1
6
-carboxy fluorescein, and removal of the pivaloyl groups
Crude 4, 25 g (0.11 mol) was dissolved in a solution of
500 mL of water, 50 g of K CO , and 90 g of (0.57 mol)
with base followed by precipitation with aqueous acid gave
material which contained >90% pure 6-carboxy 1. See
Figure 4 (inset).
2
3
KMnO . The purple solution was refluxed for 4 h and
4
allowed to cool and stand overnight. The solution was
2 4
acidified to pH 1 with 6 N H SO , and oxalic acid was added
Conclusions
until the solution became colorless. The mixture was filtered
and the aqueous phase extracted with 1000 mL of ether, and
Improved methods have been applied to the synthesis and
purification of two chlorinated fluoresceins. We hope that
these updated procedures will make 1 and 2, as well as
similar compounds, more accessible to those workers who
require them.
4
the extract was then dried over MgSO and evaporated to
give 14 g (44% yield) of 4 as a white solid, mp 226-230
8
1
°
(
C (lit 232-233 °C). H NMR (d
broad s, 3H), 8, (s, 1H).
Anal. Calcd for C
Found: C, 38.05; H, 1.68.
(and 6)-Carboxy-4,7,2′,7′-tetrachlorofluorescein (“5
6
-DMSO, δ): 14-12
9
H
4
O
6
Cl
2
2
‚1/3H O: C, 37.92; H, 1.64.
Experimental Section
5
Aqueous ammonia, sulfuric acid, acetic acid, and con-
centrated HCl were reagent grade from J.T. Baker. Ethyl
ether, 1,2-dichloroethane, dichloromethane, chloroform, ethyl
acetate, 2-propanol, toluene, acetonitrile, ethanol, methanol,
and petroleum ether were Omnisolve grade from VWR.
Aluminum trichloride, 2,5-dichloro-p-xylene, acetyl chloride,
potassium permanganate, oxalic acid, isovanillin, chlorine
gas, m-chloroperoxybenzoic acid (MCPBA), sodium hy-
droxide, potassium carbonate, sodium bicarbonate, magne-
sium sulfate, 4-chlororesorcinol, trimellitic acid anhydride,
chromatographic alumina, and methane sulfonic acid were
from Aldrich. Chromatographic silica was from Grace
Chemical Co. Elemental analyses were performed by Desert
Analytics (Tuscon, AZ), and NMR work was done by Acorn
NMR (Fremont, CA). MALDI Mass spectra were done in-
house on a Finnigan Lasermat.
and 6 TET”), 1. A tared 500 mL round-bottom flask
containing 11.4 g (41 mmol) of 5 was heated to 150° for 20
min in a fume hood. Water was observed to pass out of the
flask. The flask was sparged briefly with argon and allowed
to cool. The flask was re-weighed, and contained 10.5 g of
now dark solid. 4-Chlororesorcinol, 10.5 g (7.4 mmol), was
added, along with 100 mL of methane sulfonic acid. The
mixture was heated for 2 h at 140-150°. The dark red
mixture was cooled and poured slowly into 1500 mL of
rapidly stirred water. The brown-yellow solid was collected
by suction filtration and washed with 300 mL of water, and
air-dried for several days. The yield was 15.5 g (79%).
Reverse Phase HPLC with photodiode array detection of the
crude product showed 74% of the total peak area due to
combined dye isomers at 254 nm. MALDI MS showed
+
MH /e 512.86 amu, Calcd 512.910 amu.
Synthesis of 1. 3-Acyl-2,5-dichloro-p-xylene 4. In a 3 L
round-bottom flask with reflux condenser and magnetic
stirring under argon was added 2,5-dichloro-p-xylene (100
g, 0.571 mol) dissolved in 1400 mL of ethylene dichloride,
along with acetyl chloride (55 mL, 0.77 mol). The mixture
was heated to reflux, and aluminum trichloride (103 g, 0.77
mol) was added as a solid in small portions over 1.5 h. Reflux
was continued another 4 h. The large volume of HCl gas
which evolved was allowed to pass out of the flask into a
fume hood. The solution was cooled, and solids were
removed by filtration. The organic phase was poured into 1
L of cracked ice, and after 20 min 200 mL of concentrated
aqueous ammonia was added. The mixture was placed in a
6
-Carboxy-4,7,2′,7′-tetrachlorofluorescein Dipivalate
Diisopropylamine Salt. 1, 20 g (40 mmol), was dissolved
in 200 mL of dry pyridine and reduced to a tar by rotary
evaporation. Another 200 mL of dry pyridine was added,
and the solution was chilled to 0° C in an ice bath under
argon. Pivaloyl chloride (30 mL, 29 g, 244 mmol) was then
added dropwise over 30 min, and the black mixture was
allowed to stand and warm to room temperature overnight.
Water, 20 mL, was added dropwise over 10 min with stirring,
and the solution was evaporated to a tar by rotary evapora-
tion. The dark residue was dissolved in 400 mL of EtOAc
and washed successively with 300 mL portions of of 1 M
HCl, saturated NaHCO
phase was dried over MgSO
3
, and then 1 M HCl. The organic
and evaporated and then
4
L separatory funnel along with 400 mL of 2 M HCl, 600
mL of CH Cl , and 500 mL of water. A rod was used to
2
2
4
redissolved in 250 mL of anhydrous ethanol. Diisopropyl-
amine, 30 mL, was added, and the mixture was chilled at
gently stir the aqueous layer. (An emulsion may form if the
separatory funnel is shaken in a normal manner.) The layers
were allowed to separate overnight. The organic phase was
-20° C for 2 days. A salmon colored precipitate was
collected by filtration on a medium porosity glass frit and
the solid washed with a small amount of cold ethanol.
Vacuum drying gave 8 g of solid. The mother liquor was
concentrated to 150 mL and re-chilled, affording another 2.5
g of the same solid. The combined yield was 10.5 g, Both
crops were analytically pure.
4
dried over MgSO and evaporated to give 75 g (60% yield)
of a beige solid. A small amount of the product was purified
by column chromatography on silica; a gradient of 0-20%
CH
2
Cl
2
in pet ether over 3 L of mobile phase was used to
) 0.8 in 2% MeOH/CH
, mp 97-101 °C. H NMR (CDCl , δ): 7.25 (s, 1 H),
.5 (s, 3H), 2.3 (s, 3H), 2.1 (s, 3H).
Anal. Calcd for C10 : C, 55.33; H, 4.68. Found:
C, 56.00; H, 4.75.
,5-dicholoro-1,3,4-trimellitic acid, 5.
obtain pure 4 as tan crystals, R
f
2
-
1
Cl
2
3
2
Anal. Calcd for C37
1.79.
9 4
H39NO Cl : C, 56.75; H, 5.01; N,
H10OCl
2
Crop 1, Found: C, 56.79; H, 5.06; N,1.71.
Crop 2, Found: C, 56.69; H, 5.11; N,1.91.
2
48
•
Vol. 5, No. 1, 2001 / Organic Process Research & Development