Textbook Electron Paramagnetic Resonance Volume 25 1St Edition Bagryanskaya Ebook All Chapter PDF
Textbook Electron Paramagnetic Resonance Volume 25 1St Edition Bagryanskaya Ebook All Chapter PDF
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Published on 30 November 2016 on http://pubs.rsc.org | doi:10.1039/9781782629436-FP001
Volume 25
Electron Paramagnetic Resonance
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Editors
Victor Chechik, University of York, UK
Damien M. Murphy, Cardiff University, Cardiff, UK
Authors
Edward A. Anderson, University of Oxford, UK
Elena Bagryanskaya, N. N. Vorozhtsov Novosibirsk Institute of
Organic Chemistry SB RAS and Novosibirsk State University, Novosibirsk,
Russian Federation
Vincenzo Barone, Scuola Normale Superiore, Pisa, Italy
Jerryman Appiahene Gyamfi, Scuola Normale Superiore, Pisa, Italy
Marius M. Haugland, University of Oxford, UK
Johann P. Klare, University of Osnabrück, Germany
Olesya Krumkacheva, N. N. Vorozhtsov Novosibirsk Institute of Organic
Chemistry SB RAS, Novosibirsk State University and International
Tomography Center SB RAS, Novosibirsk, Russian Federation
Janet E. Lovett, St Andrews University, UK
S.R.A. Marque, N.N. Vorozhtsov Novosibirsk Institute of Organic Chemistry
SB RAS, Novosibirsk, Russian Federation and Aix-Marseille Université,
France
Andrea Piserchia, Scuola Normale Superiore, Pisa, Italy
Stephen Sproules, University of Glasgow, UK
Published on 30 November 2016 on http://pubs.rsc.org | doi:10.1039/9781782629436-FP001 View Online
ISBN: 978-1-78262-857-6
PDF eISBN: 978-1-78262-943-6
EPUB eISBN: 978-1-78801-089-4
DOI: 10.1039/9781782629436
ISSN: 1464-4622
A catalogue record for this book is available from the British Library
Apart from any fair dealing for the purpose of research or private study for
non-commercial purposes, or criticism or review, as permitted under the
terms of the UK Copyright, Designs and Patents Act, 1988 and the
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prior permission in writing of The Royal Society of Chemistry, or in the case of
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terms stated here should be sent to The Royal Society of Chemistry at the
address printed on this page.
Cover
Published on 30 November 2016 on http://pubs.rsc.org | doi:10.1039/9781782629436-FP007
Preface v
5 Conclusion 231
Acknowledgements 231
References 231
Spin labels containing nitroxyl radicals possess many properties that render them useful
for electron paramagnetic resonance (EPR) spectroscopy. This review describes the
relationships between the structure and properties of nitroxide spin labels, methods for
their synthesis, advances in methods for their incorporation into biomolecules, and
selected examples of applications in biomolecule structural investigations.
1 Introduction
Within the field of electron paramagnetic resonance (EPR) spectroscopy,
‘spin labelling’ describes the attachment of a radical or paramagnetic
centre (i.e. a molecule containing at least one unpaired electron spin)
onto a material of interest, which enables its investigation using para-
magnetic resonance spectroscopy. For such applications, spin labels
should ideally fulfil several criteria: the framework of the label must
stabilise the radical against redox processes; the radical must possess
desirable properties for the magnetic resonance experiment (such as
chemical stability and spin coherence persistence); and, the label must
be readily (and site-specifically) attached without structural distortion of
the system under study.
By far the largest family of spin labels are those based on the nitroxyl
(N–O ) radical, which are called nitroxide spin labels. These are typically
five- or six-membered heterocyclic derivatives of piperidine, pyrrolidine,
isoindoline, and other heterocycles containing two heteroatoms;
importantly, the nitroxyl radical is flanked by two quaternary carbon
atoms. The ‘classic’ nitroxide is the piperidine-based 2,2,6,6-tetra-
methylpiperidine 1-oxyl (TEMPO, 1, Fig. 1), which has found use in many
chemical and materials applications. This radical, in which the unpaired
electron is located mainly on the nitrogen and oxygen atoms, is stabilised
by the steric screening imparted by its four adjacent methyl groups,
which protect the radical from reduction or other processes. The lack of
a-protons also prevents the decomposition of the nitroxyl to the corres-
ponding nitrone. Some other examples of common nitroxide families
(2–7) are illustrated.
a
Chemistry Research Laboratory, University of Oxford, Oxford, OX1 3TA, UK
b
SUPA School of Physics and Astronomy, University of St Andrews, St Andrews,
KY16 9SS, UK. E-mail: jel20@st-andrews.ac.uk
1 2 3 4 5 6 7
typically about 100 MHz. Both g and Aiso are weakly sensitive to solvent
polarity and proticity, with Aiso increasing and g-values decreasing in
increasingly polar/protic solvents.22,23 This property has been used to
map membrane protein channels, and to probe changes in solvent
behaviour associated with the glass-transition temperature in water/
glycerol mixtures.23,24
Coupling of proximal nuclei (Ia0) to the electron gives rise to the
Published on 30 November 2016 on http://pubs.rsc.org | doi:10.1039/9781782629436-00001
8 9 10
11 1 12 13
dynamics when tc B1 ns. Altering the microwave frequency will alter the
timescales measured and higher frequencies will offer enhanced angular
resolution.37,38 There are many reports of using this property to map
global and local dynamics in spin-labelled proteins.38–46
1 2 3
Fig. 4 The stability of nitroxyl radicals towards reduction correlates with the size and
nature of the nitroxyl-bearing ring.
Published on 30 November 2016 on http://pubs.rsc.org | doi:10.1039/9781782629436-00001
14 15 16 17
The second general stabilising effect is that of increasing the steric bulk
of the flanking alkyl substituents which shield the radical from reduction,
or stabilise it relative to the hydroxylamine where an equilibrium exists.56
For example, tetraethyl-substituted isoindolinyl nitroxides have been
shown to be highly resistant to ascorbic acid reduction, and its stability
was further enhanced when bound to the ribose of RNA via a thiourea
linker (14, Fig. 5).57 The enhanced stability of the PROXYL framework can
be combined with such steric protection to give particularly stable radicals:
the tetraethyl-flanked PROXYL (15) remains B90% intact after two hours
exposure to ascorbate or frog oocyte cells/cell extract.51,58 Bis(spiro-
cyclohexyl) groups flanking the nitroxyl (16) also confer stability against
bioreduction, although to a lesser extent than tetraethyl substituents. This
is likely due to less effective steric shielding for a cyclohexane ring com-
pared to the more mobile ethyl groups.52,56,58,59 However, some spirocyclic
systems can confer remarkable stability: the fully substituted PROXYL (17)
was found to be exceptionally resistant to ascorbic acid reduction.60
One caveat in label design is that many of these extended alkyl chains
increase label hydrophobicity, which may cause problems for some
labelling strategies, or unwanted sample aggregation.61 The electronic
influence of substituents can also affect stability, and a careful con-
sideration of both steric and electronic effects is therefore required when
designing labels.49
and that the label can be used in a facile manner through simple and
efficient labelling procedures.
The conformations and dynamics of some spin labels attached to bio-
molecules, particularly methanethiosulfonate (MTS, 18, Fig. 6), have been
investigated computationally and experimentally through EPR analysis
and crystallography.87–99 Importantly, there is software freely available to
enable users to label their target in silico and calculate the most probable
conformations, since the conformation of the spin label tether must also
be considered when interpreting DEER measurements.100–102
Fig. 7 The temperature dependence of T11 and Tm1 for tetramethyl- (triangles) and
bis(spirocyclohexyl)-substituted (squares) pyrrolinoxyls. Image modified and reprinted
from ref. 81 with permission from Elsevier.
22
21
20 21 23
Published on 30 November 2016 on http://pubs.rsc.org | doi:10.1039/9781782629436-00001
24 25
26 9 8
the nitroxyl by mild oxidants such as MnO2 or NaNO2 (Path C);58,108 this
latter oxidation can even proceed spontaneously in the presence of
atmospheric oxygen under neutral or basic conditions.9
An alternative method for amine oxidation employs 1.5–2 equivalents
of m-CPBA.109,110 This reagent often results in a rapid and high-yielding
oxidation, which likely proceeds via path B in Scheme 2.111 It is tolerant
of other functional groups: for example, the double bond of pyrroline 24
(Scheme 3) was unaffected during nitroxyl formation (25).112 One
cautionary note in all of these oxidations is that the oxoammonium salt
22 (formed in both the Na2WO4/H2O2 and m-CPBA methodologies) has
been observed to effect oxidation of alcohols, as in the oxidation of
aminoalcohol 26 to a mixture of nitroxyl alcohol 9 and ketone 8.109,110,113
Due to the potential susceptibility of nitroxyls to reduction or oxidation
during chemical synthesis or under biological conditions, several inter-
esting protection strategies have been employed that allow a late-stage
deprotection of the nitroxyl itself. This includes the use of an O-methyl
hydroxylamine derivative 27 (Fig. 8), a robust functionality from which
the nitroxyl can be revealed on treatment with m-CPBA.114 An elegant
photolabile protecting group strategy has also been developed, where
irradiation of 28 at 405 nm delivers the nitroxyl in high yield (92%).115
This latter chemistry has been applied to a masked nitroxyl attached to a
DNA oligonucleotide at cytidine.
27 28
Published on 30 November 2016 on http://pubs.rsc.org | doi:10.1039/9781782629436-00001
29 30 35
32
33
31 36
34
37 (TEMPA) 29
42
38 40 39 41 43
44 45 19 46
Azide 46 was recently reported by our groups, and was used to label nucleic
acids in a CuAAC spin-labelling strategy.139
48 47 52
50 51
49 53
55 18 (MTS) 59 61
54 49 53 60
56 58 57 (TPA) 62
63 64 67 71
65 68
66
Published on 30 November 2016 on http://pubs.rsc.org | doi:10.1039/9781782629436-00001
69
70
72 73 74 75
76 77 78 79 80
81 82 83 84
86 88 89 91
Published on 30 November 2016 on http://pubs.rsc.org | doi:10.1039/9781782629436-00001
85 87 90
97 95 92
93
98 96 94
nitroxide uridine derivative 94 was obtained from triamine 95,167 the latter
prepared by nitration of 85, then amination through vicarious nucleophilic
substitution, and nitro reduction.167 Triamine 95 has also been employed
for the synthesis of 96 from oxidative condensation with the corresponding
benzaldehyde.168 A further approach uses iodophenyl phthalimide 97,
which is prepared from 85 by a double bromination/cyanation/hydrolysis
sequence,157,159 then condensation with p-iodoaniline. A Sonogashira
coupling strategy leads to uridine derivative 98.169
One further notable example is the cytidine analogue 99 (Scheme 11),
also known as Ç, which gives highly rigid systems in the EPR spectro-
scopic investigation of nucleic acids.166 99 was synthesised from
isoindoline o-aminophenol derivative 92 (Scheme 10) by reaction with
5-bromouridine (100) and cyclisation of intermediate bromide 101. After
conversion into a phosphoramidite building block, nucleoside derivative 99
was incorporated into nucleic acids by solid-phase synthesis under modi-
fied conditions. A free nucleobase analogue of 99 (known as ç) has been
developed using equivalent chemistry on 1-benzyl-5-bromouracil.170,171
8 102
104 103
Published on 30 November 2016 on http://pubs.rsc.org | doi:10.1039/9781782629436-00001
Scheme 12 Amino acid derivatives for two-point attachment of piperidinyl spin labels.
112 109
In the domestic animals in general the liver may become the seat
of imperfectly spherical nodules of a white, yellow or brownish white
color, varying in size from a millet seed to a pea or hazel nut, and of a
gritty consistency and feeling, from the deposition of earthy salts.
These may be seen in groups under the proper capsule, the adjacent
hepatic tissue being healthy, or atrophied, sclerosed or pigmented.
These lesions have been found most abundantly in solipeds.
Pathogenesis. The most varied doctrines have been advanced as to
the origin of these lesions. They have been attributed to the previous
presence in the liver of linguatula, echinococcus, cœnurus, oxyurus,
distoma, and other parasites (Cadeac, Mazanti, Olt, Ostertag, Gripp,
Leuckart, Ratz), to glanders, to microbian attacks (Dieckerhoff), to
minute embolic infarcts in omphalitis in the foal, or intestinal
disease in the adult (Kitt), and to obstructions by the eggs of
distomata in the biliary ducts (Galli-Vallerio). It is not improbable
that the lesion may be due to any one of these in a specific case, and
this may be ascertained by the existence of certain definite features
and conditions. Linguatula, echinococcus and cœnurus can only be
suspected in districts where these prevail, and a careful examination
of the central mass of the nodule should reveal the presence of the
indestructible hooklets, as certified for given cases by Olt, Ostertag
and Gripp. In case of nematoid worms or distomata, the eggs may
possibly be found as in the cases of Villach and Ratz, or the embryos
(Mazanti). Or there may be traces of channels formerly hollowed out
by the worms in the vicinity of the nodules, as seen by Leuckart.
Coincident tumors of the intestinal mucosa from larval nematodes,
or aneurism or emboli in the anterior mesenteric artery would
corroborate this conclusion. If distomata had started the lesions, the
distension of the gall ducts and the thickening of their walls would be
likely to indicate their former presence. Glander nodules might be
suspected from the absence of a distinct rounded or oval outline,
from the lack of a distinct, clear line of demarcation between the
nodule and the adjacent liver tissue, and by the manifestation in the
periphery of the nodule and around it of free cell proliferation,
showing the mode of progression by the invasion of new tissue. If
still active, the bacilli should be discoverable in stained scrapings or
sections. There should also be distinct indications of the lesions of
glanders in the lymph glands of the portal fissure, of the
mediastinum, of the submaxillary region and of other parts.
Heiss records an interesting case of general calcification of the
horse’s liver, with large aneurism of the abdominal aorta, mesenteric
and renal arteries. The liver was thirty-two pounds, puckered on the
surface and showed calcic degeneration of the walls of the vessels
and hepatic tissue, to such an extent that when the organ was dried it
did not add materially to its hardness. Microscopically the diseased
centres indicated minute blood clots (thrombi), with fibrinous
development and cretifaction. The lesions in this case were
attributed to multiple emboli in connection with the aneurism. It
might suggest further, microbian infection of both the aneurismal
and hepatic vessels. In another case of extensive cretifaction of the
horse’s liver reported by Cszoker, the calcified masses tended to
assume rounded forms like tubercle, and had a clear glistening
surface.
These lesions are mainly interesting in a pathological sense, and
unless they are very extensive do not give rise to appreciable
symptoms.
Treatment could only be prophylactic and directed to the removal
of the special conditions, in which the calcification originated in a
given locality.
ACTINOMYCOSIS OF THE LIVER.
Mostly in cattle from over feeding, dry feeding, inactivity. Small. Multiple.
Round, angular, lobulated. Nucleus. Composition. Dilated ducts. Atrophied or
sclerosed glandular tissue. Prevention: succulent food, water at will, open air life,
correction of local catarrh.
The spleen even more than the pancreas is so deeply seated and so
surrounded by other organs, that its diseases are not readily
appreciable by physical examination, while the absence of any special
secretion excludes the possibility of diagnostic deductions through
this channel. Even the relation of the condition of the organ to the
number of the leucocytes and red globules fails to afford trustworthy
indications of disease, since leucocytes originate in other tissues as
well as the spleen, and the destruction of red globules may take place
elsewhere. Yet an excess of eosinophile leucocytes in the blood
suggests hypertrophy or disease of the spleen, and an excess of
leucocytes in general is somewhat less suggestive of disease of this
organ (see Leucocythemia). If adenoma is further shown, in
enlargement of lymphatic glands elsewhere there is the stronger
reason to infer disease of the spleen.
The physiological relation of the spleen to the blood especially
predisposes it to diseases in which the blood is involved. The
termination of splenic capillaries, in the pulp cavities, so that the
blood is poured into these spaces and delayed there, opens the way,
not only for the increase of the leucocytes, and the disintegration of
red globules, but for the multiplication of microörganisms which
may be present in the blood, and for a poisoning (local and general)
with their toxins. Hence we explain the congestions, sanguineous
engorgements and ruptures of the spleen in certain microbian
diseases (anthrax, Southern cattle fever, septicæmia, etc.)
We should further bear in mind that the spleen is in a sense a
safety valve for the blood of the portal vein, when supplied in excess
during digestion. In this way it protects the liver against sudden and
dangerous engorgements, but it is itself subjected to extreme
alternations of vascular plenitude and relative deficiency. This may
be held to take place largely under the influence of the varying force
of the blood pressure in the portal vein, but according to the
observations of Roy on dogs and cats, it is also powerfully influenced
by muscular and nervous action. He found rhythmic contractions of
the organ due to the muscles contained in the capsule and trabeculæ,
repeating themselves sixty times per hour, and which might be
compared to tardy pulsations. He further found that electric
stimulation of the central end of a cut sensory nerve, of the medulla
oblongata, or of the peripheral ends of both splanchnics and both
vagi caused a rapid contraction of the spleen. The spleen may thus be
looked on not only as a temporary store-house for the rich and
abundant blood of the portal system of veins during active digestion,
but also as a pulsating organ acting under the control of nerve
centres in the medulla. That the various ascertained normal
functions of this viscus may be vicariously performed by others, as
shown in animals from which it has been completely extirpated, does
not contradict the occurrence of actual disease in the organ, nor the
baleful influence of certain of its diseases on the system at large.
ANÆMIA OF THE SPLEEN.
General anæmia, debility, wasting diseases, starvation, hæmorrhage, stimulus to
formation of red globules, asphyxia, electricity, cold, quinine, eucalyptus, ergot.
Symptoms: lack of eosinophile leucocytes in the blood of a debilitated subject may
lead to suspicion. Treatment: tonic, light, sunshine, pure air, exercise, nutritive
food, iron, bitters.
In cases of general anæmia the spleen is liable to be small,
shrunken, wrinkled, and when cut the surface is drier and lighter
colored than in the normal condition. This condition may be seen
after old standing debilitating diseases, but is common in animals
that have been reduced by starvation, just as the opposite condition
of hyperæmia and enlargement comes of abundance of rich food and
an active digestion. It may shrink temporarily as the result of profuse
hemorrhage, but Bizzozero and Salvioli found that several days after
such loss of blood it became enlarged and its parenchyma contained
many red nucleated hæmatoblasts. The result of hemorrhage is
therefore to stimulate the organ to enlargement and to the
resumption of its embryonic function of producing red blood
globules. Contraction of the spleen further occurs under asphyxia,
the deoxidized blood being supposed to operate through the medulla
oblongata. As already noted the spleen shrinks under stimulation of
the central end of a sensory nerve (vagus, sciatic). An induced
current of electricity applied to the skin over the spleen causes
marked contraction (Botkin). Cold, quinine, eucalyptus, ergot and
other agents also induce contraction. In the normal condition there
is an inverse ratio between the bulk of the spleen and the liver, the
enlargement of the one entailing a diminution of the other, but in
certain diseased states, such as anthrax, ague, etc., both are liable to
enlargement at the same time.
Symptoms of splenic anæmia are wanting, through a lack of
eosinophile leucocytes, in the blood of a starved or otherwise
debilitated animal, may lead to suspicion of the condition.
The treatment of such a case would be addressed rather to the
general debility which induced the splenic contraction than to the
contraction itself. Light, sunshine, pure air, exercise, grooming,