Synthesis of N-phosphoryl amino acids using bis(9-fluorenylmethyl)phosphite

Article abstract of DOI:10.1016/j.tetlet.2005.06.022

Bis(9-fluorenylmethyl)phosphite (BFMP) was found to be an effective reagent for the N-phosphorylation of various amino acid methyl esters. BFMP was prepared from N,N-diisopropyl phosphoramidous dichloride in a one-pot two-step reaction and was obtained as a crystalline solid. N-Phosphorylation of the methyl esters of seven representative amino acids with BFMP was high-yielding and generally resulted in crystalline products. Complete deprotection of both the 9-fluorenylmethylphosphosphate esters and the amino acid methyl esters was accomplished concomitantly with LiOH to give N-phosphoryl amino acids.

Full text of DOI:10.1016/j.tetlet.2005.06.022

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 5301–5303
Synthesis of N-phosphoryl amino acids using
bis(9-fluorenylmethyl)phosphite
Lisa Y. Wu and Clifford E. Berkman*
San Francisco State University, Department of Chemistry & Biochemistry, 1600 Holloway Ave., San Francisco, CA 94132, USA
Received 23 May 2005; revised 7June 2005; accepted 7June 2005
Abstract—Bis(9-fluorenylmethyl)phosphite (BFMP) was found to be an effective reagent for the N-phosphorylation of various
amino acid methyl esters. BFMP was prepared from N,N-diisopropyl phosphoramidous dichloride in a one-pot two-step reaction
and was obtained as a crystalline solid. N-Phosphorylation of the methyl esters of seven representative amino acids with BFMP was
high-yielding and generally resulted in crystalline products. Complete deprotection of both the 9-fluorenylmethylphosphosphate
esters and the amino acid methyl esters was accomplished concomitantly with LiOH to give N-phosphoryl amino acids.
ꢀ
2005 Elsevier Ltd. All rights reserved.
5
Phosphoramidates and their derivatives continue to rep-
resent an important class of biologically relevant struc-
tures. Our group has recently been interested in such
compounds as competitive inhibitors of glutamate carb-
oxypeptidases, specifically prostate-specific membrane
antigen (PSMA). The most potent inhibitor for this en-
zyme that we have identified to date is a relatively sim-
mediate. Specifically, the lack of a chromophore or a
responsiveness to stains typically used for thin layer
chromatography presented a problem in identifying
fractions that contained the desired intermediate during
purification by flash chromatography. To address these
problems, we sought a protecting group for phosphor-
amidates which could be conveniently removed under
mild basic conditions. This constraint was important
since the P–N bond is labile under acidic conditions typi-
cally used in the deprotection of tert-butyl ester protect-
ing groups as well as the neutral conditions of
hydrogenolysis in which acidic groups are revealed from
benzyl esters. The 9-fluorenylmethyl group was found to
ple molecule; N-phosphoryl glutamic acid which
1
exhibited a K value of 70 pM against PSMA. In addi-
i
tion to serving as competitive inhibitors of metallo-pep-
tidases or proteases, N-phosphoryl amino acids have
also gained a growing interest in the self-assembly of
2
polypeptides. In order to establish a convenient prepa-
6
ration of N-phosphoryl glutamic acid as well as other N-
phosphoryl amino acids, we synthesized bis(9-fluor-
enylmethyl)phosphite 1 and explored its versatility in
the phosphorylation of various amino acid methyl
esters.
be a promising protecting group for phosphate esters
and in a previous report, we established that 9-fluorenyl-
methyl esters of phosphorus could be deptrotected along
with the methyl esters of glutamic acid under mild basic
conditions. Based upon these results, and the known
reaction of H-phosphonate diesters with amines in car-
7
8
Our original method for the preparation of N-phos-
phoryl glutamic acids employed 2-cyanoethoxy groups
as base-labile protecting ligands on phosphorus that
could be concomitantly deprotected under the same con-
dition necessary to hydrolyze the methyl ester of glu-
bon tetrachloride to form phosphoramidate diesters,
we sought to establish a similar methodology for the
O
3
,4
P
tamic acid. However, this method suffered from
chromatographic challenges of the penultimate inter-
1) 9-fluorenemethanol
TEA
O
H
P
Cl
N(iPr)2
Cl
2
) H O, 1-H-tetrazole
2
2
Keywords: Phosphoryl amino acid; Phosphoramidate; Bis(9-fluorenyl-
methyl)phosphite.
1
*
Corresponding author. Tel.: +1 415 338 6495; fax: +1 415 338
384; e-mail: cberkman@sfsu.edu
2
Scheme 1. Synthesis of bis(9-fluorenylmethyl)phosphite 1.
0
040-4039/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.06.022

5302
L. Y. Wu, C. E. Berkman / Tetrahedron Letters 46 (2005) 5301–5303
10
Table 1. Synthesis of N-phosphoryl amino acids
O
P
–
O
P
CO2
O
Xaa-OMe
Xaa-OMe, CCl4
DIPEA
1
–
O–
LiOH
N
H
R
O
2
2
3
11
12
Xaa-OMe
Phosphoramidates 2
Yield (%)
N-Phosphoryl amino acids 3
Yield (%)
85
CO2–
O
P
–
H-Glu(OMe)-OMe
2a
93
98
3a
3b
O–
O–
O–
O–
–
N
H
CO2
O
CO –
O
P
2
H-Gly-OMe
H-Leu-OMe
H-Pro-OMe
H-Ser-OMe
H-Trp-OMe
2b
2c
2d
2e
2f
–
–
–
85
89
90
91
78
N
H
O
CO –
O
P
2
96
83
80
93
3c
3d
3e
3f
N
H
O
CO –
O
P
2
N
O
–
O
P
CO2
–
–
O–
O–
OH
N
H
O
–
O
P
CO2
NH
N
H
O
–
OH
O
P
CO2
H-Tyr-OMe
2g
86
3g
–
93
O–
N
H
O
synthesis of N-phosphoryl amino acids using bis(9-fluor-
enylmethyl)phosphite 1.
(S06-GM052588) and the National Cancer Institute,
U56-Program (Grant No. CA 96217). The authors
would also like to extend their gratitude to W. Tam
and NMR facility at SFSU for their expert assis-
tance.
From diisopropyl phosphoramidous dichloride, bis(9-
fluorenylmethyl)phosphite 1 (BFMP) was prepared as
9
outlined in Scheme 1. From the reaction mixture,
BFMP was conveniently purified by crystallization from
methylene chloride and hexane. The phosphorylation of
various amino acid methyl esters with BFMP to give
phosphoramidate precursors 2 (Table 1) occurred in
excellent yields. It is noteworthy to mention that the
amino acids did not require neutralization prior to
use. Phosphoramidates 2 were generally purified by crys-
tallization in high yield. The concomitant deprotection
of both the methyl esters and the 9-fluorenylmethyl
groups by LiOH proceeded smoothly and in good yield
to give the desired N-phosphoryl amino acids 3 (Table
References and notes
1
2
. (a) Rodriguez, C. E.; Lu, H.; Martinez, A. R.; Brunelle,
A.; Berkman, C. E. J. Enzym. Inhib. 2001, 16, 359; (b)
Maung, J.; Mallari, J. P.; Girtsman, T. A.; Wu, L. Y.;
Rowley, J. A.; Santiago, N. M.; Brunelle, A.; Berkman, C.
E. Bioorg. Med. Chem. 2004, 12, 4969.
. (a) Jiang, Y.; Tan, B.; Chen, Z. Z.; Liu, T.; Zhong, R. G.;
Li, Y. M.; Stewart, D. J.; Zhao, Y. F.; Jiang, H. L. Int. J.
Quantum Chem. 2003, 94, 232; (b) Xue, C. B.; Yin, Y. W.;
Zhao, Y. F. Tetrahedron Lett. 1988, 29, 1145; (c) Tan, B.;
Lee, M. C.; Cui, M.; Liu, T.; Chen, Z. Z.; Li, Y. M.; Ju,
Y.; Zhao, Y. F.; Chen, K.; Jiang, H. J. Mol. Struct.
1
). In summary, bis(9-fluorenylmethyl)phosphite is an
excellent reagent for the N-phosphorylation of amino
acid esters and useful in the preparation of N-phos-
phoryl amino acids.
(
Theochem) 2004, 672, 51; (d) Chen, Z. Z.; Li, Y. M.;
Wang, H. Y.; Du, J. T.; Zhao, Y. F. J. Phys. Chem. A
004, 108, 7686.
2
3
. (a) Lu, H.; Mlodnosky, K. L.; Dinh, T. T.; Dastgah, A.;
Lam, V. Q.; Berkman, C. E. J. Org. Chem. 1999, 64, 8698;
Acknowledgements
(
3
b) Lu, H.; Berkman, C. E. Bioorg. Med. Chem. 2001, 9,
95.
4. Lu, H.; Ng, R.; Shieh, C. C.; Martinez, A. R.; Berkman,
This work was supported by grants from the
National Institutes of Health, MBRS SCORE Program
C. E. Phosphorus Sulfur Silicon 2003, 178, 17 .

L. Y. Wu, C. E. Berkman / Tetrahedron Letters 46 (2005) 5301–5303
5303
3
7.75 (m, 2H). P NMR (CDCl
1
5
. Rodriguez, C. E.; Lu, H.; Dinh, T. T.; Mlodnosky, K. L.;
Dastgah, A.; Lam, V. Q.; Nichols, C. B.; Berkman, C. E.
Bioorg. Med. Chem. Lett. 1999, 9, 1415.
. (a) Watanabe, Y.; Nakamura, T.; Mitsumoto, H. Tetra-
hedron Lett. 1997, 38, 7407; (b) Watanabe, Y.; Nakatomi,
M. Tetrahedron 1999, 55, 9743; (c) Seeberger, P. H.; Yau,
E.; Caruthers, M. H. J. Am. Chem. Soc. 1995, 117, 1472.
. Lu, H.; Hu, Y.; Choy, C. J.; Mallari, J. P.; Villanueva, A.
F.; Arrozal, A. F.; Berkman, C. E. Tetrahedron Lett. 2001,
3
) d: 7.35. Compound 2b:
1
3
98% yield; mp 58–59 ꢁC. H NMR (CDCl ) d: 3.16–3.24
(m, 1H), 3.44 (dd, J = 6.3, 10.2 Hz, 1H), 3.65 (s, 3H), 3.62–
3.69 (t, J = 6.6 Hz, 2H), 4.18–4.34 (m, 4H), 7.22–7.40 (m,
8H), 7.51 (dd, J = 7.5, 12 Hz, 4H), 7.68–7.73 (t, J = 15 Hz,
6
3
1
4H). P NMR (CDCl
lized from methylene chloride (6 mL) and hexane (30 mL);
3
) d: 7.89. Compound 2c: crystal-
1
7
8
3
96% yield; mp 108–109 ꢁC. H NMR (CDCl ) d: 0.80–0.89
(m, 6H), 1.26–1.34 (m, 1H), 1.36–1.51 (m, 1H), 1.56–1.70
(m, 1H), 3.02 (t, J = 21 Hz, 1H), 3.60 (s, 3H), 3.70–3.84
(m, 1H), 4.08–4.31 (m, 6H), 7.22–7.41 (m, 8H), 7.47–7.57
4
2, 4313.
. (a) Istv a´ n, T.; Eszter, G. B.; L a´ szl o´ , O. Tetrahedron 1995,
1, 6797; (b) Atherton, F. R.; Opeshaw, H. J.; Todd, A. R.
¨
3
1
5
3
(m, 4H), 7.68–7.74 (m, 4H). P NMR (CDCl ) d: 7.54.
J. Chem. Soc. 1945, 660; (c) Atherton, F. R.; Todd, A. R.
J. Chem. Soc. 1947, 674; (d) Steinberg, G. M. J. Org.
Chem. 1950, 15, 637.
. Synthesis of BFMP 1. To a stirred solution of diisopropyl
phosphoramidous dichloride (9 mmol, 1.77 g) in methyl-
ene chloride (15 mL) was added a solution of 9-fluorenyl-
methanol (14.4 mmol, 2.25 g) in methylene chloride
Compound 2d: the product was purified by flash chroma-
tography (hexane–ethyl acetate, 5:2, v/v, R = 0.34); 83%
f
1
yield; mp 50–51 ꢁC. H NMR (CDCl
3
) d: 1.70–1.85 (m,
9
2H), 1.85–1.93 (m, 1H), 2.00–2.10 (m, 1H), 3.05–3.09 (m,
2H), 3.62 (s, 3H), 4.09–4.41 (m, 7H), 7.22–7.39 (m, 8H),
7.48–7.52 (m, 2H), 7.57–7.62 (m, 2H), 7.65–7.72 (m, 4H).
3
1
3
P NMR (CDCl ) d: 6.52. Compound 2e: crystallized
(
15 mL) via syringe under Ar(g) at 0 ꢁC. The reaction
from methylene chloride (5.5 mL) and hexane (13.5 mL);
1
mixture was then stirred for 3 h at room temperature. The
solvent was evaporated under reduced pressure to give a
viscous residue which was subsequently dissolved in
acetonitrile (25 mL) and chilled to 0 ꢁC. A solution of
acetonitrile (4 mL), 1-H-tetrazole (0.535 g, 7.64 mmol)
and distilled water (2 mL) were then added and the
reaction mixture stirred for 75 min. The solution was then
diluted in ethyl acetate (75 mL) and sequentially washed
with 10% HCl (2 · 75 mL), 10% Na CO (70 mL), distilled
water (75 mL) and brine (75 mL). After drying the organic
layer with MgSO , the solvent was removed in vacuo to
give a yellow oil. The crude product was purified by
crystallization from methylene chloride (10 mL) and
hexane (50 mL) to give white crystals; yield: 64%; mp
80% yield; mp 105–106 ꢁC. H NMR (CDCl ) d: 2.30 (br s,
3
1H), 3.49–3.67 (m, 6H), 4.06–4.28 (m, 7H), 7.21–7.50 (m,
3
1
3
12H), 7.67–7.69 (m, 4H). P NMR (CDCl ) d: 7.63.
Compound 2f: crystallized from methylene chloride
(6.5 mL) and hexane (30 mL); 93% yield; mp 145–
1
146 ꢁC. H NMR (CDCl
3
) d: 2.95–3.11 (m, 2H), 3.19 (t,
J = 20 Hz, 1H), 3.56 (s, 3H), 3.94–4.10 (m, 5H), 4.16–4.26
(m, 2H), 6.74 (d, J = 2.1 Hz, 1H), 7.00–7.13 (m, 2H), 7.21–
7.30 (m, 5H), 7.33–7.47 (m, 9H), 7.69–7.76 (m, 4H), 8.05
2
3
3
1
3
(s, 1H). P NMR (CDCl ) d: 7.17. Compound 2g:
4
crystallized from methylene chloride (11 mL) and hexane
1
(33.5 mL); 86% yield; mp 142–143 ꢁC. H NMR (CDCl )
3
d: 2.70 (d, J = 3.9 Hz, 2H), 3.10 (t, J = 22 Hz, 1H), 3.61 (s,
3H), 3.82–3.88 (m, 1H), 3.91–3.98 (m, 1H), 3.98–4.07(m,
3H), 4.17–4.27 (m, 2H), 6.61 (d, J = 8.1 Hz, 2H), 6.77 (d,
1
9
4
1
1
9–100 ꢁC. H NMR (CDCl
3
) d: 4.11 (t, J = 6.4 Hz, 2H),
.23–4.27(m, 4H), 6.66 (d, J = 707 Hz, 1H), 7.25–7.53 (m,
J = 8.1 Hz, 2H), 7.26–7.48 (m, 12H), 7.69–7.73 (m, 4H).
1
2H), 7.68–7.73 (m, 4H). C NMR (CDCl
3
31
3
) d: 48.6, 67.7,
3
P NMR (CDCl ) d: 7.11.
3
20.7, 125.6, 127.8, 128.6, 142.1, 143.6. P NMR (CDCl
1
3
)
12. General procedure for N-phosphoryl amino acids 3. To a
stirred solution of the bis(9-fluorenylmethyl)phosphoryl
amino acids 2 (0.106 mmol) in methanol (2 mL) was added
an aqueous solution of LiOH (1.2 M, 800 lL for 2b–g or
1.0 mL for 2a). The reaction mixture was stirred for 15 h
at room temperature and then evaporated to dryness
under reduced pressure. The residue was dissolved in
Milli-Q water, the solids were filtered and the filtrate was
again evaporated to dryness under reduced pressure to
give the products as white solids in a lithium salt form.
d: 8.42.
0. Anhydrous solvents were used in all reactions. Each amino
1
acid methyl ester was used as supplied in the acid salt
form. H, C and P NMR spectra were recorded on a
1
13
31
1
Bruker DRX 300 MHz NMR spectrometer. H NMR
chemical shifts are relative to TMS (d = 0.00 ppm), CDCl3
(
(
CD OD (d = 49.15 ppm) or CDCl3 (d = 77.23 ppm). P
NMR chemical shifts in CDCl or D O were externally
d = 7.26 ppm), CD
d = 4.87). C NMR chemical shifts are relative to
3
OD (d = 4.87and 3.31 ppm), or D
2
O
1
3
3
1
3
Yields were calculated by subtracting the known excess of
3
2
1
referenced to 85% H
O, respectively.
3
PO
4
(d = 0.00 ppm) in CDCl
3
and
LiOH in each reaction. Compound 3a: 85% yield.
NMR (D
3.44–3.51 (m, 1H). P NMR (D O) d: 8.57. Compound
H
D
2
2
O) d: 1.73–1.88 (m, 2H), 2.02–2.21 (m, 2H),
31
1
1. General procedure for N-bis(9-fluorenylmethyl)phos-
phoryl amino acids 2. To a stirred solution of bis(9-
fluorenylmethyl)phosphite (0.459 mmol), an amino acid
methyl ester (0.505 mmol) in acetonitrile (5 mL) and
carbon tetrachloride (5 mL) at 0 ꢁC under Ar was added
2
1
31
3b: 85% yield. H NMR (D
NMR (D O) d: 9.05. Compound 3c: 89% yield. H NMR
(D O) d: 0.64–0.67(m, 6H), 1.15–1.27(m, 2H), 1.32–1.40
(m, 1H), 3.25 (dd, J = 7.5, 15.9 Hz, 1H). P NMR (D O)
2
O) d: 3.08–3.14 (m, 2H).
P
1
2
2
3
1
(
g)
2
1
N,N-diisopropylethyl amine (1.01 mmol, 180 lL) via
syringe. The reaction mixture was stirred for 70 min at
room temperature. The reaction mixture was then diluted
with ethyl acetate (50 mL) and sequentially washed with
d: 8.56. Compound 3d: 90% yield. H NMR (D
1.30–1.51, (m, 3H), 1.69–1.81 (m, 1H), 2.72–2.83 (m, 2H),
2
O) d:
3
1
3.48–3.56 (m, 1H). P NMR (D O) d: 8.15. Compound
2
1
3e: 91% yield. H NMR (D O) d: 3.53–3.58 (m, 2H), 3.66–
2
3
1
1
0% HCl (50 mL), 10% NaHCO
50 mL) and brine (50 mL). After drying the organic layer
3
(50 mL), distilled water
3.73 (m, 1H). P NMR (D
2
O) d: 8.59. Compound 3f: 7 8%
O) d: 2.93–3.00 (m, 1H), 2.15–3.19 (m,
1H), 3.75 (br s, 1H), 7.04–7.17 (m, 3H), 7.37 (d, J = 6 Hz,
1
(
with MgSO , the solvent was removed in vacuo. Com-
pound 2a: crystallized from methylene chloride (5 mL)
and hexane (35 mL); 93% yield; mp 79–80 ꢁC. H NMR
(
J = 15 Hz, 2H), 3.24 (t, J = 21 Hz, 1H), 3.60 (s, 3H), 3.62–
yield. H NMR (D
2
4
3
1
1H), 7.68 (d, J = 6 Hz, 1H). P NMR (D
2
O) d: 8.37.
O) d: 2.61–2.67
(m, 1H), 2.85–2.91 (m, 1H), 3.59–3.66 (m, 1H), 6.49 (d,
1
1
Compound 3g: 93% yield. H NMR (D
2
3
CDCl ) d: 1.75–1.85 (m, 1H), 1.90–2.01 (m, 1H), 2.25 (t,
3
1
J = 6 Hz, 2H), 6.93 (d, J = 18 Hz, 2H). P NMR (D O) d:
2
3
.69 (m, 4H), 4.13–4.28 (m, 6H), 7.26–7.54 (m, 14H), 7.70–
8.27.