Fluorescent Labels
FULL PAPER
and excess of Et
lated by reversed-phase column chromatography on Polygoprep (MeCN/
1:2),yield 28 mg (97%). HPLC: =18.7 min (area 100%).
H NMR (300 MHz,[D ]DMSO + [D ]methanol,2 rotamers): d=1.88
m,4H; (CH )CH (CH )),2.75 (m,4H; ArCH
),3.07 (s, ꢁ0.5H; NMe,
rotamer 1),3.18 (s, ꢁ2.5H; NMe,rotamer 2),3.40–3.65 (m,6H;
CH NAr and NCH (CH OCO)),3.73 (m,1H; (CH )CH OCO),4.10
m,1H; (CH )CH OCO),6.78–6.96 (m,4H; H-1/8 and H-2 ’’),7.36–
.50 (m,1H; H-3 ’),7.68–7.82 (m,2H; H-4 ’/5’),8.06–8.23 ppm (m,3H;
3
N were removed in vacuo,and the title product was iso-
31 3 10 2
HR-MS (ESI,positive mode): m/z: calcd for C31H N O S : 670.1524;
+ +
found: 670.1522 [M+H] ,692.1341 [ M+Na] .
H
2
O
t
R
Acid 3c: Ester 13 (160 mg,0.228 mmol) was suspended in H
2
O (2 mL)
1
6
4
and cooled with an ice bath. Cold aq. NaOH (1m,0.6 mL) was added
dropwise to the reaction mixture,and it was stirred at 0–4 8C for 1 h.
After 50 min,HPLC displayed full conversion of the starting compound.
Aqueous HCl (1m,1.0 mL) was added,and the mixture was applied on
top of the column with 100 mL of Polygoprep 60–50 C18 equilibrated
(
2
2
A
C
H
T
R
E
U
N
G
2
2
2
2
A
C
H
T
R
E
U
N
G
2
2
A B
H
(
7
2
A B
H
with MeCN/H
MeCN/H
O 2:1 ! 4:1. Water was removed from the fractions by freeze-
drying. HPLC: t =9.4 min (area 94%,ca. 6% of the acid 3a with higher
t ). H NMR (300 MHz,[D 6]DMSO): d=1.76 (brs,4H; (CH 2)CH2-
2
O 2:1. The title compound (134 mg,86%) was eluted with
H-6’ and H-3’’); UV/Vis (water): lmax (e)=540 nm (84000), lem =560 nm,
2
ꢀ
F
fl =0.15; ESI-MS,negative mode: m/z (%): 791 (100) [MꢀH] ; HR-MS
R
1
(
32 4 13 2
ESI,positive mode): m/z: calcd for C36H N O S : 793.1480; found:
+ + +
R
7
93.1478 [M+H] ,815.1297 [ M+Na] ,831.1043 [ M+K] .
A
H
R
U
G
A
H
R
U
G
2
2
2
N-Hydroxysuccinimidyl carbonate (3d): Compound 3b (20 mg,
.030 mmol) was dissolved with dry DMAA (1 mL),di( N-succinimidyl)-
carbonate (76 mg,0.30 mmol) and Et (0.10 mL,0.72 mmol) were
added under N with stirring. The reaction mixture was left at RT for 1 h.
The solvent and excess of Et N were removed in vacuo,water (5 mL)
was added to the residue,and the title product was isolated by reversed-
3H; NMe),3.32 (m,2H; N CH
6.75 (brs,2H; H-1/8),7.38 (m,1H; H-3 ’),7.60–7.75 (m,3H; H-4 ’/5’/6’),
2
A
H
R
U
G
2
CO
2
2
H)),3.53 (brs,4H; CH NAr),
0
3
N
10.18 ppm (brs,2H; NH); ESI-MS,negative mode: m/z (%): 654 (100)
ꢀ
[MꢀH] ; HR-MS (ESI,positive mode): m/z: calcd for: C30
H N O S :
29 3 10 2
2
+
+
656.1367; found: 656.1365 [M+H] ,678.1185
[
M+Na] ,700.1005
3
+
[MꢀH+2Na] .
1
phase preparative HPLC:
300 MHz,[D ]DMSO,2 rotamers): d=1.78 (m,4H; (CH
.70 (m,4H; Ar CH ),2.80 (s,4H; (OC) CH CH (CO)),2.90 (s,1H;
NMe,rotamer 1),3.00 (s,2H; NMe,rotamer 2),3.53 (m,4H; CH NAr),
.36 (m,2H; (CH )CH OCO),6.78 (brs,2H; H-1/8),7.36–7.48 (m,1H;
H-3’),7.6–7.8 (m,3H; H-4 ’/5’/6’),10.16 ppm (brs,2H; NH); HR-MS
ESI,positive mode): m/z: calcd for C34 : 769.1480; found:
t
R
=11.6 min (area 100%). H NMR
N-Hydroxysuccinimidyl ester 3e: Acid 3c (0.13 g,0.20 mmol) was dis-
(
6
2
)CH (CH )),
2
A
H
R
U
G
2
solved in dry DMF (4 mL),TSTU (0.24 g,0.80 mmol) was added fol-
2
2
2
2
lowed by Et N (0.7 mL,10 mmol). The reaction mixture was stirred at
3
2
room temperature overnight; DMF was evaporated in vacuo,and the
title compound was isolated by preparative HPLC. Corresponding frac-
4
2
2
tions were pooled and freeze-dried. The residue was stored at ꢀ208C.
(
H
33
N
4
O
13
S
2
HPLC: t =10.4 min (coincides with the methyl ester 13; area 98%).
R
+
7
69.1479 [M+H] .
Methyl 3-(methylamino)propionate (12):
carbonylamino)-propionic acid 10 (FLUKA,2.23 g,10 mmol),Ag
7.3 g,31 mmol),MeI (8.5 g,60 mmol) and DMF (40 mL) was stirred at
room temperature in the dark for 16 h. The solvent and excess of MeI
were removed in vacuo at 508C,the residue was suspended in CH Cl
and filtered. The filtrate was washed with sat. aq. NaHCO ,brine,dried
and evaporated in vacuo to give crude 11 (2.2 g,89%). It was dissolved
in ethyl acetate (20 mL). The solution was added through a septum
under N to 0.87 g of 10% Pd/C (Merck),placed into dry Schlenk flask
equipped with a reflux condenser. The flask was filled with H and stirred
under H at atmospheric pressure and ambient temperature for 7 h. Then
the reaction mixture was flashed with N ,the catalyst was filtered off,
HR-MS (ESI,positive mode): m/z: calcd for C H N O S : 753.1531;
3
4
32
4
12 2
+
+
+
[
18]
found: 753.1530 [M+H] ,775.1351 [ M+Na] ,797.1170 [ MꢀH+2Na] .
Stock-solutions in DMF decompose slowly at room temperature; typical-
ly the content of NHS-ester 3e decreases from 95–97 to 80% in ca. 12–
A mixture of 3-(benzyloxy-
2
O
(
1
8 h (and the content of the starting acid 3c increases proportionally).
2
2
,
Rhodamines 14 and 15: A mixture of finely grounded compound 7 (2.7 g,
18 mmol) and trimellitic anhydride (4.7 g,24 mmol) was heated at 170 8C
for 3 h. Then an additional portion of 7 (2.8 g,19 mmol) and 85% aq.
3
3 4
H PO (7.5 mL) were added to the cooled reaction mixture and heating
was continued at 1708C for 3 h. After cooling,the reaction mixture was
stirred and refluxed with methanol (40 mL) for several minutes,cooled,
and kept at RT to form a precipitate for 10 h. The crude isomer 15 (1.1 g,
2
2
2
13%) was filtered off and purified (as a salt with Et
matography (CH Cl /MeOH 1:1, + 1% Et N v/v). HPLC: t
(area 98%). H NMR (600 MHz,[D ]MeOH): d=1.30 (t, J=7.5 Hz,9H;
CH (CH NEt )),1.88 (m,4H; (CH )CH (CH )),2.69 (brt, J=6.0 Hz,
4H; ArCH ),3.20 (q, J=7.5 Hz,6H; (CH )CH NEt )),3.44 (t, J=
5.7 Hz,4H; CH N),6.65 (s,2H; H-4/5),6.71 (brs,2H; H-1/8),7.48 (d,
3
N) by column chro-
2
and the filtrate was carefully evaporated at reduced pressure (50 mbar)
and 308C (bath). The crude product 12 (1.03 g,100%,content of the
2
2
3
R
=23.7 min
1
product was about 70 mol%; EtOAc: 30 mol%) was used for next step
3
A
H
R
U
G
2
2
2
2
A
H
R
U
G
2
1
without further purification. H NMR (250 MHz,CDCl
3
): d=1.68 (s,1H;
2
3
2
2
NH),2.42 (s,3H; NMe),2.50 (m,2H; (CH
NCH (CH CO Me)),3.67 ppm (s,3H,OMe).
Methyl ester13 : Rhodamine 3a (0.57 g,1.0 mmol) and TSTU (0.60 g,
.0 mmol) were placed into a dry Schlenk flask under N . Dry DMF
19 mL) and Et N (1 mL,6.8 mmol) were added through a septum,and
2
)CH
2
A
H
R
U
G
2
Me)),2.83 (m,2H;
2
A
C
H
T
R
E
U
N
G
2
2
J=7.8 Hz,1H; H-5 ’),8.42 (dd, J=1.8,7.8 Hz,1H; H-6 ’),8.90 ppm (d,
J=1.8 Hz,1H; H-2 ’); HR-MS (ESI,positive mode): m/z: calcd for
2
+
C
27
H
22
N
2
O
5
: 455.1602; found: 455.1601 [M+H] .
2
2
(
3
The filtrate was diluted with water and kept at RT for 24 h. A mixture of
the mixture was placed into an ultrasonic bath for 5 minutes and kept at
room temperature for 19 h. The solution (1.2 mL; about 0.06 mmol of ac-
tivated ester of 3a) was taken with syringe and added through a septum
to the crude compound 12 (75 mg, ꢁ0.5 mmol),placed into a dry flask.
The mixture was kept at RT for 24 h. Then the solvent was evaporated in
vacuo,and the title product was isolated from the residue by column
14 (major component) and 15 (minor component) precipitated (1.4 g,
17%). Isomer 14 was isolated as a salt with Et
raphy (CH Cl /MeOH 1:1, + 1% NEt v/v). HPLC: t
99%). H NMR (600 MHz,[D ]MeOH): d=1.30 (t, J=7.5 Hz,3.5H;
CH (CH NEt )),1.88 (m,4H; (CH )CH (CH )),2.69 (m,4H; Ar CH ),
3.20 (q, J=7.5 Hz,2.4H; (CH )CH NEt )),3.43 (m,4H; CH N),6.63 (s,
3
N by column chromatog-
2
2
3
R
=21.7 min (area
1
4
3
A
H
R
U
2
2
2
2
A
T
E
N
2
2
3
2
2
2
chromatography (R
f
=0.4,CH
2
Cl
2
/MeOH 4:1). Pure product (36 mg,
2H; H-4/5),6.71 (brs,2H; H-1/8),7.87 (brs,1H; H-2
’),8.34–8.38 (m,
m/z: calcd for
9
0%) was precipitated by adding ether to the solution in aq. MeOH).
2H; H-5’,H-6 ’); HR-MS (ESI,positive mode):
1
+
HPLC: t
R
=10.4 min (area 100%). H NMR (300 MHz,[D
)CH (CH )),2.24 (m,2H; (CH )CH (CO
),2.98 (s,3H; NMe),3.39 (m,2H; N CH (CH
.56 (m,7H; CH NAr and OMe),6.78 (brs,2H; H-1/8),7.38–7.42 (m,
H; H-3’),7.6–7.8 (m,3H; H-4 ’/5’/6’),10.18 ppm (brs,2H; NH);
6
]DMSO): d=
CH )),2.70
CO Me)),
C
27
H
22
N
2
O
5
: 455.1602; found: 455.1601 [M+H] .
1
.79 (m,4H; (CH
2
2
A
C
H
T
R
E
U
N
G
2
2
2
A
H
R
U
2
3
Rhodamine 3 f: Compound 15 (0.40 g,0.72 mmol of the adduct with
(
m,4H; Ar CH
2
2
A
H
R
U
G
2
2
Et N) was added carefully to 30% SO in H SO (5.9 g,obtained by
3
3
2
4
3
1
2
3 3 2 4
mixing of 20% SO and 65% SO in H SO ) at 0–58C. The mixture was
stirred at 08C for 15 min and kept at 48C for 14 h. Then it was poured
onto 40 g of frozen 1,4-dioxane and mixed carefully. Diethyl ether
(80 mL) was added,and after 3 h the precipitate was filtered off. The pre-
cipitate was heated under reflux in iPrOH (20 mL) for several minutes;
the suspension was cooled and diluted with diethyl ether (60 mL). The
title compound was filtered off,purified by column chromatography
1
3
C NMR (75.5 MHz,[D
ArCH ),30.8 ((CH )CH
NCH (CH CO Me)),51.4 (OMe),112.2 (C),112.8 (C),124.1 (C),127.3
6
]DMSO): d=19.0 ((CH
2
)CH
2
A
H
R
U
G
2
)),27.2
(
(
(
(
2
2
2
A
H
R
U
G
2
2
2
A
C
H
T
R
E
U
N
G
2
2
CH),128.6 (CH),129.4 (CH),129.5 (CH),130.4 (CH),131.3 (C),135.8
C),151.5 (C),153.5 (C),154.3 (C),167.4 (CO),171.4 ppm (CO); UV/
Vis (water): lmax (e)=539 nm (92400), lem =558 nm, Ffl =0.93; ESI-MS,
negative mode: m/z (%): 668 (96) [MꢀH] ,690 (100) [ Mꢀ2H+Na] ;
(CH
2
Cl
2
/MeOH 2:1) and dried in vacuo (0.28 g,63%). HPLC:
R
t =
ꢀ
ꢀ
1
2
9.7 min (area 100%). H NMR (300 MHz,D O): d=1.85 (brs,4H;
Chem. Eur. J. 2008, 14,1784 – 1792
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA,Weinheim
1791