Manipulating reduction potentials in an artificial safranin cofactor

Article abstract of DOI:10.1016/j.tetlet.2011.12.028

Safranines hold great promise as artificial flavin-like electron transfer cofactors with tunable properties. We report the design and chemical synthesis of the p-methoxy derivative of safranine O using a new synthetic route based on the Ulmann condensation. Spectroelectrochemical comparison of the purified parent safranine and this derivative demonstrates that the modification increases its two-electron reduction potential by 125 mV, or 5.75 kcal/mol. This modification also causes redshifts in the absorbance and fluorescence spectra of the cofactor, suggesting that it may find future utility in arrayed sensor applications.

Full text of DOI:10.1016/j.tetlet.2011.12.028

Tetrahedron Letters 53 (2012) 1201–1203
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Manipulating reduction potentials in an artificial safranin cofactor
Gheevarghese Raju b, Joseph Capo c, Bruce R. Lichtenstein d, Jose F. Cerda c, Ronald L. Koder a,
⇑
a Department of Physics, The City College of New York, New York, NY 10031, USA
b Department of Chemistry, The City College of New York, New York, NY 10031, USA
c Department of Chemistry, Saint Joseph’s University, Philadelphia, PA 19131, USA
d Department of Biochemistry and Biophysics, The University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Safranines hold great promise as artificial flavin-like electron transfer cofactors with tunable properties.
We report the design and chemical synthesis of the p-methoxy derivative of safranine O using a new syn-
thetic route based on the Ulmann condensation. Spectroelectrochemical comparison of the purified par-
ent safranine and this derivative demonstrates that the modification increases its two-electron reduction
potential by 125 mV, or 5.75 kcal/mol. This modification also causes redshifts in the absorbance and fluo-
rescence spectra of the cofactor, suggesting that it may find future utility in arrayed sensor applications.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 11 October 2011
Revised 7 December 2011
Accepted 8 December 2011
Available online 14 December 2011
Keywords:
Safranine
Enzyme design
Reduction potential
Ulmann condensation
The combination of natural and designed proteins with artificial
cofactors is a rapidly expanding focus of modern enzyme design
efforts.1–4 The anticipated benefit of this combination is that a
cofactor energetically and structurally optimized to perform the
catalytic task at hand will aid the enzyme design or re-engineering
effort by removing the necessity for the enzyme to ‘tune’ the cofac-
tor reactivity for the desired task.5–7 The properties to be optimized
may include reduction potentials, substrate or ligand affinity,
hydrophobicity, chemical reactivity, or photophysical properties.8
Progress to date has primarily been in the area of metallopro-
teins, especially the replacement of heme residues with synthetic
porphyrins.9–12 There are few reports of artificial proteins which
incorporate artificial organic cofactors.13,14 Despite the fact that
flavoenzymes form more than a tenth of known cofactor-contain-
ing enzymes,15 there have been no reports thus far of artificial
flavin enzymes which utilize artificial flavin-like cofactors, and
likewise no reports of the synthesis of flavin-like cofactors created
for use in such a manner. We have reported the synthesis and char-
acterization of riboflavin derivatives with differing hydrophobic-
ity,16,17 but these lack desirable properties such as changes in
reduction potential.
to create a series of flavin-like cofactors in which small changes
in the cofactor structure engender large changes in its reduction
potentials. Safranines are ideal candidates for this as the phenazine
moiety is similar in size and shape to that of isoalloxazine (see
Fig. 1). The reported two-electron reduction midpoint potential
Safranine
Riboflavin
OH
OH
HO
OH
N
N
N
H2N
N
N
O
NH2
NH
O
-2e- +2e-
-2e- +2e-
OH
OH
Reduction midpoint potentials, both one- and two-electron,
play a large role in determining the chemical reactivity of flavin
cofactors,18,19,21 and flavoproteins observed in nature have been
shown to modulate the reduction potentials of their bound cofac-
tors by more than half a volt.15 For this reason, we have set out
HO
OH
H
N
N
N
H2N
NH
N
O
NH
N
H
O
⇑
Corresponding author. Tel.: +1 212 650 5583.
E-mail address: koder@sci.ccny.cuny.edu (R.L. Koder).
Figure 1. Oxidized and two electron-reduced forms of safranine O and riboflavin.
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.12.028

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G. Raju et al. / Tetrahedron Letters 53 (2012) 1201–1203
of safranine O, ꢀ290 mV versus SHE,22 is 100 mV, or 2.3 kcal/mol
more negative than that of riboflavin.23 Furthermore the N(10)
phenyl substituent of safranine O is conjugated to the phenazine,
raising the possibility that the reduction potential of the molecule
may be simply altered by the modifications of this ring.
The safranine mauveine was the first synthetic dye, created by
Perkin in 1856.1 It was synthesized by the oxidation of crude aniline,
itself derived from the nitration and reduction of a benzene–toluene
mixture, and purified in a 5% yield from a mixture of products con-
taining a variety of oligoanilines.24 Soon after dozens of safranine
derivatives were created using similarly uncontrolled oxidative con-
densations of various arylamines and diamines.2,25 Modern indus-
trial safranine dyes are still synthesized in this manner.24
In this Letter we describe our initial effort to create a general
synthetic route toward safranine O analogues modified at the para
position of the N(10) phenyl ring using a stepwise synthesis in
which the phenyl substituent is incorporated as aniline. Given
the large number of commercially available aniline derivatives,
we hoped such a synthetic route may lead to a similarly large scope
of safranine products. Furthermore, given our recent demonstra-
tion that the chemical shift tensor of the isoalloxazine N(5) nitro-
gen in flavin compounds is very informative as to the chemical
reactivity imparted upon flavins by their environments,1,26 we de-
sired to create a route which enables the ready and inexpensive
incorporation of isotopically labeled nitrogen at the equivalent
N(5) position in the phenazine ring of these analogues.
phthalate protecting groups in a single step reaction7 forming 5 in
an 86% isolated yield. Compound 5 is cyclized with 15N-sodium ni-
trite in a 17/3 mixture of acetic acid/tetrahydrofuran at 0 °C forming
6ina55%yield. Reductivedeprotectionof6resultsinthepurplefinal
product 7 in a 40% isolated yield. The low yield in the final two steps
is compensated for in part by the fact that the isotopic label is intro-
duced in the second to last step. This greatly reduces label loss in
comparison to other possible approaches in which either the label
is introduced at the beginning, which would entail a geometrically
growing loss of expensive labeled material, or employing the extre-
mely low yield uncontrolled oxidative condensation methods cur-
rently in use.
Similar syntheses with other aniline starting materials demon-
strate that this reaction pathway is limited in scope: anilines with
methyl, cyano or hydrogen groups at the position para to the amine
had reduced yields of 52%, 24% and 38%, respectively in the con-
densation step, and all had negligible yields (<5%) of cyclization.
The latter is likely due to the fact that these are not as activating
as the methoxy for electrophilic substitution at the position meta
to the amine.
Compound 7 was compared to a commercial sample of safra-
nine O, the latter of which was purified using both normal- and
reversed-phase chromatography in succession.9 Safranines were
dissolved in 0.2 M sodium phosphate, 0.2 M NaCl, pH 7.0. Solutions
were degassed using several cycles of applied vacuum followed by
flushing with argon. Oxidized spectra were collected and then the
safranines were reduced using sodium hydrosulfite. The oxidized
and two-electron reduced spectra of 7 are both significantly red-
shifted in comparison to the base compound safranine O, and the
fluorescence emission maximum is likewise shifted by 44 nm
(see Fig. 2 parts A and B). Neither compound is fluorescent in the
reduced state. These spectral differences are advantageous, as this
means they can be separately monitored in mixed solutions of the
two species, or in mixed complexes of the two cofactors with one
or more proteins, as has been proposed for biochemical sensor
arrays.21
Ourapproachis outlinedin Scheme1 (syntheticdetails, including
product characterization, are included in the Supplementary data).
5-Iodo-2-methylaniline 1 is quantitatively protected with tert-buty-
ldicarbonate2 and then condensed with p-anisidine 3 using copper
iodide, 2,20-bipyridine and three equivalents of potassium tert-
butoxide in dry toluene forming triarylamine 4.25 Initial attempts
using 1,10-phenanthroline as a copper ligand and potassium
hydroxide as a base resulted in low yields, so the rigid phenanthro-
line ligand was replaced with the more flexible bipyridine ligand5
and the stronger, more hindered, tert-butoxide base was used to
replace potassium hydroxide.3
We performed spectroelectrochemical analysis of the two pro-
teins using an apparatus described previously.13 Briefly, the cell
is a 10 mm quartz cuvette that contains a pair of gold slides that
serve as the working electrode, a platinum wire auxiliary electrode,
and an Ag–AgCl reference microelectrode (Microelectrodes Inc.).
The next step is nitrosative cyclization.27 Direct cyclization of 4
proved unsuccessful as the acidic conditions required deprotected
theamines, formingazocompoundsfrom thefree aminesand theni-
trite. We thus replaced the Boc protecting groups with acid- stable
O
O
+
O
N
4
BocHN
2
NHBoc
Di-tert butyl dicarbonate, THF
Toluene, CuI, t-BuOK
2,2'-bipyridine
I
NH
O
NH2
I
NH2
2
1
3
Acetic acid, Phthalic
anhydride
O
N
O
N
O
O
O
N
PhtN
N
NPht
0
o C - RT, Na15NO2,
Acetic acid/THF
H2N
NH2
RT, NaBH4, Isopropanol
O
15N
O
15N
7
N
O
5
6
Scheme 1.

G. Raju et al. / Tetrahedron Letters 53 (2012) 1201–1203
1203
1
0.4
0.2
0.2
A.
B.
C.
522
625
581
0.8
0.6
0.4
0.2
0.15
538
0.1
448
0.05
500
600
700
400
500
600
-600
-400
-200
Wavelength, nm
Wavelength, nm
Figure 2. Comparative characterization of safranine O and p-methoxysafranine 7. Absorbance spectra of oxidized (black lines), reduced (grey lines) and fluorescence emission
(dotted lines) spectra of oxidized safranine O (panel A) and p-methoxysafranine 7 (panel B). Emission spectra were produced by exciting at 522 and 538 nm, respectively. (C)
Potentiometric comparison of safranine O (filled circles) and p-methoxysafranine 7 (open circles) in 0.2 M sodium phosphate, 0.2 M NaCl, pH 7.0 buffer. Lines are fits with the
Nernst equation with n = 2.0 electrons. Potentials are referenced to a Ag/AgCl2 electrode.
The gold working electrode was coated with 1-mercaptohexanol
by soaking the slides in a 1-proponal solution containing 1-merca-
ptohexanol 1% (v/v) for 20 h. The applied potential is set by using a
PWR-3 Power Module potentiostat (Bioanalytical Systems Inc.).
Spectra were collected in a PerkinElmer Lambda 35 UV/vis spec-
trometer. Safranines were dissolved in water containing 0.2 M
sodium phosphate, 0.2 M NaCl, pH 7.0. The applied potentials are
referenced against Ag/AgCl2 which is +210 mV (NHE). Solutions
were equilibrated at each potential for at least ten minutes before
spectra were collected. Absorbance values at the oxidized maxi-
mum were used to calculate the fractional oxidation at each poten-
tial and these data were fit with the Nernst equation.
ture support from P41 GM-66354 to the New York Structural Biol-
ogy Center and the NIH National Center for Research Resources to
CCNY (NIH 5G12 RR03060). B.R.L. gratefully acknowledges support
by NIH Grant GM41048.
Supplementary data
Supplementary data (synthetic details and product character-
ization) associated with this article can be found, in the online ver-
sion, at doi:10.1016/j.tetlet.2011.12.028.
References and notes
Figure 2C depicts the data obtained for the two compounds in pH
7.0 buffer solution. p-Methoxysafranine 7 has a reduction potential
125 mV higher than that of safranine O. Each displays two-state
behavior, directly transforming from the oxidized state to the two-
electron reduced state during the titration. No semiquinone spectral
intermediates are observed. The two-electron nature of these reduc-
tions are further evinced both by the presence of isosbestic wave-
lengths in each titration and by the fact that the fits to the titration
data each report a 2.0 0.1 electron reduction. Thus a protein con-
taining 7 as a cofactor will have 125 mV, or 5.75 kcal/mol, more driv-
ing force for two-electron oxidative reactions such as the oxidation
of nicotinamide cofactors such as NADH and NADPH.
In conclusion, we have developed a synthetic route to a flavin-
like cofactor, a safranine O analogue which incorporates a single
substitution at a point far removed from the phenazine headgroup
at which reduction occurs. This route enables the inexpensive incor-
poration of an isotopic reporter atom at the reduction site. This ana-
logue displays large differences in both reduction potentials and
photophysical properties from its parent compound. We are cur-
rently exploring in more detail the pH-dependent oxidation–reduc-
tion potentials of both one- and two-electron reduction of both
molecules in solution, and investigating their properties when com-
plexed with artificial proteins designed to bind and activate them.
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Acknowledgments
R.L.K. gratefully acknowledges support by the following Grants:
S06GM008168 from the National Institutes of Health, infrastruc-