Inverse electron-demand Diels-Alder (iEDDA) bioorthogonal conjugation of half-sandwich transition metallocarbonyl entities to a model protein

Article abstract of DOI:10.1002/aoc.5507

Novel transition metallocarbonyl complexes carrying a norbornene or an oxanorbornene group were synthesized by [4 + 2] cycloaddition between the organometallic maleimide dienophiles and cyclopentadiene or furan, respectively. The oxanorbornene adduct was obtained as a mixture of endo and exo isomers as confirmed by X-ray diffraction and NMR spectroscopy. The (oxa)norbornene groups further provided convenient chemical reporters to carry out inverse electron demand Diels-Alder (iEDDA) reactions with tetrazine derivatives. Detailed kinetic studies with a model tetrazine revealed that faster rates of reaction were determined with both isomers of the oxanorbornene complex with respect to the norbornene complexes. Eventually, incorporation of metallocarbonyl entities into bovine serum albumin equipped with tetrazine handles was achieved as shown by IR spectroscopy of the protein conjugates.

Full text of DOI:10.1002/aoc.5507

Received: 14 October 2019
DOI: 10.1002/aoc.5507
Revised: 3 December 2019
Accepted: 7 December 2019
F U L L P A P E R
Inverse electron-demand Diels-Alder (iEDDA)
bioorthogonal conjugation of half-sandwich transition
metallocarbonyl entities to a model protein
1
2
3
Daria Jamroz | Nathalie Fischer-Durand
| Marcin Palusiak
|
4
| Szymon Jarzy nꢀ ski1
1
Sławomir Wojtulewski
Michèle Salmain
| Marlena Stępniewska
|
2
1
| Bogna Rudolf
1
Department of Organic Chemistry,
Novel transition metallocarbonyl complexes carrying a norbornene or an
oxanorbornene group were synthesized by [4 + 2] cycloaddition between the
organometallic maleimide dienophiles and cyclopentadiene or furan, respec-
tively. The oxanorbornene adduct was obtained as a mixture of endo and exo
isomers as confirmed by X-ray diffraction and NMR spectroscopy. The (oxa)
norbornene groups further provided convenient chemical reporters to carry
out inverse electron demand Diels-Alder (iEDDA) reactions with tetrazine
derivatives. Detailed kinetic studies with a model tetrazine revealed that faster
rates of reaction were determined with both isomers of the oxanorbornene
complex with respect to the norbornene complexes. Eventually, incorporation
of metallocarbonyl entities into bovine serum albumin equipped with tetrazine
handles was achieved as shown by IR spectroscopy of the protein conjugates.
Faculty of Chemistry, University of Lodz,
91–403 Lodz, Poland
2
Sorbonne Université, CNRS, Institut
Parisien de Chimie Moléculaire (IPCM),
75005 Paris, France
3
Department of Physical Chemistry,
Faculty of Chemistry, University of Lodz,
90–236 Lodz, Poland
4
Institute of Chemistry, University of
Bialystok, Ciolkowskiego 1 K, 15–245
Bialystok, Poland
Correspondence
Bogna Rudolf, Department of Organic
Chemistry, Faculty of Chemistry,
University of Lodz, 91–403 Lodz, Poland.
Email: bogna.rudolf@chemia.uni.lodz.pl
K E Y W O R D S
iEDDA, norbornene, tetrazine, metallocarbonyl complex, bioconjugation
Funding information
European Union; Sorbonne Université,
France; Faculty of Chemistry, University
of Lodz, Poland; Centre National de
Recherche Scientifique (CNRS)
1
| INTRODUCTION
reaction owing to the release of dinitrogen during the
process through a retro-Diels-Alder reaction (Scheme 1).
Among all the bioorthogonal methodologies introduced
to date, inverse electron demand Diels-Alder (iEDDA)
reactions between 1,2,4,5-tetrazines and alkenes/alkynes
iEDDA is best accomplished using strained and/or elec-
tron-rich olefins as dienophiles affording stable
dihydropyrizadines in good yield.
[1]
appear as one of the most prominent strategies. iEDDA
combines high chemoselectivity and fast reaction rates
and what is more important, does not require metal catal-
ysis conversely to copper-catalyzed alkyne-azide cycload-
dition (CuAAC). This allows reactions to take place in
complex media even inside living cells without toxic
effects. Furthermore, iEDDA is an irreversible ligation
process conversely to normal electron demand D-A
Among the various dienophiles reported to date,
norbornenes show interesting features as they are easy to
synthesize, display very good stability in biological
medium and have a relatively small size. Rates of reac-
tion between norbornenes and tetrazines are moderate
−
1 −1
(second order rate constant in the range of 0.1 M .s or
lower) and highly dependent on electronic, steric and ste-
[
2]
reomeric factors on the norbornene side.
Appl Organometal Chem. 2020;e5507.
wileyonlinelibrary.com/journal/aoc
© 2020 John Wiley & Sons, Ltd.
1 of 12
https://doi.org/10.1002/aoc.5507

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JAMROZ ET AL.
TABLE 1 Second order rate constants of iEDDA reaction
between Tz-NHS and various norbornene derivatives
azide handles. While the protein was efficiently labeled
by copper-catalyzed reaction with the terminal alkyne
complex, the uncatalyzed strained alkyne version gave
disappointing results because of the unexpected instabil-
Second order
rate constant
[
3]
−
1 −1
Entry Temperature (K) Norbornene
k
2nd (M .s
)
ity of the metallocarbonyl reagent.
a
Some of us had previously reported the synthesis and
characterization of organometallic norbornenes (Figure 1)
1
2
3
4
5
6
7
8
9
1
296
303
296
303
303
303
303
303
303
303
Nb-OH
0.77
a
Nb-OH
1.7
resulting from the Diels-Alder reaction between CpM
b
1
0.006
0.012
0.0008
0.032
No reaction
0.14
1
(
CO) (η -N-maleimidato (M = Fe, n = 2; M = Mo, W,
n
b
[4]
1
n = 3) complexes as dienophiles and cyclopentadiene.
b
3
In our hands, metallocarbonyl norbornenes revealed infi-
nitely more stable than metallocarbonyl cyclooctynes.
We reckoned that these complexes could be suitable
reagents to perform iEDDA reactions with biomolecules
carrying the partner tetrazine handle.
b
4
b
Suc-NB
2a
2b
0.063
0.063
Surprisingly, literature reports dealing with iEDDA
for labeling of biomolecules with transition organometal-
lic complexes are still rather scarce. Recently, Lo and
coworkers reported a series of phosphorogenic dipyridyl
rhenium(I) tricarbonyl complexes carrying a tetrazine
c
0
Suc-oxNB
a
mixture of endo and exo isomers;
b
endo isomer;
c
mixture of endo and exo isomers in the proportion 2 to 1.
[
5]
handle on one of the ligands. The same team also
reported cyclometalated iridium (III)-based phos-
phorogenic complexes including a tetrazine group either
In our recent efforts to devise bioorthogonal strategies
to conjugate transition metallocarbonyl entities to pro-
teins, we first focused our attention on the [3 + 2] dipolar
azide-alkyne cycloaddition (AAC) reaction. To this aim,
we designed terminal and strained alkyne (cyclooctyne)
[
6]
appended to one of the ligands or directly involved in
[
7]
metal coordination.
In this paper we report the synthesis of three new
metallocarbonyl norbornenes by Diels-Alder reaction
5
derivatives of (η -cyclopentadienyl)iron dicarbonyl
Fp) and studied their reaction with a protein carrying
5
1
(
between [(η -Cp)M (CO) (η -N-maleimidato)] (M = Fe,
2
SCHEME 1 iEDDA between tetrazine and alkene
FIGURE 1 Structure of norbornenes used in this study
SCHEME 2 Synthesis of metallocarbonyl norbornenes 2a, 2b and 3

BIOCONJUGATION OF METALLOCARBONYL INFRARED LABELS BY CLICK CHEMISTRY
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Ru) and cyclopentadiene or furan (Scheme 2). Reactivity
studies of all the metallocarbonyl norbornenes were per-
formed on model tetrazine derivatives and bovine serum
albumin (BSA) carrying tetrazine handles.
6.03 ppm (5.97 ppm for 1) assigned to the two protons of
the olefin bond.
Maleimides also undergo [4 + 2] cycloadditions with
furan derivatives to afford oxanorbornenes. This provides
a simple strategy to protect maleimides as, conversely to
the norbornene products, it is a thermally reversible reac-
tion as the retro-Diels Alder reaction of oxanorbornenes
2
2
| RESULTS AND DISCUSSION
.1 | Synthesis of organometallic
[
10]
occurs at reasonable temperature.
The Diels-Alder
reaction between 1 and furan was performed under simi-
lar experimental conditions (Scheme 2). Two addition
products – endo and exo isomers were formed in equal
proportions as shown by TLC of the reaction mixture.
Previously, the Diels-Alder reaction of furan and
maleimide also produced a mixture of endo and exo iso-
mers which was explained by similar transition state bar-
riers for the formation of both isomers and the process
norbornenes and oxanorbornenes
Compounds 1 and 4 were synthesized by Diels-Alder
5
1
reaction between [(η -Cp)M (CO) (η -N-maleimidato)]
n
(
M = Fe, n = 2; M = Mo, n = 3) and cyclopentadiene as
[4]
previously reported. In both cases, the endo isomer was
solely obtained as shown by the molecular structure of 1.
[
4]
[
9]
The same stereochemical outcome had been previously
observed for the reaction between cyclopentadiene and
being under thermodynamic control.
The two stereoisomers were separated by column
chromatography. We found that the chemical shift of the
protons of carbons C3 (see numbering in Figure 2)
showed the largest difference between the exo and the
endo isomers. The signal for the exo isomer was shifted
upfield by 0.75 ppm. The differences in the chemical
[8]
maleimide. The preference for the endo isomer results
from the process being under kinetic control and the tran-
[9]
sition state barrier being lower for the endo adduct.
The ruthenium analog 3 was newly prepared using
5
1
the same procedure from [(η -Cp)Ru (CO) (η -N-
2
1
maleimidato)] (Scheme 2). A single product was again
shifts for the other protons were insignificant. The H
obtained which was identified as the endo isomer on the
basis of H NMR data (Figure S3) by comparison with
NMR spectra of both isomers showed a characteristic
peak around 5 ppm for Cp-H and a singlet at 6.3 ppm
(endo) and 6.45 ppm (exo) assigned to the protons of the
olefin bond. The chemical shift of the protons of carbons
C6 was observed at 5.16 ppm and 5.20 ppm for the exo
and endo isomers, respectively (Figures S2 and S3).
1
the data of 1. There was no significant difference between
1
the H NMR spectra of 3 and 1. Two doublets at
1
.66 ppm and 1.49 ppm assigned to the protons of the
methylene bridge were observed as well as a singlet at
.28 ppm assigned to the four aliphatic CH protons. Simi-
3
larly, two doublets were found at 1.43 and 1.61 ppm
together with two singlets at 3.17 and 3.23 assigned to the
four aliphatic CH protons of 1. The H NMR spectrum of
2.2 | Crystal structures description
1
Single crystals of 2a (exo) and 2b (endo) were obtained
after slow evaporation of n-heptane/chloroform (1:3)
solution at room temperature.
3
also displayed a singlet at 5.40 ppm for the 5 protons of
the Cp ring (4.97 ppm for Fp-NB) and a singlet at
FIGURE 2 Molecular structure of 2a (exo
form) in the crystal. Ellipsoids correspond to
5
0% probability. Selected bonds (Å) and angles
) Fe1-C30 2.117(3), Fe1-C31 2.1135(14), Fe1-
ꢀ
(
C32 2.088(2), Fe1-centroid 1.717, Fe1-C20 1.780
2), Fe1-N1 1.959(2), N1-C2 1.374(2), C2-C3
(
1
1
.512(3), C3-C3a 1.536(4), C3-C6 1.564(3), C6-C7
.515(3), C7-C7a 1.316(5), C2-O2 1.227(2), C20-
O20 1.145(3), C6-O3 1.448(3), Fe1-C20-O20 177.5
2), C2-N1-C2a 110.6(2), Fe1-N1-C2-O2–10.6(2)
(

4
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JAMROZ ET AL.
The molecular structures of 2a and 2b are presented
2.3 | Computational studies
in Figure 2 and Figure 3. The asymmetric unit of both
crystal structures contains halves of molecular com-
plexes, since the molecules lie on the crystallographic
symmetry mirror plane. The Fe1 atom is bonded to the
cyclopentadienyl ring (C30-C31-C32-C32a-C31a), two
carbonyl ligands (C20-O20 and C20a-O20a) and nitro-
gen atom N1 of pyrrolidine-2,5-dione ring. The bond
lengths between iron atom and the two carbonyl
ligands equal 1.780 Å for 2a and 1.787 Å for 2b. The
distance between iron atom and centroid of the cyclo-
pentadienyl ring equals 1.717 Å and 1.722 Å, respec-
tively. However, the Fe1-N1 bond is significantly longer
and equals 1.959 Å (2a) and 1.964 Å (2b). Hence the
overall coordination sphere around iron can be
described as a tetrahedron strongly deformed in direc-
tion of trigonal pyramid with Fe-Cg apical bond. A sim-
ilar coordination geometry of iron and other transition
metal was found for other cyclopentadienyl complexes
By comparing the energy of endo and exo isomers of
2 one may find that the exo isomer is energetically more
favorable by 2.96 kcal/mol. Nevertheless, experimentally,
the less energetically favorable endo isomer is also
observed, which may be explained by general effect of
[
9,15]
kinetic preference of this isomeric form.
For both
endo and exo isomers of 2 there are two possible con-
formers, that is, syn and anti, as shown in Figure 4. X-ray
studies reveal that the syn conformation of D-A product
[
16]
is more preferable in solid state conditions.
Computa-
tional analysis gives the same observation, the molecular
energy indicates the syn conformer being slightly more
stable by 1.73 kcal/mol and 0.53 kcal/mol for exo and
endo isomers, respectively. One possible explanation of
this minor energy preference would be the presence of
some extra intramolecular interaction in syn conformers
in respect to its anti-counterparts. In fact, analysis of elec-
tron density distribution allows to find such interactions
in syn conformers, as shown in Figure 4. Therefore, a
[4,11–14]
previously studied and reported by us.
The main
difference between the two isomers is the conformation
of the oxabicyclo[2.2.1]heptane ring. The conforma-
tional change is related to the rotation of the cyclo-
pentadienyl ring. The crystal structure of 2a contains a
molecular complex, where the oxabicyclo[2.2.1]heptane
ring is in the exo configuration and the C30-H30 is
directed towards the oxygen atom O3 and the distance
H30 … O3 is 2.821 Å. On the other hand, in the crystal
structure 2b the endo configuration of the oxabicyclo
[17]
bond path with corresponding bond critical point
between Cp ligand and the oxanorbornene can be found
in syn conformer of exo isomer.
This type of interaction is well characterized in the lit-
[
18]
erature.
The C-H … O bond found in syn conformer of
exo isomer has electron density characteristics typical for
other such interactions, with low value of electron den-
sity in bond critical point (ρ = 0.005 au) positive values of
2
[
2.2.1]heptane ring is observed and the cyclopentadienyl
its laplacian (r ρ = 0.018 au) and total electron energy
ꢀ
ring is rotated by 72 deg [ ].
density (H = 0.001au).
FIGURE 3 Molecular structure of 2b (endo
form) in the crystal. Ellipsoids correspond to
5
0% probability. Selected bonds (Å) and angles
ꢀ
(
) Fe1-C30 2.112(2), Fe1-C31 2.1135(14), Fe1-
C32 2.0929(14), Fe1-centroid 1.722, Fe1-C20
.7873(15), Fe1-N1 1.9624(16), N1-C2 1.3747(17),
C2-C3 1.5196(19), C3-C3a 1.536(3), C3-C6 1.564
2), C6-C7 1.519(2), C7-C7a 1.329(4), C2-O2
.2237(18), C20-O20 1.1422(18), C6-O3 1.4471
1
(
1
(
(
17), Fe1-C20-O20 177.12(12), C2-N1-C2a 111.48
16), Fe1-N1-C2-O2 1.1(2)

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FIGURE 4 Molecular
graphs of syn (upper side) and
anti (lower side) conformers of
endo (left side) and exo (right
side) isomers of 2. Larger
spheres correspond to atoms
(
yellow C, red O, pink N, blue H,
brown Fe). Small red and yellow
spheres are bond and ring
critical points. Additional weak
interactions discussed in the text
are marked with dotted lines
(
being the proper bond paths)
In syn conformation of endo complex there is also an
py-Tz was attempted in chloroform. No reaction was
observed with Ph-Tz probably owing to its poor
reactivity.
additional interaction, as indicated by electron density
analysis (see Figure 4b). This time it is a weak C-H … π
contact, which can also be considered as a specific hydro-
gen bond with typical interaction electron density charac-
[
1,21]
Conversely, the reaction of 1 and py-Tz (Scheme 3)
under exclusion of light proceeded steadily at room tem-
perature as the starting complex was fully consumed and
the characteristic pink color of py-Tz disappeared, indi-
cating completion of the reaction. Subsequent flash col-
umn chromatography afforded pure compound 5 as
yellow-green crystals. The structure of 5 was elucidated
2
teristics in bond critical point (ρ = 0.006 au, r ρ = 0.016
au, H = 0.0007au). It is worth mentioning that C-H … π
bonds are for instance responsible for stabilization of ace-
[19]
tone crystal in low temperature conditions.
Interest-
ingly, merely in the case of conformer anti of endo
isomer there are extra intramolecular contacts between
carbonyl groups of Fp entity and carbonyl imide ligand,
as shown in Figure 4c. In our previous works, we have
shown that these interactions may influence the chemical
1
13
on the basis of H and C NMR spectroscopy sup-
plemented with 2D NMR measurements. Only one set of
1
signals is observed on the H NMR spectrum in agree-
ment with the formation of a single cycloaddition prod-
uct (Figure 5). The presence of an unshielded singlet at
[4,14,20]
properties of similar complexes.
9.35 ppm assigned to N-H indicates that the reaction
2
.4 | Reactivity studies with model
product is a 1,4-dihydropyridazine and not the 4,5-tauto-
mer. The proton carried by C7 at the junction of the
dihydropyridazine ring appears as a doublet at 2.23 ppm,
with a vicinal coupling constant between H7 and H6 of
1.2 Hz. In consequence, the dihedral angle between both
tetrazines
iEDDA reaction between 1 and 3,6-di (phenyl)-1,2,4,5-
tetrazine Ph-Tz or 3,6-di (pyridin-2-yl)-1,2,4,5-tetrazine
SCHEME 3 Reaction of 1 with py-Tz

6
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JAMROZ ET AL.
1
FIGURE 5
H NMR spectrum of 5 in CDCl
3
protons is close to 90 deg according to the Karplus equa-
tion. This feature is only met for the endo/exo
configuration.
Beforehand, the second order rate constants of reaction
of the organometallic norbornenes with Tz-NHS were
determined under pseudo first-order conditions (i.e. with
an excess of dienophile) by monitoring the decrease of
absorption of Tz-NHS at 520 nm. This wavelength was
preferably chosen over the band at 267 nm (see spectrum
below) because of possible interference from the
dihydropyridazine products.
Additional signals in the aromatic region clearly indi-
cated the presence of the two pyridinyl substituents.
13
Moreover, in the C NMR spectrum signals found at
0.5 ppm (CH) and 113.3 ppm (C ) confirmed the forma-
4
q
tion of the cycloaddition product (Figure S4). The IR
spectrum of product 5 displayed typical ν (CO) bands at
For comparison, the rate of reaction between Tz-NHS
and the reference compound 2-norbornene-5-methanol
Nb-OH was measured in the same conditions (Table 1,
entries 1 and 2). To delineate the influence of the organo-
metallic substituent on the reactivity of the norbornenes,
the purely organic succinimide norbornene Suc-NB and
oxanorbornene Suc-oxNB were prepared by reaction of
maleimide and cyclopentadiene or furan, respectively fol-
−1
1
997 and 2047 cm owing to the carbonyl ligands of the
organometallic moiety.
2.5 | Kinetic studies
To introduce tetrazine handles onto the model protein
BSA, we used the commercially available N-hydro-
xysuccimide ester Tz-NHS depicted in Scheme 4 below.
[
4]
lowing the same reaction condition and their kinetics
of reaction with Tz-NHS was measured as well. Suc-NB
SCHEME 4 Introduction of tetrazine handles onto BSA and iEDDA with metallocarbonyl norbornenes

BIOCONJUGATION OF METALLOCARBONYL INFRARED LABELS BY CLICK CHEMISTRY
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was obtained as a single isomer (endo) whereas Suc-
oxNB was obtained as a mixture of endo and exo isomers
in the proportion 1 to 2.
(entry 7). Steric effects were ruled out since the more
voluminous complex of the series, that is the molybde-
num derivative was also the most reactive.
All the organometallic derivatives gave rise to much
slower rates of reaction with respect to Nb-OH. This find-
ing is in agreement with the previously noticed rate
The rate of reaction between Tz-NHS and the two ste-
reoisomers 2a and 2b revealed that this complex was the
most reactive of the series of complexes. For instance, the
rate constant of 2b was ca. five times higher than that of
1. In other words, replacement of the methylene bridge
by an oxygen bridge markedly enhanced the reactivity of
the Fp derivative. This finding is counterintuitive since
the additional electron withdrawing effect of the bridging
oxygen should decrease the reactivity of the olefin lead-
[
2]
reducing ability of electron–withdrawing substituents.
The unfavorable endo configuration of the organometal-
lic norbornenes may also play a role in the reduced reac-
tivity of the complexes. There were subtle differences of
reactivity among 1 (M = Fe), 3 (M = Ru) and
4
(M = Mo), probably reflecting their relative degree of
[
22]
electron withdrawing strength. Nonetheless, they were
ing to a smaller rate constant.
Conversely to the
still much more reactive than the organic analog Suc-Nb
for which no reaction was observed after 15 hr at 30 C
norbornene series, the rate constant of the organic analog
was similar (entry 10).
ꢀ
FIGURE 6 A) UV–visible spectra
of Tz-NHS and BSA-Tz conjugate; B)
Uv–visible spectra of BSA conjugates
after iEDDA with norbornenes

8
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JAMROZ ET AL.
ꢀ
Moreover, 2a reacted at a faster rate than 2b (entries
and 9). This is in complete agreement with previous
17 to 72 hr at 30 or 37 C depending on the norbornene
(Scheme 4). Unreacted norbornene was removed by
extensive dialysis or gel filtration. Solutions were ana-
lyzed by uv–visible spectroscopy (Figure 6B). A signifi-
cant change of the uv–vis spectrum of the protein was
observed with the disappearance of the characteristic
band at 520 nm, except for the reaction product with
3 (see Figure 6B, inset). Solutions were concentrated by
centrifugal ultrafiltration and further analyzed by FT-IR
spectroscopy by spotting them on nitrocellulose mem-
branes and subsequent air drying (Figure 7). The
corresponding spectra displayed two bands in the
2000 cm− region (Table 2) characteristic of the metal
carbonyl moieties. This indicated that the iEDDA took
place between the tetrazine handles and the organome-
tallic norbornenes. This feature and the disappearance of
the band at 520 nm are in good agreement with the cova-
lent binding of metal carbonyl groups to BSA by iEDDA.
The protein content was quantified by the colorimet-
ric BCA assay since the simple protein assay at 280 nm
gave overestimated concentration values owing to the sig-
nificant contribution of the dihydropyridazine entities to
the absorbance at 280 nm. Quantification of meta-
llocarbonyl entities was done from the intensity of the
higher wavenumber band of each complex measured on
the IR spectrum of the conjugates. The average metal-to-
8
experimental data which had been substantiated by com-
putational calculations on energy barriers.
[
22]
The steric
hindrance brought by the bulky Fp substituent may favor
the reaction with the exo isomer. The other reason for
the faster reaction rate of the exo isomer 2a could be the
rotation barrier of Fe-N bond which must be larger with
respect to 2b because of C-H … O interaction.
2
.6 | Introduction of tetrazine handles
1
on BSA
A solution of BSA was treated with a 20 molar excess of
Tz NHS at pH 7.8 (Scheme 4). After 24 hr, the solution
was dialyzed in PBS to remove unreacted tetrazine. The
uv–visible spectrum of the conjugate displays 2 maxima
at 526 and 276 nm (Figure 6A). The former one is charac-
teristic of the tetrazine moiety and gives its typical bright
pink color. It is slightly shifted to the red compared to the
starting tetrazine NHS ester. The tetrazine concentration
in BSA-Tz sample was easily deduced from the OD at
5
26 nm assuming the same extinction coefficient for Tz
bound to BSA and Tz-NHS. On the other hand, the pro-
tein concentration was measured by the colorimetric
BCA assay since the tetrazine is highly absorbing in the
2
80 nm range. The average coupling ratio was ca. 13 t-
TABLE 2 Spectroscopic analysis of BSA conjugates
etrazine entities per BSA.
Complex
1
IR, v/cm−
2050, 2002
2051, 2003
2047, 1962
2050, 1997
1
Uv–vis, λmax/nm
2
.7 | Conjugation of metallocarbonyl
277
norbornenes to BSA-Tz
2
4
3
a
277
277
BSA-Tz was treated with an excess of norbornene with
respect to BSA (initial norbornene/Tz ratio = 1.3 to 2) for
277, 520
FIGURE 7 IR spectrum of
conjugate resulting from the successive
reactions of BSA with Tz-NHS and 1

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9 of 12
BSA ratio equaled 4.8 or 4 after reaction with 1 and 2a,
respectively. For the two other norbornenes 3 and 4, the
coupling ratios were 1.8 and 0.9, respectively.
4.2 | Synthetic procedures
4.2.1 | Synthesis of 2
5
1
To argon-saturated solution of [(η -C H )Fe (CO) (η -N-
5
5
2
3
| CONCLUSIONS
maleimidato)] (100 mg, 0.366 mmol) in methanol/water
1:3 mixture (9 mL), furan (25 mg, 0.366 mmol) was
In this paper, we demonstrated that various meta-
llocarbonyl reporter entities could be introduced on the
model protein BSA by a two-step process, the second one
involving the bioorthogonal reaction between tetrazine
and norbornene handles. Subtle differences in the reac-
tivity of the metallocarbonyl norbornenes were noticed
by carrying kinetic studies with the tetrazine reagent
used for further conjugation to BSA, with the
oxanorbornene derivative affording the fastest reaction
rate with respect to the other organometallic
norbornenes. The stereochemistry of the oxanorbornene
also influenced the rate of iEDDA, with the exo isomer
displaying the fastest rate. The protein bioconjugates
added. The mixture was stirred for 24 hr at r.t. The sol-
vent was evaporated and the residue dissolved in chloro-
form and chromatographed. Yellow bands containing
two isomers were eluted with chloroform–MeOH 9:1.
1
The ratio of products was about 1:1 ( H NMR). Crystalli-
zation from CHCl /heptane (3:1) afforded yellow crystals.
3
−
1
2a: Yield (34%, 42 mg), IR ν/cm 2034, 1988 (C ≡ O) and
1
1640 (CO imide). H NMR (600 MHz, CDCl ) δ/ppm 6.45
3
(s, 2H, H7), 5.16 (s, 2H, H6), 5.05 (s, 5H, Cp) and 2.79 (s,
+
2H, H3). ESI-MS m/z 342 (M + H) . 2b: (Yield
−1
34,4%,43 mg), IR ν/cm 2034, 2003 (C ≡ O) and 1638
1
(CO imide). H NMR (600 MHz, CDCl ) δ/ppm 6.30 (s,
3
2H, Holefin), 5.20 (s, 2H, COCH), 5.00 (s, 5H, Cp), 3.54 (s,
+
were characterized by the presence of bands in the
2H, CH). ESI-MS m/z 364 (M + Na) .
1
2
000 cm− spectral range typical of the stretching vibra-
tion modes of terminal CO ligands. This bioorthogonal
labeling strategy may be further applied to any other bio-
molecule carrying a tetrazine handle.
4.2.2 | Synthesis of 3
5
1
To argon-saturated solution of [(η -C H )Ru (CO) (η -N-
5
5
2
maleimidato)] (50 mg, 0.157 mmol) in water/methanol
3:1 mixture (9 ml), cyclopentadiene (52 μL, 0.63 mmol)
was added. The reaction mixture was stirred for 24 hr at
r.t. The solvent was evaporated and the residue was dis-
solved in chloroform and chromatographed. Yellow band
containing product was eluted with chloroform–MeOH
9:1. After evaporation of the solvent, the solid was dis-
solved in chloroform and washed three times with 2.5%
4
4
| EXPERIMENTAL SECTION
.1 | Materials and instruments
5
-norbornene-2-methanol (mixture of endo and exo iso-
mers) Nb-OH, 3,6-di (pyridin-2-yl)-1,2,4,5-tetrazine and
furan were purchased from Aldrich. Cyclopentadiene
was obtained by cracking of the dimer (Sigma–Aldrich)
directly before use. Complexes [(η -Cp)M (CO) (η -N-
maleimidato)] (M = Fe, Ru) were synthesized as previ-
aq. NaOH solution. Crystallization from CHCl /heptane
3
5
1
(3:1) afforded yellow crystals. Yield (67%, 40 mg). IR
2
ν/cm− 2050, 1986 (C ≡ O) and 1640 (CO). H NMR
1
1
[11,16,23]
ously described.
Tetrazine-NHS ester was pur-
(600 MHz, CDCl ) δ/ppm 6.03 (s, 2H, Holefin), 5.40 (s, 5H,
3
chased from Click Chemistry Tools (Scottsdale, AZ,
USA). Bovine serum albumin fraction V was purchased
from Sigma. Compounds 1 and 4 were synthesized
according to a published procedure. Protein concentra-
tions were measured using the micro BCA protein assay
Cp), 3.28 (s, 4H), 1.66 (d, J = 8.5 Hz, 1H, CH ), 1.49 (d,
2
+
J = 8.5 Hz, 1H, CH ). ESI-MS m/z 386 (M + H) ,
2
+
408 (M + Na) .
[4]
kit according to the manufacturer's instructions (Pierce
4.2.3 | Synthesis of 5
1
Chemicals). H NMR were recorded in CDCl on Bruker
3
Avance III (600 Hz) spectrometer. The IR spectra of com-
pounds were recorded in KBr on a FT-IR NEXUS
Compound 1 (100 mg, 0.295 mmol), 3,6-di-2-pyridyl-
1,2,4,5-tetrazine (70 mg, 0.295 mmol) and freshly distilled
(Thermo Nicolet) spectrometer. ESI-MS spectra were
dry CHCl (5 ml) were placed in a round-bottomed flask.
3
recorded in positive mode on Varian 500-MS LC ion trap
spectrometer. All syntheses were carried out under argon.
Solvents were dried using standard procedures. Chro-
matographic purifications were performed on Silica gel
Merck 60 (230–400 mesh ASSTM).
The mixture was protected from light, stirred under
argon at room temperature and monitored by TLC until
the starting materials were fully consumed. Next the sol-
vent was evaporated and crude compound was purified
by column chromatography (chloroform/methanol 98:2).

10 of 12
JAMROZ ET AL.
Product 5 was crystallized from dichloromethane/hep-
tane to yield yellow-green crystals. Yield (68%, 97 mg). IR
NH), 6.55 (s, 2H, Holefin endo), 6.60 (s, 1H, Holefin exo)
5.36–5.27 (m, 2H, CH-CO endo), 5.23 (s, 1H, CH-CO
exo), 3.61 (dd, J = 3.6, 1.6 Hz, 2H, CH endo), 2.96 (s, 1H,
CH exo).
ν/cm− 2050, 1997 (C ≡ O). H NMR (600 MHz, CDCl )
1
1
3
2
δ/ppm 1.29 (d, JHH = 9,5 Hz, 1H, H10), 1.63 (d,
2
3
JHH = 9.5 Hz, 1H, H10`), 2.23 (d, JHH = 1.2 Hz, 1H,
H7), 3.43 (m, 2H, H6, H9), 3.66 (d, J = 4.7 Hz, 1H, H3),
.00 (d, J = 5.5 Hz, 1H, H4), 4.91 (s, 5H, Cp), 7.25–7.29
m, 2H, Harom), 7.69 (d, J = 3.7 Hz, 2H, Harom), 7.88 (td,
4
(
4.3 | Single crystal X-ray analysis
J = 7.8, 1.6 Hz, 1H, Harom), 8.31 (d, J = 7.9 Hz, 1H,
Harom), 8.59 (d, J = 4.3 Hz, 1H, Harom), 8.68 (d,
J = 3.8 Hz, 1H, Harom), 9.35 (s br, 1H, NH,). C NMR
Suitable crystals were sequentially mounted on a fiber
loop and used for X-ray measurement. X-ray data were
collected on the Oxford Diffraction SuperNova
DualSource diffractometer with use of the mono-
chromated CuKα X-ray source (λ = 1.54184). Crystals
were kept at 100 K during data collection. Data reduction
and analytical absorption correction were performed with
13
(
(
(
(
150 MHz, CDCl ) δ/ppm 40.5 (CH 2.23 ppm z 1H), 40.6
3
CH 4.01 ppm z 1H), 43.7 (CH 3.66 ppm z 1H), 45.4
CH 1.29 i 1.63 ppm z 1H), 51.6 (CH 3.39 ppm z 1H), 52.3
CH 3.49 ppm z 1H), 84.8 (Cp), 113.3 (Cq), 122.9 (Carom),
[
24]
[25]
123.0 (Carom), 123.2 (Carom), 123.3 (Carom), 133.6 (Cq),
136.6 (Carom), 137.4 (Carom), 138.5 (Cq), 148.3 (Carom),
148.5 (Carom), 150.1 (Cq), 154.0 (Cq), 190.8 (C=O), 191.0
CrysAlis PRO.
solved with the ShelXS
using Direct Methods and refined with the ShelXL
Using Olex2,
the structures were
[
26]
structure solution program
[26]
(
5
C=O), 212.3 (C ≡ O), 212.5 (C ≡ O). ESI-MS m/z
50 (M + H) .
refinement package using Least Squares minimization.
The non-hydrogen atoms were refined anisotropically.
Hydrogen atoms were introduced in calculated positions
with idealized geometry and constrained using a rigid
body model with isotropic displacement parameters
equal to 1.2 of the equivalent displacement parameter of
the parent atoms. A summary of relevant crystallographic
data is given in Table S1 The molecular geometry was
+
4
.2.4 | Synthesis of Suc-NB
To argon-saturated solution of maleimide (50 mg,
.515 mmol) in methanol/water 1:3 mixture (9 ml),
0
[
27]
cyclopentadiene (136 mg, 2.0 mmol) was added. The mix-
ture was stirred for 2 hr at r.t.. The solvent was evapo-
rated and the residue was dissolved in chloroform and
chromatographed. White band containing product was
eluted with chloroform/MeOH 9:1. After evaporation of
the solvent the solid was dissolved in chloroform and
washed three times with 2.5% aq. NaOH solution. Crys-
calculated by Mercury 3.10.
The CCDC 1876957 and
CCDC 1876958 contains the supplementary crystallo-
graphic data for this paper. The data can be obtained free
of charge from The Cambridge Crystallographic Data
Centre via http://www.ccdc.cam.ac.uk/conts/retrieving.
html
tallization from CHCl /heptane (3:1) afforded analyti-
3
1
cally pure sample. Yield (70%, 59 mg). H NMR
4.4 | Methodology of quantum-chemical
calculations
(
600 MHz, CDCl ) δ/ppm 8.01 (bs, 1H, NH), 6.22 (d, 2H,
3
J = 2.0 Hz, Holefin), 3.41–3.39 (m, 2H, C(O)-CH-CH-CH2),
.34–3.31 (m, 2H, C(O)-CH), 1.76 (d, 1H, J = 8.8 Hz,
CH ), 1.54 (d, 1H, J = 8.8 Hz, CH ).
3
Geometry optimization and energy analysis were per-
[
28]
formed with the Gaussian 09 sets of codes.
Calcula-
2
2
[
29]
tions were done at ωB97XD
conjunction with Def2TZVP basis set
For all optimized geometries the corresponding WFX
level of DFT theory in
[
30]
for all atoms.
4.2.5 | Synthesis of Suc-oxNB
[
17]
files were obtained and used for QTAIM
calculations
[
31]
To argon-saturated solution of maleimide (50 mg,
.515 mmol) in methanol/water 1:3 mixture (9 ml), furan
with AIMAll software.
0
was added (35 mg, 0.515 mmol). The mixture was stirred
for 24 hr at r.t.. The solvent was evaporated and the resi-
due dissolved in chloroform and chromatographed.
White band containing mixture of isomers was eluted
with chloroform–MeOH 9:1. Crystallization from CHCl3/
heptane (3:1) afforded Suc-oxNB as a mixture of endo
4.5 | Kinetics of reaction of Tz-NHS with
norbornenes
Stock solutions of 1, 2a and 2b (5 mM in PBS), Tz-NHS
(10 mM in DMSO), 5-norbornene-2-methanol Nb-OH
(mixture of endo and exo-isomers) (100 mM in DMSO),
3 (50 mM in DMSO) and 4 (10 mM in DMSO) were
and exo isomers in the proportion 1/2. Yield (70.5%,
1
6
0 mg) H NMR (600 MHz, MeOD) δ/ppm 7.94 (s, 1H,

BIOCONJUGATION OF METALLOCARBONYL INFRARED LABELS BY CLICK CHEMISTRY
11 of 12
prepared. Mixtures containing Tz-NHS (1 mM) and (oxa)
norbornene (4 mM) in PBS/DMSO 9:1 to 1:1 (500 μL)
were prepared in a test tube and immediately transferred
column, 10 ml, Thermo scientific) and species were
eluted with PBS. Twelve 1-ml fractions were collected
manually and the fractions containing the protein were
pooled. The solution was transferred to a centrifugal fil-
ter (Amicon Ultra – 2 ml, 30 KDa) and concentrated to
0.3 ml. Protein concentration was assayed by the micro-
BCA method.
in a plastic cuvette. The absorption spectrum (600–
ꢀ
4
50 nm) was recorded for 15 to 900 min at 20 or 30 C.
The OD at 520 nm was plotted as a function of time.
Nonlinear fitting of the data was done assuming first
order kinetics, equation (1) where kobs is the pseudo first
order rate constant.
4
.9 | Quantification of metallocarbonyl
OD520 = A + B*expð−kobs*tÞ
ð1Þ
entities by IR spectroscopy
The second order rate constant k2nd was calculated from
equation (2).
The concentration of metallocarbonyl moieties in conju-
gate samples was determined by IR analysis according to
our previously described procedure and using the follow-
ing calibration curve equations.
k2nd = kobs=½norborneneꢁ
ð2Þ
À
1*1000Á
Fp : y = 25:6 x + 0:9 y in μmol=mland x = absorbance at2050 cm−
À
À
1*1000Á
Rp : y = 33:3 x−0:43 y in μmol=mland x = absorbance at 2050 cm−
4.6 | Conjugation of tetrazine to BSA
Á
To a solution of BSA (20 mg, 300 nmol) in NaPB pH = 7.8
Mp : y = 22:5 x + 0:09 y in μmol=mland x = absorbance at 2050 cm−1*1000
(
(
1 ml), was added a solution of tetrazine-NHS ester
1.89 mg; 6.06 μmol, 20 molar eq.) in DMSO (0.2 ml). The
reaction was stirred for 24 hr at r.t. and the protein was
purified by dialysis in PBS pH 7.4 (1 day), transferred to a
centrifugal filter and concentrated to 0.5 ml. The concen-
tration of tetrazine conjugated to BSA was calculated
from the absorbance of the conjugate at 520 nm taking
ACKNOWLEDGMENTS
This work was financially supported by the University of
Lodz, Poland, Faculty of Chemistry, CNRS and Sorbonne
Université, France and by ERASMUS + programme of
the European Union for funding internships in Paris
(D. J.). Funding for this research was also provided by
EFRD in Operational Programme Development of East-
ern Poland 2007–2013 (project No. POPW.01.03.00-20-
004/11, for the Oxford Diffraction SuperNova DualSource
diffractometer). Dr Vincent Corcé is gratefully acknowl-
edged for helpful discussion regarding the NMR spectra.
−
1
−1
ε = 370 M .cm . Protein concentration was assayed by
the microBCA method.
4.7 | Conjugation of 1 to BSA-Tz
To a solution of BSA-Tz in PBS (2.9 mg; 44 nmol;
0
.125 ml) was added 1 (0.46 mg, 1.36 μmol; 31 eq.) in PBS
ꢀ
(0.375 ml). After stirring for 24 hr at 30 C, the mixture
ORCID
was purified by dialysis in PBS pH 7.4 (1 day), then the
solution was transferred to a centrifugal filter (Amicon
Ultra – 2 ml, 30 KDa) and concentrated to 0.3 ml. Protein
concentration was assayed by the microBCA method.
Nathalie Fischer-Durand https://orcid.org/0000-0002-
4715-5662
Marcin Palusiak https://orcid.org/0000-0002-0032-0878
Sławomir Wojtulewski https://orcid.org/0000-0001-
5
535-4525
Szymon Jarzy nꢀ ski https://orcid.org/0000-0002-1558-
98X
4.8 | Conjugation of other norbornenes
0
to BSA-Tz
Michèle Salmain https://orcid.org/0000-0003-3039-5659
Bogna Rudolf https://orcid.org/0000-0003-0319-0535
To a solution of BSA-Tz in PBS (0.9 mg; 13.6 nmol,
0
.05 ml) was added a solution of norbornene in DMSO
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