Strain-Promoted Reaction of 1,2,4-Triazines with Bicyclononynes

Article abstract of DOI:10.1002/chem.201502397

Strain-promoted inverse electron-demand Diels-Alder cycloaddition (SPIEDAC) reactions between 1,2,4,5-tetrazines and strained dienophiles, such as bicyclononynes, are among the fastest bioorthogonal reactions. However, the synthesis of 1,2,4,5-tetrazines is complex and can involve volatile reagents. 1,2,4-Triazines also undergo cycloaddition reactions with acyclic and unstrained dienophiles at elevated temperatures, but their reaction with strained alkynes has not been described. We postulated that 1,2,4-triazines would react with strained alkynes at low temperatures and therefore provide an alternative to the tetrazine cycloaddition reaction for use in in vitro or in vivo labelling experiments. We describe the synthesis of a 1,2,4-triazin-3-ylalanine derivative fully compatible with the fluorenylmethyloxycarbonyl (Fmoc) strategy for peptide synthesis and demonstrate its reaction with strained bicyclononynes at 37°C with rates comparable to the reaction of azides with the same substrates. The synthetic route to triazinylalanine is readily adaptable to late-stage functionalization of other probe molecules, and the 1,2,4-triazine-SPIEDAC therefore has potential as an alternative to tetrazine cycloaddition for applications in cellular and biochemical studies.

Full text of DOI:10.1002/chem.201502397

DOI: 10.1002/chem.201502397
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Chemical Biology |Hot Paper|
Strain-Promoted Reaction of 1,2,4-Triazines with Bicyclononynes
[
a]
Katherine A. Horner, Nathalie M. Valette, and Michael E. Webb*
Abstract: Strain-promoted inverse electron-demand Diels–
Alder cycloaddition (SPIEDAC) reactions between 1,2,4,5-tet-
razines and strained dienophiles, such as bicyclononynes,
are among the fastest bioorthogonal reactions. However, the
synthesis of 1,2,4,5-tetrazines is complex and can involve
volatile reagents. 1,2,4-Triazines also undergo cycloaddition
reactions with acyclic and unstrained dienophiles at elevated
temperatures, but their reaction with strained alkynes has
not been described. We postulated that 1,2,4-triazines would
react with strained alkynes at low temperatures and there-
fore provide an alternative to the tetrazine cycloaddition re-
action for use in in vitro or in vivo labelling experiments. We
describe the synthesis of a 1,2,4-triazin-3-ylalanine derivative
fully compatible with the fluorenylmethyloxycarbonyl (Fmoc)
strategy for peptide synthesis and demonstrate its reaction
with strained bicyclononynes at 378C with rates comparable
to the reaction of azides with the same substrates. The syn-
thetic route to triazinylalanine is readily adaptable to late-
stage functionalization of other probe molecules, and the
1,2,4-triazine-SPIEDAC therefore has potential as an alterna-
tive to tetrazine cycloaddition for applications in cellular and
biochemical studies.
Introduction
of cycloaddition has now been applied in multiple applications
[
4]
[6,8]
including intracellular imaging, in vivo imaging,
live label-
[
7]
Strain-promoted inverse electron-demand Diels–Alder cycload-
dition (SPIEDAC) between 1,2,4,5-tetrazines and strained al-
kenes and alkynes are the fastest known bioorthogonal conju-
gation reaction. For example, tetrazines (Scheme 1, X=N) react
with strained cycloalkynes to form pyridazine products via a se-
quential inverse electron-demand hetero-Diels–Alder (ihDA)
ling of cell-surface antigens, as well as the modification of
[
5]
cells with nanomaterials for clinical diagnostics.
Although tetrazine SPIEDAC reactions are rapid and efficient,
production of functionalised tetrazine scaffolds remains syn-
thetically challenging in comparison to triazine alternatives.
Unsymmetrical aromatic and aliphatic tetrazines can either be
[1]
[2,9]
retro-Diels–Alder (rDA) cascade. Because this class of reac-
tions proceeds with rate constants ranging from 1–
accessed inefficiently through the Pinner synthesis,
or via
S Ar reaction of a preformed symmetrical tetrazine, and a cor-
N
5
À1 À1[2,3]
[10–14]
1
0 m
s
and gives no toxic by-products, they have
responding multistep synthesis.
The more efficient opti-
[4–8]
become the reaction of choice for in vivo studies.
This class
mised routes to substituted tetrazines involve volatile precur-
sors and intermediates; for example, the use of anhydrous hy-
drazine in the metal-mediated synthesis of aliphatic tetrazines
[
15]
described by Yang et al., or the “highly energetic” 3,6-dihy-
drazino-1,2,4,5-tetrazine intermediate in the synthesis of 3,6-di-
[
14]
chloro-1,2,4,5-tetrazine. In some cases, the tetrazine deriva-
tives required for late-stage functionalisation are readily de-
composed, for example, 3,6-dimethyldicarboxylate-1,2,4,5-tetra-
Scheme 1. SPIEDAC between a tetrazine (1, X=N) or a triazine (1, X=CH)
and a strained cycloalkyne 2. Rate-determining [4+2] cycloaddition be-
tween 1 and 2 led to the highly strained bicyclic adduct 3, which undergoes
a cycloreversion yielding pyridazine (4, X=N) or pyridine-(4, X=CH) prod-
ucts and releasing dinitrogen.
zine is prone to acid-promoted rearrangement and slowly de-
[16]
composes upon warming
and 3,6-dimethylthio- and 3,6-
dichlorotetrazines are incompatible with organometallic spe-
[
17]
cies. Furthermore, some tetrazines are prone to either hy-
[
9]
drolysis or decomposition into the corresponding pyrazoles
or thiazoles when exposed to endogenous cellular nucleo-
[
a] K. A. Horner, Dr. N. M. Valette, Dr. M. E. Webb
School of Chemistry and Astbury Centre for Structural Molecular Biology
University of Leeds, Woodhouse Lane, LS2 9JT Leeds (UK)
E-mail: m.e.webb@leeds.ac.uk
[
18]
philes.
Cycloaddition reactions have also been reported to occur
between 1,2,4-triazines (Scheme 1, X=CH) and dienophiles to
[
19,20]
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201502397.
form dihydropyridine and pyridine derivatives.
The earliest
examples of 1,2,4-triazine cycloaddition reactions involved
ꢀ
2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
[21]
their conjugation to simple nitriles. More recently, attention
This is an open access article under the terms of the Creative Commons At-
tribution License, which permits use, distribution and reproduction in any
medium, provided the original work is properly cited.
has been focused on exploiting the reaction to access a range
of polycyclic and fused heterocycles through tethered triazine
Chem. Eur. J. 2015, 21, 14376 – 14381
14376 ꢀ 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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[20,22]
alkyne/alkene scaffolds.
Although this cycloaddition is now
commonly used in elegant synthetic routes to provide com-
plex pyridyl-containing structures, the need for elevated tem-
peratures (mostly exceeding 1008C) and extended reaction
times means that it has never been considered for cellular ap-
plications. 1,2,4-Triazine and 1,2,4,5-tetrazine conjugations are
controlled by the HOMOdienophile–LUMOdiene gap; dienophiles
with a high degree of ring strain reduce the activation energy
of the [4+2]cycloaddition RDS by raising the HOMOdienophile
and decreasing the distortion energy needed to reach the
[23,24]
cycloaddition transition state.
To date, nearly all examples
of 1,2,4-triazine cycloaddition reactions involve open chain and
[25]
unstrained cyclic dienophiles. Until recently, there were no
reports of a 1,2,4-triazine SPIEDAC reaction with strained
[26,27]
dienophiles other than norbornadiene.
Based on the understanding that strained dienophiles in-
crease the reaction rate for cycloaddition with 1,2,4,5-tetra-
zines, we proposed that the conjugation of a strained cyclo-
alkane/alkyne with a 1,2,4-triazine derivative would also pro-
ceed without the need for elevated temperatures, and could
thus offer an alternative to 1,2,4,5-tetrazine SPIEDAC for use in
a range of in vitro and in vivo applications. We envisaged that
this alternative, although slower, would obviate the need for
toxic and volatile precursors and increase the range of probe
molecules that could be generated. Herein, we report the syn-
thesis of a new 1,2,4-triazinylalanine (TrzAla) derivative 13
(
Scheme 2iii), which is compatible with the fluorenylmethyloxy-
carbonyl (Fmoc) solid-phase peptide synthesis (SPPS) strategy,
demonstrate its incorporation into a model probe peptide 14
and determine the rate of reaction of these compounds to
a representative dienophile bicyclononyne 19. This reaction
has the potential for direct application for conjugation to gen-
etically incorporated bicyclononyne-containing amino acids in
future applications.
Results and Discussion
Our initial aim was to synthesise a triazine with a functional
group handle suitable for rapid derivatisation of target mol-
ecules. We initially synthesised 6-substituted 3-amino-1,2,4-tri-
azines 5a–c (Scheme 2i) with the aim of generating molecules
of the form 8 via amide bond coupling. However, the low nu-
cleophilicity of the exocyclic amine meant that we were able
to generate the protected succinylamidotriazine 7 in only 1%
yield over two steps. In general, the de novo synthesis of tri-
azines by controlled condensation between aminoguanidine
and glyoxal derivatives (Scheme 2i) is low yielding and gives
mixtures of isomers; however, the aminotriazine derivative 5a
is now commercially available in large quantities, and we
therefore used this as the basis for subsequent reactions (Sche-
me 2ii).
Scheme 2. i) Condensation of aminoguanidine and a variety of substituted
glyoxal derivatives to make substituted 3-amino-1,2,4-triazines 5a–c and
subsequent conversion in low yield to amide linked scaffolds. ii) Conversion
of commercial 3-amino-1,2,4-triazine 5a to 3-iodo-1,2,4-triazine 9 by direct
iodination. iii) Synthetic route from serine methyl ester 10 to Fmoc-compati-
ble building block Fmoc-TrzAla-OH 13 and subsequent incorporation into
a peptide.
egy (Scheme 2iii) that utilised iodotriazine 9 as the electrophil-
ic coupling partner. Iodotriazine 9 was formed through diazo-
tisation of aminotriazine with isopentyl nitrite in diiodome-
Jackson and co-workers have reported the synthesis of
enantiomerically pure pyridylalanine amino acids through pal-
ladium-catalysed cross-coupling of serine-derived organozinc
[
30]
thane (Scheme 2ii). Fmoc-iodoalanine methyl ester 11 was
[
28]
synthesised in three steps from serine methyl ester 10.
[28,29]
reagents with halopyridyl derivatives.
We postulated that
For Negishi cross-coupling, commercial zinc dust was acti-
vated with iodine in anhydrous DMF. The use of a dipolar
aprotic solvent, such as DMF prevents coordination of the car-
Negishi-type cross-coupling could be extended to include
other N-heterocycles, and therefore devised a synthetic strat-
Chem. Eur. J. 2015, 21, 14376 – 14381
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bamate carbonyl group to zinc, and therefore promotes b-
elimination (by coordination to the carbonyl methyl ester) to
[31]
form the organozinc reagent. Zinc insertion of iodoalanine
methyl ester 11 was complete after two hours at room tem-
perature (determined by loss of the iodoalanine by TLC), and
the subsequent cross-coupling with iodotriazine 9 was per-
formed using catalytic amounts of palladium(II) acetate and 2-
dicyclohexylphosphino-2’,6’-dimethoxybiphenyl (SPhos). This
gave the desired triazinylalanine methyl ester 12 in a yield of
6
9%. Limited attempts to optimise this coupling by increased
catalyst loading and the alternative palladium catalyst
tris(dibenzylideneacetone)dipalladium(0)) did not lead to sig-
nificant increases in yield. Demethylation of the methyl ester
2 to give the free acid 13 was carried out using trimethyltin
(
1
[
32]
hydroxide in a yield of 27%. In this case, the overall isolated
yield is limited by observable degradation of the triazines 12
and 13 at the elevated temperatures required for deprotection.
(
Note that base-catalysed deprotection with LiOH leads to con-
comitant Fmoc-group removal.) To demonstrate the compati-
bility of the protected triazinylalanine with the conditions for
the Fmoc strategy for solid-phase peptide synthesis, we gener-
ated a simple fluorescein isothiocyanate (FITC)-labelled peptide
14 using Rink amide resin and on-resin fluorescence labelling
in 37% yield following HPLC purification.
We next evaluated suitable cycloaddition partners to couple
to our 1,2,4-triazine derivatives 12 and 13. There are a number
of strained cyclic dienophiles reported for tetrazine SPIEDAC
conjugations (with rate constants spanning five orders of mag-
[
2,3]
nitude).
We initially investigated the cycloaddition reaction
Scheme 3. i) Attempted reaction of triazinylalanine methyl ester 12 with nor-
bornene derivatives 15 and 16 led to no observable product formation. ii)
Synthesis of benzoylated bicyclononyne 19 from single diastereoisomer of
bicyclononene 17. iii) Reaction of protected Fmoc-TrzAla-Ome 12 with pro-
tected bicyclononylyl benzoate 19 gave a mixture of diastereoisomers 20a
and b at 378C.
with norbornene, which reacts with tetrazine with a relatively
À1 À1 [2]
slow rate constant (1–10m s ). We used the methyl ester
2 for rate determination to avoid potential interference from
the free acid in 13. We first synthesised norbornenyl-lysine 15
1
[2]
according to the procedure of Lang et al. , but could not
detect formation of reaction product between 12 and 15 fol-
lowing prolonged incubation up to 808C (below the tempera-
ture at which we had observed thermal degradation of the tri-
azines; Scheme 3i). To ensure that the unprotected norbornen-
yl-lysine 15 was not interfering with our analysis, we confirmed
that unfunctionalised norbornene 16 also did not react under
these conditions.
idyl derivatives 20a and b in 38% yield after 12 h at 378C to-
gether with unreacted triazine 12 (Scheme 3iii). In this case,
the reaction was limited by apparent degradation of the bicy-
clononyne coupling partner (as was determined by NMR).
Next, we assessed the reaction rate at lower concentrations by
determining the rate of product formation by HPLC, using the
purified mixture of authentic 20a and b as a concentration
standard (Figure 1). Product formation in MeCN was measured
over approximately 18 h using 1 mm 12 and increasing con-
centrations of the bicyclononyne 19. Global fitting of the data
The unhindered, strained bicyclo[6.1.0]nonynes react more
rapidly with tetrazines with approximate rate constants of
2.5
3.5 À1 À1 [3]
1
0 –10 m s ; only exceeded by the rates of reaction to
trans-cyclooctenes. We generated benzoyl-protected bicyclo-
nonyne 19 (Scheme 3ii) by adaptation of the synthetic route of
gave an estimate for the second-order rate constant k of
2
[
33]
À2 À1
À1
Dommerholt et al.;
following rhodium-catalysed cyclo-
2.30Æ0.03 ”10
m min . Compounds 12 and 19 are poorly
propanation of 1,5-cyclooctadiene to yield a mixture of the
anti- and syn-bicyclononene ethyl esters 17 and 17b, anti-bi-
cyclononene ethyl ester 17 was converted to bicyclononylol
water soluble, preventing us from carrying out the analogous
experiment in water; however, the reaction rate in 10% H O/
2
MeCN (see the Supporting Information) increased slightly to
À2 À1
À1
18 (Scheme 3ii) by sequential reduction, dibromination and
3.0Æ0.03 ”10
m min suggesting that the rate in bio-
double elimination, followed by protection using benzoyl
logical media will be slightly higher, consistent with other ex-
[34]
[35,36]
chloride to give alkyne 19.
amples of this class of reaction.
À3 À1 À1
We initially assessed the reaction of the protected bicyclo-
nonyne 19 with 12 at high concentration (65 mm in dichloro-
methane). Incubation of an equimolar mixture of the two reac-
tion components yielded a 1:1 mixture of the bicyclononapyr-
Comparison of this rate of reaction (0.3–0.5”10 m s ) to
other reactions in this class
[
37]
suggests that although the
cycloaddition of triazines with bicyclononynes is much slower
than the corresponding reaction of tetrazines, it is comparable
Chem. Eur. J. 2015, 21, 14376 – 14381
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Experimental Section
General chemical experimental details procedures for synthesis of
compounds 5a, b, c, 6, 7, 10, 11, 14, 17a, 18 and 19, and proto-
cols for rate determination can be found in the Supporting Infor-
mation.
Synthesis of 3-iodo-1,2,4-triazine (9)
Isoamyl nitrite (42 mL, 300 mmol, 14 equiv) was added to a stirred
solution of 3-amino-1,2,4-triazine 5a (2 g, 20 mmol, 1 equiv) in
[30]
diiodomethane (ca. 40 mL).
The turbid orange mixture was
stirred at 558C for 4 h, allowed to cool, filtered at the pump and
concentrated as far as possible in vacuo to leave the product and
residual unreacted diiodomethane (ca. 30 mL). The remaining fil-
trate was applied to a silica column and purified by column chro-
matography eluting with 3:1 hexanes/EtOAc. The resultant orange
solid was dissolved in 1,4-dioxane and lyophilised to give the prod-
Figure 1. Rate of reaction of triazinylalanine methyl ester (TrzAla) 12 (1 mm)
with bicyclononyne (BCN) 19 determined by HPLC measurement of forma-
tion of products 20a and b. Rate data were globally fitted to second-order
uct (1.25 g, 6.04 mmol, 30%) as a flocculent orange solid. R (3:1
F
1
hexanes/EtOAc) 0.29; H NMR (500 MHz, CDCl ): d=9.26 (1H, d,
3
3
3
13
kinetics (rate=k
2
[BCN][TrzAla]) under the assumption of low substrate con-
J
2.1 Hz, H ), 8.38 ppm (1H, d,
J
2.2 Hz, H5); C NMR
HÀH
6
HÀH
sumption during the measured time course.
(
3
2
125 MHz, CDCl ): d=149.20 (C ), 148.37 ppm (C ); n˜
450, 3417 cm (NH2 stretch); MS (ES): m/z calcd for C H IN :
07.9293 [M+H]; found: 207.9366; HPLC (5–95% A): retention
(solid):
3
À1
5
6
max
3
3
3
with other known bioorthogonal reactions, such as the Stau-
dinger ligation. With suitable reaction partners, it has the po-
tential to have comparable rates to the strain-promoted add-
ition of azides to alkynes. Very recently, Kamber et al. have re-
ported a complementary study of the reaction between 1,2,4-
time 1.41 min, 100%.
Synthesis of Fmoc-TrzAla-OMe (9H-fluoren-9-yl)methyl (S)-1-
(methoxycarbonyl)-2-(1,2,4-triazin-3-yl)ethylcarbamate (12)
[
25]
To an oven-dried two-neck flask, zinc dust (1.16 g, 18 mmol,
triazin-6-yl derivatives and strained trans-cyclooctenes. Over
3
equiv) was added; the flask was evacuated, dried with a flame
a range of triazine substrates, they observe reaction rates of
[28]
and purged with nitrogen three times. The flask was allowed to
cool to room temperature, dry DMF (18 mL) and iodine (225 mg,
À2 À1 À1
between 1 and 7”10
m s —approximately 30-fold higher
than those we have determined. This ratio is similar to the ap-
proximately 15-fold difference in rate observed for the reaction
of tetrazines with bicyclononyne and trans-cyclooctene sub-
0
.89 mmol, 0.15 equiv) were added in quick succession. The solu-
tion became orange, and after two minutes returned to grey. After
15 min, iodoalanine 11 (2.67 g, 5.9 mmol, 1 equiv) was added, fol-
lowed immediately by iodine addition (225 mg, 0.89 mmol, 0.15
equiv), and the mixture was stirred at room temperature. After two
hours, zinc activation was shown to be complete by TLC (2:1 hex-
anes/EtOAc) and 3-iodo-1,2,4-triazine (9; 1.59 g, 7.68 mmol,
[3]
strates by Lang et al. and Kamber et al. observations are
therefore fully consistent with our observed reaction rates.
1
.3 equiv), palladium(II) acetate (33 mg, 0.15 mmol, 0.025 equiv)
and 2-dicyclohexylphosphino-2’,6’-dimethoxybiphenyl (SPhos)
121 mg, 0.30 mmol, 0.05 equiv) were added to the flask in quick
Conclusion
(
We have defined a route to 1,2,4-triazin-3-yl-linked amino acids
compatible with conventional peptide-synthesis strategies
using readily available and inexpensive starting materials as
precursors. The alkyl triazine reacts readily with the strained bi-
cyclononyne dienophile at 378C indicating that it is suitable
for protein-labelling applications. The synthetic strategy adopt-
ed can be readily adapted to generate triazine-linked scaffolds
at a late stage. The amino acid is similar in structure to a range
of tyrosine-based scaffolds that have been genetically incorp-
orated into proteins in response to an amber codon using
succession. The flask was heated to 508C and stirred for five hours,
the reaction was allowed to cool and filtered through a Celite pad,
which was washed several times with CH Cl . The resultant solution
was concentrated in vacuo, and the pale orange solid was purified
by column chromatography on silica gel, eluting initially with 4:1
hexanes/EtOAc and then EtOAc. The combined fractions were con-
centrated in vacuo and lyophilised to give Fmoc-TrzAla-OMe
2
2
2
7
(
+
1.66 g, 4.12 mmol, 69%) as a flocculent, pale orange solid. [a] =
D
1
4.12 (c=0.19, CH Cl ); R (EtOAc) 0.72; H NMR (500 MHz, CDCl ):
2 2 F 3
3
d=9.36 (1H, s, Tz-H ), 8.81 (1H, d, J
m, Fmoc-H ), 7.58 (2H, t,
Fmoc-H ), 7.31 (2H, t,
2.4, Tz-H ), 7.79–7.73 (2H,
5
7.4, Fmoc-H ), 7.43–7.37 (2H, m,
6
HÀH
3
J
4
HÀH 1
7.3, Fmoc-H ), 5.98 (1H, d,
[38,39]
3
3
evolved tyrosyl-tRNA synthetases.
We hypothesise that
J
J
8.5,
HÀH
3
HÀH
2
that it will be possible to identify such systems as has been re-
cently demonstrated for triazinylphenylalanine by Kamber
NH), 5.07–4.96 (1H, m, H
a
), 4.45–4.35 (2H, m, CHCH
2
), 4.21 (1H, t,
3
13
J
6.97, CHCH ), 3.79–3.72 ppm (5H, m, H and OCH ); C NMR
HÀH
2
b
3
[
25]
(125 MHz, CDCl ): d=171.7 (CO
3
2
Me) 166.4 (Tz-C ), 155.8 (OCO.NH),
3
et al. But because strategies to incorporate bicyclononyne-
1
1
6
51.9 (Tz-C5/6), 147.4 (Tz-C5/6), 143.7 (Fmoc-C5/6), 140.7 (Fmoc-C5/6
)
[
40]
containing amino acids into proteins are already established,
27.8 (Fmoc-C ), 127.1 (Fmoc-C ), 125.2 (Fmoc-C ), 120.0 (Fmoc-C ),
3
2
1
4
this is not a limiting factor for application to site-specific label-
ling in an in vitro or in vivo context.
7.4 (C ), 67.1 (CHCH ), 53.1 (C ), 51.9 (OCH ), 47.0 ppm (CHCH );
b
2
a
3
3
À1
n˜ max (solid): 3049 and 2950 cm (NH stretch), 1715 (CO); MS (ES):
m/z calcd for C H N O : 405.1557 [M+H]; found: 405.1560; HPLC
22
21
4
4
(
5–95% B): retention time 2.93 min, 100%.
Chem. Eur. J. 2015, 21, 14376 – 14381
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Synthesis of Fmoc-TrzAla-OH (9H-fluoren-9-yl)methyl (S)-1-
carboxy)-2-(1,2,4-triazin-3-yl)ethylcarbamate (13)
Acknowledgements
(
K.A.H. was supported by a studentship from BBSRC. N.M.V. and
M.E.W. were supported by funding from EPSRC for research
(EP/I013083/1) and equipment (EP/K03135X/1). We would like
to thank Martin Huscroft for assistance with HPLC assay devel-
opment and Christian Hedberg for recommended conditions
for Negishi-type cross-coupling.
Fmoc-TrzAla-OMe (12; 300 mg, 0.74 mmol, 1 equiv) and trimethyl-
tin hydroxide (402 mg, 2.2 mmol, 3 equiv) were dissolved in an-
hydrous C H Cl (9 mL) and heated at reflux for 2.5 h, until the de-
2
4
2
[32]
protection was shown to be complete by TLC (EtOAc). The reac-
tion was allowed to cool to room temperature and quenched with
H O (15 mL). The organic layer was extracted with CH Cl (3”
2
2
2
2
0 mL), and the combined organic layers were washed with H O
2
(
1”20 mL), brine (2”20 mL), dried (MgSO ) and concentrated in
4
vacuo. The orange oil was purified by column chromatography on
silica gel (95:4:1 CH Cl /MeOH/AcOH) and lyophilised to give
Keywords: bioorthogonal chemistry · chemical biology ·
strain-promoted reactions · synthetic methods · triazines
2
2
Fmoc-trzAla-OH (78 mg, 0.19 mmol, 27%) as a pale yellow, floccu-
2
D
7
lent solid. [a] = +4.90 (c=0.10, CH Cl ); R (CH Cl /MeOH/AcOH)
2
2
F
2
2
1
0
.26; H NMR (500 MHz, CDCl ): d=9.18 (1H, s, Tz-H ), 8.63 (1H, s,
Tz-H ), 7.75 (2H, d, J
.1, Fmoc-H ), 7.39 (2H, t, J
Fmoc-H ), 6.05 (1H, d, J
3
6
3
3
4
[1] A.-C. Knall, C. Slugovc, Chem. Soc. Rev. 2013, 42, 5131–5142.
7.5, Fmoc-H ), 7.57 (2H, dd, J
7.4, J
5
HÀH
4
HÀH
HÀH
[2] K. Lang, L. Davis, J. Torres-Kolbus, C. Chou, A. Deiters, J. W. Chin, Nat.
3
3
3
7.3, Fmoc-H ), 7.30 (2H, t, J
7.4,
1
HÀH
3
HÀH
Chem. 2012, 4, 298–304.
3
8.3. NH), 5.04–4.97 (1H, m, H ), 4.39
2
HÀH
a
[3] K. Lang, L. Davis, S. Wallace, M. Mahesh, D. J. Cox, M. L. Blackman, J. M.
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Angew. Chem. Int. Ed. 2010, 49, 2869–2872; Angew. Chem. 2010, 122,
2931–2934.
2
3
3
(
2H, dd, J
17.1, J
9.1, CHCH ), 4.22 (1H, t, J
5.5, H ), 3.76 ppm (1H, d, J
7.1, CHCH ),
HÀH
HÀH
2
HÀH
2
3
3
13
3
.79 (1H, d, J
5.0, Hb); C NMR
HÀH
b
HÀH
(
1
1
(
1
125 MHz, CDCl ): d=156.1 (OCONH), 149.1 (Tz-C5/6), 148.0 (Tz-C5/6),
3
43.8 (Tz-C ), 143.7 (Fmoc-C5/6), 141.3 (Fmoc-C5/6) 127.7 (Fmoc-C3),
3
[5] J. B. Haun, N. K. Devaraj, S. A. Hilderbrand, H. Lee, R. Weissleder, Nat.
27.1 (Fmoc-C ), 125.1 (Fmoc-C ), 120.0 (Fmoc-C ), 67.3 (C ), 67.1
2
1
4
b
Nanotechnol. 2010, 5, 660–665.
CHCH ), 52.2 (C ), 39.0 ppm (CHCH ); n˜ (solid): 3379 (OH stretch),
2 a 3 max
À1
[6] R. Rossin, P. R. Verkerk, S. M. van den Bosch, R. C. M. Vulders, I. Verel, J.
Lub, M. S. Robillard, Angew. Chem. Int. Ed. 2010, 49, 3375–3378; Angew.
Chem. 2010, 122, 3447–3450.
714 cm (CO); MS (ES): m/z calcd for C H N O : 391.1401 [M+
21 19 4 4
H]; found: 391.1401; HPLC (5–95% B): retention time 2.87 min,
00%.
1
[
[
[
7] N. K. Devaraj, R. Upadhyay, J. B. Hatin, S. A. Hilderbrand, R. Weissleder,
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3
20, 664–667.
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Synthesis of methyl (2S)-3-[2-((1S*,8R*,9R*)-9-benzoyloxyme-
thylbicyclo[6.1.0]nona [4,5-c]pyridyl)-2-((1S)N-(9-fluorenyl-
methoxycarbonyl)amino)propionate (20a and b)
2
[
To a solution of (Z,1S,8R,9r)-bicyclo[6.1.0]non-4-ene-9-ylmethanol
6
(
19; 66 mg, 0.26 mmol, 1 equiv) in CH Cl (2 mL), Fmoc-TrzAla-OMe
[12] Y.-H. Gong, F. Miomandre, R. MØallet-Renault, S. BadrØ, L. Galmiche, J.
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2
2
(12; 105 mg, 0.26 mmol, 1 equiv) in CH Cl (2 mL) was added, and
2
2
the reaction was stirred at 378C for 16 h, after which time, com-
plete consumption of (Z,1S,8R,9r)-bicyclo[6.1.0]non-4-ene-9-ylme-
thanol was observed (4:1 hexanes/EtOAc). The orange solution was
concentrated in vacuo and purified by column chromatography on
silica gel (5% MeOH in CH Cl ), the resultant pale yellow solid was
[
[
14] D. E. Chavez, M. A. Hiskey, J. Energ. Mater. 1999, 17, 357–377.
15] J. Yang, M. R. Karver, W. Li, S. Sahu, N. K. Devaraj, Angew. Chem. Int. Ed.
2
012, 51, 5222–5225; Angew. Chem. 2012, 124, 5312–5315.
16] D. L. Boger, R. S. Coleman, J. S. Panek, F. X. Huber, J. Sauer, J. Org. Chem.
985, 50, 5377–5379.
2
2
[
dissolved in dioxane and lyophilised to give the product (62 mg,
1
2
7
0
0
5
.09 mmol, 38%) as a pale yellow flocculent solid. [a] = +4.7 (c=
D
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1
.11, CH Cl ); R (25:1 hexanes/EtOAc) 0.09; H NMR ([D ]CH Cl ,
2
2
F
2
2
2
00 MHz); d=8.26–8.18 (1H, m, H ), 8.08–7.96 (2H, m, Bz-H ), 7.79
9
2
3
4
(
2H, dd, J
7.6, J
4.0 Hz, Fmoc-H ), 7.67–7.55 (3H, m, Bz-H
4
[19] J. Balcar, G. Chrisam, F. X. Huber, J. Sauer, Tetrahedron Lett. 1983, 24,
HÀH
HÀH
4
1
481–1484.
20] B. R. Lahue, S.-M. Lo, Z.-K. Wan, G. H. C. Woo, J. K. Snyder, J. Org. Chem.
004, 69, 7171–7182.
and Fmoc-H ), 7.53–7.43 (2H, m, Bz-H ), 7.43–7.38 (2H, m, Fmoc-
H ), 7.37–7.28 (2H, m, Fmoc-H ), 7.03–6.93 (1H, m, H ), 6.64–6.55
(
4
1
3
[
3
2
8
2
1H, m, NH), 4.83–4.73 (1H, m, H ), 4.43–4.29 (2H, m, Fmoc-CHCH ),
a 2
[
[
[
21] E. C. Taylor, L. G. French, J. Org. Chem. 1989, 54, 1245–1249.
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6854.
.25 (1H, t, J=7.1 Hz, CHCH ), 4.12–3.98 (2H, m, Bz-COOCH ), 3.54
2
2
2
4
3
(
1H, ddd,
J
17.0,
J
12.1,
J
5.6 Hz, H ), 3.31 (1H, ddd,
HÀH
HÀH
HÀH
HÀH b
2
4
3
J
16.0, J
11.4, J
4.3 Hz, H ), 3.05–2.92 (2H, m, H ), 2.87–
b 3
HÀH
HÀH
2
0
.72 (2H, m, H ), 2.64–2.44 (2H, m, H ), 1.48–1.33 (2H, m, H ), 0.95–
[24] J. Sauer, D. K. Heldmann, J. Hetzenegger, J. Krauthan, H. Sichert, J.
Schuster, Eur. J. Org. Chem. 1998, 2885–2896.
3
’
4
4’
1
3
.88 (1H, m, H ), 0.85–0.70 ppm (2H, m, H ); C NMR (125 MHz,
5
4a
CDCl3): d=172.8 (COOCH3), 166.8 (Bz-COCH2), 156.5 (Fmoc-
COONH), 155.0 (C ), 152.3 (C ), 146.1 (C ), 144.4 (Fmoc-C ), 141.6
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Prescher, J. Am. Chem. Soc. 2015, DOI: 10.1021/jacs.5b05100.
2
2a
9
4a
[
[
[
26] V. N. Kozhevnikov, D. N. Kozhevnikov, O. V. Shabunina, V. L. Rusinov,
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(
Fmoc-C ), 136.6 (C ), 133.1 (Fmoc-C ), 131.0 (Bz-C ), 129.8 (Bz-C ),
1a 7a 1 1 2
1
28.7 (Bz-C ), 128.0 (Fmoc-C ), 127.4 (Fmoc-C ), 125.5 (Bz-C ), 124.5
3 3 2 4
(C ), 120.3 (Fmoc-C ), 68.8 (C ), 67.2 (Fmoc-CHCH ), 53.1 (COOCH ),
8 4 5’ 2 3
3688.
5
2
2.5 (C ), 47.6 (Fmoc-CHCH ), 35.9 (C ), 33.9 (C ), 29.0 (C ), 26.7 (C ),
a 2 b 3 4 5
28] S. Tabanella, I. Valancogne, R. F. W. Jackson, Org. Biomol. Chem. 2003, 1,
À1
2.4 (C ); n˜ (solid): 3335 (NH stretch), 1714 cm (CO); MS (ES):
4
a
max
4254–4261.
m/z calcd for C H N O : 631.2803 [M+H]; found 631.2814; HPLC
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3
9
39
2
6
(
5–95% B): 4.75 min, 100%.
Chem. Eur. J. 2015, 21, 14376 – 14381
www.chemeurj.org
14380 ꢀ 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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Received: June 20, 2015
1
3518.
Published online on August 13, 2015
Chem. Eur. J. 2015, 21, 14376 – 14381
www.chemeurj.org
14381 ꢀ 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim