (5.00 g, 21.1 mmol) in carbon
tetrachloride (450 ml) containing iodosobenzene diace-
tate (10.20 g, 26.9 mmol) and iodine (5.35 g, 21.1 mmol)
was irradiated with two 100-W tungsten lamps for 2 h at
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278
N. YAMAUCHI and S. ENDOH
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8) All new compounds were characterized by 1H-NMR,
IR and elemental analyses (or HR-MS). For example, 8:
1H-NMR (90 MHz) ꢂH (CDCl3): 1.00 (2H, m, CH22H),