The Photocatalyzed Thiol-ene reaction: A New Tag to Yield Fast, Selective and reversible Paramagnetic Tagging of Proteins

Article abstract of DOI:10.1002/cphc.202000071

Paramagnetic restraints have been used in biomolecular NMR for the last three decades to elucidate and refine biomolecular structures, but also to characterize protein-ligand interactions. A common technique to generate such restraints in proteins, which do not naturally contain a (paramagnetic) metal, consists in the attachment to the protein of a lanthanide-binding-tag (LBT). In order to design such LBTs, it is important to consider the efficiency and stability of the conjugation, the geometry of the complex (conformational exchanges and coordination) and the chemical inertness of the ligand. Here we describe a photo-catalyzed thiol-ene reaction for the cysteine-selective paramagnetic tagging of proteins. As a model, we designed an LBT with a vinyl-pyridine moiety which was used to attach our tag to the protein GB1 in fast and irreversible fashion. Our tag T1 yields magnetic susceptibility tensors of significant size with different lanthanides and has been characterized using NMR and relaxometry measurements.

Full text of DOI:10.1002/cphc.202000071

Accepted Article
Title: The photo-catalysed thiol-ene reaction: a new tag to yield fast,
selective and irreversible paramagnetic tagging of proteins
Authors: Maxime Denis, Charlotte Softley, Stefano Giuntini, Matteo
Gentili, Enrico Ravera, Giacomo Parigi, Marco Fragai,
Grzegorz Popowicz, Michael Sattler, Claudio Luchinat, Linda
Cerofolini, and Cristina Nativi
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To be cited as: ChemPhysChem 10.1002/cphc.202000071
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ChemPhysChem
10.1002/cphc.202000071
The photo-catalysed thiol-ene reaction: a new tag to yield fast,
selective and irreversible paramagnetic tagging of proteins
a,b
c,d
b,e
a
Maxime Denis , Charlotte Softley , Stefano Giuntini , Matteo Gentili , Enrico
b,e
b,e
b,e
d
c,d
Ravera , Giacomo Parigi , Marco Fragai , Grzegorz Popowicz , Michael Sattler ,
b,e
e*
b*
Claudio Luchinat , Linda Cerofolini , Cristina Nativi
a
b
Giotto Biotech, S.R.L, Via Madonna del piano 6, 50019, Sesto Fiorentino (FI) Italy ; Department of
Chemistry “Ugo Schiff”, University of Florence, Via della Lastruccia 3, 50019, Sesto Fiorentino (FI),
c
Italy; Biomolecular NMR, Department Chemie, Technical University of Munich, Lichtenbergstrasse 4,
d
8
5747, Garching, Germany, Institute of Structural Biology, Helmholtz Center Munich, Neuherberg,
e
Germany, Magnetic Resonance Center (CERM), University of Florence, and Consorzio
Interuniversitario Risonanze Magnetiche di Metalloproteine (C.I.R.M.M.P), Via L. Sacconi 6, 50019
Sesto FIorentino (FI), Italy
*to whom correspondence should be addressed: cerofolini@cerm.unifi.it, cristina.nativi@unifi.it
Paramagnetic restraints have been used in biomolecular NMR for the last three
decades to elucidate and refine biomolecular structures, but also to characterize
protein-ligand interactions. A common technique to generate such restraints in
proteins, which do not naturally contain a (paramagnetic) metal, consists in the
attachment to the protein of a lanthanide-binding-tag (LBT).
In order to design such LBTs, it is important to consider the efficiency and
stability of the conjugation, the geometry of the complex (conformational exchanges
and coordination) and the chemical inertness of the ligand. Here we describe a photo-
catalyzed thiol-ene reaction for the cysteine-selective paramagnetic tagging of
proteins. As a model, we designed an LBT with a vinyl-pyridine moiety which was used
to attach our tag to the protein GB1 in fast and irreversible fashion. Our tag T1 yields
magnetic susceptibility tensors of significant size with different lanthanides and has
been characterized using NMR and relaxometry measurements.
1
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ChemPhysChem
10.1002/cphc.202000071
paramagnetic by attaching metal
binding peptides or organic small
INTRODUCTION
Nuclear magnetic resonance
NMR) is a powerful technique used for
the study of biomacromolecules in
solution. Indeed, it offers the
opportunity to investigate structure,
behavior, internal motions and
synthetic
ligands
chelating
(
paramagnetic metal ions[28–33]. The idea
is to specifically attach the metal at a
chosen position to monitor protein
dynamics or protein-protein and
[34]
protein-ligand interactions .
mechanism of action of proteins, as well
as their interactions with small
molecules and other biomolecules at
To generate such restraints, one
often relies on chemically synthesized
[28]
the atomic level[
1,2]
lanthanide-binding tags (LBTs) . An
important feature of LTBs is their ability
to react quickly and selectively with a
given amino acid in the protein. In
addition, it is desirable that the metal
coordination is rigid and avoids the
presence of stereoisomers upon protein
conjugation, which may complicate
NMR data analysis. Apart from non-
natural amino acids, paramagnetic tags
.
Structure calculation by NMR
mainly relies on the collection of short-
range distance restraints (up to ~5-6 Å)
provided by the time-consuming and
troublesome analysis of NOESY
spectra. The use of long-range
paramagnetic distance restraints (up to
~
40 Å), such as pseudo-contact shifts
PCS), induced by a paramagnetic ion,
has been widely proposed to help in de
(
generally
target
cysteines.
The
standard strategy is the conjugation by
formation of a disulfide bond: this
technique has been widely used but it is
[
3–
novo structure determination by NMR
8
]
or in the refinement of pre-existing X-
ray structures [
9–11]
. In this way, a more
hampered by the stability of the bond
reliable model describing the protein in
[35,36]
formed
. Thus, in recent years,
solution can be obtained from the
much effort has been concentrated on
the design of paramagnetic tags that
are stable under reducing conditions
crystal structure [
12,13]
.
Furthermore, PCS restraints,
together with residual dipolar coupling
[
37,38]
once attached to the protein
which could also be used in cells . For
this purpose, copper-catalyzed
azide−alkyne “click” cycloadditions,
which, however, require the
introduction of non-natural amino acids
, and
[39]
(
RDC) restraints that originate from the
same paramagnetic susceptibility
tensor, have been exploited in the
analysis of the internal dynamics of
(
multi-domain proteins [
14–21]
and in the
investigation of the interaction of
proteins with their partners or ligands
[40]
i.e. p- azido-phenylalanine
in the
protein sequence), and thiol-ene
[22–24]
.
[41]
coupling (TEC)
(which relies on
One approach to incorporate
existing or mutated cysteine residues)
have emerged as useful alternative
tools in protein chemical conjugation. In
particular, thiol-ene coupling creates,
through a radical mechanism, a stable
thioether C-S bond between the free
thiol of a cysteine and a double bonded
paramagnetic metal ions into metallo-
proteins is via the exchange of the
naturally occurring diamagnetic metal
[25–27]
ion
. As a more widely applicable
alternative, any protein, not necessarily
binding metal ions, can be made
2
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ChemPhysChem
10.1002/cphc.202000071
carbon in an irreversible fashion. This
reaction can be spontaneous but may
We have tested the new tag with
GB1 as a model protein with a single
engineered cysteine residue. The effect
of the designed LBT, chelating different
[42]
also be photo-catalyzed . There are a
few examples of the use of this reaction
for the paramagnetic tagging of
3+
3+
paramagnetic metal ions (Lu , Yb
[43]
3+
proteins
but the reactions are slow
and Dy ), on the resonances of the
protein has been investigated by
(
overnight). Here, we describe a new
vinyl-pyridine-based paramagnetic tag
and its conjugation to proteins via the
presented photo-catalyzed thiol-ene
reaction to demonstrate that this
reaction increases the applicability of
paramagnetic tagging by making it
suitable for proteins that are not stable
for extended periods of time in vitro.
solution
NMR.
Relaxometry
measurements were also performed to
obtain information on the lanthanide
coordination sites in T1(Ln).
RESULTS AND DISCUSSION
To design LBTs, it is important to
consider the geometry of the complex
and the chemical inertness of the
Synthesis of T1
In order to synthesize T1, a picolinic-
acid-like intermediate bearing the
double-bond for conjugation and a
leaving group for cyclen alkylation was
needed. A was designated as a key
intermediate in the synthesis. Derivative
A was synthesized from commercial
chelidamic acid (fig. 1). The carboxylic
acids of chelidamic acid were protected
as methyl esters and position 4 was
brominated with tetrabutylammonium
[44]
ligand . ((2S,2’S,2’’S,2’’’S)-1,1’,1’’,1’’’-
1,4,7,10-tetraazacyclo
dodecane-1,4,7,10-tetrayl)tetrakis
propan-2-ol)), (S)-THP has been
proposed as simple yet
enantiomerically pure platform for the
(
(
a
[45]
[46]
design of LBTs . Hence, we have
designed a THP-like LBT (T1) to be
conjugated to proteins via TEC through
a vinyl-pyridine group, as the single
point of attachment.
bromide (TBAB) and P
2 5
O .
Br
N
Br
OH
c
a,b
O
O
O
O
O
N
N
OH
OH
O
O
OH
O
d
e
O
O
N
N
Cl
O
OH
O
A
Figure 1: Synthesis of intermediate A
a. H
2
SO , MeOH, 18 h, 98%. b. P , TBAB, Tol, 3 h, 97%. c. NaBH , MeOH, 75% d. trivinyl-
4
2
O
5
4
boroxin, K PO , JohnPhos, Pd(dba)
3
4
2
, dioxane, 2 h, 68% e. MsCl, DIPEA, DCM, 95%
3
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ChemPhysChem
10.1002/cphc.202000071
NH HN
NH HN
NH
N
a
N
O
N
NH HN
O
O
Cl
O
A
B
b,c
OH
OH
N
N
N
Ln3+
d
N
N
N
N
N
HO
HO
N
O
N
O
HO
O
HO
HO
T1
C
Figure 2: Synthesis of T1
a. DCM, 3 days b. (S)-propylen oxide, MeOH, 4 days, quant. c. LiOH, THF/H
2
O, 18 h, quant.
d. LnCl nH O, H O/MeCN
3
2
2
The reduction of a single methyl ester
was easily achieved thanks to the
peculiar reactivity of pyridine 2,6
diesters. The double bond in position 4
was then introduced via a Suzuki-like
stable under many conditions and was
engineered to bear one cysteine. Vinyl
picolinic acids have been shown to
react through TEC with cysteines
[
43],[48]
overnight by Su et al
, but this
[47]
coupling
and the benzylic hydroxyl
reaction is quite slow (overnight). To
reduce the reaction time, we used a
photo-activated radical initiator, 2,2-
dimethoxy-2-phenylacetophenone
(DPAP), which has been shown to
catalyze TEC when activated with UV at
365 nm.
was subsequently replaced by a
chloride as a good leaving group, giving
A with a yield of 50% calculated over 5
steps.
The intermediate A was then added to
an excess of cyclen to isolate the mono
functionalized cyclen derivative B (fig.
To a solution of GB1, 5 equiv. of tag and
2
). B was subsequently alkylated using
5
equiv. of DPAP were added. The
mixture was irradiated by a UV lamp at
65 nm for 1 hour after which the
sample was analyzed by NMR. We
(
S)-propylene oxide and the methyl
ester was deprotected to yield C.
Lanthanide-chelation was achieved
quantitatively, by refluxing overnight C
3
3+
3+
used T1 loaded with Lu , Yb and
Dy . The protein tagging was
in the presence of LnCl
O/MeCN mixture.
3
salts in a
3+
H
2
1
15
evaluated recording 2D H- N HSQC
spectra which showed after 1 hour a
conjugation of 80% (fig. 3). The reaction
yield was evaluated from the relative
intensity of the signals of free and
Conjugation to GB1 T53C
In order to test our LBT we chose the
GB1 T53C mutant as a model system.
GB1 is a small globular protein which is
1
15
conjugated protein. In each 2D H- N
HSQC, only one set of new peaks was
4
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ChemPhysChem
10.1002/cphc.202000071
S-GB1
OH
OH
DPAP
nm
N
N
N
Ln3+
N
N
N
Ln3+
UV 365
N
N
+
GB1-SH
HO
HO
1h
N
O
N
O
80%
O
O
HO
HO
Figure 3: Tagging of GB1 with T1 using the thiol-ene reaction.
observed, indicating that no side-
reaction or protein degradation took
place. We can assume that the reaction
rate depends on the accessibility of the
cysteine and on the stability of the
corresponding thiyl species and may
require protein-specific optimization.
We can indeed expect that buried
cysteines might not react under these
conditions. For example, attempts to
signals, which shifted after the addition
of the diamagnetic compound
correspond to residues located close to
the tagging-site (c.f. S.I. fig. S2). We
note, that for each cross-peak in the 2D
1
15
H– N HSQC spectrum of diamagnetic
samples only a single cross-peak is
observed for the paramagnetic species.
We therefore conclude that T1 is indeed
present as a single stereoisomer or a
single conformation, giving rise to a
single set of paramagnetically shifted
peaks.
functionalize another protein, the small
N-terminal domain of PEX14[
49,50]
were
apparently hampered by the reduced
solvent accessibility of the target
cysteine on the protein surface and
steric hindrance of the tag (see
Supporting Information for more
details). More radical initiator/UV
wavelength couples could also be
explored.
The PCS is described as a function of
both the distance and orientation of the
electron-nuclear spin vector and the
magnitude of the anisotropy tensor
[51,52]
(
ꢀꢁ)
, according to the equation
1
2ꢃꢄ3
ꢂpc
=
2
�Δꢅ (3cos ꢈ − 1)
ꢆꢇ
1
3
2
2
+
Δꢅ sin ꢈcos2ꢊꢋ
ꢉℎ
Evaluation of paramagnetic effects
where r, ꢈ and ꢊ are the spherical
coordinates of the nucleus in the frame
in which the anisotropy tensor is
diagonal and has its origin at the metal
position. Δꢅꢆꢇ and Δꢅꢉℎ are the axial
and rhombic components of the tensor,
defined as
The tagging of the T53C mono-cysteine
variant of the GB1 protein with
paramagnetic [T1(Yb)] and [T1(Dy)]
1
15
resulted in PCS in the 2D H– N HSQC
spectrum (fig. 4). As expected, the two
paramagnetic metal ions provide shifts
[25]
in opposite directions . In order to
differentiate between the chemical shift
perturbation stemming from the tagging
itself and the paramagnetic contribution
to the shifts (PCS), we also tagged GB1
with the diamagnetic [T1(Lu)]. The NMR
ꢅ + ꢅ
ꢇꢇ
ꢍꢍ
Δꢅꢆꢇ = ꢅꢌꢌ −
2
Δꢅꢉℎ = ꢅ − ꢅ
ꢇꢇ
ꢍꢍ
5
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ChemPhysChem
10.1002/cphc.202000071
1
15
Figure 4: 2D H– N HSQC of GB1 T53C tagged with [T1(Lu)] (black), [T1(Yb)] (blue) and
T1(Dy)] (red), acquired at 700 MHz and 298 K. In the insert, in the top-left corner, a zoom on
[
residue G41 is shown. Interestingly, [T1(Yb)] and [T1(Dy)] provide shifts in the same directions
for W43, because this residue is located at the interface between the negative and positive
PCSs iso-surfaces.
The program FANTEN[53] was used to
obtain the best fit ꢀꢁ tensor (consisting
of 5 parameters: Δꢅ , Δꢅ , the three
Euler angles defining the frame in which
the tensor is diagonal) and the distance
of the metal ion to the nuclear spins
observed.
calculations, the position of the metal
was obtained. New NMR signals could
then be assigned, taking advantage of
the PCS values predicted by the
program for the other nuclei, so that a
ꢆꢇ
ꢉℎ
N
total of 36 H PCSs were obtained for
the [T1(Yb)] tagged protein.
First, the resonances of 19 signals in
the spectra of the protein tagged with
The assignment of the spectrum of the
protein tagged with [T1(Dy)] was
obtained by comparison with the
assigned spectra of the [T1(Yb)] and
[T1(Lu)] tagged proteins (fig. 4); in this
way 32 PCS were obtained for [T1(Dy)].
[T1(Yb)] were unambiguously assigned,
and the PCS were evaluated. The
anisotropy tensor for the Yb3+ tagged
protein was determined from the best fit
N
of these 19 H PCS to the X-ray
The PCS originating from [T1(Yb)] and
[54]
structure (PDB 1IGD) , using the
program FANTEN. During these tensor
[T1(Dy)] were first analyzed separately
(
fig. S3). The metal positions obtained
6
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ChemPhysChem
10.1002/cphc.202000071
from the two sets of data were similar
the metal is found at 8.6 and 8.1 Å,
from the Cβ of the cysteine residue, for
T1(Dy)] and [T1(Yb)], respectively), as
well as the direction of the main axes of
the best fit tensors (fig. 5A and 5B).
with the expected covalent structure of
the tag. The orientations and
magnitudes of the two tensors were
almost unaffected with respect to the
values obtained from the separate fits,
thus showing high consistency between
the two sets of paramagnetic data
(
[
(
Table 1 and fig. 5). The two tensors are
almost coaxial, with angles of 11.5°
between the two Z-axes, 5° between
the two X-axes and 12° between the two
Y-axes.
Table 1. Tensor parameters calculated with
the program FANTEN using the PCS
values measured with [T1(Yb)] and
[
T1(Dy)] implemented separately or jointly
in the evaluation of the metal position.
-
-
Metal
Yb
PCS
source
Yb
Q
factor
∆χax (10
∆χrh (10
3
2
3
32
3
m )
1.91 ±
.02
-6.51 ±
.02
m )
0.68 ±
0.25
2.54 ±
0.89
0.061
0.072
0.064
0.072
0
Dy
Dy
Figure 5. Graphical representation of the
magnetic susceptibility anisotropy tensor
0
Yb
Yb &
Dy
Yb &
Dy
1.83 ±
0.03
-0.59 ±
0.28
orientations. The x,
y
and
z
axes
Dy
-6.81 ±
0.21
3.06 ±
0.89
(corresponding in turn to the directions with
the smallest, intermediate and largest
magnetic susceptibility) are represented as
red, yellow and blue arrows, respectively.
The panels A and C show the orientation of
Yb tensors when only its own PCS dataset
was considered, and when the datasets of
Yb and Dy were both taken into account in
FANTEN, respectively. The panels B and D
show the orientation of the Dy tensors when
only its PCS-data-set was considered, and
when data sets of Yb and Dy where both
The agreement between experimental
and back-calculated PCS obtained from
the fit of both sets of data is very good,
with Q factors of 0.064 and 0.072, for
[T1(Yb)] and [T1(Dy)], respectively (fig.
S4). The PCS iso-surfaces for the two
metals are represented in figure 6.
It has been shown that some
LBTs can be immobilized on the protein
surface by an electrostatic interaction
between the lanthanide cage and a
carboxylate belonging to an Asp or Glu
taken
into
account
in
FANTEN,
respectively.
Then, the two sets of PCSs were
evaluated jointly by constraining both
metals to reside in the same position.
The final position of the metal, obtained
by FANTEN (8.3 Å from the Cβ of the
cysteine residue), is very close to the
positions found from the sets of data
analyzed separately, and compatible
[43,55]
residue
.
This interaction may
decrease the tag mobility and therefore
increase the magnitude of the magnetic
susceptibility anisotropies. In the case
of GB1 T53C, there are two
carboxylates at distances of 9 and 11 Å
7
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ChemPhysChem
10.1002/cphc.202000071
Figure 6. Graphical representation of PCS iso-surfaces of 1 (blue) and -1 (red) ppm obtained
3+
3+
for Yb -T1 (A) and Dy -T1 (B) using the program FANTEN.
from the cysteine (E42, E56),
respectively. We therefore expected to
see such a stabilizing interaction taking
place between our tag and our protein.
However, we find that the axial
anisotropies of the two tensors are 4-5-
fold reduced compared to the values
expected for the anisotropies of the
magnetic susceptibility tensors of the
in agreement with the small tensor
anisotropies observed. We suppose
that in T1 the lanthanide ions have a
coordination number of 9 and have no
further coordination site with which to
interact with the carboxylate positioned
on the protein surface.
The sizable mobility of the tag may be
attributed to the two rotatable bonds,
after the pyridine moiety, that tether the
tag to the protein surface, which are
absent in similar tags reported in the
3+
3+
Yb and Dy ions usually calculated
from rigid systems containing these
paramagnetic ions[
56,57]
. This likely
indicates that some motional averaging
of the magnetic susceptibility tensors
occurred, so that the best fit tensors
result from the averaging of the
[58]
literature (such as DO3MA ). An
additional methyl group on the pyridine
moiety (i.e. in position 3 or 5) could have
possibly restrained the rearrangement
of the pyridine and stabilize the tag.
Moreover, a different attachment site on
the protein surface could have affected
differently the tensor parameter values
magnetic
susceptibility
anisotropy
tensors due to tag mobility.
Interestingly, the position of the metal
calculated with FANTEN is relatively far
from the protein surface. This
observation corroborates the initial
design of the tag to not interact with the
protein surface, thus being very mobile,
because
of
different
possible
protein/tag interaction (steric hindrance
and/or hydrophobic interactions) which
might rigidify the tag.
8
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ChemPhysChem
10.1002/cphc.202000071
Relaxometry
5
4
3
2
1
In order to characterize the coordination
of the lanthanides in T1, Fast Field
Cycling (FFC) relaxometry experiments
were performed[
59–62]
. The H nuclear
1
magnetic relaxation dispersion (NMRD)
profiles of the [T1(Gd)] complex in water
solution at 10, 25 and 37 °C are shown
in figure 7. The profiles are
characterized by dispersions somewhat
smoother than predicted by the
Lorentzian spectral density function;
however, any attempt to reproduce
them by considering inner sphere and
outer-sphere contributions failed (with
the diffusion coefficients constrained to
values in the range expected for water
solutions) unless the distance of closest
approach between paramagnetic ion
and diffusive water molecules was
larger than 8 Å. Therefore, no sizable
T1(Gd)
10 °C
2
3
5 °C
7 °C
0
0
.01
0.1
1
10
100
Proton Larmor frequency (MHz)
Figure 7: NMRD Profiles of [T1(Gd)]
measured at different temperatures.
The best fit parameters are reported in
Table 2 and the corresponding profiles
are shown in figure 7. Due to the low
sensitivity to the correlation time τ
electron relaxation, the values of τ
contribution
from
outer-sphere
v
for
relaxation is apparent, and the profiles
were fit by including two protons at a
v
were kept fixed to values typically
observed in gadolinium complexes
distance r
distance r
1
and other two protons at a
3
+
2
from the Gd ion (r
1
and r
2
[
63,64]
.
Although there exists some
covariance among the different
parameters, the analysis indicates that
the lifetime of the two protons at r
.05 Å, τM(1), is as long as several
were left free to be adjusted in the best
fit analysis).
Table 2. Best fit parameters obtained from
the NMRD profiles of [T1(Gd)], shown in
figure 7. Contributions from two protons in
1
=
3
microseconds (ca. 4 µs at 25 °C). The
analysis also shows contributions from
fast exchanging second-sphere water
molecules, with a lifetime on the
picosecond timescale. The number of
these second-sphere water molecules
is totally covariant with the metal-proton
the
first-coordination
and
second-
coordination spheres were considered.
3
35
7 °C
25 °C
57
10 °C
112
τ
R
(ps)
∆
t
0
.023
distance r ; if one water molecule is
considered, r is about 3.5 Å.
−
1
2
(
cm )
2
τ
v
(ps)
(Å)
15
20
3.05
25
r
1
Fits of equivalent quality, however, can
be obtained also for increasing values
τ
M(1)
3
8
.4
.8
3.8
3.48
18
4.6
(
µs)
of r
1
(and decreasing values of r
2
).
r
2
(Å)
M(2)
When the condition r
1
= r = 3.3 Å is met,
2
τ
51
(
ps)
9
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ChemPhysChem
10.1002/cphc.202000071
almost indistinguishable best fit profiles
are calculated, the complex
reorientation time τ , the transient ZFS
and the lifetimes τM(1) and τM(2) being
somewhat increased (τ = 84 ps, ∆
CONCLUSIONS
R
∆
t
In this work, we present the synthesis
and the performance of an enantiopure
paramagnetic
spectroscopy, designed to efficiently
conjugate proteins under mild
R
t
=
-1
tag
for
NMR
0
.027 cm , τM(1) = 5.3 µs and τM(2) = 28
ps, at 25 °C).
The fast exchanging second-sphere
water protons may be related to one or
more water molecules hydrogen
bonded in positions allowing for a large
mobility.
conditions using UV irradiation. In
particular, we have demonstrated that
UV-catalyzed TEC is applicable to the
paramagnetic tagging of GB1 T53C
with a high yield and short reaction
times. The creation of the stable
thioether bond required much shorter
The presence of two protons
exchanging in the microsecond time
scale at 3.05 Å, i.e. at the distance
expected for the protons of a water
molecule regularly coordinated to the
reactions
times
than
previously
described methods[43,67].
The conjugation involves the protein’s
cysteine side-chain and forms thioether
3+
Gd ion, or at a somewhat larger
distance (such as 3.3 Å), could be
related to one water molecule
hydrogen-bonded to the oxygen atoms
of hydroxyl groups or of the carboxylate
group. Its lifetime is much longer
bonds
without
affecting
the
paramagnetic properties of the tag.
Being easily obtained, and in high
yields, the formation of non-reducible
thioether bonds is convenient and
makes this strategy a method of choice
in the field of paramagnetic tagging,
opening the way to development of
more novel and rigid paramagnetic
tags. Those tags could be applied to the
study of larger systems, where the
PCSs analysis could be complemented
by the use of residual dipolar couplings
(
several microseconds) than the lifetime
of the water molecule coordinated to the
3+
Gd ion in DO3A-like or DOTA-like
complexes (on the sub-microsecond
timescale), likely because of the
bipositive charge of this complex which
disfavours water exchange with respect
to neutral or negative complexes[
65,66]
. A
long lifetime of this water molecule
indicates a relatively “compact” and
stable environment surrounding the
and
paramagnetic
relaxation
[68,69]
enhancements measurements
.
3+
Gd
ion, which may prevent the
replacement of the coordinated water
by negatively charged groups present
on the protein surface. This is in
agreement with the observed tag
mobility that affects the magnitude of
EXPERIMENTAL SECTION
Organic synthesis
All the reagents were purchased from
Sigma-Alrich, except cyclen which was
purchased from Chematech. Small-
molecules NMR experiments were
recorded on a Bruker AVANCE II 500
the
PCS-determined
tensor.
Alternatively, these slowly exchanging
protons may be the hydrogen atoms of
the three hydroxyl groups coordinated
1
MHz ( H Larmor frequency) at 298 K.
3+
to the Gd ion.
HPLC was performed with an Agilent
10
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ChemPhysChem
10.1002/cphc.202000071
1
200 Sries with ZORBAX 300SB-18
with the program CARA[70] (ETH
Zürich).
The spectra were collected using a
protein concentration of ~ 180 µM in
buffered solution (20 mM sodium
phosphate pH 7.5).
analytica and semi-preparative. Mass
spectrometry was performed on a
Thermo-Fisher LTQ-XL ESI.
Formation of Lanthanide Complexes
C (70 mg) and LnCl
were dissolved in 6 mL H
50/50). The pH was adjusted to 7 and
3
nH
2
O (100 mg)
The assignment of GB1 was taken from
the literature .
[71]
2
O/MeCN
(
the mixture was refluxed. Chelation was
typically quantitative overnight, as
shown by LC/MS analysis. Reaction
mixtures were purified via semi-
preparative HPLC.
The PCS values were calculated from
the difference in the value of chemical
shift of each amino acid peak between
the paramagnetic [T1(Yb) or T1(Dy)]
and diamagnetic [T1(Lu)] 2D H- N
HSQC spectra acquired.
1
15
Protein Conjugation
The fitting of the PCS tensor was
carried out using the program
GB1 T53C was expressed and purified
as reported in the supplementary
information. Prior to conjugation, the
protein was buffer-exchanged into NaPi
[
53]
FANTEN .
ACKNOWLEDGMENTS
2
0 mM, pH 7.5, then concentrated to
1
80 μM. 5 equivalents each of DPAP
and [T1(Ln)] were added to the protein,
as well as 10% D2O. The mixture was
transferred into a 5-mm NMR tube and
argon was gently bubbled through the
solution for 5 minutes. The tube was
placed under a UV Lamp (UVGL-55
Mineralight 26W) at 365 nm for 1 h, after
which the sample was measured using
NMR spectroscopy and the spectra
analyzed. Excess of small molecules
were then washed away by buffer
exchange and NMR spectra were
recorded again.
This worked received funding from the
European Union’s framework program
for Research and Innovation Horizon
2
020 (2014-2020) under the Marie–
Slodowska Curie Grant agreement
number 675555, Accelerated Early-
staGe drug dIScovery (AEGIS). The
authors also acknowledge MIUR Italy
PRIN
2012SK7ASN,
“Progetto
Dipartimenti di Eccellenza 2018–2022”
to the Department of Chemistry “Ugo
Schiff”, the Fondazione Cassa di
Risparmio di Firenze, the COST Action
CA15209 (EURELAX) and iNEXT,
Grant number 653706, funded by the
Horizon 2020 program of the European
Union.
NMR measurements and PCS
analysis
All the experiments were acquired on a
Bruker
AVANCE
NEO
NMR
1
spectrometer operating at 700 MHz ( H
Larmor frequency) at 298 K equipped
with a 5 mm TCI 3 channels HCN cryo-
probehead. All the spectra were
processed with the Bruker TopSpin
Keywords Paramagnetic tags, photo-
catalyzed thiol-ene reaction, lanthanide
coordination,
Biomolecular
NMR,
protein structure refinement.
4
.0.7 software package and analysed
11
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ChemPhysChem
10.1002/cphc.202000071
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10.1002/cphc.202000071
Table of content.
A fast and irreversible tagging of GB1 T53C mutant, through the creation of a thioether
bond between the tag alkene and the free thiol of the cysteine, has been obtained
using for the first time a photo-catalyzed thiol-ene reaction. The paramagnetic effects
on the protein NMR spectra were measured and analysed and the tag coordination
studied through relaxometry studies.
16
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