Fluorescence labeling of amino acids and peptides with 7-aminocoumarins

Article abstract of DOI:10.1055/s-0031-1291145

Pechmann condensation is a straightforward protocol for the synthesis of alkynyl- and azido-substituted 7-(dialkylamino)coumarins, which can be coupled to amino acid and peptide derivatives by copper-catalyzed [3+2]-cycloaddition (click reaction) in high yield. This allows the introduction of these efficient fluorescence labels into biologically relevant molecules. Georg Thieme Verlag Stuttgart ? New York.

Full text of DOI:10.1055/s-0031-1291145

PAPER
▌2005
Paper
Fluorescence Labeling of Amino Acids and Peptides with 7-Aminocoumarins
Fluorescence Labeling of Amino Acids and Peptides
Lisa Wirtz,a Dagmar Auerbach,b Gregor Jung,b Uli Kazmaier*a
a
Institut für Organische Chemie, Universität des Saarlandes, 66123 Saarbrücken, Germany
Fax +49(681)3022409; E-mail: u.kazmaier@mx.uni-saarland.de
b
Biophysikalische Chemie, Universität des Saarlandes, 66123 Saarbrücken, Germany
Received: 13.03.2012; Accepted after revision: 18.04.2012
with a β-keto ester in the presence of a strong acid such as
Abstract: Pechmann condensation is a straightforward protocol for
sulfuric acid. Although this approach is suitable for a wide
the synthesis of alkynyl- and azido-substituted 7-(dialkylami-
range of phenols, it is critical for aminophenols, probably
because of protonation and deactivation of the aromatic
no)coumarins, which can be coupled to amino acid and peptide de-
rivatives by copper-catalyzed [3+2]-cycloaddition (click reaction)
in high yield. This allows the introduction of these efficient fluores- ring by the strong acid. Therefore, we recently developed
cence labels into biologically relevant molecules.
a very mild procedure using chlorotriisopropoxytitani-
um(IV), which is especially suitable for the synthesis of
Key words: amino acids, click chemistry, cycloaddition, couma-
rins, fluorescence labeling, peptides
the required 7-(dialkylamino)coumarins (Scheme 1).18
R
R
In the life sciences, fluorescence microscopy is an impor-
tant and powerful tool for the three-dimensional imaging
of tissues and living cells.1 A wide range of fluorescence
dyes can be used for the labeling of proteins and drugs, al-
lowing the investigation of cellular processes and target
identification.2 Besides the BODIPYs3 and fluoresceine
derivatives,4 coumarins also play a major role. Excellent
fluorescence quantum yields are obtained with coumarin
derivatives bearing electron-donating substituents at the
7-position, such as the 7-(dialkylamino)coumarins.5
Therefore, these derivatives are appropriate candidates for
the development of fluorescence labels.6 These labels
have to be coupled with biomolecules such as peptides
and proteins.7 In general, functionalized amino acid side
chains, as found in cysteine,8 lysine,9 or tyrosine,10 are
ideal candidates for selective labeling. In addition, the
Huisgen–Meldal–Sharpless reaction,11 a copper-cata-
lyzed [3+2]-cycloaddition between an azide and an al-
kyne, was developed to an attractive labeling strategy
during the last years.12 At the beginning, this technique
could only be applied in vitro, because of the cytotoxicity
of copper in the μM range. But in the meanwhile a proto-
col has been developed to reduce the copper levels, so that
this method has become biocompatible.13
ClTi(Oi-Pr)3
O
+
toluene, Δ
68–86%
N
OH
N
O
O
MeOOC
Scheme 1 Synthesis of 7-(dialkylamino)coumarins via modified
Pechmann synthesis
Herein we report on the synthesis of coumarin-based
‘clickable’ fluorescence labels and their application in the
modification of amino acids and peptides. The synthesis
of alkyne- and azide-substituted coumarins is shown in
Scheme 2. According to a procedure developed by
Masamune et al.19 pent-4-ynoic acid was first activated
with 1,1′-carbonyldiimidazole (CDI) and subsequently
coupled with the potassium salt of monomethyl malonate
in the presence of magnesium chloride. The desired β-
keto ester 1 was obtained in good yield and directly sub-
jected to a modified Pechmann condensation using 8-hy-
droxyjulodin as phenol component. The Pechmann
condensation delivered the alkynylated coumarin deriva-
tive 2 in high yield. For the synthesis of analogous azido-
coumarin we started with the previously described
aminoethyl derivative using a diazo-transfer reagent.20
The required triflyl azide was freshly prepared before
use.21 Unfortunately, the expected azide 3 was obtained
only in modest yield. Therefore, we decided to switch to a
more straightforward approach using again the modified
Pechmann condensation. Starting from commercially
available 4-chloroacetoacetate under standard conditions,
the chlorinated coumarin 4 was obtained in acceptable
yield. Subsequent nucleophilic substitution with sodium
azide gave rise to azidocoumarin 5.
For an application of this click chemistry,14 the biological
target has to be modified by introduction of an alkyne
group, while the fluorescence label has to contain an
azide, or the other way around. Because of our previous
work on natural product synthesis,15 we became interested
in the fluorescence labeling of peptides for biological
studies.16
While a wide range of reactions can be used for the syn-
thesis of coumarins, the Pechmann condensation is prob-
ably the most popular approach.17 Here, a phenol reacts
With these coumarin derivatives in hand we next focused
on the synthesis of azide- and alkyne-substituted amino
acid and peptide derivatives. Two alkynylated peptides 7
and 8 could be obtained easily starting from N-Boc-pro-
tected serine (Scheme 3). Deprotonation with an excess of
sodium hydride and propargylation with propargyl bro-
SYNTHESIS 2012, 44, 2005–2012
Advanced online publication: 05.06.2012
0
0
3
9
-
7
8
8
1
1
4
3
7
-
2
1
0
X
DOI: 10.1055/s-0031-1291145; Art ID: SS-2012-T0265-OP
© Georg Thieme Verlag Stuttgart · New York

2006
L. Wirtz et al.
PAPER
can easily be removed with water, providing the required
peptides in pure form.22
MeOOC
COOK
O
CDI, MgCl2
THF, r.t., 16 h
82%
COOH
COOMe
To get access to some amino acid and peptide derived
azides we started on the one hand with N-Boc-protected
leucinol and dipeptide alcohol 11 (Scheme 4). Reaction
with mesyl chloride gave rise to mesylate 9 in excellent
yield, which could be converted into the required azide
10. We observed that 9 is not a very stable compound and
should not be stored for long. It should be coupled directly
after preparation. Even worse was the situation in the case
of the dipeptide alcohol 11. Also here, the mesylate was
nicely formed, but the decomposition started immediate-
ly. Therefore, the crude product was directly reacted with
sodium azide to give 12.
1
OH
N
N
O
O
ClTi(Oi-Pr)3
toluene, Δ
87%
2
N3
NH2
O
CuSO4
TfN3, K2CO3
CH2Cl2/H2O
29%
N
O
O
N
O
MsCl, Et3N
3
OMs
OH
CH2Cl2, r.t., 3 h
96%
BocHN
N3
BocHN
Cl
9
Cl
O
ClTi(Oi-Pr)3
NaN3
+
toluene, Δ
DMF, 60 °C, 16 h
73%
N
OH
N
O
O
BocHN
71%
MeOOC
10
4
N3
Ph
Ph
1) MsCl, Et3N
CH2Cl2, r.t., 3 h
H
N
H
N
NaN3, DMF
91%
2) NaN3, DMF
60 °C, 16 h
CbzHN
N3
CbzHN
OH
N
O
O
O
O
38% (2 steps)
12
11
5
Scheme 2 Synthesis of ‘clickable’ aminocoumarins
TfN3, CuSO4, K2CO3
CH2Cl2/H2O
79%
N3
COOMe
Ph
H2N
COOMe
13
O
OH
1) NaH, DMF, 0 °C
Ph
O
Br
2)
, r.t., 3 h
BocHN
COOH
BocHN
COOH
TfN3
CuSO4, K3CO3
H
N
H
N
70%
6
COOMe
COOMe
H2N
N3
CH2Cl2/H2O
94%
O
O
O
H
N
14
Ile-OMe
COOMe
BocHN
T3P, Et3N, EtOAc
–78 °C to r.t., 16 h
90%
Scheme 4 Synthesis of amino acid and peptide azides
7
On the other hand, we introduced the azide functionality
into the N-terminus of amino acids and dipeptides as illus-
trated for azides 13 and 14. In contrast to the previous re-
action, the diazo transfer with triflyl azide, starting from
the corresponding free amines, provided the azides 13 and
14 in high yield and in pure form.
O
O
Ph
O
H
N
Val-Phe-OMe
BocHN
N
H
COOMe
T3P, Et3N, EtOAc
–78 °C to r.t., 16 h
76%
8
Now we had building blocks in hand with ‘clickable’
functionalities at the N-terminus, the previous C-termi-
nus, as well as in the side chain. The results obtained in the
subsequent click reactions are summarized in Table 1. We
started our investigations with the acetylenic coumarin 2
and the leucine-derived azide 10. Under standard coupling
conditions, by using 10 mol% each of copper(II) sulfate
and sodium ascorbate, a very sluggish reaction was ob-
served at room temperature. Even after six days a yield of
only 25% was obtained.
Scheme 3 Synthesis of acetylenic amino acids and peptides
mide provided ether 6 in acceptable yield, without signif-
icant amounts of propargyl ester formed.
Initial peptide couplings were performed with N,N′-dicy-
clohexylcarbodiimide, but here some side products were
obtained, which could not be separated easily from the de-
sired product. Therefore, we switched to propylphosphon-
ic anhydride (T3P). In this case, possible side products
Synthesis 2012, 44, 2005–2012
© Georg Thieme Verlag Stuttgart · New York

PAPER
Fluorescence Labeling of Amino Acids and Peptides
2007
Table 1 Fluorescence Labeling of Amino Acids and Peptides via Huisgen–Meldal–Sharpless Reaction
CuSO4 (10 mol%)
Na ascorbate (10 mol%)
R1
R2
N
R1
+
R2 N3
N
N
DMSOûH2O, 80 °C, 16 h
Entry
Alkyne
Azide
Product
Yield (%)
N
N
N
NHBoc
1
10
15
78
2
O
N
N
N
N
O
O
O
O
N
N
N
NH
O
2
3
12
13
16
17
78
77
2
2
Bn
O
NHCbz
N
N
N
COOMe
O
N
Ph
N
N
4
14
18
75
2
O
HN
O
COOMe
N
N
N
O
NHBoc
5
5
19
84
7
O
N
O
O
NH
COOMe
N
N
N
O
NHBoc
O
N
O
O
6
5
20
77
8
NH
O
HN
COOMe
Ph
© Georg Thieme Verlag Stuttgart · New York
Synthesis 2012, 44, 2005–2012

2008
L. Wirtz et al.
PAPER
mL/mmol) was added and the whole solution was poured into a
stirred sat. potassium sodium tartrate solution (10 mL/mmol) until
the phases separated. The aqueous layer was extracted with CH2Cl2
(3 × 15 mL). The combined organic layers were dried (Na2SO4), the
solvent was removed in vacuo, and the crude product was purified
by flash chromatography (silica gel, hexane–EtOAc).
The situation changed significantly when we heated the
reaction mixture to 80 °C overnight. A clean conversion
was observed and the triazole derivative 15 was isolated
in 78% yield (entry 1). Exactly the same result was ob-
tained with the dipeptide-derived azide 12 (entry 2). Steric
hindrance does not seem to play a significant role. Even
with azides 13 and 14 with the azido group at the α-posi-
tion of an amino acid, comparable results were obtained
(entries 3 and 4).
8-But-3-ynyl-2,3,5,6-tetrahydro-1H,4H-11-oxa-3a-azaben-
zo[de]anthracen-10-one (2)
According to the general aminocoumarin procedure, 8-hydroxyju-
lodin (378 mg, 2.00 mmol) and β-keto ester 1 (308 mg, 2.00 mmol)
were allowed to react in the presence of Ti(Oi-Pr)3Cl (4 mL, 4.00
mmol); this gave 2 after flash chromatography (silica gel, hexane–
EtOAc 7:3) as a yellow solid.
Last but not least, we changed the components, using
azide 5 and the propargyl ethers 7 and 8. The same picture
resulted: also here the yields were in the region of 80%
(entries 5 and 6). In all examples investigated so far, the
yields were in the range of 75–84%, and only the 1,4-di-
substituted triazoles are formed.
Yield: 508 mg (1.73 mmol, 87%); mp 125–127 °C.
1H NMR (400 MHz, CDCl3): δ = 1.93–2.00 (m, 4 H, 3-H, 14-H),
2.04 (t, J = 2.6 Hz, 1 H, 19-H), 2.54 (td, J = 7.4, 2.6 Hz, 2 H, 17-H),
2.77 (t, J = 6.3 Hz, 2 H, 16-H), 2.86–2.90 (m, 4 H, 4-H, 13-H),
3.22–3.27 (m, 4 H, 2-H, 15-H), 5.94 (s, 1 H, 9-H), 6.97 (s, 1 H, 6-H).
13C NMR (100 MHz, CDCl3): δ = 17.9, 20.5, 20.6, 21.6, 27.8, 30.6,
49.5, 49.9, 69.9, 82.5, 107.0, 107.4, 107.6, 118.0, 121.0, 145.8,
151.3, 154.1, 162.4.
In conclusion, we could show that ‘clickable’ derivatives
of coumarin fluorescence dyes are ideally suited to label
amino acids and peptides in constantly high yield. Steric
factors seem to play no significant role, as nearly the same
results are obtained independent of the coupling position.
Further applications of this protocol are currently under
investigation.
HRMS (CI): m/z calcd for C19H19NO2 [M]+: 293.1416; found:
293.1427.
Anal. Calcd for C19H19NO2 (293.36): C, 77.79; H, 6.53; N, 4.77.
Found: C, 77.54; H, 6.71; N, 4.69.
The products were purified by flash chromatography on silica gel
(0.063–0.2 mm) or on Flashsystem Reveleris® (UV- and ELSD-de-
tector, Grace). Mixtures of EtOAc and hexanes were generally used
as eluents. Analysis by TLC was carried out on commercially pre-
coated silica gel 60 TLC-PET plates (Fluka). Visualization was ac-
8-Chloromethyl-2,3,5,6-tetrahydro-1H,4H-11-oxa-3a-azaben-
zo[de]anthracen-10-one (4)
According to the general aminocoumarin procedure, 8-hydroxyju-
lodin (6.27 g, 33.2 mmol) and methyl 4-chloroacetoacetate (5.00 g,
33.2 mmol) were allowed to react in the presence of Ti(Oi-Pr)3Cl
(66 mL, 66.0 mmol); this gave 4 after flash chromatography (silica
gel, hexane–EtOAc, 7:3) as a yellow solid.
1
complished with UV light or KMnO4 solution. H and 13C NMR
spectroscopic analysis was performed on a Bruker Avance II 400
(400 MHz) spectrometer. HRMS were measured with at Finnigan
MAT 95S mass spectrometer at the Institute of Organic Chemistry
at Saarland University. Elemental analyses were carried out at the
Department of Chemistry at Saarland University.
Yield: 6.83 g (23.6 mmol, 71%); mp 188–190 °C.
1H NMR (400 MHz, CDCl3): δ = 1.95–1.99 (m, 4 H, 3-H, 14-H),
2.79 (t, J = 6.5 Hz, 2 H, 4-H), 2.88 (t, J = 6.5 Hz, 2 H, 13-H), 3.24–
3.29 (m, 4 H, 2-H, 15-H), 4.54 (s, 2 H, 16-H), 6.13 (s, 1 H, 9-H),
7.02 (s, 1 H, 6-H).
3-Oxo-hept-6-ynoic Acid Methyl Ester (1)23
A suspension of MgCl2 (938 mg, 9.86 mmol) and the potassium salt
of hydrogen methyl malonate (2.22 g, 14.2 mmol) in anhyd THF
(15 mL) was stirred for 4 h at 50 °C. In a second flask, CDI (1.85 g,
11.4 mmol) was added in portions to a solution of pent-4-ynoic acid
(930 mg. 9.48 mmol) in anhyd THF (20 mL) at 5 °C. Then the mix-
ture was stirred for 1 h at r.t. To the methylmagnesium malonate
suspension, the aforementioned imidazolide solution was added
dropwise at r.t. The reaction mixture was stirred at r.t. overnight,
concentrated under reduced pressure, and dissolved in EtOAc (20
mL). The suspension thus obtained was washed with 1 M KHSO4,
NaHCO3, and brine (15 mL each). The organic layer was dried
(Na2SO4) and the solvent was evaporated in vacuo. The crude prod-
uct was purified by flash chromatography (silica gel, hexane–
EtOAc, 8:2); this gave 1 as a pale yellow oil.
13C NMR (100 MHz, CDCl3): δ = 20.5, 20.6, 21.5, 27.8, 41.6, 49.5,
49.9, 106.0, 107.0, 108.3, 118.3, 121.1, 146.1, 150.0, 151.5, 162.1.
HRMS (CI): m/z calcd for C16H16ClNO2 [M]+: 289.0870; found:
289.0888.
8-Azidomethyl-2,3,5,6-tetrahydro-1H,4H-11-oxa-3a-azaben-
zo[de]anthracen-10-one (5)
NaN3 (306 mg, 4.70 mmol) was added to a solution of the chlorinat-
ed coumarin 4 (1.14 g, 3.92 mmol) in acetone (250 mL), and the re-
action mixture was stirred overnight. The reaction control showed
that the reaction was incomplete. Therefore, NaN3 (306 mg, 4.70
mmol) was added again and the mixture was stirred overnight at
35 °C. The solvent was removed under vacuum and the crude prod-
uct was purified by flash chromatography (silica gel, hexane–
EtOAc, 9:1, 8:2); this gave 5 as an orange solid.
Yield: 1.20 g (7.78 mmol, 82%).
1H NMR (400 MHz, CDCl3): δ = 1.96 (t, J = 2.7 Hz, 1 H, 1-H), 2.47
(td, J = 7.2, 2.7 Hz, 2 H, 3-H), 2.81 (t, J = 7.2 Hz, 2 H, 4-H), 3.48
(s, 2 H, 6-H), 3.74 (s, 3 H, 8-H).
13C NMR (100 MHz, CDCl3): δ = 12.8, 41.6, 48.9, 52.4, 69.0, 82.4,
167.3, 200.4.
Yield: 1.05 g (3.54 mmol, 91%); mp 148–152 °C.
1H NMR (400 MHz, CDCl3): δ = 1.94–2.00 (m, 4 H, 3-H, 14-H),
2.77 (t, J = 6.3 Hz, 2 H, 4-H), 2.89 (t, J = 6.3 Hz, 2 H, 13-H), 3.24–
3.29 (m, 4 H, 2-H, 15-H), 4.40 (s, 2 H, 16-H), 6.09 (s, 1 H, 9-H),
6.92 (s, 1 H, 6-H).
13C NMR (100 MHz, CDCl3): δ = 20.4, 20.6, 21.5, 27.8, 49.5, 49.9,
51.0, 106.1, 107.0, 107.8, 118.3, 120.9, 146.1, 148.7, 151.5, 162.0.
HRMS (CI): m/z calcd for C16H16N4O2 [M]+: 296.1273; found:
296.1294.
Aminocoumarins 2 and 4; General Procedure
A 1 M solution of Ti(Oi-Pr)3Cl (2.00 equiv) was added to a suspen-
sion of 8-hydroxyjulodin (1.00 equiv) and the corresponding β-keto
ester (1.00 equiv) in toluene (3 mL/mmol). The mixture was heated
to reflux overnight. After the mixture had cooled to r.t., CH2Cl2 (10
Synthesis 2012, 44, 2005–2012
© Georg Thieme Verlag Stuttgart · New York

PAPER
Fluorescence Labeling of Amino Acids and Peptides
2009
(S)-2-[(tert-Butoxycarbonyl)amino]-3-(prop-2-ynyloxy)propa-
noic Acid (6)
1H NMR (400 MHz, CDCl3): δ = 0.84 und 0.90 (2d, J = 6.8 Hz, 6
H, 12-H, 13-H), 1.44 (s, 9 H, 1-H), 2.15 (m, 1 H, 11-H), 2.46 (t,
J = 2.3 Hz, 1 H, 8-H), 3.06 (dd, J = 13.9, 6.4 Hz, 1 H, 16-H), 3.06
(dd, J = 13.9, 6.0 Hz, 1 H, 16-H), 3.63 (dd, J = 9.3, 6.4 Hz, 1 H, 5-
H), 3.69 (s, 3 H, 22-H), 3.89 (dd, J = 9.3, 4.3 Hz, 1 H, 5-H), 4.10 (d,
J = 2.3 Hz, 2 H, 6-H), 4.25–4.30 (m, 2 H, 4-H, 10-H), 4.84 (br s, 1
H, 15-H), 5.40 (br s, 1 H, NHSer), 6.55 (br s, 1 H, NHPhe), 6.83 (d,
J = 8.6 Hz, 1 H, NHVal), 7.08–7.29 (m, 5 H, 18-H, 19-H, 20-H).
13C NMR (100 MHz, CDCl3): δ = 17.6, 19.1, 28.2, 30.5, 37.8, 52.2,
53.2, 54.0, 58.5, 58.6, 69.4, 75.3, 78.9, 80.4, 127.1, 128.6, 129.2,
135.7, 155.5, 170.0, 170.3, 171.
HRMS (CI): m/z calcd for C26H38N3O7 [M + H]+: 504.2710; found:
504.2713.
N-Boc-L-serine (5.00 g, 24.4 mmol) was dissolved in DMF (37 mL)
and the solution was cooled to 0 °C. NaH (60% w/w) dispersion in
mineral oil (2.16 g, 53.7 mmol) was added over 15 min and the re-
action mixture was stirred for 1 h at 0 °C. 3-Bromopropyne (3.00
mL, 3.99 g, 26.8 mmol) was added dropwise over 15 min and the
reaction mixture was stirred for 1 h at 0 °C. Afterwards the ice bath
was removed and stirring continued for 3 h at r.t. The solvent was
evaporated and the residue was dissolved in H2O (50 mL). The so-
lution was washed with Et2O (3 × 20 mL) and acidified to pH 2 by
addition of 3 M aq HCl. The resulting mixture was extracted with
EtOAc (5 × 20 mL). The combined organic extracts were dried
(Na2SO4) and the solvent was evaporated. The crude product was
purified by flash chromatography (silica gel, hexane–EtOAc–
AcOH, 1:1:0.01); this afforded 6 as a pale yellow, viscous oil.
Anal. Calcd for C26H37N3O7 (503.59): C, 62.01; H, 7.41; N, 8.34.
Found: C, 62.08; H, 7.28; N, 8.21.
Yield: 4.15 g (17.1 mmol, 70%).
(S)-2-[(tert-Butoxycarbonyl)amino]-3-methylpentyl Methane-
sulfonate (9)
1H NMR (400 MHz, CDCl3): δ = 1.44 (s, 9 H, 1-H), 2.46 (t, J = 2.3
Hz, 1 H, 8-H), 3.80 (dd, J = 9.3, 3.4 Hz, 1 H, 5-H), 3.98 (dd, J = 9.3,
2.4 Hz, 1 H, 5-H), 4.17 (d, J = 2.3 Hz, 2 H, 6-H), 4.48 (m, 1 H, 4-
H), 5.41 (d, J = 8.4 Hz, 1 H, NH), 10.03 (br s, 1 H, COOH).
13C NMR (100 MHz, CDCl3): δ = 28.3, 53.7, 58.7, 69.5, 75.3, 78.7,
80.4, 155.7, 175.1.
A solution of N-Boc-protected leucinol (1.00 g, 4.60 mmol) and
Et3N (1.45 g, 14.3 mmol) in absolute CH2Cl2 (23 mL) was cooled
to 0 °C and MsCl (790 mg, 6.90 mmol) was added dropwise. The
mixture was stirred 3 h at 0 °C. After dilution with CH2Cl2 (25 mL),
the mixture was washed with 1 M KHSO4 (20 mL), sat. aq NaHCO3
(2 × 20 mL), H2O, and brine (25 mL each). The organic layer was
dried (Na2SO4) and the solvent was removed in vacuo. The crude
product was purified by flash chromatography (silica gel, hexane–
EtOAc, 7:3); this gave 9 as a colorless oil (unstable compound).
(S)-2-{(S)-2-[(tert-Butoxycarbonyl)amino]-3-(prop-2-ynyl-
oxy)propanoylamino}-4-methylpentanoic acid Methyl Ester (7)
A 50% solution of T3P in EtOAc (9.2 mL, 4.93 g, 15.5 mmol) was
added to a solution of 6 (2.50 g, 10.3 mmol), leucine methyl ester
(1.50 g, 10.3 mmol), and DIPEA (5.4 mL, 3.99 g, 30.9 mmol) in
EtOAc (150 mL) at 0 °C. The reaction mixture was stirred over-
night at r.t. For workup, the mixture was quenched with brine and
the aqueous layer was extracted with CH2Cl2 (2 × 25 mL). The com-
bined organic extracts were washed with 1 M HCl and brine (25 mL
each) and dried (Na2SO4). The solvent was removed under vacuum
and the crude product was purified by flash chromatography (silica
gel, hexane–EtOAc, 7:3); this gave 7 as a colorless, viscous oil.
Yield: 1.30 g (4.42 mmol, 96%).
1H NMR (400 MHz, CDCl3): δ = 0.90–0.96 (m, 6 H, 6-H, 8-H),
1.16 (m, 1 H, 5-H), 1.45 (s, 9 H, 1-H), 1.51–1.66 (m, 2 H, 7-H), 3.02
(s, 3 H, 10-H), 3.70 (br s, 1 H, 4-H), 4.24–4.31 (m, 2 H, 9-H), 4.63
(d, J = 7.7 Hz, 1 H, NH).
13C NMR (100 MHz, CDCl3): δ = 11.1, 15.4, 25.2, 28.3, 35.6, 37.4,
53.8, 69.7, 79.8; Boc carbonyl not observed.
HRMS (CI): m/z calcd for C12H24NO5S [M]+: 295.1453; found:
293.1474.
Yield: 3.42 g (9.23 mmol, 90%).
1H NMR (400 MHz, CDCl3): δ = 0.91 (d, J = 6.5 Hz, 6 H, 13-H),
1.43 (s, 9 H, 1-H), 1.52–1.64 (m, 3 H, 11-H, 12-H), 2.44 (t, J = 1.4
Hz, 1 H, 8-H), 3.63 (dd, J = 9.3, 6.4 Hz, 1 H, 5-H), 3.70 (s, 3 H, 15-
H), 3.90 (dd, J = 9.3, 4.0 Hz, 1 H, 5-H), 4.16 (d, 2 H, 6-H), 4.28 (br
s, 1 H, 10-H), 4.60 (m, 1 H, 4-H), 5.39 (d, J = 8.7 Hz, 1 H, NHSer),
6.85 (br s, 1 H, NHIle).
13C NMR (100 MHz, CDCl3): δ = 21.9, 22.8, 24.7, 28.2, 41.5, 50.6,
52.2, 53.6, 58.6, 69.3, 75.1, 78.8, 80.3, 155.4, 169.9, 172.9.
HRMS (CI): m/z calcd for C18H30N2O6 [M + H]+: 371.2182; found:
371.2215.
tert-Butyl (S)-1-Azido-3-methylpentan-2-ylcarbamate (10)
NaN3 (2.22 g, 34.2 mmol) was added to a solution of 9 (1.30 g, 4.40
mmol) in absolute DMF (44 mL). After the reaction mixture had
been stirred for 15 h at 60 °C, the cooled mixture was diluted with
H2O (100 mL) and EtOAc (100 mL). The aqueous layer was ex-
tracted with EtOAc (3 × 20 mL). Afterwards the combined organic
layers were washed with H2O (3 × 20 mL) and brine (20 mL). The
organic layer was dried (Na2SO4) and the solvent was removed un-
der vacuum. The crude product was purified by flash chromatogra-
phy (silica gel, hexane–EtOAc, 8:2); this gave 10 as a colorless
solid.
Anal. Calcd for C18H30N2O6 (370.44): C, 58.36; H, 8.16; N, 7.56.
Found: C, 57.86; H, 7.68; N, 7.29.
Yield: 782 mg (3.23 mmol, 73%); mp 48–50 °C. The azide is stable
and can be stored at r.t.
(S)-2-{(S)-2-[(tert-Butoxycarbonyl)amino]-3-(prop-2-ynyl-
oxy)propanoylamino}-3-methylbutanoylamino]-3-phenylpro-
panoic Acid Methyl Ester (8)
1H NMR (400 MHz, CDCl3): δ = 0.89–0.93 (m, 6 H, 6-H, 8-H),
1.12 (m, 1 H, 5-H), 1.45–1.58 (m, 11 H, 1-H, 7-H), 3.42 (m, 2 H, 9-
H), 3.58 (br s, 1 H, 4-H), 4.55 (d, J = 7.3 Hz, 1 H, NH).
13C NMR (100 MHz, CDCl3): δ = 11.3, 15.5, 25.2, 28.4, 36.3, 52.9,
54.4, 79.6, 155.5.
HRMS (CI): m/z calcd for C11H22N4O4 [M]+: 243.1821; found:
243.1793.
A 50% solution of T3P in EtOAc (4.6 mL, 2.43 g, 7.65 mmol) was
added to a solution of 6 (1.24 g, 5.10 mmol), valyl-phenylalanine
methyl ester (1.42 g, 5.10 mmol), and DIPEA (2.7 mL, 1.98 g, 15.3
mmol) in EtOAc (45 mL) at 0 °C. The reaction mixture was stirred
overnight at r.t. For workup, the mixture was quenched with brine
(25 mL) and the aqueous layer was extracted with CH2Cl2 (2 × 20
mL). The combined organic extracts were washed with 1 M HCl
and brine (25 mL each) and dried (Na2SO4). The solvent was re-
moved under vacuum and the crude product was purified by flash
chromatography (silica gel, hexane–EtOAc, 7:3, 6:4); this gave 8 as
a colorless solid.
Anal. Calcd for C11H22N4O4 (242.32): C, 54.52; H, 9.15; N, 23.12.
Found: C, 54.52; H, 8.77; N, 22.94.
Benzyl (S,S)-1-(1-Hydroxypropan-2-ylamino)-1-oxo-3-phenyl-
propan-2-ylcarbamate (11)
NaBH4 (446 mg, 11.8 mmol) and LiCl (500 mg, 11.8 mmol) in
EtOH (14 mL) were added to a solution of N-Cbz-phenylalanyl-
alanine methyl ester (2.27 g, 5.90 mmol) in THF (7 mL). The reac-
Yield: 1.95 g (3.87 mmol, 76%); mp 87–90 °C.
© Georg Thieme Verlag Stuttgart · New York
Synthesis 2012, 44, 2005–2012

2010
L. Wirtz et al.
PAPER
tion mixture was stirred overnight at r.t. For workup, the mixture
was hydrolyzed with 10% aq citric acid (40 mL) and the solvent was
removed under reduced pressure. The residue was dissolved in
EtOAc (50 mL) and washed with H2O and brine (20 mL each). The
organic layer was dried (Na2SO4) and the solvent was removed in
vacuo. The crude product was purified by flash chromatography
(silica gel, hexane–EtOAc, 1:1, EtOAc); this gave 11 as a colorless
solid.
MeOH (6 mL/mmol amino acid). After addition of the TfN3 solu-
tion, the reaction was stirred overnight at r.t. The organic solvents
were removed under reduced pressure. The residue was extracted
with EtOAc (3 × 20 mL), the combined organic layers were dried
(Na2SO4), and the solvent was evaporated under vacuum.
8-(2-Azidoethyl)-2,3,5,6-tetrahydro-1H,4H-11-oxa-3a-azaben-
zo[de]anthracen-10-one (3)
According to the Wong procedure, 8-(2-aminoethyl)-2,3,4,5-tetra-
hydro-1H,4H-11-oxa-3a-azabenzo[de]anthracen-10-one (533 mg,
1.87 mmol) and TfN3 were allowed to react in the presence of 1 M
CuSO4 solution (20 μL, 1 mol%) and K2CO3 (388 mg, 2.81 mmol).
After workup, 3 was obtained without further purification as a yel-
low oil.
Yield: 1.68 g (4.71 mmol, 80%); mp 110–114 °C.
1H NMR (400 MHz, CDCl3): δ = 1.04 (d, J = 6.8 Hz, 3 H, 15-H),
2.15 (br s, 1 H, OH), 2.99 (dd, J = 13.5, 8.1 Hz, 1 H, 8-H), 3.11 (dd,
J = 13.5, 6.3 Hz, 1 H, 8-H), 3.22 (dd, J = 11.2, 5.2 Hz, 1 H, 16-H),
3.41 (dd, J = 11.2, 3.6 Hz, 1 H, 16-H), 3.94 (m, 1 H, 14-H), 4.36 (m,
1 H, 7-H), 5.08 (s, 2 H, 5-H), 5.51 (d, J = 5.2 Hz, 1 H, NHPhe), 5.86
(d, J = 5.9 Hz, 1 H, NHAla), 7.07–7.37 (m, 10 H, 1-H, 2-H, 3-H, 10-
H, 11-H, 12-H).
13C NMR (100 MHz, CDCl3): δ = 16.7, 39.0, 47.6, 56.6, 66.1, 67.1,
127.1, 128.1, 128.2, 128.5, 128.8, 128.8, 136.1, 136.5, 156.0, 170.8.
HRMS (CI): m/z calcd for C20H24N2O4 [M]+: 356.1735; found:
356.1707.
Yield: 169 mg (0.94 mmol, 29%).
1H NMR (400 MHz, CDCl3): δ = 1.93–2.00 (m, 4 H, 3-H, 14-H),
2.77 (t, J = 6.3 Hz, 2 H, 4-H), 2.85–2.93 (m, 4 H, 13-H, 16-H),
3.23–3.28 (m, 4 H, 2-H, 15-H), 3.58 (t, J = 7.3 Hz, 2 H, 17-H), 5.92
(s, 1 H, 9-H), 6.95 (s, 1 H, 6-H).
13C NMR (100 MHz, CDCl3): δ = 20.4, 20.5, 21.5, 27.7, 31.0, 49.4,
49.9, 49.9, 107.0, 107.5, 107.8, 118.2, 121.0, 146.0, 151.3, 152.4,
162.3.
Anal. Calcd for C20H24N2O4 (356.42): C, 67.40; H, 6.79; N, 7.86.
Found: C, 67.17; H, 6.94; N, 7.71.
HRMS (CI): m/z calcd for C17H18N4O2 [M]+: 310.1430; found:
310.1437.
Benzyl (S,S)-1-(1-Azidopropan-2-ylamino)-1-oxo-3-phenylpro-
pan-2-ylcarbamate (12)
(S)-2-Azido-3-methylpentanoic Acid Methyl Ester (13)
According to the Wong procedure, isoleucine methyl ester (145 mg,
1.00 mmol) and TfN3 were allowed to react in the presence of 1 M
CuSO4 solution (10 μL, 1 mol%) and K2CO3 (207 mg, 1.50 mmol).
After workup, 13 was obtained without further purification as a col-
orless oil.
A solution of 11 (500 mg, 1.40 mmol) and Et3N (440 mL, 4.34
mmol) in absolute CH2Cl2 (27 mL) was cooled to 0 °C before MsCl
(241 mg, 2.10 mmol) was added dropwise. The mixture was stirred
for 3 h at 0 °C. After dilution with CH2Cl2, the mixture was washed
with 1 M KHSO4 (29 mL), sat. NaHCO3 (2 × 29 mL), H2O (29 mL),
and brine (29 mL). The organic layer was dried (Na2SO4) and the
solvent was removed under vacuum. The methanesulfonate was in-
stable. Therefore, the crude product (200 mg, 0.46 mmol) was sub-
sequently dissolved in absolute DMF (5 mL), and NaN3 (232 mg,
3.58 mmol) was added. After the reaction mixture had been stirred
for 15 h at 60 °C, the cooled mixture was diluted with H2O and EtO-
Ac (10 mL each). The aqueous layer was extracted with EtOAc
(3 × 30 mL). The combined organic layers were washed with H2O
(3 × 20 mL) and brine (25 mL). The organic layer was dried
(Na2SO4) and the solvent was removed under vacuum. The crude
product was purified by flash chromatography (silica gel, hexane–
EtOAc, 7:3, 6:4); this gave 12 as a colorless, viscous oil.
Yield: 133 mg (0.79 mmol, 79%).
1H NMR (400 MHz, CDCl3): δ = 0.88–0.96 (m, 6 H, 3-H, 5-H),
1.23 (m, 1 H, 4-H), 1.51 (m, 1 H, 4-H) 1.95 (m, 1 H, 2-H), 3.73 (s,
3 H, 7-H), 3.78 (br s, 1 H, 1-H).
13C NMR (100 MHz, CDCl3): δ = 11.5, 15.8, 25.0, 37.1, 51.8, 67.2,
170.7.
(S,S)-2-(2-Azido-3-phenylpropanoylamino)-3-methylpentanoic
Acid Methyl Ester (14)
According to the Wong procedure, phenylalanyl isoleucine methyl
ester (292 mg, 1.00 mmol) and TfN3 were allowed to react in the
presence of 1 M CuSO4 solution (10 μL, 1 mol%) and K2CO3 (207
mg, 1.50 mmol). After workup, 14 was obtained without further pu-
rification as a colorless oil.
Yield: 204 mg (0.53 mmol, 38%). The azide is stable and can be
stored at r.t.
1H NMR (400 MHz, CDCl3): δ = 1.06 (d, J = 6.8 Hz, 3 H, 15-H),
2.96–3.32 (m, 3 H, 8-H, 16-H), 3.80 (m, 1 H, 16-H), 4.07 (m, 1 H,
14-H), 4.41 (m, 1 H, 7-H), 5.08 (s, 2 H, 5-H), 5.44 (d, J = 6.6 Hz, 1
H, NHPhe), 5.79 (br s, 1 H, NHAla), 7.11–7.35 (m, 10 H, 1-H, 2-H, 3-
H, 10-H, 11-H, 12-H).
13C NMR (100 MHz, DMSO-d6): δ = 17.2, 38.5, 47.4, 56.8, 65.1,
66.1, 126.7, 128.0, 128.2, 128.5, 128.9, 129.6, 137.5, 138.2, 156.0,
171.1.
Yield: 299 mg (0.94 mmol, 94%).
1H NMR (400 MHz, CDCl3): δ = 0.69 (d, J = 6.9 Hz, 3 H, 10-H),
0.81 (t, J = 7.4 Hz, 3 H, 12-H), 0.98 (m, 1 H, 11-H), 1.25 (m, 1 H,
11-H,) 1.71 (m, 1 H, 9-H), 2.99 (dd, J = 14.1, 7.8 Hz, 1 H, 2-H),
2.26 (dd, J = 14.1, 4.1 Hz, 1 H, 2-H), 3.66 (s, 3 H, 14-H), 4.18 (dd,
J = 7.8, 4.1 Hz, 1 H, 1-H), 4.44 (dd, J = 8.7, 5.0 Hz, 1 H, 8-H), 6.66
(d, J = 8.4 Hz, 1 H, NH), 7.18–7.24 (m, 5 H, 4-H, 5-H, 6-H).
13C NMR (100 MHz, CDCl3): δ = 11.5, 15.2, 25.0, 37.7, 38.4, 52.2,
56.4, 65.4, 127.2, 128.6, 129.6, 135.9, 168.1, 171.8.
HRMS (CI): m/z calcd for C16H23N4O3 [M]+: 319.1770; found:
319.1784.
HRMS (CI): m/z calcd for C20H23N5O3 [M]+: 381.1801; found:
381.1819.
Wong Method for the Preparation of Azides 3, 13, and 14; Gen-
eral Procedure
TfN3 was freshly prepared prior to each reaction. NaN3 (9.80 equiv)
was dissolved in H2O (0.16 mL/mmol). At 0 °C an equal volume of
CH2Cl2 was added. Afterwards Tf2O (1.99 equiv) was added drop-
wise to the vigorously stirred solution. After stirring of the mixture
for 2 h at 0 °C, the aqueous phase was extracted with CH2Cl2 (2 × 1
mL/mmol Tf2O). The combined organic phases were washed with
sat. aq NaHCO3 and used without further purification. The amine
(1.00 equiv), 1 M CuSO4 in H2O (1 mol%), and K2CO3 (1.50 equiv)
were dissolved/suspended in H2O (3 mL/mmol amino acid) and
Triazoles 15–20 by Copper-Catalyzed Click Chemistry; Gener-
al Procedure
A freshly prepared 1 M aq solution of sodium ascorbate (10 mol%)
and 1 M CuSO4 solution (10 mol%) were added to a suspension of
the appropriate azide (1.00 equiv) and alkyne (1.50 equiv) in
DMSO–H2O (4:1; 4 mL). The heterogeneous mixture was stirred
vigorously overnight at 80 °C. The reaction mixture was diluted
with CH2Cl2 (20 mL) and afterwards washed with H2O and brine
(20 mL each). The organic layer was dried (Na2SO4), the solvent
Synthesis 2012, 44, 2005–2012
© Georg Thieme Verlag Stuttgart · New York

PAPER
Fluorescence Labeling of Amino Acids and Peptides
2011
was evaporated under vacuum, and the crude product was purified
by flash chromatography (silica gel or flash system, hexane–EtO-
Ac).
13C NMR (100 MHz, DMSO-d6): δ = 10.5, 15.1, 20.0, 20.1, 20.9,
24.2, 24.4, 27.0, 30.3, 36.6, 42.0, 48.6, 49.1, 52.5, 66.5, 105.6,
106.4, 107.0, 117.7, 121.6, 122.2, 145.4, 145.5, 150.6, 155.7, 160.7,
168.8.
HRMS (CI): m/z calcd for C26H32N4O4 [M]+: 464.2424; found:
464.2430.
tert-Butyl (S)-2-Methyl-1-{4-[2-(10-oxo-2,3,5,6-tetrahydro-
1H,4H-11-oxa-3a-azabenzo[de]anthracen-8-yl)ethyl]-1,2,3-tri-
azol-1-ylmethyl}butylcarbamate (15)
According to the general click-chemistry procedure, azide 10 (97.0
mg, 0.40 mmol) and fluorescent alkyne 2 (176 mg, 0.60 mmol) were
allowed to react. After workup, the crude product was purified by
flash chromatography (silica gel, hexane–EtOAc, 9:1, 7:3, 1:1); this
gave 15 as a pale brown solid.
Methyl (S,S)-3-Methyl-2-({4-[2-(10-oxo-2,3,5,6-tetrahydro-
1H,4H-11-oxa-3a-azabenzo[de]anthracen-8-yl)ethyl]-1,2,3-tri-
azol-1-yl}-3-phenylpropanoylamino)pentanoate (18)
According to the general click-chemistry procedure, azide 14 (96.0
mg, 0.30 mmol) and fluorescent alkyne 2 (132 mg, 0.45 mmol) were
allowed to react. After workup, the crude product was purified by
flash chromatography (silica gel, hexane–EtOAc, 7:3, CH2Cl2–
EtOAc, 98:2); this gave 18 as a yellow-brown, viscous oil.
Yield: 168 mg (0.31 mmol, 78%); mp 205–208 °C.
1H NMR (400 MHz, CDCl3): δ = 0.92 (t, J = 7.3 Hz, 3 H, 25-H),
0.99 (d, J = 6.7 Hz, 3 H, 23-H), 1.18 (m, 1 H, 22-H), 1.38 (s, 9 H,
28-H), 1.51 (m, 2 H, 24-H), 1.94–2.00 (m, 4 H, 3-H, 14-H), 2.77 (t,
J = 6.3 Hz, 2 H, 4-H), 2.88 (t, J = 6.5 Hz, 2 H, 13-H), 2.98–3.07 (m,
4 H, 16-H, 17-H), 3.22–3.27 (m, 4 H, 2-H, 15-H), 3.83 (m, 1 H, 21-
H), 4.39–4.49 (m, 2 H, 20-H), 4.67 (d, J = 8.7 Hz, 1 H, NH), 5.86
(s, 1 H, 9-H), 7.06 (s, 1 H, 6-H), 7.35 (s, 1 H, 19-H).
13C NMR (100 MHz, CDCl3): δ = 11.2, 15.6, 20.5, 20.7, 21.6, 24.7,
25.0, 27.7, 28.3, 31.3, 36.3, 49.4, 49.9, 51.3, 55.0, 79.7, 107.0,
107.1, 107.8, 118.1, 121.4, 121.8, 145.8, 146.1, 151.3, 155.5, 155.5,
162.5; Boc carbonyl not observed.
Yield: 138 mg (0.23 mmol, 75%)
1H NMR (400 MHz, DMSO-d6): δ = 0.75–0.80 (m, 6 H, 29-H, 31-
H), 1.06 (m, 1 H, 30-H), 1.29 (m, 1 H, 30-H), 1.77 (m, 1 H, 28-H),
1.86–1.91 (m, 4 H, 3-H, 14-H), 2.69 (t, J = 6.4 Hz, 2 H, 4-H), 2.77
(t, J = 6.4 Hz, 2 H, 13-H), 2.91–2.98 (m, 4 H, 16-H, 17-H), 3.14–
3.19 (m, 4 H, 2-H, 15-H), 3.26 (dd, J = 14.0, 9.2 Hz, 1 H, 21-H),
3.44 (dd, J = 14.0, 6.1 Hz, 1 H, 21-H), 3.60 (s, 3 H, 33-H), 4.39 (dd,
J = 8.2, 5.4 Hz, 1 H, 27-H), 5.57 (dd, J = 9.0, 6.3 Hz, 1 H, 20-H),
5.79 (s, 1 H, 9-H), 6.97–7.14 (m, 6 H, 6-H, 23-H, 24-H, 25-H), 7.56
(s, 1 H, 19-H), 7.70 (d, J = 8.2 Hz, 1 H, NH).
13C NMR (100 MHz, DMSO-d6): δ = 11.1, 15.3, 20.0, 20.1, 21.0,
24.0, 24.7, 27.0, 30.3, 36.3, 37.5, 48.6, 49.2, 51.8, 56.6, 63.2, 105.7,
106.2, 107.1, 117.8, 121.6, 121.7, 126.7, 128.2, 128.9, 136.0, 145.3,
145.5, 150.7, 155.7, 160.9, 167.9, 171.4.
HRMS (CI): m/z calcd for C30H41N5O4 [M]+: 535.3159; found:
535.3129.
Benzyl (S,S)-1-(1-Methyl-2-{4-[2-(10-oxo-2,3,5,6-tetrahydro-
1H,4H-11-oxa-3a-azabenzo[de]anthracen-8-yl)ethyl]-1,2,3-tri-
azol-1-yl}ethylcarbamoyl)-2-phenylethylcarbamate (16)
According to the general click-chemistry procedure, azide 12
(114.0 mg, 0.30 mmol) and fluorescent alkyne 2 (132 mg, 0.45
mmol) were allowed to react. After workup, the crude product was
purified by flash chromatography (silica gel, hexane–EtOAc, 1:1,
EtOAc); this gave 16 as a yellow-brown, viscous oil.
HRMS (CI): m/z calcd for C35H41N5O5 [M]+: 611.3108; found:
611.3091.
Methyl (S)-2-((S)-2-tert-Butoxycarbonylamino-3-{1-[(10-oxo-
2,3,5,6-tetrahydro-1H,4H-11-oxa-3a-azabenzo[de]anthracen-8-
yl)methyl]-1H-1,2,3-triazol-4-ylmethoxy}propanoylamino)-4-
methylpentanoate (19)
Yield: 158 mg (0.23 mmol, 78%)
According to the general click-chemistry procedure, azide 5 (116
mg, 0.39 mmol) and fluorescent alkyne 7 (111 mg, 0.30 mmol) were
allowed to react. After workup, the crude product was purified by
flash chromatography (flash system, hexane–EtOAc, gradient 7:3 to
0:1); this gave 19 as a yellow-brown, viscous oil.
1H NMR (400 MHz, DMSO-d6): δ = 0.97 (m, 3 H, 22-H), 1.87–1.96
(m, 4 H, 3-H, 14-H), 2.73–2.86 (m, 4 H, 4-H, 13-H), 2.93–3.10 (m,
6 H, 16-H, 17-H, 25-H), 3.18–3.25 (m, 4 H, 2-H, 15-H), 4.15–4.39
(m, 4 H, 20-H, 21-H, 24-H), 4.97 (s, 2 H, 31-H), 5.52 (d, J = 7.3 Hz,
1 H, NHPhe), 5.66 (s, 1 H, 9-H), 6.55 (d, J = 6.9 Hz, 1 H, NHAla),
7.04 (s, 1 H, 6-H), 7.18–7.29 (m, 11 H, 19-H, 27-H, 28-H, 29-H, 33-
H, 34-H, 35-H).
13C NMR (100 MHz, DMSO-d6): δ = 17.2, 19.9, 20.1, 20.9, 24.1,
26.9, 30.5, 37.4, 44.9, 48.6, 49.1, 53.1, 56.1, 65.1, 105.6, 106.1,
107.0, 117.7, 121.5, 122.8, 126.1, 126.9, 127.8, 129.1, 137.0, 138.1,
145.2, 145.4, 150.6, 155.6, 155.8, 160.8, 171.0.
Yield: 168 mg (0.25 mmol, 84%)
1H NMR (400 MHz, DMSO-d6): δ = 0.85 (d, J = 6.5 Hz, 6 H, 29-
H), 1.36 (s, 9 H, 24-H), 1.46–1.62 (m, 3 H, 27-H, 28-H), 1.87–1.91
(m, 4 H, 3-H, 14-H), 2.70–2.73 (m, 4 H, 4-H, 13-H), 3.23–3.28 (m,
4 H, 2-H, 15-H), 3.51–3.64 (m, 5 H, 16-H, 31-H), 4.21 (m, 1 H, 21-
H), 4.29 (m, 1 H, 26-H), 4.56 (br s, 2 H, 20-H), 5.33 (s, 1 H, 9-H),
5.79 (s, 2 H, 19-H), 6.67 (d, J = 8.2 Hz, 1 H, NHSer), 7.02 (s, 1 H, 6-
H), 7.94 (s, 1 H, 17-H), 8.20 (d, J = 7.7 Hz, 1 H, NHIle).
13C NMR (100 MHz, DMSO-d6): δ = 19.8, 19.9, 20.8, 21.1, 22.7,
23.9, 27.0, 28.0, 48.6, 49.0, 49.1, 50.1, 51.7, 54.1, 63.5, 69.7, 78.1,
105.2, 105.3, 105.6, 117.9, 121.3, 124.7, 144.2, 145.9, 150.3, 150.6,
155.0, 160.4, 169.9, 172.6.
HRMS (CI): m/z calcd for C39H42N6O5 [M]+: 674.3217; found:
674.3176.
Methyl (S)-3-Methyl-2-{4-[2-(10-oxo-2,3,5,6-tetrahydro-
1H,4H-11-oxa-3a-azabenzo[de]anthracen-8-yl)ethyl]-1,2,3-tri-
azol-1-yl}pentanoate (17)
According to the general click-chemistry procedure, azide 13 (51
mg, 0.30 mmol) and fluorescent alkyne 2 (132 mg, 0.45 mmol) were
allowed to react. After workup, the crude product was purified by
flash chromatography (silica gel, hexane–EtOAc, 7:3, 1:1); this
gave 17 as a yellow-brown, viscous oil.
HRMS (CI): m/z calcd for C34H46N6O8 [M]+: 666.3377; found:
666.3375.
Methyl (S)-2-[(S)-2-((S)-2-tert-Butoxycarbonylamino-3-{1-[(10-
oxo-2,3,5,6-tetrahydro-1H,4H-11-oxa-3a-azabenzo[de]anthra-
cen-8-yl)methyl]-1H-1,2,3-triazol-4-ylmethoxy}propanoylami-
no)-3-methylbutanoylamino]-3-phenylpropanoate (20)
According to the general click-chemistry procedure, azide 5 (116
mg, 0.39 mmol) and fluorescent alkyne 8 (151 mg, 0.30 mmol) were
allowed to react. After workup, the crude product was purified by
flash chromatography (flash system, hexane–EtOAc, gradient 7:3 to
0:1); this gave 20 as a yellow-brown, viscous oil.
Yield: 108 mg (0.23 mmol, 77%).
1H NMR (400 MHz, DMSO-d6): δ = 0.77 (d, J = 7.4 Hz, 3 H, 24-
H), 0.88 (t, J = 6.8 Hz, 3 H, 22-H), 0.95 (m, 1 H, 23-H), 1.08 (m, 1
H, 23-H), 1.86–1.89 (m, 4 H, 3-H, 14-H), 2.27 (m, 1 H, 21-H), 2.68–
2.74 (m, 4 H, 4-H, 13-H), 2.97–3.03 (m, 4 H, 16-H, 17-H), 3.20–
3.24 (m, 4 H, 2-H, 15-H), 3.69 (s, 3 H, 26-H), 5.23 (d, J = 8.7 Hz, 1
H, 20-H), 5.75 (s, 1 H, 9-H), 7.15 (s, 1 H, 6-H), 8.01 (s, 1 H, 19-H).
Yield: 185 mg (0.23 mmol, 77%)
© Georg Thieme Verlag Stuttgart · New York
Synthesis 2012, 44, 2005–2012

2012
L. Wirtz et al.
PAPER
120, 9133. (c) Triola, G.; Brunsveld, L.; Waldmann, H.
J. Org. Chem. 2008, 73, 3646. (d) Wittrock, S.; Becker, T.;
Kunz, H. Angew. Chem. Int. Ed. 2007, 46, 5226; Angew.
Chem. 2007, 119, 5319. (e) Jonkheijm, P.; Weinrich, D.;
Köhn, M.; Engelkamp, H.; Christianen, P. C. M.; Kuhlmann,
J.; Maan, J. C.; Nüsse, D.; Schröder, H.; Wacker, R.;
Breinbauer, R.; Niemeyer, C. M.; Waldmann, H. Angew.
Chem. Int. Ed. 2008, 47, 4421; Angew. Chem. 2008, 120,
4493.
1H NMR (400 MHz, DMSO-d6): δ = 0.74 und 0.79 (2d, J = 6.6 Hz,
6 H, 28-H, 29-H), 1.36 (s, 9 H, 24-H), 1.88–1.94 (m, 5 H, 3-H, 14-
H, 27-H), 2.69–2.73 (m, 4 H, 4-H, 13-H), 2.91 (dd, J = 13.8, 8.7 Hz,
1 H, 32-H), 2.99 (dd, J = 13.8, 6.1 Hz, 1 H, 32′-H), 3.23–3.27 (m, 4
H, 2-H, 15-H), 3.54–3.58 (m, 5 H, 16-H, 38-H), 4.17–4.23 (m, 2 H,
21-H, 26-H), 4.45 (m, 1 H, 31-H), 4.53 (br s, 2 H, 20-H), 5.33 (s, 1
H, 9-H), 5.78 (s, 2 H, 19-H), 7.09 (d, J = 8.2 Hz, 1 H, NHSer), 7.16–
7.26 (m, 6 H, 6-H, 34-H, 35-H, 36-H), 7.57 (d, J = 8.9 Hz, 1 H, NH-
Val), 8.15 (s, 1 H, 17-H), 8.45 (d, J = 7.1 Hz, 1 H, NHPhe).
13C NMR (100 MHz, DMSO-d6): δ = 17.5, 18.8, 19.8, 19.9, 20.8,
27.0, 28.0, 48.6, 49.0, 49.1, 51.6, 53.4, 54.5, 56.8, 63.5, 78.3, 105.2,
105.3, 105.6, 117.9, 121.3, 124.8, 126.4, 128.1, 128.8, 136.9, 144.2,
145.8, 150.3, 150.6, 155.2, 160.4, 169.4, 170.7, 171.6.
HRMS (CI): m/z calcd for C42H54N7O9 [M + H]+: 800.3978; found:
800.3980.
(9) McFarland, J. M.; Francis, M. B. J. Am. Chem. Soc. 2005,
127, 13490.
(10) (a) Kodadek, T.; Duroux-Richard, I.; Bonnafous, J. C.
Trends Pharmacol. Sci. 2005, 26, 210. (b) McFarland, J. M.;
Joshi, N. S.; Francis, M. B. J. Am. Chem. Soc. 2008, 130,
7639. (c) Tilley, S. D.; Francis, M. B. J. Am. Chem. Soc.
2006, 128, 1080.
(11) (a) Huisgen, R. Angew. Chem., Int. Ed. Engl. 1963, 2, 565;
Angew. Chem. 1963, 75, 604. (b) Rostovtsev, V. V.; Green,
L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem. Int. Ed.
2002, 41, 2596; Angew. Chem. 2002, 114, 2708. (c) Tornoe,
C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67,
3057. (d) Chan, T. R.; Hilgraf, R.; Sharpless, K. B.; Fokin,
V. V. Org. Lett. 2004, 6, 2853. (e) Rodionov, V. O.;
Presolski, S. I.; Diaz, D. D.; Fokin, V. V.; Finn, M. G. J. Am.
Chem. Soc. 2007, 129, 12705.
Acknowledgment
Financial support from the Deutsche Forschungsgemeinschaft is
gratefully acknowledged.
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synthesis.SnuIomprifgtooatrinSugpIoi
nnfomartit
(12) Sharpless, K. B.; van der Eycken, E. QSAR Comb. Sci. 2007,
26, 1110.
References
(13) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem. Int.
Ed. 2001, 40, 20046; Angew. Chem. 2001, 113, 205.
(14) Amo, D. S. D.; Wang, W.; Jiang, H.; Besanceney, C.; Yan,
A. C.; Levy, M.; Liu, Y.; Marlow, F. L.; Wu, P. J. Am. Chem.
Soc. 2010, 132, 16893.
(1) (a) Valeur, B.; Brochon, J.-C. New Trends in Fluorescence
Spectroscopy: Applications to Chemical and Life Sciences;
Springer: Berlin, 2001. (b) Goldys, E. M. Fluorescence
Applications in Biotechnology and Life Science; Wiley:
Hoboken, 2009.
(2) (a) Hama, Y.; Urano, Y.; Koyama, Y.; Gunn, A. J.; Choyke,
P. L.; Kobayashi, H. Clin. Cancer Res. 2007, 13, 6335.
(b) Dillingham, M. S.; Wallace, M. I. Org. Biomol. Chem.
2008, 6, 3031. (c) Neef, A. B.; Schultz, C. Angew. Chem. Int.
Ed. 2009, 48, 1498; Angew. Chem. 2009, 121, 1526.
(3) (a) Sunahara, H.; Urano, Y.; Kojima, H.; Nagano, T. J. Am.
Chem. Soc. 2007, 129, 5597. (b) Ulrich, G.; Ziessel, R.;
Harriman, A. Angew. Chem. Int. Ed. 2008, 47, 1184; Angew.
Chem. 2008, 120, 1202.
(4) (a) James, T. D.; Sandanayake, K. R. A. S.; Shinkai, S.
Angew. Chem. Int. Ed. 1994, 33, 2207; Angew. Chem. 1994,
106, 2287. (b) Ueno, T.; Urano, Y.; Setsukinai, K.-I.;
Takakusa, H.; Kojima, H.; Kikuchi, K.; Ohkubo, K.;
Fukuzumi, S.; Nagano, T. J. Am. Chem. Soc. 2004, 126,
14079.
(15) (a) Ullrich, A.; Chai, Y.; Pistorius, D.; Elnakady, Y. A.;
Herrmann, J. E.; Weissman, K. J.; Kazmaier, U.; Müller, R.
Angew. Chem. Int. Ed. 2009, 47, 4422; Angew. Chem. 2009,
121, 4486. (b) Ullrich, A.; Herrmann, J.; Müller, R.;
Kazmaier, U. Eur. J. Org. Chem. 2009, 6367. (c) Chai, Y.;
Pistorius, D.; Ullrich, A.; Weissman, K. J.; Kazmaier, U.;
Müller, R. Chem. Biol. 2010, 17, 296. (d) Burkhart, J. L.;
Müller, R.; Kazmaier, U. Eur. J. Org. Chem. 2011, 3050.
(16) Burkhart, J. L.; Kazmaier, U. RSC Adv. 2012, in press.
(17) (a) v. Pechmann, H. Ber. Dtsch. Chem. Ges. 1884, 17, 929.
(b) Sethna, S. M.; Phadke, R. Org. React. 1953, 7, 1.
(18) Wirtz, L.; Kazmaier, U. Eur. J. Org. Chem. 2011, 7062.
(19) Brooks, D. W.; Lu, L. D. L.; Masamune, S. Angew. Chem.,
Int. Ed. Engl. 1979, 18, 72; Angew. Chem. 1979, 91, 76.
(20) Alper, P. B.; Hung, S.-C.; Wong, C.-H. Tetrahedron Lett.
1996, 37, 6029.
(21) (a) Lundquist, J. T. IV.; Pelletier, J. C. Org. Lett. 2001, 3,
781. (b) Ahsanullah; Schmieder, P.; Kühne, R.; Rademann,
J. Angew. Chem. Int. Ed. 2009, 48, 5042; Angew. Chem.
2009, 121, 5143.
(22) Klose, J.; Bienert, M.; Mollenkopf, C.; Wehle, D.; Zhang,
C.-W.; Carpino, L. A.; Henklein, P. Chem. Commun. 1999,
1847.
(5) Maeda, M. Laser Dyes; Academic Press: New York, 1984.
(6) Yee, D. Y.; Balsanek, V.; Sames, D. J. Am. Chem. Soc. 2004,
126, 2282.
(7) Sletten, E. M.; Bertozzi, C. R. Angew. Chem. Int. Ed. 2009,
48, 6974; Angew. Chem. 2009, 121, 7108.
(8) (a) Bernardes, G. J. L.; Chalker, J. M.; Errey, J. C.; Davis, B.
G. J. Am. Chem. Soc. 2008, 130, 5052. (b) Dondoni, A.
Angew. Chem. Int. Ed. 2008, 47, 8995; Angew. Chem. 2008,
(23) Balog, A.; Curran, D. P. J. Org. Chem. 1995, 60, 337.
Synthesis 2012, 44, 2005–2012
© Georg Thieme Verlag Stuttgart · New York