Model systems for flavoenzyme activity: An investigation of the role functionality attached to the C(7) position of the flavin unit has on redox and molecular recognition properties

Article abstract of DOI:10.1039/b900269n

We describe the role functionality attached to the C(7) position of a family of flavin derivatives has in tuning their redox and recognition properties and the subsequent exploitation of two of these derivatives as a three-component electrochemically cont

Full text of DOI:10.1039/b900269n

View Article Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/chemcomm | ChemComm
Model systems for flavoenzyme activity: an investigation of the role
functionality attached to the C(7) position of the flavin unit has on
redox and molecular recognition propertieswz
Stuart T. Caldwell,a Louis J. Farrugia,a Shanika Gunatilaka Hewage,a Nadiya Kryvokhyzha,a
Vincent M. Rotello*b and Graeme Cooke*a
Received (in Cambridge, UK) 6th January 2009, Accepted 26th January 2009
First published as an Advance Article on the web 11th February 2009
DOI: 10.1039/b900269n
We describe the role functionality attached to the C(7) position
of a family of flavin derivatives has in tuning their redox and
recognition properties and the subsequent exploitation of two
of these derivatives as a three-component electrochemically
controllable molecular switch.
positions have been used to quantify the role structural
modification of this type has in controlling their physical pro-
perties in aqueous and non-aqueous media.6 However, in
these investigations the role functionality attached to the non-
biomimetic C(7) position has largely been overlooked. In this
communication, we report the role functionality attached to the
C(7) position of the flavin unit has in modulating both the redox
and recognition properties. We then use this modulation to
create a three-component electrochemically controllable mole-
cular switch from flavin derivatives 1 and 6 and complementary
diamidopyridine (DAP) derivative 7.
Flavoenzymes are a class of proteins that catalyse a range of
redox transformations and mediate one- and two-electron
transfer processes in biological systems.1 Supramolecular inter-
actions between the flavin cofactor and the apoenzyme modulate
the redox potential of the flavin moiety by over 500 mV, tuning
the redox properties of the flavin units to match their biological
function.2 X-Ray crystallography of flavoenzymes has provided
important information regarding the nature and relative posi-
tion of functionality in the flavoenzyme active site; however,
little information is provided relating to the role individual
interactions play in modulating flavin properties.3 In other
studies, the apoenzymes of flavoenzymes have been reconsti-
tuted with artificial flavins to probe the role non-covalent
interactions around the flavin binding site play in modulating
the reactivity and properties of the cofactor.4
A family of synthetic flavin derivatives 1–6 have been
synthesised with a range of electron donating and withdrawing
functionality attached to the C(7) position (see ESIw). Slow
evaporation of solvent from a solution of 1 dissolved in
toluene resulted in the formation of X-ray quality crystals.y
The X-ray structure of compound 1 is shown in Fig. 1, and
unequivocally confirms the presence of a –NMe2 group
attached to the C(7) position of the flavin. In the solid state,
the flavin units self-assemble via two point hydrogen bonding
between the imide moieties of adjacent flavin units, resulting in
a dimeric structure.
We have investigated the complexation of flavin derivatives
1–6 with complementary derivative 7 using NMR titrations. It
has previously been shown that DAP derivative 7 serves as an
effective host for flavin derivatives and mimics the three
point hydrogen bonding interactions with the complementary
residues of the apoenzyme.7 The titrations resulted in a
smooth downfield shift in the hydrogen attached to N(3) of
the flavin upon addition of aliquots of 7. The titration data
were successfully fit to a 1 : 1 binding isotherm using non-
linear curve fitting methods to afford the association constants
(Ka) provided in Table 1. It is clear from these data that the
electronic characteristics of the functionality attached to the
Small molecule model system studies of flavoenzymes provide
insight into biological processes while simultaneously providing
access to new functional molecular systems. Synthetic model
systems have been utilised to explore the role of non-covalent
interactions in modulating redox properties of flavins.5 Further-
more, structurally modified flavin units in their C(7) and C(8)
a Glasgow Centre for Physical Organic Chemistry, WestCHEM,
Department of Chemistry, Joseph Black Building, University of
Glasgow, Glasgow, UK G12 8QQ. E-mail: graemec@chem.gla.ac.uk
b Department of Chemistry, University of Massachusetts at Amherst,
Amherst, MA 01002, USA
z Electronic supplementary information (ESI) available: Synthesis of
derivatives 1–6. CCDC 715445. For ESI and crystallographic data in
CIF or other electronic format see DOI: 10.1039/b900269n
Fig. 1 X-Ray crystal structure of flavin 1. Hydrogen bonding inter-
actions are highlighted in light blue. The molecular structure was
visualised using Mercury 1.4.2.
ꢀc
This journal is The Royal Society of Chemistry 2009
1350 | Chem. Commun., 2009, 1350–1352

View Article Online
Table 1 Calculated and LFER predicted binding and electrochemical properties of flavins 1–6
DGbinding exp.b/
DGbinding LFERc/
DGredox exp.d/
DGredox, LFERc/
Ka/Mꢁ1a
E, SWV/V
kcal molꢁ1
kcal molꢁ1
kcal molꢁ1
kcal molꢁ1
1
2
3
4
5
6
446 ꢂ 22
455 ꢂ 23
460 ꢂ 23
482 ꢂ 24
434 ꢂ 22
436 ꢂ 22
ꢁ1.40
ꢁ1.36
ꢁ1.33
ꢁ1.21
ꢁ1.15
ꢁ1.10
ꢁ3.61
ꢁ3.61
ꢁ3.63
ꢁ3.66
ꢁ3.60
ꢁ3.60
ꢁ2.53
ꢁ3.15
ꢁ3.63
ꢁ6.60
ꢁ7.50
ꢁ9.60
32.29
31.36
30.67
27.90
26.52
25.37
31.78
31.15
30.67
27.70
26.81
25.70
a
b
c
Determined in CDCl3 at 298 K. Calculated using: DG = ꢁRTlnKa. Calculated using: DG(X,Y) = rmsm(X) + rpsp(Y) + DG(H,H), where
rm = ꢁ6.9 kcal molꢁ1 sꢁ1 and rp = ꢁ4.6 kcal molꢁ1 sꢁ1
.
Calculated using: DG = ꢁnFE.
d
Fig. 3 Graphical representation of the good correlation between
experimentally determined and LFER predicted DG values for redox
properties of 1–6. Inset shows the near-zero slope of the experi-
mentally determined and LFER predicted DG values for binding of
1–6 and 7.
ꢁ
are subsequently reoxidised at a less negative potential than 1rad
and 2radꢁ. However, it must be stressed that the intensity of the
second reoxidation wave is suppressed compared to the electro-
chemical data obtained for flavin derivatives featuring electron
donating functionality attached to C(8).6a This e–c–e process is
largely suppressed with derivatives 5 and 6, suggesting that the
electron withdrawing nature of the –CN and –NO2 groups
significantly lowers the pKa of their radical anions to below that
of the imide of the oxidised forms of these derivatives.6a
Fig. 2 CVs of compound 1 (blue line) and compound 6 (red line)
recorded in CH2Cl2 (B1 mmol). Scan rate = 100 mV sꢁ1
.
C(7) position plays little role in modulating the electron
density of the carbonyl oxygen atoms O(2) and O(4) of the
flavin moiety, and thus the binding efficiency with com-
plementary DAP derivative 7.
We next investigated the electrochemical properties of deri-
vatives 1–6 dissolved in CH2Cl2 and upon addition of excess 7
using cyclic (CV) and square wave voltammetries (SWV)
(Fig. 2 and Table 1). In contrast to the recognition properties,
functionality attached to the C(7) position plays an important
role in controlling the redox properties of the flavin unit.
Indeed, upon exchanging the electron donating –NMe2 group
of compound 1 with an electron withdrawing –NO2 group of
compound 6, over a 300 mV change in the reduction potential
to a less negative value was observed. In each case the addition
of DAP derivative 7 resulted in a near-identical ꢁ100 (ꢂ10)
mV shift in the reduction of the flavin unit (compared to the
free flavin derivative), indicating that significant stabilisation
of the flavin radical anion state occurs upon binding to DAP.7
Interestingly, for derivative 1, and to a lesser extent derivative
2, a single reduction wave and two distinct reoxidation waves
We have used linear free energy (LFER) relationships to
further corroborate our experimental work and gain further
insight into the role functionality attached to the C(7) position
of the heterocycle has upon the redox and recognition pro-
perties of the flavin.6a The experimental and LFER predicted
values for host–guest binding and redox properties were deter-
mined using our previously described methodology and are
summarised in Table 1 and represented graphically in Fig. 3,
6a
also see ESI.z Good correlation exists between the experi-
mental and LFER predicted DG values for the electrochemical
properties of the flavin derivatives, further supporting the
importance electronic effects of functionality attached to C(7)
have in modulating redox properties. In contrast, a slope of
essentially zero was observed for the plot of experimental versus
LFER predicted DG values for host–guest binding, indicating
that the electronic characteristics of functionality attached at
the C(7) position do not significantly influence recognition of
DAP. However, the CV studies suggest that the electronic
effects may weakly influence the acidity of the N(5) moiety of
the radical anions derived from derivatives 1 and 2. We
attribute the differing role functionality attached to the C(7)
position has in modulating recognition and redox properties as
follows. Functional groups attached to the C(7) position of
flavin are inductively connected to the O(2) and O(4) carbonyls
were observed for these reductions.8 The first reductꢁion wave can
ꢁ
be attributed to the reversible formation of 1rad and 2rad
,
whereas the second reoxidation waves are due to an electro-
chemical–cꢁhemical–electrochemical (e–c–e) process where some
of the
species formed at the electrode surface rapidly
rad
deprotonates the flavin in the bulk solution. The protonated
flavin radicals (1radH and 2radH) formed in this process undergo a
further one-electron reduction at the working electrode surface to
form the fully reduced flavin anions (1redHꢁ and 2redHꢁ), which
ꢀc
This journal is The Royal Society of Chemistry 2009
Chem. Commun., 2009, 1350–1352 | 1351

View Article Online
Thus, in this system electrochemistry not only provides a direct
readout process for the DAP–flavin binding event through the
change in voltage of the flavin reduction process but also provides
a means of modulating the host–guest binding event.
In conclusion, we have shown that the electronic effects of
functionality attached to the C(7) position of flavin derivatives
play an important role in modulating the redox properties of the
unit. In contrast, they do not significantly modulate the hydro-
gen bonding affinity for receptor 7. The similar binding efficien-
cies of the oxidised flavin derivatives 1 and 6 with compound 7
provide direct access to a prototype three-component redox
controlled switch whereby the differing onset of the radical
anion states of derivatives 1 and 6 can be sequentially read by
the application of increasing negative potential. These results
pave the way for the formation of new flavin-based molecular
devices addressed by electrochemically controlled host–guest
complexation. The results of our investigations in this area will
be reported in due course.
Fig. 4 Schematic representation of the three-component electro-
chemical switch derived from 1, 6 and 7.
GC gratefully acknowledges the EPSRC for supporting this
work and The Royal Society of Edinburgh for a Support Research
Fellowship. VR acknowledges the NSF (US) CHE-0808945.
Notes and references
w This article is dedicated to Prof. Seiji Shinkai on the occasion of his
65th birthday.
y Crystal data for 1: 4(C16H19N5O2)ꢃC7H8: Mr = 1345.58, Z = 1,
ꢀ
triclinic, P1, a = 10.3010(4), b = 11.8400(5), c = 16.1447(7) A,
a = 106.983(2), b = 108.228(2), g = 106.322(2)1, V = 1631.48(12) A3,
T = 100(2) K, Nmeas = 39465, Nind = 5795, Rint = 0.041, Robs
Fig. 5 Cyclic voltammogram of an equimolar mixture of 1 and 6
(B1 mmol) before (—) and after (ꢃ ꢃ ꢃ) the addition of DAP 7
(B10 mmol). Inset shows CV for cycles between ꢁ0.2 and ꢁ1.6 V.
(all data) = 0.041 (0.068), wR2 (all data) = 0.096 (0.104).
obs
Scan rate 100 mV sꢁ1
.
1 (a) K. Stevenson and V. Massey, in Flavins and Flavoproteins. 1996,
ed. C. Willams, University of Calgary, Calgary, 1997;
(b) Chemistry and Biochemistry of Flavoenzymes, ed. F. Muller,
CRC Press, Boca Raton, 1991, vol. 1–3.
2 For examples see: V. Massey and P. Hemmerich, in The Enzymes,
ed. P. D. Boyer, Academic Press, New York, 1976, vol. 12,
pp. 191–240R. Swenson, G. Krey and M. Eren, in Flavins and
Flavoproteins, ed. D. Edmondson, D. McKormick de Gruyter,
Berlin, 1987, pp. 98–107.
that dictate binding affinity via the ‘‘meta’’ position of the
phenyl unit. In contrast, the C(7) position is in direct resonance
communication with the central redox core of the flavin unit,
thereby significantly influencing its redox properties.
In recent years, an enormous amount of effort has been
directed towards the development of molecular switches.9
Flavin–DAP systems are attractive moieties for developing
switches as it is well established that the specific DAP–flavin
recognition process affords a write state that can be read
electrochemically due to the shift in flavin reduction potential
that accompanies the hydrogen bonding mediated stabilisation
of the flavin radꢁ state.7 Systems 1–7 offer the exciting ability to
create a novel three-component write–read molecular switch,10
whereby the binding between 7 and oxidised flavins can be
sequentially electrochemically read by adjusting the redox
window of the reduction process (Fig. 4). To test this hypo-
thesis we have investigated the formation of a prototype
three-component switch from derivatives 1, 6 and 7. The CV
of an equimolar solution of 1 and 6 is shown in Fig. 5
and clearly shows that the disparate onset of their radicꢁal
3 S. Ghisla and V. Massey, Biochem. J., 1986, 239, 1.
4 For examples see: (a) M. L. Ludwig, L. M. Schopfer,
A. L. Metzger, K. A. Pattridge and V. Massey, Biochemistry,
1990, 29, 10364; (b) Y. V. S. N. Murthy, Y. Meah and
V. Massey, J. Am. Chem. Soc., 1999, 121, 5344.
5 For recent reviews see: (a) A. Niemz and V. M. Rotello, Acc. Chem.
Res., 1999, 32, 44; (b) V. M. Rotello, Curr. Opin. Chem. Biol., 1999,
3, 747; (c) Y. Yano, Rev. Heteroat. Chem., 2000, 22, 151.
6 For recent examples see: (a) Y.-M. Legrand, M. Gray, G. Cooke and
V. M. Rotello, J. Am. Chem. Soc., 2003, 125, 15789; (b) J. J. Hasford
and C. J. Rizzo, J. Am. Chem. Soc., 1998, 120, 2251;
(c) D. E. Edmondson and S. Ghisla, in Flavins and Flavoproteins,
ed. S. Ghisla, P. Kroneck P. Macheroux and H. Sund, Rudolf
Weber, Agency For Scientific Publications, Berlin, 1999, pp. 71–76.
7 E. Breinlinger, A. Niemz and V. M. Rotello, J. Am. Chem. Soc.,
1995, 117, 5379.
8 A. Niemz and V. M. Rotello, J. Am. Chem. Soc., 1997, 119, 6833.
9 Molecular Switches, ed. B. L. Feringa, Wiley-VCH, Weinheim, 2001.
10 For a previous example of a three-component flavin-based mole-
cular switch see: R. Deans, A. Niemz, E. C. Breinlinger and
V. M. Rotello, J. Am. Chem. Soc., 1997, 119, 10863.
anion states allows the sequential formation of 1radꢁ and 6rad
.
Upon the addition of DAP and cycling the applied potential
between ꢁ0.3 and ꢁ2.4 V, both of the redox waves are shifted
to a more positive potential (100 mV), which corresponds to a
11 The redox-based enhancement in recognition can be calculated
using a thermodynamic cycle, which can be expressed mathemati-
1/2(bound)ꢁE1/2(unbound))
cally using: Ka(red)/Ka(ox)
=
e(nF/RT)(E
.
new Ka of B22 000 Mꢁ1 for 7.1rad and 7.6rad
.
By adjusting
ꢁ
ꢁ 11
Ka(red) and Ka(ox) are the association constants in the reduced
and oxidised forms, and E1/2(bound) and E1/2(unbound) are the
half-wave redox potentials in the receptor bound and unbound
states.
the applied voltage to cycle between ꢁ0.2 and ꢁ1.6 V it is possible
to only read the 7.6 binding event in the presence of derivative 1.
ꢀc
This journal is The Royal Society of Chemistry 2009
1352 | Chem. Commun., 2009, 1350–1352