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C. Yu, J. W. Taylor / Bioorg. Med. Chem. 7 (1999) 161±175
ester in 200 mL of anhydrous dioxane and benzene (1/1)
was added into the mixed anhydride mixture. The reac-
tion solution was stirred for 3 days at rt and for 4 h at
50 ꢀC. The mixture was then evaporated, and the resi-
due was dissolved in ethyl acetate. This solution was
washed with 5% (w/v) NaHCO3, water, 5% (w/v) citric
acid, and water, then dried over Na2SO4, and evapo-
rated. The residue was recrystallized from chloroform
andꢀhexanes, two times: Yield 7.1 g (59%); mp 115±
9-Fluorenylmethyl-N-Boc-4-aminophenylacetate (Boc-
APA-OFm). N-Boc-4-aminophenylacetic acid (Boc-
APA-OH) was prepared by same procedure as that for
Boc-ABA-OH. Boc-APA-OH (7.58 g, 30 mmol) was
then dissolved in 300 mL of dioxane and DCM (1/1). 9-
Fluorenemethanol (5.89 g, 30 mmol), DCC (6.80 g,
33 mmol), and DMAP (37 mg, 0.3 mmol) were then
added. The mixture was stirred for 10 h at rt. The DCU
was removed by ®ltration, and then the solvents were
evaporated. The residue was dissolved in DCM and
washed with 5% (w/v) NaHCO3, water, 5% (w/v) citric
acid, and water, then dried over MgSO4 and evapo-
rated. The residue was recrystallized from chloroform
1
117 C; H NMR (CDCl3) d 9.4 (br, 1H), 8.10 (d, 2H),
7.72±7.85 (m, 4H), 7.65 (d, 2H), 7.30±7.50 (m, 4H),
5.35±5.45 (m, 1H), 4.60 (d, 2H), 4.40 (t, 1H), 4.1±4.3 (m,
1H), 2.4±2.6 (m, 2H), 2.2±2.4 (m, 1H), 1.8±2.0 (m, 1H),
1.50 (s, 9H), 1.47 (s, 9H); Elemental analysis calcd for
C35H40O7N2: C, 69.98; H, 6.71; N, 4.66. Found: C,
69.44; H, 6.82; N, 5.04.
and hexanes: Yield 10.1 g (78.1%); mp 134±136 ꢀC; H
1
NMR (DMSO-d6) d 9.32 (s, 1H), 7.87 (d, 2H), 7.20±7.52
(m, 8H), 7.06 (d, 2H), 4.35 (d, 2H), 4.20 (t, 1H), 3.60 (s,
2H), 1.47 (s, 9H).
Nꢀ-Boc-(L)-Glu(ABA-OFm)-OH. Na-Boc-(l)-Glu(ABA-
OFm)-OBut (6.0 g, 10.0 mmol) was dissolved in 100 mL
of TFA. The solution was stirred for 1 h at rt. The TFA
was then removed by evaporation. DCM (100 mL) was
added and evaporated again. The residue was dried
under vacuum. Di-t-butyl dicarbonate (3.27 g, 15 mmol)
and DIEA (3.87 g, 30 mmol) was dissolved in 100 mL
of dioxane. The solution of the TFA salt of H-
Glu(ABA-OFm)-OH (10 mmol) in 200 mL of dioxane
and water (2/1) was added, dropwise, over 3 h at rt.
The solution was stirred for another hour at rt, then
neutralized by 10% (w/v) citric acid. Most of dioxane
was evaporated. 5% (w/v) citric acid (200 mL) was
added, and the mixture was extracted with ethyl acetate
(3Â200 mL). The organic layer was washed with 5%
(w/v) citric acid and water, dried over Na2SO4, and then
evaporated. The residue was precipitated from ethyl
acetate and hexanes two times. The crude product was
then puri®ed by silica gel ¯ash chromatography (eluent:
CHCl3/MeOH/AcOH, 20/1/1): Yield 3.5 g (64.3%); mp
Nꢀ-t-Boc-(L)-Glu(APA-OFm)-OBut. O-9-Fluorenyl-
methyl-N-Boc-4-aminophenylacetate (8.59 g, 20 mmol)
was dissolved in 100 mL of TFA. The solution was stir-
red for 1 h at rt. The TFA was evaporated. DCM
(200 mL) was added and evaporated again. The residue
was then dried under vacuum. Boc-(l)-Glu-OtBu
(6.07 g, 20 mmol) and DIEA (2.60 g, 20 mmol) were dis-
solved in 100 mL of anhydrous benzene. The solution
was cooled to 5 ꢀC. Trimethylacetyl chloride (2.40 g,
20 mmol) was added. This solution was stirred for 2 h at
0 ꢀC and 2 h at rt. The TFA salt of O-9-¯uorenylmethyl-
4-aminophenylacetate (20 mmol) was dissolved in
200 mL of anhydrous dioxane and benzene (2/1). DIEA
(5.83 g, 45 mmol) was added, and then the above mixed
anhydride solution was ®ltered into the solution of O-9-
¯uorenylmethyl-4-aminophenylacetate. This mixture
was stirred for 3 days at rt and 2 h at 50 ꢀC. The solvents
were evaporated. The oil residue was dissolved in ethyl
acetate and washed with 5% (w/v) NaHCO3, water, 5%
(w/v) citric acid, and then water, dried over Na2SO4,
and evaporated. The residue was recrystallized from
ethyl acetate and hexanes: Yield 9.3 g (75.6%); mp 77±
sublimes at 90 ꢀC; H NMR (CDCl3) d 8.90±8.95 (br,
1
1H), 8.04 (d, 2H), 7.6±7.8 (m, 6H), 7.30±7.42 (m, 4H),
5.60 (d, 1H), 4.58 (d, 2H), 4.30±4.50 (m, 2H), 2.50±2.65
(br, 2H), 2.28±2.45 (m, 1H), 2.0±2.2 (m, 1H), 1.47 (s,
80 ꢀC; H NMR (CDCl3) d 8.95 (s, 1H), 7.75 (d, 2H),
1
.
9H); Elemental analysis calcd for C31H32O7N2 H2O: C,
66.18; H, 6.09; N, 4.98. Found: C, 66.24; H, 6.03; N,
4.75.
7.60 (d, 2H), 7.35±7.50 (m, 4H), 7.15±7.30 (m, 4H), 5.40
(d, 1H), 4.40 (d, 2H), 4.15±4.32 (m, 2H), 3.65 (s, 2H),
2.45 (t, 2H), 2.20±2.35 (m, 1H), 1.80±1.95 (m, 1H), 1.48
(s, 9H), 1.46 (s, 9H).
Nꢀ-t-Boc-(L)-2,3-diaminopropionic acid (Boc-Dap-
OH).32±34 Boc-Dap-OH was prepared from Na-Boc-(l)-
Asn-OH by the same proceduꢀre as that for Boc-Dab-
OH: Yield 76.5%; mp 203±205 C (lit. 198±200 ꢀC dec;33
205±205.5 ꢀC34); 1H NMR (CD3OD) d 4.06 (t, 1H),
3.10±3.20 (two d, 2H), 1.47 (s, 9H); Elemental analysis
Nꢀ-t-Boc-(L)-Glu(APA-OFm)-OH. Na-t-Boc-(l)-Glu-
(APA-OFm)-OtBut (7.4 g, 12.0 mmol) was dissolved in
100 mL of TFA. The solution was stirred for 30 min at
rt. The TFA was evaporated, then 20 mL of 4 N HCl in
dioxane was added. The precipitate was washed with
diethyl ether. The solid was dissolved in 150 mL of
dioxane and water (2/1). Di-t-butyl dicarbonate (3.3 g,
15 mmol) and DIEA (3.88 g, 30 mmol) were added, and
the solution was stirred for 2 h at rt. The solvents were
evaporated. The residue was dissolved in ethyl acetate.
The solution was washed with 5% (w/v) citric acid and
water, dried over MgSO4 and evaporated. The solid was
recrystallized from ethyl acetate and hexanes. The pro-
duct was further puri®ed by silica gel ¯ash chromato-
graphy (eluent: CHCl3/MeOH/AcOH, 20/1/1): Yield
.
calcd for C8H16N2O4 1/2 H2O): C, 45.06; H, 8.04; N,
13.14. Found: C, 45.11; H, 8.07; N, 13.02.
Nꢀ-t-Boc-Nꢂ-Fmoc-(L)-2,3-diaminopropionic acid (Boc-
Dap[Fmoc]-OH).34 Boc-Dap(Fmoc)-OH was prepared
from Boc-Dap-OH by same procedure as that for Boc-
Dab(Fmoc)-OH: Yield 71.2%; mp sublimes at above
85 ꢀC (lit. sublimes at 75±100 ꢀC34); 1H NMR (CD3OD)
d 7.79 (d, 2H), 7.65 (d, 2H), 7.27±7.45 (m, 4H), 4.15±
4.35 (m, 4H), 3.35±3.70 (m, 2H), 1.42 (s, 9H); Elemental
analysis calcd for C23H26O6N2: C, 64.78; H, 6.15; N,
6.57. Found: C, 64.35; H, 6.09; N, 6.72.
ꢀ
1
4.1 g (61.2%); mp sublimes at above 88 C; H NMR
(CDCl3) d 8.62 (s, 1H), 7.74 (d, 2H), 7.32±7.66 (m, 6H),