A convenient synthesis of new chromophoric tetracyanobutadiene-scaffolded peptides via a dipolar [2+2] cycloaddition-cycloreversion reaction

Article abstract of DOI:10.1016/j.tetlet.2011.05.136

Herein, we report a novel approach for the synthesis of π-conjugated peptide-based donor-acceptor (D-π-A) chromophores, by reacting electron-rich alkynes with tetracyanoethylene. The desired tetracyanobutadiene-scaffolded peptides were obtained in good yields with various optical properties, λ_max: 321-492 nm, ε: 21,000-65,000 mol^-1 dm³ cm^-1 depending on the substitution pattern of the cyanobutadiene scaffold.

Full text of DOI:10.1016/j.tetlet.2011.05.136

Tetrahedron Letters 52 (2011) 4021–4025
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
A convenient synthesis of new chromophoric tetracyanobutadiene-scaffolded
peptides via a dipolar [2+2] cycloaddition–cycloreversion reaction q
Dirk T. S. Rijkers a, , François Diederich b
⇑
a Division of Medicinal Chemistry and 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:
Herein, we report a novel approach for the synthesis of
p-conjugated peptide-based donor–acceptor
Received 24 March 2011
Revised 19 May 2011
Accepted 27 May 2011
Available online 2 June 2011
(D-
anobutadiene-scaffolded peptides were obtained in good yields with various optical properties, kmax
321–492 nm,
: 21,000–65,000 molꢀ1 dm3 cmꢀ1 depending on the substitution pattern of the cyanobut-
adiene scaffold.
p-A) chromophores, by reacting electron-rich alkynes with tetracyanoethylene. The desired tetracy-
:
e
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Acetylenes
Cycloaddition
Donor–acceptor chromophores
p-Conjugated peptides
Peptides
Peptidomimetics
Peptides play a pivotal role in maintaining homeostasis in a liv-
ing organism, among others, as hormones, signaling molecules, en-
zyme inhibitors. Functionalization of such peptides with reporter
groups (fluorescent or chromophoric tags) is an important and of-
ten applied tool in chemical biology, to monitor the fate of the la-
beled peptide, thereby gaining information of its biological
function, and also for studying the molecular basis of disease.1
Such fluorescent/chromophoric labels are introduced post-syn-
thetically, by featuring amino or thiol specific conjugation reac-
tions.2 However, in recent years, the development of novel
chemoselective bioconjugation reactions, for example, native
chemical ligation,3 the Staudinger ligation,4 oxime/hydrazone liga-
tion,5 and the Cu(I)-catalyzed cycloaddition between azides and al-
kynes,6 has led to a renewed interest in biocompatible, site-specific
introduction of fluorophores, chromophores, biotin, metal chela-
tors and other biophysical probes. This renewed interest has re-
sulted in many new peptide-derived constructs as tools in
chemical biology for studying signal transduction and post-trans-
lational modification pathways in a living organism. Herein, we
report on a novel approach for the synthesis of tetracyanobutadi-
ene-scaffolded peptides that represent a new class of intense pep-
tide-based chromophores (Fig. 1).
To access this new class of
p-conjugated peptidic donor–accep-
tor (D- -A) chromophores, the recently developed reaction be-
p
tween electron-rich alkynes with electron-deficient ethylenes has
been applied.7 This dipolar [2+2] cycloaddition-cycloreversion
reaction leads, with a high degree of regio- and stereoselectivity,
to a cyanobutadiene-scaffold, which is an intense chromophore
and its optical properties can be fine-tuned by variation of the
conjugation by the substituents.
p-
The syntheses started with N-monoalkylation of aniline deriva-
tive 1 (Scheme 1). Despite the weak nucleophilicity of the amine,
an efficient alkylation was obtained with one equivalent of ethyl
bromoacetate in the presence of anhydrous Na2CO3 in dry DMF
at 95 °C, and the N-monoalkylated product was obtained in 70%
yield.8 In the next step, N-methylation required at least three days
of stirring in DMF/Na2CO3 at room temperature, since the low boil-
ing point (41–43 °C) of iodomethane did not allow reaction at an
elevated temperature. The N,N-dialkylated aniline 2 was obtained
in an excellent yield of 98%. Saponification of ethyl ester 2 pro-
ceeded smoothly, and it turned out that during aqueous work-up
the intermediate acid was stable in solution, but unstable as a solid
since evaporation of the solvent resulted in the formation of an
intractable black precipitate. Therefore, the transformation into
the corresponding hydroxysuccinimide ester was performed di-
rectly in the solvent used for the aqueous extraction. Active ester
3 was obtained, as a stable solid, in good yield (77%) after
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).
⇑
Corresponding author. Tel.: +31 (0)6 2026 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.2011.05.136

4022
D. T. S. Rijkers, F. Diederich / Tetrahedron Letters 52 (2011) 4021–4025
N
N
N
N
N
N
PEPTIDE
PEPTIDE
PEPTIDE
PEPTIDE
PEPTIDE
N
N
PEPTIDE
colorless (protected) peptides
with an
intensely colored
electron-rich alkyne moiety
(protected) peptide adducts
N
N
N
N
N
PEPTIDE
N
PEPTIDE
H
N
10
-12
13-14
PEPTIDE
N
N
N
N
in bold: buta-1,3-diene scaffold as an intense chromophore
Figure 1. Reaction of electron-rich alkynes with TCNE results in intensely colored tetracyanobutadiene-scaffolded peptide-based chromophores.
1. 1 M NaOH in THF, rt, 60 min
2. aqueous work-up at pH 4
3. DCC/HONSu in EtOAc, 0 oC to rt, 16 h
overall yield: 77%
1. ethyl bromoacetate/Na2CO3
in DMF, 95 oC, 16 h (70%)
2. MeI/Na2CO3 in DMF,
rt, 3 d (98%)
O
NH2
N
O
1
3
2
O
O
HCl.H-Val-OMe/DIPEA
in CH2Cl2, rt, 16 h (97%)
O
O
N
O
N
N
N
H
O
O
O
4
N
O
N
H
a.) CuCl/TMEDA in acetone/O2
rt, 16h (35%)
O
4
5
b.) [PdCl2(Ph3P)2]/CuI
CH3CN/(i-Pr)2NH/O2, rt, 90 min (91%)
O
H
N
O
N
O
Scheme 1. Synthesis of alkyne 4, and its corresponding dialkyne 5.
recrystallization from 2-propanol. Subsequent N-acylation of H-
peptides.13 The acylation of dipeptide H-Val-Ile-OMe by active es-
ter 3 to afford alkyne 6 proceeded without any problems, a high
yield was achieved. During the dimerization of 6 into 7, a gel
formed and the reduced solubility of dialkyne 7 was reflected in
the lower isolated yield compared to compound 5. Tripeptide
HClꢁH-Val-Ile-Ala-OMe was converted into alkyne 8 by reaction
with active ester 3 and DIPEA as the base. During the acylation,
the reaction mixture turned gradually into a gel, alkyne 8 was spar-
ingly soluble in CHCl3, prone to aggregation. After purification by
column chromatography, and alkyne 8 was obtained in 56% yield.
Next, the dimerization step required DMF as a co-solvent and dial-
kyne 914 was obtained in a high yield (81%); this compound precip-
itated from the solution and was isolated by filtration. It turned out
that dialkyne 9 was almost insoluble in common solvents such as
MeOH, CHCl3 or DMF, and only poorly soluble in DMSO.
A series of tetracyanobutadienes 10–14 was prepared by react-
ing tetracyanoethylene (TCNE) with a suitable alkyne (4, 6, 8) or
dialkyne (5, 7), as shown in Scheme 3. Donor-substituted, elec-
tron-rich alkynes reacted smoothly with electron-poor ethylenes
in an atom-economic, one-step transformation in nearly quantita-
tive yield to give the tetracyanobutadiene (TCBD) framework. This
reaction is a formal [2+2] cycloaddition toward a cyclobutene
a
a
a
Val-OMe gave the N -aryl dipeptide 4 [N -(4-ethynylphenyl)-N -
(methyl)-Gly-Val-OMe]9 as an oil in an excellent yield of 97%.
A versatile approach for oxidative acetylenic homocoupling was
described by Hay in the early 1960s.10,11a In the presence of a com-
plex formed by Cu(I)/N,N,N0,N0-tetramethylethylenediamine (TME-
DA) as the catalyst and dioxygen, alkynes undergo smooth
dimerization in nearly quantitative yield. However, in the case of
alkyne 4, a rather disappointing yield of 35% of the dimer 5 was
achieved (Scheme 1). In this context, it should be mentioned that
in an attempt to couple 4 to dimethyl 2,3-dibromofumarate with
[PdCl2(PPh3)2]/CuI under Sonogashira conditions, dialkyne 5 was
isolated as a side product in 68% yield. This prompted us to attempt
the Pd/Cu co-catalyzed homocoupling of terminal alkynes11b for
the synthesis of 5. Gratifyingly, alkyne 4 underwent smooth dimer-
ization into 512 in an excellent yield of 91% (Scheme 1).
Next, dialkynes 7 and 9 were synthesized (Scheme 2), essen-
tially according to the same approach as described for dialkyne 5.
Since the peptide sequence was derived from the C-terminus (res-
idues 40–42) of the highly amyloidogenic Alzheimer Ab (1–42)
peptide, the synthesis of 7–9 was rather challenging due to the re-
stricted solubility, high intrinsic aggregation properties of these

D. T. S. Rijkers, F. Diederich / Tetrahedron Letters 52 (2011) 4021–4025
4023
[PdCl2(PPh3)2]/CuI
CH3CN/(i-Pr)2NH/O2
rt, 60 min (71%)
HCl.H-Val-Ile-OMe/DIPEA
in CH2Cl2, rt, 16 h (96%)
O
O
H
N
N
3
N
H
O
O
O
O
6
H
N
N
N
H
O
O
7
HCl.H-Val-Ile-Ala-OMe/DIPEA
in CH2Cl2, rt, 16 h (56%)
O
H
N
N
O
O
N
H
O
O
O
H
N
N
O
N
H
N
H
O
O
H
N
O
O
N
O
8
N
H
N
H
O
O
9
[PdCl2(PPh3)2]/CuI
CH3CN/DMF/(i-Pr)2NH/O2
rt, 16 h (81%)
O
O
H
H
N
N
O
N
N
H
O
O
Scheme 2. Synthesis of alkynes 6 and 8, and their corresponding dialkynes 7 and 9.
N
N
N
N
O
O
N
N
N
N
R
R
solvent
16 h, rt
R =
R =
10 [~Val-OMe (84%)]
~Val-OMe (4)
11 [~Val-Ile-OMe (79%)]
12 [~Val-Ile-Ala-OMe (45%)]
~Val-Ile-OMe (
6)
N
N
~Val-Ile-Ala-OMe (
8
)
O
N
N
O
N
)
R
N
N
N
R
N
N
solvent
16 h, rt
R =
~Val-OMe (
R =
13 [~Val-OMe (quant)]
R
N
N
O
R
5
N
N
~Val-Ile-OMe (
7
)
14 [~Val-Ile-OMe (75%)]
O
Scheme 3. The [2+2] cycloaddition reaction of TCNE with peptide-derived alkynes.
intermediate, that undergoes an electrocyclic ring-opening to give
the TCBDs (Scheme 4).15
sight into the efficiency of this reaction, alkyne 8 was dissolved in
CDCl3, the course of the reaction was followed by 1H NMR spec-
troscopy by monitoring the decrease of the HC„ signal (d = 2.99),
the increase of the ꢂCH@C(CN)2 signal (d = 8.01) relative to the
OCH3 signal (d = 3.74).17 By means of this approach, a conversion
of 77% after a 16 hour reaction time could be calculated, this value
was in the range of the isolated yields of compounds 10 and 11.
Alkynes 4 and 6 were smoothly converted into their corre-
sponding TCBDs which were isolated after purification by column
chromatography in 84% and 79% yield for 1016 and 11, respectively.
The reaction of 8 with TCNE resulted in a precipitate from which
TCBD 12 was isolated in a modest yield of 45%. To obtain more in-
N
N
N
N
N
C
N
N
N
N
N
R3
N
N
N
R3
N
R3
R3
R1
R1
N
N
R2
R1
R2
R1
N
N
N
N
R2
R2
Scheme 4. Mechanism of the [2+2] cycloaddition–cycloreversion reaction.

4024
D. T. S. Rijkers, F. Diederich / Tetrahedron Letters 52 (2011) 4021–4025
(Utrecht University) is acknowledged for critically reading the man-
uscript and for his suggestions, and enthusiastic support.
References and notes
1. (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.
2. Dent, A. H.; Aslam, M. Bioconjugation, Protein Coupling Techniques for the
Biomedical Sciences; Macmillan: London, 2000. Chapter 2.
3. Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H. Science 1994, 266, 776.
4. Saxon, E.; Bertozzi, C. R. Science 2000, 287, 2007.
5. Gaertner, H. F.; Rose, K.; Cotton, R.; Timms, D.; Camble, R.; Offord, R. E.
Bioconjugate Chem. 1992, 3, 262.
6. (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.
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.
Figure 2. UV–vis spectra (in CHCl3 at T = 298 K) of 10 (black round dotted line:
8. Kruijtzer, J. A. W.; Hofmeyer, L. J. F.; Heerma, W.; Versluis, C.; Liskamp, R. M. J.
Chem. Eur. J. 1998, 4, 1570.
1
l
M); 13 (solid black line: 1
lM, red line: 2 lM, blue line: 20 lM, green line:
a
a
45
lM).
9. N -(4-Ethynylphenyl)-N -(methyl)-glycyl-valine methyl ester (4): Rf = 0.33
(Et2O);
+51.8 (c = 0.49 in CHCl3); 1H NMR (300 MHz, CDCl3, 25 °C):
d = 0.73 (d, 3J(H,H) = 6.9 Hz, 3H; CH3 Val), 0.86 (d, 3J(H,H) = 6.9 Hz, 3H; c0CH3
½ ꢃ
a 2D0
c
Tetracyanobutadienes 10–12 were obtained as purple-black solids
with metallic luster, and were stable in air at ambient
temperatures.
The addition of TCNE to a solution of dialkynes 5 and 7 (in
CH2Cl2, and CHCl3, respectively) resulted immediately in a color
change of the reaction mixture, and compounds 1318 and 14 were
isolated as dark-red colored solids in quantitative and 75% yields,
respectively. Since dialkyne 9 was not soluble in solvents that were
inert toward TCNE its corresponding TCBD could not be obtained.
The UV–vis spectra of compounds 10 and 13 are shown in Fig-
Val), 2.12 (m, 1H; bCH Val), 2.99 (s, 1H; HC„), 3.08 (s, 3H; NCH3), 3.69 (s, 3H;
OCH3), 3.83 (d, 3J(H,H) = 18 Hz, 1H; CH2 Gly), 3.96 (d, 3J(H,H) = 38.4 Hz, 1H; CH2
a
Gly), 4.55 (dd, 3J(H,H) = 4.8 Hz, 3J(H,H) = 9 Hz, 1H;
aCH Val), 6.66 (d,
3J(H,H) = 9 Hz, 2H; arom H), 6.75 (d, 3J(H,H) = 9 Hz, 1H; amide NH), 7.39 (d,
3J(H,H) = 9 Hz, 2H; arom H); 13C NMR (75.5 MHz, CDCl3, 25 °C): d = 171.8,
169.5, 148.5, 133.3, 112.6, 111.4, 84.0, 75.6, 58.1, 56.7, 52.2, 39.7, 31.1, 19.1,
17.6; UV–vis (CHCl3): kmax (e
) = 282 nm (36,750 molꢀ1 dm3 cmꢀ1); ESMS calcd
for C17H22N2O3: 302.16, found: m/z 303.25 [M+H]+, 321.30 [(M+H2O)+H]+.
10. Hay, A. S. J. Org. Chem. 1962, 27, 3320.
11. For a review on acetylenic (homo)coupling, see: (a) Siemsen, P.; Livingston, R.
C.; Diederich, F. Angew. Chem., Int. Ed. 2000, 39, 2632; We used the following
approach: (b) Batsanov, A. S.; Collings, J. C.; Fairlamb, I. J. S.; Holland, J. P.;
Howard, J. A. K.; Lin, Z.; Marder, T. B.; Parsons, A. C.; Ward, R. M.; Zhu, J. J. Org.
Chem. 2005, 70, 703.
ure 2. An absorption maximum at k = 321 nm (
e
21,000 molꢀ1
12. Dialkyne (5): Rf = 0.40 (EtOAc/Et2O 1:1); mp 131–132 °C; ½a D20
ꢃ
+110.5 (c = 0.41
dm3 cmꢀ1) was observed for tetracyanobutadiene 10, while tetracy-
anobutadiene 13 showed a maximum at k = 492 nm with a high mo-
in CHCl3); 1H NMR (300 MHz, CDCl3, 25 °C): d = 0.73 (d, 3J(H,H) = 6.9 Hz, 6H;
c
CH3 Val), 0.86 (d, 3J(H,H) = 6.9 Hz, 6H; c0CH3 Val), 2.14 (m, 2H; bCH Val), 3.09
(s, 6H; NCH3), 3.69 (s, 6H; OCH3), 3.85 (d, 3J(H,H) = 18 Hz, 1H; CH2 Gly), 3.98 (d,
lar extinction coefficient of 65,000 molꢀ1 dm3 cmꢀ1 19
.
The molar
extinction coefficient was found to be highly concentration depen-
dent, as shown for compound 13. At c = 1 M, a molar extinction
coefficient of 65,000 molꢀ1 dm3 cmꢀ1 at kmax = 492 nm was found,
while at c = 45 M,
had dropped to 30,000 molꢀ1 dm3 cmꢀ1. This
3J(H,H) = 39 Hz, 1H; CH2 Gly), 4.55 (dd, 3J(H,H) = 4.8 Hz, 3J(H,H) = 9 Hz, 2H;
aCH
Val), 6.66 (d, 3J(H,H) = 9 Hz, 4H; arom H), 6.71 (d (overlapping signal), 2H;
amide NH), 7.40 (d, 3J(H,H) = 9 Hz, 4H; arom H); 13C NMR (75.5 MHz, CDCl3,
25 °C): d = 171.8, 169.3, 148.9, 133.7, 112.6, 111.1, 81.7, 73.0, 58.0, 56.7, 52.2,
l
39.7, 31.1, 19.1, 17.6; UV–vis (CHCl3): kmax (e) = 368 (59,000), 343 (77,000),
l
e
322 nm (73,500 molꢀ1 dm3 cmꢀ1); ESMS calcd for C34H42N4O6: 602.31, found:
m/z 603.50 [M+H]+.
concentration dependency was a strong indication of (peptide)
aggregation, and this effect was more pronounced by elongation of
the peptide sequence, from ꢂGly-Val-OMe, to ꢂGly-Val-Ile-OMe
and ꢂGly-Val-Ile-Ala-OMe (data not shown).
13. Peptide synthesis was performed in solution according to: Ray, S.; Das, A. K.;
Drew, M. G. B.; Banerjee, A. Chem. Commun. 2006, 4230.
14. Dialkyne (9): Mp 266 °C (dec); 1H NMR (300 MHz, DMSO-d6, 25 °C): d = 0.77–
0.84 (m, 24H;
c
CH3 Val/c0CH3 Val (4 ꢄ 3H)/
cCH3 Ile (2 ꢄ 3H)/dCH3 Ile
(2 ꢄ 3H)), 1.05 (m, 2H; c0CH2 Ile), 1.24 (d, 3J(H,H) = 7.2 Hz, 6H; bCH3 Ala),
In conclusion, a new class of peptide-based chromophores has
been described featuring the [2+2] cycloaddition–cycloreversion
reaction between an electron-rich alkyne and tetracyanoethylene,
resulting in intensely-colored peptide constructs with high molar
extinction coefficients. This chemistry can be considered as a mod-
el study for bioorthogonal modification of peptides with imaging
chromophores possessing tunable optical properties. Since the
cyanobutadiene scaffold has been functionalized with peptides
that have a strong b-sheet propensity, these newly designed pep-
tide chromophores may lead to the development of imaging probes
for amyloid deposits.
1.40 (m, 2H; c0CH2 Ile), 1.68 (m, 2H; bCH Ile), 1.94 (m, 2H; bCH Val), 3.01 (s, 6H;
NCH3), 3.58 (s, 6H; OCH3), 4.06 (m, 4H; CH2 Gly), 4.22 (m, 6H;
aCH Val
(2 ꢄ 1H)/
a
CH Ile (2 ꢄ 1H)/
a
CH Ala (2 ꢄ 1H)), 6.60 (d, 3J(H,H) = 9 Hz, 4H; arom
H), 7.28 (d, 3J(H,H) = 9 Hz, 4H; arom H), 7.84 (d, 3J(H,H) = 8.7 Hz, 2H; amide
NH), 7.98 (d, 3J(H,H) = 9 Hz, 2H; amide NH), 8.38 (d, 3J(H,H) = 6.6 Hz, 2H; amide
NH); 13C NMR (125 MHz, DMSO-d6, 25 °C): d = 133.9, 112.5, 57.7, 56.6, 55.0,
51.7, 47.8, 39.6, 37.0, 30.7, 24.3, 19.3, 18.1, 16.8, 15.1, 11.1 (16 lines out of 24
based on an HSQC); ESMS calcd for C52H74N8O10: 970.55, found: m/z 971.80
[M+H]+, 993.55 [M+Na]+.
15. This reaction is another example of ‘click chemistry’, see: Kolb, H. C.; Finn, M.
G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40, 2004.
a
a
16. N -[4-(1,1,4,4-Tetracyanobuta-1,3-dien-2-yl)phenyl]-N -(methyl)-glycyl-valine
methyl ester (10):20 Rf = 0.38 (EtOAc/Et2O 1:1 v/v), Rf = 0.42 (CHCl3/MeOH 95:5
v/v), Rf = 0.58 (CH2Cl2/EtOAc 1:1 v/v); mp 140–141 °C; 1H NMR (300 MHz,
CDCl3, 25 °C): d = 0.76 (d, 3J(H,H) = 6.9 Hz, 3H;
cCH3 Val), 0.87 (d,
Acknowledgements
3J(H,H) = 6.9 Hz, 3H; c0CH3 Val), 2.15 (m, 1H; bCH Val), 3.24 (s, 3H; NCH3),
3.71 (s, 3H; OCH3), 4.03 (d, 3J(H,H) = 18 Hz, 1H; CH2 Gly), 4.14 (d,
3J(H,H) = 34.2 Hz, 1H; CH2 Gly), 4.55 (dd, 3J(H,H) = 4.8 Hz, 3J(H,H) = 8.7 Hz,
D.T.S.R. gratefullyacknowledgestheETHZürichResearchCouncil
for financial support, and Utrecht University for facilitating a sabbat-
ical leave at the ETHZ. Dr. Milan Kivala (Friedrich-Alexander-Univer-
sität, Erlangen-Nürnberg, Germany) is acknowledged for his interest
and contributions to the scientific discussions with respect to this
work. Dr. Johan Kemmink (Utrecht University) is thanked for
measuring the 13C HSQC of dialkyne 9. Professor Rob M. J. Liskamp
1H;
a
CH Val), 6.40 (d, 3J(H,H) = 8.7 Hz, 1H; amide NH), 6.82 (d, 3J(H,H) = 9 Hz,
2H; arom H), 7.48 (d, 3J(H,H) = 9 Hz, 2H; arom H), 8.03 (s, 1H; HC@C(CN)2); 13
C
NMR (75.5 MHz, CDCl3, 25 °C): d = 171.7, 167.8, 159.8, 155.2, 153.2, 131.8,
119.4, 112.8, 112.7, 111.5, 108.7, 97.6, 82.9, 57.0, 56.8, 52.4, 39.9, 31.1, 19.1,
17.7; UV–vis (CHCl3): kmax
(e) = 553 (6800), 463 (6000), 321 nm
(21,000 molꢀ1 dm3 cmꢀ1); ESMS calcd for C23H22N6O3: 430.18, found: m/z
463.55 [(M+CH3OH)+H]+, 485.55 [(M+CH3OH)+Na]+.
17. The residual solvent peak CHCl3 (7.26 ppm) at T = 298 K was used as reference.

D. T. S. Rijkers, F. Diederich / Tetrahedron Letters 52 (2011) 4021–4025
4025
18. 1,1,4,4-Tetracyanobutadiene (13):20 Rf = 0.44 (CH2Cl2/MeOH 95:5 v/v); 1H NMR
(300 MHz, CDCl3, 25 °C): d = 0.76 (d, 3J(H,H) = 6.9 Hz, 3H; CH3 Val), 0.77 (d,
107.5, 89.8, 88.6, 77.7, 77.2, 57.0, 56.8, 56.6, 52.4, 39.9, 39.8, 31.1, 19.1, 17.7,
17.6 (31 lines out of 36); UV–vis (CHCl3): kmax (e) = 492 (65,000), 400 nm
3J(H,H) = 6.9 Hz, 3H; CH3 Val), 0.87 (d, 3J(H,H) = 6.9 Hz, 6H;
c
CH3/c0CH3 Val),
(33,700 molꢀ1 dm3 cmꢀ1); ESMS calcd for C40H42N8O6: 730.81, found: m/z
731.50 [M+H]+, 753.45 [M+Na]+, 1462.45 [2M+H]+.
2.14 (m, 2H; bCH Val), 3.20 (s, 3H; NCH3), 3.24 (s, 3H; NCH3), 3.71 (s, 6H;
OCH3), 3.97 (d, 3J(H,H) = 18.3 Hz, 1H; CH2 Gly), 4.04 (d, 3J(H,H) = 18 Hz, 1H; CH2
Gly), 4.09 (d, 3J(H,H) = 33.3 Hz, 1H; CH2 Gly), 4.20 (d, 3J(H,H) = 32.1 Hz, 1H; CH2
19. In comparison with commonly used fluorophores, among others, 5-
carboxyfluorescein:
nitrobenz-2-oxa-1,3-diazole
e
= 74,000 molꢀ1 dm3 cmꢀ1
,
kmax = 495 nm; 4-chloro-7-
Gly), 4.53 (ddd, 3J(H,H) = 0.9 Hz, 3J(H,H) = 4.8 Hz, 3J(H,H) = 9.0 Hz, 2H;
aCH
(NBD-Cl):
e
= 9800 molꢀ1 dm3 cmꢀ1
,
Val), 6.38 (d, 3J(H,H) = 9.0 Hz, 1H; amide NH), 6.44 (d, 3J(H,H) = 9.0 Hz, 1H;
amide NH), 6.70 (d, 3J(H,H) = 9 Hz, 2H; arom H), 6.79 (d, 3J(H,H) = 9.3 Hz, 2H;
arom H), 7.51 (d, 3J(H,H) = 9 Hz, 2H; arom H), 7.78 (d, 3J(H,H) = 9.3 Hz, 2H;
arom H); 13C NMR (75.5 MHz, CDCl3, 25 °C): d = 171.7, 168.1, 167.6, 161.4,
153.4, 151.9, 149.3, 135.9, 132.2, 123.0, 119.5, 113.5, 112.6, 112.5, 112.0, 110.8,
kmax = 336 nm.
See also: www.invitrogen.com/site/us/en/home/References/Molecular-Probes-
The-Handbook.html; last visited on March 7, 2011.
20. Due to the intense color of the TCBDs an accurate ½a D20
ꢃ
value could not be
measured.