Diazo group as a new chemical reporter for bioorthogonal labelling of biomolecules

Article abstract of DOI:10.1039/c4ra08861a

Diazoacetyl groups have been shown to undergo spontaneous cycloaddition reactions with strained alkenes and alkynes. The rates of reaction are similar to the equivalent reactions of azide groups. This allows diazo groups to be used as bioorthogonal reporter groups. This is demonstrated by fluorescent labelling of a diazoacetylated protein and of cell-surface glycans via metabolic incorporation of a diazoacetylated sugar. This journal is

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Diazo group as a new chemical reporter for bioorthogonal labelling of biomolecules
Laia Josa-Culleré, Yelena A. Wainman, Kevin M. Brindle and Finian J. Leeper
RSC Adv., 2014, 4,
DOI: 10.1039/
Diazoacetyl groups undergo spontaneous cycloaddition with strained alkenes and alkynes and can be
bioorthogonal reporter groups labelling proteins and glycans.

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Diazo group as a new chemical reporter for
bioorthogonal labelling of biomolecules
Cite this: DOI: 10.1039/x0xx00000x
Laia Josa-Culleré,a Yelena A. Wainman,a Kevin M. Brindleb and Finian J. Leepera
Diazoacetyl groups have been shown to undergo spontaneous cycloaddition reactions with
strained alkenes and alkynes. The rates of reaction are similar to the equivalent reactions of
azide groups. This allows diazo groups to be used as bioorthogonal reporter groups. This is
demonstrated by fluorescent labelling of a diazoacetylated protein and of cell-surface
glycans via metabolic incorporation of a diazoacetylated sugar.
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
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most widely used because of their chemical stability and small
size.
Introduction
For in vivo molecular imaging, small molecules have better
access to extravascular compartments and show better clearance
of unbound material than macromolecules such as antibodies
and lectins. Their use as imaging agents requires them to target
selectively the desired biomolecule. One way to achieve this is
to use the bio-orthogonal chemical reporter strategy, in which
the target of interest is labelled in a two-step process. Firstly, a
bio-orthogonal chemical reporter is incorporated into the target
of interest using the cell’s own biosynthetic machinery. This
can be achieved by attaching a suitable reporter to substrates
that can be metabolised by the cell. For example, some
unnatural amino acids can be incorporated into proteins or
monosaccharides bearing bioorthogonal functional groups can
be introduced into cell-surface glycans by enzymes that tolerate
the unnatural motif. Secondly, the chemical reporter is then
reacted with a small molecule probe that has a complementary
bioorthogonal reactive moiety. The bioorthogonal reaction
partners should react rapidly under physiological conditions (37
°C, pH 6-8) and remain inert to other biological functional
groups, they should form a stable and non-toxic product and
have no or inert by-products. The bioorthogonal reporter
strategy has proved to be a powerful tool to image glycans,1-4
proteins5-7 and lipids8.
The reaction of diazo compounds with alkenes and alkynes
has been known for many years22 yet little attention has been
paid to its potential as a new chemical reporter group. In this
paper, we present a proof-of-principle study where we first
evaluated the rate of cycloaddition reactions of a diazo
compound with a strained alkene and an alkyne. We also
demonstrated the bioorthogonality of the diazo group by
labelling a protein and tested the incorporation of diazo-bearing
sugars into cell-surface glycans.
Results and discussion
Kinetic studies with the diazo group
The highly strained (E,E)-1,5-cyclooctadiene (COD) 2 has been
reported to perform very efficient [3+2] cycloadditions with
1,3-dipoles and inverse-electron-demand Diels Alder reactions
with tetrazines, making it suitable for applications in chemical
ligation.23 In order to test whether the diazo group could be
used as a chemical reporter in a two-step labelling process, its
reaction with (
formed with tetrazine was explored. (
from ( )-COD via a three-step phosphine oxide-mediated
olefin inversion.24 It was found to react cleanly with ethyl
diazoacetate by a [3+2] cycloaddition to give pyrazoline
E
,
E
)-COD followed by reaction of the adduct
E,E
)-COD was prepared
Z,Z
The number of functional groups that have been shown to
possess all the requisites to be useful chemical reporters is
limited. This group comprises peptide motifs that can be tagged
with small-molecule imaging probes,5,9 ketones and aldehydes
that can be ligated with hydrazine and hydroxylamine
derivatives,1,6,10 azides that can be modified with phosphines or
reactive alkynes,7,8,11-13 terminal alkynes that can be tagged
with azides,14 and strained alkenes15-20 and isonitriles21 that can
be ligated with tetrazines. Amongst these, azides have been the
1
3
after tautomerisation (Fig. 1). The second-order rate constant of
this reaction was measured by UV spectroscopy (Fig. S3a†) in
pentane at room temperature to be 0.045 ± 0.002 M-1 s-1. The
rate constant of the second [3+2] cycloaddition, giving 4a and
4b, was measured by 1H NMR spectroscopy in CD3OD at room
temperature and is two orders of magnitude lower, which can
be explained by the relief of ring strain once the first trans
double bond of
2 has reacted. Thus pyrazoline 3 can be
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N
N
N
CO2Et
HN
CO2Et
N2
CO2Et
CO2Et
HN
HN
1
2
H
H
H
H
H
H
CO2Et
N2
pentane
0.045 M-1 s-1
CD3OD
1.8 x 10-4 M-1 s-1
1
+
H
H
H
H
3
NH
HN
EtO2C
CO2Et
N
N
MeO
MeO
OMe
OMe
MeOH, 206 M-1 s-1
CDCl3
0.043 M-1 s-1
4a
4b
N
N
N
N
N
CO2Et
HN
HO
H
H
N
N
7
5
N
N
CO2Et
EtO2C
HN
NH
N
H
H
N
MeO
OMe
MeO
MeO
OMe
OMe
N
N
+
6
MeO
OMe
HO
HO
8a
8b
Fig. 1 Cycloaddition of ethyl diazoacetate with (E,E)-COD
measured rate constants.
2 and TMDIBO 7 and inverse-electron-demand Diels Alder with tetrazine 5, with
obtained in almost quantitative yield by reaction of equimolar and 37 °C. 1H NMR spectroscopy showed no significant
amounts of and
. The inverse-electron-demand Diels Alder reaction after 24 h (see ESI, Fig. S5†), indicating that
reaction of the remaining trans double bond of with tetrazine diazoesters do not react with biologically relevant functional
to give the dihydropyridazine adduct was then measured by groups (thiols, carboxylic acids and amines).
1
2
3
5
6
UV spectroscopy in MeOH at room temperature and proceeded,
as expected, at a much faster rate, 206 ± 6 M-1 s-1.
In order to demonstrate the use of the diazo group as a
bioorthogonal labelling agent, its ability to label proteins was
Therefore, using
2 as an intermediary, two consecutive click tested. A solution of lysozyme was incubated with the
reactions can ligate a diazo ester to a tetrazine, which could diazoacetate NHS ester 927 for 3 h to prepare the diazo-
potentially bear an imaging agent. The rates of these two click modified protein (Fig. 2). The protein was then purified by gel
reactions are of the same order of magnitude as for an azide filtration, labelled with different concentrations of a TMDIBO-
group reacting with first
faster and the second one 2-fold slower. However, because of gel. Fluorescence imaging of the gel showed concentration-
the lack of solubility in water and volatility of , it cannot be dependent labelling of the protein, co-localised with the protein
used as a reagent with a diazo chemical reporter in biological bands as shown by Coomassie Blue staining. (see Fig. S7†).
systems. Therefore, we used instead an alternative [3+2] We next studied the application of the diazo group for the
cycloaddition with TMDIBO , a stable dibenzocyclo-octyne metabolic labelling of cell-surface glycans. Tetra- -acetyl-
2 and then 5, the first one being 2-fold fluorescein complex 10 and then run on a SDS-polyacrylamide
2
7
O
developed previously by our group and used for imaging of galactosamine hydrochloride (Ac4GalNH3Cl 12) was prepared
cell-surface azido sugars.25 Its reaction with ethyl diazoacetate in anomerically pure form from galactosamine 11 by the three-
1
1
to give 8a and
b
was followed by H NMR spectroscopy in step process of Faroux-Corlay et al.,28 involving protection of
CDCl3 at room temperature and proved to have almost exactly the amine as its imine with anisaldehyde. The diazoacetyl
the same rate as for the cycloaddition of with ( )-COD derivative (Ac4GalNDiaz, 13) was then synthesised using NHS
with a second-order rate constant of 0.043 ± 0.004 M-1 s-1. This ester
(Fig. 3A). In metabolic labelling of glycans, the sugar
-peracetylated form as
1
E
,
E
2,
9
cycloaddition of diazo compounds with strained alkynes has precursor is normally administered in
O
only recently been reported by McGrath and Raines.26
this increases cellular uptake; the sugars are deacylated inside
the cell by endogenous esterases.29
Bioorthogonal labelling of diazo-modified sugars and proteins
The bioorthogonality of the diazo group was tested by
incubating ethyl diazoacetate with 10 mM glutathione at pH 7.4
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pathway or of glycan biosynthesis, even though the diazoacetyl
group is smaller than the azidoacetyl group. However it is a
different shape, preferring a planar conformation, and this may
be detrimental to binding to some enzymes. Alternatively it
may be that the
N-diazoacetylgalactosamine is diverted into a
different pathway, involving either enzymic degradation or
incorporation into some intracellular entity. It may well be that
other diazo-labelled sugars would be better incorporated - the
enzymes required for incorporation of N-acetylmannosamine
Fig. 2. Fluorescence-labelling of diazo-modified lysozyme. The star
represents the fluorophore (fluorescein)
Lewis lung carcinoma (LL2) cells were incubated with a
medium containing 0 or 200 µM of the diazo sugar for 24 h. To
detect diazo groups incorporated into the cell-surface glycans,
the cells were washed, treated with TMDIBO-PEG-biotin (Fig.
3B) for 30 min, washed again, treated with Neutravidin-
Dylight680 for 15 min, washed again, and analysed by flow
cytometry. This two-step labelling procedure, labelling first
with biotin and then visualising this with fluorescently labelled
neutravidin gives an improved signal-to-background ratio
compared to a one-step procedure using, for example, a
TMDIBO-fluorophore conjugate such as 10 30
Furthermore,
.
Neutravidin, being a large protein, is completely impermeable
to the cells. Therefore any increase in fluorescence must be due
to diazo groups on the cell-surface and not intracellular diazo
groups.
The flow cytometry results showed a clear increase in
fluorescence in cells incubated with the diazosugar compared to
untreated cells, As shown in Fig. 3C. the mean fluorescence
almost doubled (see ESI† for a representative histogram). This
shows that some diazo groups have survived and been
incorporated into cell-surface glycans. However the level of
labelling is much lower than one would typically expect with
the equivalent azido sugar.25 The reason(s) for this are not
known at present. N-diazoacetylgalactosamine may be a poor
substrate for one or more enzymes of the galactosamine salvage
Fig. 3. (A) Synthesis of tetra-O-acetyl-N-diazoacetylgalactosamine 13;
(B) Detection of diazo-modified glycans on LL2 cancer cells surface
(C) Flow cytometry results. M.F.I. = mean fluorescence intensity in the far-
red channel. Error bars represent standard deviation for three repeats.
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