5
(
1
151 MHz, CD OD / D O) δ (ppm) = 119.7 (
A
CH
C
),
C
1
E
20
P
.6TE(C
D
), MA(C
N
H
U
),
S
52
C
.5
R
(CIP
H
T
), 60.5 (CH), 112.1-112.5 (m, C ), 113.4 (C ),
3 q q
3
2
q
2
30.2 (CH), 132.3 (CH), 149.8 (C ), 151.2 (C ).
116.5 (CH), 127.2 (C ), 131.5 (CH), 132.5 (CH), 136.4-138.5 (m,
q
q
q
2
× C ), 139.6-141.6 (m, C ), 143.5-145.2 (m, 2 × C ), 153.6
q q q
+
Titanium(III) chloride (1.00 mmol, 1 mL of a ca. 1 M solution
(C ), 170.6 (C ), 172.1 (C ); MS (EI) m/z (%): 403 (5) [M ], 345
q q q
in 3 N hydrochloric acid) was added dropwise by a syringe pump
to a vigorously stirred, degassed mixture of methyl L-tyrosinate
hydrochloride (2) (3.00 mmol, 695 mg) and 2,4-dinitrophenyl-
diazonium tetrafluoroborate (5) (1.00 mmol, 282 mg) in hydro-
chloric acid (3 N, 3 mL) under nitrogen atmosphere within 10-
(19), 344 (100), 313 (23), 302 (20), 274 (13), 273 (71), 88 (32),
+
6
4
0 (14), 43 (34); HRMS (EI) calcd. for C H F NO [M ]:
18 14 5 4
03.0843, found: 403.0845.
4-Fluorophenyldiazonium chloride
1
5 minutes. After stirring for 10 more minutes, the pH of the
A degassed solution of 4-fluoroaniline (10.0 mmol, 0.96 mL)
in hydrochloric acid (3 N, 10 mL) and water (10 mL) was treated
at 0 °C with a degassed solution of sodium nitrite (10.0 mmol,
crude mixture was adjusted to a value of 8-9 by the use of satd.
aqueous sodium carbonate. Threefold extraction with ethyl
acetate (3 × 75 mL), washing of the combined organic phases
with a satd. aqueous solution of sodium chloride, drying over
sodium sulfate and concentration in vacuo led to the crude
product. Purification by column chromatography (silica gel,
CH Cl /MeOH = 25:1→10:1) gave methyl L-3-(2,4-dinitro-
0
.69 g) in water (5 mL) by dropwise addition over a period of 20
minutes, followed by additional stirring for 30 minutes at 0 °C.
The mixture was diluted with further hydrochloric acid (3
0 mL) and water (15 mL) for a final concentration of (0.2
N,
1
(
M
2
2
10 mmol / 50 mL)).
phenyl)tyrosinate (6).
AcHN-(3-(4-fluorophenyl)-Tyr)-Lys-OMe 16
R = 0.1 (CH Cl /MeOH 25:1); colorless solid; yield 139 mg
f
2
2
1
(
38%);
H
NMR (600 MHz, CDCl3) δ (ppm) = 2.86 (dd,
Conditions A (Scheme 2): To a solution of AcHN-Tyr-Lys-
OMe (14) (18.4 µmol, 8.80 mg) in water (1 mL) titanium(III)
chloride (168 µmol, 126 µL, in hydrochloric acid,
1.33 mmol/mL) was added. Under argon atmosphere at 30° C a
solution of 4-fluorophenyldiazonium chloride (0.2 , 84.0 µmol,
J = 7.8 Hz, J = 13.9 Hz, 1 H), 3.10 (dd, J = 6.7 Hz, J = 14.1 Hz,
1
7
H), 3.75 (s, 3 H), 3.76-3.80 (s, 1 H), 6.53 (d, J = 8.8 Hz, 1 H),
.05-7.07 (m, 2 H), 7.58 (d, J = 8.5 Hz, 1 H), 8.41 (dd,
2 M
13
J = 2.4 Hz, J = 8.5 Hz, 1 H), 8.76 (d, J = 2.3 Hz, 1 H); C NMR
M
(
1
1
(
(
151 MHz, CDCl ) δ (ppm) = 39.4 (CH ), 51.9 (CH ), 55.0 (CH),
420 µL) was added over 20 minutes and the mixture was stirred
for further 25 minutes at this temperature. The reaction mixture
was washed with diethyl ether (3 × 2.5 mL) and the water phase
was lyophilized. After removal of titanium salts by solid phase
3
2
3
15.5 (CH), 119.5 (CH), 123.2 (CH), 126.5 (CH), 128.0 (Cq),
29.9 (CH), 131.5 (C ), 133.7 (CH), 139.6 (C ), 146.3 (C ), 149.2
q
q
q
+
C ), 152.7 (C ), 174.9 (C ); MS (EI) m/z (%): 361 (2) [M ], 302
q q q
1
16), 274 (9), 273 (17), 256 (21), 181 (10), 89 (23), 88 (100), 33
extraction, the water phase was lyophilized again and H NMR
+
(
3
12); HRMS (EI) calcd. for C H N O [M ]: 361.0910, found:
spectrum was recorded of the crude reaction mixture. The ratio of
starting material to product was determined as 1:1. Purification
by preparative HPLC (solvents A (water 0.1% formic acid) and B
16
15
3
7
61.0909.
Methyl L-3-(pentafluorophenyl)tyrosinate (9) (Scheme 1)
(
acetonitrile, 0.1% formic acid): from 5 to 50% B at 35 min and
A solution of pentafluoroaniline (5.00 mmol, 916 mg) in dry
to 100% B at 55 min, followed by a wash and re-equilibration
acetonitrile (1 mL) is added dropwise to a mixture of pulverized
nitrosyl tetrafluoroborate (5.00 mmol, 915 mg) in dry acetonitrile
step (total run time was 61 min), led to 16 as white powder.
Conditions B (Scheme 2): Dipeptide AcHN-Tyr-Lys-OMe
(1 mL) at –30 °C over a period of 30 minutes. After stirring for 1
(5.2 µmol, 2.5 mg) was dissolved in phosphate buffer (30.0 mg
hour at –30 °C, dry dichloromethane (7.5 mL) was added and the
resulting precipitate was filtered off. Drying in vacuo led to the
desired pentafluorophenyldiazonium tetraflurorborate (8).
NaH PO , 35.8 mg Na HPO in 0.55 mL deuterated water with
2
4
2
4
diluted hydrochloric acid (2 M), pH 4-5) and under air
atmosphere was added 4-fluorophenylazocarboxylate (25 µmol)
(synthesized as described in ref. 26a) in a acetonitrile-d -
deuterated water solution (150 µL / 50 µL) over 60 minutes. H
13
Yellow solid; yield 1.07 g (76%); C NMR (151 MHz,
3
1
CD CN) δ (ppm) = 96.1 (m, C ), 139.8 (d, J = 260.5 Hz,
3
q
CF
2
× C ), 149.0 (dd, J = 15.6 Hz, J = 282.4 Hz, C ), 154.4 (d,
NMR spectrum was recorded from the crude reaction mixture.
The ratio of starting material to product was determined as 6.3:1.
q
CF
CF
q
19
JCF = 279.3 Hz, 2 × Cq);
F
NMR (282 MHz, CD CN)
3
δ (ppm) = –120.1, –123.7, –151.9, –153.0.
1
H NMR (600 MHz, CD OD): δ (ppm) = 1.42-1.49 (m, 2 H),
3
A
solution of sodium iodide (0.50 mmol, 75 mg) in
1.58-1.72 (m, 3 H), 1.84-1.89 (m, 1 H), 1.92 (s, 3 H), 2.84-2.91
(m, 3 H), 3.02 (dd, J = 6.5 Hz, J = 13.9 Hz, 1 H), 3.63 (s, 3 H),
4.44 (dd, J = 4.7 Hz, J = 9.7 Hz, 1 H), 4.50 (dd, J = 6.6 Hz,
J = 8.5 Hz, 1 H), 6.81 (d, J = 8.2 Hz, 1 H), 7.04 (dd, J = 2.3 Hz,
J = 8.3 Hz, 1 H) 7.09 (t, JHF = 8.9 Hz, J = 8.9 Hz, 2 H), 7.15 (d,
J = 2.2 Hz, 1 H), 7.56 (dd, JHF = 5.5 Hz, J = 8.9 Hz, 2 H); LC-
acetonitrile (1 mL) was added dropwise by a syringe pump to a
mixture of the doubly protected tyrosine derivative 7 (0.50 mmol,
19 mg) and pentafluorophenyldiazonium tetrafluoroborate (8)
1.00 mmol, 282 mg) in acetonitrile (5.8 mL) under nitrogen
atmosphere within 15 minutes. After addition of an aqueous
solution of sodium thiosulfate (0.05 M, 30 mL), the reaction
mixture was extracted with ethyl acetate (3 × 50 mL). The
combined organic phases were washed with satd. aqueous
solution of sodium chloride, dried over sodium sulfate and
concentrated in vacuo. Purification by column chromatography
1
(
+
MS (ESI) m/z: 460.25 [M+H] . HRMS (ESI) calcd. for
+
C H FN O [M+H] 460.2242, found: 460.2242.
24
30
3
5
H N-Arg-Arg-Pro-(3-(4-fluorophenyl)-Tyr)-Ile-Leu-OH 19
2
(
Table 3)
(silica gel, EtOAc/hexanes = 3:1) gave the desired product 9.
To a solution of NT(8-13) triformiate (18) (10.5 µmol,
1
0.4 mg) in water (1 mL) titanium(III) chloride (92.0 µmol,
69.2 µL, in 2 hydrochloric acid, 1.33 mmol/mL) was added.
Under argon atmosphere at 30° C the 4-fluorophenyldiazonium
chloride solution (0.2 , 48.0 µmol, 240 µL) was added over 20
R = 0.4 (EtOAc/hexanes 3:1); colorless solid; yield 46 mg
f
1
M
(
2
23%); H NMR (360 MHz, CDCl ): δ (ppm) = 1.98 (s, 3 H),
3
.98 (dd, J = 6.2 Hz, J = 14.1 Hz, 1 H), 3.12 (dd, J = 5.7 Hz,
J = 14.1 Hz, 1 H), 3.74 (s, 3 H), 4.88 (dd, J = 6.0 Hz,
J = 14.1 Hz, 1 H), 6.10 (d, J = 8.1 Hz, 1 H), 6.83 (d, J = 8.3 Hz,
M
minutes and the reaction mixture was stirred for further 25
minutes. The reaction mixture was washed with diethyl ether
(3 × 2.5 mL) and the water phase was lyophilized. After removal
of titanium salts by solid phase extraction, the water phase was
1
1
H), 6.90 (d, J = 2.1 Hz, 1 H), 7.07 (dd, J = 2.2 Hz, J = 8.4 Hz,
13
H); C NMR (151 MHz, CDCl ): δ (ppm) = 22.8 (CH ), 37.2
3
3