Model systems for flavoenzyme activity: Intramolecular self-assembly of a flavin derivative via hydrogen bonding and aromatic interactions

Article abstract of DOI:10.1039/b809762c

We have synthesised a flavin derivative incorporating functionalities that promote intramolecular self-assembly via hydrogen bonding and aromatic interactions. The Royal Society of Chemistry.

Full text of DOI:10.1039/b809762c

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Model systems for flavoenzyme activity: intramolecular self-assembly
of a flavin derivative via hydrogen bonding and aromatic interactionsw
Stuart T. Caldwell,a Graeme Cooke,*a Shanika G. Hewage,a Suhil Mabruk,a
Gouher Rabani,a Vincent Rotello,b Brian O. Smith,c
Chandramouleeswaran Subramanib and Patrice Woiseld
Received (in Cambridge, UK) 10th June 2008, Accepted 23rd July 2008
First published as an Advance Article on the web 1st August 2008
DOI: 10.1039/b809762c
We have synthesised a flavin derivative incorporating function-
alities that promote intramolecular self-assembly via hydrogen
bonding and aromatic interactions.
moiety and NAP in the absence of hydrogen bonds, whereas
compounds 3 and 4 were used as controls in NMR, UV-vis
and fluorescence spectroscopy experiments. Compound 3 will
allow us to explore the physical properties in the absence of
supramolecular interactions, whereas compound 4 will allow
us to investigate the role intramolecular hydrogen bonding
interactions have in modulating flavin properties.
Flavoenzymes are proteins that catalyse a variety of biological
processes such as redox transformations, signal transduction
and electron-transfer.1 Non-covalent interactions (e.g. hydro-
gen bonding and p-stacking) between the flavin cofactor and
the apoenzyme have been shown to modulate the redox
properties of the flavin unit by over 500 mV.2 Although the
flavin co-factor is usually non-covalently bonded to the
apoenzyme, in some flavoenzymes the flavin unit is covalently
linked to the polypeptide. In these systems, the flavin unit is
usually attached through the C8 position of the flavin to the
cysteine, tyrosine or histidine moieties of the peptide back-
bone.3 The flavin cofactor of triethylamine dehydrogenase is
covalently attached to the apoenzyme through a C6 thioether
link.4
The synthesis of compounds 1–4 is reported in the ESI.w
The solution properties of flavin derivatives 1–4 were charac-
terised by 1H NMR, 2D NOESY, UV-vis and fluorescence
spectroscopies. In all cases, non-polar solvents (e.g. chloro-
form and dichloromethane) were used for these experiments to
more accurately mimic the polarity of the microenvironment
surrounding the flavin moiety of a typical flavoenzyme.6 1
H
NMR spectroscopy of flavin 1 revealed upfield shifts for the
flavin and NAP moieties and downfield shifts of the flavin
imide N–H and two amide N–H groups attached to the DAP
moiety (compared to controls 3 and 5) (Fig. 2). The upfield
shifts of the aromatic signals are consistent with the expected
ring current effects for face-to-face stacking between the flavin
and NAP units.8 The downfield shifts for the N–H moieties are
in line with hydrogen bonding interactions between the flavin
and DAP moieties.6,7
In our continuing studies to develop model systems for
flavoenzymes, and in particular mimics for flavoenzymes
where the cofactor is covalently bound to the apoenzyme,
we wanted to develop a model system to study the combined
effects of intramolecular aromatic5 and hydrogen bonding
interactions have on the physical properties of the flavin unit.
Compound 1 was designed to allow us to explore these dual
effects as the diamidopyridine moiety (DAP) is known to form
complementary hydrogen bonds with the imide unit of the
flavin,6,7 and the electron rich naphthalene (NAP) unit should
participate in aromatic interactions with the electron deficient
flavin core (Fig. 1).8 Molecular modeling studies clearly pre-
dicted the formation of an intramolecular self-assembled
structure through hydrogen bonding and aromatic interac-
tions (see ESIw). Compound 2 should allow us to explore the
role of intramolecular aromatic interactions between the flavin
The 1H NMR spectra of flavin 1 recorded over 1.0 Â 10À2 M
to 5.3 Â 10À4 M in CDCl3 displayed a limited concentration
dependence. Therefore, it is unlikely that supramolecular
a Glasgow Centre for Physical Organic Chemistry, WestCHEM,
Department of Chemistry, University of Glasgow, Joseph Black
Building, Glasgow, UK G12 8QQ
b Department of Chemistry, University of Massachusetts at Amherst,
Amherst, MA 01002, USA
c IBLS, University of Glasgow, Glasgow, UK G12 8QQ
d
´
´
Laboratoire de Chimie Organique et Macromoleculaire, Universite
des Sciences et Technologies de Lille, 59655 Villeneuve d’Ascq,
France. E-mail: graemec@chem.gla.ac.uk
w Electronic supplementary information (ESI) available: Full syn-
thetic details for the preparation of 1–5 and characterization of their
physical properties. See DOI: 10.1039/b809762c
Fig. 1 Structures of derivatives 1–5.
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Fig. 2 Partial 1H NMR spectra of compounds 3, 5, 2 and 1 recorded at B31 mM in CDCl3.
oligomers are formed to any great extent, and intramolecular Therefore, in accordance with the data obtained for derivative
interactions are likely to predominate under the conditions
examined. Further evidence of intramolecular aromatic inter-
actions in compound 1 were obtained from the NOESY
spectrum, which shows weak intensity long range couplings
between the aromatic flavin hydrogens and the aromatic NAP
hydrogens (Fig. 3). Cross-peaks between the flavin unit and the
ethylene glycol ‘‘arms’’ of the naphthalene unit were also
observed, indicating that the ‘‘arms’’ are wrapped around the
flavin unit.
1, face-to-face aromatic stacking interactions appear to occur
between the flavin and NAP moieties. Interestingly, we also
observed a downfield shift (B0.5 ppm) of the flavin imide
hydrogen compared to compound 3. For compound 4, the
imide of the flavin moiety appeared at a more downfield
position compared to control 3, indicating the formation of
hydrogen bonds to the DAP moiety. The 1H NMR spectra of
The 1H NMR spectrum of flavin 2 recorded in CDCl3 shows
upfield shifts in the signals arising from the NAP unit com-
pared to the signals obtained for the same protons of 5
(Fig. 2). A similar upfield shift was also observed for the
aromatic flavin protons compared to control compound 3.
Fig. 3 Partial NOESY spectrum of 1 showing cross peaks between
flavin proton a (y-axis) and naphthalene protons and ethlylene glycol
‘‘arms’’ (x-axis). Recorded in CDCl3 using a mixing time of 450 ms.
Fig. 4 Fluorescence spectra of compounds 3 (—), 4 (– –), 2 (–Á –Á) and
1 (ÁÁÁÁ) recorded in chloroform (B4 Â 10À6 M). Inset shows an
expansion of the fluorescence spectra of derivatives 4, 2 and 1.
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around 80 mV towards a more positive potential. Thus the
electrochemical data indicate that intramolecular complemen-
tary hydrogen bonding interactions appear to have a more
profound role in stabilizing the flavin radical anion state than
intramolecular aromatic interactions.11
In conclusion, we have developed a new flavin model system
that has allowed us to probe the combined role intramolecular
hydrogen bonding and aromatic interactions have in modulat-
ing the physical properties of the flavin moiety. Aromatic
interactions appear to play a more important role in perturb-
ing the optical and fluorescence properties, whereas hydrogen
bonding appears to play a more important role in tuning the
electrochemical properties of the flavin. We are currently using
this motif to tune the properties of synthetic flavoenzymes,12
and to develop new self-assembling polymeric systems. Our
endeavours in these areas will be reported in due course.
Fig. 5 Cyclic voltammetry of compounds 1 ( ), 2 ( ), 3 ( ) and
4 ( ) recorded in dichloromethane (B1 Â 10À4 M). Scan rate =
Notes and references
100 mV sÀ1
.
1 (a) Flavins and Flavoproteins 1996, ed. K. Stevenson, V. Massey
and C. Willams, University of Calgary, Calgary, 1997; (b) Chemis-
try and Biochemistry of Flavoenzymes, ed. F. Muller, CRC Press,
Boca Raton, 1991, vol. 1–3.
2 (a) For examples see: V. Massey and P. Hemmerich in, The Enzymes,
ed. P. D. Boyer, Academic Press, New York, 1976, vol. 12,
pp. 191–240; (b) R. Swenson, G. Krey and M. Eren, Flavins and
Flavoproteins, ed. D. Edmondson and D. McKormick de Gruyter,
Berlin, 1987, pp. 98–107.
3 D. E. Edmondson and R. De Francesco, Chemistry and Bio-
chemistry of Flavoenzymes, ed. F. Muller, CRC Press, Boca Raton,
FL, 1991, vol. 1, pp. 73–103.
4 D. J. Steenkamp and F. S. Mathews, Chemistry and Biochemistry
of Flavoenzymes, ed. F. Muller, CRC Press, Boca Raton, FL, 1991,
vol. 2, pp. 395–424.
5 (a) C. A. Hunter, K. R. Lawson, J. P. Perkins and C. J. Urch,
J. Chem. Soc., Perkin Trans. 2, 2001, 651; (b) E. A. Meyer, R. K.
Castellano and F. Diederich, Angew. Chem., Int. Ed., 2003, 42,
1210.
6 For recent reviews see: (a) A. Niemz and V. M. Rotello, Acc.
Chem. Res., 1999, 32, 44; (b) V. M. Rotello, Curr. Opp. Chem.
Biol., 1999, 3, 747; (c) Y. Yano, Rev. Heteroatom Chem., 2000, 22,
151.
7 A. S. F. Boyd, J. B. Carroll, G. Cooke, J. F. Garety, B. J. Jordan,
S. Mabruk, G. Rosair and V. M. Rotello, Chem. Commun., 2005,
2468.
8 For examples of foldamers fabricated from 1,5-dialkyloxynaph-
thalene units see: (a) A. J. Zych and B. L. Iverson, J. Am. Chem.
Soc., 2000, 122, 8898; (b) M. S. Cubberley and B. L. Iverson,
J. Am. Chem. Soc., 2001, 123, 7560–7563; (c) G. J. Gabriel,
S. Storey and B. L. Iverson, J. Am. Chem. Soc., 2005, 127, 2637.
9 E. C. Breinlinger and V. M. Rotello, J. Am. Chem. Soc., 1997, 119,
1165.
flavins 2 and 4 recorded over 3.6 Â 10À2 M to 1.8 Â 10À3 M in
CDCl3 displayed limited concentration dependence, suggest-
ing that intramolecular interactions are likely to predominate
under the conditions examined.
When compounds 1 or 2 were dissolved in non-polar
solvents such as chloroform or toluene red–orange solutions
were obtained for these derivatives, whereas derivatives 3 and
4 displayed the standard yellow colour typical of flavin
derivatives dissolved in these solvents (see ESIw). When more
polar solvents were used (e.g. DMF, DMSO) the colour of
derivatives 1 and 2 changed to yellow. Thus the data are
consistent with the more polar solvents disrupting aromatic
interactions between the naphthalene and flavin moieties of
derivatives 1 and 2, and thus suggest that donor–acceptor
interactions are responsible for the complexation processes.7b
Due to the complex nature of the UV-vis spectra recorded in
chloroform, it was not possible to determine whether charge-
transfer bands could be observed. The fluorescence of the
flavin nucleus is very sensitive to supramolecular interactions.9
The fluorescence spectra of flavins 1–4 recorded in chloroform
show dramatic fluorescence quenching for derivatives 1, 2 and
4 (with respect to flavin 3), further suggesting that the func-
tionality attached to the N(10) of the flavin unit are participat-
ing in supramolecular interactions (Fig. 4).
We have investigated the solution electrochemistry of deri-
vatives 1–4 using cyclic voltammetry (CV) in CH2Cl2 (Fig. 5).
Derivative 3 afforded CV data in accordance with flavin
derivatives recorded in this solvent.10 CV data for compound
2 showed that the addition of a NAP moiety had very limited
effect on the electrochemical properties of the flavin unit.
However, the addition of DAP units to the N(10) side chain,
as in derivatives 1 and 4, resulted in a near identical shift of
10 E. Breinlinger, A. Niemz and V. M. Rotello, J. Am. Chem. Soc.,
1995, 117, 5379.
11 For a discussion of the combined roles aromatic stacking and
hydrogen bonding have in controlling the redox properties of
flavin units in intermolecular complexes see: M. Gray, A. J.
Goodman, J. B. Carroll, K. Bardon, M. Markey, G. Cooke and
V. M. Rotello, Org. Lett., 2004, 6, 385.
12 B. J. Jordan, G. Cooke, J. F. Garety, M. A. Pollier, N. Kryvokhyzha,
A. Bayir, G. Rabani and V. M. Rotello, Chem. Commun., 2007, 1248.
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4128 | Chem. Commun., 2008, 4126–4128