Amino acid bioconjugation via iClick reaction of an oxanorbornadiene-masked alkyne with a MnI(bpy)(CO)3-coordinated azide

Article abstract of DOI:10.1039/c4cc07892f

The catalyst-free room temperature iClick reaction of an unsymmetrically 2,3-disubstituted oxanorbornadiene (OND) as a "masked" alkyne equivalent with [Mn(N₃)(bpy CH₃,CH₃)(CO)₃] leads to isolation of a phenylalanine ester bioconjugate, in which the model amino acid is linked to the metal moiety via a N-2-coordinated triazolate formed in a cycloaddition-retro-Diels-Alder (crDA) reaction sequence, in a novel approach to bioorthogonal coupling reactions based on metal-centered reactivity.

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ARTICLE TYPE
Amino acid bioconjugation via iClick reaction of an oxanorbornadiene-
masked alkyne with a MnI(bpy)(CO)3-coordinated azide
Lucas Henry,a Christoph Schneider,a Benedict Mützel,a Christoph Nagel,a Katharina Fucke,a,b
and Ulrich Schatzschneider*a
5
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
The catalyst-free room temperature iClick reaction of an unsymmetrically 2,3-disubstituted
oxanorbornadiene (OND) as a "masked" alkyne equivalent with [Mn(N3)(bpyCH3,CH3)(CO)3] leads to
isolation of a phenylalanine ester bioconjugate, in which the model amino acid is linked to the metal
10 moiety via a N-2-coordinated triazolate formed in a cycloaddition-retro-Diels-Alder (crDA) reaction
sequence, in a novel approach to bioorthogonal coupling reactions based on metal-centered reactivity.
The site-selective modification of bio(macro)molecules is a key
technology in the field of chemical biology. The plethora of
context, Veige et al. introduced the term "iClick" (inorganic
click) reaction for the cycloaddition of a metal-coordinated azide
functional groups present in peptides and proteins as well as 50 and an alkyne coupling partner or vice versa.13
15 oligonucleotides and carbohydrates but also other bioactive
molecules requires specific bioorthogonal conjugation methods to
introduce novel functionality to such systems.1 Several different
types of reactions, most of them based on "click" chemistry,2 are
now commonly utilized in this context. The most prominent is
20 certainly the copper-catalyzed azide-alkyne cycloaddition
(CuAAC), which selectively leads to 1,4-substituted triazoles.3
However, the toxicity of copper is a concern for in vitro or in vivo
applications and thus, catalyst-free variants have been developed,
in particular based on cycloalkynes, in a reaction which has been
25 termed strain-promoted azide-alkyne cycloaddition (SPAAC).4
Other methods include the oxime ligation of aldehydes with
aminoxy groups5 or the inverse-electron demand Diels-Alder
reaction of strained alkenes with tetrazines.6
Scheme 1 Comparison of standard copper-catalyzed azide-alkyne
cycloaddition reaction (CuAAC, top) and catalyst-free iClick reaction in
the metal coordination sphere (bottom).
55
Besides their versatility, applications of iClick reactions in a
biological context are still very rare. The groups of Gray and
Metzler-Nolte reported on the coupling of linear phosphane
gold(I) azides of the general formula [(R3P)AuN3] to alkyne-
functionalized dendrimers and peptides, respectively,14 which
When applied to metal complexes, these bioorthogonal
30 coupling reactions usually take place in the periphery of the
60 leads to the gold-phosphane fragment coordinated to a carbon
instead of a nitrogen atom of the triazolate. Another very
interesting approach, recently described by Mirica et al., is the
iClick reaction of electron-poor alkynes with metal-azide
fragments of enzyme active site models.15 A major hurdle in the
65 general application of this method to bioconjugation, however, is
due to the fact that with the exception of the gold-based systems
mentioned above, there seems to be a general requirement for the
use of alkynes with strongly electron-withdrawing substituents
like dimethyl acetylenedicarboxylate (DMAD) to ensure smooth
70 coupling under mild conditions. This is a significant drawback,
since such systems are very prone to Michael-type addition of
nucleophiles like thiols and amines, which are highly abundant in
biological systems.
8
ligand sphere,7, at a position pointing away from the metal
center. As an alternative, we are interested in novel, generally
applicable methods for the conjugation of functional
(organo)metal compounds to bio(macro)molecules which are
35 based on an inherently metal-centered reactivity, which allows to
tune the reaction kinetics by proper combination of metal center
and (co)ligands. Metal-coordinated azides are a particularly
attractive choice since they are easily accessible by ligand
exchange from the corresponding halide complexes and might
40 react with organic alkyne coupling partners already utilized in the
context of other conjugation methods, leading to functionalized
triazolate complexes in a catalyst-free process (Scheme 1). Some
early work on cycloaddition reactions in the metal coordination
sphere was summarized by Frühauf and additional applications
45 were reviewed more recently.9 Current research activities in the
field, albeit still very limited, are especially focused on gold(I),10
molybdenum(II),11 and ruthenium(II) compounds.12 In this
Therefore, we wanted to explore "masked" alkynes in the form
75 of functionalized oxanorbornadienes (ONDs) as a promising
alternative.16 In this context, 2,3-dicarboxy-7-oxanorbornadiene
dimethylester is easily accessible by Diels-Alder reaction of furan
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with dimethyl acetylenedicarboxylate (DMAD). However, the
selective mono-functionalization of this compound turned out to
coordinated azide ligand at 2049 cm-1 disappeared. In addition,
the 19F NMR signal of the CF3 group in the masked alkyne
be difficult, since both ester groups are equally labile towards 40 coupling partner serves as a sensitive NMR spectroscopic marker.
hydrolysis, giving rise to inseparable product mixtures (data not
shown). Therefore, we chose to replace one of the carboxylates
by a trifluoromethyl group and prepared the corresponding
oxanorbornadiene (OND) ester 6 in a four-step procedure from
While it appears at -62.9 ppm in ester 6 and is unaffected by
hydrolysis to free acid 7, the reaction with metal azide complex
14 leads to a shift by 3.0 ppm, with product 15 now showing a
single 19F NMR peak at -59.9 ppm. Single crystals suitable for X-
5
bromoacetic acid ethyl ester 1 and 2-methylfuran (Scheme S1). 45 ray structure analysis could be obtained by diffusion of n-hexane
Hydrolysis with dilute hydrochlorid acid led to acid 7, which was
into a dichloromethane solution of crude 15. This confirmed the
successful tandem cycloaddition/retro-Diels-Alder (crDA)
reaction16 of oxanorbornadiene 6 with metal azide 14 (Supporting
information, Figure S1 and Table S1). However, in contrast to the
50 gold(I) azides,10, 14 where the metal is bound to the C4 carbon
atoms of the triazole ring, the product of the reaction with the
10 then coupled to the amino group of phenylalanine methyl ester 9,
serving as a model system for functional oligopeptides, using
EDCl and DMAP as the coupling agents, leading to conjugate 10
(Scheme 2).
Inspired by our ongoing interest in the biological activity of
15 manganese(I) tricarbonyl complexes as molecular probes and
delivery agents for carbon monoxide7, 17 and further stimulated by
the almost complete absence of reports on Mn azide compounds
in iClick reactions,18-20 [Mn(N3)(bpyCH3,CH3)(CO)3] 14 was
chosen as the coupling partner for ONDs 6 and 10. This complex
20 was easily prepared from manganese pentacarbonyl bromide 11
and 4,4'-dimethyl-2,2'-bipyridine 12 via the bromide complex
[MnBr(bpyCH3,CH3)(CO)3] 13 by halide abstraction with silver
triflate followed by addition of sodium azide (Scheme 2).
manganese(I)-coordinated azide is
a
nitrogen-coordinated
triazolate with the metal bound to the central N-2 nitrogen atom.
55
Fig. 1 Molecular structure of 16 with atomic displacement ellipsoids
drawn at the 50% probability level.
Encouraged by these results, the OND-phenylalanine
bioconjugate 10 was used as a model system for more complex
bio(macro)molecules and reacted with azide complex 14 under
60 similar conditions as identified above. Stirring at room
temperature in dichloromethane for 7 d under exclusion of light
to avoid decomposition of the Mn(CO)3 moiety led to isolation of
a yellow product in 67% yield. Again, the IR spectrum of 16
showed the absence of the azide band at around 2050 cm-1 and
65 the C≡O peaks only marginally shifted compared to the starting
material to 2025 and 1933 cm-1. Two new bands were observed in
the product at 1740 and 1680 cm-1 and assigned to the
phenylalanine ester and amide C=O vibrations, respectively. The
19F NMR also shows only a single peak at -59.6 ppm, which is
70 shifted relative to the OND starting material by 6.2 ppm but at a
position comparable to the corresponding CF3 signal in 15. The
X-ray crystal structure confirms the connectivity of the
phenylalanine-triazolate conjugate 16 resulting from the crDA
reaction of 10 with 14 via the N-2 nitrogen atom of the aromatic
75 heterocycle (Fig. 1). The octahedral coordination of the Mn(I)
center is completed by the bpy ligand with a compressed N(4)-
Mn(1)-N(5) angle of 79.05(8)° and three carbonyl ligands in a
facial arrangement. No significant differences are seen in the Mn-
C and C-O bond distances for the carbonyl groups trans to the
80 triazolate vs. the bipy N donor atoms. Furthermore, bond lengths
25
Scheme 2 Synthesis of manganese(I) azide complex 14 and room-
temperature catalyst-free iClick reaction with "masked" alkynes 6 and 10
to give triazolate 15 and the corresponding amino acid bioconjugate 16.
Stirring of a slight excess of 6 with 14 in dichloromethane at
room temperature for 5 d under exclusion of light led to the
30 isolation of a light yellow solid product after removal of the
solvent, which was further purified by recrystallisation or column
chromatography. The symmetrical and antisymmetrical C≡O
stretching vibrations experienced only a small shift from 2006,
1927, and 1886 cm-1 in starting material 14 to 2026, 1929, and
35 1912 cm-1 in product 15, which also showed an additional peak at
1727 cm-1 at essentially the same position as the ester C=O
vibration in coupling partner 6. Furthermore, the signal due to the
2
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in the crystal structures of 15 and 16 are comparable (Table S2).
azide complex to prepare
a
novel triazolate-linked
One notable difference between the two compounds, however, is 60 Mn(bpy)(CO)3-phenylalanine bioconjugate by simple stirring of
the orientation of the mean plane of the triazolate (N(1)-N(2)-
N(3)-C(4)-C(5)) relative to the pseudomirror plane between the
two halves of the bpy ligand formed by the manganese center, the
N(2) nitrogen of the triazolate and the mid-point of the central C-
the two reactants at room temperature for an extended period of
time. Symmetrical binding via the triazolate N-2 nitrogen atom
was demonstrated by X-ray crystallography, which additionally
revealed weak interactions between the Phe phenyl ring and some
5
C bond of the bipy. In 15, the triazolate is oriented almost 65 bipy methyl and ring protons responsible for the observed solid-
perpendicular to this central axis of the bpy and essentially
bisects the C(2)-Mn(1)-C(3) angle with a deviation of only 7.0°.
state conformation.
10 In contrast, for 16, this angle is increased to 59.1° and the
triazolate mean plane is now in an almost eclipsed orientation
relative to the C(3)-O(3) carbonyl ligand. A hydrogen bond exists
between the ester carbonyl O(5) atom and the H(19b) proton of
one of the bipyridine methyl groups with a O(5)-C(19) distance
15 of 3.463 Å and a O(5)-H(19b)-C(19) angle of 125.6°. Since there
are additional short intermolecular contacts, it is difficult to
discern how much this intramolecular interaction contributes to
the stabilization of the observed conformation. Only one other X-
ray crystal structure of a triazolate coordinated to a fac-Mn(CO)3
20 moiety is known in the literature,19 with the octahedral ligand
sphere of the metal however completed by a P-N chelating
1-dimethylamino-2-diphenylphosphinoethane ligand instead of a
N-N coordinated bipy as in 15 and 16. Other closely related
compounds also structurally characterized retain the
25 Mn(bpy)(CO)3 coordination sphere but incorporate a sixth axial
imidazole21 or thiazole22 ligand. Finally, the X-ray crystal
structure of a tetrazolate complex with an Mn(CO)3(P-P) core is
based on the 1,2-bis(diethylphosphino)ethane ligand.18 Besides
these variations in the axial N ligand (triazolate vs. tetrazolate vs.
30 imidazole vs. thiazole) and the nature of the chelator (N-N vs. P-
N vs. P-P), there are very little differences in the Mn-N and M-C
as well as C-O distances.
Fig. 2 Conformational energy diagram of 15 for variation of the torsion
angle between the triazolate mean plane and the bpy C2-C2' axis
calculated with DFT (RI-BP86, def2-TZVP/def2-TZVP/J).
70
The stay of L. Henry in Würzburg was supported by the Erasmus
mobility program and AMIE (Aide à la Mobilité Internationale
des Étudiants) of the Région Ile de France and performed in the
context of his M1 year research project at the ENS Chemistry
75 Department in Paris and UPMC. We thank H. Pfeiffer, A. Belz,
and S. Pai for some preliminary experiments and assistance
related to this work.
To further study the conformational preference of 15, a relaxed
surface scan was carried out with DFT at the RI-BP86 def2-
35 TZVP/def2-TZVP/J level of theory. The perpendicular
orientation of the triazolate mean plane relative to the central C2-
C2' axis of the bpy ligand with the CF3 group pointing towards
the bpy turned out to be the lowest energy conformation.
However, the parallel arrangement of the triazolate and the C2-
40 C2' axis is higher in energy by only about 0.1 kcal mol-1. A much
Notes and references
a Institut für Anorganische Chemie, Julius-Maximilians-Universität
80 Würzburg, Am Hubland, D-97074 Würzburg, Germany. Fax: 49 931
3184605; Tel: 49 931 3183636; E-mail: ulrich.schatzschneider@uni-
wuerzburg.de
b School of Medicine, Pharmacy and Health, Durham University Queen’s
campus, Stockton-on-Tees, TS17 6BH, United Kingdom
85 † Electronic Supplementary Information (ESI) available: synthetic
procedues, X-ray crystal structure of 15, crystallographic data, and
relevant bond distances and angles for 15 and 16. CCDC 951372 (15) and
951371 (16). For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/b000000x/
less favorable orientation results from
a
perpendicular
arrangement of the triazolate relative to the C2-C2' axis, but with
the ester group pointing towards the bpy ligand, which is higher
in energy by 0.9 kcal mol-1 compared to the one with the CF3
45 aligned this way. Not surprisingly, the four possible
conformations with an eclipsed orientation of the triazolate and a
carbonyl ligand are transition states in the interconversion
between the four minimum structures. However, the ones with
the CF3 group pointing towards the bpy are still somewhat lower
50 in energy (by 0.3 kcal mol-1) than those with the ester moiety in
that orientation. With a maximum barrier height of 1.3 kcal mol-1,
the rotation around the Mn-N2 axis is much more facile than, for
example, in ethane (E ~ 2.9 kcal mol-1)23 and thus the
conformations are expected to freely interconvert at room
55 temperature.
90
1. C. R. Bertozzi and E. M. Sletten, Angew. Chem. Int. Ed., 2009, 48,
6974; R. K. V. Lim and Q. Lin, Chem. Commun., 2010, 46, 1589; A.
El-Sagheer and T. Brown, Chem. Soc. Rev., 2010, 39, 1388; M.
Boyce and C. R. Bertozzi, Nature Methods, 2011, 8, 638; M. King
95
and A. Wagner, Bioconjugate Chem., 2014, 25, 825; K. Lang and J.
W. Chin, Chem. Rev., 2014, 114, 4764.
2. H. C. Kolb, M. G. Finn and K. B. Sharpless, Angew. Chem. Int. Ed.,
2001, 40, 2004; J. E. Moses and A. D. Moorhouse, Chem. Soc. Rev.,
2007, 36, 1249; P. Thirumurugan, D. Matosiuk and K. Jozwiak,
Chem. Rev., 2013, 113, 4905.
In summary, we have utilized a catalyst-free iClick reaction of an
unsymmetrically 2,3-disubstituted oxanorbornadiene (OND) as a
"masked" alkyne equivalent with a manganese(I) tricarbonyl
100
3. M. Meldal and C. W. Tomoe, Chem. Rev., 2008, 108, 2952; V. Hong,
S. I. Presolski, C. Ma and M. G. Finn, Angew. Chem. Int. Ed., 2009,
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