A R T I C L E S
Adams et al.
2
H, H-1′, 8′), 7.06 (m, 1H, H-4), 7.5 (m, 2H, H-5,6), 8.0 (m, 1H, H-7).
material (by TLC) to give baseline product and by the weight gain of
the isolated product.
-
ES-MS (90% MeOH, 0.04% NH
for C20 As 512.1.
Resorufin 4,5-Bis(mercuric trifluoroacetate); Bis(trifluoroacetato-
κO)[µ-(7-hydroxy-3H-phenoxazin-3-one-4,5-diyl)]dimercury. Re-
sorufin sodium salt (59 mg, 0.25 mmol) was added with stirring to a
solution of mercuric oxide (120 mg, 0.50 mmol) in trifluoroacetic acid
3
) -ve mode: (M - 1) 511.2. Calcd
H O
10 7
2
4
,5-Bis(1,3,2-dithiarsolan-2-yl)-3,6-dihydroxy-9H-xanthen-
. Dimercurated intermediate 1H NMR (d
6
-
9
-one, HoXAsH-EDT
2
DMSO) 7.07 (d, 2H, H-2,7), 7.97 (d, 2H, H-1,8) 10.7 (s, 2H, OH).
1
Biarsenical H NMR (d
6 2
-DMSO) 3.3 (m, part obscured by H O, EDT),
6
.91 (d, 2H, H-2, 7), 8.00 (d, 2H, H-1, 8), 11.4 (br s, 2H, OH) ES-MS
(2 mL) at room temperature. After overnight reaction, the dark red
+
(
90% MeOH, 1% HOAc) +ve mode: (M + 1) 560.6. Calcd for
As 559.8. Complex with AcWDCCPGCCK-NH ES-MS
50% MeOH, 1% HOAc) +ve mode: (M + 1) 1427.5, (M + 2)2+
solution deposited a solid. The reaction mixture was evaporated and
diluted with water (20 mL), and the precipitate was collected by
C
(
17
H
14
O
4
S
2 4
2
+
filtration, dried in vacuo over P
O
2 5
to give a dark red powder. Yield,
-DMSO) 6.97 (d, 2H, J ) 8.8 Hz), 7.04
s, 1H, OH), 7.26 (d, 2H, 8.7 Hz).
2
,5-Bis(1,3,2-dithiarsolan-2-yl)-resorufin; ReAsH-EDT . Resoru-
7
1
0
2 12 16 4
14.1, deconvolved to MW of 1426.28. Calcd for C55H64As N O S
426.27. Fluorescence quantum yield of peptide complex in pH 7 buffer,
.12.
1
1
(
32 mg (63%). H NMR: (d
6
4
4
,5-Bis(1,3,2-dithiarsolan-2-yl)-2,8-dichloro-3,6-dihydroxy-9H-
fin bis(mercuric trifluoroacetate) (73 mg, 87 µmol) was suspended in
dry NMP (1.5 mL) under argon. Arsenic trichloride (Caution: toxic,
handle only in a fume-hood!) (170 µL, 4.0 mmol), palladium acetate
1
xanthen-9-one, CHoXAsH-EDT
NMR (d -DMSO) 8.01 (s, 2H, H-1,8), 10.97 (s, 2H, OH). Biarsenical
H NMR (d -DMSO) 3.5 (EDT peaks part obscured by H O), 7.71 (br
2
. Dimercurated intermediate H
6
1
6
2
(few mg), and DIEA (140 µL, 1.6 mmol) were added, and the reaction
3
s, 2H, OH), 8.05 (s, 2H, H-1,8). ES-MS (50% MeOH, 0.1% NH ) -ve
mixture was stirred at 60-70 °C for 2 h. After cooling, the reaction
mixture was poured into acetone -0.25 M phosphate buffer pH 7 (25
mL, 1:1 v/v), treated with excess 1,2-ethanedithiol (0.5 mL), extracted
1-
2 2 4 4
mode: (M - 1) 626.7. Calcd for C17H12As Cl O S 627.7. UV-vis
-1
-1
(
pH 7 buffer) λmax 389 ꢀmax 33600 M cm . Complex with AcWD-
CCPGCCK-NH
2
ES-MS (50% MeOH, 1% HOAc) +ve mode: (M +
Cl 1496.1. Fluorescence
with CHCl
3
(3 × 30 mL), dried over Na
2
SO
4
, evaporated, and purified
+
1
)
1497.0. Calcd for C55
H
62As
2
2 12 16 4
N O S
by chromatography on silica (20 g packed in toluene, eluted with 10%
EtOAc-toluene). Care should be taken not to evaporate purified
fractions excessively as this leads to partial loss of EDT. After trituration
with 95% EtOH, the product was obtained as a dark red-purple solid
quantum yield of peptide complex in pH 7 buffer, 0.35.
4
′,5′-Bis(1,2,3-dithioarsolan-2-yl)-2′,7′-dibromofluorescein,Br
2
AsH-
1
EDT
2
. Dimercurated intermediate H NMR (d
6
-DMSO) 1.85 (s, 3H,
OAc), 1.86 (s, 3H, OAc), 7.26 (s, 2H, H-1′,8′), 7.32 (m, 1H, H-7),
(
16 mg) with the evaporated EtOH yielding a further 2 mg. Yield (39%).
1
7
.53 (m, 2H, H-5,6), 7.68 (d, 1H, H-4). Biarsenical, H NMR
-DMSO) 3.5 (EDT peaks part obscured by H O), 6.85 (s, 1H, H-1′),
.02 (s, 1H, H-8′), 7.42 (dd, 1H, H-7), 7.82 (m, 2H, H-5,6), 8.12 (dd,
1
H NMR (CDCl
3
): 3.49 (m, 8H, S-CH
2
), 6.22 (d, 2H, J ) 8.3 Hz),
) -ve mode:
544.8. Complex with
ES-MS (50% MeOH, 1% HOAc)
ve mode: (M + 3) 766.5. Calcd for C88 2297.6.
Fluorescence quantum yield of peptide complex in pH 7 buffer,
(d
6
2
6
(
.30 (d, 2H, J ) 8 Hz). ES-MS (90% MeOH, 0.04% NH
3
7
1
8
-
M - 1) 543.8. Calcd for C16
3 2 4
H13NO As S
1
-
H, H-4). ES-MS (50% MeOH, 0.1% NH
21.9. Calcd for C24 Br S 822.7. Complex with AcWEAAAR-
ES-MS (50% MeOH, 1% HOAc) +ve mode:
Br 2576.1 (av).
3
) -ve mode: (M - 1)
AcWEAAAREACCRECCARA-NH
2
H16As
2
2 5
O
3
+
+
2 27 29 4
H121As N O S
EACCRECCARA-NH
2
+
(
M + 3)3 859.2. Calcd for C96
H124As
2
2 26 31 4
N O S
0
.2.
Fluorescence quantum yield of peptide complex in pH 7 buffer, 0.18.
′,5′-Bis(1,2,3-dithioarsolan-2-yl)-2′-bromofluorescein, BrAsH-
EDT . Biarsenical NMR (CDCl ) 3.6 (m, 8H, S-CH ), 6.52 (d, 1H,
H-7′), 6.61 (d, 1H, H-8′), 6.90 (s,1H, H-1′), 7.21 (dd, 1H, H-7), 7.69
m, 2H, H-5,6), 8.03 (dd, 1H, H-4), 9.90 (s, 1H, 3′-OH), 10.55 (s, 1H,
4
2
,5-Bis(arsenoso)resorufin, (ReAsHO). ReAsH-EDT (1 mM in
4
THF) was treated with an equal volume of 2 mM mercuric acetate in
0 mM aqueous TFA. The THF was removed by evaporation with a
stream of N , and the product was collected by centrifugation, washed
2
3
2
1
2
(
with water, dissolved in DMSO, filtered, and stored frozen. ES-MS
1+
-
6′-OH). ES-MS (50% MeOH) +ve mode (M + 1) 744.3. Calcd for
BrO 743.4. Complex with AcWEAAAREACCREC-
CARA-NH ES-MS (50% MeOH, 1% HOAc) +ve mode: (M + 3)
832.4. Calcd for C96 2497.2 (av). Fluorescence
(
50% MeOH, 0.2% NH
As NO 392.86.
General Synthesis of Biarsenical Derivatives of Dyes. 3,6-
3
) -ve mode: (M - 1) 391.6. Calcd for C12
5
H -
C
24
H
17As
2
5 4
S
2
5
3
+
2
3
9
40
2 31 4
H125As BrN26O S
Dihydroxyxanthone, 2,7-dichloro-3,6-dihydroxyxanthone, 2′-bro-
mofluorescein (prepared by fusion of 4-bromoresorcinol and phthalic
anhydride at 190 °C; purified as the diacetate), 2′,7′-dibromofluores-
quantum yield of peptide complex in pH 7 buffer, 0.27.
4′,5′-Bis(1,2,3-dithioarsolan-2-yl)-thiofluorescein, tFlAsH-EDT
2
.
41
42
1
cein, thiofluorescein (3′,6′-dihydroxyspiro[isobenzofuran-1(3H),9′-
Biarsenical, H NMR (CDCl
3
2
) 3.56 (m, 8H, S-CH ), 6.64 (d, 2H,
43
[9H]thioxanthene]-3-one), thionol (7-hydroxy-3H-phenothiazin-3-one),
H-2′,7′), 7.09 (d, 2H, H-1′,8′), 7.3 (m,1H, H-7), 7.63 (m, 2H, H-5,6),
4
4
and 2,7-dibromoresorufin (2,8-dibromo-7-hydroxy-3H-phenoxazin-
-one) were synthesized according to literature methods. 5,(6)-
7.92 (m, 1H, H-4). ES-MS (50% MeOH, 1% HOAc) +ve mode: (M
+ 1)1 680.7. Calcd for C24
+
H
18As
O
S
679.8. Complex with AcWD-
ES-MS (50% MeOH, 1% HOAc) +ve mode: (M +
2) 819.4. Calcd for C64 1636.19. Fluorescence
3
2
4
5
Sulfofluorescein was purchased from Molecular Probes. Standard
mercuration conditions used mercury bis(trifluoroacetate) in TFA or
CCDECCK-NH
2
2
+
H
2 12 20 5
70As N O S
mercury diacetate in acetic acid and transmetalation with AsCl
3
and
quantum yield of peptide complex in pH 7 buffer, <0.01.
was as described above for the preparation of ReAsH-EDT . Biarseni-
2
4′,5′-Bis(1,2,3-dithioarsolan-2-yl)-fluorescein-5(6)-sulfonic acid,
1
1
cal derivatives of dyes were characterized by H NMR, ES-MS of their
EDT complexes, and by ES-MS of the purified peptide complex.
Comparable analysis of the dimercurial intermediate was often greatly
hindered by their insolubility in any suitable solvents, although reaction
progress could be conveniently monitored by the conversion of starting
sFlAsH-EDT . Dimercurated intermediate, H NMR (D O + Na -
2
2
2
CO ): 1.85 (s, 3H, OAc), 1.86 (s, 3H, OAc), 6.62 (d, 2H, H-2′,7′), 7.0
3
(m, 1H, H-7), 7.04 (d, 2H, H-1′,8′), 7.5-8.1 (m’s, 2H, H-4,(5,6)).
1
Biarsenical, H NMR (d -DMSO) 3.5 (m, obscured by water), 6.64 (d,
6
2H, H-2′,7′), 7.03 (d, 2H, H-1′,8′), 7.23 (m, 1H, H-7), 7.72 (m, 1H,
3
H-5(6)), 8.09 (m, 1H, H-4). ES-MS (50% MeOH, 0.2% NH ) -ve
mode: (M - 1) 742.7. Calcd for C24H18As O S 743.80.
2 8 5
(
39) Grover, P. K.; Shah, G. D.; Shah, R. C. J. Chem. Soc. 1955, 3982-3985.
40) Kurduker, R.; Subba Rao, N. V. Proc. Indian Acad. Sci., Part A 1963, 57,
1-
(
2
80-287.
4,5-Bis(1,2,3-dithioarsolan-2-yl)-thionol, ThAsH-EDT
2
. Biarseni-
(41) Sandin, R. B.; Gillies, A.; Lynn, S. C. J. Am. Chem. Soc. 1939, 61, 2919-
1-
cal, ES-MS (50% MeOH, 0.2% NH ) -ve mode: (M - 1) 559.8.
3
2
921.
Calcd for C16
NH
Calcd for C56
peptide complex in pH 7 buffer, <0.01.
H
13As
2
NO
2
S
5
560.8. Complex with AcWDCCDECCK-
(
42) Pfoertner, K. H. J. Chem. Soc. Perkin Trans. 2 1991, 523-526.
(
43) Houston, D. F.; Kester, E. B.; DeEds, F. J. Am. Chem. Soc. 1949, 71, 3819-
ES-MS (50% MeOH, 1% HOAc) +ve mode: (M + 2) 759.9
2+
2
3
822.
2 13 18 5
H65As N O S 1517.16. Fluorescence quantum yield of
(
44) Afanas’eva, G. B.; Viktorova, T. S.; Pashkevich, K. I.; Postovskii, I. Y.
Khim. Geterotsikl. Soedin. 1972, 3, 348-353.
6074 J. AM. CHEM. SOC.
9
VOL. 124, NO. 21, 2002