Design of spiro[2.3]hex-1-ene, a genetically encodable double-strained alkene for superfast photoclick chemistry

Article abstract of DOI:10.1021/ja5012542

Reactive yet stable alkene reporters offer a facile route to studying fast biological processes via the cycloaddition-based bioorthogonal reactions. Here, we report the design and synthesis of a strained spirocyclic alkene, spiro[2.3]hex-1-ene (Sph), for an accelerated photoclick chemistry, and its site-specific introduction into proteins via amber codon suppression using the wild-type pyrrolysyl-tRNA synthetase/tRNA_CUA pair. Because of its high ring strain and reduced steric hindrance, Sph exhibited fast reaction kinetics (k₂ up to 34 000 M^-1 s^-1) in the photoclick chemistry and afforded rapid (<10 s) bioorthogonal protein labeling.

Full text of DOI:10.1021/ja5012542

Communication
pubs.acs.org/JACS
Open Access on 03/04/2015
Design of Spiro[2.3]hex-1-ene, a Genetically Encodable Double-
Strained Alkene for Superfast Photoclick Chemistry
Zhipeng Yu and Qing Lin*
Department of Chemistry, State University of New York at Buffalo, Buffalo, New York 14260, United States
S
* Supporting Information
ABSTRACT: Reactive yet stable alkene reporters offer a
facile route to studying fast biological processes via the
cycloaddition-based bioorthogonal reactions. Here, we
report the design and synthesis of a strained spirocyclic
alkene, spiro[2.3]hex-1-ene (Sph), for an accelerated
photoclick chemistry, and its site-specific introduction
into proteins via amber codon suppression using the wild-
type pyrrolysyl-tRNA synthetase/tRNACUA pair. Because
of its high ring strain and reduced steric hindrance, Sph
exhibited fast reaction kinetics (k2 up to 34 000 M−1 s−1)
in the photoclick chemistry and afforded rapid (<10 s)
bioorthogonal protein labeling.
Figure 1. Structures of cyclopropene derivatives and their optimized
geometries. The LUMO orbitals are rendered on the structures, with
the LUMO energies indicated in eV. Calculations were performed
using the B3LYP method at the 6-311++G** level in a vacuum at 298
K. The dihedral angles between the cyclopropene plane of symmetry
line (in blue) and the indicated C4−H or C4−O bond are shown.
he use of strain, a classic concept in organic chemistry,1 to
Taccelerate bioorthogonal reactions has attracted a lot of
interest recently, particularly in the cycloaddition reactions.2
Some prominent examples include strain-promoted azide−
alkyne cycloaddition,3 nitrone−alkyne cycloaddition,4 tetra-
zine−alkene cycloaddition,5 and tetrazole−alkene cycloaddition
(“photoclick chemistry”).6 With rare exceptions,7 the majority
of strained substrates serve as dipolarophiles or dienophiles,
such as cyclooctyne and its variants,3 thiacycloheptyne,8 trans-
cyclooctene,5a norbornene,5b and cyclopropene and its
derivatives.6b,9 To further enhance the reactivity of these
strained substrates, additional conformational controls have
been devised, such as the fusion of a second ring. For example,
Fox and co-workers elegantly designed a cyclopropane-fused
trans-cyclooctene that adopts the higher-energy “half-chair”
conformation and affords a second-order rate constant, k2, of
22 000 M−1 s−1 in the inverse electron-demand Diels−Alder
reaction with 3,6-di(2-pyridyl)-s-tetrazine in methanol, 18 times
faster than the parent trans-cyclooctene.10 Similarly, van Delft
and co-workers ingeniously fused the cyclopropane ring to
cyclooctyne to generate bicyclo[6.1.0]non-4-yn-9-ylmethanol
(BCN), which exhibited a rate acceleration of 70-fold (k2 = 0.14
M−1 s−1) over cyclooctyne in the nitrone−alkyne cycloaddition
reaction.11 A recent report by Chin and Fox et al. revealed that
BCN is also an excellent substrate for the tetrazine ligation
reaction with a 3,6-di(2-pyridyl)-s-tetrazine derivative (k2 = 437
M−1 s−1).12
which may cause considerable steric clash with the aryl
substituents of the incoming nitrile imine along the reaction
coordinate. Therefore, we envisioned that the cycloaddition
reaction could be accelerated if we decrease this steric
hindrance. Indeed, computational studies suggested that this
type of steric hindrance is a major impediment in obtaining fast
reaction kinetics in the dibenzocyclooctyne-mediated cyclo-
addition reaction.13 Here, we report the design, synthesis, and
genetic encoding of spiro[2.3]hex-1-ene, an unprecedented
stable cyclopropene derivative that showed superior reactivity
in photoclick chemistry.
To relieve the steric repulsion, we considered four basic
cyclopropene structures, as shown in Figure 1. DFT
calculations indicated that the LUMO energies of these four
structures followed the order of 1 < 3 < 2 ≈ 4, suggesting that
cyclopropene 1 has the smallest HOMO−LUMO gap, which
may lead to the fastest cycloaddition reaction.14 On the other
hand, the addition of an exocyclic 3- or 4-membered ring (3
and 4) significantly altered the projections of the hydrogens at
the C4 position; the dihedral angles between the cyclopropene
ring symmetry line and the C−H bond increased to 122.5° and
79.5°, respectively. Considering the size of H vs O, the steric
hindrance presented by these four cyclopropene structures
toward an incoming nitrile imine dipole should follow the order
of 3 < 4 < 2 < 1. Taking these factors together, the reactivity
We previously reported the synthesis of a stable and
genetically encodable 3,3-disubstituted cyclopropene6b (1 in
Figure 1) as a privileged substrate for photoclick chemistry (k2
= 58 M−1 s−1 with 2-(p-methoxyphenyl)-5-phenyltetrazole
(Tet-1) in 1:1 acetonitrile/PBS). The small ring size forces C3-
substituents into close proximity with the cyclopropene π-bond,
Received: February 5, 2014
Published: March 4, 2014
© 2014 American Chemical Society
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trend should follow the order of 3 > 4 > 2, with the position of
cyclopropene 1 uncertain because of the two opposing effects.
To experimentally determine the reactivity trend, we set out
to prepare three new cyclopropene derivatives based on the
structures of 2−4 (Scheme 1). The 3,3-dialkyl-substituted
strain energy (∼90 kcal/mol) in this spirocyclic system.17 The
extensive search for an alternative base to effect the elimination
was not successful, suggesting the spiropentene might be
unstable at room temperature. In this regard, spiro[2.3]hexene
should offer a balance between stability and reactivity because
of its larger ring size. The synthesis of spiro[2.3]hex-1-ene 23
started with cyclopropanation of the commercially available 3-
methylene-cyclobutanecarbonitrile to produce the diaster-
eomers 18a and 18b in 7:6 ratio with a combined yield of
76% (Scheme 1c). These two diastereomers were separated and
allowed to proceed in parallel in subsequent transformations:
(i) reduction of the nitrile group to the alcohol through
sequential treatments of DIBAL and NaBH4; (ii) protection of
the alcohol by ethyl vinyl ether; and (iii) mono-debromination
to give mono-bromo-spirohexane 21a/21b (Scheme 1c). To
our satisfaction, the elimination reactions with 21a/21b
proceeded smoothly to generate spiro[2.3]hex-1-ene 22 with
excellent yields. A succinimidyl carbonate derivative 24 was
then prepared for the crystallographic study.
Scheme 1. Synthesis of Cyclopropene Derivatives
The crystal structures of cyclopropene 11 and spiro[2.3]hex-
1-ene 24 were obtained (see Tables S1 and S2 in the
Supporting Information (SI) for crystal data and structural
refinement), allowing us to compare them with 3-methyl-3-
cyclopropenecarboxylic acid that was determined previously6b
(Figure 2). From the top view, the three cyclopropene rings are
Figure 2. Crystal structures of the three cyclopropene derivatives
viewed from the side and the top. The carbamate group in 11 and the
carbonate group in 24 are omitted for clarity.
essentially identical, with the CC bond length of 1.27−1.28
Å and the opposing bond angle of approximately 50°. However,
from the side view the bond angle between two C3-substituents
decreased dramatically from 113.5° for 3-methyl-3-cyclo-
propenecarboxylic acid and 112.3° for 11 to 92.3° for
spiro[2.3]hex-1-ene 24. As expected, the cyclobutane ring in
24 pulls the C3-substituents away from the π-faces of the
cyclopropene ring, resulting in reduced steric hindrance.
To examine whether reduced steric hindrance in spiro[2.3]-
hex-1-ene leads to faster cycloaddition reaction, we performed
pairwise comparison studies in which a mixture of cyclo-
propenes were incubated with Tet-1 in CD3CN and
competitive formation of the pyrazoline products was
cyclopropene 9 was obtained from ethyl-3-methylbut-3-enol
(Scheme 1a) via the following key steps: (i) cyclopropanation
with the in situ-generated dibromocarbene in the presence of
phase-transfer catalyst cetyltrimethylammonium bromide
(CTAB) to form dibromocyclopropane 6; (ii) Ti(OiPr)4-
catalyzed mono-debromination to form bromocyclopropane 7;
and (iii) base-mediated elimination to generate the 3,3-
dialkylated cyclopropene 8. For X-ray structural determination,
crystalline carbamate analogue 11 was also prepared. In parallel,
the synthesis of spiro[2.2]pentene began with the ethoxyethyl-
protected (methylenecyclopropyl)carbinol 14 (Scheme 1b),
prepared using a published procedure.15 Subsequent cyclo-
propanation and mono-debromination proceeded smoothly to
afford a diastereomeric mixture of 1-bromo-spiro[2.2]pentane
16 in an overall yield of 36%. However, treatment of 16 with
potassium tert-butoxide in DMSO did not produce the desired
spiro[2.2]pentene 17 as reported,16 presumably due to the high
18
1
monitored by H NMR (Figure 3). The reactions proceeded
cleanly in the NMR tube (Figures S4 and S5 in SI) upon
photoirradiation with a hand-held 302 nm UV lamp (UVM,
0.16 A, 2.3 mW/cm2). Based on the characteristic pyrazoline
proton signals, spirohexene 22 was about 17 times more
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Figure 4. M06-2X/6-311+G(d,p)-optimized transition-state structures
for the cycloaddition of the nitrile imine with 1, 2 (simplified 8), and
5-methylspiro[2.3]hex-1-ene 22d (simplified 22) in water at 298 K.
The bond lengths (in Å) at the saddle point are marked on the TS
structures. The activation energies, ΔG°⧧, are in kcal/mol, and the
single imaginary frequencies, vi, are in cm−1. The relative rate
constants, krel, were computed on the basis of ΔΔG°⧧.
genetically encoding structurally diverse lysine derivatives,21 we
suspected that spiro[2.3]hexene could be similarly introduced
into proteins site-specifically using this system. Accordingly, we
prepared Nε-(spiro[2.3]hex-1-ene-5-methoxycarbonyl)-L-lysine
(SphK) from 24 (Scheme S4 in SI) and found that SphK is
stable toward excess glutathione (Figure S8 in SI). To our
delight, SphK was efficiently incorporated into superfolder GFP
(sfGFP) carrying an amber codon at position 2 in BL21(DE3)
cells expressing the wild-type PylRS/tRNACUA pair. The SphK-
encoded sfGFP (sfGFP-S2SphK) was purified in a yield of 4.9
mg/L (Figure S9 in SI). For comparison, the CpK-encoded
sfGFP (sfGFP-S2CpK) was expressed at 4.1 mg/L when an
engineered PylRS/tRNACUA pair previously reported to charge
Nε-acryloyl-L-lysine22 was employed (Figure S10 in SI).
Subsequent kinetic studies revealed that sfGFP-S2SphK reacted
with Tet-1 in PBS/CH3CN (2:1) with k2 = 1850 218 M−1
s−1, about 9 times faster than sfGFP-S2CpK (k2 = 206 17
M−1 s−1) (Figure 5; Figures S11 and S12 in SI). To eliminate
the inhibitory effect of Cl−, we also ran the reaction in the Cl−-
free phosphate buffer/CH3CN (2:1) and found k2 to be 7000
320 M−1 s−1, about 4 times faster than in Cl−-containing PBS/
CH3CN (2:1) buffer (Figure 5b; Figure S13 in SI). To
completely remove CH3CN from the solvent system, we
synthesized a water-soluble Tet-2 carrying a sulfonic acid group
(Scheme S5 in SI; structure shown in Figure 5a). In the
fluorescence-based verification study, Tet-2 maintains excellent
reactivity toward spirohexene 22 (k2 = 34 000 1300 M−1 s−1
in CH3CN/phosphate buffer (1:1); Figure S14 in SI). When
sfGFP-SphK was treated with Tet-2 in phosphate buffer under
identical photoirradiation conditions, the second-order rate
Figure 3. Competitive cycloadditions of Tet-1 with pairs of
cyclopropene dipolarophiles in CD3CN at 25 °C. (a) Reaction
scheme. (b,c) Selected regions of H NMR of the reaction mixtures
before and after 302 nm photoirradiation, showing the characteristic
proton signals for pyrazoline 25−27. See Figures S1−S5 in SI for
spectrum assignment and ratio determination details.
1
reactive than cyclopropene 1 (Figure 3b) and 4 times more
reactive than cyclopropene 8 (Figure 3c). A separate HPLC-
based kinetic study of the cycloaddition of Tet-1 with
spirohexene 22 in PBS/ACN (1:1) gave a cycloaddition rate
constant of 890 51 M−1 s−1 (Figure S6 in SI), about 15 times
faster than the reaction of cyclopropene 1 under the same
condition.6b Furthermore, when Cl−-free phosphate buffer/
ACN (1:1) was used as the solvent, the cycloaddition rate
constant increased to 2600 180 M−1 s−1 (Figure S7 in SI),
consistent with a recent report that the nitrile imine−alkene
cycloaddition is extremely fast in the absence of Cl−.19 To
understand the basis of the reactivity trend among the
cyclopropene derivatives, we conducted a DFT-based search
of the transition states (TSs) for the cycloaddition reactions
(Figure 4).20 We found that for cyclopropene 1 the exo TS is
favored over the endo TS by 2.0 kcal/mol, presumably due to
increased steric hindrance and lack of secondary interactions (a
result of orthogonal arrangement of the nitrile imine π system
and the carbonyl π system) in the endo TS. Compared with
cyclopropene 1, cyclopropene 8 and spirohexene 22 showed
lower activation barriers, which led to 2.3 and 15 times faster
cycloaddition reaction, respectively (Figure 4). These results
agree well with the NMR-based competition studies (Figure 3).
Since the Methanosarcina mazei pyrrolysyl-tRNA synthetase
(PylRS)/tRNACUA pair has shown tremendous versatility in
constant, k2, was determined to be 10 420
810 M−1 s−1
(Figure 5b; Figure S15 in SI), in a range close to the tetrazine
ligation (k2 = 22 000 M−1 s−1 for protein substrates as measured
by a fluorescence-based assay).10
In summary, we have synthesized a biocompatible spirocyclic
alkene reporter that is stable at ambient conditions and yet
highly reactive toward tetrazoles in photoclick chemistry.
Crystallographic analysis and computational studies indicated
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determination for compounds 11 and 24 (Cambridge
Structural Database accession numbers CCDC 983641 and
983642, respectively).
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1
AUTHOR INFORMATION
Corresponding Author
qinglin@buffalo.edu
(18) Initial studies of the individual cycloaddition reactions by H
■
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge the National Institutes of Health
(GM 085092) and the National Science Foundation (CHE-
1305826) for financial support. The FT-ICR mass spectrometer
used in this study was supported through a grant from the NIH
National Center for Research Resources (S10RR029517). We
thank Prof. Wenshe Liu at Texas A&M University for
generously providing us the plasmids pEvol-PylT-PylRS,
pEvol-AcrKRS, and pET-sfGFPS2TAG, and William Brennes-
sel at the University of Rochester for X-ray structural
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