Design, synthesis, and characterization of fluorescent cobalamin analogues with high quantum efficiencies

Article abstract of DOI:10.1021/ol900401z

Cobalamin tethered to fluorescein or Rhodamine 6G has been synthesized and characterized. The fluorophore is conjugated to the ribose-5-OH of cobalamin through a rigid linker to prevent the fluorophore from folding back through space and interacting with

Full text of DOI:10.1021/ol900401z

ORGANIC
LETTERS
2009
Vol. 11, No. 12
2499-2502
Design, Synthesis, and Characterization
of Fluorescent Cobalamin Analogues
with High Quantum Efficiencies
Manfai Lee and Charles B. Grissom*
Department of Chemistry, UniVersity of Utah, 315 South 1400 East,
Salt Lake City, Utah 84112-0850
grissomc@chem.utah.edu
Received February 26, 2009
ABSTRACT
Cobalamin tethered to fluorescein or Rhodamine 6G has been synthesized and characterized. The fluorophore is conjugated to the ribose-
5′-OH of cobalamin through a rigid linker to prevent the fluorophore from folding back through space and interacting with the corrin ring of
cobalamin. This increases the fluorescence quantum yield. This new family of cobalamin analogues may be suitable for use as tumor markers
to tag cancer cells for surgical resection.
Cancer cells exhibit an unusually high requirement for
cobalamin (vitamin B12) to support DNA synthesis prior to
cell division.1 Cobalamin analogues with a fluorophore
attached at the ꢀ-axial ligand position of Co(III) or the ribose-
5′-hydroxyl moiety of cobalamin have proved useful as
probes of vitamin B12 trafficking. Furthermore, they show
promise as visual indicators of tumor micromargins in murine
xenograft models of human tumors.2 While the nascent
generation of fluorescent cobalamin conjugates was useful
as a tool to explore the cellular biology of vitamin B12, the
earliest fluorescent cobalamin conjugates reported suffer from
severe fluorescence quenching due to overlap of the elec-
tronic orbital of cobalamin and the excited state of the
fluorophore.3,4
Herein, we report the design, synthesis, and characteriza-
tion of a new generation of fluorescent cobalamin analogues
that incorporate a rigid linker to thrust the fluorophore away
from the corrin ring and enforce an orthogonal relationship
between the electronic dipoles of the fluorophore and
cobalamin.
The overarching goal of this work is to develop highly
fluorescent molecular imaging agents that are targeted to
rapidly dividing cancer cells by the cobalamin moiety and
retain the ability to interact with the cobalamin transport
protein, transcobalamin. Highly fluorescent cobalamin ana-
logues that meet these criteria may be useful as intraoperative
imaging agents to help surgeons delineate cancerous tissue
from healthy tissue during surgical resection.
While some of the previous CobalaFluor fluorescent
cobalamin analogues that were constructed by attaching the
fluorophore directly to the Co(III) atom at the ꢀ-axial ligand
(1) (a) Hogenkamp, H. P. C.; Collins, D. A.; Grissom, C. B.; West,
F. G. Chemistry and Biochemistry of B12; John Wiley and Sons: New York,
1999; pp 385. (b) Kunze, S.; Zobi, F.; Kurz, P.; Spingler, B.; Alberto, R.
Angew. Chem., Int. Ed. 2004, 43, 5025. (c) Flodh, H. Acta Radiol. Suppl.
1968, 284, 1. (d) Hall, C. A. J. Lab. Clin. Med. 1984, 103, 70. (e) Collins,
D. A.; Hogenkamp, H. P. C.; O’Connor, M. K.; Naylor, S.; Benson, L. M.;
Hardman, R.; Thorson, L. M. Mayo Clin. Proc. 2000, 75, 568.
(2) Cannon, M. J. Ph. D. Thesis. University of Utah, Salt Lake City,
UT, 2001.
(3) Horton, R. A. Ph. D. Thesis. University of Utah, Salt Lake City,
UT, 2004.
(4) Smeltzer, C. C.; Cannon, M. J.; Pinson, P. R.; Munger Jr, J. D.;
West, F. G.; Grissom, C. B. Org. Lett. 2001, 3, 799.
10.1021/ol900401z CCC: $40.75
Published on Web 05/14/2009
 2009 American Chemical Society

position had respectable fluorescent quantum yields (φf) in
the range of 0.01-0.1,4 these compounds suffer from
photochemical instability because of the low Co-C bond
dissociation energy of ∼37 kcal/mol that falls well within
the range of visible photons.5 To solve this problem, we
chose to activate the 5′-OH of the cobalamin R-ribofuranotide
for conjugation to the fluorophore.
Scheme 1. Synthesis of the
Cobalamin-trans-1,4-diaminocyclohexane Complex
Chemical modification of cobalamin must not disrupt the
binding interactions between the synthetic analogue and the
proteins required for endocytosis and intracellular traffick-
ing.6 Modification of the ribose moiety of cobalamin is
reported to be well tolerated by the plasma cobalamin binding
protein, transcobalamin (TC) and the enteric cobalamin
binding protein, intrinsic factor (IF).7
On the basis of preliminary experiments with flexible
linkers of increasing length to separate the ribose-5′-hydroxyl
group and the fluorophore, an increase in φf was observed
as the distance separating the corrin ring and the fluorophore
was increased, but the maximum increase in φf achieved was
only 5-fold. This is likely due to the flexible linker allowing
an intramolecular folding and π-stacking interaction between
the corrin ring and the fluorophore.
To reduce the intramolecular dynamical movement that
allows for fluorescence quenching, we incorporated a rigid
linker shown in red (Figure 1) in the design of the synthetic
analogues.
The synthesis of the common precursor 2 is shown in
Scheme 1. Derivatization of the ribose-5′-OH of cyanoco-
balamin with 1,1′-carbonyl-di-(1,2,4-triazole) (CDT) gives
intermediate 2 in 75% yield. Nucleophilic attack by trans-
1,4-diaminocyclohexane yields 3 in 60% yield. The use of
CDT as an acylating agent for activation of the ribose-5′-
OH is effective even in multigram preparations (Scheme 1).
We also investigated the use of a less expensive acylating
agent, 1,1′-carbonyldiimidazole (CDI); however, the results
were less satisfactory with reaction yields of less than 5%.
5(6)-Carboxyfluorescein was synthesized using a modified
literature procedure.8 A mixture of resorcinol and 1,2,4-
benzenetricarboxylic acid was heated to reflux in concen-
trated methanesulfonic acid. The neat reaction conditions
dramatically increase the rate of reaction and give 5(6)-
carboxyfluorescein in 99% yield.
Figure 1. trans-1,4-Diaminocyclohexane was used as a rigid
molecular scaffold to link cobalamin and the pendant fluorophore.
The linker orients the fluorophore away from the corrin ring of
cobalamin to minimize the intramolecular dynamical interaction,
thereby increasing the overall fluorescence quantum yield of the
fluorophore.
Reaction of 5(6)-carboxyfluorescein with N-hydroxysuc-
cinimide and N-(3-dimethylaminopropyl)-N′-ethylcarbodi-
imide hydrochloride (EDAC) gave compound 4.9 The
activated ester of fluorescein was used in subsequent reac-
We have synthesized cobalamin-fluorescein 5 (Scheme 2)
and cobalamin-Rhodamine 6G 7 (Scheme 3) conjugates as
exemplars of rigidly tethered cobalamin-fluorophore conju-
gates.
(7) (a) Pathare, P. M.; Wilbur, D. S.; Heusser, S.; Quadros, E. V.;
McLoughlin, P.; Morgan, A. C. Bioconjugate Chem. 1996, 7, 217. (b)
Wuerges, J.; Garau, G.; Geremia, S.; Fedosov, S. N.; Petersen, T. E.;
Randaccio, L. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 4386.
(5) Martin, B. D.; Finke, R. G. J. Am. Chem. Soc. 1992, 114, 585.
(6) (a) Fedosov, S.; Laursen, N. B.; Moestrup, S. K.; Nexo, E.; Petersen,
T. E.; Jensen, E. O.; Berglund, L. Biochemistry 2004, 42, 15095. (b)
Fedosov, S.; Dedosova, N. U.; Nexo, E.; Petersen, T. E. J. Biol. Chem.
2000, 275, 11791.
(8) This procedure yields an isomeric mixture of the 5- and 6-carboxy-
fluoresceins. Use of this mixture of isomers is common in the construction
of fluorescein-based imaging agents. (a) Orndroff, W. R.; Hemmerm, A.
J. Am. Chem. Soc. 1927, 49, 1272. (b) Sun, W.-C.; Gee, K. R.; Klaubert,
D. H.; Haughland, R. P. J. Org. Chem. 1997, 62, 6469.
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Scheme 2
.
Synthesis of 5
Table 1. Fluorescence Quantum Yield Values
compd
λmax ex, nm
λmax em, nm
φf
5
7
481
530
528
554
0.10 ( 0.01
0.20 ( 0.01
tions without further purification. The final coupling of
cobalamin and fluorescein gives 5 with an overall yield of
22% after prep-HPLC purification. The reactions involving
fluorescein were carried out in dim red light to reduce the
adventitious photobleaching of the fluorophore.
Derivatization of Rhodamine 6G at the ester moiety by
substitution of an amide bond for the ester was carried out
by refluxing Rhodamine 6G with Al(CH3)3 with trans-1,4-
diaminocyclohexane (Scheme 3).10 Compound 6 was purified
by prep-HPLC. The 1° NH2 group of 6 is a good nucleophile
for conjugation to biomolecules. Final coupling of 6 with 2
yields 7.
The optical properties of 5 and 7 are reported in Table 1.
Compound 5 exhibits an emission maximum at 528 nm (λex
Scheme 3. Synthesis of 7
Figure 2. Absorption and fluorescence spectra of 5 and 7. The 358
nm peak is characteristic of the corrin ring. The excitation
wavelengths for 5 and 7 were 481 and 530 nm, respectively.
Org. Lett., Vol. 11, No. 12, 2009
2501

) 480 nm) and φf ) 0.10, whereas compound 7 exhibits an
emission maximum at 554 nm (λex ) 530 nm) and φf )
0.20. The quantum yields for both compounds were obtained
by measuring the integrated fluorescence emission, optical
density, and refractive index, and referencing these values
to the reported values for fluorescein (φf ) 0.92) or
Rhodamine 6G (φf ) 0.95) (Table 1; additional details are
provided in Supporting Information). In contrast to the
nascent generation of fluorescent cobalamin analogues,
neither 5 nor 7 undergo adventitious photodegradation at the
Co-C bond because the fluorophore is now attached to the
ribose ring of cobalamin.4
We are investigating the binding kinetics and thermody-
namics for the interaction of 5 and 7 with the cobalamin
transport proteins TC and IF to determine their applicability
as tumor imaging agents. The typical rates of dissociation
for cobalamin from TC and IF are on the order of 10-7 s-1.
Experiments are also being carried out to assess the ability
of each analogue to enter human cancer cells and to assess
the fidelity of cellular accumulation relative to the naturally
occurring cobalamins.
In summary, we have demonstrated the rational design and
synthesis of two members of a new class of photostable
fluorescent cobalamin analogues with fluorescein and
Rhodamine 6G as the reporter groups. The use of a
cyclohexane ring as a rigid linker orients the fluorophore
away from the corrin ring of cobalamin to minimize through
space intramolecular interactions, thereby increasing the
overall fluorescence quantum yield relative to the analogous
fluorophore tethered to cobalamin with a flexible linker. The
cobalamin analogues reported herein may be suitable for use
as intraoperative tumor markers to guide surgeons during
tumor resection, ultimately improving the overall outcome
of cancer surgery.
Acknowledgment. We gratefully acknowledge financial
support from NIH grant CA121743, the University of Utah
Research Foundation, and Vestan, Inc.
Supporting Information Available: Full experimental
procedures, and characterization data for compounds 1-7
and the method for determining the fluorescence quantum
yields. This material is available free of charge via the
Internet at http://pubs.acs.org.
(9) Adamczyk, M.; Fishpaugh, J. R.; Heuser, K. J. Bioconjugate Chem.
1997, 8, 253.
(10) (a) Nguyen, T.; Francis, M. B. Org. Lett. 2003, 5, 3245. (b)
Adamczyk, M.; Grote, J. J. Bioorg. Med. Chem. Lett. 2000, 10, 1539. (c)
Adamczyk, M.; Grote, J. Synth. Commun. 2001, 31, 2681.
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