A caged cyanide

Article abstract of DOI:10.1039/c2pp05359d

A photoactivatable caged cyanide, 1-(2-nitrophenyl)ethyl (NPE) cyanide, was synthesized, which upon irradiation in the near UV releases cyanide. It is demonstrated that the compound can be used to induce formation of the Fe ^III-CN^- complex in the heme protein nitrophorin 4 from Rhodnius prolixus.

Full text of DOI:10.1039/c2pp05359d

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COMMUNICATION
A caged cyanide
Markus Knipp,*a Johanna J. Taing,a Chunmao Hea and Cristiano Viappiani*b
Received 26th October 2011, Accepted 23rd February 2012
DOI: 10.1039/c2pp05359d
A photoactivatable caged cyanide, 1-(2-nitrophenyl)ethyl
(NPE) cyanide, was synthesized, which upon irradiation in
the near UV releases cyanide. It is demonstrated that the
compound can be used to induce formation of the FeIII–CN−
complex in the heme protein nitrophorin 4 from Rhodnius
prolixus.
Rapid mixing techniques allow an insight into the kinetics of
fast bimolecular reactions down to the millisecond time-scale.
However, when reactions should be observed that are faster than
this limit, more advanced and specialised techniques are
required. Photoactivatable caged compounds represent a method
to generate an active compound in situ from an inactive precur-
Scheme 1 Reaction mechanism for the photolysis of 1-(2-nitrophenyl)
ethyl sulfate 1 and cyanide 2.3
sor by flash photolysis.1 Here we describe a novel caged cyanide
for studies of fast kinetics of heme proteins. Cyanide is an inter-
esting effector for studies with heme proteins, as it can readily
form very stable FeIII–CN− complexes which provide a useful
model for retrieving structural information on liganded species.2
More relevant, exposure of {FeNO}6† (or other complexes) to
excess cyanide can be used to displace NO (or other ligands) to
follow the associated kinetics.
The preparation of a caged cyanide was derived from the 1-(2-
nitrophenyl)ethyl (NPE) compounds which were used for the
generation of rapid H+ release. The principle of action is pre-
sented in Scheme 1 for the sulphate 1,3 which has been success-
fully applied to study the acid unfolding of myoglobin.3a,4
Electronic excitation leads to the formation of a nitronic acid
(pKa ∼ 3.5),3b,4,5 which then decays to a bicyclic intermediate
by nucleophilic attack of the nitronic acid on the Cα–CN.6 Upon
release of the leaving group X− ring opening occurs to yield the
product 3.
Scheme 2 Synthesis of caged cyanide 2.
the bromide in 1-(1-bromo)ethyl-2-nitrobenzene 4 with CN−, so
that a 2-cyanoalkyl is obtained in ortho position to the nitro
group (Scheme 2). The compound is obtained in good yields.‡
The concomitant appearance of H+ expected on the basis of
the reaction shown in Scheme 1 should then be neutralized by
the application of a strong enough buffered solution. Photode-
composition of 2 in 100 mM sodium phosphate buffer at pH =
7.0 by near UV (355 nm) light leads to absorption changes,
reported in Panel A of Fig. 1, which are very similar to those
observed for the related compound 1. The spectral changes upon
irradiation are identical when 2 is irradiated in 100 mM sodium
acetate buffer at pH = 5.5 (data not shown). Panel B in Fig. 1
compares the time courses for formation and decay of the aci-
nitro reaction intermediates, observed for 1 and 2. Experiments
were performed using a previously described setup.7 The absor-
bance at 400 nm for 1 rises with a biexponential kinetics, due to
formation of the nitronic acid followed by its deprotonation.3b
Decay of intermediate occurs with a pH-dependent lifetime, in
keeping with acid catalysis of this reaction, which results in shift-
ing the equilibrium towards the nitronic acid, so favoring for-
mation of the bicyclic intermediate.3b The observed aci-nitro
decay kinetics sets a lower limit for the time constant for CN−
release which, at neutral pH is 10 ms and at pH 5.5 becomes
300 μs.
In the previous approach, the sulfate derivative 1 was used to
achieve large concentrations of H+ after photolysis, thanks to the
2−
very weak base character of SO4 which assures the full avail-
ability of the H+ as the active species.3a In the approach reported
herein, CN− was introduced as the leaving group X, which rep-
resents also the desired active species. The synthesis of the
caged cyanide 2 was performed by nucleophilic substitution of
aMax-Planck-Institut für Bioanorganische Chemie, Stifstrasse 34-36,
D-45470 Mülheim an der Ruhr, Germany. E-mail: mknipp@
mpi-muelheim.mpg.de; Fax: +49 (0)208 306 3591; Tel: +49 (0)208 306
3671
bDipartimento di Fisica, Università degli Studi di Parma, and NEST,
Istituto Nanoscienze-CNR, viale delle Scienze 7A, 43124 Parma, Italy.
E-mail: cristiano.viappiani@fis.unipr.it; Fax: +39 0521 905223;
Tel: +39 0521 905208
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Fig. 2 Change in the absorbance spectrum of 2 μM of NP4 in 50 mM
sodium phosphate buffer containing 2 mM of 2. The sample was stirred
continuously in a quartz cuvette and illuminated with 200 and 400
flashes of a nanosecond 355 nm Nd:YAG laser (32 mJ per puls). From
these data, the efficiency of the photoinduced release of CN− is esti-
mated to be in the order of ∼10−4
.
hemes. The affinity of CN− to NP4 is very high (Keq(CN−) = 2.5
0.1 × 108 M−1 at pH = 7.5).¶
When a solution of NP4[FeIII] containing 2 is illuminated
with the 355 nm output of the third harmonic of a nanosecond
Nd:YAG laser, the absorbance spectrum is converted into that of
NP4[FeIII–CN−] (Fig. 2). In particular, the absorbance at
403 nm, diagnostic of NP4[FeIII] decreases while the absorption
at 416 nm, typical of NP4[FeIII–CN−], concomitantly increases.
Our findings suggest that the photodissociation of the caged
cyanide 2 occurs in low yield, and leads to decomposition of the
compound on time scales comparable to those observed for the
related caged sulphate 1. Nevertheless, it is possible to estimate
Fig. 1 A. Absorption spectra of caged cyanide 2 in buffered (100 mM
sodium phosphate) aqueous solution before and after exposure to an
increasing number of 355 nm, 28 mJ laser shots. B. Time-resolved
absorbance changes at 400 nm following exposure to single, 355 nm,
28 mJ laser shots for NPE caged sulfate 1 (a, c) and caged cyanide 2 (b,
d) in 100 mM sodium phosphate buffer at pH = 7.0 (black curves) and
in 100 mM sodium acetate buffer at pH = 5.5 (gray curves). Absorbance
changes are normalized for the absorbance at 355 nm. In the case of
caged cyanide 2, the very small signals prevented the monitoring of the
aci-nitro intermediate formation occurring on the short nanosecond time
scale, which is observed for caged sulfate 1 (curves a, c).
2−
that, under experimental conditions where 1 mM of SO4
release was observed,3b,4 the concentration of released CN−
would exceed 60 μM. This concentration is large enough to
allow NO displacement experiments from heme proteins, where
CN− must be added to large excess of the protein.
Since the chromophoric properties of 1 and of 2 are essentially
identical, the former can be used as an actinometer and the data
in Fig. 1 can be exploited to estimate the yield for formation of
the aci-nitro intermediate. Using the deprotonation yield deter-
mined for 1 (0.47)3a as a reasonable estimate for the yield of for-
mation of the aci-nitro intermediate, the yield for formation of
the aci-nitro intermediate of 2 is ∼0.03. This represents an upper
limit for the quantum yield for decomposition of 2 to release
CN−.
Conclusions
A new caged cyanide compound 2 was synthesized, using the 1-
(2-nitrophenyl)ethyl chromophore as a photosensitive protecting
group. This preliminary characterization demonstrates that the
compound is fully functional and can lead to release of CN−
upon excitation with near UV light. However, the photorelease
efficiency is rather low; thus, the development of a next gener-
ation of caged cyanide will aim at the increase of the photosensi-
tivity by the insertion of suitable phenyl ring substituents.
To test the capability of 2 to release CN− upon photoexcitation
with near UV light, we have monitored formation of the [FeIII–
CN−] species starting from the [FeIII] complex of recombinant
nitrophorin 4 (NP4).§ Nitrophorins comprise a unique class of
heme proteins that originate from the saliva of the blood sucking
insect Rhodnius prolixus. Their purpose is the storage and trans-
port of NO from the insect saliva to the victim’s blood where the
iron is formally kept in the +3 oxidation state8 with rather large
affinities (Keq(NO) = 7.9 0.1 × 106 M−1 at pH = 7.5).9 Like
ferriheme–NO complexes, CN− binds in a linear axial fashion to
ferric hemes, thus resembling a structural model for {FeNO}6
Acknowledgements
The authors like to thank Leslie Currell and Dagmar Merkel for
technical support. Financial support was from the Max Planck
Society (MPG, to M. K.), from the German Academic Exchange
Service (DAAD), Project No. 50728589 (to M.K.), and from the
Vigoni program (to C.V.).
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Notes and references
†According to the widely applied notation of Enemark and Feltham.10
‡Synthesis of 2-(2-nitrophenyl)propionitrile (1.9 g, 57% light yellow
crystals). The substrate 1-(1-bromo)ethyl-2-nitrobenzene 4 was syn-
thesized as described.11 4.3 g (19 mmol) of 4 was dissolved in DMF and
4 g (61 mmol) of KCN were added. The solution was stirred over night
in the dark at 35 °C. Afterwards, the solution was filtered and DMF was
removed under reduced pressure. The product was dissolved in 100 mL
of ethyl acetate and extracted 3 times with 300 mL of water and 3 times
with 300 mL of saturated NaCl. After drying with Na2SO4, the product
was purified by silica gel chromatography using 9 : 1 cyclohexane/ethyl
acetate. mp 40 °C (from ethyl acetate); λmax(100 mM phosphate buffer
pH 7.5)/nm 263 (ε/dm−3 mol−1 cm−1 2860); λmax(MeOH)/nm 253 (ε/
dm−3 mol−1 cm−1 3 870), ∼338sh; NMR: δH(400 MHz; CDCl3;
(CH3)4Si) 1.66 (3 H, d, CH3), 4.69 (1 H, q, CHCN), 7.48 (1 H, t, Ph–
C5H), 7.67 (1 H, t, Ph–C4H), 7.75 (1 H, d, Ph–C6H), 7.98 (1 H, d, Ph–
C3H); δ13C(400 MHz; CDCl3; CDCl3) 20.9 (CH3), 27.9 (CHCN), 120.6
(CN), 127.2 (Ph–C5), 129.2 (Ph–C4), 129.5 (Ph–C6), 132.1 (Ph–C1),
134.1 (Ph–C3), 147.3 (Ph–C2).
̲
§Recombinant NP4 was expressed in Escherichia coli, purified, and
reconstituted as was previously described.12
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This journal is © The Royal Society of Chemistry and Owner Societies 2012