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654 J. Chem. SOC. (C), 1970

Reaction Paths t o Pure 5- and 6-Aminofluorescein. Pitfalls encountered

in Conventional Synthesis Procedures
By Roger M. McKinney" and Frederick C. Churchill, II, Technical Development Laboratories, Laboratory
Division, National Communicable Disease Center, Health Services and Mental Health Administration,
Public Health Service, U.S. Department of Health, Education, and Welfare, Savannah, Georgia 31402

Previously unrecognized pitfalls in the published procedure for the preparation of 5-and 6-isothiocyanatofluores-
ceins were examined. Hydrolysis of 5-nitrofluorescein diacetate with hot ethanol saturated with sodium hydroxide
gavevarying amounts of 5,5"-azoxydifluorescein in addition to 5-nitrofluorescein. The use of methanol and sodium
hydroxide was found to give pure 5- and 6-nitrofluorescein from the corresponding diacetate. Further, reduction
of 5-nitrofluorescein with hydrogen and W-2 Raney nickel gave a mixture of 5-aminofluorescein and 9-(4-amino-
2-carboxyphenyl)-3,6-dihydroxyxanthene. A sodium sulphide-sodium hydrosulphide reduction system reduced
5-nitrofluorescein cleanly to the amine without reductive lactone cleavage. Synthesis and characterization of
Published on 01 January 1970 on http://pubs.rsc.org | doi:10.1039/J39700000654

5,5"-azoxydifluorescein,9-(4-amino-2-carboxyphenyl)-3,6-dihydroxyxanthene,and related fluorescein and xan-

thene derivatives are described.

THE5- and 6-isothiocyanatofluoresceins are widely used action medium. When methanol and sodium hydroxide
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as fluorescent protein labels in immunofluorescence tech- was used rather than ethanol and sodium hydroxide,
niques. Preparation and separation of the two nitro- the acetyl groups were readily removed to give pure
fluorescein isomers obtained by condensation of 4-
nitrophthalic acid with resorcinol followed by reduction
to the amines, conversion into the isocyanates, and
subsequent labelling of immune serum globulin with
these fluorescent dyes was first described by Coons and
Kap1an.l Riggs et aL2 reported the reduction of the
diacetate of isomer I (5-nitrofluorescein diacetate) (la),
to the amine diacetate (lb) with Raney nickel, and con-
version into the isothiocyanate (lc) by reaction with
6\5 V
' =O
4

thiophosgene. Because of its greater stability, the
isothiocgnate has generally been used in immuno-
fluorescence work, rather than the isocyanate.
R1 R2
t
0 (2)
a; R = H . b; R = A c
(Id) with no reduction to (2a) even with a prolonged
period under reflux. Ethanol with sodium hydroxide a t
65" (b.p. of methanol) slowly reduced (Id) to (2a),
indicating that ethanol is the stronger reducing alcohol

7a=0
6
e
a;
b.
cf
d;
e;
Ac

I-I.
H
NO,

NO,
NH,
F:
under these conditions. Methanol and sodium methoxide
with (Id) in a sealed tube a t 100" for 16 hr. was found to
give (2a) in 95% yield, thus indicating that methanol
will reduce the nitro-group of (Id) when more vigorous
reaction conditions are used.
I n our hands, the direct reduction of (la) did not give
The hydrolysis of (la) with hot ethanol saturated with
a satisfactory yield of (lb). When hydrogen and Raney
sodium hydroxide was found to give varying amounts
nickel reduction of (Id) was carried out under carefully
of a by-product (5,5"-azoxydifluorescein) which could not
controlled conditions so as t o interrupt the reduction
be satisfactorily reduced to 5-aminofluorescein (le) by
when 3.0 moles of hydrogen had been consumed per mole
the usual reduction methods (sodium sulphide-sodium
of (Id) a good yield of (le) was obtained. However, when
hydrosulphide ; hydrogen and Raney nickel). Indeed,
the hydrogen and Raney nickel reduction of (Id) was
when (la) was heated under reflux for 5 min. with ethanol
allowed to proceed until no further uptake of hydrogen
saturated with sodium hydroxide, the only product
was observed, the principal product was (3a). This
isolated was 5,5"-azoxydifluorescein (2a), rather than the
expected 5-nitrofluorescein (Id). This was characterized compound was characterized as the triacetate of the
methyl ester (3b) which is crystalline and easily purified.
as the crystalline tetra-acetate (2b). An investigation
of the effects of varying the conditions of the reaction It is apparent that successful reduction of (Id) to (le)
with hydrogen and Raney nickel requires more precise
showed that the extent of reduction to (2a) is lessened
when ethanolysis or hydrolysis of the acetyl groups is control than has previously been indicated. When pure
carried out a t lower than reflux temperature. Re- (Id), obtained by methanol and aqueous sodium
duction is also lessened by addition of water to the re- hydroxide hydrolysis of (la), was heated under reflux
H. S. Corey, jun., and F. C. Churchill, 11, Natztre, 1966, 212,
1 A. H. Coons and M. H. Kaplan, J. Exp. Med., 1950, 91, 1. 1040.
2 J. L. Riggs, R.J. Seiwald, J. H. Burckhalter, C. M. Downs, R. M. McKinney, J. T. Spillane, and G. W. Pearce, J . Ovg.
and T.G. Metcalf, Amer. J. Pathology, 1958, 34, 1081. Chem., 1962, 27, 3986.

Org.
with an aqueous solution of sodium sulphide and sodium fluxing acetic anhydride (125 ml.) during 2 hr. The tetra-
hydrosulphide it was reduced cleanly to the amine (le). acetate (2b) crystallized out overnight and was washed with
This reduction is performed very simply on any desired acetic anhydride. Two more recrystallizations gave the
scale, and the possibility of lactone cleavage is avoided. product (4.26 g.) which tenaciously held acetic anhydride.
Published on 01 January 1970 on http://pubs.rsc.org | doi:10.1039/J39700000654
a ; R1 = 11, R2 = 11, R3 = H
b; R1 == Ac, R2 = Me, R3 = Ac
It was demonstrated that 6,6”-azoxydifluoresein can be
prepared from 6-nitrofluoresceinunder conditions similar
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to those used to prepare the 5-isomer. Also, pure
6-nitrofluorescein was prepared from the diacetate by
hydrolysis with sodium hydroxide and methanol.
Sodium sulphide-sodium hydrosulphide reduction satis-
factorily reduces 6-nitrofluorescein to the a ~ n i n e . ~
EXPERIMENTAL
The 5-nitrofluorescein diacetate (la), m.p. 225.0-225.5”
(corr.), and 6-nitrofluorescein diacetate, m.p. 216.0-216-5”
(corr.),were made essentially by a literature meth0d.l T.1.c.
was performed with Silica Gel F-254 Merck pre-coated
analytical glass plates with an inorganic fluorescent
indicator. Ascending elution was used in each case, with a
solvent system containing 5.0 ml. of methanol per 150 ml.
of diethyl ether saturated with water. N.m.r. spectra were
obtained in deuteriodimethylsulphoxide (DMSO).
5,6”-A zoxydipuorescein (2a).-Method 1. Compound (la)
(10 g.) was added to 95% ethanol (200 ml.) previously
saturated with sodium hydroxide at reflux temperature
[ l l % (wfv) sodium hydroxide as determined by titration
against standard hydrochloric acid). The reaction mixture
was heated gently under reflux for 10 min. after
which it was diluted with 1 1. of water and acidified
with acetic acid. An amorphous yellow precipitate was
produced upon acidification which, overnight , changed
colour to a dark red. The product was washed with water
and dried in vacuo a t room temperature; yield of (2a)
7.25 g. ; t.1.c. showed the major spot, which was orange, a t
RF 0-07, while a minor yellow impurity was found a t RP
0.22. The i.r. spectrum (DMSO) showed no evidence of an
NO, absorption peak in the 1540 cm.-l region, indicating
complete reduction of the nitro-group. The 5,5”-azoxy-
difluorescein (2a) was characterized as the tetra-acetate (2b).
Method 2. Compound (Id) (8 g.) was treated with a
solution of sodium methoxide [from sodium (8 g.) and
methanol (350 ml.)] in a sealed flask a t 100”for 16 hr. The
reaction mixture, diluted with water (3 1.) and acidified with
acetic acid, was heated on a steam-bath to give a light yellow
amorphous precipitate; this also became dark red with time.
The product was collected and dried as in Method 1 to give
(2a) (7.1 g., 95%) ; the i.r. spectrum again indicated the dis-
appearance of the nitro-group. The t.1.c. showed a major
spot a t RF 0-07 and minor impurities a t RF 0.13 and 0.25.
5,5”-Azoxydi,fluorescein Tetra-acetate (2b).-Compound
(2a) (5-3 g.) prepared by Method 1 was acetylated with re-
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655
A sample (250 mg.) of this, crystallized from benzene and
dried a t 100” in vacuo overnight, yielded (2b) (193 mg.), did
not melt below 300”; t.l.c., colourless spot R p 0.54; , ,v
(KBr) 1480sh (azoxy), 1330 (azoxy), 1770 (acetate CEO,
lactone G O ) , 1421 and 1370 cm.-I (CH,) (Found: C, 66.2;
H, 3.65; N, 3.45. C,,H,,,N,O,, requires C, 65.9; H, 3.45; N,
3.25%). An n.m.r. spectrum of (2b) was not obtained be-
cause of its low solubility. A sample, therefore, of (2b) was
hydrolysed to ( 2 4 with methanol and aqueous sodium
hydroxide; compound (2a) had the following n.m.r. signals :
6 10.2 (s, 4H, OH), 8-96 (d, lH, J 2 Hz, 4-H), 8.87 (d, lH, J
2 Hz, 4”-H), 8-73 (meta split d, l H , J 2 Hz, 8 Hz, 6-H), 8.40
(meta split d, lH, J 2 Hz, 8 Hz, 6”-H), 7.63 (d, l H , J 8 Hz,
7-H), 7.53 (d, lH, J 8 Hz, 7”-H), and 7.00-6.50 (m, 12H,
xanthene ring protons).
6,6”-A xoxydiJEuorescein.-Reaction of 6-nitrofluorescein
diacetate (9.22 g.) in the manner of Method 2 above, gave
6,6”-azoxydifluorescein (6.1 g., 71%). The product gave
an orange spot in the t.1.c. a t RF 0.05 with a minor yellow
impurity a t Rp 0.08. The i.r. spectrum showed the
absence of an NO, absorption peak a t 1540 cm.-l.
6,6”-Axoxyd~fEuorescein Tetra-acetate. - 6,6”-Azoxydi-
fluorescein (1.0 g.) crystallized as the tetra-acetate from
acetic anhydride (120 ml.). The yield of tetra-acetate was
0.37 g.; it did not melt below 300”; t.l.c., colourless spot
Rp 0.57; vmX. (KBr) 1770 (acetate G O , lactone G O ) ,
1480sh (azoxy), 1330 (azoxy), 1421, and 1370 cm.-1 (CH,)
(Found: C, 66.0; H, 3.75; N, 3.0%. C,,H30N201, re-
quires C, 65-9; H, 3-45; N, 3025%); an n.m.r. spectrum
of the tetra-acetate was not obtained because of its low
solubility. A sample was hydrolysed to 6,6”-azoxydi-
fluorescein with methanol and aqueous sodium hydroxide.
The n.ni.r. of 6,6”-azoxydifluorescein was not as simple as
that for the corresponding 5,5”-isomer (2a), because of over-
lapping spectra of the phthalyl hydrogens; however 6 of the
18 aromatic protons were in the range 6 8.5-7.5, well below
the 12 protons of the upper xanthene rings which resonate
between 6 6.75 and 6.40.
5-NitrofEuorescein.-Compound (la) (1 g.) was dissolved
with heating in a mixture of 10% aqueous sodium hydroxide
(10 ml.) and methanol (50 ml.). The mixture was then
diluted with water (150 ml.) and acidified with acetic acid
to give an amorphous yellow Precipitate, which, when the
solution was heated, gave an orange precipitate. The
product was collected, washed with water, and dried in
vacuo at room temperature; the yield of (Id) was 0.81 g.
(99%). The product was chromatographically pure, with
an R p of 0.33 for the orange spot, the same as for an authen-
tic sample in the standard t.1.c. system. The i.r. (DMSO)
showed an intense NO, band a t 1540 cm.-l.
6-Nitrofluorescein.-6-Nitrofluorescein diacetate ( 1 g.)
was dissolved with heating in a mixture of 10% aqueous
sodium hydroxide (10 ml.) and methanol (50 ml.). The
reaction mixture was diluted with water (150 ml.) and acidi-
fied with acetic acid to give an amorphous orange precipitate
which changed to a bright red colour when the mixture was
heated. The yield of 6-nitrofluorescein was 0-80 g. (98%).
The product was chromatographically pure, with RP 0.33,
or the same as for the 5-isomer. The i.r. (DMSO) showed
an intense NO2 band a t 1540 cmF1.
Reduction of 5-Nitrojhovescein (Id) t o 5-AnzinofEuorescein

656
(le).--Method 1. Reduction was carried out with a sodium
sulphide-sodium hydrosulplide solution as described
p r e v i ~ u s l y . ~The yield of (le) from 37-7 g. (0.1 mole) of
(Id) after 2 recrystallizations from 6% hydrochloric acid
and subsequent precipitation from aqueous sodium
hydroxide with acetic acid was 27-7 g. (80%). The
product was pure by t.l.c., giving a spot a t RF 0.26.
Method 2. Compound (Id) (3.77 g., 0.01 mole) in
absolute ethanol was shaken with W-2 Raney nickel5 a t
room temperature [lo ml. of settled suspension in ethanol
(230 ml.) a t pressures not exceeding 60 lb./sq. in.]. After
70 min., the theoretical quantity of hydrogen had been
consumed. The solution was filtered and the filtrate was
evaporated to dryness. The crude product was collected
Published on 01 January 1970 on http://pubs.rsc.org | doi:10.1039/J39700000654
and dried a t 100" in vacuo overnight; the recovery was
3.32 g. (96% of theoretical). A t.1.c. was run on the
product using the normal system. The sole, yellow spot,
was found a t RF 0.26, corresponding to (le).
9- (4-Anzino-2-carboxyphenyE)-3,6-dihydroxyxanthene(3a).
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-Compound (Id) (7.54 g., 0.02 mole) was hydrogenated as
above, except that the reaction was allowed to proceed until
the uptake of hydrogen (4 mol.) ceased. The nickel was
filtered off and the product was taken to dryness under
reduced pressure; the residue weighed 6.78 g.
The i.r. spectrum (DMSO) of the crude product showed
the major carbonyl absorption band a t 1690 cm.-l which
corresponds with an aromatic carboxylic acid. A very
weak band a t 1750 cm.-l is attributed to the lactone
carbonyl of the small amount of (le) present. T.1.c. showed
the major spot a t RF 0.08 due to (3a) and a minor spot a t
Rp 0.26 due to (le).
9- (4-A cetaunido-2-methoxyca~bo~zyZ~henyl) -3,6-diacetoxy-
xanthene (3b).-A solution of compound (3a) (5.46 g.) in
methanol (1 25 ml.) was saturated with dry hydrochloric
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J. Chem. SOC.(C), 1970
acid; the mixture was then heated under reflux for 2.5 hr.
Removal of methanol a t room temperature under reduced
pressure, left the crude methyl ester (5.63 g.). A sample
(1.0 g . ) of this in acetic anhydride (25 ml.) was stirred and
saturated with dry hydrochloric acid ; complete dissolution
occurred within ca. 20 min. After 16 hr. a t room tem-
perature the acetic anhydride was removed under reduced
pressure and the residue was dissolved in methanol (10 ml.) .
Crude compound (3b) (0.94 g.) crystallized out and was
twice recrystallized from methanol to give the pure product
(0.37 g.), m.p. 221.0-221.5° (corr.), i.r. (KBr), 1755
(acetate and lactone C=O), 1713 (methyl ester G O ) , 1665
(amide I), 1540 cm.-l (amide 11); n.m.r., 6 10.2 (s, lH, N-H),
8.10 (d, lH, J 2 Hz, lower ring position 3), 7.58 (meta split d,
lH, J 2 Hz, 8 Hz, lower ring position 5), 7.10-6.60 (m, 7H,
lower ring position 6 and upper ring aromatic protons),
6.15 (s, lH, upper ring position 9), 2.25 (s, 6H, O-acetyl),
2.03 (s, 3H, N-acetyl) (Found: C, 66.35; H, 4.8; N,
2.8%. C2,H,,N0, requires C, 66.25; H, 4-75; N, 2.85%).
Reduction of5-Nitrofluorescein Diacetate (la) with Hydrogen
and Raney Nickel.-A sample of (la) (4.62 8.)was reduced
with Raney nickel by the standard method; 3.0 mol. of
hydrogen were absorbed. T.1.c. analysis of the hydrolysate
(rnethanol-aqueous sodium hydroxide) of the product
demonstrated the presence of (3s) ( R p 0.08),(le) ( R p 0.26)
and (Id) ( R p 0.33) together with two minor impurities.
Thus, in the reduction of 5-nitrofluorescein diacetate (1a)
lactone ring reduction occurs a t a rate competitive with
nitro-group reduction.
We thank Dr. James W. Miles for suggesting the solvent
system which was used for t.1.c. analysis.
[9/1112 Received, June 30th, 19691
6 R. Mozingo, Org. Syntlz., 1941, 21, 15.