A convenient [2+2] cycloaddition-cycloreversion reaction for the synthesis of 1,1-dicyanobuta-1,3-diene-scaffolded peptides as new imaging chromophores

Article abstract of DOI:10.1016/j.tetlet.2011.10.084

We report on the chemoselective coupling between colorless peptide fragments functionalized with a mutually reactive electron-rich N ^α-(4-ethynylphenyl)-N^α-(methyl)-glycyl- and an electron-deficient [4-(2,2-dicyanovinyl)]benzoyl moie

Full text of DOI:10.1016/j.tetlet.2011.10.084

Tetrahedron Letters 52 (2011) 6963–6967
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
A convenient [2+2] cycloaddition–cycloreversion reaction for the synthesis
of 1,1-dicyanobuta-1,3-diene-scaffolded peptides as new imaging
chromophores q
Dirk T.S. Rijkers a, , Fernando de Prada López a, Rob M.J. Liskamp a, François Diederich b
⇑
a Medicinal Chemistry & Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Department of Pharmaceutical Sciences, Faculty of Science,
Utrecht University, Universiteitsweg 99, 3584 CG Utrecht, The Netherlands
b Laboratorium für Organische Chemie, ETH-Zürich, Hönggerberg HCI, Wolfgang-Pauli-Strasse 10, CH-8093 Zürich, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 July 2011
Revised 29 September 2011
Accepted 17 October 2011
Available online 20 October 2011
We report on the chemoselective coupling between colorless peptide fragments functionalized with a
mutually reactive electron-rich N -(4-ethynylphenyl)-N -(methyl)-glycyl- and an electron-deficient
[4-(2,2-dicyanovinyl)]benzoyl moiety. The resulting donor-substituted 1,1-dicyanobuta-1,3-dienes
a
a
represent a new class of orange-red colored (kmax = 450–500 nm, with molar extinction coefficients (e)
above 5,000 molꢀ1 dm3 cmꢀ1) peptide-based imaging chromophores.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Cycloaddition
Dicyanovinyl derivatives
Donor–acceptor chromophores
p-Conjugated peptides
Peptides
Peptidomimetics
Labeled peptides are important tools in chemical biology to ob-
tain information on their biological function in, for example, cellu-
lar communication processes as well as to study the molecular
basis of diseases.1 The functionalization of peptides with biophys-
ical reporter groups such as fluorophores, strongly absorbing
chromophores, biotin, or spin-labels, is most often performed via
post-synthetic chemoselective conjugation reactions, involving
amino, thiol, or azide specific reactions.2 An alternative approach
for the installation of fluorescent or chromophoric organic mole-
cules is the coupling of non-fluorescent/chromophoric precursor
molecules to obtain fluorescence/chromophoric properties in the
final product.3 A versatile method to achieve this makes use of
the Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC),4 to enable
modulation of the emission response of the newly synthesized
fluorophores/chromophores.5
tetracyanoethylene (TCNE) to obtain 1,1,4,4-tetracyanobuta-1,3-
diene-scaffolded peptides as a new class of p-conjugated peptidic
donor–acceptor chromophores.6,7 Here, we report on the use of
a
N -[(4-(2,2-dicyanovinyl)]benzoyl moiety, as an alternative to
TCNE, for N-terminal peptide modification, to modulate the optical
properties, to increase peptide diversity, and to access 1,1-dicya-
nobuta-1,3-diene-scaffolded peptides as chromophores, in a single
final assembly step, as shown schematically in Figure 1.
a
As a model compound, N -{[4-(2,2-dicyanovinyl)]benzoyl}-ala-
nine ethyl ester (3a)8 was synthesized (Scheme 1). 4-Formylbenzoic
acid (1) was preactivated with benzotriazol-1-yloxy-tris-(dimethyl-
amino)-phosphonium hexafluorophosphate/N,N-diisopropylethyl-
amine (BOP/DIPEA) prior to the addition of HClꢁH-Ala-OEt, to avoid
imine formation between the aldehyde and the amine,9 and amide
2a was obtained in a high yield (93%). In the next step, an Al2O3-cat-
alyzed Knoevenagel condensation10 was performed in the presence
of malononitrile in THF under reflux, which gave dicyanovinyl com-
pound 3a in good yield (76%) after purification by column chroma-
tography. In a similar approach, valine derivative 3b, and the
dicyanovinyl compounds 3c and 3d, containing a dipeptide and a
protected dipeptide, respectively, were synthesized in satisfactory
yields ranging between 55% and 61% (Scheme 1).
To extend the repertoire for installing a chromophoric property
into non-chromophoric precursor molecules, we recently reported
on a formal [2+2] cycloaddition–cycloreversion reaction between
peptide-functionalized electron-rich alkynes and electron-deficient
q
Parts of this research have been presented at the 3rd EuCheMS Chemistry
Congress (Nuremberg, Germany, August 2010) and at the 31st European Peptide
Symposium (Copenhagen, Denmark, September 2010).
In an initial attempt to perform the [2+2] cycloaddition–cyclo-
reversion with these peptide congeners, N,N-dimethyl-4-ethyny-
laniline (4)11 was reacted with dicyanovinyl derivative 3a in
⇑
Corresponding author. Tel.: +31 (0) 62 026 0572; fax: +31 (0) 30 253 6655.
E-mail address: D.T.S.Rijkers@uu.nl (D.T.S. Rijkers).
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.10.084

6964
D. T. S. Rijkers et al. / Tetrahedron Letters 52 (2011) 6963–6967
PEPTIDE
PEPTIDE
PEPTIDE
PEPTIDE
colorless (protected) peptide fragments
with mutually reactive groups
colored (protected) peptide adducts
N
N
H
N
N
H
N
N
H
PEPTIDE
R1
N
H
N
H
PEPTIDE
PEPTIDE
H
R2
5a-d
7a-d
O
O
in bold: the substituted buta-1,3-diene scaffold
Figure 1. Mutually reactive groups result in strongly absorbing 1,1-cyanobuta-1,3-diene-scaffolded peptide-based chromophores.
CH2(CN)2
Al2O3 in THF
reflux, 90 min
N
N
O
O
BOP/DIPEA/amine
CH2Cl2, 0 oC to rt, 16 h
R
OH
R
O
O
1
2a
-
d
3a-d
O
amine: H-Ala-OEt
amine: H-Val-OMe
amine: H-Ala-Phe-OMe
amine: H-Lys(Boc)-Ala-OtBu
O
O
O
O
H
H
H
H
R =
N
N
N
O
N
O
R =
R =
R =
O
O
N
H
N
H
O
O
H
N
O
2b (49%)
3b (59%)
2c (86%)
3c (55%)
2d (70%)
3d (61%)
2a (93%)
3a (76%)
O
Scheme 1. Synthesis of the building blocks to obtain dicyanovinyl derivatives 3a–d.
Table 1
tide-based azides and alkynes.13 Therefore, the reaction was per-
formed in CH2Cl2 at 60 °C under microwave irradiation (entry 5)
over 20 min and 5a was isolated in 40% yield, a remarkable in-
crease in yield in a significantly shorter reaction time compared
to entry 1. Finally, a quantitative yield of 5a was obtained by run-
ning the reaction in CH2Cl2 at 75 °C under microwave irradiation
(entry 6) for 60 min using excess of the alkyne (1.2 equiv) to facil-
itate purification by column chromatography.
Reaction optimization between alkyne 4 and dicyanovinyl derivative 3a to yield
dicyanobutadiene 5a
Entry
Solvent
T (°C)
Time
Isolated yield (%)
1
2
3
4
5
6
CH2Cl2
DMF
DMA
DMA
CH2Cl2
CH2Cl2
25
100
100
130
60 (MW)
75 (MW)
4 d
16 h
16 h
16 h
20 min
60 min
19
79
67
Intractable material
40
Quant.
These optimized reaction conditions were used for the synthe-
sis of 1,1-dicyanobuta-1,3-diene-scaffolded peptides 5b–d (alkyne
4 + dicyanovinyl derivatives 3b–d) and 7a14–d (alkyne 615 + dicy-
anovinyl derivatives 3a–d), as shown in Scheme 2. The highest
yields were obtained with alkyne 4 (48–100%), while the cycload-
dition products of alkyne 6 were obtained in yields ranging from
17% to 70%, partly due to the increasing bulkiness of the peptide
moieties and the low solubility and rather difficult purification of
the obtained dicyanobutadienes. Figure 2 shows the aromatic re-
gion of the 1H NMR spectrum of 5b16 indicating the E-selectivity
of the cycloaddition–cycloreversion reaction, resulting from the
torquoselectivity of the latter step,7d since 3J-values of 15.7 Hz
for both olefinic protons were observed.
CH2Cl2 at room temperature. Under these conditions, product for-
mation was rather slow, and after 4 days of stirring, compound
5a12 was isolated in 19% yield as an intense purple-red solid (entry
1, Table 1, Scheme 2). Apparently, dicyanovinyl derivatives are less
reactive toward electron-rich alkynes than TCNE, and this reaction
needed to be optimized. The results are shown in Table 1. Running
the reaction in DMF at 100 °C7d for 16 h improved the efficiency
and 5a was isolated in 79% yield (entry 2). To avoid the presence
of amines due to any decomposition of DMF at elevated tempera-
tures, N,N-dimethylacetamide (DMA) was used as an alternative
(entries 3 and 4). Unfortunately, no further improvements were
observed; moreover, running the reaction at 130 °C led to an
intractable reaction mixture. This made us try microwave irradia-
tion as an alternative way of heating, based on our positive
experience with the Cu(I)-catalyzed cycloaddition between pep-
The UV–Vis spectra (in CHCl3) of compounds 5a and 7a are
shown in Figure 3A. Dicyanobutadiene 5a showed an absorption
maximum at k = 358 nm with a high molar extinction coefficient
(e
) of 25,000 molꢀ1 dm3 cmꢀ1, and a second, less intense maxi-
mum, at k = 475 nm (
e
8,000 molꢀ1 dm3 cmꢀ1). A solution of com-
pound 5a was orange-red in color while as a solid, this class of

D. T. S. Rijkers et al. / Tetrahedron Letters 52 (2011) 6963–6967
6965
N
N
H
3a-d
optimized
reaction conditions
Table 1
N
H
N
R
4
5a
-d
N
N
H
O
3a-d in CH2Cl2
MW, 75 oC, 60 min
O
O
H
N
H
N
O
N
O
N
O
H
O
O
R
6
7a-
d
O
O
O
O
H
O
H
H
N
H
N
N
O
N
O
R =
N
N
H
O
H
O
O
H
N
O
5b (quant.)
7b (56%)
5c (48%)
7c (21%)
5d (84%)
7d (17%)
5a (quant.)
7a (70%)
O
Scheme 2. The [2+2] cycloaddition reaction of dicyanovinyl compounds with alkynes.
Figure 2. Aromatic region of the 1H NMR spectrum of 5b (see Ref. 12 for a complete assignment) indicating the E-selectivity of the cycloaddition–cycloreversion reaction.
peptide chromophores was found to have a purple-black color
with a typical metallic luster.17 The orange-red color originates
from the longer-wavelength band for which charge-transfer char-
acter has been previously established.7c,d This charge-transfer
character is supported by a reversible quenching experiment:
protonation of the anilino donor with trifluoroacetic acid (TFA)
eliminates this absorption band, whereas it is fully restored upon
neutralization with triethylamine (Et3N) as is shown in Figure 3B.
moiety, which tempers the electron-donating properties of the aniline
nitrogen.
In conclusion, the synthesis of a new class of peptide-based
chromophores has been described via the [2+2] cycloaddition–
cycloreversion reaction between an electron-rich alkyne and an
electron-deficient ethylene species, resulting in colored peptide
scaffolds, ranging from yellow to orange-red, with high molar
a
extinction coefficients. In principle, building blocks such as N -
a
a
The absorption spectrum of 7a indicated
a
maximum at
(4-ethynylphenyl)-N -(methyl)-glycine or N -[4-(2,2-dicyanovi-
k = 358 nm with a molar extinction coefficient of 17,000 molꢀ1
nyl)benzoyl]-glycine would be ideally suited for peptide modifica-
tion to access this novel class of p-conjugated peptides as donor–
acceptor chromophores. Finally, this chemistry can be considered
as a model study for a novel bioconjugation approach to biologi-
cally relevant peptides in which the imaging chromophores are in-
stalled during bioconjugation as the final reaction step.
dm3 cmꢀ1, while the second maximum was hypsochromically
shifted, as compared to 5a, by 40 nm to k = 435 nm (
e
5,100 molꢀ1
dm3 cmꢀ1) (Fig. 3A), which is also apparent from the bright-yellow
color of the solution. This relatively large hypsochromic shift (blue
shift) maybe explained by the
r-acceptor character of the a-carbonyl

6966
D. T. S. Rijkers et al. / Tetrahedron Letters 52 (2011) 6963–6967
Figure 3. (A) UV–Vis spectra (in CHCl3 at T = 298 K) of 5a (red line: 23
l
M) and 7a (blue line: 15
lM); (B) Reversible charge-transfer quenching of 5a: (red line: 23 lM in
CHCl3), after the addition of TFA (10
l
L) (blue line), after neutralization with Et3N (10 L) (green line).
l
11. 4-Ethynyl-N,N-dimethylaniline is commercially available; CAS [17573-94-3].
Acknowledgments
a
12. N -{4-[(1E)-4,4-dicyano-3-[4-(dimethylamino)phenyl]buta-1,3-dien-1-yl]benzoyl}-
alanine ethyl ester (5a):18 Rf = 0.62 (CH2Cl2/MeOH, 95:5 v/v), Rf = 0.66 (EtOAc/
Et2O, 1:1 v/v), Rf = 0.37 (Et2O); Rt = 38.8 min;19 mp 136–140 °C; 1H NMR
(300 MHz, CDCl3, 25 °C): d = 1.32 (t, 3J(H,H) = 7.2 Hz, 3H; CH3), 1.53 (d,
3J(H,H) = 7.2 Hz, 3H; bCH3 Ala), 3.10 (s, 6H; N(CH3)2), 4.22 (q, 3J(H,H) = 7.2 Hz,
D.T.S.R. gratefully acknowledges the ERC Advanced Grant No.
246637 (‘OPTELOMAC’) for financial support, and Utrecht Univer-
sity for facilitating the sabbatical leave at the ETHZ. Dr. Milan
Kivala (Friedrich-Alexander Universität, Erlangen-Nürnberg, Ger-
many) is acknowledged for his interest and scientific discussions
in relation to this work.
2H; OCH2), 4.78 (quin, 3J(H,H) = 7.2 Hz, 1H; CH Ala), 6.75 (d, 3J(H,H) = 9 Hz, 2H;
a
arom H), 6.79 (overlapping signal, 1H; amide NH), 7.01 (d, 3J(H,H) = 15.6 Hz,
1H; @CH), 7.40 (d, 3J(H,H) = 9 Hz, 2H; arom H), 7.54 (d, 3J(H,H) = 15.6 Hz, 1H;
@CH), 7.60 (d, 3J(H,H) = 8.4 Hz, 2H; arom H), 7.82 (d, 3J(H,H) = 8.4 Hz, 2H;
arom H); 13C NMR (75.5 MHz, CDCl3, 25 °C): d = 172.9, 170.0, 165.5, 152.7,
146.2, 137.6, 135.6, 131.6, 128.4, 127.7, 126.9, 119.5, 114.8, 114.0, 111.3, 78.2,
77.2, 61.8, 48.7, 40.1, 18.8, 14.3; UV–Vis (CHCl3): kmax (e) = 475 (8,350),
References and notes
358 nm (25,750 molꢀ1 dm3 cmꢀ1); ESMS calcd for C26H26N4O3: 442.20, found
m/z 443.40 [M+H]+.
1. For selected reviews, see: (a) Tung, C.-H. Biopolymers (Peptide Science) 2004, 76,
391; (b) Rao, J.; Dragulescu-Andrasi, A.; Yao, H. Curr. Opin. Biotechnol. 2007, 18,
17; (c) Lee, S.; Xie, J.; Chen, X. Chem. Rev. 2010, 110, 3087.
13. Rijkers, D. T. S.; van Esse, G. W.; Merkx, R.; Brouwer, A. J.; Jacobs, H. J. F.; Pieters,
R. J.; Liskamp, R. M. J. Chem. Commun. 2005, 4581.
14. Compound 7a: Rf = 0.58 (CH2Cl2/MeOH, 9:1 v/v), Rf = 0.26 (CH2Cl2/MeOH, 95:5
2. Dent, A. H.; Aslam, M. In Bioconjugation, Protein Coupling Techniques for the
Biomedical Sciences; Macmillan: London, 2000; pp 50–100. Chapter 2.
3. Herein, we use ‘chromophore’/‘non-chromophore’ in terms to indicate that it
absorbs visible light, irrespective of its property to absorb UV light.
4. (a) Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057; (b)
Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int. Ed.
2002, 41, 2596; (c) Meldal, M.; Tornøe, C. W. Chem. Rev. 2008, 108, 2952.
5. For a review, see: (a) Le Droumaguet, C.; Wang, C.; Wang, Q. Chem. Soc. Rev. 2010,
39, 1233; for selected papers, see: (b) Zhou, Z.; Fahrni, C. J. J. Am. Chem. Soc. 2004,
126, 8862; (c) Jarowski, P. D.; Wu, Y.-L.; Schweizer, W. B.; Diederich, F. Org. Lett.
2008, 10, 3347; (d) Bag, S. S.; Kundu, R. J. Org. Chem. 2011, 76, 3348; (e) Qi, J.; Han,
M.-S.; Chang, Y.-C.; Tung, C.-H. Bioconjugate Chem. 2011, 22, 1758.
v/v), Rf = 0.07 (EtOAc/hexane, 1:1 v/v); Rt = 38.5 min;19 mp 149–153 °C; 1H
NMR (300 MHz, CDCl3, 25 °C): d = 0.81 (d, 3J(H,H) = 6.6 Hz, 3H;
c CH3 Val),
0.92 (d, 3J(H,H) = 6.6 Hz, 3H; c0CH3 Val), 1.32 (t, 3J(H,H) = 7.2 Hz, 3H; CH3),
1.55 (d, 3J(H,H) = 7.2 Hz, 3H; bCH3 Ala), 2.17 (m, 1H; bCH Val), 3.20 (s, 3H;
NCH3), 3.73 (s, 3H; OCH3), 3.97 (d, 3J(H,H) = 18 Hz, 1H; CH2 Gly), 4.09 (d,
3J(H,H) = 35.1 Hz, 1H; CH2 Gly), 4.27 (q, 3J(H,H) = 7.2 Hz, 2H; OCH2), 4.59 (dd,
3J(H,H) = 4.5 Hz, 3J(H,H) = 9 Hz, 1H;
a
CH Val), 4.78 (quin, 3J(H,H) = 7.2 Hz, 1H;
a
CH Ala), 6.64 (d, 3J(H,H) = 9.3 Hz, 1H; amide NH Val), 6.80 (d,
3J(H,H) = 7.2 Hz, 1H; amide NH Ala), 6.85 (d, 3J(H,H) = 9.3 Hz, 2H; arom H),
6.96 (d, 3J(H,H) = 15.6 Hz, 1H; @CH), 7.40 (d, 3J(H,H) = 9.3 Hz, 2H; arom H),
7.57 (d, 3J(H,H) = 15.6 Hz, 1H; @CH), 7.61 (d, 3J(H,H) = 8.1 Hz, 2H; arom H),
7.82 (d, 3J(H,H) = 8.1 Hz, 2H; arom H); 13C NMR (75.5 MHz, CDCl3, 25 °C):
d = 173.1, 172.1, 170.0, 169.0, 165.6, 151.5, 146.8, 137.5, 136.0, 131.4, 128.6,
127.9, 126.7, 122.1, 114.2, 113.4, 112.6, 61.8, 57.6, 56.8, 52.3, 48.7, 39.7,
6. Rijkers, D. T. S.; Diederich, F. Tetrahedron Lett. 2011, 52, 4021.
7. (a) Michinobu, T.; May, J. C.; Lim, J. H.; Boudon, C.; Gisselbrecht, J.-P.; Seiler, P.;
Gross, M.; Biaggio, I.; Diederich, F. Chem. Commun. 2005, 737; (b) Michinobu, T.;
Boudon, C.; Gisselbrecht, J.-P.; Seiler, P.; Frank, B.; Moonen, N. P.; Gross, M.;
Diederich, F. Chem. Eur. J. 2006, 12, 1889; (c) Jarowski, P. D.; Wu, Y.-L.; Boudon,
C.; Gisselbrecht, J.-P.; Gross, M.; Schweizer, W. B.; Diederich, F. Org. Biomol.
Chem. 2009, 7, 1312; (d) Wu, Y.-L.; Jarowski, P. D.; Schweizer, W. B.; Diederich,
F. Chem. Eur. J. 2010, 16, 202.
31.2, 22.7, 19.1, 18.7, 17.6, 14.1; UV–Vis (CHCl3): kmax (e) = 435 (5,100),
358 nm (17,000 molꢀ1 dm3 cmꢀ1); ESMS calcd for C33H37N5O6: 599.68, found
m/z 600.40 [M+H]+, 622.70 [M+Na]+.
15. The synthesis of alkyne 6 is described in Ref. 6
a
16. N -{4-[(1E)-4,4-dicyano-3-[4-(dimethylamino)phenyl]buta-1,3-dien-1-yl]benzoyl}-
a
valine methyl ester (5b): Rf = 0.08 (CH2Cl2/MeOH, 98:2 v/v), Rf = 0.14 (EtOAc/
hexane, 2:1 v/v), Rf = 0.30 (EtOAc/hexane, 1:1 v/v); Rt = 38.8 min;19 mp 163–
165 °C; 1H NMR (300 MHz, CDCl3, 25 °C): d = 1.03 (t, 3J(H,H) = 5.8 Hz, 6H; CH3
Val), 2.32 (m, 1H; bCH Val), 3.10 (s, 6H; N(CH3)2), 3.79 (s, 3H; OCH3), 4.80 (m,
8. N -[4-(2,2-Dicyanovinyl)benzoyl]-alanine ethyl ester (3a): Rf = 0.68 (CH2Cl2/
MeOH, 95:5 v/v), Rf = 0.33 (CH2Cl2/MeOH, 98:2 v/v); mp 112–117 °C;
½ ꢂ
a 2D0 = +59.1 (c = 0.33, CHCl3); 1H NMR (300 MHz, CDCl3, 25 °C): d = 1.30 (t,
3J(H,H) = 7.2 Hz, 3H; CH3), 1.51 (d, 3J(H,H) = 7.2 Hz, 3H; bCH3 Ala), 4.23 (q,
a
CH Val), 6.66 (d, 3J(H,H) = 8.5 Hz, 1H; amide NH), 6.76 (d, 3J(H,H) = 9 Hz,
3J(H,H) = 7.2 Hz, 2H; OCH2), 4.75 (quin, 3J(H,H) = 7.2 Hz, 1H;
aCH Ala), 7.03 (d,
1H;
2H; arom H), 7.02 (d, 3J(H,H) = 15.7 Hz, 1H; @CH), 7.39 (d, 3J(H,H) = 9 Hz, 2H;
arom H), 7.54 (d, 3J(H,H) = 15.7 Hz, 1H; @CH), 7.59 (d, 3J(H,H) = 8.5 Hz, 2H; arom
H), 7.81 (d, 3J(H,H) = 8.5 Hz, 2H; arom H); 13C NMR (75.5 MHz, CDCl3, 25 °C):
d = 172.5, 170.0, 166.2, 152.8, 146.2, 135.8, 131.6, 128.4, 127.8, 127.0, 119.5,
114.0, 111.3, 57.5, 52.3, 40.0, 31.6, 18.9, 17.9 (19 lines out of 22); UV–Vis
3J(H,H) = 7.2 Hz, 1H; amide NH), 7.83 (s, 1H; @CH), 7.92 (m, 4H; arom H); 13C
NMR (75.5 MHz, CDCl3, 25 °C): d = 172.7, 164.7, 158.4 (2 lines), 138.5, 133.1,
130.5, 128.0, 113.1, 112.1, 84.7, 61.8, 48.8, 18.4, 14.1; UV–Vis (CHCl3): kmax
(e
) = 314 nm (28,000 molꢀ1 dm3 cmꢀ1); ESMS calcd for C16H15N3O3: 297.31,
found m/z 298.65 [M+H]+.
(CHCl3): kmax (e
) = 475 (9,800), 358 nm (31,000 molꢀ1 dm3 cmꢀ1); ESMS calcd
9. Graffner-Nordberg, M.; Fyfe, M.; Brattsand, R.; Mellgård, B.; Hallberg, A. J. Med.
Chem. 2003, 46, 3455.
10. Cabello, J. A.; Campelo, J. M.; Garcia, A.; Luna, D.; Marinas, J. M. J. Org. Chem.
1984, 49, 5195.
for C27H28N4O3: 456.54, found m/z 457.45 [M+H]+.
17. In comparison with commonly used fluorophores such as 4-chloro-7-nitrobenz-2-
oxa-1,3-diazole (NBD-Cl):
e
= 9,800 molꢀ1 dm3 cmꢀ1, kmax = 336 nm.

D. T. S. Rijkers et al. / Tetrahedron Letters 52 (2011) 6963–6967
6967
See also: www.invitrogen.com/site/us/en/home/References/Molecular-Probes-
19. HPLC analysis was performed on a Phenomenex Luna C8 column (particle size:
The-Handbook.html; last visited on September 29, 2011.
5 lm, pore size 100 Å, 250 ꢃ 4.6 mm) using a linear gradient starting from
18. Due to the deeply colored solution of dicyanobutadienes, an accurate ½a D20
ꢂ
buffer A (0.1% TFA in CH3CN/H2O, 5:95 v/v) to buffer B (0.1% TFA in CH3CN/H2O,
95:5 v/v) over 60 min at a flow rate of 1 mL/min.
value could not be measured.