Synthesis of probes with broad pH range fluorescence

Article abstract of DOI:10.1016/S0960-894X(03)00411-6

Reaction of a rhodamine 2′-ester with an excess of alkyldiamines provides amino-functionalized rhodamine spirolactams, which when subsequently conjugated with carboxyfluorescein, provides probes which are fluorescent at acidic, neutral, and basic pH ranges.

Full text of DOI:10.1016/S0960-894X(03)00411-6

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2327–2330
Synthesis of Probes with Broad pH Range Fluorescence
Maciej Adamczyk* and Jonathan Grote
Department of Chemistry (D9MD), Abbott Diagnostics Division, Abbott Laboratories, 100 Abbott Park Road,
Abbott Park, IL 60064-6016, USA
Received 27 February 2003; revised 17 April 2003; accepted 22 April 2003
Abstract—Reaction of a rhodamine 20-ester with an excessof alkyldiamines provides amino-functionalized rhodamine spirolactams, which
when subsequently conjugated with carboxyfluorescein, provides probes which are fluorescent at acidic, neutral, and basic pH ranges.
# 2003 Elsevier Science Ltd. All rights reserved.
Recent reports of fluorescent probes attest to their con-
tinued utility in the study of complex biological sys-
tems.1ꢀ5 In particular, there is a need for new probes
which display fluorescence over a broad pH range, since
the lack of a signal resulting from fluorescence attenua-
tion due to environmental factors such as pH can
appear similar to the lack of accessibility of the probe to
a particular environment.6ꢀ8 Carboxyfluorescein, for
example, suffers from the fact that in some relatively
acidic intracellular environments its fluorescence inten-
sity is reduced due to closure to the non-fluorescent
spirolactone form.8 In contrast to fluorescein, the
recently disclosed rhodamine spirolactams9 exist in a
fluorescent quinone form at acidic or neutral pH, and a
non-fluorescent spiro form at basic pH. The com-
plementarity of the pH ranges of these two labels at which
fluorescence is displayed suggests the intriguing possibility
that the two labels could together serve as a pair of
fluorophores with broad pH range utility. Furthermore,
covalent linkage of the two labels would alleviate any
relative migratory aptitude or permeability differences
between the labels if individually used, and provide a sin-
gle conjugate which would be fluorescent in almost any
physiological pH environment. This report describes the
synthesis and properties of such conjugates.
For our study, rhodamine 6G was chosen due to its
lipophilicity and permeability in cell membranes.5 Initi-
ally, we tested the reactivity of rhodamine 6G with
alkyldiamines (Table 1). In all cases, reaction of the
rhodamine ester substrate with excess diamine did not
produce detectable quantities of the bis-substituted
product (ESMS), apparently since the unreacted alkyl-
amine of the mono-substituted product cannot attain a
suitably uncongested orientation to the 9-position for
reaction with a second rhodamine ester.9 This regio-
selectivity allowed the use of excess diamine, which
eliminated the need for supplemental tertiary amine
base for neutralization of the rhodamine salts. The
reactions also showed rapid conversion (complete in <2
hr in all cases) to the amides (1a–f). Direct purification
of the reaction mixture by HPLC using trifluoroacetic
acid (TFA) as the aqueous modifier, followed by lyo-
philization, produced good yields of the desired amino
functionalized amides as red solids in their open qui-
none forms. For NMR analysis, the individual products
*Corresponding author. Tel.: +1-847-937-0225; fax: +1-847-938-
8927; e-mail: maciej.adamczyk@abbott.com
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00411-6

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M. Adamczyk, J. Grote / Bioorg. Med. Chem. Lett. 13 (2003) 2327–2330
Table 1. Reaction of rhodamine 6G with diamines
Entry
Amine
Product
Isol. yield (%)
79
1Hexanediamine
2
3
4
5
6
Ethylenediamine
Bis(oxyethylamino)ethane
p-Diaminoxylene
83
89
74
75
89
Bis(aminoethyl)amine
Tris(aminoethyl)amine
were neutralized with saturated aqueous sodium bicar-
bonate to fully convert each derivative into its symme-
trical, off-white non-fluorescent spirolactam form, which
could be isolated by extraction with dichloromethane.
13C NMR analysis confirmed interconversion to the
closed spirolactam form, by observation of a signal at
64–65 ppm, assignable to the sp3 hybridized 9-position
carbon attached to the nitrogen atom of the amide.10
Fig. 1) and its corresponding conjugate (e.g., 2d, Fig.
2) both demonstrated a strong pH dependence and a
good correlation between the rhodamine portion of
the two spectra, indicating that conjugation had little
As shown in Table 2, reaction of the amines 1a–f with
6-carboxyfluorescein N-hydroxysuccinimide active ester
(6CFAE, diisopropylethylamine, DMF) produced the
target probes. In all cases, isolation of the product was
achieved by preparative reversed phase HPLC with
TFA as the aqueous modifier, producing the conjugates
as red solids (rhodamine open, fluorescein closed). The
1
structures of the probes were elucidated by ESMS, H
NMR, and 13C NMR. The amine salts were neutralized
for NMR analysis, resulting in degenerate aromatic
resonances due to the interconversion of the rhodamine
to its closed form.11 Additionally, probe 2e was synthe-
sized by reaction of the carboxyfluorescein derivative of
1e with succinic anhydride. Two probes (2e and 2f)
equipped with functionality for bioconjugation were
thus prepared.
Figure 1. UV spectrum of spirolactam 1d.
Each fluorescein conjugate and its amino functionalized
rhodamine amide precursor was analyzed by UV spec-
troscopy at a fixed concentration and at three different
pH values (pH 4, pH 6, and pH 8) in phosphate buffers.
The spectra of each precursor (e.g., spirolactam 1d,
Figure 2. UV spectrum of conjugate 2d.

M. Adamczyk, J. Grote / Bioorg. Med. Chem. Lett. 13 (2003) 2327–2330
2329
Table 2. Conjugation of amino rhodamine amides with carboxyfluorescein N-hydroxysuccinimide active ester
Entry
1
Amine
Product
Isol. yield (%)
42
1a
2
3
4
5
6
1b
1c
1d
1e
1f
25
59
45
30a
32
aTwo step yield: yield of conjugation 44%; yield of succinylation 68%.
effect on the interconversion of the rhodamine spiro-
lactam. Examination of these spectra revealed that the
conjugates all demonstrated one lmax near 497 nm at
pH 8 and above, consistent with the fluorescein label
existing in its open form, and another lmax near 540
nm at pH 4 and below, consistent with the rhodamine
being in its open form.9 The solutions were visibly pink at
pH 4 (from the rhodamine chromophore) and yellow at
pH 8 (from the fluorescein chromophore). Solutions at all
pH values were visibly fluorescent.
spectra of the probes displayed a lmax-emis of 561–562
nm, red shifted relative to rhodamine 6G (lmax-emis 554).
The relative quantum yields varied from 0.35–0.82
(Table 4). The emission spectra at pH 8 demonstrated
approximately the same lmax-emis as 6-carboxy-
fluoresceine (517 nm), and quantum yields of 0.19–0.64.
In conclusion, the conjugation of amino functionalized
rhodamine spirolactams to carboxyfluorescein provides
a variety of new fluorescent conjugates under mild con-
ditions. All of the conjugates synthesized generate
fluorescent signals across a broad pH range, making
them useful as fluorescent probes at a variety of pH
values, or conversely as indicators of pH, based on the
quantity and wavelength of fluorescence observed.
The extinction coefficients of each precursor and its
conjugate were determined at pH 4 and pH 8 by serial
dilution in the corresponding buffers (Table 3). Inter-
estingly, despite a change in the linker length (1a vs 1b),
hydrophilicity (vs 1c), rigidity (vs 1d), or the presence of
charged groups (vs 1e, 1f), all precursors showed rea-
sonably good agreement in their extinction coefficients
at the two pH values tested. Similarly, the conjugates all
showed approximately equivalent extinction coefficients
at the two pH values tested.
Table 3. Extinction coefficients of rhodamine spirolactam precursors
and conjugates at their lmax
Precursor pH 4a=e pH 8a=e Conjugate pH 4b=e pH 8c=e
1a
1b
1c
1d
1e
1f
67,700
60,900
68,800
60,200
57,000
63,500
1500
500
750
1000
1000
400
2a
2b
2c
2d
2e
2f
36,200
32,800
35,100
27,500
26,300
27,700
66,000
67,900
69,600
54,900
62,400
58,800
The rhodamine values of the conjugates are attenuated
when compared to their precursors, as are the fluor-
escein values of the conjugates, when compared to the
extinction coefficient of 6-carboxyfluorescein at pH 8
(lmax 492 nm, e=80,000).
a530 nm.
b540 nm.
c497 nm.
The probes were also analyzed by fluorescence emission
spectroscopy12 at pH 4 and pH 8. At pH 4, the emission

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M. Adamczyk, J. Grote / Bioorg. Med. Chem. Lett. 13 (2003) 2327–2330
Table 4. Quantum yields of new probesa
10. Representative example: Rhodamine 6G hydrochloride
(100 mg, 209 mmol) was dissolved in anhydrous DMF (500
mL), and hexanediamine (135 mg, 300 mol%) was added.
After stirring for 12 h, the reaction mixture was purified
directly by preparative reversed phase HPLC (CH3CN/0.05%
aqueous TFA mixtures). Concentration on a rotovap, fol-
lowed by lyophilization, provided 122 mg (79%) of 1a: exci-
tation lmax 530 nm; e530=67,700 (pH 4). Neutralization
(saturated NaHCO3/CH2Cl2) and concentration provided an
off-white solid: 1H NMR (DMSO-d6) d 7.74 (m, 1H), 7.48 (m,
2H), 6.97 (m, 1H), 6.24 (s, 2H), 6.03 (s, 2H), 5.05 (t, 2H,
J=5.2 Hz), 3.26 (s, 4H), 3.11 (m, 3H), 2.92 (m, 2H), 2.41 (t,
2H, J=6.8 Hz), 1.84 (s, 6H), 1.19 (t, 6H, J=6.7 Hz), 0.95 (m,
6H); 13C NMR (DMSO-d6) d 166.7, 153.3, 151.0, 147.6, 147.6,
132.5, 130.8, 128.2, 127.6, 123.6, 122.1, 118.1, 118.1, 104.9,
95.5, 64.2, 41.0, 37.5, 31.9, 27.6, 26.3, 25.8, 17.0, 14.1; ESMS
513.3 (M+H)+.
11. To a solution of 1a (50 mg, 68 mmol) and 6-carboxy-
fluorescein N-hydroxysuccinimide active ester (34 mg, 71
mmol) in anhyd DMF was added diisopropylethylamine (41
mL, 350 mol%). After stirring for 14 hr, the reaction mix-
ture was purified directly by preparative reversed phase
HPLC (CH3CN/0.05% aqueous TFA mixtures). Concen-
tration on a rotovap, followed by lyophilization, provided
28 mg (42%) of 2a: excitation lmax 540 nm, e540=36,200
(pH 4); excitation lmax 497 nm, e497=66,000 (pH 8). Neu-
tralization (pH 6 phosphate buffer) and concentration pro-
vided an off-white solid: 1H NMR (d6DMSO) d 8.43 (m,
1H), 8.01 (d, 1H, J=8.0 Hz), 7.91(d, 1H, J=7.9 Hz), 7.77
(m, 1H), 7.47 (m, 2H), 6.98 (m, 1H), 6.53 (s, 1H), 6.49 (s,
1H), 6.22 (s, 2H), 6.05 (m, 6H), 5.02 (m, 2H), 3.08 (m,
4H), 2.91 br, 2H), 1.82 (s, 6H), 1.17 (m, 6H), 0.95 (m, 6H);
13C NMR (DMSO-d6) d 169.0, 166.7, 165.1, 153.3, 151.0,
147.6, 132.5, 130.9, 130.1, 128.2, 127.6, 123.6, 122.2, 118.2,
109.1, 104.9, 102.5, 95.6, 64.2, 37.5, 28.9, 26.1, 17.0, 14.1;
ESMS 871.5 (M+H)+.
Probe
pH 4b
pH 8c
2a
2b
2c
2d
2e
2f
0.410.24
0.45
0.35
0.82
0.52
0.27
0.19
0.64
0.35
0.23
0.39
aValues are the average of duplicate determinations.
bRelative to rhodamine 6G.
cRelative to 6-carboxyfluoresceine.
Probes 2e and 2f also contain an amine or acid func-
tionality, respectively, making them suitable for cova-
lent attachment to generate a variety of bioprobes.13
Since different rhodamine esters and fluoresceins would
be serve as suitable substrates for this reaction, con-
siderable flexibility in the designer’s fine-tuning of the
fluorescence properties of these broad range fluoro-
phores can be achieved.
References and Notes
1. Abugo, O. O.; Nair, R.; Lakowicz, J. R. Anal. Biochem.
2000, 279, 142.
2. el Jastimi, R.; LaFleur, M. Biospectroscopy 1999, 5, 133.
3. Lan, Z.-H.; Qian, X.; Giese, R. J. Chrom. A 1999, 831, 325.
4. Scaduto, R. C., Jr.; Grotyohann, L. W. Biophys. J. 1999,
76, 469.
5. Mandala, M.; Serck-Hanssen, G.; Martino, G.; Helle, K. B.
Anal. Biochem. 1998, 18, 69.
6. Robey, R. B.; Ruiz, O.; Santos, A.; Ma, J.; Kear, F.; Wang,
L.-J.; Li, C.-J.; Bernardo, A. A.; Arruda, J. A. L. Biochemistry
1998, 37, 9894.
12. Fluorescence emission spectra were obtained on an ISS
PC1spectrofluorometer (Champaign, IL).
7. Lee, R. J.; Wang, S.; Turk, M. J.; Low, P. S. Biosci. Rep.
1998, 18, 69.
8. Handbook of Fluorescent Probes and Research Chemicals,
6th ed.; Haugland, R.P., Ed.; Molecular Probes: Eugene, OR,
1996; p 51.
13. In a trial study, probe 2e was conjugated to an eledoisin
related hexapeptide using TBTU as a coupling reagent. Pur-
ification by reversed-phase HPLC provided a conjugate that
demonstrated 1:1 incorporation (ESMS), and displayed fluo-
rescence properties similar to those of the probe described
above (pH 4: lmax-abs 539, lmax-emis 561; pH 8: lmax-abs 499;
lmax-emis 517).
9. Adamczyk, M.; Grote, J. Bioorg. Med. Chem. Lett. 2000,
10, 1539.