Synthesis of N-Fmoc 3-(4-(di-(tert-butyl)phosphonomethyl)-phenyl)pipecolic acid as a conformationally constrained phosphotyrosyl mimetic suitably protected for peptide synthesis

Article abstract of DOI:10.1016/S0040-4020(02)01421-7

Phosphonomethylphenylalanine (Pmp, 2) has shown wide utility as a hydrolytically stable phosphtyrosyl (pTyr, 1) mimetic, particularly in Src homology 2 (SH2) domain-binding peptides. (2S,3R)-3-(4-(phosphonomethyl)phenyl)pipecolic acid (3) represents a var

Full text of DOI:10.1016/S0040-4020(02)01421-7

Tetrahedron 58 (2002) 10423–10428
Synthesis of N-Fmoc 3-(4-(di-(tert-butyl)phosphonomethyl)-
phenyl)pipecolic acid as a conformationally constrained
phosphotyrosyl mimetic suitably protected for peptide synthesisq
Ding-Guo Liu,a Xiang-Zhu Wang,a Yang Gao,a Bihua Li,b Dajun Yangb
and Terrence R. Burke, Jr.a
,
*
aLaboratory of Medicinal Chemistry, Center for Cancer Research, NCI at Frederick, Frederick, MD 21702, USA
bDepartment of Internal Medicine, Division of Hematology and Oncology, the University of Michigan, Ann Arbor, MI 48109, USA
Received 26 February 2002; accepted 30 October 2002
Abstract—Phosphonomethylphenylalanine (Pmp, 2) has shown wide utility as a hydrolytically stable phosphtyrosyl (pTyr, 1) mimetic,
particularly in Src homology 2 (SH2) domain-binding peptides. (2S,3R)-3-(4-(phosphonomethyl)phenyl)pipecolic acid (3) represents a
variant of Pmp having f and x1 torsion angles constrained through incorporation into the piperidinyl ring structure contained within pipecolic
acid. Reported herein is the enantioselective preparation of 3, in an orthogonally protected form (4) suitable for use in peptide synthesis.
Stereochemistries at both the 2- and 3-positions are derived inductively from a single chiral center provided by the commercially available
Evans chiral auxiliary, (4S)-4-benzyl-1,3-oxazolidin-2-one. Incorporation of 4 into a Grb2 SH2 domain-directed tripeptide (18) showed that
Grb2 SH2 domain-binding affinity was reduced relative to the parent Pmp-containing tripeptide (19). Although conformational constraint did
not enhance affinity in this case, novel amino acid analogue 4 may serve as a useful tool for the induction of defined phosphotyrosyl geometry
in peptides directed at other signal transduction targets. q 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction
reported on the development of conformationally restricted
pTyr mimetics for use in peptide-based inhibitors.13,14 As an
entry into this area of investigation, we therefore designed
analogue 3 as a variant of Pmp, wherein restriction of x1
and f angles is achieved by a 3-carbon bridge between the
b-carbon and the a-nitrogen. The resulting 3-(4-phos-
phonomethylphenyl)pipecolic acid derivative bears the 2S
a-amino configuration, which is the more potent SH2
domain-binding enantiomer of Pmp.15,16 Molecular model-
ing studies of analogue 3 docked in an SH2 domain pTyr-
binding pocket mandate 3R-stereochemistry. Our utilization
of pipecolic acid adds to recent reports where this nucleus
has been used for construction of constrained amino acid
analogues.17–19 Herein is reported the synthesis of N-Fmoc-
protected 4 as a conformationally constrained pTyr mimetic
suitably protected for peptide synthesis (Fig. 1).
Binding of flexible peptide ligands to target proteins can be
negatively affected by entropy terms arising when specific
binding geometries must be achieved from random
populations of solution conformations. To minimize such
penalties and thereby achieve higher affinities, amino acid
analogues have been developed, which either restrict
peptide backbone or side chain torsion angles.2,3 Phospho-
tyrosyl residues (pTyr, 1) provide critical recognition
elements in a variety of cellular signal transduction
processes. Accordingly, pTyr mimetics can serve important
roles in the development of signaling inhibitors.4,5 Among
pTyr mimetics, phosphonomethyl phenylalanine (Pmp, 2),
has proven useful in the preparation Src of homology 2
(SH2) domain-binding ligands,6–10 particularly for Grb2
SH2 domain-directed agents, where Pmp exhibits binding
affinity nearly equal to parent pTyr.11 We have pre-
viously detailed synthesis of conformationally constrained
monomeric pTyr mimetics which approximate binding
geometries observed in the X-ray structure of a p56lck
SH2 domain-bound pTyr residue.12 However, little has been
q See ref. 1.
Keywords: tripeptide; inhibitor; mimetic.
*
Corresponding author. Fax: þ1-301-846-6033;
e-mail: tburke@helix.nih.gov
Figure 1. Structures of pTyr and selected pTyr mimetics.
0040–4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(02)01421-7

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D.-G. Liu et al. / Tetrahedron 58 (2002) 10423–10428
Scheme 1. Reagents and conditions: (a) Pd(OAc)2, tri-o-tolylphosphine, NEt3, reflux (90% yield); (b) allylmagnesium bromide, CuBr·SMe2, THF, 2788C
(80% yield); (c) (i) BH3, THF, 08C, (ii) NaBO3·H2O, H2O (90% yield, two steps); (d) TBDMS-Cl, imidazole, DMF (73% yield); (e) (i) KHMDS, trisyl azide,
2788C; (ii) HOAc, KOAc, 30–358C (35% yield); (f) HF·pyridine, THF, 08C (87% yield); (g) oxalyl chloride, DMSO, NEt3, 2788C–room temperature;
(h) H2/10% Pd·C, MeOH (39% yield, two steps); (i) 1N NaOH, 08C; CO2, Fmoc-OSu (47% yield).
2. Results and discussion
nolysis of the chiral auxillary to provide 14 directly in 39%
yield. Finally, hydrolysis of the methyl ester and in situ
Fmoc-amino protection gave desired final product 4 in 47%
yield.
Our synthetic approach (Scheme 1) was predicated on our
recently reported synthesis of 3-phenylpipecolic acid,19
wherein introduction of chirality at both the 2- and
3-positions is achieved through stereochemical induction
originating from commercially available Evans reagent.20
As previously reported,21 cinnamoyl derivative 7 is
obtained by Heck reaction of phosphonate 5 with acryl-
amide 6, which was prepared from (S)-(2)-4-benyl-2-
oxazolidinone.22 Michael addition of allylmagnesium
bromide in the presence of copper bromide·dimethyl sulfide
complex leads to 8 (80% yield), which was converted to
primary alcohol 9 by hydroboration/oxidation23 (90%
yield). Silylation yields 10, which upon electrophilic Ca
asymmetric azidation by the method of Evans,21,24 yields
11. Removal of silyl protection through treatment with
HF·pyridine25 (use of tetrabutylammonium fluoride resulted
in partial cleavage of the chiral auxiliary) gave free alcohol
12 in 87% yield. Sequential Swern oxidation26 of 12 to
aldehyde 13 was followed by hydrogenation over 10% Pd·C
in MeOH (40 psi H2). Similar to our recent report,19 this
protocol achieved ring closure with concomitant metha-
Pmp-containing tripeptide 19 (Scheme 2) previously has
been shown to have high Grb2 SH2 domain-binding
affinity27 and to exhibit interesting biological effects in
whole cells driven by Grb2-dependent signaling path-
ways.28 In order to evaluate the potential utility of 4,
peptide 18 was prepared as a variant of 19 bearing
conformationally restricted Pmp analogue 3. Synthesis of
18 was achieved by standard Fmoc-protocols (Scheme 2)
similar to that reported for the preparation of parent 19.27
The resulting peptide was examined for Grb2 SH2 domain
binding potency using an extracellular ELISA-based
assay.29 As shown in Fig. 2, peptide 18 exhibited an
approximate 200-fold reduction in binding potency
relative to parent 19. Although enhanced binding affinity
was not achieved, the utility of 4 as a new amino acid
analogue suitably protected for peptide synthesis has been
demonstrated. The extensive variety of contexts in which
pTyr residues serve important roles in cellular signal

D.-G. Liu et al. / Tetrahedron 58 (2002) 10423–10428
10425
Scheme 2. Reagents and conditions: (a) HOBT, DPCDI, DMF (quantitative); (b) (i) piperidine, acetonitrile; (ii) (tert-BuO)COCOCl, N(i-Pr)2Et, DMF
(77% yield); TFA, SiEt3, CH2Cl2 (79% yield).
Figure 2. ELISA Grb2 SH2 domain-binding data for compounds 18 and 19, performed as described in Section 3.

10426
D.-G. Liu et al. / Tetrahedron 58 (2002) 10423–10428
transduction, may render 4 of potential value for the
development of pharmacological tools directed at other
systems.
and brine, dried (Na2SO4), filtered and evaporated. Crude
product was purified by silica gel flash chromatography
(from EtOAc–hexane, 1:1 to MeOH–CHCl3, 3:100) to
afford 9 as white solid (3.56 g, 90%). Mp 98–998C; [a]2D0¼
þ35.3 (c 0.60, CHCl3); 1H NMR (250 MHz, CDCl3) d
7.14–7.35 (9H, m), 4.48 (1H, m),4.05 (2H, m), 3.60 (2H, t,
J¼6.4 Hz), 3.38 (1H, m), 3.21 (3H, m), 3.0 (2H, d, J¼
21.3 Hz), 2.67 (1H, dd, J¼13.2, 10.3 Hz), 1.41 (9H, s), 1.40
(9H, s), 1.30–1.85 (4H, m). IR (neat) 2980, 1782, 1698,
1172 cm21. FABMS (þVE) m/z 574 (MHþ). Anal. calcd
for C31H44NO7P·0.5H2O: C 63.90, H 7.78, N 2.40. Found: C
63.91, H 7.62, N 2.36.
3. Experimental
3.1. Evaluation of Grb2 SH2 domain binding using
ELISA techniques
A biotinated phosphopeptide encompassing the Grb2 SH2
domain-binding sequence derived from the SHC protein,
was bound at 20 ng/mL to 96-well plates, overnight.
Nonspecific interactions were inhibited by 5% bovine
serum albumin containing TBS. Samples of recombinant
purified Grb2 SH2-GST fusion protein were pre-incubated
with serial of dilutions of inhibitor peptides prior to addition
into each well. After extensive washing with 0.1% bovine
serum albumin in TBS, bound Grb2 SH2 domain protein
was detected using anti-GST antibodies and goat anti-
mouse antibody conjugated to alkaline phosphatase.
Quantitation of bound alkaline phosphatase was achieved
by a colorimetric reaction employing para-nitrophenyl
phosphate as substrate.
3.2.3. (4S)-3-((3S)-3-(4-((Bis-(tert-butyl)phosphono)-
methyl)phenyl)-6-(1,1,2,2-tetramethyl-1-silapropoxy)-
hexanoyl)-4-benzyl-1,3-oxazolidin-2-one (10). To
a
solution of 9 (722 mg, 1.26 mmol) in DMF (5 mL) were
added tert-butyldimethylsilyl chloride (TBDMS-Cl)
(230 mg, 1.51 mmol) and imidazole (214 mg, 3.16 mmol)
and the mixture was stirred overnight at room temperature.
Solvent was evaporated and the resulting residue was
partitioned between EtOAc (30 mL) and brine, dried
(Na2SO4), filtered and evaporated. Crude product was
purified by silica gel flash chromatography (EtOAc–hexane
1:1) to afford 10 as a colorless oil (712 mg, 82%); [a]2D0¼
þ37.5 (c 0.99, CHCl3); 1H NMR (250 MHz, CDCl3) d
7.13–7.35 (9H, m), 4.45 (1H, m), 3.95–4.08 (2H, m), 3.55
(2H, t, J¼6.4 Hz), 3.41 (1H, m), 3.11–3.24 (3H, m), 2.99
(2H, d, J¼21.4 Hz), 2.66 (1H, dd, J¼13.2, 10.3 Hz), 1.60–
1.85 (2H, m), 1.35–1.45 (2H, m), 1.40 (9H, s), 1.39 (9H, s),
0.87 (9H, s), 0.01 (6H, s). IR (neat) 2980, 1782, 1698,
984 cm21. FABMS (þVE) m/z 482 (MHþ). Anal. calcd for
C37H58NO7SiP: C, 64.60; H, 8.50; N, 2.04. Found: C, 64.88;
H, 8.56; N, 2.01.
3.2. Synthesis
3.2.1. (4S)-3-((3S)-3-(4-((Bis-(tert-butyl)phosphono)-
methyl)phenyl)hex-5-enoyl)-4-benzyl-1,3-oxazolidin-2-
one (8). To a slurry of CuBr·SMe2 (4.44 g, 21.6 mmol) in
THF (150 mL), was added a solution of allylmagnesium
bromide (43.2 mL, 43.2 mmol) at 2788C under argon, and
the resulting mixture was stirred at 2788C (1.5 h). A
solution of 721 (7.38 g, 14.4 mmol) in THF (150 mL) was
added at 2788C, and stirring was continued (2.5 h) then the
reaction was quenched by addition of saturated NH4Cl.
The mixture was extracted with EtOAc (3£100 mL) and the
combined organic extracts were washed with H2O and
brine, dried (Na2SO4), filtered and evaporated to provide
crude product. Purification by silica gel flash chroma-
tography (EtOAc–hexane from 1:2 to 1:1) afforded 8
(6.40 g, 80%). Mp 83–848C; [a]2D0¼þ49.3 (c 1.08, CHCl3);
1H NMR (400 MHz, CDCl3) d 7.35–7.20 (3H, m), 7.18–
7.11 (6H, m), 5.67 (1H, ddt, J¼17.0, 10.2, 7.0 Hz), 5.03–
4.92 (2H, m), 4.44 (1H, m), 4.04–3.96 (2H, m), 3.40–3.28
(2H, m), 3.24–3.16 (2H, m), 2.96 (2H,d, J¼21.3 Hz), 2.63
(1H, dd, J¼13.2, 9.8 Hz), 2.41 (2H, t, J¼7.0 Hz), 1.37
(9H, s), 1.36 (9H, s). IR (neat) 2980, 1782, 1698,
1172 cm21. FABMS (þVE) m/z 556 (Mþ1). Anal. calcd
for C31H42NO6P: C 67.01, H 7.62, N 2.52. Found: C 66.71,
H 7.73, N 2.48.
3.2.4. 1-((4S)-2-Oxo-4-benzyl(1,3-oxazolidin-3-yl))-
(2S,3R)-2-azido-3-(4-((bis-(tert-butyl)phosphono)-
methyl)phenyl)-6-(1,1,2,2-tetramethyl-1-silapropoxy)-
hexan-1-one (11). To a solution of KHMDS (3 mL, 0.5 M
in toluene, 1.50 mmol) in THF (6 mL), was added a solution
of 10 (870 mg, 1.26 mmol) in THF (6 mL) at 2788C under
argon and stirring was continued at 2788C (40 min). To
this was added a cooled (2788C) solution of trisyl azide
(585 mg, 1.89 mmol) in THF (3 mL). The mixture was
stirred at 2788C for 2 min then the reaction was quenched
by addition of acetic acid (0.36 mL, 6.3 mmol) and KOAc
(1.24 g, 12.6 mmol) and the mixture was stirred at 30–358C
(3 h). The mixture was diluted with EtOAc, washed with
saturated NaHCO3, H2O and brine. The solvent was dried
(Na2SO4) and evaporated. Crude product was purified by
silica gel flash chromatography (EtOAc–hexane from 1:3 to
1:1) to afford 11 as a colorless oil (319 mg, 35% yield);
[a]2D0¼þ96.5 (c 1.59, CHCl3); 1H NMR (400 MHz, CDCl3)
d 7.26 (4H, m), 7.11 (5H, m), 5.24 (1H, d, J¼10.2 Hz), 3.97
(1H, m), 3.85 (1H, dd, J¼9.2, 1.9 Hz), 3.58 (1H, m), 3.56
(2H, t, J¼6.4 Hz), 3.13 (1H, dd, J¼13.3, 3.0 Hz), 3.05 (1H,
t, J¼10 Hz), 2.93 (2H, d, J¼21.3 Hz), 2.66 (1H, dd, J¼13.2,
9.8 Hz), 2.14 (1H, m), 1.82 (1H, m), 1.30–1.43 (2H, m),
1.41 (9H, s), 1.36 (9H, s), 0.88 (9H, s), 0.02 (6H, s). IR
(neat) 2931, 2103 (–N3), 1783, 1739, 1699, 1240,
3.2.2. (4S)-3-((3S)-6-Hydroxy-3-(4-((bis-(tert-butyl)phos-
phono)methyl)phenyl) hexanoyl)-4-benzyl-1,3-oxazo-
lidin-2-one (9). To a solution of 8 (3.84 g, 6.91 mmol) in
THF (36 mL), was added BH3 (6.91 mL, 1.0 M in THF,
6.91 mmol) at 08C under argon, then the mixture was stirred
at 08C (1 h). To the mixture were added H2O (36 mL) and
NaBO3·H2O (690 mg, 6.91 mmol) and the mixture was
stirred at room temperature (3 h). The mixture was diluted
with EtOAc and the organic phase was collected and
combined with EtOAc extracts (3£40 mL) of the aqueous
phase. The combined organic layers were washed with H2O
977 cm21
.
FABMS (þVE) m/z 673 (MHþ2C4H8).
Anal. calcd for C37H57N4O7PSi: C 60.97, H 7.88, N 7.69.
Found: C 61.27, H 8.15, N 7.22. HR-FABMS calcd for

D.-G. Liu et al. / Tetrahedron 58 (2002) 10423–10428
10427
C29H42N4O7PSi (MHþ22[C4H8]): 617.2560. Found:
617.2546.
The pH was adjusted to 6.5 using 2 M KHSO4 at 08C, then
dioxane was evaporated and the solution was diluted with
H2O. The resulting mixture was acidified to pH 5 using 2 M
KHSO4, extracted with EtOAc (3£20 mL) and evaporated
to give a residue, which was purified by silica gel flash
chromatography (MeOH–CHCl3 from 1:50 to 1:10) to
afford title compound 4 as a white solid (12 mg, 47%). Mp
3.2.5. 1-((4S)-2-Oxo-4-benzyl(1,3-oxazolidin-3-yl))-
(2S,3R)-2-azido-6-hydroxy-3-(4-((bis-(tert-butyl)phos-
phono)methyl)phenyl)hexan-1-one (12). To a solution of
11 (60 mg, 0.098 mmol) in THF (3 mL) in a plastic vial was
added HF·pyridine (0.1 mL) at 08C and the mixture was
stirred at 08C (30 min), then at room temperature (3 h). The
mixture was cooled to 08C, diluted with EtOAc and
neutralized with saturated NaHCO3 until generation of
carbon dioxide ceased. The mixture was extracted with
EtOAc (3£15 mL) and evaporated. Crude product was
purified by silica gel flash chromatography (from EtOAc–
hexane, 1:1 to MeOH–CHCl3, 3:100) to afford 12 as a white
solid (44 mg, 87%). Mp 52–558C; [a]2D3¼þ126.8 (c 0.57,
CHCl3); 1H NMR (250 MHz, CDCl3) d 7.13–7.33 (9H, m),
5.33 (1H, d, J¼9.8 Hz), 4.03 (1H, m), 3.90 (1H, d, J¼
7.7 Hz), 3.62 (1H, m), 3.60 (2H, t, J¼6.4 Hz), 2.90–3.21
(3H, m), 2.94 (2H, d, J¼21.3 Hz), 2.67 (1H, dd, J¼13.2,
9.8 Hz), 2.17 (1H, m), 1.85 (1H, m), 1.30–1.43 (2H, m),
1.42 (9H, s), 1.37 (9H, s). IR (neat) 2102 (–N3), 1780,
980 cm21. FABMS (þVE) m/z 615 (MHþ). Anal. calcd for
C31H43N4O7P: C 60.57, H 7.05, N 9.11. Found: C 60.21, H
7.16, N 8.91.
1
102–1048C; H NMR (400 MHz, CDCl3) d 7.74 (2H, m),
7.57 (2H, m), 7.20–7.40 (8H, m), 5.29 (3/5H, s), 5.11 (2/5H,
s), 4.46 (2H, m), 4.31 (3/5H, m), 4.23 (2/5H, m), 4.07
(1H, m), 3.69 (1H, m), 3.29 (3/5H, m), 3.15 (2/5H, m), 3.07
(1H, s), 3.02 (1H, s), 1.82–1.92 (2H, m), 1.40–1.55
(2H, m), 1.41 (18H, s). FABMS (þVE) m/z 634 (MHþ).
HR-FABMS calcd for C36H45NO7P (MHþ): 634.2934.
Found: 634.2872.
3.2.8. Tripeptide 16. To a solution of 4 (12 mg, 0.019
mmol) in DMF (1 mL) were added HOBt (3 mg, 0.019
mmol) and 1,3-diisopropylcarbodiimide (3 mL, 0.019
mmol), and the mixture was stirred at room temperature
(30 min). Dipeptide 1527 (8.1 mg, 0.019 mmol) was added
and the mixture was stirred at room temperature (overnight).
Solvent was evaporated under high vacuum and residue was
purified by silica gel flash chromatography (MeOH–CHCl3
from 1:50 to 1:20) to afford tripeptide 16 as a colorless oil
1
(20 mg, quantitative). H NMR (400 MHz, CDCl3) d 8.02
3.2.6. Methyl (2S,3R)-3-(4-((bis-(tert-butyl)phosphono)-
methyl)phenyl)piperidine-2-carboxylate (14). To a solu-
tion of oxalyl chloride (44 mL, 0.50 mmol) in CH2Cl2
(1 mL), was added a solution of DMSO (71 mL, 1.00 mmol)
in CH2Cl2 (1 mL) at 2788C. The mixture was stirred at
2788C (20 min), then a solution of 12 (62 mg, 0.10 mmol)
in CH2Cl2 (2 mL) was added and the mixture was stirred at
2788C (40 min). To this was added triethylamine (140 mL)
and stirring was continued at 2788C (20 min). The mixture
was allowed to warm to room temperature, then it was
diluted with EtOAc and washed with H2O, brine and dried
over anhydrous Na2SO4. Filtration and evaporation gave a
residue, which was filtered through a short silica gel column
to provide crude aldehyde 13. This was dissolved in MeOH
(10 mL) and hydrogenated at room temperature over 10%
Pd·C (30 mg) 40 psi H2 overnight. The mixture was filtered
through celite, evaporated and residue was purified by silica
gel flash chromatography (CH3OH–CHCl3 from 1:100 to
1:6) to afford 14 as a colorless oil (17 mg, 39%); [a]¼þ32.4
(1H, d, J¼7.8 Hz), 7.98 (1H, s), 7.79 (2H, t, J¼7.8 Hz), 7.74
(2H, d, J¼7.4 Hz), 7.65 (1H, m), 7.57 (1H, m), 7.53 (2H, d,
J¼7.0 Hz), 7.23–7.45 (8H, m), 7.11 (2H, d, J¼6.3 Hz),
6.94 (2H, d, J¼7.4 Hz), 6.27 (1H, s), 5.42 (1H, s), 4.66 (1H,
m), 4.46 (1H, m), 4.37 (1H, m), 4.22 (1H, t, J¼6.6 Hz), 4.14
(1H, m), 3.81 (2H, m), 3.28–3.40 (3H, m), 3.02–3.11 (2H,
m), 2.93 (d, J¼21.5 Hz, 2H), 2.52 (1H, dd, J¼15.2, 5.0 Hz),
1.90–2.10 (4H, m), 1.75–1.86 (3H, m), 1.66 (1H, m), 1.32–
1.56 (8H, m), 1.40 (9H, s), 1.37 (9H, s). FABMS (þVE) m/z
1040 (MHþ).
3.2.9. Tripeptide 17. To a solution of 16 (20 mg,
0.019 mmol) in acetonitrile was added piperidine (15 mL,
0.152 mmol), the mixture was stirred at room temperature
(4 h) then solvent was evaporated to provide a residue,
which was purified by silica gel flash chromatography (from
MeOH–CHCl3¼1:100 to NH4OHaq–MeOH–CHCl3¼
1:5:50) to give Fmoc-deprotected intermediate (14 mg).
This was dissolved in DMF (1.5 mL) and to this were added
successively diisopropylethylamine (12 mL, 0.068 mmol)
and tert-butyloxalyl chloride (6.4 mL, 0.051 mmol). The
mixture was stirred at room temperature (overnight), then
DMF was evaporated under high vacuum and residue was
purified by silica gel flash gel chromatography (MeOH–
CHCl3 from 1:50 to 1:20) to afford 17 as a colorless oil
1
(c 0.73, CHCl3); H NMR (400 MHz, CDCl3) d 7.20 (2H,
dd, J¼8.2, 2.4 Hz), 7.09 (2H, d, J¼8.2 Hz), 3.52 (1H, d,
J¼10.4 Hz), 3.36 (3H, s), 3.26 (1H, m), 2.97 (2H, d,
J¼21.5 Hz), 2.73–2.80 (2H, m), 2.20 (1H, br), 1.98 (1H,
m), 1.64–1.82 (3H, m), 1.41 (9H, s), 1.40 (9H, s) ppm. IR
(neat) 3542, 2979, 1735, 1170 cm21. FABMS (þVE) m/z
426 (MHþ). HR-FABMS calcd for C22H37NO5P (MHþ):
426.2409. Found 426.2381.
1
(14 mg, 77%). H NMR (400 MHz, CDCl3) d 8.06 (1H, d,
J¼8.1 Hz), 7.81 (1H, d, J¼7.6 Hz), 7.74 (1H, d, J¼8.0 Hz),
7.68 (1H, dd, J¼6.0, 3.3 Hz), 7.54 (1H, t, J¼5.4 Hz), 7.35–
7.49 (3H, m), 7.28 (m, 2H), 7.13 (2H, dd, J¼8.2, 2.3 Hz),
6.92 (2H, d, J¼8.0 Hz), 6.74 (1H, s), 6.34 (1H, s), 5.50 (1H,
s), 5.07 (1H, d, J¼4.9 Hz), 4.71 (1H, dt, J¼8.0, 5.1 Hz),
3.50 (1H, m), 3.35–3.47 (3H, m), 3.25 (1H, td, J¼13.3,
4.7 Hz), 3.10–3.17 (2H, m), 2.96 (d, J¼21.4 Hz, 2H), 2.92
(1H, dd, J¼15.3, 5.1 Hz), 2.55 (1H, dd, J¼15.3, 5.1 Hz),
1.92–2.18 (5H, m), 1.82–1.92 (2H, m), 1.32–1.70 (8H, m),
1.56 (9H, s), 1.43 (9H, s), 1.40 (9H, s). FABMS (þVE) m/z
946 (MHþ).
3.2.7. (2S,3R)-1-((Fluoren-9-ylmethyl)oxycarbonyl)-3-
(4-((bis-(tert-butyl)phosphono) methyl)phenyl)piperi-
dine-2-carboxylic acid (4). To a solution of 14 (17 mg,
0.040 mmol) in a mixture of dioxane (3 mL) and H2O
(2 mL) was added at 08C, a mixture of 1N NaOH in dioxane
(1:1:0.6 mL) and the mixture was stirred at 08C (2 h). The
mixture was buffered by addition of a small quantity of dry
ice, then Fmoc-OSu (13.5 mg, 0.040 mmol) was added and
the mixture was stirred at room temperature (overnight).

10428
D.-G. Liu et al. / Tetrahedron 58 (2002) 10423–10428
4. Burke, Jr. T. R.; Yao, Z.-J.; Smyth, M. S.; Ye, B. Curr. Pharm.
Des. 1997, 3, 291–304.
3.2.10. Tripeptide 18. To a solution of 17 (14 mg, 0.015
mmol) in a mixture of dichloromethane (1.5 mL) and TFA
(1.5 mL), was added triethylsilane (7.1 mL, 0.044 mmol)
and the mixture was stirred at room temperature (3 h), then
solvent was evaporated. The resulting residue was triturated
with ether (10 mL) to give a light gray suspension, which
was collected by centrifugation. The procedure was
repeated two times, then the resulting light gray solid was
purified by HPLC [Vydac Protein and Peptide C18 column
(250 mm£20 mm dia.); flow rate¼10 mL/min.; linear
gradient from 5% B to 60% B over 25 min then 60% B to
95% B over 5 minutes; solvent A, 0.1% aqueous TFA;
solvent B, 0.1% TFA in acetonitrile]. Product was collected
from 22.5 to 23.8 min to afford 18 as a white solid (9.1 mg,
79%). Analytical HPLC [Vydac Protein and Peptide C18
column (250 mm£10 mm dia.); flow rate¼2 mL/min.;
linear gradient from 5% B to 60% B over 10 min then
60% B to 70% B over 20 min; solvent A, 0.1% aqueous
TFA; solvent B, 0.1% TFA in acetonitrile] indicated a major
peak at t¼20.7 with a shoulder at 22.5 min (ratio of 80:20).
The main peak and the shoulder were separately
collected and each provided an identical elution profile
upon re-injection. This suggested that the shoulder was
potentially a stable conformational isomer of the main peak.
Mp 203.58C (dec). 1H NMR (400 MHz, D2O) d 8.18 (1H, d,
J¼8.8 Hz), 8.00 (1H, d, J¼7.3 Hz), 7.87 (1H, d, J¼8.1 Hz),
7.48–7.68 (4H, m), 7.22 (2H, d, J¼7.3 Hz), 7.03 (2H, d,
J¼8.1 Hz), 4.70 (1H, d, J¼8.8 Hz), 4.61 (1H, m), 3.43–
3.49 (2H, m), 3.31–3.38 (2H, m), 3.22 (1H, m), 3.17 (2H, t,
J¼8.0 Hz), 2.94 (1H, s), 2.89 (1H, s), 2.84 (1H, dd, J¼15.4,
5.9 Hz), 2.70 (1H, dd, J¼15.4, 8.8 Hz), 1.98–2.07 (2H, m),
1.71–1.91 (4H, m), 1.53–1.65 (4H, m), 1.31–1.47 (4H, m),
5. Burke, Jr. T. R.; Yao, Z.-J.; Liu, D.-G.; Voigt, J.; Gao, Y.
Biopolymers 2001, 60, 32–44.
6. Sawyer, T. K. Biopolymers 1998, 47, 243–261.
7. Broadbridge, R. J.; Sharma, R. P. Curr. Drug Targets 2000, 1,
365–386.
8. Cody, W. L.; Lin, Z. W.; Panek, R. L.; Rose, D. W.; Rubin,
J. R. Curr. Pharm. Des. 2000, 6, 59–98.
9. Vu, C. B. Curr. Med. Chem. 2000, 7, 1081–1100.
10. Muller, G. Top. Curr. Chem. 2001, 211, 17–59.
11. Fretz, H.; Furet, P.; Garcia-Echeverria, C.; Rahuel, J.;
Schoepfer, J. Curr. Pharm. Des. 2000, 6, 1777–1796.
12. Burke, Jr. T. R.; Barchi, Jr. J. J.; George, C.; Wolf, G.;
Shoelson, S. E.; Yan, X. J. Med. Chem. 1995, 38, 1386–1396.
13. Liu, W. Q.; Carreaux, F.; Meudal, H.; Roques, B. P.; Garbay-
Jaureguiberry, C. Tetrahedron 1996, 52, 4411–4422.
14. Davidson, J. P.; Lubman, O.; Rose, T.; Waksman, G.; Martin,
S. F. J. Am. Chem. Soc. 2002, 124, 205–215.
15. Domchek, S. M.; Auger, K. R.; Chatterjee, S.; Burke, T. R.;
Shoelson, S. E. Biochemistry 1992, 31, 9865–9870.
16. Burke, Jr. T. R.; Smyth, M. S.; Otaka, A.; Nomizu, M.; Roller,
P. P.; Wolf, G.; Case, R.; Shoelson, S. E. Biochemistry 1994,
33, 6490–6494.
17. Souers, A. J.; Ellman, J. A. J. Org. Chem. 2000, 65,
1222–1224.
18. Davis, F. A.; Zhang, H.; Lee, S. H. Org. Lett. 2001, 3,
759–762.
19. Liu, D.-G.; Gao, Y.; Wang, X.; Kelley, J. A.; Burke, Jr. T. R.
J. Org. Chem. 2002, 67, 1448–1452.
20. Evans, D. A.; Britton, T. C.; Ellman, J. A.; Dorow, R. L. J. Am.
Chem. Soc. 1990, 112, 4011–4030.
1.05–1.12 (2H, m). IR (neat) 3318, 1635, 1534, 1205 cm21
.
21. Burke, T. R.; Liu, D. G.; Gao, Y. J. Org. Chem. 2000, 65,
6288–6291.
FABMS (þVE) m/z 776 (M2Hþ). HR-FABMS calcd for
C39H48N5O10P: 776.30626. Found: 776.30866.
22. Both (S)-(2)- and (R)-(þ) 4-benzyl-2-oxazolidinone can be
purchase from Aldrich Chemical Corp., S.L., MO.
23. Kabalka, G. W.; Shoup, T. M.; Goudgaon, N. M. J. Org.
Chem. 1989, 54, 5930–5933.
Acknowledgements
24. Evans, D. A.; Evrard, D. A.; Rychnovsky, S. D.; Fru¨h, T.;
Whittingham, W. G.; DeVries, K. M. Tetrahedron Lett. 1992,
33, 1189–1192.
Appreciation is expressed to Dr James Kelley of the LMC
for mass spectral analysis. Work was supported in part by
the Susan G. Komen Breast Cancer Foundation (D. Yang).
25. Nicolaou, K. C.; Webber, S. E. Synthesis 1986, 3, 453–461.
26. Mancuso, A. J.; Huang, S. L.; Swern, D. J. Org. Chem. 1978,
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