Unexpected reactivity of the 2′-carboxyl functionality in rhodamine dyes

Article abstract of DOI:10.1016/j.tetlet.2017.03.054

Contrary to previous literature reports, the reactivity of the 2′ carboxylic acid in rhodamine dyes was found to be much more reactive than anticipated. Typically, the 4′- or 5′-carboxy functionality in dicaboxyrhodamines is targeted for bioconjugation use due to the supposed unreactivity of the 2′ carboxylic acid. Reactive esters of 2′-carboxyrhodamine dyes lacking the 4′(5′)-carboxy substitutions permit a simplified synthesis of single isomer dyes but possess similar reactivities useful for labeling studies.

Full text of DOI:10.1016/j.tetlet.2017.03.054

Accepted Manuscript
Unexpected reactivity of the 2’-carboxyl functionality in rhodamine dyes
Richard A. Haack, Susan Gayda, Richard J. Himmelsbach, Sergey Y. Tetin
PII:
S0040-4039(17)30359-3
DOI:
http://dx.doi.org/10.1016/j.tetlet.2017.03.054
TETL 48755
Reference:
Tetrahedron Letters
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Revised Date:
Accepted Date:
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14 March 2017
17 March 2017
Please cite this article as: Haack, R.A., Gayda, S., Himmelsbach, R.J., Tetin, S.Y., Unexpected reactivity of the
2’-carboxyl functionality in rhodamine dyes, Tetrahedron Letters (2017), doi: http://dx.doi.org/10.1016/j.tetlet.
2017.03.054
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Unexpected reactivity of the 2’-carboxyl
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functionality in rhodamine dyes
Richard A. Haack, Susan Gayda, Richard J. Himmelsbach and Sergey Y. Tetin

1
Tetrahedron Letters
Unexpected reactivity of the 2’-carboxyl functionality in rhodamine dyes
Richard A. Haacka,, Susan Gaydab, Richard J. Himmelsbacha and Sergey Y. Tetinb
aOrganic Chemistry Process Design, Abbott Diagnostics Division, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, Il 60064-6016, USA
b Molecular Binding Characterization, Abbott Diagnostics Division, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, Il 60064-6016, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Contrary to previous literature reports, the reactivity of the 2’ carboxylic acid in rhodamine dyes
was found to be much more reactive than anticipated. Typically, the 4’- or 5’-carboxy
functionality in dicaboxyrhodamines is targeted for bioconjugation use due to the supposed
unreactivity of the 2’ carboxylic acid. Reactive esters of 2’-carboxyrhodamine dyes lacking the
4’(5’)-carboxy substitutions permit a simplified synthesis of single isomer dyes but possess
similar reactivities useful for labeling studies.
Available online
Keywords:
Rhodamine
Bioconjugation
Fluorescent
Fluorescein
Xanthene
2009 Elsevier Ltd. All rights reserved.
———
 Corresponding author. Tel.: +0-000-000-0000; fax: +0-000-000-0000; e-mail: author@university.edu

2
Tetrahedron
Introduction
photo-efficient RDs. Due to the challenging separations required
during their synthesis, isomerically pure, 4’- or 5’-caboxyphenyl
RDs are limited, extremely expensive13 and many are sold only
as a mixture of the two regioisomers.5 Therefore, we began a
program in our laboratories to prepare RDs tailored to our
specific needs.
Xanthene dyes (fluoresceins, rhodols and rhodamines)
containing a reactive linker functionality for bioconjugation are
used extensively as labels, tags and probes to study biological
systems.1 In the xanthene dye family, the spectroscopic
properties of rhodamine dyes’ (RDs) and their conjugates’
absorption/emission properties show less pH sensitivity and
possess greater photostability compared to their fluorescein
relatives.2 The classic synthesis of reactive symmetrical RDs for
use in conjugation chemistry employs reaction of trimellitic
anhydride and an aminophenol that generates two regioisomeric
dicarboxylic acids that are difficult to separate (Scheme 1).
Initially, we utilized the method described by Shmanai
et al4 which condenses trimellitic anhydride and 3-
(dimethylamino)phenol in toluene in the absence of strong acid
catalysts to produce
a
mixture of two regioisomeric
benzophenones (1a and 1b). These are easily separable on a large
scale by crystallization (Scheme 2).
Scheme 2. Condensation of trimellitic anhydride under non-
acidic conditions.4
Scheme 1. Classic synthesis of symmetrical 4’(5’)
regioisomeric RDs. Other numbering systems are also employed
in the literature.3-5
Either isolated benzophenone reacts with
aminophenols under acidic conditions
a
variety of
producing
“unsymmetrical” single isomer RDs. Structural features of the
aminophenol used in the reaction alter the spectroscopic and
brightness properties of the resulting dye, which allows
tailoring/tuning of the RD’s with desired properties.3,14 For
example, benzophenone 1a reacts with 2,2,4-trimethyl-1,2-
dihydroquinolin-7-ol (2)15 in 50% sulfuric acid at 100 °C with
concomitant sulfonation of the allylic methyl group occurring
under these reaction conditions7 yielding 4’-carboxy-RD 3
(Scheme 3).
Typically, the
4’- or 5’-carboxy functionality in
dicarboxyrhodamines (or a mixture of both)6 is targeted for
bioconjugation since the 2’ carboxylic acid is generally observed
as difficult to activate, possessing low chemical reactivity due to
steric hindrance of the xanthene system”.5,7,8 Activation of the
2’-carboxyl in these systems usually requires harsh reagents such
as phosphorus oxychloride, which are often incompatible with
other functionality in the molecule.5,7,9 Single isomer RDs are
preferable for biological studies because the 4’ or 5’ dicarboxy
isomeric dyes often possess differing labeling specificities and
photophysical properties when conjugated to biomolecules.10
Also, purifications and spectroscopic characterizations during
chemical synthesis of the dye and dye/linker combinations are
greatly simplified when operating with a single isomer. Another
reason the 2’ position is undesirable for direct conjugation is that
reaction with a primary amine (e.g. lysine residue in a protein)
produces a secondary amide, which cyclizes under neutral to
basic conditions to produce a non-fluorescent spiroamide product
(Figure 1).5,8
Scheme 3. Condensation of benzophenone 1a with
aminophenol 2. (1a + 1.2 eq. 2, 50% H2SO4, 125 °C, 2 hr,
reverse phase HPLC purification, Yield 3 10.4%)
During our initial attempt to prepare the 4’-mono-
pentafluorophenyl active ester of RD
3
utilizing
pentafluorophenyl trifluoroacetate/pyridine/DMF16 (Scheme 4
left), UPLC-MS analysis showed the formation of a 1:1 mixture
of two products with the same mass corresponding to two
isomeric mono pentafluorophenyl esters, a small amount of the
diester product and unreacted starting material 3 within the first
10 minutes of the reaction (Figure 2 top). After 1 hour, the
reaction mixture contained a 1:1 mixture of unreacted RD 3 and
the diester product 4 as well as a “steady state” 1:1 mixture of
two monoester products (Figure 2 middle). After 24 hours, both
the starting material and the two monoester products were
consumed and diester 4 was the major product (Figure 2 bottom).
Figure 1. Non-fluorescent spirolactam formation from 2’-
carboxyamido RDs derived from a primary amine.
Experimental Findings
High definition immunoassay (HDIA)11 and single molecule
detection12 research projects in our laboratories required the
preparation of large quantities of photophysically customizable,

3
Bioconjugation studies typically require a single point of
attachment of the RD making the reactive diesters unsuitable for
this purpose since the 2’ and 4’(5’) esters react non-selectively
with amines. For example, the pentafluorophenyl diester 4 shows
no chemoselectivity with amines18,19 providing a mixture of both
monoamide products as well as the diamide product 6a/6b/7
(Figure 4) Thus, we further examined the 2’-carboxyl reactivity
of RDs and fluoresceins.
Figure 4. Example of mono and diamide products obtained
from the dipentafluorophenyl ester 4.
Figure 2. Pentafluorophenyl active ester formation after 10
minutes (top), 1 hour (middle) and 24 hours (bottom).
Reaction monitored by UPLC-MS17
Confirmation of Reactivity
To ascertain the scope of the 2’ carboxyl group’s reactivity,
we examined the 2’-monocarboxylic acid in both rhodamine and
fluorescein dyes. 4’(5’)-carboxytetramethyl rhodamine (8)
(mixture of 4’ and 5’ position regioisomers) and rhodamine B
(10) were treated with pentafluorophenyl trifluoroacetate in
pyridine/DMF at room temperature. UPLC-MS analysis of each
reaction indicated that after only 10 minutes of contact, the
diester 9 and the 2’ ester 11 had formed quantitatively (Scheme
5).
Scheme 4. Unexpected diester products 4 and 5 produced
from 3 utilizing pentafluorophenyl trifluoroacetate and TSTU
respectively.
The reactivity profile for the 2’ and 4’ carboxyl groups was
further explored by using N,N,N’,N’-tetramethyl-O-(N-
succinimidyl)-uronium tetrafluoroborate (TSTU)5 to generate the
corresponding NHS diester 5 (Scheme 4 right). The unexpected
facile activation of the 2’carboxyl group of RD 3 lies in contrast
to literature reports indicating that the 2’ carboxylic acid is
unreactive due to steric hindrance of the bulky xanthene ring
system and its activation requires highly reactive reagents.5,8
Additionally, it has been reported that either the 4’ or 5’ carboxyl
groups may be easily converted to active esters and conjugated in
the presence of the unreactive 2’-carboxylic acid.8 Also, the low
reactivity of the 2’ carboxylic acid in RD systems is exemplified
in a publication describing activation of a compound similar to
RD 3 but containing only a 2’ carboxylic acid (Figure 3), This
RD required the highly reactive reagent, O-(7-azabenzotriazol-1-
Scheme 5. Pentafluorophenyl active ester formation 4’(5’)-
carboxyrhodamine 8 (top) and rhodamine B 10 (bottom).
We then subjected 4’-carboxyfluorescein (12) and fluorescein
(15) to the same conditions (Scheme 6). UPLC-MS analysis of
the 4‘-carboxyfluorescein reaction showed only a single mono-
pentafluorophenyl ester, presumably 4’-13, without a trace of
diester 14 and fluorescein gave no detectable 2’-ester 16. These
results clearly contrast the reactivity differences of the 2’-
carboxy functionality in the fluorescein and rhodamine dye
families.
-
yl)-N,N,N’,N’-tetramethyluronium-PF6 (HATU) and required
overnight reaction run at 40-50 °C.7
Figure 3. Literature example of a 2’-carboxyrhodamine

4
Tetrahedron
Figure 6. Nonequivalent sulfonylmethy 1H signals of RD 3.29
It is apparent that the 2’carboxyl of RDs is not as sterically
hindered as previously claimed due to its positioning above the
xanthene plane. The 2’-position appears to be as reactive as the
4’ or 5’ positions, making it a suitable point for conjugation in
fluorescent studies.
Scheme 6. Pentafluorophenyl active ester formation 4’-
carboxyfluorescein (top) and fluorescein (bottom).
For example, we prepared an unsymmetrical RD with phthalic
anhydride to produce benzophenone 1714 which lacks the 4’(5’)-
carboxyl moiety, alleviating the need to separate regioisomers.
The benzophenone 17 was condensed with hydroxy-
Discussion
These results demonstrate the high reactivity of the 2’-
carboxyl group of RDs and the inertness of the 2’-carboxyl group
of fluorescein dyes. This suggests that in polar aprotic organic
solvents, fluoresceins probably exist in the closed,
spirolactone/zwitterionic form, rendering their 2’-carboxyl inert
(Figure 5).20-22 Contrarily, RDs exist in the open form through a
broad pH (3-14) range in polar organic solvents.23,24 This
explanation is consistent with our observation that when either
fluorescein, 12 or 15, is dissolved in the reaction solvents (DMF
containing pyridine), both give a pale straw colored and weakly
fluorescent solution indicating the non-fluorescent spirolactone
form predominates under the reaction conditions. However, upon
acidification with 1N hydrochloric acid, these DMF solutions
become bright yellow and are highly fluorescent when irradiated
with a handheld long wave UV lamp (365 nm) indicating the
presence of the fluorescent open form under these non-reaction
conditions. In contrast, rhodamines 8 and 10, under the same
conditions give bright red, highly fluorescent solutions which
remain unchanged upon acidification, indicating the fluorescent
open form predominates under the reaction conditions.
dihydroquinoline 2 in 50% sulfuric acid at 125 °C, providing RD
18 (Scheme 7).
Scheme 7. Synthesis of unsymmetrical-RD 18 lacking the
4’(5’)-carboxyl (same reaction conditions as in Schemes 2
and 3 above. Yield 18 11%).
Also, although the low reactivity of the 2’-carboxyl in the RD
series has been attributed to steric hinderance,8 molecular models
show that the phenyl bearing the 2’ carboxyl is essentially
perpendicular to the xanthene system due to the congestion of the
1,8-peri hydrogens.25,26 Also, X-ray studies have shown that the
spirolactone or inner salt of the carboxylate/xanthene cation
exists in the crystal state (Figure 5).27,28 This situates the 2’-
carboxyl above the plane of the xanthene system (Figure 5).
RD 3 and RD 18 possess photo-physical properties similar to
the commercially available RD, Alexa Fluor 546.(Figure 7).30
Figure 5. Fluorescein spirolactone (left) and RD (right).
Proton NMR confirmed the perpendicularity of the phenyl of
RD 3 as its NMR spectrum clearly shows the hindered rotation of
the phenyl ring as the two hydrogens of the sulfonylmethyl group
are non-equivalent due to the chirality of the hindered system. An
AB quartet centered at ~3.61 ppm (Figure 6) is observed showing
the non-equivalency. This splitting pattern has been observed in a
structurally similar, symmetrical RD-disulfonic acid as well.7
Figure 7. Absorption/emission properties of RD 3 and RD 18
(top). Fluorescence intensity compared to Alexa Fluor 546
(bottom).

5
(2)
Panchuk-Voloshina, N.; Haugland, R. P.;
For fluorescent bioconjugation studies, a secondary amine
must be used to react with the 2’ position to prevent
spirolactamization.5,8 Secondary amines such as piperazine, N-
methyl-6-aminocaproic acid, N,N’-dimethylethylenediamine
(DMEDA) have been employed by us and others8 for this
purpose. For example, we prepared a biotinylated-RD 19
containing a PEG linker. Reaction of RD 18-pentafluorophenyl
ester with excess DMEDA followed by reaction of 18-DMEDA
with a biotinylated PEG pentafluorophenyl ester provided highly
fluorescent PEG-linked biotinylated-RD 19 (Scheme 8). The dye
possesses essentially identical absorption/emission properties as
that of the core RD 18 and the 2’-tertiary amide linkage makes it
insensitive to pH changes.5,8 The uses and properties of this dye
will be presented elsewhere.
Bishop-Stewart, J.; Bhalgat, M. K.; Millard, P. J.; Mao, F.; Leung,
W. Y.; Haugland, R. P. The journal of histochemistry and
cytochemistry : official journal of the Histochemistry Society 1999,
47, 1179.
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Trans. 1 1994, 2967.
(4)
Corrie, J. E. T.; Craik, J. S. J. Chem. Soc., Perkin
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Johnson, I. S., Michelle, Ed. 2010, p 35.
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Belov, V. N.; Bossi, M. L.; Folling, J.;
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Boyarskiy, V. P.; Belov, V. N.; Medda, R.; Hein,
B.; Bossi, M.; Hell, S. W. Chemistry 2008, 14, 1784.
(9)
Yuan, L.; Lin, W.; Feng, Y. Organic &
biomolecular chemistry 2011, 9, 1723.
(10)
Kamino, S.; Ichikawa, H.; Wada, S.; Horio, Y.;
Usami, Y.; Yamaguchi, T.; Koda, T.; Harada, A.; Shimanuki, K.;
Arimoto, M.; Doi, M.; Fujita, Y. Bioorganic & medicinal chemistry
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Ruan, Q.; Skinner, J. P.; Tetin, S. Y. Biophysical
Journal 2012, 102, 193a.
(12)
Valeur, B.; Berberan-Santos, M. N. In Molecular
Fluorescence; Wiley-VCH Verlag GmbH & Co. KGaA: 2012, p
360.
(13)
Alexa Fluor® 488 NHS ester mixture of 4’/5’
Scheme 8. Preparation of PEG-linked biotin-RD 19: (i. RD
9, pentafluorophenyl trifluoacetate, pyridine, DMF, room
temperature; ii. excess DMEDA; iii. pegylated biotin acid19,
pentafluorophenyl trifluoacetate, pyridine, DMF; iv. product
of step i. + product of step iv., DIEA, DMF) 75% overall
yield from 18.
isomers $299/mg from ThermoFisher (2017 price).
(14)
Sauers, R. R.; Husain, S. N.; Piechowski, A. P.;
Bird, G. R. Dyes Pigm. 1987, 8, 35.
(15)
Heller, E.; Lautenschlaeger, W.; Holzgrabe, U.
Tetrahedron Lett. 2009, 50, 1321.
(16)
Hermanson, G. T. In Bioconjugate Techniques
(Second Edition); Academic Press: New York, 2008, p 169.
Conclusion
(17)
C18 1.7 um 2.1
water/acetonitrile/0.1% formic acid, gradient 10:90-->90:10.
(18) No selelectivity was observed when utilizing 1
or 2 equivalents of amine.
Waters Acquity UPLC system with SQ detector.
x
50 mm column. ESI+ ionization.
Whilst the origin of the notion that the 2’-caboxyl group of
rhodamine dyes is difficult to activate due to steric hindrance of
the bulky xanthene remains unclear, it may be a carryover which
stems from the 2’-carboxyl’s inertness in the fluorescein series.
We have found that, contrary to many discussions published on
its reactivity, the 2’-carboxyl group of the rhodamine dye system
can be easily activated with mild activating agents clearly
demonstrating its availability for reaction equal to that of the less
hindered 4’ or 5’ positions.
We demonstrated that the need for a “reactive” 4’ or 5’-
carboxyl group is unnecessary as an attachment point for
conjugation. Synthesis of RDs lacking the 4’ or 5’ carboxylic
acids and utilization of the mildly activated 2’ position as an
attachment point greatly simplifies synthesis and purification of
RDs and eliminates the need for tedious isomer separations when
single isomers are required.
(19)
(20)
Available from Quanta BioDesign Ltd.
Martin, M. M.; Lindqvist, L. J. Lumin. 1975, 10,
381.
(21)
McHedlov-Petrossyan, N. O.; Kukhtik, V. I.;
Alekseeva, V. I. Dyes Pigm. 1994, 24, 11.
(22)
(23)
Zanker, V.; Peter, W. Chem. Ber. 1958, 91, 572.
Ramette, R. W.; Sandell, E. B. J. Am. Chem. Soc.
1956, 78, 4872.
(24)
With the exception that in nonpolar solvents,
such as benzene or ether, the spirolactone form predominates. see
reference 23.
(25)
1981, 79, 347.
(26)
Lett. 1978, 57, 526.
(27)
Arbeloa, I. L.; Ojeda, P. R. Chem. Phys. Lett.
Sadkowski, P. J.; Fleming, G. R. Chem. Phys.
Qu, J.-Q.; Wang, L.-F.; Li, Y.-Z.; Sun, G.-C.;
Acknowledgments
The authors gratefully acknowledge Stefan Hershberger, Brian
Bax, Qiaoqiao Ruan (Abbott Laboratories) and Ian Marsden
(Abbvie Laboratories) for their many helpful discussions.
Zhu, Q.-J.; Xia, C.-G. Synth. React. Inorg. Met.-Org. Chem. 2001,
31, 1577.
(28)
Wang, X.-Q.; Sun, Y.; Long, Y.-C. Huaxue
Xuebao 2000, 58, 1173.
(29)
1H NMR spectra taken in Methanol-d4 (TMS)
References and notes
on a Varian 400 mHz spectrometer.
(30)
The absorption spectra were measured on Cary
* All new compounds exhibited analytical data (NMR and HRMS)
consistent with their assigned structures. The yields reported are for
chromatographically pure samples.
UV-Vis spectrophotometer. All fluorophores were diluted in PBS to
various concentrations. Corresponding
spectrum of each sample was excited at 540nm and measured on
Florolog spectrofluorometer. Total fluorescence intensity of each
sample was plotted as a function of its absorption value at 540nm.
fluorescence emission
(1)
Hermanson, G. T. In Bioconjugate Techniques
(Second Edition); Academic Press: New York, 2008, p 396.

6
Tetrahedron
Highlights





Unexpected reactivity of the 2’-carboxyl
functionality in rhodamine dyes was found.
Pentafluorophenyl esters of 2'-
carboxyrhodamine are mildly prepared.
A “reactive” 4’(5’)-carboxyl is unnecessary
as an attachment point for conjugation.
Rhodamine dyes lacking the 4’(5’) carboxyl
produces a single isomer.
No tedious isomer separations when single
isomers are required.