Synthesis of individual glutamate-containing phosphonamidothionate stereoisomers

Article abstract of DOI:10.1016/S0040-4039(01)00746-8

The acquisition of the individual stereoisomers of chiral phosphonothioic acids is anticipated to reveal the significance of phosphorous stereochemistry upon the inhibition of metallocarboxypeptidases as well as their utility as chiral and stereoselective inhibitory probes. Two methods for preparing individual glutamate-containing phosphonamidothioic acid diastereomers have been identified. One method achieves the resolution through fractional crystallization of the intermediate 9-fluorenemethoxy phosphonamidates while the second does so thorough chromatographic resolution of intermediate β-(acylmercapto)ethyl phosphonamidothiolates.

Full text of DOI:10.1016/S0040-4039(01)00746-8

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 4313–4316
Synthesis of individual glutamate-containing
phosphonamidothionate stereoisomers
Haiyan Lu, Ying Hu, Cindy J. Choy, Jeremy P. Mallari, Alina F. Villanueva, Annamarie F. Arrozal
and Clifford E. Berkman*
San Francisco State University, Department of Chemistry & Biochemistry, 1600 Holloway Ave., San Francisco, CA 94132, USA
Received 8 March 2001; accepted 3 May 2001
Abstract—The acquisition of the individual stereoisomers of chiral phosphonothioic acids is anticipated to reveal the significance
of phosphorus stereochemistry upon the inhibition of metallocarboxypeptidases as well as their utility as chiral and stereoselective
inhibitory probes. Two methods for preparing individual glutamate-containing phosphonamidothioic acid diastereomers have
been identified. One method achieves the resolution through fractional crystallization of the intermediate 9-fluorenemethoxy
phosphonamidates while the second does so through chromatographic resolution of intermediate b-(acylmercapto)ethyl phospho-
namidothiolates. © 2001 Elsevier Science Ltd. All rights reserved.
Recent research efforts in our laboratory have been
aimed at developing potent competitive inhibitors for
the general class of enzymes known as glutamate car-
boxypeptidases (GCP). Examples of such metallopepti-
dase include N-acetylated-alpha-linked-acidic dipepti-
dase (NAALADase),1 prostate-specific membrane anti-
gen (PSMA),2 pteroylpoly-glutamate hydrolase (PPH),3
and carboxypeptidase G (CPG).4 The acquisition of
inhibitors for such enzymes is expected to further the
understanding of the biological role of these metallocar-
boxypeptidases as well as to serve in the elucidation of
germane active site features. In addition, inhibitors of
CPG2, an enzyme closely related to CPG and one for
which the crystal structure is known, have been recently
sought for the use in inhibiting non-tumor-localized
enzyme in ADEPT strategies.5
Based upon our preliminary evidence, compounds con-
taining the phosphonamidothionate motif (Fig. 1) show
strong promise as potent tetrahedral-intermediate
analog inhibitors of metallopeptidases with the unique
value of probing enzyme active-site architecture with
complementary chiral phosphorus centers.6 The basis
for the enhanced inhibitory potency of such com-
pounds, especially against zinc–metallopeptidases, is
presumably due to favorable zinc–sulfur interactions
within enzyme active sites.7
The focus of the present study was to explore method-
ology which would allow for the procurement of the
individual diastereomers of phosphonamidothionates
such as 1 and 2. With the acquisition of such individual
stereoisomers, the significance of phosphorus stereo-
chemistry of chiral phosphonothioic acids upon the
inhibition of metallocarboxypeptidases as well as their
utility as chiral and stereoselective inhibitory probes
can be ultimately addressed.
For the purposes of this work, two specific analogs 1
and 2 were targeted as representative glutamate-con-
taining phosphonamidothionates. The rationale for the
selection of the hydrocarbon ligands to phosphorus was
based upon the known capability of some GCP’s to
hydrolyze a variety of alkyl and aryl amides of glutamic
acid.4 Indeed, we recently noted that for this glutamate
carboxypeptidase, both methotrexate and N-[4-(4-nitro-
phenyl)butanoyl] glutamic acid exhibited very similar
Figure 1. Phosphonamidothionate inhibitors of GCP.
Keywords: chiral phosphonothioic acids; chiral phosphonamidothio-
lates; glutamate; b-(acylmercapto)ethyl; 9-fluorenemethoxy.
* Corresponding author. Tel.: 415-338-6495; fax: 415-338-2384;
e-mail: cberkman@sfsu.edu
kinetic profiles as substrates, the later substrate having
8
a slightly lower Km and higher Vmax
.
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)00746-8

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H. Lu et al. / Tetrahedron Letters 42 (2001) 4313–4316
In a previous report, phosphonamidothionates 1 and 2
were prepared utilizing a 2-cyanoethoxy ligand as a
protecting group on phosphorus. Although this group
could be conveniently removed simultaneously with the
glutamate methyl esters in mild basic conditions, it
afforded no chromatographic resolution of its synthetic
precursors.6 It was envisioned that a larger ligand could
impart either greater chromatographic resolution of the
chiral phosphorus center or allow synthetic intermedi-
ates to exists as solids allowing for separation of phos-
phorus diastereomers through fractional crystallization.
The selection of the phosphorus protecting group was
further constrained by the requirement of being
removed conveniently with the glutamate methyl esters
under mild basic conditions in a final step. It was noted
that the 9-fluorenemethoxy group had been previously
used as a phosphorus protecting group9 with the poten-
tial for meeting the established selection criteria. As
such, it was incorporated into the synthetic strategy
outlined in Scheme 1.
Phosphonamidates 3 and 4 were conveniently prepared
via the 1H-tetrazole catalyzed two-step reaction, which
could be fractionally recrystallized to provide both
individual diastereomers (3a, 3b and 4a, 4b) with good
yields.10 Stereospecific thionation with Lawesson’s
reagent was envisioned to provide the individual respec-
tive isomers (5a, 5b and 6a, 6b) with retention of the
phosphorus configuration,11 which could be further
deprotected with LiOH to provide the individuals 1 and
2. This tactic of sequential thionation12 and
deprotection13 worked as planned for phenyl congener
2 (Scheme 1). However, problems were encountered in
the thionation step with Lawesson’s reagent for the
butyl analog. It was found that, the conversion of the
individual isomers of 3a and 3b to 5a and 5b, respec-
tively, did not occur with absolute stereospecificity but
occurred with approximately 10% configuration inver-
sion at the phosphorus center giving a mixture of two
isomers.
Scheme 1. Synthesis and resolution of O-(9-fluorenemethyl)
phosphonamidothionates.
The problem of the slight racemization of the phospho-
rus configuration that occurred during the respective
thionation of 3a and 3b to 5a and 5b, was solved by
submitting these 90:10 diastereomeric mixtures of 5 to a
two-step deprotection procedure (Scheme 2). Following
treatment of 5 with quinine in refluxing methanol, the
9-fluorenemethoxy group was removed and the result-
ing quinine salt mixture was recrystallized to give the
single isomers 7a and 7b.14 Final deprotection with
LiOH afforded the target individual isomers 1a and 1b
as single diastereomers (Scheme 2).13
Scheme 2. Quinine resolution of phosphonamidothionates.
While working towards the preparation of a related
series of phosphonamidothiolates, it was discovered
and 10a, 10b) by silica-gel chromatography. Treatment
of 9 and 10, respectively, with quinine in refluxing
methanol removed the protecting group from the sulfur
ligand to phosphorus.14 Subsequent treatment with
LiOH/MeOH hydrolyzed the glutamate methyl esters
to afford 1 and 2 as single isomers.13
that
the
individual
diastereomers
of
b-
(acylmercapto)ethyl phosphonamidothiolates 9 and 10
were readily resolved by column chromatography. The
phosphonamidothiolates
9
and 10 were readily
prepared15 from the corresponding phosphonyl dichlo-
ride in a sequential reaction with CH3COS(CH2)2SH16
and Glu(OMe)2 and were subsequently and conve-
niently resolved into two single diastereomers (9a, 9b
It should be noted that the two-step deprotection pro-
cedure (first with quinine then with LiOH) provides 1
and 2 in essentially pure form without any undesired

H. Lu et al. / Tetrahedron Letters 42 (2001) 4313–4316
4315
Table 1. 31P NMR chemical shift data
formation of the corresponding phosphonamidothion-
ate; the major byproduct when 9 or 10 was treated with
LiOH directly without the pre-treatment with quinine.
Based upon these observations, it was hypothesized
that direct treatment of 9 or 10 with LiOH resulted in
significant displacement of CH3COS(CH2)2SH rather
than the desired deprotection. Therefore, when 9 or 10
was first treated with quinine in methanol, the resultant
thiophosphonamidate anion served to protect the phos-
phorus center from the attack of hydroxide in the
subsequent deprotection with LiOH.
Compound
R
31P NMR (l)
1a
1b
2a
2b
3a
3b
4a
4b
5a
5b
6a
6b
7a
7b
8a
8b
9a
9b
10a
10b
n-Bua
n-Bua
Pha
69.02
69.31
56.28
57.28
35.66
36.24
22.58
23.39
91.11
91.61
78.11
78.33
70.48
70.82
58.80
59.13
50.02
52.41
37.38
39.21
Pha
n-Bu
n-Bu
Ph
Ph
n-Bu
n-Bu
Ph
It is noteworthy to mention that the resolution of the
individual isomers was conveniently monitored by 31P
NMR (Table 1). All ‘a’ and ‘b’ isomers were identified
as intermediates in the synthesis of 1a or 2a and 1b or
2b, respectively. Determination of the absolute configu-
ration of the phosphorus center in these isomers is
currently underway.
Ph
n-Bua
n-Bua
Pha
Pha
n-Bu
n-Bu
Ph
In summary, the preparation of the individual
diastereomers of glutamate-containing phosphonami-
dothioic acids has been achieved through two methods;
fractional crystallization and chromatography. The suc-
cess of these methods relied upon the 9-fluorene-
methoxy or the b-(acetylmercapto)ethylthio groups as
phosphorus protecting ligands. Although targets 1 and
2 could be obtained as individual isomers by the frac-
tional recrystallization method (Schemes 1 and 2), this
procedure was quite time consuming. On the other
hand, the procurement of the individual diastereomers
of 1 and 2 through the chromatographic resolution of
phosphonamidothiolates 9 and 10 (Scheme 3) was rapid
and more convenient. With the acquisition of the indi-
vidual stereoisomers of compounds such as 1 and 2, the
significance of phosphorus stereochemistry of chiral
phosphonothioic acids upon the inhibition of metallo-
carboxypeptidases as well as their utility as chiral and
stereoselective inhibitory probes can now be examined.
Ph
a Chemical shifts were obtained in CD3OD and externally referenced
to H3PO (85%) in CD3OD, all others were obtained in CDCl3 and
externally referenced to H3PO (85%) in CDCl3.
Acknowledgements
This work was supported in part by grants from the
National Institutes of Health, MBRS SCORE Pro-
gram-NIGMS (Grant No. S06 GM52588-04) and the
MBRS-RISE Program-NIGMS (Grant No. GM59298-
02). A ‘Research Infrastructure in Minority Institu-
tions’ award from the National Center for Research
Resources with funding from the Office of Research on
Minority Health, National Institutes of Health (Grant
No. RR11805-02), and fellowships from the Depart-
ment of Defense Science Scholars Program (Grant No.
6-93472).
References
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J. Biol. Chem. 1987, 262, 14498.
Scheme 3. Synthesis and resolution of b-(acylmercapto)ethyl
phosphonamidothionates.
2. Carter, R. E.; Feldman, A. R.; Coyle, J. T. Proc. Natl.
Acad. Sci. USA 1996, 93, 749.

4316
H. Lu et al. / Tetrahedron Letters 42 (2001) 4313–4316
N-[hydroxy(n-butyl)phosphinothioyl]-
3. Reisenauer, A. M.; Krumdieck, C. L.; Hlasted, C. H.
L
-glutamic acid tri-
lithium salt (1a). 1H NMR (CD3OD): l 0.80 (t, J=7.2
Hz, 3H), 1.25 (m, 2H), 1.40–1.59 (m, 2H), 1.61–1.73 (m,
2H), 1.74–1.92 (m, 2H), 2.08–2.27 (m, 2H), 3.58 (dt,
J=10.8 Hz, J=6.3 Hz, 1H); 13C NMR (CD3OD): l
12.96, 23.87 (d, J=18.70 Hz), 26.43, 32.74, 34.12, 37.54
(d, J=93.98 Hz), 56.98, 180.91 (d, J=3.38 Hz), 181.99;
FABHRMS (M−Li) calcd 294.0729. Found 294.0728 for
C9H15Li2NO5PS.
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10. Experimental procedures for compounds 3 and 4. A
solution of 9-fluorenemethanol (0.53 g, 2.7 mmol) and
DEA (0.52 ml, 3.0 mmol) in benzene (6.0 ml) was added
via syringe to a stirring solution of alkylphosphonic
dichloride (0.42 ml, 3.0 mmol) and 1H-tetrazole (0.02 g,
0.27 mmol) in benzene (10 ml) under an argon atmo-
sphere at 4°C. The resulting solution was warmed to
room temperature and stirred for 3 h, followed by the
N-[hydroxy(n-butyl)phosphinothioyl]-L-glutamic acid tri-
lithium salt (1b). 1H NMR (CD3OD): l 0.85 (t, J=7.5
Hz, 3H), 1.31 (m, 2H), 1.51–1.76 (dm, 4H), 1.80–1.96 (m,
2H), 2.13–2.31 (m, 2H), 3.64 (dt, J=11.1 Hz, J=6.0 Hz,
1H); 13C NMR (CD3OD): l 14.43, 25.10 (d, J=18.45
Hz), 27.91, 34.22 (d, J=3.75 Hz), 35.64, 39.44 (d, J=
119.18 Hz), 58.40, 182.38 (d, J=4.65 Hz), 183.32;
FABHRMS (M−Li) calcd 294.0729. Found 294.0705 for
C9H15Li2NO5PS.
N-[hydroxy(n-butyl)phosphinothioyl]-L-glutamic acid tri-
lithium salt (2a). 1H NMR (CD3OD): l 1.57–1.77 (m,
2H), 1.88–2.14 (dm, 2H), 3.27 (dt, J=6.3 Hz, J=5.7 Hz,
1H), 6.98–7.08 (m, 3H), 7.57–7.64 (dm, 2H); 13C NMR
(CD3OD): l 34.19, 35.35, 58.50, 128.54, 128.71, 130.20,
131.80, 131.94, 144.51 (d, J=128.6 Hz), 181.62 (d, J=
5.10 Hz), 183.69; FABHRMS (M−Li) calcd 314.0416.
Found 314.0433 for C11H11Li2NO5PS.
N-[hydroxy(n-butyl)phosphinothioyl]-L-glutamic acid tri-
lithium salt (2b). 1H NMR (CD3OD): l 1.45–1.58 (m,
2H), 1.81–1.83 (m, 2H), 3.28 (dt, J=6.0 Hz, J=2.8 Hz,
1H), 6.96–7.02 (m, 3H), 7.57–7.64 (dm, 2H); 13C NMR
(CD3OD): l 33.72 (d, J=4.65 Hz), 35.55, 58.55, 128.45,
128.62, 130.24, 131.92, 132.04, 144.40 (d, J=131.4 Hz),
182.18 (d, J=3.75 Hz), 183.27. FABHRMS (M−Li) calcd
314.0416. Found 314.0411 for C11H11Li2NO5PS.
addition of a solution of L-glutamic acid dimethyl ester
(0.57 g, 3.2 mmol) and DEA (0.59 ml, 3.4 mmol) in
benzene (6.0 ml). The solution was stirred for another 3 h
and then filtered and concentrated in vacuo to afford a
yellow oil, which was purified by flash chromatography
(EtOAc:hexane 2:1, v/v) to give the ester 3 (62%) and 4
(60%). Phosphonamidates 3 and 4 were fractionally
recrystallized from acetone and hexane to afford the
individual diastereoisomers, respectively.
14. Experimental procedures for compounds 7 and 8. Reflux-
ing a solution of 5, 9 or 10 (0.45 mmol) and quinine (146
mg, 0.45 mmol) in methanol (5 ml) for 24 h followed by
concentration in vacuo gave a yellow oil, which was
recrystallized from acetone and hexane (3:10 v/v) to give
compounds 7 from 5 or 9 and 8 from 10.
15. Experimental procedures for compounds 9 and 10. Into a
flask charged with 1H-tetrazole (0.02 g, 0.3 mmol) and
alkylphosphonic dichloride (3.3 mmol) in benzene (10.0
mL) were added sequentially ethanedithiol monoacetate
(0.41 g, 3.0 mmol) and a solution of diisopropylethyl-
amine (0.52 mL, 3.0 mmol) in benzene (5.0 mL) via
syringe at 4°C under an argon atmosphere. The solution
was allowed to warm to room temperature and stirred
until ethanedithiol monoacetate was consumed (approxi-
mately 3 h) as monitored by TLC. Glutamic acid
dimethyl ester (0.58 g, 3.3 mmol) and DEA (0.59 mL, 3.4
mmol) in benzene (5.0 mL) was added dropwise to the
reaction mixture and allowed to stir for an additional 3 h.
The reaction mixture was concentrated in vacuo and
purified by flash chromatography to give 9 (65%) and 10
(60%) [EtOAc:hexane 4:1 v/v (9a: Rf=0.35. 9b: Rf=0.25.
10a: Rf=0.36. 10b: Rf=0.27)].
11. Jackson, J. A.; Berkman, C. E.; Thompson, C. M. Tetra-
hedron Lett. 1992, 33, 6061.
12. Experimental procedures for compounds 5 and 6. Reflux-
ing a solution of 3 or 4 (0.66 mmol) and Lawesson’s
reagent (0.37 mmol) in toluene (16 ml) for about 2 h
followed by concentration in vacuo gave a yellow oil,
which was purified by flash chromatography to give 5
(89%) and 6 (84%) [CH2Cl2: EtOAc 50:1 v/v (5a: Rf=
0.43. 5b: Rf=0.42. 6a: Rf=0.46. 6b: Rf=0.44)].
13. General experimental procedures for compounds 1 and 2.
Phosphonamidothionates 7 and 6 or 8 (0.1 mmol) were
dissolved in methanol (0.6 mL), into which was added a
1.0 M aqueous solution of LiOH (0.36 mL). The solution
was stirred at room temperature for 15 h and then
filtered. The solvent was evaporated in vacuo to give 1
and 2 as white residue, which was further purified by
re-suspension in anhydrous methanol and filtration (0.2
mm Teflon membrane) to provide the desired pure
trilithium salts 1 (68%) and 2 (81%).
16. Wiesler, W. T.; Caruthers, M. H. J. Org. Chem. 1996, 61,
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.