Differentiation of chiral phosphorus enantiomers by 31P and 1H NMR spectroscopy using amino acid derivatives as chemical solvating agents

Article abstract of DOI:10.1016/j.tetasy.2007.06.012

The ability of commercially available amino acid derivatives, especially Fmoc-Trp(Boc)-OH, to differentiate enantiomers of chiral phosphonates, phosphinates, phosphates, phosphine oxides, and phosphonamidates is demonstrated with ³¹P, ¹³

Full text of DOI:10.1016/j.tetasy.2007.06.012

Tetrahedron: Asymmetry 18 (2007) 1391–1397
Differentiation of chiral phosphorus enantiomers by 31P and 1H NMR
spectroscopy using amino acid derivatives as chemical solvating agents
Yingchun Li and Frank M. Raushel*
Department of Chemistry, Texas A&M University, PO Box 30012, College Station, TX 77842-3012, USA
Received 29 March 2007; revised 4 June 2007; accepted 6 June 2007
Abstract—The ability of commercially available amino acid derivatives, especially Fmoc-Trp(Boc)-OH, to differentiate enantiomers of
chiral phosphonates, phosphinates, phosphates, phosphine oxides, and phosphonamidates is demonstrated with 31P, 13C, and 1H NMR
spectroscopy. The chiral differentiation provided a rapid and convenient method for measuring the enantiomeric purity of these phos-
phorus compounds.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
rationally designed site-directed mutant enzymes have pro-
vided a convenient method for the kinetic resolution of
racemic phosphinate, phosphonate, and phosphate esters
through the stereoselective hydrolysis of a single enantio-
mer.13–15 These enzyme libraries have facilitated the
isolation of enantiomerically pure substrates of either ste-
reochemistry, since mutant enzymes have been identified
where the stereoselectivity is either enhanced or inverted
relative to the wild type enzyme.16,17 The absolute configu-
rations of the unhydrolyzed products could be reliably
predicted based upon the known stereoselectivity of the
wild type enzyme and the characterized mutant vari-
ants.17,18 However, experimental determination of the pre-
cise enantiomeric purity of the isolated phosphinate,
phosphonate, and phosphate ester products is not trivial.
For example, the enantiomeric purity of the four stereoiso-
mers of pinacolyl 4-nitrophenyl methylphosphonate and
the two enantiomers of 4-acetylphenyl methyl phenyl-
phosphonate were resolved with much effort by chiral
HPLC methods and chiral electrophoresis, respec-
tively.13,19 A more rapid and convenient method is needed,
as more of such enantiomers are routinely obtained by
enzymatic methods.
Chiral phosphorus oxide derivatives 1 are a class of com-
pound that have been widely utilized in both chemistry
and biology. Chiral phosphine oxides and phosphinate
monoesters are important precursors to the corresponding
phosphines that are used as chiral ligands for metal cata-
lysts in modern asymmetric transformations.1–4 The enan-
tiomers of chiral phosphonate and phosphate esters are
differentially toxic to multi-cellular organisms and these
compounds are used as agricultural pesticides and chemical
weapons.5–7 These applications have stimulated efforts
directed at the synthesis, resolution, and determination of
the enantiomeric purity of chiral phosphorus com-
pounds8–12 (Chart 1).
O
P
O
P
O
P
Y
H
N
O
SH
SH
X
Z
R1
OH
R2
1
2
3
4
An attractive alternative method for the determination of
the chiral purity of these compounds is the use of 31P
and 1H NMR spectroscopy with a chiral solvating agent.20
The potential advantages of using chemical solvating
agents for the differentiation of enantiomers, relative to
other methods, such as the use of lanthanide complexes
or the formation of covalent diastereomer derivatives with
chiral reagents, are well recognized.21 Chiral methyl phenyl
Chart 1.
Recent advances in the characterization of the catalytic
properties of the bacterial phosphotriesterase (PTE) and
*
Corresponding author. Tel.: +1 979 845 3373; fax: +1 979 845 9452;
e-mail: raushel@tamu.edu
0957-4166/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2007.06.012

1392
Y. Li, F. M. Raushel / Tetrahedron: Asymmetry 18 (2007) 1391–1397
phosphinothioic acid 2 and t-butyl phenyl phosphinothioic
acid 3 have been successfully used to discriminate between
the enantiomers of chiral phosphorus compounds includ-
ing the amide and ester derivatives of phosphonic and
phosphinic acids.21–27 However, these chiral reagents are
not commercially available.
FBTrp is presented in Figure 1A. At saturating concentra-
tions of FBTrp the maximum difference in chemical shift is
approximately 38 ppb and the data are consistent with the
formation of a 1:1 complex between compound 7 and
FBTrp.
To determine if the solvating agent preferentially binds one
of the two enantiomers over the other, enantiomerically
pure (RP)-7 and (SP)-7 were titrated separately with FBTrp
in CDCl3. The values of Kd for the complexes formed
between (RP)-7 or (SP)-7 and FBTrp were calculated by a
non-linear least squares analysis to be 21 1 and
19 1 mM, respectively, from a fit of the data to Eq. 1
for the formation of a 1:1 complex (Fig. 1B). In Eq. 1,
dob is the observed chemical shift for (RP)-7 or (SP)-7; dA
is the chemical shift of (RP)-7 or (SP)-7 in the absence of
FBTrp; dAB is the chemical shift for the complex of (RP)-
7 or (SP)-7 with FBTrp; ‘A’ is the total concentration of
(RP)-7 or (SP)-7; ‘B’ is the total concentration of FBTrp;
and Kd is the corresponding dissociation constant
Thioic acids 2 and 3 are apparently able to form transient
diastereomeric complexes in solution with chiral phospho-
rus esters but the specific mode of interaction is not pre-
cisely known. In an effort to mimic the molecular
interactions in these complexes with commercially avail-
able chiral acids, we chose N-acyl substituted amino acids
4. Modified amino acids have been used as chemical solvat-
ing agents for the enantiodiscrimination of chiral a-aryl-
alkylamines.28 The rationale for the choice of 4 as a
potential complexing agent for the differentiation of enan-
tiomers such as 1 is that a broad range of modified amino
acids are readily available and the free carboxylic acid may
function in a manner similar to the thioic acid in com-
pounds 2 and 3. The formation of hydrogen bonded com-
plex 5 might be the origin of the differentiation in the
chemical shifts between enantiomers.21,22 In this respect,
chiral carboxylic acids, such as 4, may form similar com-
plexes in solution as presented in complex 6. Compounds
of the general structure 4 are readily available as inexpen-
sive commercial derivatives of amino acids used in solid
phase peptide synthesis.29 Herein we report that 31P and
1H spectroscopy in the presence of protected L-amino acid
derivatives can differentiate between the enantiomeric
forms of chiral phosphorus oxide derivatives (Chart 2).
dob ¼ dA þ ðdAB ꢀ dAÞ ꢁ fðA þ B þ KdÞ
2
1=2
ꢀ ½ðA þ B þ KdÞ ꢀ 4ABꢂ g=ð2AÞ
ð1Þ
These results indicate that the binding strength between
FBTrp and either of the two enantiomers of 7 is approxi-
mately the same. This conclusion is consistent with the
observation that the 31P NMR signals for the two diaste-
reomers in the racemic mixture are of the same linewidth
and signal intensity. Therefore, the differentiation of the
two enantiomers by FBTrp is attributed to the intrinsic dif-
ference in the chemical shift of the two diastereomeric com-
plexes formed between FBTrp and (RP)-7 or (SP)-7. The
upfield signal in the 31P NMR spectra was assigned to
(RP)-7 and the downfield resonance to (SP)-7 by the addi-
tion of the corresponding pure enantiomers separately into
the racemic mixture in the presence of FBTrp. The NMR
spectra of (RP)-7 or (SP)-7 alone in the presence of FBTrp
demonstrated that the chiral purity of these compounds,
prepared via a kinetic enzymatic resolution, exceeded an
enantiomeric excess of 98%.
O
H
O
X
Y
Z
H
O
X
Y
Z
P
O
S
P
P
R2
O
R''
NH
R1
R'
5
6
Chart 2.
For racemic 7, the differentiation between the two enantio-
mers can easily be measured by 31P NMR spectroscopy,
but no separation was detectable by 1H NMR spectro-
scopy. In addition to FBTrp and N-Fmoc-L-phenylalanine,
other N-protected L-amino acids were assessed for the
differentiation of racemic 7. Most of them are unable to
discriminate between the two enantiomers and none gave
a better separation than FBTrp when measured by 31P
NMR or 1H NMR spectroscopy. A change in solvent from
chloroform-D to benzene did not improve the resolution.
2. Results and discussion
To test the potential of N-acyl L-amino acid derivatives as
chiral solvating agents for the differentiation of the chiral
phosphorus compounds, we added N-Fmoc-L-phenyl-
alanine to a solution of racemic 4-acetylphenyl cyclohexyl
methylphosphonate 7 (see Table 1). Two signals of moder-
ate separation without significant line broadening were
observed in the 31P NMR spectrum in CDCl3 at room
temperature (Fig. 3C). N-Fmoc-N0-Boc-L-tryptophan
(FBTrp), which has a larger side-chain group, was used
next and a better resolution between the two enantiomers
was obtained. To optimize the conditions for the differenti-
ation of the two enantiomers with this compound, we ti-
trated compound 7 with FBTrp in CDCl3 at room
temperature. The observed dependence of the separation
in the chemical shift (Dd in ppb) between the two enantio-
mers of 7 in the presence of an increasing concentration of
The ability of FBTrp to differentiate between the enantio-
mers of compounds, which are chiral at a phosphorus cen-
ter was further tested with the diasteromeric mixture of the
methyl phosphonate diester 8 (Fig. 2F). In the presence of
FBTrp, each of the four stereoisomers of 8 exhibited nearly
baseline resolved 31P NMR resonances as shown in Figure
2A. For the assignment of these resonances to specific dia-
stereomers, each of the four isomers was added separately
into the complete mixture of stereoisomers in the presence

Y. Li, F. M. Raushel / Tetrahedron: Asymmetry 18 (2007) 1391–1397
1393
Table 1. Differences in the chemical shifts between enantiomers induced upon the addition of 50 mM FBTrp in CDCl3 at 25 °C
Entry
X
Y
Z
Dd (ppb) 31P (NMR)
Dd (ppb)a 1H (NMR)
O
7
CH3–
28
0
0
O
O
O
O
O
O
O
8 (SPSC, RPRC)
8 (RPSC, SPRC)
9
CH3–
CH3–
CH3–
46
17
20
O
0
3
O
O
O
O
O
O
10
11
12
CH3–
CH3–
CH3–
0
13
54
5
3
0
O
O
O
O
O
O
13
14
15
16
CH3CH2–
CH3CH2–
CH3O–
51
0
0
7
2
0
O
CH3CH2O–
8
O
O
O2N
O
O
CH3O–
5
O
17
18
CH3O–
CH3–
0
3b
10
O
O
H2N–
46
O
O
19
20
CH3–
H2N–
80, 17
0
6, 4
6
CH3–
H3C
O
a When the separation of the proton resonance are indicated, the specific hydrogens are highlighted in bold font.
b In the presence of Fmoc-serine(trityl)-OH.
of FBTrp (Fig. 2B–E). The two most downfield signals
were assigned to enantiomeric pair of (SPSC)-8 and
(RPRC)-8 with the (SPSC)-8 isomer as the most downfield
resonance. The two upfield resonances were assigned to
(SPRC)-8 and (RPSC)-8 with (RPSC)-8 as the most upfield
resonance. Upon complexation with FBTrp, the chemical
shifts of the two diasteromers moved about 0.65 ppm.
The separation between the NMR signals of the enantio-
meric pair, (SPSC)-8 and (RPRC)-8, is 46 ppb, which is sig-
nificantly larger than the 17 ppb separation between the
enantiomeric pair of (SPRC)-8 and (RPSC)-8. This example
demonstrates that the separation of the NMR signals upon
the addition of FBTrp is dependent upon the identity of the
functional groups and stereochemical orientation distant
from the phosphorus center.
The generality of using FBTrp to differentiate between chi-
ral phosphorus compounds was tested in the resolution of
phosphonates 7–11, phosphinates 12–14, phosphates
15–17, phosphonamidates 18–19, and phosphine oxides
1
20–21 by both 31P and H NMR spectroscopy. The results
are summarized in Table 1. Racemic phosphonate com-
pounds with alkoxy groups smaller than cyclohexyl and
pinacolyl, 9–11, were utilized. For racemic compounds 9

1394
Y. Li, F. M. Raushel / Tetrahedron: Asymmetry 18 (2007) 1391–1397
40
30
20
10
0
A
F
28.1
28.0
27.9
27.8
27.2
27.1
27.0
26.9
(SpSc)-8
E
(RpSc)-8
D
(SpRc)-8
C
B
0
50
100
150
200
(RpRc)-8
FBTrp (mM)
B
A
28.2
28.0
27.8
28.7
28.6
28.5
27.8
27.7
27.6
PPM
Figure 2. Assignment of the 31P NMR signals for the individual
enantiomers within a racemic mixture of compound 8. (A) Spectrum of
diastereomeric 8 in the presence of FBTrp. (B) Addition of authentic
(RPRC)-8 enantiomer into diastereomeric 8. (C) Addition of (SPRC)-8
enantiomer into diastereomeric 8. (D) Addition of (RPSC)-8 enantiomer
into diastereomeric 8. (E) Addition of (SPSC)-8 enantiomer into diaste-
reomeric 8. (F) Spectrum of diastereomeric 8 in the absence of FBTrp.
27.6
27.4
27.2
24
20
16
150
100
12
50
the differences in the 31P NMR spectra for these resonances
are 54 and 51 ppb in the presence of FBTrp, about twice
that observed for the phosphonate esters. However, no dif-
ferentiation was observed in the 1H NMR spectra of either
12 or 13. For compound 14, no distinction between the two
enantiomers was observed in the 31P NMR spectrum but a
0
Figure 1. (A) Separation of the 31P NMR signals (Dd in ppb) for racemic
compound 7 (47.5 mM in 0.6 mL CDCl3) after the addition of FBTrp in
CDCl3 at 25 °C. (B) Binding of variable amounts of (SP)-7 with variable
amounts of FBTrp in CDCl3 at 25 °C. The data were fit to Eq. 1 with a Kd
of 19 mM.
1
separation of 7 ppb was measured in the H NMR spec-
trum of compound 14. The protons from the methyl group
of the ethoxy substituent, which is a triplet in the absence
of FBTrp, are further separated from one another with a
separation of 7 ppb.
and 11, the 31P resonances for the two enantiomers were
distinguishable from one another with differences in chem-
ical shift of 20 and 13 ppb, respectively. However, there
was no observable difference in the chemical shifts for the
individual enantiomers of 10 by 31P NMR spectroscopy.
Nevertheless, for the three phosphonate esters 9–11, the
1H NMR spectra revealed distinct resonances for the
methyl group directly attached to the phosphorus core.
Fully esterified phosphates were examined with compounds
15–17. These compounds showed the weakest separation in
the 31P NMR spectra in the presence of FBTrp. For com-
pounds 15 (Fig. 3D) and 16, the separation in chemical
shift values for the individual enantiomers are 8 and
5 ppb, respectively. For compound 15, the protons of the
1
In the absence of FBTrp, the H NMR signals for these
1
protons were observed as a doublet with a separation of
16 Hz due to spin coupling with the adjacent phosphorus.
In the presence of FBTrp, each of the resonances for the
methyl phosphonate group in 9–11 was further separated
into resonances for each of the enantiomers with separa-
tions of 3, 5, and 3 ppb, respectively.
methoxy group are separated by 2 ppb in the H NMR
spectrum but for compound 16 no separation was observed
in the presence of FBTrp. For compound 17, there was no
separation in either the 31P or 1H NMR spectra in the pres-
ence of FBTrp. To determine if other amino acid deriva-
tives could differentiate between the two enantiomers of
compound 17, we tested 18 other protected amino acids.
We found that Fmoc-serine (trityl)-OH (FTSer) gave a
moderate separation of 3 ppb for the proton resonances
Compounds 12–14 were assessed as examples of racemic
phosphinate esters. For compounds 12 (Fig. 3B) and 13,

Y. Li, F. M. Raushel / Tetrahedron: Asymmetry 18 (2007) 1391–1397
1395
O
O
P
O
O
O
P
1
2
5
15
D
NO2
21
3
4
6.70
6.65
6.60
6.55
6.50
O
6.45
Chart 3.
O
P
O
O
observed for the protons of the methyl group, which is at-
1
tached directly to the phosphorus center. In the H NMR
C
spectrum of compound 21 (Fig. 4A–D), the resonances
for the proton attached to C2, centered at ꢃ5.91 ppm
and coupled to the phosphorus core with JP–H of 25 Hz
(Fig. 4A), were further split with a separation of 19 ppb
in the presence of FBTrp (Fig. 4B). The resonance for
the methyl group of 21 is a singlet at 2.04 ppm (Fig. 4C)
in the absence of FBTrp but is a doublet with a separation
of 9 ppb in the presence of FBTrp (Fig. 4D). The two pro-
tons attached to C5 gave a complex set of resonances in the
range of 2.26–2.13 ppm (Fig. 4C) that are split into two sets
of resonances at 2.40–2.29 ppm and 2.25–2.16 ppm in the
presence of FBTrp (Fig. 4D). No separation for the reso-
nances of the protons attached to C4 was observed.
7
O
28.30
28.25
28.20
28.15
28.10
O
P
O
B
12
O
45.70
45.65
36.70
45.60
36.65
45.55
36.60
45.50
45.45
O
O
P
NH2
For compound 21, the 13C NMR spectra were also re-
corded in the presence and absence of FBTrp (Fig. 5A–
D). The 13C NMR resonance for C3 of compound 21, cen-
tered at 164.96 ppm with JP–C = 25.3 Hz as a doublet
(Fig. 5A), divided into two pairs of doublets with a separa-
tion of 42 ppb in the presence of FBTrp (Fig. 5B). The 13C
A
18
36.75
36.55
36.50
ppm
Figure 3. 31P NMR spectra for racemic mixtures of four types of chiral
phosphorus compounds utilized in this investigation in the presence of
FBTrp. (A) Phosphonamidate 18. (B) Phosphinate ester 12. (C) Methyl
phosphonate ester 7. (D) Phosphate triester 15.
CH
3
of the methoxy group, while no separation of the 31P NMR
signal was observed.
C
(C5)H
A
and CH
3
(C2)H
2
(C5)H
2
Compounds 18 and 19 were examined as examples for
phosphonamidates. In the presence of FBTrp in CDCl3,
both of these chiral compounds exhibited good separation
of the 31P NMR signals. The separation for compound 18
is 46 ppb for 31P (Fig. 3A) and 10 ppb for the hydrogens of
the methyl group. In the 31P NMR spectrum of the diaste-
reomeric mixture of 19, two pairs of signals centered at
36.70 and 36.55 ppm were separated by 80 and 17 ppb,
2.4
2.3
2.2
2.1
2.0
ppm
5.96 5.92 5.88 5.84
CH
3
B(C2)H + FBTrp
D
1
respectively. In the H NMR spectrum, the differences in
the chemical shifts for the protons of the methyl group
are 6 and 4 ppb for each enantiomeric pair. The absolute
configurations corresponding to these resonances have
not been determined.
(C5)H
(C5)H
2
2
The final chiral phosphorus compounds tested were the
phosphine oxides 20 and 21 (Chart 3). Neither compound
showed separation in their 31P NMR spectrum in the pres-
ence of FBTrp, although complexation of the two com-
pounds with FBTrp was indicated by a 4 ppm downfield
change in the chemical shift. In the H NMR spectrum
of compound 20, a separation of 6 ppb was observed for
the protons of the methoxy group, but no separation was
2.0 ppm
2.4
2.3
2.2
2.1
6.00 5.96 5.92
Figure 4. 1H NMR spectra for the protons at C2, C5, and methyl group of
compound 21. (A) 1H NMR signal for the proton at C2 in the absence
of FBTrp. (B) 1H NMR resonance for the proton at C2 in the presence of
FBTrp. (C) 1H NMR signal for the protons at C5 and the methyl group
of 21 in the absence of FBTrp. (D) Same as in C but in the presence of
FBTrp.
1

1396
Y. Li, F. M. Raushel / Tetrahedron: Asymmetry 18 (2007) 1391–1397
3. Conclusion
C
A
C3
Me
In conclusion, we have found that FBTrp is a versatile
chemical solvating agent that can be used for the differen-
tiation of chiral phosphorus centers in compounds that
include phosphine oxides, phosphinates, phosphonates,
1
phosphates, and phosphonamidates by 31P, 13C, and H
NMR spectroscopy. The separations of the resonances that
are induced by FBTrp in the 31P NMR spectra are substan-
tial except for the chiral phosphates and phosphine oxides,
for which the separation is relatively small. This observa-
tion has allowed the determination of the enantiomeric
purity for chirally enriched phosphorus compounds. Since
FBTrp is commercially available and relatively inexpen-
sive, it might be used as a convenient new chemical solvat-
ing agent for other chiral compounds that may include
chiral sulfoxides.30 FBTrp or other amino acid derivatives
may also be applied in the development of new methods for
the rapid and convenient determination of enantiomeric
excess.31
21.4
21.2
21.0
165.5
165.0
164.5
B
D
C3+FBTrp
Me+FBTrp
4. Experimental
4.1. General
21.4
166.0
21.2
21.0
167.0
166.5
Figure 5. 13C NMR spectra for C3 and the methyl group of compound 21.
(A) 13C NMR signal for C3 in the absence of FBTrp. (B) Same as in A but
in the presence of FBTrp. (C) 13C NMR signal of the methyl group in the
absence of FBTrp. (D) Same as in C but in the presence of FBTrp.
1
All of the 31P and H NMR experiments were carried out
with a Varian Inova-400 Broad Band Spectrometer unless
mentioned otherwise. For the 31P NMR spectra, the acqui-
sition time was set to 5–8 s with a 2 s delay. Aqueous phos-
phoric acid (85%) was used as an external reference. For
titration of the single enantiomers of (RP)-7 or (SP)-7,
aqueous phosphoric acid (85%) in a sealed capillary was
inserted into the NMR tube as an internal reference. For
collection of 1H and 13C NMR spectra, standard parameters
were used. The amino acid derivatives Fmoc-Trp(Boc)-
OH, Fmoc-Ser(Trityl)-OH were used as purchased from
EMD Biosciences. (4-Methoxyphenyl)methylphenylphos-
phine oxide 20 was purchased from ASDI Inc. 3-Methyl-
1-phenyl-2-phospholene 1-oxide 21 was purchased from
TCI America. The racemic phosphinates, phosphonates,
and phosphates ester were prepared following known pro-
cedures.13 Phosphonamidates 18 and 19 were made and
characterized as described below. Enantiomeric phospho-
nates were obtained by enzymatic resolution of their race-
mic mixtures.19
resonances for the methyl group, centered at 21.22 ppm
with JP–C = 17.2 Hz as a doublet (Fig. 5C), split into two
pairs of doublets with a separation of 25 ppb in the
presence of FBTrp (Fig. 5D). However, no separation
was observed for the 13C resonances of C2, C4, or C5.
A comparison of atypical separation in the 31P NMR
spectra induced by FBTrp for the five types of chiral phos-
phorus compounds tested in this investigation is presented
in Table 1. The magnitude of the chemical shift differences
between the pairs of enantiomers are: phosphinates ꢃ
phosphonamidates > phosphonates > phosphates > phos-
phine oxides. The downfield chemical shift changes in the
31P NMR spectra suggest the complexation of amino acid
derivatives with the phosphorus compounds. The discrim-
ination between enantiomers by the modified amino acids
is attributed to the chemical shift differences of the diaste-
romeric complexes because the dissociation constants for
the binding of the (RP)- and (SP)-enantiomers of com-
pound 7 to FBTrp are essentially identical. However,
we do not understand why some enantiomers can be differ-
entiated by 31P NMR and others by 1H or 13C NMR spec-
troscopy. A direct correlation of the absolute configuration
at phosphorus relative to the chemical shift of the diaste-
reomeric complex formed between each enantiomer and
FBTrp has not been determined. The direct interaction be-
tween these phosphorus compounds and FBTrp is expected
to be facilitated by hydrogen bonding. In this respect,
FBTrp might function as a versatile chemical solvating
4.2. Cyclohexyl methylphosphonamidate 18
To a solution of cyclohexanol (10 mmol) in ethyl ether
(20 mL) in a dry ice/acetone bath was added butyl lithium
(10 mmol, 2.5 M in hexanes). To the suspension was added
a solution of methylphosphonic dichloride (10 mmol) in
ethyl ether (40 mL). The mixture was stirred for 20 min in
a dry ice/acetone bath and then at room temperature for
1 h. After removal of the solvent, concentrated aqueous
ammonia (20 equiv) was added to the residue and the mix-
ture was stirred at room temperature for 30 min. After the
ammonia and water were removed under reduced pressure,
the residue was resuspended in ethyl ether, filtered, and
washed with ethyl ether. A solution of ethyl ether was col-
lected and condensed to dryness. Recrystallization from
1
agent in the H NMR spectroscopy for the enantio-differ-
entiation of chiral compounds, such as alcohols and
amines.

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ethyl ether yielded the desired compound 18 as colorless
1
crystals in 80% yield. H NMR (CDCl3, d in ppm): 4.50–
4.35 (2H, m); 2.83 (2H, br); 2.05–2.14 (14H, m). 31P (CDCl3,
d in ppm, 85% aqueous H3PO4 as external reference): 32.73.
4.3. Pinacolyl methylphosphonamidate 19
The diasteromeric mixture was prepared in an 85% yield
following the procedure described for 18. 1H NMR
(CDCl3, d in ppm): 4.25–4.17 (1H, CH, m); 2.96, 2.91
(2H, NH2, br s), 1.49, 1.51 (3H, CH3–P, two doublets,
JP–H = 16.7 Hz); 1.27, 1.24 (3H, CH3–C, two doublets,
JH–H = 6.70 Hz); 0.87, 0.88 (9H, t-butyl, two singlets).
31P (CDCl3, d in ppm, 85% aqueous H3PO4 as external ref-
erence): 33.11; 32.77.
18. Hermann, J. C.; Ghanem, E.; Li, Y.; Raushel, F. M.; Irwin, J.
J.; Shoichet, B. K. J. Am. Chem. Soc. 2006, 128, 15882–
15891.
Acknowledgment
19. Li, W.-S.; Lum, K. T.; Chen-Goodspeed, M.; Sogorb, M. A.;
Raushel, F. M. Bioorg. Med. Chem. 2001, 9, 2083–2091.
20. Parker, D. Chem. Rev. 1991, 91, 1441–1457.
21. Omelanczuk, J. O.; Mikolajczyk, M. Tetrahedron: Asymmetry
1996, 7, 2687–2694.
This work was supported in part by the NIH (GM 68550).
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