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PRACTICAL SYNTHETIC PROCEDURES
Synthesis of Regioisomerically Pure 5-Functionalized 2¢,7¢-Dichlorofluoresceins
Aminodichlor fluoresceinDerivatives C. Castro, Andrey Malakhov, Kevin Burgess*
Juan
Department of Chemistry, Texas A & M University, P.O. Box 30012, College Station, TX 77842, USA
Fax +1(979)8458839; E-mail: burgess@tamu.edu
Received 12 November 2008; revised 10 December 2008
PSP
154
No
Abstract: Regioisomerically pure 2¢,7¢-dichloro-5-nitrofluorescein was isolated on a multigram scale, and used as an intermediate for the
generation of three compounds that might be used for labeling biomolecules, namely the corresponding amino, azido, and (4,6-dichloro1,3,5-triazin-2-ylamino) derivatives.
Key words: fluorescence, chromophores, condensation, heterocycles, regioselectivity
NO2
NO2
NO2
Cl
O
HO
OH
O
neat, 200 °C, 24 h
(ii) Ac2O, 150 °C, 4 h
(iii) recrystallization
O
O
Cl
O
K2CO3
MeOH–THF (1:1)
Cl
CO2H
Cl
Cl
H2, Raney Ni
O
MeOH, 5% H2O
25 °C, 1.5 h
3
25 °C, 4 h
AcO
OAc
O
HO
O
1 48%
2 90%
59%
Cl
N
N3
NH2
Cl
N
Cl
HO
O
Cl
(i) NaNO2, H2O–HCl (1:1)
EtOH, 0 °C, 1 h
O
(ii) addition of NaN3, H2O,
0–25 °C, 6 h
NH
Cl
5
CO2H
N
N
N
CO2H
Cl
2'
7'
CO2H
Cl
Cl
N
Cl
Cl
Cl
MeOH, CH2Cl2, 0 °C, 2 h
HO
4 47%
O
3
O
HO
O
O
5 86%
Scheme 1
Fluorescein undergoes transitions between various ionization states at physiological pH levels [Figure 1 (a)]. Quantum yields and extinction coefficients of fluorescein
therefore may change significantly as the pH of the solvent varies. Consequently, fluorescein is non-ideal, or at
least, difficult to use, for many quantitative biochemical
experiments that rely on intensity of fluorescence.
The phenolic hydroxy groups of 2¢,7¢-dichlorofluorescein
are more acidic than those of the parent compound because of inductive stabilization of the phenolate form.
2¢,7¢-Dichlorofluorescein therefore is completely ionized
at pH levels near physiological and even slightly below.1
Figure 1 (b) shows data recorded for this paper wherein
the pH of a 2¢,7¢-dichlorofluorescein solution was varied
around physiological levels; the fluorescence of the solution remained approximately stable until the pH of the
medium fell to 5 and below. This dye, therefore, has some
attributes that fluorescein does not. To be widely useful,
SYNTHESIS 2009, No. 7, pp 1224–1226xx. 209
Advanced online publication: 06.03.2009
DOI: 10.1055/s-0028-1088010; Art ID: M11408SS
© Georg Thieme Verlag Stuttgart · New York
however, probes such as these must have functional
groups that allow them to be attached to biomolecules.
This usually involves preparation of derivatives as regioisomeric mixtures then separation via recrystallization.2
This paper reports application of this kind of approach to
the syntheses of the 5-amino, 5-azido, and 5-(4,6-dichloro-1,3,5-triazin-2-ylamino) derivatives 3, 4, and 5. Literature searches have only yielded patented mention of a 5and 6-aminofluorescein compounds.3
Separation of the isomers of 2¢,7¢-dichloro-5-nitrofluorescein (2) was pivotal to the success of this project
(Scheme 1). Fortunately, 3-nitrophthalic anhydride could
be condensed with 4-chlororesorcinol on a large scale to
give a mixture of the 5- and 6-nitro isomers. Treatment of
this mixture with acetic anhydride gave the diacetate 1.
This material was recrystallized from acetic anhydride to
give the 5-nitro compound. After three recrystallizations
the material was 96% pure (estimated by 1H NMR); this
may be adequate for many applications. After six recrystallizations the procedure gave 46% of the target material
of more than 99% purity (HPLC). The protocol could con-
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(i)
PRACTICAL SYNTHETIC PROCEDURES
Aminodichlorofluorescein Derivatives
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All chemicals were obtained from commercial suppliers and used
without further purification. Chromatography on silica gel was performed using a forced flow of the indicated solvent on EM reagents
silica gel 60 (230–400 mesh). CH2Cl2 was dried/degassed by passing it down an alumina column. 1H and 13C NMR spectra were recorded on an Inova Instrument at 500 MHz (1H), 125 MHz (13C) or
75 MHz (13C) as indicated relative to internal solvent peaks.
Figure 1 (a) Ionization states of fluorescein at various pH levels; (b)
pH dependence of fluorescence intensity for fluorescein vs 2¢,7¢-dichlorofluorescein
veniently be performed to give 35 grams of the desired
product.
An ammonia solution in ethanol (or methanol) (or K2CO3,
MeOH–THF) was used for removal of the acetate groups
from the lactone 1, which gave the free, ring-opened form
2. This intermediate is then subjected directly to reduction
of the nitro group via treatment with hydrogen and Raney
nickel to give the free amine 3.4
Amine 3 could be functionalized in several ways to enable
it to be conjugated to biomolecules. Two approaches were
chosen to illustrate this: formation of an azide 4 and conversion into the dichlorotriazine derivative 5. The azide 4
is suitable for copper-mediated azide-to-alkyne coupling
reactions.5 Compound 5 is of particular interest to us for
its ability to add nucleophilic groups sequentially.6 The
fluorescein analogue of this compound7 has been used extensively by us as a linker to form bivalent peptidomimetics.8 However, we have found that direct binding assays
based on measurement of fluorescence for these mimics
requires very careful control of pH.
The procedures outlined in this report are scalable to preparative quantities. Syntheses of the 5-nitro- 2 and 5 amino-2¢,7¢-dichlorofluorescein (3) are easily obtained in
multigram scale (ca. 35 g or above). The subsequent derivatization into the azidodichlorofluorescein 4 is also
scalable, although in this report we just give a representative procedure.
3¢,6¢-Diacetoxy-2¢,7¢-dichloro-5-nitrospiro[isobenzofuran1(3H),9¢-[9H]xanthen]-3-one (1)
4-Nitrophthalic anhydride (11.6 g, 60.4 mmol) and 4-chlororesorcinol (17.46 g, 120.8 mmol) were heated to 200 °C for 24 h; the mixture was allowed to cool to r.t. before venting the flask. A dark
orange/brown solid was obtained and dissolved in a sufficient
amount of 1 M NaOH (ca. 1.0 L). This soln was filtered and the resulting filtrate carefully acidified to pH 2.5 using concd HCl in an
ice bath. An orange precipitate was obtained, this mixture was allowed to stand in an ice bath for ca. 1 h before filtering. The orange
solid was collected by filtration and dissolved in 25% i-PrOH–
CHCl3, dried (Na2SO4), filtered, and concentrated in vacuo. The orange solid obtained was dried under vacuum and Ac2O (300 mL)
was then added and the soln refluxed at 150 °C for 4 h. It was then
concentrated in vacuo and fresh Ac2O (ca. 150 mL) was added. The
soln was heated then allowed to crystallize. This recrystallization
was repeated at least six more times to increase the purity of the desired isomer to give 1 (16.1 g, 48%) as a white crystalline solid; mp
(dihydroxy derivative) 210 °C (dec); Rf = 0.73 (60% EtOAc–hexanes).
1
H NMR (500 MHz, CDCl3): d = 8.91 (d, J = 2.2 Hz, 1 H), 8.60 (dd,
J = 8.7, 2.2 Hz, 1 H), 7.42 (d, J = 8.7 Hz, 1 H), 7.21 (s, 2 H), 6.87
(s, 2 H), 2.23 (s, 6 H)
13
C NMR (125 MHz, CDCl3): d = 168.0, 166.2, 156.8, 149.6, 149.1,
130.8, 128.7, 128.7, 127.3, 125.7, 123.3, 121.6, 116.1, 113.3, 80.9,
20.8.
HRMS (ES): m/z [M + Li]+ calcd for C24H13Cl2LiNO9: 536.0127;
found: 536.0121
Anal. Calcd for C24H13Cl2NO9: C, 54.36; H, 2.47; O, 27.16. Found
C, 54.29; H, 2.53; O, 27.41
2¢,7¢-Dichloro-5-nitrofluorescein (2)
2¢,7¢-Dichloro-5-nitrofluorescein 3¢,6¢-diacetate 1 (2.4 g, 4.5 mmol)
and K2CO3 (2.5 g, 18.1 mmol) were combined and stirred in
MeOH–THF (1:1, 50 mL) at 25 °C for 4 h. [An alcoholic NH3 soln
was also used as in the synthesis of compound 3]. The dark brown/
orange soln obtained was filtered and concentrated in vacuo. The
obtained solid was dissolved in H2O and carefully acidified to pH
2.5 using 1 M HCl. The precipitate obtained was filtered, dissolved
in 25% i-PrOH–CHCl3, washed with brine (2 × 200 mL) and H2O
(2 × 200 mL), dried (Na2SO4), filtered, and concentrated in vacuo to
give 2 (1.84 g, 90%) as a brick red color solid; mp ca. 250 °C (dec);
Rf = 0.59 (10% MeOH–CH2Cl2).
1
H NMR (500 MHz, CD3OD): d = 8.85 (d, J = 2.2 Hz, 1 H), 8.62
(dd, J = 8.4, 2.3 Hz, 1 H), 7.51 (d, J = 8.4 Hz, 1 H), 6.86 (s, 2 H),
6.78 (s, 2 H).
13
C NMR (75 MHz, CD3OD): d = 168.6, 157.9, 152.5, 151.2, 131.4,
129.7, 127.2, 122.3, 119.3, 114.5, 111.3, 105.1.
HRMS (ES): m/z [M – H]– calcd for C20H8Cl2NO7: 443.9756;
found: 443.9837.
Synthesis 2009, No. 7, 1224–1226
© Thieme Stuttgart · New York
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Fluorescence spectra were recorded on a Cary Eclipse spectrophotometer. The slit width was 5 nm for both excitation and emission.
Non-degassed, aqueous buffers and a 10-mm quartz cuvette were
used. Dilute solns (0.01 < A < 0.05) were used to minimize reabsorption effects.
PRACTICAL SYNTHETIC PROCEDURES
J. C. Castro et al.
Anal. Calcd for C20H9Cl2NO7: C, 53.59; H, 2.47; N, 3.13. Found: C,
53.58; H, 2.74; N, 2.87.
5-Amino-2¢,7¢-dichlorofluorescein (3)
2¢,7¢-Dichloro-5-nitrofluorescein 3¢,6¢-diacetate 1 (1.0 g, 1.88
mmol) was dissolved in MeOH–THF (5:1, 20 mL). To this soln, 2
M NH3 in MeOH (5 mL) was added and the mixture was allowed to
stir at 25 °C for 1 h, after which time TLC showed completion of the
reaction. It was then concentrated under vacuum. The obtained residue was dissolved in 5% H2O–MeOH (15 mL). To this soln, Raney
Ni (110 mg, 1.88 mmol) was added and stirred under H2 (1 bar) for
1.5 h. (It is important to stop the reaction immediately after the starting material has disappeared). The mixture was filtered through
Celite and concentrated under vacuum. It was further purified by
column chromatography (silica gel, MeOH–CH2Cl2, 1:1), and the
obtained dark red solid recrystallized (EtOH–i-PrOH, ~1:1, ca. 7–
10 mL) to give 3 (460 mg, 59%) as fine red crystals; mp ca. 125 °C
(dec); Rf = 0.63 (30% MeOH–CH2Cl2).
1
H NMR (500 MHz, DMSO-d6): d = 11.06 (s, 1 H), 7.04 (d, J = 2.1
Hz, 1 H), 7.00 (dd, J = 2.1, 8.2 Hz, 1 H), 6.95 (d, J = 8.2 Hz, 1 H),
6.88 (m, 2 H), 6.66 (m, 2 H), 5.19 (br, 2 H).
13
C NMR (125 MHz, DMSO-d6): d = 169.6, 155.6, 150.0, 139.6,
128.8, 127.9, 124.9, 124.9, 122.9, 116.7, 112.2, 107.5, 104.3, 95.0.
2¢,7¢-Dichloro-5-(4,6-dichloro-1,3,5-triazin-2-ylamino)fluorescein (5)
A soln of 5-amino-2¢,7¢-dichlorofluorescein (3, 900 mg, 2.16 mmol)
in MeOH (25 mL) was added dropwise into a soln of cyanuric chloride (518 mg, 2.81 mmol) in CH2Cl2 (20 mL) at 0 °C. The mixture
was stirred at 0 °C for 2 h. Then 12 M HCl (0.7 mL) was added and
the reaction stirred at 0 °C for 1 h. The mixture was concentrated under vacuum. It was further purified by column chromatography (silica gel, d = 2 cm, h = 15 cm, MeOH–CH2Cl2, 1: 9 + 0.1% AcOH)
to give 5 (740 mg, 60%) as a dark yellow solid; mp ca. 320 °C (dec);
Rf = 0.61 (10% MeOH–CH2Cl2 + 0.1% AcOH).
1
H NMR (500 MHz, DMSO-d6): d = 11.61 (s, 1 H), 11.12 (s, 1 H),
8.33 (d, J = 1.7 Hz, 1 H), 7.96 (dd, J = 1.7, 8.5 Hz, 1 H), 7.39 (d,
J = 8.5 Hz, 1 H), 6.91 (s, 2 H), 6.74 (s, 2 H).
13
C NMR (125 MHz, DMSO-d6): d = 171.2, 170.5, 169.7, 169.6,
168.6, 164.6, 155.9, 150.8, 139.9, 129.1, 129.1, 129.0, 127.6, 125.3,
116.1, 110.1, 104.3.
MS (MALDI-TOF): m/z [M – H]– calcd for C23H9Cl4N4O5: 564.16;
found: 564.84.
Anal. Calcd for C23H10Cl4N4O5: C, 48.97; H, 1.79; N, 9.93. Found:
C, 49.05; H, 2.20; N, 9.92.
MS (MALDI-TOF): m/z [M – H]– calcd for C20H10Cl2NO5: 414.00;
found: 413.99.
Acknowledgment
Anal. Calcd for C20H11Cl2NO5: C, 57.71; H, 2.66; N, 3.37. Found C,
57.78; H, 3.11; N, 3.24.
Mass spectral data was collected by the TAMU/LBMS-Applications Laboratory directed by Dr. Shane Tichy. Support for this work
was provided by The National Institutes of Health (HG 01745) and
by The Robert A. Welch Foundation.
5-Azido-2¢,7¢-dichlorofluorescein (4)
5-Amino-2¢,7¢-dichlorofluorescein (3, 15 mg, 0.036 mmol) was dissolved in H2O–HCl (1:1, 2 mL) and abs EtOH (1 mL), sonicated until dissolved, and cooled to 0 °C in an ice bath. To this soln, NaNO2
(5 mg, 0.072 mmol) dissolved in H2O (0.5 mL) was added dropwise
to the previous soln. The reaction was placed under N2 and allowed
to stir for 1 h until TLC indicated the reaction was complete. At this
point, NaN3 (3 mg, 0.043 mmol) dissolved in H2O (0.1 mL) was
added dropwise and the soln was allowed to warm slowly to 25 °C
while stirring for 6 h. It was then poured into 25% i-PrOH–CHCl3
(20 mL) and washed with brine (2 × 50 mL), dried (anhyd Na2SO4),
filtered, and concentrated under vacuum. It was further purified by
column chromatography (silica gel, 10% MeOH–CH2Cl2) to give 4
(8 mg, 47%) as an orange solid; mp ca. 159 °C (dec); Rf = 0.59
(10% MeOH–CH2Cl2).
1
H NMR (500 MHz, CDCl3): d = 7.69 (d, J = 1.9 Hz, 1 H), 7.33 (dd,
J = 8.3, 2.2 Hz, 1 H), 7.14 (d, J = 8.3 Hz, 1 H), 6.94 (s, 2 H), 6.72
(s, 2 H).
13
C NMR (125 MHz, 10% CD3OD–CDCl3): d = 168.5, 151.3,
143.1, 128.7, 128.5, 126.5, 126.1, 115.3, 110.8, 104.0, 90.9. (some
signals overlap)
HRMS (ESI): m/z [M – H]– calcd for C20H8Cl2N3O5: 439.9846;
found: 439.9841.
Synthesis 2009, No. 7, 1224–1226
© Thieme Stuttgart · New York
References and Notes
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