The reaction solution was stirred for 4.5 h at room temperature
after the addition was complete and ethyl acetate (150 mL) was
added. The solution was washed with 5% citric acid, saturated
NaHCO and saturated NaCl, and dried over Na SO . Solvent
7.4 Hz, 1H), 7.90 (d, J = 7.2 Hz, 2H), 7.74 (d, J = 7.4 Hz, 2H),
7.54-7.30 (m, 6H), 7.03 (s, 1H), 6.78 (s, 1H), 5.24 (m, 1H), 4.94
(s, 1H), 4.25 (1H, covered by the solvent peak), 4.00-3.80 (m,
3H), 3.74-3.61 (m, 4H), 3.46-3.18 (4H, covered by the solvent
3
2
4
1
3
was removed by evaporation in vacuo to give a yellow solid.
peak), 2.50 (s, 2H), 2.12-2.09 (m, 2H), 1.88-1.60 (m, 4H);
C
6
Flash silica gel column chromatography purification
NMR (50 MHz, DMSO-d ) 174.6, 174.4, 171.9, 156.7, 144.7,
1
(
CH Cl :CH OH, 20:1) gave a yellow solid (456 mg, 76%); H
144.6, 141.5, 128.4, 127.8, 126.1, 120.8, 66.5, 64.1, 58.3, 55.1,
2
2
3
+
NMR (200 MHz, 1% CD OD/CDCl ) 7.75-7.69 (m, 3H), 7.57-
49.1, 47.5, 32.3, 31.5, 28.5; LC-MS (ESI+) 626.1 [MH] , 628.1,
3
3
7
5
.50 (m, 4H), 7.37-7.21 (m, 14H), 7.05 (d, J = 7.8 Hz, 1H), 5.40-
.36 (m, 1H), 5.05 (s, 2H), 4.30 (d, J = 6.6 Hz, 2H), 4.17-4.00
406.1, 368.0 (100%); HRMS (FAB+) m/z calc’d for
+
C H N O PCl [MH] 626.1702, found 626.1680.
2
7
35
5
6
2
1
3
(m, 4H), 2.22-1.80 (m, 6H), 1.53 (d, J = 3.8 Hz, 6H); C NMR
H-Gln-NH-CPA (4). cis-(2R, 4R)-14 (24 mg, 0.038 mmol) or
(
50 MHz, 1% CD OD/CDCl ) 176.5, 176.1, 172.1, 156.7,
3
3
trans-(2R, 4R)-14 (10 mg, 0.016 mmol) was treated with 10%
diethyl amine (DEA) in DMF (3 mL) for 4.5 h. The solution was
evaporated to dryness under reduced pressure at room
temperature. The solid residue was washed with ethyl ether to
give a white solid of cis-(2R, 4R)-4 (13 mg, 91%) or trans-(2S,
1
1
6
54.9, 149.0, 144.0, 143.8, 141.3, 138.5, 135.1, 133.4, 128.6,
28.4, 128.2, 127.8, 127.2, 125.7, 125.2, 120.0, 77.5, 69.8, 67.1,
4.5, 55.1, 54.4, 47.1, 47.0, 38.6, 32.2, 31.4, 28.2, 27.2; IR
(
7
3
CDCl ) 1744.2, 1662.9, 1530.8, 1451.3, 1395.7, 1361.7, 1265.0,
3
-1 + +
55.0 cm ; LC-MS (ESI+) 788.2 [M+Na] , 766.2 [MH] , 558.1,
68.0 (100%).
4
4
R)-4 (14 mg, 95%); LC-MS (ESI+) 428.1, 426.1, 408.0, 406.0,
04.0 [MH] (100%).
+
Fmoc-Gln-gHse-NH (13). To a solution of 12 (600 mg, 0.784
2
General methods for peptide synthesis. All of the peptides
mmol) in 10 mL of acetic acid and methanol (1:9) was added
0% Pd-C. The solution was stirred under H balloon at room
were synthesized by Merrifield solid-phase peptide synthesis
protocols using a double-coupling protocol on a bubbler peptide
synthesizer. The resins for the peptide assembly were 2-methoxy-
-alkoxybenzyl alcohol (SASRIN, 200-400 mesh) resin,
purchased from Bachem (Torrance, CA), or 4-
1
2
temperature for 3 h. The catalyst was filtered through Celite and
the filtrate was concentrated in vacuo. The residue was purified
by flash silica gel column chromatography (CH Cl :CH OH, 20:1
4
2
2
3
1
to 5:1) to give a white solid (315 mg, 87%); mp 115 C (dec.); H
hydroxymethylphenoxy (WANG-type HMP) resin, purchased
from Advanced Chemtech (Louisville, KY). N-Fmoc-protected
amino acids of the L-configuration and reagents were purchased
from Advanced Chemtech. Side-chain protection was Ser(But)
and Lys(Cl-Z). The capping group was introduced via
monobenzyl succinate or monobenzyl glutarate. C-terminal
amino acid was loaded using DMAP/DIC protocol for SASRIN
resin, and HOBt/DMAP/DIC protocol for WANG resin, and 3-
fold excess of activated protected amino acid was used for each
coupling and recoupling, using NMP as solvent. Following
completion of the assembly on the resin support, the N-terminal
Fmoc group was removed via the standard 25% piperidine/NMP
protocol, followed by washing 5 times with NMP and
introduction of N-terminal capping group. Deprotection and
removal of the peptide from the resin support were effected using
90% TFA in CH Cl for both SASRIN and WANG resin. The
NMR (200 MHz, CD OD) 7.84 (d, J = 1.2 Hz, 2H), 7.68 (d, J =
3
6
.6 Hz, 2H), 7.45-7.30 (m, 4H), 5.22 ( t, J = 6.6 Hz, 1H), 4.42 (d,
J = 3.6 Hz, 1H), 3.39 (d, J = 3.6 Hz, 1H), 4.28-4.13 (m, 2H),
3
.77-3.69 (m, 2H), 2.38 (t, J = 7.4 Hz, 2H), 2.10-1.92 (m, 4H);
1
3
C NMR (50 MHz, CD OD) 177.2, 172.8, 156.6, 143.4, 143.2,
3
1
3
1
40.7, 126.9, 126.3, 124.3, 119.1, 66.3, 56.2, 55.9, 53.8, 33.3,
0.1, 26.9; IR (KBr) 3298.6 (br), 1668.6, 1540.4, 1449.7, 1248.1,
052.0, 739.4 cm ; LC-MS (ESI+) 463.1, [M+Na] , 424.1
-
1
+
(100%), 368.1.
Fmoc-Gln-4-NH-CPA (14). At 0 C, to a stirred solution of
bis(2-chloroethyl) phosphoramidic dichloride (185 mg, 0.693
mmol) in anhydrous THF (25 mL) under N was added a solution
2
of 12 (254 mg, 0.577 mmol) in NMP (25 mL) and TEA (195 L,
1
.39 mmol). The reaction mixture was stirred at 0 C for 30 min
and at room temperature for 48 h. The solution was filtered to
remove the white precipitate and evaporated at reduced pressure.
The residue was purified by flash silica gel column
chromatography (CH Cl :CH OH, 40:1) to give Fmoc-Gln-NH
2
2
2
benzyl esters of the capping groups were not affected under these
conditions. After removal of solvents under reduced pressure, the
peptides were purified by preparative HPLC on reverse-phase
radial compression C18 column (Waters, 15 µm, 100 Å, 5 x 30
cm). A step gradient was generated from 1 L each of successively
increasing concentration of mobile phase (solvent A, 0.1%
TFA/H O; solvent B, 0.1% TFA/CH CN). A flow rate of 9
2
2
3
and two diastereoisomers. The starting material was recovered in
1
1
(
5% yield. Fmoc-Gln-NH : white solid (38 mg, 18%); H NMR
2
6
200 MHz, DMSO-d ) 7.90 (d, J = 7.8 Hz, 2H), 7.74 (d, J = 7.5
Hz, 2H), 7.47-7.30 (m, 5H), 4.25 (s, 2H), 4.07 (br s, 2H), 3.94-
2
3
3
1
.80 (m, 2H), 3.36 (br s, 2H), 2.20-2.12 (t, J = 8.4 Hz, 2H), 1.95-
.74 (m, 2H); LC-MS (ESI+) 390.0, 368.0 [MH] (100%); cis-
mL/min was used to elute the peptide. Detection was performed
by monitoring the UV absorbance at 220 nm. Homogeneous
product fractions (>98% pure) were pooled and freeze-dried. The
homogeneity of the peptides was demonstrated by analytical
reverse phase HPLC using C18 column. Identities were confirmed
by mass spectral analysis.
+
(
2R,4R)-14 (the faster eluting isomer): white solid (44 mg, 12%);
H NMR (400 MHz, DMSO-d6) 8.44 (d, J = 3.4 Hz, 1H), 7.77
d, J = 3.8 Hz, 2H), 7.63-7.60(m, 2H), 7.43 (d, J = 4.0 Hz, 1H),
1
(
7
6
.30 (t, J = 3.6 Hz, 2H), 7.27 (s, 1H), 7.22 (t, J = 3.8 Hz, 2H),
.64 (s, 1H), 5.13-5.11 (m, 1H), 4.99 (d, J = 8.6 Hz, 1H), 4.33
Monobenzyl Succinate. To a solution of succinic anhydride
(
3
(
dd, J = 9.2, 4.4 Hz, 1H), 4.15-4.07 (m, 3H), 4.05-3.98 (m, 1H),
.87 (dd, J = 7.1, 3.6 Hz, 1H), 3.55 (t, J = 3.6 Hz, 4H), 3.23-3.14
m, 4H), 2.05 (t, J = 4 Hz, 2H), 1.93-1.89 (m, 1H), 1.77-1.67 (m,
(1.0 g, 10 mmol) in DMF (4 mL) was added benzyl alcohol (0.94
mL, 9.09 mmol) and DIEA (1.93 mL, 11 mmol) at 0 C. The
reaction mixture was stirred at room temperature overnight and
was evaporated in Speed-vac. The residue was dissolved in ethyl
acetate (50 mL) and washed with saturated NaCl (10 mL x 2).
1
3
2
1
6
3
H), 1.58 (d, J = 6.8 Hz, 1H); C NMR (50 MHz, DMSO-d6)
74.5, 172.3, 156.6, 144.6, 144.6, 141.5, 128.4, 127.9, 126.1,
6.4, 63.8, 58.4, 54.6, 48.8 (d, J = 15.2 Hz), 47.4, 43.1, 32.1,
3
1
6
The organic solution was extracted with aqueous NaHCO (5 M,
3
0.4, 28.7; P NMR (162 MHz, DMSO-d ) 9.15 (s); LC-MS
+
5 mL x 3) and the aqueous extractions were combined, acidified
to pH 4 by adding citric acid (5 M), extracted with ethyl acetate
(ESI+) 626.1 [MH] , 628.1, 406.0, 368.0 (100%); HRMS (FAB)
+
m/z calc’d for C H N O PCl [MH] 626.1702, found
6
solid (20 mg, 6%); H NMR (200 MHz, CD OD) 8.48 (d, J =
2
7
35
5
6
2
(30 mL x 3). The EtOAc extractions were combined, washed
26.1721; trans-(2S, 4R)-14 (The slower eluting isomer): white
1
with saturated NaCl, and dried over Na SO . Solvent was
2
4
3