Reaction Rates of Various N-Acylenamines in the Inverse-Electron-Demand Diels–Alder Reaction

Article abstract of DOI:10.1002/ejoc.201800094

In light of the bioorthogonal inverse-electron-demand Diels–Alder strategy, an extended investigation into the effects of ring strain and electron inductive effects on the reactivity of the N-acylenamine core towards tetrazine has been carried out. Through a comparative study between N-acylazetines, N-vinylcarbamates and an N-vinylamide it was shown that ring strain has a more significant effect on reaction rate than electron donation. A significantly improved synthetic route is reported for the preparation of an N-acylazetine biorthogonal tag we have invented previously.

Full text of DOI:10.1002/ejoc.201800094

A Journal of
Accepted Article
Title: Reaction Rates of Various N-Acylenamines in the Inverse-
Electron-Demand Diels-Alder Reaction
Authors: Sander B Engelsma, Thomas C van den Ende, Hermen S.
Overkleeft, Gijs A van der Marel, and Dmitri Filippov
This manuscript has been accepted after peer review and appears as an
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content of this Accepted Article.
To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201800094
Link to VoR: http://dx.doi.org/10.1002/ejoc.201800094
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10.1002/ejoc.201800094
European Journal of Organic Chemistry
FULL PAPER
Reaction Rates of Various N-Acylenamines in the Inverse-
Electron-Demand Diels-Alder Reaction
Sander B. Engelsma, Thomas C. van den Ende, Hermen S. Overkleeft, Gijsbert A. van der Marel*, and
Dmitri V. Filippov*
this knowledge may lead to improved probes for the tetrazine
ligation strategy.
Abstract: In light of the bioorthogonal inverse-electron-demand
Diels-Alder strategy, an extended investigation into the effects of
ring-strain and electron inductive effects on the reactivity of the N-
acylenamine core towards tetrazine has been carried out. Through a
comparative study between N-acylazetines, N-vinylcarbamates and
an N-vinylamide it was shown that ring-strain has a more significant
effect on reaction rate than electron donation.
A significantly
improved synthetic route is reported for the preparation of N-
acylazetine biorthogonal tag we invented previously.
Introduction
The inverse-electron-demand Diels-Alder (IEDDA) reaction
between (cyclic) alkenes and tetrazines has been well-studied
for several decades.[1–7] Renewed interest arose in 2008, due to
the breakthrough of tetrazine ligation in the field of bioorthogonal
chemistry.[8,9] Bioorthogonal ligation handles require an intricate
balance between several physical properties; preferably the
handle should be sterically small in size and be stable under
physiological conditions, while maintaining high reactivity
towards tetrazines. An additional beneficial property is the
moderate-to high hydrophilicity to assist the water solubility of
the functionalized chemical probe. By now, a strong positive
correlation had been established between the ring-strain within
cyclic alkenes and the enhanced reactivity towards tetrazines.[5]
Contrary, significantly less effort has been directed towards the
effects of the alkene electron density on reactivity. Recently we
reported[10] a new ligation handle featuring an N-acylazetine as
the reactive IEDDA partner (e.g. 1 in Figure 1), which was
designed to utilize both ring-strain and electron donating
properties to enhance reactivity towards tetrazines. An N-
acylazetine tag functionalized as p-nitrophenyl active ester was
synthesized and successfully applied in an activity-based protein
profiling experiment. These advancements warranted further
investigation into the structure-reactivity relationship of the N-
alkene structure.
Figure 1. The N-acylazetines 1 and 2 and N-vinyl derivatives 3-5.
N-vinylcarbamates (3 and 4) and vinylamide (5) can be
considered as linear analogues of the original cyclic N-
acylazetine (1) and thereby potential suitable for IEDDA
reactions with tetrazine derivatives. A possible tautomerization
equilibrium between the enamine and imine forms, as in 3,
makes N-vinyl groups both electrophilic at the α-carbon and
nucleophilic at β-carbon.[11] This property make vinylamides
susceptible to hydrolysis, or polymerization in either acid
catalyzed or radical mediated processes.[12,13] N-methylation of
N-vinyl derivative suppresses tautomerization and makes the
corresponding N-methyl-N-vinylamides more stable (i.e. 4). In
addition, disconnection of the N-acylazetine core between the N-
CH2 and the N-vinyl-β-CH leads to commercially available N-
methyl-N-vinylamide (5). To ensure the necessary water
solubility in the kinetic experiments, morpholine was
incorporated in the N-acylazetines (1-2) and N-vinylcarbamates
(3-4).
Results and Discussion
The earlier described synthesis of N-acylazetine 1 consists of six
reaction steps and lacks overall efficiency and scalability.[10]
Therefore, another method to synthesize 1 was explored
(Scheme 1). Commercially available 3-hydroxyazetidine 6 was
selected as starting compound, and the tosyl group was used
instead of the mesyl group as a more potent and UV-detectable
leaving group. Boc-protection of the amine followed by tosylation
of the hydroxyl group in 6 provided protected azetidine 7 in 72%
yield over two steps. To prevent potential displacement of the
We here describe the synthesis of two N-acylazetines (1, 2,
Figure 1) and two N-vinylcarbamates (3, 4) and the assessment
of their stability in water and reactivity towards tetrazines. By
comparing the reaction-rate constants of the IEDDA reactions of
N-acylazetines 1 and 2 with N-vinyl derivatives (3-5) insight can
be gained about the contribution of both ring-strain and electron
donating effects on their reactivity towards tetrazines. Ultimately
tosyl group the Boc-group in
toluenesulfonic acid.
7 was cleaved using p-
[a]
Leiden Institute of Chemistry, Leiden University
Department of Bioorganic Synthesis
Einsteinweg 55, 2333 CC Leiden (The Netherlands)
E-mail: marel_g@lic.leidenuniv.nl; filippov@chem.leidenuniv.nl
Homepage: http://biosyn.lic.leidenuniv.nl/
Supporting information for this article is given via a link at the end of
the document.
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10.1002/ejoc.201800094
European Journal of Organic Chemistry
FULL PAPER
reagents. However, switching to bis(p-nitrophenyl)carbonate
resulted in cleaner and more consistent conversions. Treatment
of p-nitrophenyl esters 10a and b with morpholine gave target N-
acylazetines 1 and 2 in high yield. Alternatively, one could
consider converting the N-acylazetine containing carboxylic
acids to the amides in a “one-pot” procedure. We considered
this approach to be impractical and did not investigate it.
The route of synthesis toward N-vinylcarbamates 3 and 4 makes
use of the Curtius rearrangement of acryl azide (12), in situ
generated from acryloyl chloride (11) and sodium azide. Upon
heating azide 12 rearranges into vinyl isocyanate (13), which
can be transformed into N-vinylcarbamates by coupling with
alcohol of choice. A small amount of base and radical scavenger,
such as hydroquinone or phenothiazine, is needed to suppress
dimerization and radical polymerization. In practice, a solution of
acryl azide 12 in toluene was prepared and slowly added to a
reaction mixture containing 2-morpholinoethanol, pyridine and
hydroquinone in toluene at 100 ˚C, providing N-vinylcarbamate 3
in 29% isolated yield. The vinyl nitrogen was methylated with
sodium hydride and methyl iodide in THF at 0 ˚C, to give N-
methyl-N-vinylcarbamate 4.
The instability of N-vinylcarbamates brings about the question
whether these compounds would survive the conditions of the
kinetic experiments and future bioorthogonal tagging reactions.
The aqueous stability of N-acylazetine 2 and N-vinylcarbamates
3 and 4 was evaluated by dissolving the respective compounds
in deuterated water and monitoring the solution over a period of
13 hours at 37 ˚C with 1H-NMR spectroscopy. After the full
duration, secondary N-vinylcarbamate 3 showed 4% hydrolysis
of the vinyl group, as determined by integration of the formyl
hydrogen (δ = 9.74) resulting from the formed acetaldehyde.
Fortunately, this rate of decomposition proved to be insignificant
within the 30 minutes timeframe required for the kinetic
Scheme 1. Synthesis of N-acylazetines 1, 2 and N-vinylcarbamates 3, 4.
Reagents and conditions: [a] i: Boc2O, TEA, MeOH, 0 ˚C, 2h, used crude. [b]
TsCl, TEA, DCM, 2h, 72% over 2 steps. [c] pTsOH, DCE, reflux, 16h, 79% [d]
Succinic- or glutaric anhydride, K2CO3, MeCN, reflux, 6h, 50% 9a, 70% 9b. [e]
KOtBu, DMF, 2h. ii: bis(p-nitrophenyl)carbonate, 16h, 80% 10a, 69% 10b. [f]
morpholine, DCM, 1h, 84% 1, 100% 2. [g] NaN3, H2O/toluene (1:1), 6h. [h] 2-
morpholinoethanol, pyridine, hydroquinone, toluene, 100 ˚C, 1.5h, 29% [j] NaH,
THF, MeI, 0 ˚C, 1.5h, 75%.
Subsequent purification by crystallization from MeOH yielded
azetidine 8 in 80%. Treatment of 8 with both succinic and
glutaric anhydride was carried out under the agency of
potassium carbonate in refluxing acetonitrile, to give the four-
and five-carbon spacer 9a, and 9b respectively. Next, the key
elimination was initiated by the addition of a solution of KOtBu in
THF. The presence of the tosyl in 9a, and 9b improved solubility
while the reaction proceeded readily at room temperature. The
respective N-acylazetine intermediates were treated in situ with
bis(p-nitrophenol)carbonate to provide linkable handles 10a
(80%) and 10b (69%) in good yields over two steps. Initially, the
p-nitrophenol was introduced using DIC or EDC as coupling
experiments (Table 1). N-acylazetine
2 and N-methyl-N-
vinylcarbamate 4 proved to be stable during the experiment. In
addition, the N-acylazetine group stability was evaluated in the
presence of 100 mM of ethanethiol and 2-aminoethanol, to
mimic the physiological conditions used during in vivo activity-
based protein profiling experiments. Again, no decomposition
occurred.
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10.1002/ejoc.201800094
European Journal of Organic Chemistry
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Table 1: Reaction-rates for addition of dienophiles 1-5 to tetrazine 14. The first-order reaction rates were calculated from three data sets each
using all measurement intervals up until t1/2. The errors ranges are the standard deviations derived from the generated first-order rate data. Half-
life values are calculated from first-order rate constant: t1/2 = ln(2) / k.
Dienophile
T (˚C)
20
1st Order (s-1)
2nd Order (M-1·s-1)
6.88 · 10-1 ± 6.23 · 10-2
1.21 ± 1.42 · 10-2
Half-life (s)
169
4.10 · 10-3 ± 3.71 · 10-4
7.11 · 10-3 ± 8.95 · 10-5
4.40 · 10-3 ± 1.57 · 10-4
7.78 · 10-3 ± 4.25 · 10-5
1.27 · 10-3 ± 2.04 · 10-5
1.97 · 10-3 ± 9.34 · 10-5
7.53 · 10-4 ± 8.54 · 10-6
1.63 · 10-3 ± 4.65 · 10-5
3.12 · 10-4 ± 2.16 · 10-6
5.05 · 10-4 ± 1.68 · 10-6
1
2
3
4
5
37
97
20
7.50 · 10-1 ± 2.62 · 10-2
1.32 ± 7.40 · 10-3
157
37
89
20
2.16 · 10-1 ± 3.48 · 10-3
3.34 · 10-1 ± 1.59 · 10-2
1.27 · 10-1 ± 1.44 · 10-3
2.76 · 10-1 ± 7.90 · 10-3
5.27 · 10-2 ± 3.67 · 10-4
8.54 · 10-2 ± 2.88 · 10-4
544
37
351
20
920
37
424
20
2223
1372
37
With the N-acylazetines 1, 2 and N-vinyl derivatives 3, 4, 5 in
hand, the stage was set for determining the rates of IEDDA
reactions with tetrazine 14 (Table 1). The conditions we opted to
apply for the kinetic experiments were similar to those used by
Devaraj et al in their work featuring the 1-methylcyclopropene
ligation handle.[14] Pseudo-first-order and second-order reaction-
rates were assessed by reacting a 20-fold excess of the
respective dienophile (1-5) with tetrazine 14 in 12% aqueous
DMSO. The rate of tetrazine consumption was measured by
monitoring the characteristic tetrazine absorption at 517 nm.
Each experiment was conducted thrice both at room
temperature (20 ˚C) and at body temperature (37 ˚C). The
results are summarized in Table 1. The availability of N-
acylazetine 1 and 2 allows the determination of the influence of
the spacer length on the IEDDA reaction rate. The outcome
indicates that shortening the spacer length by one carbon as in 1
results in a minor decrease of the reaction rate. This result could
be explained by the increased proximity of the electron
withdrawing carbonyl group to the azetine moiety. However,
in electron density by progressing from an N-vinylamide to an N-
vinylcarbamate. Here the results show that an approximate 2.8-
fold decrease in half-life time, considerably less impactful than
the effects induced by ring-strain. Methylation at the nitrogen of
3 into secondary N-vinylamide 4 only induces a minor (1.5-fold)
increase in half-life time. Additional support for our assertion that
the electronic density effects make only a minor contribution to
the reactivity differences has been provided by the calculated[15]
energies of occupied frontier molecular orbitals (HOMO’s) for
model N-acylenamines (Supporting Information, pages S32-
S36).
Conclusions
An investigation into the contribution of ring-strain and electron
donating effects upon the IEDDA reactivity of N-acylenamines
towards tetrazines is described. To this end, an improved
synthesis toward N-acylazetines 1, 2 is presented while N-
vinylcarbamates 3, 4 could be accessed through the Curtius
rearrangement of acryl azide. The reaction rate constants were
determined for N-acylazetine 1 and 2 and N-vinyl compounds 3-
5 at 20 ˚C and 37 ˚C. Comparison between the reaction rates of
N-acylazetine 2 and N-vinylamide 5 shows a 15-fold higher
reaction rate for the four-membered ring. The influence of the
differences in electron donation between a N-vinylamide and a
given the small size of this effect and
a relatively big
measurement uncertainty, this result has to be treated with
caution. Comparison of the reaction-rates for N-acylazetine 2
and N-vinylamide 5 shows the influence of ring-strain on the
IEDDA reaction rate. Evidently, ring strain contributes
significantly to the reaction rate, as the acyclic N-vinylamines
show (both at 20 ˚C and 37 ˚C) a 15-fold increase in half-life
time. A similar comparison between 4 and 5 reflects the increase
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10.1002/ejoc.201800094
European Journal of Organic Chemistry
FULL PAPER
N-vinylcarbamate was significantly less, resulting in a 2.8-fold
increase in reaction rate for the latter.
charged with p-toluenesulfonic acid (90.9 mmol, 17.3g, 1.1 eq)
and refluxed for 20 hours. The reaction mixture was
concentrated in vacuo. The crude product was crystalized from
MeOH, yielding compound 8 as a white crystalline substance
1
(65 mmol, 25.9 g, 79%). H NMR (400 MHz, CDCl3) δ 9.05 (bs,
Experimental Section
1H), 8.94 (bs, 1H), 7.70 (d, J = 8.2 Hz, 4H), 7.29 (d, J = 8.1 Hz,
2H), 7.19 (d, J = 7.8 Hz, 2H), 5.06 (t, J = 6.2 Hz, 1H), 4.25 (bs,
1H), 4.13 (bs, 1H), 2.42 (s, 3H), 2.39 (s, 3H). 13C NMR (101
MHz, CDCl3) δ 146.07, 141.26, 132.03, 130.38, 129.39, 128.17,
125.92, 67.85, 53.37, 21.86, 21.55.
All solvents and reagents were obtained commercially and used
as received. Reactions were executed at ambient temperatures
unless stated otherwise. Reactions were monitored by TLC-
analysis, spraying with varying stains; an aqueous solution of
cerium molybdate ((NH4)6Mo7O24·4H2O 25 g/L), an aqueous
solution of potassium permanganate (5 g KMnO4, 25 g K2CO3
per L) or an ethanolic solution bromocresol (0.4 g in 1 L, addition
of 0.1 M NaOH(aq) until the solution turns blue). Column
chromatography was performed on silica gel (40-63 µm). 1H and
13C-APT spectra were recorded on a Bruker AV-400 (400 MHz),
Bruker DMX-600 (600 MHz) or Bruker BioSpin (850 MHz). All
present 13C-APT spectra are proton decoupled. The high
resolution mass spectrometry were recorded by direct injection
(2 µL of a 2 µM solution in water/acetonitrile; 50/50; v/v and
0.1% formic acid) on a mass spectrometer (Thermo Finnigan
LTQ Orbitrap) equipped with an electrospray ion source in
positive mode (source voltage 3.5 kV, sheath gas flow 10,
capillary temperature 250 ˚C) with resolution R = 600000 at m/z
400 (mass range m/z = 120-400).
4-Oxo-4-(3-(tosyloxy)azetidin-1-yl)butanoic
acid
(9a)
Compound 8 (20.0 mmol, 5.58 g, 1.1 eq) was co-evaporated
with dioxane, redissolved in MeCN (200 mL), and put under
argon atmosphere. Succinic anhydride (18.2 mmol, 1.82 g, 1 eq)
was added to the reaction mixture, followed by potassium
carbonate (45.5 mmol, 6.3 g, 2.5 eq) and the reaction mixture
was refluxed for 6 hours. Reaction progression was monitored
by TLC, using a bromocresol stain to visualize the produced
carboxylic acid. The reaction mixture was diluted with water (200
mL) and Amberlite-H+ (IR120, ±70 g) was added until the pH fell
below 3. The solution was filtered and the residual MeCN was
removed in vacuo. The water layer was extracted twice with
EtOAc. The organic layers were combined, dried over
magnesium sulfate, filtered and concentrated in vacuo. The
crude product was purified by column chromatography. The
crude product was purified by column chromatography (5% »
10% EtOH in DCM), yielding compound 9a as a white crystalline
substance (9.1 mmol, 3.09 g, 50%). 1H NMR: (400 MHz, CDCl3)
δ 7.80 (d, J = 8.3 Hz, 2H), 7.39 (d, J = 8.2 Hz, 2H), 5.08 (ddd, J
= 11.1, 6.9, 4.3 Hz, 1H), 4.42 (dd, J = 9.4, 7.5 Hz, 1H), 4.30 –
4.08 (m, 2H), 3.94 (dd, J = 11.5, 4.0 Hz, 1H), 2.71 – 2.61 (m,
2H), 2.48 (s, 3H), 2.35 (t, J = 6.8 Hz, 2H). 13C NMR: (101 MHz,
CDCl3) δ 171.99, 145.99, 130.37, 128.07, 76.84, 67.22, 57.38,
55.23, 28.81, 26.20. HRMS: Calculated for C14H18NO6S+
328.08548 [M+H]+; found 328.08477.
tert-Butyl 3-(tosyloxy)azetidine-1-carboxylate (7) A solution of
3-hydroxyazetidine hydrochloride (115 mmol, 10.55 g, 1 eq) and
Et3N (161 mmol, 22.5 mL, 1.4 eq) in MeOH (115 mL) was
prepared at 0 ˚C. Boc2O (126.5 mmol, 27.6 g, 1.1 eq) was added
and the ice-bath was removed. After 5 hours of stirring, the
reaction mixture was concentrated in vacuo, redissolved in DCM
and washed twice with water. The water layers were combined
and extracted twice with DCM. The organic layers were
combined, dried with magnesium sulfate, filtered and
concentrated in vacuo. The intermediate Boc-hydroxyazetidine
was used without further purification. An ice-cooled solution of
the Boc-protected intermediate and Et3N (172.5 mmol, 24 mL,
1.5 eq) in dry DCM (100 mL) was prepared under argon
atmosphere. p-toluenesulfonyl chloride (138 mmol, 26.3 g, 1.2
eq) was added in eight portions over 2 hours and the reaction
mixture was stirred overnight. The reaction mixture was washed
with water twice and the combined aqueous layers were
extracted thrice with DCM. The organic layers were combined,
dried over magnesium sulfate, filtered and concentrated in
vacuo. The crude product was purified by column
chromatography (5% » 10% EtOAc in pentane), yielding tosylate
4-Nitrophenyl
4-(azet-1(2H)-yl)-4-oxobutanoate
(10a)
Compound 9a (9.1 mmol, 3.09 g, 1 eq) was co-evaporated with
dioxane, redissolved in dry DMF (45.5 mL) and put under argon
atmosphere. Next, a 1 M solution of potassium tert-butoxide in
THF (20 mL, 2.1 eq) was added to the reaction mixture and the
reaction was stirred for 1 hour. Subsequently the reaction
mixture was charged with bis(p-nitrophenol)carbonate (10 mmol,
3.01 g, 1.1 eq) and left stirring for an additional 3 hours. The
reaction mixture was diluted with EtOAc and washed twice with
10% aqueous sodium bicarbonate, twice with water and once
with brine. The combined organic layer were dried with MgSO4,
filtered and concentrated in vacuo. The crude product was
purified with column chromatography (50% » 100% EtOAc in
1
7 as a yellow oil. (82.6 mmol, 27.7 g, 72% over two steps). H
NMR: (300 MHz, CDCl3) δ 7.75 (d, J = 8.1 Hz, 2H), 7.34 (d, J =
8.1 Hz, 2H), 4.97 (ddd, J = 10.8, 6.6, 4.3 Hz, 1H), 4.14 – 4.01 (m,
2H), 3.97 – 3.82 (m, 2H), 2.43 (s, 3H), 1.38 (s, 9H). 13C NMR:
(75 MHz, CDCl3) δ 155.86, 145.61, 132.91, 130.17, 127.92,
80.23, 67.84, 56.28, 28.30, 21.74. HRMS: Calculated for
C15H22NO5S+ 328.12132 [M+H]+; found 328.12128.
pentane), yielding compound 10a as
a yellow crystalline
substance (7.3 mmol, 2.0 g, 80%). 1H NMR: (400 MHz, CDCl3) δ
8.26 (d, J = 9.2 Hz, 2H), 7.31 (d, J = 9.1 Hz, 2H), 6.91 (s, 0.5H),
6.71 (s, 0.5H), 5.75 (d, J = 5.3 Hz, 1H), 4.61 (s, 1H), 4.48 (s,
1H), 2.97 (t, J = 6.6 Hz, 2H), 2.75 (t, J = 6.5 Hz, 1H), 2.66 (t, J =
6.5 Hz, 1H). 13C NMR: (101 MHz, CDCl3) δ 170.76, 165.26,
164.88, 155.48, 145.32, 137.43, 136.64, 125.22, 122.55, 114.19,
Azetidin-3-yl 4-methylbenzenesulfonate (8) A solution of
compound 7 (82.6 mmol, 27.1g, 1 eq) in DCE (165 mL) was
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10.1002/ejoc.201800094
European Journal of Organic Chemistry
FULL PAPER
113.93, 77.16, 58.67, 56.91, 29.13, 29.03, 26.74, 25.76. HRMS:
in pentane), yielding compound 10b[10] as a yellow crystalline
substance (6.1 mmol, 1.78 g, 69%). 1H NMR: (400 MHz, CDCl3)
δ 8.26 (d, J = 9.2 Hz, 2H), 7.31 (d, J = 9.1 Hz, 2H), 6.91 (s,
0.5H), 6.71 (s, 0.5H), 5.75 (d, J = 5.3 Hz, 1H), 4.61 (s, 1H), 4.48
(s, 1H), 2.97 (t, J = 6.6 Hz, 2H), 2.75 (t, J = 6.5 Hz, 1H), 2.66 (t,
J = 6.5 Hz, 1H). 13C NMR: (101 MHz, CDCl3) δ 170.76, 165.26,
164.88, 155.48, 145.32, 137.43, 136.64, 125.22, 122.55, 114.19,
113.93, 77.16, 58.67, 56.91, 29.13, 29.03, 26.74, 25.76.
Calculated for C13H13N2O5+ 277.08245 [M+H]+; found 277.08191.
1-(Azet-1(2H)-yl)-4-morpholinobutane-1,4-dione (1) A solution
of compound 10a (1.03 mmol, 232 mg, 1 eq) and morpholine
(3.09 mmol, 0.27 mL, 3 eq) in DCM (2.6 mL) was prepared and
left stirring for 3 hours. The reaction mixture was directly purified
by column chromatography (79%/1% → 50%/5% Acetone/EtOH
in DCM), yielding compound 1 as a off-white crystal (0.86 mmol,
193 mg, 84%). 1H NMR: (400 MHz, CDCl3) δ 6.87 (s, 0.5H), 6.72
(s, 0.5H), 5.70 (d, J = 13.9 Hz, 1H), 4.61 (s, 1H), 4.42 (s, 1H),
3.65 (dt, J = 8.9, 4.9 Hz, 4H), 3.61 – 3.55 (m, 2H), 3.54 – 3.43
(m, 2H), 2.67 (t, J = 6.2 Hz, 2H), 2.66 – 2.58 (m, 1H), 2.52 (t, J =
6.6 Hz, 1H). 13C NMR: (101 MHz, CDCl3) δ 170.27, 166.58,
166.25, 137.27, 137.10, 113.71, 113.48, 66.80, 66.53, 58.72,
56.57, 45.73, 42.04, 27.73, 27.59, 26.91, 25.97. HRMS:
Calculated for C11H17N2O3+ 225.12392 [M+H]+, found 225.12336.
1-(5-Morpholino-5-oxopentanoyl)azetidin-3-yl
4-
methylbenzenesulfonate (2) A solution of compound 10b (0.68
mmol, 198 mg, 1 eq) in DCM (1.7 mL), was charged with
morpholine (2.04 mmol, 0.18 mL, 3 eq) and left stirring for 2
hours. The reaction mixture was directly purified by column
chromatography (20%/1% » 45%/5% Acetone/EtOH in DCM),
yielding compound 2 as a yellow oil (0.68 mmol, 158 mg, 100%).
1H NMR: (400 MHz, CDCl3) δ 6.87 (s, 0.5H), 6.67 (s, 0.5H), 5.74
– 5.63 (m, 2H), 4.55 – 4.49 (m, 1H), 4.41 (s, 1H), 3.68 – 3.60 (m,
4H), 3.58 (d, J = 5.0 Hz, 2H), 3.50 – 3.44 (m, 2H), 2.39 (t, J =
7.1 Hz, 3H), 2.29 (t, J = 6.8 Hz, 1H), 2.01 – 1.90 (m, 2H). 13C
NMR: (101 MHz, CDCl3) δ 171.16, 167.25, 166.73, 137.41,
137.18, 113.53, 66.96, 66.76, 58.72, 56.59, 46.01, 41.95, 32.15,
32.11, 31.24, 30.08, 20.61, 20.49. HRMS: Calculated for
C12H19N2O3+ 239.13902 [M+H]+; found 239.13900.
5-Oxo-5-(3-(tosyloxy)azetidin-1-yl)pentanoic
acid
(9b)
Compound 8 (14.0 mmol, 5.58g, 1.1 eq) was co-evaporated with
dioxane, redissolved in MeCN (140 mL) and put under argon
atmosphere. Glutaric anhydride (12.7 mmol, 1.45 g, 1 eq) was
added to the reaction mixture, followed by potassium carbonate
(31.8 mmol, 4.48 g, 2.5 eq) and the reaction mixture was
refluxed for 6 hours. Reaction progression was monitored by
TLC, using a bromocresol stain to visualize the produced
carboxylic acid. The reaction mixture was diluted with water (200
mL) and Amberlite-H+ (IR120, ±50 g) was added until the pH fell
below 3. The solution was filtered and the residual MeCN was
removed in vacuo. The water layer was extracted twice with
EtOAc. The organic layers were combined, dried over
magnesium sulfate, filtered and concentrated in vacuo. The
crude product was purified by column chromatography (3% »
5% EtOH in DCM), yielding compound 9b as a white crystalline
substance (8.9 mmol, 3.06 g, 70%). 1H NMR: (400 MHz, CDCl3)
δ 7.81 (d, J = 8.3 Hz, 2H), 7.40 (d, J = 8.1 Hz, 2H), 5.08 (tt, J =
6.8, 4.2 Hz, 1H), 4.45 – 4.33 (m, 1H), 4.26 – 4.13 (m, 2H), 3.93
(dd, J = 11.5, 4.3 Hz, 1H), 2.49 (s, 3H), 2.42 (t, J = 7.0 Hz, 2H),
2.17 (t, J = 7.3 Hz, 2H), 1.92 (p, J = 7.2 Hz, 2H). 13C NMR: (101
MHz, CDCl3) δ 177.76, 172.54, 145.84, 132.58, 130.24, 127.93,
67.11, 57.23, 54.93, 32.93, 30.29, 21.78, 19.57. HRMS:
Calculated for C15H20NO6S+ 342.10058 [M+H]+; found
342.10045.
Acryloyl azide (12) Acryloyl chloride (10 mmol, 0.81 mL, 1 eq)
in toluene (1.3 mL) was added to an ice-cooled solution of NaN3
(683 mg, 10.5 mmol, 1.05 eq) in water (2.5 mL). The ice-bath
was removed and the two-layered reaction mixture was stirring
vigorously for 4 hours. Toluene was added (1.3 mL) and the
mixture was poured into a separation funnel and washed twice
with saturated aqueous sodium bicarbonate, twice with water
and once with brine. The organic layer was dried over
magnesium sulfate, filtered and the solution was stored
overnight at 4 ˚C. The product was used in solution without
further purification.
2-Morpholinoethyl vinylcarbamate (3)
A
solution of
hydroquinone (55 mg, 0.5 mmol, 0.05 eq), pyridine (36 μL, 0.5
mmol, 0.5 eq), N-ethoxymorpholine (20 mmol, 2.4 mL, 2 eq) in
toluene (2 mL) was put under argon atmosphere and heated to
100 ˚C. The prepared solution of acryloyl azide in toluene was
added dropwise over one hour. After complete addition, the
reaction mixture was stirred for an additional 30 minutes at 100
˚C, before being allowed to cool to RT. The mixture was filtered
and concentrated in vacuo. The crude product was purified by
4-Nitrophenyl 5-oxo-5-(3-(tosyloxy)azetidin-1-yl)pentanoate
(10b) Compound 9b (8.9 mmol, 3.04 g, 1 eq) was co-evaporated
with dioxane, redissolved in dry DMF (44.5 mL), and put under
argon atmosphere. Next, a 1 M solution of potassium tert-
butoxide in THF (18.7 mL, 2.1 eq) was added to the reaction
mixture and left stirring for 1 hour. Subsequently the reaction
mixture was charged with bis(p-nitrophenol)carbonate (9.8 mmol,
2.95 g, 1.1 eq) and left stirring for another 3 hours. The reaction
mixture was diluted with EtOAc and washed twice with 10%
aqueous sodium bicarbonate, twice with water and once with
Brine. The combined organic layer were dried with magnesium
sulfate, filtered and concentrated in vacuo. The crude product
was purified with column chromatography (50% » 100% EtOAc
column chromatography (0%
» 5% EtOH/DCM), yielding
vinylcarbamate 3 as a yellow oil. (2.85 mmol, 570 mg, 29% over
two steps). 1H NMR: (400 MHz, CDCl3) δ 7.19 (s, 1H), 6.62 (dt, J
= 15.8, 10.0 Hz, 1H), 4.41 (d, J = 15.8 Hz, 1H), 4.21 (d, J = 8.9
Hz, 1H), 4.20 – 4.13 (m, 2H), 3.71 – 3.61 (m, 4H), 2.60 – 2.52
(m, 2H), 2.44 (s, 4H). 13C NMR: (101 MHz, CDCl3) δ 153.68,
129.99, 93.23, 66.69, 61.80, 57.33, 53.68. HRMS: Calculated for
C6H17N2O3+ 201.12392 [M+H]+; found 201.12337.
2-Morpholinoethyl methyl(vinyl)carbamate (4) Sodium
hydride (60% in mineral oil, 2.4 mmol, 96 mg, 1 eq) was added
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10.1002/ejoc.201800094
European Journal of Organic Chemistry
FULL PAPER
potion-wise to a solution of vinylcarbamate 3 (2.4 mmol, 480 mg,
1 eq) in dry THF (8 mL), under argon atmosphere. The reaction
mixture was sonicated until hydrogen evolution stopped. The
reaction mixture was cooled to 0 ˚C and methyl iodide (2.52
mmol, 0.16 mL, 1.05 eq) was added. The reaction mixture was
stirred for 1.5 hours, quenched with Et3N·HCl and concentrated
in vacuo. The crude product was purified by column
chromatography (2% » 5% EtOH in DCM), yielding compound 4
as a yellow liquid. (1.81 mmol, 387 mg, 75%). 1H NMR: (400
MHz, CDCl3) δ 7.24 – 7.01 (m, 1H), 4.37 – 4.15 (m, 4H), 3.76 –
3.59 (m, 4H), 3.01 (s, 2H), 2.65 (t, J = 5.8 Hz, 2H), 2.54 – 2.38
(m, 4H). 13C NMR: (101 MHz, CDCl3) δ 134.20, 133.51, 91.95,
66.86, 63.42, 57.25, 53.80, 30.23. HRMS: Calculated for
C7H19N2O3+ 215.13957 [M+H]+; found 215.13911.
Acknowledgements
This work was supported by an ECHO-grant (711.011.015) from
Netherlands Organisation for Scientific Research (NWO).
Keywords: IEDDA • tetrazine • reaction rates • N-acylazetine •
N-acylenamine
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By measuring the rate of the
cycloaddition reaction of N-
Cycloaddition of N-acylenamines
Sander B. Engelsma, Thomas C. van
den Ende, Hermen S. Overkleeft,
Gijsbert A. van der Marel*, and Dmitri V.
Filippov*
acylazetines, N-vinylcarbamates and
an N-vinylamide we show that ring-
strain has a more significant effect on
the reaction rate than does the
electron donation.
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Reaction Rates of Various N-
Acylenamines in the Inverse-
Electron-Demand Diels-Alder
Reaction
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