J HH ) 7.4 Hz, 2H), 7.39 (t, J HH ) 7.2 Hz, 2H), 7.30 (t, J HH ) 7.4
Hz, 2H), 5.99 (d, J HH ) 9.1 Hz, 1 H), 4.42 (m, 2H), 4.35 (dd, J HH
) 2.2 and 6.3 Hz, 1H), 4.30 (m, 1H), 4.24 (t, J HH ) 7.2 Hz, 1H),
3.07 (bs, 1H), 1.52 (s, 9H), 1.29 (d, J HH ) 6.6 Hz, 3 H). 13C NMR
(125.8 MHz, CDCl3) δ ppm: 170.4, 157.0, 143.9, 143.7, 141.3,
127.7, 127.1, 125.2, 120.0, 82.5, 68.2, 67.2, 59.8, 47.1, 28.0, 20.0.
ESI-MS: [MNa]+ 420.1770 (obsd), 420.1781 (calcd).
131.0, 128.2, 127.8, 127.5, 125.6, 120.4, 115.9, 83.1, 62.5, 55.8,
47.5, 38.0, 28.4. ESI-MS: [MH]+ 460.2115 (obsd), 460.2118
(calcd).
N-r-F m oc-p h osp h o(1-n itr op h en yleth yl-2-cya n oeth yl)-L-
tyr osin e ter t-Bu tyl Ester . N-R-Fmoc-L-tyrosine tert-butyl ester
(440 mg, 0.96 mmol) was dissolved in dry THF (4 mL) in a round-
bottom flask provided with 4 Å molecular sieves (400 mg). In a
separate flask was dissolved 1H-tetrazole (115 mg, 1.63 mmol)
in dry THF (4 mL) followed by O-1-(2-nitrophenyl)-ethyl-O′-â-
cyanoethyl-N,N-diisopropylphosphoramidite (600 mg, 1.63 mmol).
After 5 min activation the mixture was added over the Fmoc-
L-tyrosine tert-butyl ester solution, and the resulting mixture
was allowed to stir in the dark overnight under argon. The
reaction mixture was filtered over Celite and concentrated under
reduced pressure, and the residue was redissolved in CH2Cl2
(25 mL) and washed with 1% NaHCO3 (2 × 25 mL). The
combined organic layers were dried with Na2SO4 and concen-
trated under reduced pressure. The oily residue was redissolved
in dry CH2Cl2 (25 mL), and tert-butyl hydroperoxide was added
dropwise over the solution (300 µL of 5-6 M solution in decane).
The reaction was stirred for 1 h at room temperature and then
washed with 2% NaHCO3 (2 × 50 mL). The organic layer was
dried with Na2SO4 and concentrated under reduced pressure.
The resulting residue was purified by flash column chromatog-
raphy (1:1 hexanes/EtOAc, Rf ) 0.19) to give the product (442
mg) in 63% yield. 1H NMR (400 MHz, MeOD) δ ppm: 7.75-
7.41 (m, 7H), 7.34 (t, 2H, J HH ) 7.4 Hz), 7.25 (t, 2H, J HH ) 6.2
Hz), 7.19 (d, 1H, J HH ) 8.5 Hz), 7.12 (d, 2H, J HH ) 8.5 Hz), 7.05
(d, 1H, 8.3 Hz), 6.96 (dd, 1H, J HH ) 2.7, 8.6 Hz), 6.11-6.15 (m,
N-r-F m oc-p h osp h i(1-n itr op h en yleth yl-2-cya n oeth yl)-L-
th r eon in e ter t-Bu tyl Ester . In a round-bottom flask under
argon was dissolved N-R-Fmoc-L-threonine tert-butyl ester (1.35
g, 3.39 mmol) in 16 mL of anhydrous THF, and the solution was
allowed to stir. In
a pear-shaped flask under argon were
dissolved O-1-(2-nitrophenyl)-ethyl-O′-â-cyanoethyl-N,N-diiso-
propylphosphoramidite (3.74 g, 10.2 mmol) and 4,5-dicyano-
imidazole (1.20 g, 10.20 mmol) in 16 mL of anhydrous THF, and
the solution was allowed to mix for several minutes in the dark.
The phosphoramidite solution was added into the stirring amino
acid solution via cannula under argon positive pressure and
allowed to stir at room temperature in the dark overnight, under
inert conditions. The reaction was judged complete by disap-
pearance of the starting material and appearance of the product.
The crude mixture was concentrated under reduced pressure and
redissolved in EtOAc (100 mL). The solution was washed with
10% NaHCO3 (2 × 100 mL) and then brine (1 × 100 mL). The
crude material was dried over Na2SO4, filtered, concentrated
under reduced pressure, and used immediately in the following
reaction.
N-r-F m oc-p h osp h o(1-n itr op h en yleth yl-2-cya n oeth yl)-L-
th r eon in e ter t-Bu tyl Ester . N-R-Fmoc-phospho(1-nitrophenyl-
ethyl-2-cyanoethyl)-L-threonine tert-butyl ester was synthesized
as described for N-R-Fmoc-phospho(1-nitrophenylethyl-2-cyano-
ethyl)-L-serine tert-butyl ester (8). The product was purified by
silica gel flash chromatography (1:3 hexanes/EtOAc, Rf ) 0.38)
to give the product in 74% yield over two steps. 1H NMR (300
MHz, CDCl3) δ ppm: 7.96 (m, 1H), 7.80-7.60 (m, 6H), 7.53-
7.33 (m, 5H), 6.12 (m, 1H), 5.45 (m, 1H), 5.03 (m, 2H), 4.44 (m,
2H), 4.24 (m, 3 H), 2.69 (m, 2H), 1.75 (m, 3H), 1.47 (m, 9H),
1.34 (m, 3H). 13C NMR (125.8 MHz, CDCl3) δ ppm: 168.5, 168.2,
156.8, 147.1, 143.8, 141.5, 136.9, 134.1, 129.4, 128.0, 127.9, 127.8,
127.3, 125.3, 124.8, 120.2, 116.4, 83.6, 73.9, 67.5, 67.4, 62.5, 58.9,
47.3, 28.1, 24.4, 19.8, 18.4. 31P NMR (121.5 MHz, CDCl3) δ ppm:
-3.30, -3.20. ESI-MS: [MNa]+ 702.2194 (obsd), 702.2187
(calcd).
1H), 4.28-4.08 (m, 6H), 3.08-2.82 (m, 2H), 2.78 (t, 2H, J HH
)
5.7 Hz), 1.70-1.66 (m, 3H), 1.39 (s, 9H). 13C NMR (101 MHz,
MeOD) δ ppm: 172.5, 158.3, 148.4, 145.3, 142.6, 137.7, 136.2,
135.2, 132.1, 130.5, 129.0, 128.9, 128.3, 126.4, 125.7, 121.1, 121.0,
120.9, 118.3, 83.1, 75.3, 68.0, 64.9, 57.5, 48.4, 37.9, 28.4, 24.6,
20.1. 31P NMR (121.5 MHz, CDCl3) δ ppm: 7.763, 7.762, -7.40,
-7.46, -7.50. ESI-MS: [MH]+ 742.2541 (obsd), 742.2524 (calcd).
N-r-F m oc-p h osp h o(1-n itr op h en yleth yl-2-cya n oeth yl)-L-
t yr osin e (3). N-R-Fmoc-phospho(1-nitrophenylethyl-2-cyano-
ethyl)-L-tyrosine tert-butyl ester (440 mg, 590 µmol) was dis-
solved in dry CH2Cl2 (8 mL) in a 25 mL round-bottom flask, and
the resulting solution was cooled to 0 °C. TFA (8 mL) was slowly
added over the solution, and the resulting mixture was allowed
to stir in the dark for 1 h at room temperature under argon.
The solvents were removed under reduced pressure, and the
residue was redissolved in chloroform and concentrated again
to eliminate the residues of TFA. The residue was purified by
flash column chromatography (1%AcOH/5% MeOH/CH2Cl2, Rf
) 0.22) to give the product 3 (329 mg) in 81% yield. 1H NMR
(400 MHz, CDCl3) δ ppm: 9.70 (broad s, 1H), 7.95-7.93 (d, 1H,
J HH ) 7.1 Hz), 7.77-7.70 (m, 3H), 7.65-7.58 (m, 3H), 7.45-
7.37 (m, 3H), 7.27-7.32 (m, 2H), 7.24-6.99 (m, 4H), 6.25 (m,
1H), 5.54 (m, 1H), 4.65 (broad s, 1H), 4.47 (broad s, 2H), 4.35
(broad s, 1H), 4.21 (broad s, 2H), 3.1 (m, 2H), 2.50 (m, 2H), 1.71
(dd, 3H J HH ) 2.6, 3.6 Hz). 13C NMR (101 MHz, CDCl3) δ ppm:
173.5, 156.0, 149.1, 146.9, 143.9, 141.5, 136.8, 134.2, 134.1, 131.1,
129.2, 127.8, 127.3, 125.2, 124.7, 120.2, 120.1, 120.1, 116.2, 74.4,
67.2, 63.0, 54.7, 47.3, 37.1, 24.3, 19.6. 31P NMR (121.5 MHz,
CDCl3) δ ppm: -7.74. ESI-MS: [MH]+ 686.1888 (obsd), 686.1898
(calcd).
N-r-F m oc-p h osp h o(1-n itr op h en yleth yl-2-cya n oeth yl)-L-
th r eon in e (2). N-R-Fmoc-phospho(1-nitrophenylethyl-2-cyano-
ethyl)-L-threonine was synthesized as described for N-R-Fmoc-
phospho(1-nitrophenylethyl-2-cyanoethyl)-L-serine (1). The product
was purified by silica gel flash chromatography (EtOAc, then
9:1 EtOAc/MeOH with 1% AcOH, Rf ) 0.18) to give 2 in 64%
yield. 1H NMR (300 MHz, CDCl3) δ ppm: 7.97-7.26 (m, 12H),
6.21 (bs, 1H), 6.13 (m, 1H), 5.62 (m, 1H), 5.01 (m, 2H), 4.51-
4.11 (m, 5H), 2.70 (m, 2H), 1.75 (m, 3H), 1.30 (m, 3H). 13C NMR
(125.8 MHz, CDCl3) δ ppm: 170.1, 156.9, 147.1, 143.8, 141.5,
137.2, 134.2, 129.4, 128.0, 127.9, 127.3, 125.3, 124.8, 120.2, 116.6,
73.8, 67.6, 67.1, 63.0, 58.1, 47.3, 19.8, 18.5. 31P NMR (121.5 MHz,
CDCl3) δ ppm: -3.91. ESI-MS: [MNa]+ 662.2857 (obsd), 662.2877
(calcd).
Tyr osin e Bu ild in g Block (3). N-r-F m oc-L-tyr osin e ter t-
Bu tyl Ester . N-R-Fmoc-L-tyrosine (440 mg, 1.10 mmol) was
placed in a dry 25 mL round-bottom flask in CH2Cl2/THF (4 mL,
4:1) and cooled to 0 °C under argon, and tert-butyl trichloro-
acetimidate (720 mg, 3.28 mmol) was added over the resulting
solution. The reaction mixture was allowed to stir overnight at
room temperature. CH2Cl2 (100 mL) was added, and the solution
was washed with 2.5% NaHCO3 (2 × 50 mL). The organic layer
was dried with Na2SO4 and concentrated. The residue was
purified by flash column chromatography (1:1 EtOAc/hexanes,
Rf ) 0.72) to give the desired product as a sticky solid (342 mg)
Ack n ow led gm en t. This research was supported by
the NIH (GM64346 Cell Migration Consortium) and
Merck Research Laboratories. M.E.V. thanks the Hu-
man Frontier Science Program Foundation for his Long
Term Postdoctoral Fellowship. We also acknowledge
NSF Award Nos. DBI-9729592 and CHE-9808061 and
NIH No. 1S10RR13886-01 for support of the NMR
facility.
Su p p or t in g In for m a t ion Ava ila b le: 1H NMR and 13C
NMR spectra for all compounds; general experimental infor-
mation. This material is available free of charge via the
Internet at http://pubs.acs.org.
in 68% yield. 1H NMR (400 MHz, MeOD) δ ppm: 7.70 (d, J HH
)
7.5 Hz, 2H), 7.55 (d, J HH ) 7.4 Hz, 2H), 7.33 (m, 2H), 7.25 (m,
2H), 7.06 (d, J HH ) 8.3 Hz, 2H), 6.78 (d, J HH ) 8.3 Hz, 2H), 4.36-
4.25 (m, 3H), 4.09 (m, 1H), 3.05 (m, 2H), 1.47 (s, 9H). 13C NMR
(101 MHz, MeOD) δ ppm: 171.5, 156.4, 155.7, 144.3, 141.7,
J O0344891
6798 J . Org. Chem., Vol. 68, No. 17, 2003