Stereoselective route to 15N-labeled-β-deuterated amino acids: Synthesis of (2S,3R)-[3-2H,15N]-phenylalanine

Article abstract of DOI:10.1016/S0957-4166(02)00487-1

(2S,3R)-[3-²H,¹⁵N]-Phenylalanine hydrochloride was synthesized utilizing ¹⁵N-labeled 8-phenylmenthylhippurate as a chiral glycine equivalent. The key step in the synthesis was the alkylation of the glycine template with (S)-(+)-benzyl-α-d-mesylate. The doubly labeled alkylation product was obtained in 89% yield with 92% de at the α-carbon and 74% de at the β-carbon. The nature of the electrophile employed in the alkylation step significantly affects the stereochemical outcome at the β-carbon. Hydrolysis of the alkylation product under acidic conditions followed by recrystallization from isopropanol yielded the title compound as the hydrochloride salt. Analysis of the (-)-camphanamide derivative of the final product by NMR spectroscopy and HPLC revealed a 76% de at the α-carbon and a 72% de at the β-carbon. The synthetic strategy described represents a simple yet versatile route to chirally deuterated β-methylene unit containing amino acids.

Full text of DOI:10.1016/S0957-4166(02)00487-1

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 13 (2002) 1893–1900
15
Stereoselective route to N-labeled-b-deuterated amino acids:
synthesis of (2S,3R)-[3-2H,15N]-phenylalanine
Derek W. Barnett,a Michael J. Panigotb and Robert W. Curley, Jr.a,*
aThe Ohio State University, Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, Columbus,
OH 43210, USA
bDepartment of Chemistry & Physics, Arkansas State University, State University, Arkansas 72467, USA
Received 9 July 2002; accepted 15 August 2002
Abstract—(2S,3R)-[3-2H,15N]-Phenylalanine hydrochloride was synthesized utilizing 15N-labeled 8-phenylmenthylhippurate as a
chiral glycine equivalent. The key step in the synthesis was the alkylation of the glycine template with (S)-(+)-benzyl-a-d-mesylate.
The doubly labeled alkylation product was obtained in 89% yield with 92% de at the a-carbon and 74% de at the b-carbon. The
nature of the electrophile employed in the alkylation step significantly affects the stereochemical outcome at the b-carbon.
Hydrolysis of the alkylation product under acidic conditions followed by recrystallization from isopropanol yielded the title
compound as the hydrochloride salt. Analysis of the (−)-camphanamide derivative of the final product by NMR spectroscopy and
HPLC revealed a 76% de at the a-carbon and a 72% de at the b-carbon. The synthetic strategy described represents a simple yet
versatile route to chirally deuterated b-methylene unit containing amino acids. © 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction
deuterated at the b-carbon. These materials will permit
the stereospecific NMR assignments of the prochiral
b-methylene protons, which will aid conformational
studies of amino acid residue side-chains and conforma-
tional analysis of receptor-bound peptide ligands.11–17
This report details our efforts on the synthesis of a
doubly labeled phenylalanine derivative.
Isotopically labeled amino acids are valuable biochemi-
cal tools that are used in many different areas of
research. They are frequently employed in mechanistic
enzymology to study biosynthetic pathways and the
stereochemical course of enzyme-mediated processes.1–3
Another field that relies extensively on these materials is
protein structure determination using NMR methods,
which can be greatly facilitated by the incorporation of
deuterium labeled amino acids.4–8 Stereospecific 1H
NMR assignments of prochiral protons can be
obtained using chiral deuteration, and these assign-
ments significantly ease obtaining coupling constant
information and distance constraints, and thus aid con-
formational analysis of the molecules under study. Pre-
vious work from our laboratory has led to the synthesis
of (2R)-[2-2H,15N]-glycine for NMR studies of FK506
binding protein.9,10 Once incorporated into the protein,
stereospecific NMR assignments of the glycine a-pro-
tons of FK506 binding protein bound to the immuno-
suppressant ascomycin were obtained using 15N-edited
TOCSY (TOtally Correlated SpectroscopY) experi-
ments. As an extension of this previous work, we are
currently interested in developing a general route to
15N-labeled amino acids that are stereoselectively
Several synthetic routes to diasterotopically b-deuter-
ated phenylalanine derivatives are known. These routes
can generally be characterized as methods that utilize
reduction of dehydroamino acids or halogenated
derivatives,18–23 or methods that rely on enzymatic steps
to introduce chirality and/or resolve stereoisomers.24,25
The method reported herein relies on the alkylation of
a labeled glycine equivalent with an enantiotopically
deuterated electrophile.
Alkylation of glycine equivalents is well precedented
and represents a common route to a-amino acid deriva-
tives.26–38 Excellent stereochemical control of the
nascent carbonꢀcarbon bond can be achieved with the
use of the appropriate chiral protecting groups, estab-
lishing the desired stereochemistry at the a-carbon. If
an enantiotopically deuterated electrophile were
employed in the alkylation reaction, it would be possi-
ble to introduce asymmetry at the b-carbon during
construction of the C2ꢀC3 bond, assuming an SN2
* Corresponding author. Tel.: 614-292-7628; fax: 614-292-2435;
e-mail: curley.1@osu.edu
0957-4166/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(02)00487-1

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D. W. Barnett et al. / Tetrahedron: Asymmetry 13 (2002) 1893–1900
reaction mechanism (Fig. 1). This methodology has
several features that make it an attractive approach to
these materials. The availability of labeled glycine in
multiple isotopomeric forms allows for the incorpora-
tion of different labeling patterns into the final product
that may be beneficial to certain NMR experiments. In
addition, the strategy is versatile, allowing synthetic
access to all stereoisomers at C2 and C3 by varying the
chirality of the protecting groups and electrophile.
Finally, the method should be general enough to allow
for the synthesis of other b-methylene unit containing
amino acids.
2. Results and discussion
As demonstrated by McIntosh and co-workers, N-ben-
zoyl glycinate (hippurate) esters undergo C-alkylation
stereoselectively when esterified with the appropriate
chiral alcohol.39–41 With (−)-8-phenylmenthol serving as
the chiral discriminator, formation of the C2ꢀC3 bond
proceeds with high stereoselectivity to give the precur-
sor to the L
-amino acid.41 Under these same reaction
conditions, the C-alkylation of a diastereomeric lithium
enolate derived from 15N-labeled 8-phenylmenthylhip-
purate with a chiral benzylic electrophile would yield a
Figure 1. Retrosynthesis of stereoselectively b-deuterated a-amino acids by alkylation of a glycine equivalent. The protecting
groups of the glycine equivalent control the stereochemistry at the a-carbon. The use of an enantiotopically deuterated electrophile
introduces asymmetry at the b-carbon provided an SN2 mechanism.
Figure 2. Synthesis of stereoselectively b-deuterated 15N-labeled phenylalanines using 8-phenylmenthylhippurates as chiral glycine
equivalents.

D. W. Barnett et al. / Tetrahedron: Asymmetry 13 (2002) 1893–1900
1895
It was believed that the bromide would be the preferred
electrophile due to its greater reactivity. This would
result in a higher chemical yield of the amino acid
derivative and, perhaps, enhanced stereoselectivity at
C3. However, use of any of the halides for the alkyla-
tion reaction would require two formal inversions of
configuration at the benzylic center, one for the func-
tional group interconversion from 4 and one for the
alkylation reaction. The use of the mesylate derivative
would have the apparent advantage of only requiring
one formal inversion during the process. However, the
impact, if any, of its attenuated reactivity in the alkyla-
tion reaction on C3 stereochemistry in the product was
unknown. Deuterated electrophiles 6a–d (Table 1) were
prepared from 4 by halogenation reactions known to
proceed with inversion of configuration47–49 and 6e was
synthesized by methyl sulfonate ester formation. The
labeled electrophiles were used to alkylate (−)-8-phenyl-
menthylhippurate 5 and the products 7 were analyzed
by 1H NMR spectroscopy to investigate C2 and C3
stereochemistry.
doubly labeled phenylalanine derivative (Fig. 2). The
use of the enantiomeric 8-phenylmenthyl alcohols
would allow the generation of both 2-position epimers.
Additionally, the chirality of the electrophile would
impact the stereochemistry at the benzylic position,
permitting both 3-position epimers to be synthesized.
However, incorporating and maintaining chirality at
the sensitive benzylic position throughout the synthesis
was a concern. Therefore, the synthesis of the chirally
deuterated electrophile and its properties in the ensuing
alkylation chemistry were of paramount importance.
Initial efforts focused on investigating electrophile reac-
tivity in hopes of optimizing the yield and stereoselec-
tivity of the alkylation reaction. Model reactions were
performed using several unlabelled benzylic elec-
trophiles and (−)-8-phenylmenthylhippurate. This
model study established benzyl bromide as the most
reactive electrophile of those investigated, alkylating the
chiral enolate at −78°C with high stereoselectivity. Ben-
zyl mesylate and benzyl chloride each alkylated the
chiral substrate at −42 and −15°C, respectively, with
equally good stereoselectivity at the a-carbon. In all
cases, the alkylation products were obtained in approx-
imately 90–92% de with the (2S)-epimer being preferred
as expected.41 Benzyl tosylate did not undergo the
alkylation even at room temperature, perhaps due to
increased steric congestion. These results suggested that
the two halides and mesylate were candidates for fur-
ther studies as deuterated electrophiles.
The first electrophile investigated was the labeled benzyl
bromide 6a, prepared using triphenylphosphine and
carbon tetrabromide.48 Unfortunately, product 7a
showed a 1:1 ratio of stereoisomers at the benzylic
position as assessed by NMR spectroscopy (Table 1,
Fig. 3b). Likewise the use of 6b, the labeled bromide
prepared using modified Mitsunobu conditions,49
resulted in similarly poor selectivity in the formation of
7b. However, 7c obtained from the chloride prepared
using triphenylphosphine and carbon tetrachloride,
showed a slight stereoselectivity (3R/3S=2:3). Investi-
gating other chlorination conditions to perhaps
enhance this modest selectivity, the modified Mit-
sunobu conditions were employed to generate 6d, which
resulted in a selectivity of approximately 9:1 in favor of
the (3S)-epimer of 7d (Table 1, Fig. 3c). It was expected
that mesylate 6e would yield the (3R)-epimer of 7 as the
major product since 6e was of opposite configuration
from 6d. Indeed, 7e showed a similar degree of selectiv-
ity (approximately 9:1) in favor of the (3R)-epimer
(Table 1, Fig. 3d). These results were very encouraging
and demonstrated that a high degree of stereoselectivity
could be introduced at the b-carbon while constructing
the C2ꢀC3 bond to give the desired a-carbon stereoiso-
mer. The racemization observed in 7a/b synthesized
from the bromides may be due to the inherent
enhanced reactivity of the electrophile. It is feasible to
suggest that the labeled benzyl bromide was reactive
enough to undergo bromide displacement reactions that
ultimately resulted in racemization of the electrophile.
Examination of the literature revealed that other
researchers have observed similar findings using the
triphenylphosphine and carbon tetrabromide bromina-
Scheme 1 depicts the synthesis of (S)-(+)-benzyl-a-d
alcohol 4, which served as the precursor for deuterated
electrophile synthesis. This strategy, which was based
on the generation of a deuterated aldehyde42 and its
subsequent asymmetric reduction with a chiral borane
reagent,43 was the method of choice for several reasons.
The chemistry proceeded exceptionally well on a large
scale with excellent deuterium incorporation and repre-
sented a simple way to obtain these materials in a cost
effective manner. Treating benzaldehyde with morpho-
line perchlorate and KCN gave morpholineacetonitrile
1. Proton-deuteron exchange was accomplished by
reaction of 1 with NaH and D2O to yield the deuterated
2
analog 2 with 99% H incorporation as determined by
1H NMR spectroscopy. Acid hydrolysis gave 3 which
was then reduced asymmetrically with (R)-Alpine
Borane® to yield 4 with greater than 96% ee as deter-
mined by 1H NMR analysis of the (−)-camphanate
ester.44 It was critical to avoid degradation of the
benzylic stereogenic center in the subsequent conversion
to an electrophilic species. Therefore, sulfonate esters
and halides obtained via stereospecific processes45,46
were investigated as potential electrophiles.
2
Scheme 1. Synthesis of 4. Reagents and conditions: (a) Morpholine, perchloric acid, KCN, (91%); (b) NaH, D2O, (99%, 99% H
incorporation); (c) 2N HCl, (93%); (d) (R)-Alpine Borane®, (87, 96% ee).

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D. W. Barnett et al. / Tetrahedron: Asymmetry 13 (2002) 1893–1900
Table 1. Evaluation of deuterated electrophiles
E+
Y
X
Configuration of 6a–e
Alkylation reaction temp. (°C)
Product
(3R):(3S) in 7a–e
6a
6b
6c
6d
6e
Ph3P/CBr4
Mitsunobu
Ph3P/CCl4
Mitsunobu
Br
Br
Cl
Cl
R
R
R
R
S
−78
−78
−15
−15
−42
7a
7b
7c
7d
7e
1:1
1:1
2:3
1:9
9:1
DMAP/MsCl/NEt3
OMs
Figure 3. Benzylic region of 1H NMR spectra of 7 (400 MHz) Xc*=(−)-8-phenylmenthyl. (a) Undeuterated compound from
authentic -phenylalanine. (b) Compounds 7a and 7b, showing a 1:1 ratio of stereoisomers at b-carbon. (c) Compound 7d,
showing (3R/3S) of approximately 1:9. (d) Compound 7e, showing (3R/3S) of approximately 9:1.
L
tion of optically active alcohols.50 The attenuated reac-
tivity of the chlorides and mesylate may have been
sufficient enough to prevent this racemization, which
ultimately led to the observed stereoselectivity. Based
on these results, the mesylate was selected as the elec-
trophile for the synthesis of the doubly labeled material,
however, these results clearly indicate that the labeled
chloride could be used to generate the 3-epimer.
Shown in Scheme 2 are the key steps in the synthesis of
(2S,3R)-[3-2H,15N]-phenylalanine hydrochloride 10.
15N-(−)-8-Phenylmenthylhippurate 8 was obtained from

D. W. Barnett et al. / Tetrahedron: Asymmetry 13 (2002) 1893–1900
1897
Scheme 2. Synthesis of 10. Reagents and conditions: (a) DMAP, MsCl, NEt3, THF, 0°C, (86%); (b) i. 2 equiv. LDA, 2 equiv.
TMEDA, THF, −78°C, ii. 1.1 equiv. 6e, −42°C, 12 h, (89%); (c) 6N HCl, reflux in sealed bottle, 36 h, (86%).
the benzoylation of 15N-glycine followed by esterifica-
tion with (−)-8-phenylmenthol.51 Mesylate 6e was
obtained from 4 in 86% yield using DMAP catalyzed
conditions. Treatment of 8 with 2 equiv. of LDA and
TMEDA followed by the addition of 6e afforded the
doubly labeled alkylation product 9 in 89% yield with
92% de at C2 and 74% de at C3.
3. Conclusions
A simple yet versatile strategy for the chemical synthe-
sis of 15N-labeled stereoselectively b-deuterated amino
acids has been presented. These labeled compounds are
valuable research tools that find use in NMR or in
mechanistic enzymology studies. The utility of the
method was demonstrated by the synthesis of 10. The
key step in the synthesis was the alkylation of an
isotopically labeled glycine equivalent with an enan-
tiotopically deuterated electrophile. Several deuterated
benzylic electrophiles were evaluated in the alkylation
reaction and it was determined that 6d/e resulted in
higher stereoselectivities at C3 in 7 than did 6a/b. These
results suggest that the alkylation reaction proceeds
predominantly by an SN2 type mechanism since much
of the optical activity present in 4 is ultimately trans-
ferred into the alkylation products. These results also
demonstrate that asymmetry can be introduced at C3
during the stereoselective construction of the C2ꢀC3
bond.
In early model reactions with unlabelled compounds,
the (2R)-epimer of the alkylation product was obtained
in diastereomerically pure form from the alkylation
product mixture by crystallization from hexanes/ethyl
acetate and was used as a standard for HPLC analyses.
Thus, fractional crystallization represents a way to
obtain further enriched mixtures of the (2S)-epimer of
9.
Acid hydrolysis afforded 10, which was purified by
recrystallization from an isopropanol/water mixture
(Fig. 4). The hydrolysis of the hindered benzamide of 9
proved to be problematic requiring long reaction times
under reflux conditions in a closed vessel. Stereochemi-
cal analysis of the final product 10 revealed only partial
degradation of the enantiomeric purity at the a-carbon
relative to 9. Analysis of the (−)-camphanamide methyl
ester52 of 10 by HPLC showed 76% de at the 2-position.
Deuterium NMR studies of the (−)-camphanamide
derivative (free acid) of 10 revealed a 72% de at the
3-position which is consistent with 9. The loss of stereo-
chemical integrity at the a-carbon during the hydrolysis
was attributed to the harsh conditions employed in the
deprotection.
The versatility of the strategy allows for the synthesis of
all stereoisomers at the a and b carbons as well as for
the incorporation of different isotopic labeling patterns
through the use of isotopically labeled glycine equiva-
lents. Efforts are underway to further optimize and
extend this strategy to other b-methylene unit contain-
ing amino acids. The current deprotection protocol
resulted in partial racemization at the a-stereocenter. It
should be possible to avoid this stereochemical degra-
dation by the use of alternative deprotection condi-
1
Figure 4. Partial H NMR (400 MHz) spectrum of 10.

1898
D. W. Barnett et al. / Tetrahedron: Asymmetry 13 (2002) 1893–1900
tions. In addition, alternative glycine equivalents are
being investigated in an effort to further enhance the
stereoselectivity of the alkylation reaction.
cooled to 0°C and D2O (15 mL, 0.83 mol) was carefully
added. The mixture was then stirred for 30 min at 0°C,
acidified (pH 1–2) by the addition of freshly distilled
SOCl2, and poured onto ice with stirring. The resulting
white precipitate was collected, washed with water, and
dried to yield a white crystalline solid (9.91 g, 99%; 99%
2H incorporation determined by 1H NMR): mp 67–
68°C (lit. 69–70°C);42 IR (cm−1): 1118 (s), 2864 (m),
4. Experimental
4.1. General methods
1
1453 (m), 1015 (m), 862 (m), 700 (m), 2229 (w); H
Cambridge Isotope Laboratories supplied 15N-glycine
and D2O. All moisture sensitive reactions were per-
formed using oven or flame-dried glassware cooled
under argon and were maintained under an argon
atmosphere. Anhydrous THF was obtained by distilla-
tion from sodium/potassium benzophenone ketyl imme-
diately prior to use. Lithium diisopropylamide
concentrations were determined by titration against
diphenylacetic acid. Thin layer chromatography was
performed using silica gel 60 F254 aluminum-backed
plates. Column chromatography was performed using
silica gel 60 (70–230 mesh). Melting points were deter-
mined using a Thomas–Hoover capillary apparatus and
are uncorrected. Analytical scale HPLC analyses were
performed using a Beckman Ultrasphere ODS column
(4.6×250 mm) using UV detection. Semi-preparative
HPLC was performed using a Zorbax ODS column
(9.4×250 mm) with UV detection. Infrared spectra were
acquired as films on AgCl plates. Nuclear magnetic
resonance spectra were acquired at 400 MHz (1H) and
100 MHz (13C) unless otherwise indicated and refer-
enced to non-deuterated solvent resonances. 15N NMR
spectra were recorded at 40.5 MHz and referenced
externally to a 15N-glycine sample in D2O (31.5 ppm).
Deuterium NMR spectra were recorded at 92 MHz.
Mass spectrometry was performed using a Micromass
QTOF spectrometer.
NMR (CDCl3, l): 2.42 (t, 4H, J=4.4 Hz), 3.51–3.60
(m, 4H), 7.15–7.28 (m, 3H), 7.38–7.40 (m, 2H); 13C
NMR (CDCl3, l): 49.75, 61.85 (t, J=22.1 Hz), 66.60,
115.00, 127.82, 128.74, 128.97, 132.46; HRMS-ES (m/
z): [M+Na]+ calcd for C12H13DN2ONa 226.1082; found
226.1070.
4.4. Benzaldehyde-formyl-d, 3
A mixture of 2 (5 g, 25 mmol) and 2N HCl (60 mL)
was heated under reflux for 12 h under an argon
atmosphere after which the resulting two phase mixture
was cooled to room temperature and extracted with
diethyl ether. The combined organic phases were then
washed with saturated NaHCO3, water, and brine. The
organic solution was dried over MgSO4, filtered, and
concentrated under reduced pressure at ambient tem-
perature to yield a slightly yellow oil that was used
without further purification (2.45 g, 93%; 98% 2H incor-
1
1
poration determined by H NMR): H NMR (CDCl3,
l): 7.39–7.68 (m, 3H), 7.87 (d, 2H, J=4.9 Hz), 10.01 (s,
residual CHO).
4.5. (S)-(+)-Benzyl-a-d alcohol, 443
A THF solution of R-Alpine-Borane® (0.5 M, 70 mL;
35 mmol) was added to 3 (2.3 g, 21.5 mmol). The
mixture was stirred at room temperature for 12 h and
then heated under reflux for 1.5 h. After cooling to
room temperature, acetaldehyde (5 mL) was added and
the mixture was stirred for 30 min after which the
solvent was removed. Some of the pinene by-products
were then removed using a rotary evaporator under
high vacuum at 50°C for 5 h. The remaining orange oil
was dissolved in diethyl ether (75 mL) and cooled to
0°C. Ethanolamine (2.1 g, 35 mmol) was added and the
mixture was stirred for 30 min at 0°C during which
time a white solid precipitated from the mixture. The
mixture was filtered and washed with diethyl ether. The
filtrate was concentrated and the resulting oil was dis-
solved in 10% aq. MeOH and washed several times
with heptane. Isopropanol was added to the methanolic
fraction and the solution was concentrated on a rotary
evaporator. The resulting yellow oil was chro-
matographed on silica gel (95:5 hexanes/ethyl acetate)
to yield 2.0 g of colorless oil (87, >96% ee): h2D1 +1.2°
neat);43 IR (cm−1): 3349 (s), 2924 (s), 1452 (s), 1406 (s),
1320 (s), 1043 (s), 1024 (s), 723 (s), 697 (s), 3029 (m),
4.2. a-Phenyl-4-morpholineacetonitrile, 142
Perchloric acid (70%; 9.5 mL, 0.11 mol) was added
dropwise to a stirred solution of morpholine (20 mL) at
0°C followed by the addition of benzaldehyde (11.5 g,
0.11 mol). The mixture was heated to 70°C for 4 h after
which time, a solution of KCN (7.8 g, 0.12 mol; 5 mL
H2O) was added. After heating to 90°C for 1 h, the
contents were poured onto ice with stirring. The result-
ing light yellow precipitate was collected, washed with
water, and recrystallized from absolute ethanol to yield
white needles (18.8 g, 91%): mp 67–68°C (lit. 68–
70°C);53 IR (cm−1): 1117 (s), 2857 (m), 1453 (m), 1007
1
(m), 866 (m), 701 (m), 2227 (w); H NMR (CDCl3, l):
2.27–2.38 (m, 4H), 3.45–3.57 (m, 4H), 4.65 (s, 1H),
7.15–7.22 (m, 3H), 7.34–7.36 (m, 2H); 13C NMR
(CDCl3, l): 49.79, 62.31, 66.55, 115.03, 127.80, 128.61,
128.79, 132.55; HRMS-ES (m/z): [M+Na]+ calcd for
C12H14N2ONa 225.1004; found 225.0991.
4.3. a-Phenyl-4-morpholineacetonitrile-a-d, 254
1
1205 (m), 2137 (w), 1949 (w); H NMR (CDCl3, l):
Sodium hydride (95%; 2.5 g, 99 mmol) was carefully
added to a THF solution of 1 (10 g, 50 mmol; dried
under high vacuum at 40°C for 12 h) and the mixture
was heated to 40°C for 1 h. The reaction mixture was
1.90 (s, 1H), 4.64 (t, 1H, J=1.8 Hz), 7.26–7.37 (m, 5H);
13C NMR (CDCl3, l): 64.20 (t, J=22.1 Hz), 126.79,
127.23, 128.21, 140.64; HRMS-EI (70 eV) m/z: M+
calcd for C7H7DO 109.0637; found 109.0569.

D. W. Barnett et al. / Tetrahedron: Asymmetry 13 (2002) 1893–1900
1899
4.6. Stereochemical analysis of 4
1H), 2.70–2.79 (m, residual CH), 3.00 (d, J=5.8 Hz,
1H), 4.23 (dd, J=7.5 Hz, 6.5 Hz, 1H), 4.81 (dt, J=10.8
Hz, 4.3 Hz, 1H), 6.29 (dd, J=91.1 Hz, 7.5 Hz, 1H),
7.00–7.69 (m, 15H); 13C NMR (CDCl3, l): 21.73, 23.89,
26.45, 28.85, 29.31, 31.29, 34.48, 36.91 (t, J=19.2 Hz),
39.49, 41.58, 50.41, 53.16, 53.29, 76.21, 125.25, 125.41,
126.66, 126.98, 128.08, 128.23, 128.51, 129.53, 131.49,
134.34, 134.43, 136.20, 151.54, 166.52 (d, J=16.2 Hz),
170. 83; 15N NMR (CHCl3, l): 112.01 (d, J=91.1 Hz);
HRMS-ES (m/z): [M+Na]+ calcd for C32H36D15NO3Na
508.2828; found 508.2806.
1
The enantiomeric purity of 4 was determined using H
NMR spectroscopy of the (−)-camphanate ester deriva-
tive.44 Briefly, 4 was treated with excess (1S)-(−)-cam-
phanic chloride and K2CO3 in toluene. The product
was purified using preparative TLC and analyzed by
1
NMR: H NMR (800 MHz, CDCl3, l): 5.35 (broad s,
major), 5.39 (broad s, minor), major/minor >98:2.
4.7. (S)-(+)-Benzyl-a-d mesylate, 6e
Methanesulfonyl chloride (0.66 mL, 8.5 mmol) was
added to a THF solution of 4 (750 mg, 6.87 mmol),
DMAP (84 mg, 0.69 mmol), and NEt3 (1.43 mL, 10.3
mmol) at 0°C. The reaction mixture was stirred at 0°C
for 1 h and then diluted with water and extracted with
ethyl acetate. The solvent was evaporated and the crude
product was chromatographed on silica gel (1:1 hex-
anes/ethyl acetate) to yield 1.1 g of colorless oil (86%):
4.9. Stereochemical analysis of 9
Retention times for HPLC analyses were determined
using phenylalanine standards prepared from authentic
L
- and D,L-phenylalanine. The amino acids were ben-
zoylated and the crude products esterified with (−)-8-
phenylmenthol.51 Enantiomerically pure (2R)-epimer
was obtained from fractional crystallization of the race-
mate mixture containing solution.
1
[h]2D1 +0.5 (c 12.8, ethyl acetate); H NMR (250 MHz,
CDCl3, l): 2.87 (s, 3H), 5.21 (t, 1H, J=1.7 Hz),
7.35–7.45 (m, 5H); 13C NMR (62.9 MHz, CDCl3, l):
38.39, 71.23 (t, J=23.1 Hz), 128.90, 129.42; HRMS-ES
(m/z): [M+Na]+ calcd for C8H9DSO3Na 210.0327;
found 210.0319.
4.10. (2S,3R)-[3-2H, 15N]-Phenylalanine hydrochloride,
10
Chromatographed 9 was heated with 6N HCl under
reflux in a sealed vessel for 36 h after which the reaction
mixture was extracted with ethyl acetate. TLC analysis
of the organic phases revealed unreacted 9, which was
isolated and re-submitted to the hydrolysis conditions.
This process was repeated until the organic phase was
devoid of starting material. The aqueous phases were
pooled, diluted with isopropanol, and concentrated to
give crude 10 which was recrystallized from iso-
propanol/water (86%): mp 190–200°C dec.; [h]2D0 −10.75
4.8. (2S,3R)-N-Benzoyl-[3-2H,15N]-phenylalanine-(−)-8-
phenylmenthyl ester, 9
Diisopropylamine (0.43 mL, 3.05 mmol) was added to a
THF solution of 2.5 M n-BuLi (1.22 mL, 3.1 mmol) at
−78°C with stirring. After 15 min, TMEDA (0.46 mL,
3.1 mmol) was added and the mixture was allowed to
warm to room temperature. After cooling back to
−78°C, a THF solution of 8 (600 mg, 1.52 mmol) was
added via cannula. The resulting yellow solution was
stirred at −78°C for 1 h and then warmed to −42°C. A
THF solution of 6e (300 mg, 1.60 mmol) was then
added via cannula and the reaction mixture was stirred
at −42°C for 12 h. The reaction was quenched at −42°C
with 1N HCl (10 mL) and allowed to warm to room
temperature. After stirring for 30 min, the phases were
separated and the aqueous phase was extracted with
ethyl acetate. The organic phases were combined,
washed with water, diluted with isopropanol, and con-
1
(c 5.0, H2O); H NMR (D2O, l): 2.98–3.04 (m, residual
1H), 3.14 (broad triplet, 1H), 3.96 (d, J=5.2 Hz, 1H),
7.16–7.29 (m, 5H); 13C NMR (D2O/MeOH, l): 36.34 (t,
J=19.6 Hz), 55.92 (d, J=7.7 Hz), 128.47, 129.80,
130.00, 135.22, 173.52; 15N NMR (D2O, l): 39.80;
HRMS-ES (m/z): [M−Cl]+ calcd for C9H11D15NO2
168.0901; found 168.0897.
4.11. Stereochemical analysis of 10
The enantiomeric purity of 10 was assessed using the
(−)-camphanamide methyl ester derivative.52 HPLC
analysis of the derivatized material showed a 76% de at
the 2-position, and deuterium NMR studies revealed a
72% de at the 3-position.
1
centrated to give an orange oil. HPLC and H NMR
analysis of the crude product revealed 92% de at the
2-position with the (2S)-epimer as the major product
and 74% de at the 3-position. The crude product was
partially purified on silica gel (85:15 hexanes/ethyl ace-
tate) yielding 9 as a mixture of the (2S)- and (2R)-
epimers (660 mg, 89%). A portion of the mixture was
then further purified using semi-preparative HPLC
(80% MeOH/H2O) to yield 83 mg of a white solid
which was used for all analytical measurements: [h]D21
−12.1 (c 4.1, ethyl acetate); HPLC (85% MeOH/H2O,
u=254 nm) tR(2S,3R/S)=20.0 min, tR(2R,3R/S)=15.8 min;
IR (cm−1): 2955 (s), 2924 (s), 1729 (s), 1666 (s), 1479 (s),
700 (s), 3360 (m), 3060 (m), 1602 (m), 1497 (m), 1368
(m), 1210 (m); 1H NMR (CDCl3, l): 0.81–1.02 (m, 5H),
1.03–1.31 (m, 8H), 1.68 (d, J=12.5 Hz, 1H), 1.83 (dt,
J=13.8 Hz, 3.4 Hz, 2H), 2.09 (dt, J=11.3 Hz, 3.4 Hz,
Acknowledgements
We gratefully acknowledge financial support of this
work by the National Science Foundation (NSF MCB
9723642). The authors thank Dr. Charles E. Cottrell
and the OSU Campus Chemical Instrument Center
(CCIC) for help with the high field (600 and 800 MHz)
NMR experiments. In addition, the authors are appre-
ciative of the assistance provided by Mr. John W.
Fowble.

1900
D. W. Barnett et al. / Tetrahedron: Asymmetry 13 (2002) 1893–1900
28. Williams, R. M. Synthesis of Optically Active h-Amino
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