The fluorescence labelling of primary amines with perylenetetracarboxdiimides

Article abstract of DOI:10.1002/ejoc.200700234

The synthesis of perylenetetracarboxdiimide-labelled aldehydes is described as well as their condensation with primary amines. Thus, a highly fluorescent and light-fast fluorescence labelling process has been demonstrated. Application of this method to bi

Full text of DOI:10.1002/ejoc.200700234

FULL PAPER
DOI: 10.1002/ejoc.200700234
The Fluorescence Labelling of Primary Amines with
Perylenetetracarboxdiimides
Heinz Langhals,*[a] Thomas Becherer,[a] Jörg Lindner,[a] and Andreas Obermeier[a]
Keywords: UV/Vis spectroscopy / Fluorescent probes / Amines / Dyes / Pigments / Schiff bases
The synthesis of perylenetetracarboxdiimide-labelled alde-
hydes is described as well as their condensation with primary
amines. Thus, a highly fluorescent and light-fast fluores-
cence labelling process has been demonstrated. Application
of this method to biological targets is also reported.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2007)
N-(1-hexylheptyl)perylene anhydride imide[17] was success-
ful, however, the acetal 1a was very labile and hydrolysed
during the usual workup to the aldehyde 1c. Thus, the
methyl acetal is a good starting material for a one-step syn-
thesis of the aldehyde 1c (Scheme 1).
Introduction
Fluorescence labelling of biological targets is becoming
more and more important.[1] Many biologically active struc-
tures contain primary amino groups which can be labelled
by condensation reactions. Such primary amines react easily
with aldehydes, particularly aromatic aldehydes, to form
aldimines[2,3] in high yields. Fluorophores with aldehyde an-
chor groups are interesting reagents for such labelling.
We prepared the more stable ethylene acetal 4[16] for
long-term storage of the aldehyde, reduced it analogously
to the amine 6 and condensed it with N-(1-hexylheptyl)per-
ylene anhydride imide to form the acetal 1b. Standard chro-
matographic purification of 1b (silica gel 60; CHCl3/etha-
nol, 40:1) caused hydrolysis to the aldehyde 1c, as was
found for 1a. Even chromatographic separation with basic
alumina caused partial hydrolysis and the formation of the
alcohol 1f. This may be a consequence of the reduction of
1c by the ethanol used for elution or alternatively the basic
alumina may cause a Cannizzaro reaction of 1c. However,
the corresponding carboxylic acid could not be isolated. On
the other hand, chromatographic purification of the raw
material with the deactivated silica Florisil allowed the iso-
lation of the acetal 1b, a material that allows for the long-
term storage of 1c. The aldehyde 1c can be liberated from
1b by simple column chromatographic filtration (silica gel
60; CHCl3/ethanol, 40:1).
Results and Discussion
We started with the highly photostable,[4–7] highly fluo-
rescent[8] and chemically inert[9] perylenetetracarboxdi-
imides 1[10,11] as the fluorophore and attached the long-
chain sec-alkyl substituent 1-hexylheptyl (“swallow-tail”
substituent[12,13]) to one of the nitrogen atoms of 1 (R1) to
render the material soluble. A group containing an aromatic
aldehyde attached to the other nitrogen atom of 1 (R2)
would be a suitable anchor for labelling.
The labelling group in 1c is close to the chromophore
and there may be interference for some substrates by inter-
action with the chromophore. Thus, to avoid this, we sepa-
rated the labelling aldehyde group from the chromophore
by an additional phenyl group to provide a longer spacer
with low flexibility. Therefore, the bromo nitrile 7 was cou-
pled with the boronic aldehyde 8 in a Suzuki reaction[18]
with tetrakis(triphenylphosphane)palladium to form the bi-
phenyl aldehyde 9. The latter was protected as the ethylene
acetal 10, reduced to the amine 11 and condensed with N-
(1-hexylheptyl)perylene anhydride imide to form 1d. The
acetal 1d could be obtained by column chromatography
with the deactivated silica Florisil or alternatively with silica
gel 60 using dichloromethane/methanol (40:1) as eluent,
We started the synthesis with 4-cyanobenzaldehyde (2)
and protected the aldehyde group as the dimethyl acetal
3
[14,15] because a free amino group would cause self-conden-
sation. The cyano group was reduced[16] with lithium alu-
minium hydride to the amine 5. Condensation of 5 with the
[a] Department of Chemistry, LMU University of Munich
Butenandtstr. 13, 81377 Munich, Germany
Fax: +49-89-2180-77640
E-mail: Langhals@lrz.uni-muenchen.de
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
4328
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2007, 4328–4336

Fluorescence Labelling of Primary Amines
FULL PAPER
Scheme 1. Synthesis of the perylene dye 1c.
whereas common silica gel 60, CHCl3/ethanol (40:1) al- densed with 2-aminoacetaldehyde dimethyl acetal and 4-
lowed deprotection to yield the free aldehyde 1e, similarly aminobutyraldehyde diethyl acetal to form 1h and 1i,
to the behaviour of 1b (Scheme 2).
respectively. The lower reactivity of the aliphatic acetals was
Simple aliphatic aldehydes were prepared for compari- indicated by the fact that the dimethyl acetal 1g was ob-
son. The condensation of the readily accessible perylene- tained by standard acidic workup without hydrolysis so that
3,4:9,10-tetracarboxylic 3,4-anhydride monopotassium salt the preparation of the aldehyde by hydrolysis requires an
with 4-aminobutyraldehyde diethyl acetal was successful additional step. The ethyl acetal of 1i proved to be more
with direct hydrolysis of the acetal during workup yielding labile to acid so that hydrolysis proceeded directly to 1i.
the corresponding aldehyde anhydride; however, the con-
The condensation of 1c and 1e with primary aromatic
densation of this aldehyde with branched aliphatic amines amines was first verified with aniline to form the corre-
to form compounds such as 1i proved to be problematic. sponding aldimines 12a and 13a, respectively, in 70% yields.
Thus, a route analogous to that used for the preparation of Remarkably, even aliphatic amines such as butylamine re-
1c was preferred in which the N-(1-hexylheptyl)perylene- acted readily to form aldimines; see for example 12b and
3,4:9,10-tetracarboxylic 3,4-anhydride 9,10-imide was con- 13b, respectively.
Eur. J. Org. Chem. 2007, 4328–4336
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4329

H. Langhals, T. Becherer, J. Lindner, A. Obermeier
FULL PAPER
Scheme 2. Synthesis of the perylene dye 1e.
The aliphatic aldehydes 1h and 1i underwent condensa-
tion with butylamine to form the highly fluorescent, but
readily hydrolysable aldimines in low yields of about 15%
with m/z = 669 and 697, respectively. The condensation of
Moreover, the labelling of peptides with 1c and 1e is pos-
1h with aniline proceeded with an even lower yield of 10% sible, as was shown by the reaction of catalase. N-Methyl-
(m/z = 689) and no aldimine could be detected for the con- pyrrolidone gave the best results and the labelling could be
densation of 1i with aniline by mass spectrometry.
further supported by the addition of dicyclohexylcarbodi-
The procedure for labelling had to be modified for more imide (DCC). Orange-to-dark-red peptides were formed,
hydrophilic biologically important amines such as amino the coloration of which could not be removed by hydrophi-
acids. Ethanol proved to be a sufficiently good solvent for lic or lipophilic solvents such as chloroform. This indicates
both the amino acids and the perylene-labelled aldehydes. a covalent linkage of the dye to the peptide.
Thus, we condensed 4-aminobenzoic acid as the amino acid
All aldimines from 12a to 13d exhibit UV/Vis absorption
with the aldehydes 1c and 1e and obtained the imines 12c and strong fluorescence spectra very similar to the starting
and 13c, respectively. N-Methylpyrrolidone proved to give material; see the comparison of the UV/Vis spectra of 12b
more satisfying results for the even more polar natural α- and a simple aliphatically substituted reference shown in
amino acids; phenylalanine was used as an example to pre- Figure 1. The photostability of the labelled amines is rela-
pare 12d and 13d, respectively.
tively high, as has been found for other perylene dyes. The
4330
www.eurjoc.org
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2007, 4328–4336

Fluorescence Labelling of Primary Amines
FULL PAPER
for 2 h with stirring, cooled, treated with ethanol (50 mL), precipi-
tated with aqueous 2 m HCl (150 mL), collected by vacuum fil-
tration, thoroughly washed with distilled water and dried in air at
110 °C for 16 h to obtain 1a. The splitting to form 1c proceeded
by purification by column chromatography (silica gel; chloroform/
ethanol, 40:1). Compound 1c was obtained after an orange forerun
as an intense red-to-orange band and was dissolved in a small
amount of chloroform and precipitated with acetonitrile. Yield:
785 mg (76%). Rf (silica gel; CHCl3/EtOH, 40:1) = 0.29. IR (ATR):
chemical stability of the labelling linkage is, for example,
high enough for chromatographic separation. The fluores-
cence of the labelled peptide is not as pronounced as that
of labelled amino acids which may be a consequence of the
aggregation of chromophores in the highly loaded macro-
molecular peptide.
ν = 2953.8 (m), 2923.0 (s), 2855.3 (m), 1697.4 (s), 1646.4 (vs),
˜
1610.0 (m), 1593.4 (s), 1577.3 (m), 1506.9 (w), 1436.2 (m), 1403.9
(m), 1378.2 (w), 1336.1 (s), 1301.7 (w), 1249.8 (m), 1212.4 (w),
1199.7 (w), 1168.2 (m), 1125.2 (w), 1106.0 (w), 987.0 (w), 849.6 (w),
823.9 (w), 808.9 (m), 774.3 (w), 742.6 (m), 723.1 (w), 631.4 (w)
1
3
cm–1. H NMR (600 MHz, CDCl3, 25 °C): δ = 0.82 [t, J(H,H) =
6.9 Hz, 6 H, 2ϫCH3], 1.16–1.38 (m, 16 H, 8ϫCH2), 1.82–1.92 (m,
2 H, β-CH2), 2.20–2.30 (m, 2 H, β-CH2), 5.14–5.22 (m, 1 H, α-
3
CH), 5.48 (s, 2 H, NCH2), 7.70 (d, J = 8.2 Hz, 2 H, CHaryl), 7.85
(d, J = 8.3 Hz, 2 H, CHaryl), 8.68 (m, 8 H, CHaryl), 9.98 (s, 1 H,
3
CHO) ppm. 13C NMR (150 MHz, CDCl3, 25 °C): δ = 14.3, 22.8,
27.1, 29.4, 29.9, 32.0, 32.6, 43.8, 55.1, 123.0, 123.2, 123.6, 126.6,
129.6, 129.8, 130.2, 132.1, 135.2, 135.9, 144.0, 163.6, 192.1 ppm.
UV/Vis (CHCl3): λmax (ε) = 459.1 (18600), 491.0 (51400), 527.4
(85800) nm; fluorescence (CHCl3): λmax (Irel) = 534.5 (1.00), 578.0
(0.50) nm; fluorescence quantum yield {CHCl3, λexc = 491 nm,
E491nm = 0.0212 cm–1, reference: 2,9-bis(1-hexylheptyl)anthra[2,1,9-
def:6,5,10-dЈeЈfЈ]diisoquinoline-1,3,8,10-tetraone with Φ = 1.00}: Φ
= 1.00. MS (DEI+, 70 eV): m/z (%) = 690 (33) [M]+, 508 (100) [M –
C13H26]+, 374 (14) [M – C21H35O2]+, 346 (19) [M – C22H34NO2]+,
44 (15) [CH2NO]+. HMRS: calcd. for C45H42N2O5 [M]+ 690.309;
found 690.308. C45H42N2O5 (690.9): calcd. C 78.24, H 6.13, N 4.06;
found C 78.06, H 6.15, N 4.07.
Figure 1. Normalised UV/Vis absorption (left) and fluorescence
(right) spectra of 13a (thick line) and 14 (thin line) in chloroform.
2-[4-(1,3-Dioxolan-2-yl)benzyl]-9-(1-hexylheptyl)anthra[2,1,9-
def:6,5,10-dЈeЈfЈ]diisoquinoline-1,3,8,10-tetraone (1b) and Hydrolysis
To Form 4-[9-(1-Hexylheptyl)-1,3,8,10-tetraoxo-3,8,9,10-tetrahydro-
1H-anthra[2,1,9-def:6,5,10-dЈeЈfЈ]diisoquinolin-2-ylmethyl]benzal-
dehyde (1c) and the Isolation of 2-(1-Hexylheptyl)-9-(4-hydroxy-
methylbenzyl)anthra[2,1,9-def:6,5,10-dЈeЈfЈ]diisoquinoline-1,3,8,10-
tetraone (1f): 9-(1-Hexylheptyl)-2-benzopyrano[6Ј,5Ј,4Ј:10,5,6]an-
thra[2,1,9-def]isoquinoline-1,3,8,10-tetraone (600 mg, 1.05 mmol),
imidazole (14.0 g) and a microspatulum of zinc acetate [Zn(OAc)2·
2H2O] were homogenized and heated under argon (deep-red solu-
tion at 140 °C), treated with 6 (370 mg, 2.06 mmol), stirred for 4 h,
quenched by the addition of ethanol (50 mL), precipitated with 2 n
aqueous HCl, cooled, collected by vacuum filtration, washed thor-
oughly with distilled water, dried in air (110 °C, 16 h, 740 mg,
96%), purified by column chromatography (Florisil; chloroform/
ethanol, 60:1), dissolved in chloroform and precipitated with aceto-
nitrile. Yield: 12 mg of 1b (16%) (alternatively 60% yield of 1b with
silica gel 60; dichloromethane/methanol, 40:1). M.p. Ͼ250 °C. Rf
Conclusions
Aldehydes 1c and 1e are useful reagents for labelling both
lipophilic primary amines and hydrophilic amino acids and
their use can even be extended to the labelling of biological
macromolecules.
Experimental Section
General: IR spectra: Perkin Elmer 1420 Ratio Recording Infrared
Spektrometer, FT 1000. UV/Vis spectra: Varian Cary 5000 and
Bruins Omega 20. Fluorescence spectra: Perkin Elmer FS 3000
(totally corrected). NMR spectra: Varian Vnmrs 600 (600 MHz).
Mass spectra: Finnigan MAT 95. Perylene-3,4:9,10-tetracarboxylic
bis(anhydride) was obtained from CIBA Speciality Chemicals,
other staring materials from Aldrich.
(silica gel; CH Cl ) = 0.23. IR (ATR): ν = 2954.5 (m), 2921.7 (s),
˜
2
2
2854.0 (s), 2360.6 (w), 1683.2 (s), 1648.1 (vs), 1592.5 (s), 1575.8
(m), 1506.3 (w), 1465.9 (w), 1436.4 (w), 1402.4 (m), 1338.8 (s),
1306.4 (w), 1249.5 (m), 1203.6 (w), 1173.8 (m), 1126.3 (w), 1109.8
(w), 1083.9 (m), 1019.9 (w), 981.7 (w), 944.6 (w), 852.0 (w), 809.9
2-[4-(Dimethoxymethyl)benzyl]-9-(1-hexylheptyl)anthra[2,1,9-
def:6,5,10-dЈeЈfЈ]diisoquinoline-1,3,8,10-tetraone (1a) and Splitting
(s), 780.0 (w), 744.7 (m), 666.7 (w), 647.5 (w) cm–1 1H NMR
.
of the Acetal To Form 4-[9-(1-Hexylheptyl)-1,3,8,10-tetraoxo- (600 MHz, CDCl3, 25 °C): δ = 0.82 [t, 3J(H,H) = 6.9 Hz, 6 H,
3
3,8,9,10-tetrahydro-1H-anthra[2,1,9-def:6,5,10-dЈeЈfЈ]diisoquinolin-
2-ylmethyl]benzaldehyde (1c): 9-(1-Hexylheptyl)-2-benzopyr-
ano[6Ј,5Ј,4Ј:10,5,6]anthra[2,1,9-def]isoquinoline-1,3,8,10-tetraone
(860 mg, 1.50 mmol), imidazole (17.0 g) and a microspatulum of
zinc acetate [Zn(OAc)2·2H2O] were homogenized, heated under ar-
gon at 140 °C, treated with 5 (460 mg, 2.55 mmol), heated at 140 °C
2ϫCH3], 0.82 [t, J(H,H) = 7.0 Hz, 6 H, 2ϫCH3], 1.18–1.38 (m,
16 H, 8ϫCH2), 1.54 (s, 1 H, OH), 1.83–1.91 (m, 2 H, β-CH2),
2.21–2.29 (m, 2 H, β-CH2), 4.66 [d, 3J(H,H) = 3.9 Hz, 2 H,
CH2OH], 5.15–5.22 (m, 1 H, α-CH), 5.40 (s, 2 H, NCH2), 7.34 [d,
3J(H,H) = 8.1 Hz, 2 H, CHaryl], 7.58 [d, 3J(H,H) = 8.2 Hz, 2 H,
CHaryl], 8.57–8.72 (m, 8 H, CHaryl) ppm. 13C NMR (150 MHz,
Eur. J. Org. Chem. 2007, 4328–4336
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4331

H. Langhals, T. Becherer, J. Lindner, A. Obermeier
FULL PAPER
CDCl3, 25 °C): δ = 14.3, 22.8, 27.1, 29.4, 32.0, 32.6, 43.7, 55.0,
65.5, 103.7, 123.2, 123.3, 123.4, 126.6, 126.8, 129.4, 129.7, 129.8,
[C2H4O]+. HMRS: calcd. for C53H50N2O6 [M]+ 810.367; found
810.368.
131.9, 135.2, 137.6, 138.2, 163.6 ppm. UV/Vis (CHCl3): λmax (Erel.
)
4Ј-[9-(1-Hexylheptyl)-1,3,8,10-tetraoxo-3,8,9,10-tetrahydro-1H-
anthra[2,1,9-def:6,5,10-dЈeЈfЈ]diisoquinolin-2-ylmethyl]biphenyl-4-carb-
aldehyde (1e): Column separation (silica gel; chloroform/ethanol,
40:1) of 1d (300 mg, 0.370 mmol) gave an orange forerun. The in-
tensely reddish-orange main fraction was concentrated, dissolved
in the minimal amount of chloroform and precipitated with aceto-
nitrile. Yield: 248 mg (0.323 mol, 87%). Bright light-red solid, m.p.
= 459.4 (0.22), 490.4 (0.60), 527.2 (1.00) nm; fluorescence (CHCl3):
λmax (Irel.) = 534.5 (1.00), 576.0 (0.51) nm; fluorescence quantum
yield {CHCl3, λexc = 490 nm, E490nm = 0.0137 cm–1, reference: 2,9-
bis(1-hexylheptyl)anthra[2,1,9-def:6,5,10-dЈeЈfЈ]diisoquinoline-
1,3,8,10-tetraone with Φ = 1.00}: Φ = 1.00. HMRS: calcd. for
C47H46N2O6 [M]+ 734.336; found 734.337. Column separation with
silica gel (chloroform/ethanol, 40:1) results directly in the splitting
of the acetal to form 1c as an intense reddish-orange band after
having removed a forerun and was dissolved in chloroform and
precipitated with acetonitrile. Yield: 470 mg (74%) of 1c; for spec-
troscopic data see below. Column separation of the crude acetal
with neutral alumina (chloroform/ethanol, 40:1) gave a weakly
orange forerun and the main fraction. Further column chromatog-
raphy of the latter with silica gel (chloroform/ethanol, 60:1) allowed
pure aldehyde 1c to be obtained and then 1f. Yield: 25 mg. Red
powder, m.p. Ͼ250 °C. Rf (silica gel; CHCl3/ethanol, 40:1) = 0.15.
Ͼ250 °C. R (silica gel; CHCl /EtOH, 40:1) = 0.28. IR (ATR): ν =
˜
f
3
2952.5 (m), 2924.0 (s), 2854.9 (m), 1691.9 (s), 1650.2 (vs), 1592.6
(s), 1577.7 (m), 1506.5 (w), 1456.1 (w), 1434.7 (w), 1403.6 (m),
1378.0 (w), 1332.6 (s), 1247.0 (m), 1214.8 (w), 1169.3 (m), 1125.8
(w), 1106.2 (w), 1003.6 (w), 987.8 (w), 849.6 (w), 808.2 (m), 782.0
(w), 748.8 (w), 740.3 (w), 606.5 (w) cm–1. 1H NMR (600 MHz,
CDCl3, 25 °C, TMS): δ = 0.82 [t, 3J(H,H) = 7.0 Hz, 6 H, 2ϫCH3],
1.18–1.38 (m, 16 H, 8ϫCH2), 1.83–1.91 (m, 2 H, β-CH2), 2.21–
2.29 (m, 2 H, β-CH2), 5.15–5.22 (m, 1 H, α-CH), 5.46 (s, 2 H,
NCH2), 7.58–7.61 (m, 2 H, CHaryl), 7.67–7.72 (m, 4 H, CHaryl),
7.91–7.94 (m, 2 H, CHaryl), 8.59–8.71 (m, 8 H, CHperylene), 10.0 (s,
1 H, CHO) ppm. 13C NMR (150 MHz, CDCl3, 25 °C, TMS): δ =
14.3, 22.8, 27.2, 29.4, 32.0, 32.6, 43.7, 55.1, 123.2, 123.5, 126.6,
126.8, 127.7, 127.8, 129.7, 129.8, 129.9, 130.5, 132.0, 135.3, 135.4,
IR (ATR): ν = 3500.0 (br, w), 2953.8 (w), 2923.6 (m), 2855.6 (w),
˜
2360.4 (m), 2340.5 (m), 1693.4 (s), 1649.9 (vs), 1593.8 (s), 1576.1
(m), 1507.2 (w), 1437.4 (w), 1403.8 (m), 1344.3 (s), 1250.2 (m),
1173.3 (m), 1128.9 (w), 1018.0 (m), 852.2 (w), 824.5 (w), 809.9 (s),
784.1 (w), 753.1 (m), 667.9 (w), 645.9 (w), 629.4 (w) cm–1. 1H NMR
137.7, 139.3, 147.0, 163.7, 192.1 ppm. UV/Vis (CHCl3): λmax (Erel.
)
3
(600 MHz, CDCl3, 25 °C): δ = 0.82 (t, J = 6.5 Hz, 6 H, 2ϫCH3),
= 459.2 (0.22), 490.4 (0.60), 527.0 nm (1.00); fluorescence (CHCl3):
λmax (Irel.) = 535.2 (1.00), 576.5 nm (0.50); fluorescence quantum
yield {CHCl3, λexc = 490 nm, E490nm = 0.0132 cm–1, reference: 2,9-
bis(1-hexylheptyl)anthra[2,1,9-def:6,5,10-dЈeЈfЈ]diisoquinoline-
1,3,8,10-tetraone with Φ = 1.00}: Φ = 1.00. MS (DEI+, 70 eV):
m/z (%) = 766 (21) [M]+, 584 (100) [M – C13H26]+, 346 (55) [M –
C28H38NO2]+, 195 (14) [C14H11O]+. HMRS: calcd. for C53H50N2O6
[M]+ 766.340; found 766.339.
1.28 (m, 16 H, CH2), 1.57 (s, 1 H, OH), 1.87 (m, 2 H, α-CH2), 2.23
(m, 2 H, α-CH2), 4.66 (s, 2 H, CH2OH), 5.18 (m, 1 H, α-CH), 5.40
3
(s, 2 H, NCH2), 7.34 (d, J = 8.1 Hz, 2 H, CHaryl), 7.58 (d, 3J =
8.1 Hz, 2 H, CHaryl), 8.68 (m, 8 H, CHaryl) ppm. 13C NMR
(150 MHz, CDCl3, 25 °C): δ = 14.3, 22.8, 27.2, 29.4, 32.0, 32.6,
43.7, 55.0, 65.4, 123.2, 123.3, 123.4, 126.6, 127.4, 129.6, 129.7,
131.9, 135.2, 136.8, 140.5, 163.6 ppm. HMRS: calcd. for
C45H44N2O5 [M]+ 692.325; found 692.323.
2-(2,2-Dimethoxyethyl)-9-(1-hexylheptyl)anthra[2,1,9-def:6,5,10-
dЈeЈfЈ]diisoquinoline-1,3,8,10-tetraone (1g): 9-(1-Hexylheptyl)-2-
benzopyrano[6Ј,5Ј,4Ј:10,5,6]anthra[2,1,9-def]isoquinoline-1,3,8,10-
tetraone (3.00 g, 5.23 mmol), aminoacetaldehyde dimethyl acetal
(820 mg, 7.79 mmol) and imidazole (6 g) were allowed to react as
was described for 1c (yield: 3.3 g, 96%) and purified by column
chromatography (silica gel; CHCl3/acetic acid, 10:1). Yield: 2.96 g
(86%). M.p. 274 °C. Rf (silica gel, CHCl3) = 0.1. Rf (silica gel;
CHCl3/acetic acid, 10:1) = 0.87; Rf (alumina; CHCl3) = 0.52. IR
2-[4Ј-(1,3-Dioxolan-2-yl)biphenyl-4-ylmethyl]-9-(1-hexylheptyl)-
anthra[2,1,9-def:6,5,10-dЈeЈfЈ]diisoquinoline-1,3,8,10-tetraone (1d):
9-(1-Hexylheptyl)-2-benzopyrano[6Ј,5Ј,4Ј:10,5,6]anthra[2,1,9-def]-
isoquinoline-1,3,8,10-tetraone (400 mg, 0.697 mmol), 11 (350 mg,
1.37 mmol), imidazole (10.0 g) and a microspatulum of zinc acetate
[Zn(OAc)2·2H2O] under argon were allowed to react as was de-
scribed for 1a. Yield: 550 mg (0.599 mmol, 86%). Bright light-red
solid, m.p. Ͼ250 °C. Rf (silica gel; CHCl3/ethanol, 40:1) = 0.27. IR
(ATR): ν = 2956.4 (m), 2923.8 (s), 2855.8 (m), 1692.2 (s), 1653.5
˜
(KBr): ν = 3444 (s), 2965 (m), 2929 (m), 2857 (m), 1734 (w), 1700
˜
(vs), 1594.0 (s), 1578.2 (m), 1505.3 (w), 1457.1 (w), 1434.7 (w),
1403.9 (w), 1378.4 (w), 1351.8 (m), 1330.5 (s), 1246.7 (m), 1215.6
(w), 1170.5 (w), 1124.5 (w), 1107.6 (w), 1071.2 (w), 1026.0 (w),
1006.3 (w), 977.1 (w), 855.5 (w), 809.7 (m), 743.5 (m), 633.6 (w)
(s), 1660 (s), 1636 (s), 1595 (s), 1580 (m), 1457 (m), 1437 (m), 1405
(s), 1345 (s), 1255 (m), 1124 (m), 1066 (m), 1050 (m), 860 (w), 810
(s), 746 (s) cm–1. UV (CHCl3): λmax (ε) = 527 (84700), 489 (50800),
458 (19400) nm; fluorescence (CHCl3): λmax = 532, 570 nm. 1H
NMR (CDCl3): δ = 0.81 (t, 6 H,2 CH3), 1.28 (mc, 16 H,8 CH2),
1.88 (mc, 2 H, 1 α-CH2), 2.21 (mc, 2 H, 1 α-CH2), 3.46 (s, 3 H,1O
CH3), 3.49 (s, 3 H, 1O CH3), 4.36 (mc, 2 H,1 CH2), 4.94 (t, 1 H, 1
CH), 5.20 (m, 1 H, CH), 8.40 (mc, 8 H, aromatic) ppm. 13C NMR
(CDCl3): δ = 14.02, 22.59, 27.00, 29.23, 31.77, 32.38, 40.42, 53.29,
54.87, 100.35, 122.54, 122.68, 125.86, 129.01, 131.04, 133.73,
134.21, 163.00 ppm. MS (70 eV): m/z (%) = 662 (1), 661 (5), 660
(10) [M]+, 629 (6) [M – OCH3]+, 572 (6) [M – OCH3 – C3H5O]+,
cm–1 1H NMR (600 MHz, CDCl3, 25 °C, TMS): δ = 0.82 [t,
.
3J(H,H) = 7.0 Hz, 6 H, 2ϫCH3], 1.18–1.38 (m, 16 H, 8ϫCH2),
1.83–1.91 (m, 2 H, β-CH2), 2.21–2.29 (m, 2 H, β-CH2), 4.02–4.16
(m, 4 H, 2 ϫ CH2O), 5.15–5.22 (m, 1 H, α-CH), 5.45 (s, 2 H,
NCH2), 5.84 (s, 1 H, CHO2), 7.49–7.73 (m, 8 H, CHaryl), 8.58–
8.72 (m, 8 H, CHperylene) ppm. 13C NMR (150 MHz, CDCl3, 25 °C,
TMS): δ = 14.3, 22.8, 27.2, 29.4, 32.0, 32.6, 43.7, 55.1, 63.9, 123.2,
123.5, 126.6, 126.8, 127.1, 127.3, 127.5, 129.8, 129.9, 130.5, 132.0,
135.3, 135.4, 137.7, 139.3, 147.0, 163.7 ppm. UV/Vis (CHCl3): λmax
(Erel) = 459.6 (0.22), 490.8 (0.60), 527.6 (1.00); fluorescence
(CHCl3): λmax (Irel) = 535.5 (1.00), 578.5 (0.52), 628.5 nm (0.12);
479 (4), 447 (5), 404 (7), 403 (5), 391 (5), 390 (9), [M – OCH3
–
C3H5O – C13H26]+, 373 (4), 76 (3), 75 (100), 55 (2), 31 (4), 28 (1).
C41H44N2O6 (660.4): calcd. C 74.55, H 6.66, N 4.24; found C 74.39,
H 6.61, N 4.43.
fluorescence quantum yield {CHCl3, λexc = 490 nm, E490nm
=
0.0302 cm–1, reference: 2,9-bis(1-hexylheptyl)anthra[2,1,9-
def:6,5,10-dЈeЈfЈ]diisoquinoline-1,3,8,10-tetraone with Φ = 1.00}: Φ
[9-(1-Hexylheptyl)-1,3,8,10-tetraoxo-3,8,9,10-tetrahydro-1H-anthra-
= 1.00. MS (DEI+, 70 eV): m/z (%) = 810 (45) [M]+, 628 (62) [M – [2,1,9-def:6,5,10-dЈeЈfЈ]diisoquinolin-2-yl]acetaldehyde (1h): 1g
C13H26]+, 585 (78) [M – C15H13O2]+, 556 (100) [M – C16H30O2]+,
346 (57) [M – C30H42NO3]+, 167 (34) [M – C40H39N2O6]+, 44 (39) with 6 n HCl (20 mL), stirred at 40 °C for 4 h, cooled at room
(1.50 g, 2.27 mmol) was dispersed in 1,4-dioxane (50 mL), treated
4332
www.eurjoc.org
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2007, 4328–4336

Fluorescence Labelling of Primary Amines
FULL PAPER
temperature, diluted with distilled water (200 mL), collected by vac-
uum filtration (D4 glass filter), thoroughly washed with distilled
water, dried in vacuo at 30 °C for 5 h, purified by column
chromatography (silica gel; CHCl3/acetic acid, 10:1) (removing a
small forerun), concentrated in vacuo, precipitated with distilled
water, collected by vacuum filtration, thoroughly washed until neu-
tral and dried in vacuo at 40 °C for 5 h. Yield: 1.14 g (82%). M.p.
244 °C. Rf (silica gel; CHCl3) = 0.08; Rf (silica gel; CHCl3/acetic
uum filtration, washed with 8% KCl and 2% K2CO3 until colour-
less washings, dissolved in hot distilled water (500 mL), filtered
though a D4 glass filter to remove solids, precipitated by acidifying
with concd. HCl, collected by vacuum filtration (D4 glass filter),
thoroughly washed with distilled water, dried in air at 100 °C for
8 h (yield: 3.8 g, 82%), and extractively recrystallised[20] from chlo-
roform. Yield: 3.0 g (65%). Rf (silica gel; CHCl3/acetic acid, 10:1)
= 0.61. IR (KBr): ν = 3450 (w), 3090 (w), 2962 (w), 2915 (w), 2890
˜
acid, 10:1) = 0.8. IR (KBr): ν = 3445 (m), 3095 (w), 2955 (m), 2928 (w), 1796 (s), 1734 (s), 1698 (s), 1658 (s), 1616 (w), 1594 (s), 1580
˜
(s), 2856 (m), 1735 (m), 1698 (s), 1658 (s), 1615 (w), 1595 (s), 1579
(m), 1508 (w), 1457 (m), 1437 (m), 1405 (s), 1375 (m), 1345 (s),
(m), 1505 (w), 1455 (w), 1435 (w), 1405 (m), 1355 (w), 1321 (m),
1298 (m), 1271 (w), 1243 (w), 1151 (w), 1125 (m), 1069 (w), 1044
1302 (w), 1252 (m), 1174 (m), 1130 (w), 1107 (w), 1085 (w), 1055 (w), 1025 (m), 855 (w), 810 (s), 739 (m) cm–1. UV (CHCl3): λmax
(w), 1035 (w), 958 (w), 854 (m), 810 (s), 747 (m) cm–1. 1H NMR (ε) = 526 (80200), 489 (50400), 459 (18900) nm. MS (70 eV): m/z
(CDCl3): δ = 0.85 (t, 6 H, 2 CH3), 1.28 (mc, 16 H, 8 CH2), 1.92
(mc, 2 H, 1 α-CH2), 2.24 (mc, 2 H, 1 α-CH2), 5.05 (s, 2 H, 1 CH2), 413 (16), 404 (10), 392 (22), 391 (38) [M – C4H6O]+, 374 (6), 345
5.19 (mc, 1 H, CH), 8.25 (mc, 8 H, perylene), 9.79 (s, 1 H, aldehyde) (12), 341 (7), 333 (6), 320 (5), 319 (14), 274 (8), 248 (17), 124 (13),
ppm. 13C NMR (CDCl3): δ = 14.03, 22.59, 27.00, 29.23, 31.77, 64 (9), 44 (100), 36 (16), 28 (27). C28H15NO6 (461.4): calcd. C
(%) = 462 (2), 461 (6) [M]+, 433 (36) [M – CO]+, 417 (7), 416 (23),
37.37, 54.93, (C-1 1-hexylheptyl), 49.61 (CH2), 122.06, 122.48,
123.00, 125.75, 125.86, 129.01, 129.12, 131.19, 133.46, 134.54, (C-
10 perylene), 162.47 (C=O), 194.03 (aldehyde) ppm. UV (CHCl3):
λmax (ε) = 527.3 (91500), 490.1 (58200), 458.7 (26900) nm; fluores-
cence (CHCl3): λmax = 535, 574 nm. MS (70eV): m/z (%) = 616 (5),
615 (22), 614 (52) [M]+, 597 (8), 4451 (5), 434 (18), 433 (53), 432
(50), 415 (6), 406 (7), 405 (35), 404 (100), 403 (9), 390 (7), 376 (8),
359 (8), 345 (5), 125 (3), 111 (6), 97 (9), 73 (10), 57 (14), 41 (9), 28
(7).
72.88, H 3.28, N 3.04; found C 72.10, H 3.32, N 2.87.
2-(1-Hexylheptyl)-9-[4-(phenyliminomethyl)benzyl]anthra[2,1,9-
def:6,5,10-dЈeЈfЈ]diisoquinoline-1,3,8,10-tetraone (12a). Method 1: 1c
(35.0 mg, 50.7 μmol) was dissolved in chloroform (3 mL), treated
with MgSO4 (300 mg) and dropwise with freshly distilled aniline
(0.462 mL, deep-violet solution), heated at 60 °C for 3 h, stirred at
room temperature for 12 h, separated from the solid by filtration
(washed with chloroform until colourless), precipitated with meth-
anol, collected by vacuum filtration and dried in air at 110 °C for
16 h. Yield: 25.0 mg (32.6 μmol, 64%). Method 2: 1c (45.0 mg,
4-[9-(1-Hexylheptyl)-1,3,8,10-tetraoxo-3,8,9,10-tetrahydro-1H-
anthra[2,1,9-def:6,5,10-dЈeЈfЈ]diisoquinolin-2-yl]butyraldehyde (1i): 65.1 μmol) was dissolved in chloroform (5 mL), treated with molec-
9-(1-Hexylheptyl)-2-benzopyrano[6Ј,5Ј,4Ј:10,5,6]anthra[2,1,9-def]-
isoquinoline-1,3,8,10-tetraone (3.30 g, 5.75 mmol), 4-aminobutyr-
aldehyde diethyl acetal (1.85 g, 11.5 mmol) and imidazole (5 g)
were allowed to react as was described for 1c (400 mL of ethanol
and 400 mL of 2 n HCl, yield: 3.45 g, 93%) and purified by column
chromatography (silica gel; CHCl3/acetic acid, 10:1). Yield: 2.72 g
(74%). M.p. 257 °C, Rf (silica gel; CHCl3/acetic acid, 10:1) = 0.53;
ular sieves (4 Å; 1.00 g) and with freshly distilled aniline (0.593 mL,
65.1 μmol), stirred at room temperature for 12 h, separated from
the solid by filtration, concentrated in vacuo, dissolved in a small
amount of chloroform and precipitated with methanol. Yield:
38.0 mg (49.6 μmol, 76%). Method 3: 1c (17.0 mg, 24.6 μmol) was
dissolved in freshly distilled aniline (3 mL), treated with MgSO4
(300 mg), stirred at room temperature for 12 h, separated from the
solid by filtration and precipitated with methanol. Yield: 16.0 mg
(23.2 μmol, 94%). Red solid, m.p. Ͼ250 °C. Rf (silica gel; CHCl3/
R (silica gel; CHCl ) = 0.01. IR (KBr): ν = 2955 (m), 2928 (m),
˜
f
3
2855 (m), 1697 (s), 1658 (s), 1616 (w), 1595 (s), 1579 (m), 1506 (w),
1457 (w), 1440 (m), 1405 (s), 1343 (s), 1287 (w), 1249 (m), 1171
(w), 1135 (w), 1110 (w), 1060 (w), 850 (w), 810 (s), 745 (s) cm–1.
ethanol, 40:1) = 0.22. IR (ATR): ν = 2954.1 (m), 2923.6 (s), 2855.4
˜
(m), 1694.4 (s), 1648.5 (vs), 1592.5 (s), 1577.1 (m), 1506.6 (w),
1H NMR (CDCl3): δ = 0.9 (t, 6 H, 2 CH3), 1.25 (mc, 16 H,8 CH2), 1484.0 (w), 1434.8 (m), 1402.9 (m), 1335.3 (s), 1301.6 (w), 1248.8
1.95 (mc, 2 H, α-CH2), 2.10 (mc, 2 H, CH2), 2.22 (mc, 2 H, α-CH2), (m), 1170.2 (m), 1124.1 (w), 1105.6 (w), 980.4 (w), 912.1 (w), 846.5
2.6 (mc, 2 H, CH2), 4.18 (mc, 2 H, CH2), 5.18 (mc,1 H, CH), 8.2 (w), 808.5 (m), 794.5 (w), 780.2 (w), 759.1 (w), 743.1 (m), 693.5
(mc, 8 H, aromatic), 9.8 (t, 1 H, CHO) ppm. 13C NMR (CDCl3):
δ = 14.03, 22.58, 26.64, 29.22, 30.86, 31.77, 32.36, 39.65, 41.38,
(w), 644.5 (w), 620.2 (w) cm–1. 1H NMR (600 MHz, CDCl3, 25 °C):
3
δ = 0.83 [t, J(H,H) = 7.0 Hz, 6 H, 2ϫCH3], 1.18–1.39 (m, 16 H,
54.88, 122.48, 122.56, 122.60, 122.76, 129.15, 130.61, 130.90, 8ϫCH2), 1.85–1.92 (m, 2 H, β-CH2), 2.20–2.30 (m, 2 H, β-CH2),
131.34, 133.50, 133.60, 133.87, 134.18, 162.91, 162.99, 201.23 ppm. 5.16–5.22 (m, 1 H, α-CH), 5.44 (s, 2 H, NCH2), 7.15–7.23 (m, 3 H,
UV (CHCl3): λmax (ε) = 526 (85900), 489 (51800), 458 (19200) nm; CHaryl), 7.34–7.39 (m, 2 H, CHaryl), 7.64–7.68 (m, 2 H, CHaryl),
fluorescence (CHCl3): λmax = 532, 572 nm. MS (70 eV): m/z (%) = 7.85–7.88 (m, 2 H, CHaryl), 8.41 (s, 1 H, CHN), 8.49–8.69 (m, 8 H,
644 (0.6), 643 (2), 642 (6) [M]+, 614 (5), 588 (3), 587 (13), 586 (30), CHaryl) ppm. 13C NMR (150 MHz, CDCl3, 25 °C, TMS): δ = 14.3,
569 (4), 461 (4) [M – C13H26]+, 433 (5), 432 (12), 415 (4), 406 (14),
405 (53), 404 (100) [M – C13H26 – C3H4O]+, 390 (10) [M – C13H26
22.8, 27.2, 29.5, 32.0, 43.8, 55.1, 121.1, 123.1, 123.2, 123.4, 126.1,
126.5, 126.6, 129.2, 129.3, 129.6, 129.7, 131.9, 134.4, 135.1, 135.8,
–
C3H4O – CH2]+, 359 (6), 69 (5), 55 (8), 44 (12), 41 (7), 28 (4). 140.6, 152.3, 160.1, 163.5 ppm. UV/Vis (CHCl3): λmax (Erel.) =
C41H42N2O5 (642.4): calcd. C 76.64, H 6.53, N 4.34; found C 76.75,
H 6.36, N 4.37.
459.6 (0.22), 490.8 (0.60), 527.6 (1.00) nm; fluorescence (CHCl3):
λmax (Irel.) = 534.0 (1.00), 577.0 (0.50) nm; fluorescence quantum
yield {CHCl3, λexc = 490 nm, E490nm = 0.0157 cm–1, reference: 2,9-
bis(1-hexylheptyl)anthra[2,1,9-def:6,5,10-dЈeЈfЈ]diisoquinoline-
1,3,8,10-tetraone with Φ = 1.00}: Φ = 1.00. MS (DEI+, 70 eV):
m/z (%) = 765 (20) [M]+, 583 (100) [M – C13H26]+, 346 (11) [M –
C28H39N2O]+. HMRS: calcd. for C51H47N3O4 [M]+ 765.357; found
765.358.
4-(1,3,8,10-Tetraoxo-2-benzopyrano[6Ј,5Ј,4Ј:10,5,6]anthra[2,1,9-
def]isoquinolin-9-yl)butyraldehyde: 1,3-Dioxo-1H,3H-perylo-
[3,4-cd]pyran-8,9-dicarboxylic acid, monopotassium salt[19] (4.50 g,
10.0 mmol) was added with stirring and cooling (0 °C) to a solution
of 4-aminobutyraldehyde diethyl acetal in distilled water (50 mL),
stirred at room temperature for 2 h, then at 90 °C for 2 h, quenched
by the addition of 2 n HCl (50 mL), allowed to stand for 1 h, col-
lected by vacuum filtration, thoroughly washed with distilled water,
treated with 10% KOH (50 mL) at 90 °C for 1 h, collected by vac-
2-(1-Hexylheptyl)-9-{4-[4-(phenyliminomethyl)phenyl]benzyl}-
anthra[2,1,9-def:6,5,10-dЈeЈfЈ]diisoquinoline-1,3,8,10-tetraone (13a):
1e (28.0 mg, 36.5 μmol), freshly distilled aniline (0.330 mL,
Eur. J. Org. Chem. 2007, 4328–4336
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4333

H. Langhals, T. Becherer, J. Lindner, A. Obermeier
FULL PAPER
36.5 μmol), chloroform (5 mL) and MgSO4 (400 mg) were allowed
to react analogously to the procedure used to prepare 12a (5 h,
60 °C). Yield: 22.0 mg (72%). Red solid, m.p. Ͼ250 °C. Rf (silica
ture, precipitation with acetonitrile). Yield: 17.0 mg (20.7 μmol,
59%). Dark-red solid, m.p. Ͼ250 °C. Rf (silica gel; CHCl3/EtOH,
40:1) = 0.23. IR (ATR): ν = 2953.3 (m), 2924.2 (s), 2854.9 (m),
˜
gel; CHCl /EtOH, 40:1) = 0.23. IR (ATR): ν = 2953.8 (m), 2922.9
1694.4 (s), 1653.1 (vs), 1592.7 (s), 1577.7 (m), 1496.6 (w), 1456.9
(w), 1434.6 (m), 1403.4 (m), 1377.8 (w), 1332.4 (vs), 1248.4 (m),
1216.3 (w), 1170.2 (m), 1124.2 (w), 1106.1 (w), 1004.0 (w), 982.9
(w), 851.3 (w), 808.1 (m), 782.4 (w), 747.7 (w), 665.7 (w), 636.1 (w)
˜
3
(s), 2854.8 (m), 1695.4 (s), 1655.6 (vs), 1593.7 (s), 1577.5 (m),
1554.6 (w), 1496.0 (w), 1483 (w), 1435.7 (m), 1379.2 (w), 1336.3 (s),
1249.9 (m), 1171.1 (m), 1123.5 (w), 1107.9 (w), 981.8 (w), 910.1
(w), 849.9 (w), 838.2 (w), 808.1 (m), 782.5 (w), 768.2 (w), 747.7
cm–1
.
1H NMR (600 MHz, CDCl3, 25 °C, TMS): δ = 0.82 [t,
(m), 723.5 (w), 693.4 (w), 634.2 (w), 588.7 (w) cm–1
.
1H NMR 3J(H,H) = 7.0 Hz, 6 H, 2ϫCH3], 0.94 [t, J(H,H) = 7.4 Hz, 3 H,
3
3
(600 MHz, CDCl3, 25 °C, TMS): δ = 0.82 [t, J(H,H) = 7.0 Hz, 6
H, 2ϫCH3], 1.18–1.38 (m, 16 H, 8ϫCH2), 1.83–1.91 (m, 2 H, β-
CH2), 2.20–2.29 (m, 2 H, β-CH2), 5.17–5.22 (m, 1 H, α-CH), 5.47
(s, 2 H, NCH2), 7.20–7.25 (m, 3 H, CHaryl), 7.37–7.41 (m, 2 H,
CH3,butyl], 1.18–1.43 (m, 18 H, 9 ϫ CH2), 1.65–1.71 (m, 2 H,
CH2,butyl), 1.83–1.91 (m, 2 H, β-CH2), 2.21–2.29 (m, 2 H, β-CH2),
3
3.62 [t, J(H,H) = 6.8 Hz, 2 H, NCH2,butyl], 5.15–5.22 (m, 1 H, α-
CH), 5.45 (s, 2 H, NCH2), 7.56–7.61 (m, 4 H, CHaryl), 7.62–7.68
CHaryl), 7.59–7.63 (m, 2 H, CHaryl), 7.65–7.73 (m, 4 H, CHaryl), (m, 2 H, CHaryl), 7.74–7.77 (m, 2 H, CHaryl), 8.28 (s, 1 H, CHN),
7.91–7.96 (m, 2 H, CHaryl), 8.47 (s, 1 H, CHN), 8.60–8.73 (m, 8 H,
8.59–8.72 (m, 8 H, CHperylene) ppm. 13C NMR (150 MHz, CDCl3,
CHperylene) ppm. 13C NMR (150 MHz, CDCl3, 25 °C, TMS): δ = 25 °C, TMS): δ = 14.1, 14.3, 20.7, 22.8, 27.2, 29.4, 32.0, 32.6, 33.2,
14.3, 22.8, 27.2, 29.4, 32.0, 32.6, 43.7, 55.0, 121.1, 123.2, 123.3,
123.5, 126.2, 126.6, 127.5, 127.6, 129.4, 129.5, 129.8, 129.9, 132.0,
134.5, 135.2, 135.4, 137.1, 139.9, 143.9, 152.3, 160.1, 163.7 ppm.
UV/Vis (CHCl3): λmax (Erel.) = 459.6 (0.21), 490.8 (0.59), 527.6
(1.00) nm; fluorescence (CHCl3): λmax (Irel.) = 536.0 (1.00), 578.8
(0.38), 629.0 (0.12) nm; fluorescence quantum yield {CHCl3, λexc
43.7, 55.0, 61.8, 123.2, 123.3, 123.5, 126.6, 126.8, 127.4, 127.5,
128.7, 129.7, 129.8, 131.9, 135.2, 135.6, 136.8, 140.1, 143.0, 160.5,
163.7 ppm. UV/Vis (CHCl3): λmax (Erel.) = 459.8 (0.22), 490.8
(0.60), 527.6 (1.00) nm; fluorescence (CHCl3): λmax (Irel.) = 534.5
(1.00), 576.0 (0.50) nm; fluorescence quantum yield {CHCl3, λexc
= 490 nm, E490nm = 0.0167 cm–1, reference: 2,9-bis(1-hexylheptyl)-
= 490 nm, E490nm = 0.0198 cm–1, reference: 2,9-bis(1-hexylheptyl)- anthra[2,1,9-def:6,5,10-dЈeЈfЈ]diisoquinoline-1,3,8,10-tetraone with
anthra[2,1,9-def:6,5,10-dЈeЈfЈ]diisoquinoline-1,3,8,10-tetraone with
Φ = 1.00}: Φ = 1.00. MS (DEI+, 70 eV): m/z (%) = 841 (34) [M]+,
659 (100) [M – C13H26]+, 346 (49) [M – C34H43N2O]+, 104 (25)
[C7H6N]+. HMRS: calcd. for C57H51N3O4 [M]+ 841.388; found
841.387.
Φ = 1.00}: Φ = 1.00. MS (DEI+, 70 eV): m/z (%) = 821 (22) [M]+,
640 (38) [M – C13H26]+, 374 (28) [M – C31H47N2]+, 346 (60) [M –
C32H47N2O]+, 206 (100) [M – C40H43N2O4]+, 167 (49) [C13H11]+,
84 (70) [C5H10N]+. HMRS: calcd. for C55H55N3O4 [M]+ 821.419;
found 821.418.
2-[4-(Butyliminomethyl)benzyl}-9-(1-hexylheptyl)anthra[2,1,9- 4-[(1-{4-[9-(1-Hexylheptyl)-1,3,8,10-tetraoxo-3,8,9,10-tetrahydro-
def:6,5,10-dЈeЈfЈ]diisoquinoline-1,3,8,10-tetraone (12b): 1c (30.0 mg, 1H-anthra[2,1,9-def:6,5,10-dЈeЈfЈ]diisoquinolin-2-ylmethyl]phenyl}-
43.4 μmol), 1-butylamine (0.430 mL, 43.4 μmol), chloroform
(5 mL) and MgSO4 (400 mg) were allowed to react analogously to
the procedure used to prepare 12a (4 h, 50 °C). Yield: 14.0 mg
(43%). Red solid, m.p. 245 °C. Rf (silica gel; CHCl3/EtOH, 40:1) =
methylidene)amino]benzoic Acid (12c): 1c (25.0 mg, 36.2 μmol) was
dissolved in a mixture of chloroform/ethanol (10:4, 5 mL), treated
with MgSO4 (400 mg) and with 4-aminobenzoic acid (20.0 mg,
146 μmol), heated at 70 °C for 1 h, stirred at room temperature for
12 h, separated from the solid by filtration, concentrated, dissolved
0.22. IR (ATR): ν = 2955.1 (m), 2924.0 (s), 2855.5 (s), 1693.9 (s),
˜
1647.0 (vs), 1592.7 (s), 1576.5 (s), 1506.4 (w), 1458.0 (w), 1435.2 in chloroform (low solubility) and precipitated with acetonitrile.
(m), 1402.9 (m), 1335.0 (s), 1303.2 (w), 1249.0 (m), 1170.4 (m), Yield: 11.0 mg (38%). Dark-red solid, m.p. Ͼ250 °C. Rf (silica gel;
1125.6 (w), 1106.3 (w), 983.0 (w), 849.6 (w), 809.4 (m), 794.8 (w), CHCl3/ethanol, 40:1) = 0.09; Rf (silica gel; CHCl3/ethanol, 10:1) =
779.4 (w), 743.9 (m), 631.0 (w), 586.8 (w) cm–1
.
1H NMR
0.57. IR (ATR): ν = 3307.5 (w), 2066.0 (w), 2951.8 (m), 2924.1 (s),
2854.7 (m), 1693.7 (s), 1651.2 (vs), 1592.4 (vs), 1577.0 (s), 1506.9
˜
3
(600 MHz, CDCl3, 25 °C, TMS): δ = 0.82 [t, J(H,H) = 7.0 Hz, 6
3
H, 2ϫCH3], 0.91 [t, J(H,H) = 7.4 Hz, 3 H, CH3,butyl], 1.18–1.38 (w), 1435.1 (m), 1403.8 (m), 1378.2 (w), 1333.4 (vs), 1247.8 (m),
(m, 18 H, 9ϫCH2), 1.61–1.68 (m, 2 H, CH2,butyl), 1.83–1.91 (m, 2 1167.9 (m), 1125.7 (w), 1103.5 (w), 1013.9 (w), 981.5 (w), 889.2 (w),
3
H, β-CH2), 2.21–2.29 (m, 2 H, β-CH2), 3.58 [t, J(H,H) = 6.9 Hz,
851.0 (w), 808.6 (m), 775.1 (w), 743.8 (w), 696.9 (w), 625.1 (w)
2 H, NCH2,butyl], 5.15–5.22 (m, 1 H, α-CH), 5.42 (s, 2 H, NCH2), cm–1
.
1H NMR (200 MHz, CDCl3, 25 °C, TMS): δ = 0.83 [t,
7.58–7.64 (m, 2 H, CHaryl), 7.66–7.72 (m, 2 H, CHaryl), 8.22 (s, 1 H,
CHN), 8.56–8.72 (m, 8 H, CHperylene) ppm. 13C NMR (150 MHz,
CDCl3, 25 °C, TMS): δ = 14.1, 14.3, 20.6, 22.8, 27.1, 29.4, 29.9,
32.0, 32.6, 33.2, 43.8, 55.0, 123.2, 123.5, 123.6, 126.6, 126.7, 129.5,
129.6, 129.8, 130.3, 131.9, 132.1, 135.2, 163.6 ppm. UV/Vis
(CHCl3): λmax (Erel.) = 459.4 (0.22), 490.8 (0.60), 527.4 (1.00) nm;
fluorescence (CHCl3): λmax (Irel.) = 536.0 (1.00), 577.2 (0.50) nm;
3J(H,H) = 7.0 Hz, 6 H, 2ϫCH3], 1.16–1.39 (m, 16 H, 8ϫCH2),
1.76–1.98 (m, 2 H, β-CH2), 2.13–2.38 (m, 2 H, β-CH2), 5.10–5.27
(m, 1 H, α-CH), 5.49 (s, 2 H, NCH2), 7.15–7.23 (m, 2 H, CHaryl),
7.64–7.77 (m, 2 H, CHaryl), 7.81–7.95 (m, 2 H, CHaryl), 8.41 (s, 1
H, CHN), 8.60–8.83 (m, 8 H, CHperylene) ppm. UV/Vis (CHCl3):
λmax (Erel.) = 459.8 (0.22), 491.0 (0.60), 527.6 (1.00) nm; fluores-
cence (CHCl3): λmax (Irel.) = 534.8 (1.00), 578.0 (0.50) nm; fluores-
cence quantum yield {CHCl3, λexc = 490 nm, E490nm = 0.0132 cm–1,
reference: 2,9-bis(1-hexylheptyl)anthra[2,1,9-def:6,5,10-dЈeЈfЈ]diiso-
quinoline-1,3,8,10-tetraone with Φ = 1.00}: Φ = 1.00. MS (DEI+,
fluorescence quantum yield {CHCl3, λexc = 490 nm, E490nm
=
0.0257 cm–1, reference: 2,9-bis(1-hexylheptyl)anthra[2,1,9-
def:6,5,10-dЈeЈfЈ]diisoquinoline-1,3,8,10-tetraone with Φ = 1.00}: Φ
= 1.00. MS (DEI+, 70 eV): m/z (%) = 745 (24) [M]+, 564 (25) 70 eV): m/z (%) = 810 (23) [M]+, 627 (100), [M – C13H26]+, 346
M – C13H26]+, 346 (18) [M – C27H43N2O]+, 173 (29) [M –
(23) [M – C29H39N2O3]+, 238 (17) [M – C37H35N2O4]+, 137 (46)
C3 7 H3 6 N2 O4 ]+ , 130 (100) [C9 H8 N]+ . HMRS: calcd. for [C7H7NO2]+, 120 (44) [C7H4O2]+, 91 (23) [C6H5N]+. HMRS: calcd.
C49H51N3O4 [M]+ 745.388; found 745.383.
for C52H47N3O6 [M]+ 809.346; found 809.348.
2-{4-[4-(Butyliminomethyl)phenyl]benzyl}-9-(1-hexylheptyl)anthra-
[2,1,9-def:6,5,10-dЈeЈfЈ]diisoquinoline-1,3,8,10-tetraone (13b): 1e
(28.0 mg, 36.5 μmol), 1-butylamine (0.700 mL, 70.4 μmol), chloro-
form (5 mL) and MgSO4 (400 mg) were allowed to react analo-
gously to the procedure used to prepare 12a (12 h, room tempera-
2-(4-{4-[(4-Carboxyphenyl)iminomethyl]phenyl}benzyl)-9-(1-hexyl-
heptyl)anthra[2,1,9-def:6,5,10-dЈeЈfЈ]diisoquinoline-1,3,8,10-tetraone
(13c): 1e (25.0 mg, 32.6 μmol), a mixture of chloroform/ethanol
(10:4, 5 mL), MgSO4 (400 mg) and 4-aminobenzoic acid (17.8 mg,
130 μmol) were allowed to react analogously to the procedure used
4334
www.eurjoc.org
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2007, 4328–4336

Fluorescence Labelling of Primary Amines
FULL PAPER
to prepare 13a (dissolved in chloroform and precipitated with
methanol, low solubility). Yield: 4.00 mg (14%). Dark-red solid,
m.p. Ͼ250 °C. Rf (silica gel; CHCl3/ethanol, 40:1) = 0.07; Rf (silica
dЈeЈfЈ]diisoquinolin-2-ylmethyl]benzaldehyde (1c). Method 1: Com-
pound 1c (20 mg, 28.9 μmol) and a microspatulum of dicyclohex-
ylcarbodiimide (DCC) were added with stirring to a mixture of
bovine catalase (bison liver, 40.0 mg) and molecular sieves (4 Å;
300 mg) in NMP (20 mL) at 40 °C, stirred for 60 h, cooled to room
gel; CHCl /ethanol, 10:1) = 0.47. IR (ATR): ν = 3305.9 (br, w),
˜
3
3068.3 (w), 2951.4 (m), 2924.7 (s), 2954.9 (m), 1693.5 (s), 1651.5
(vs), 1591.9 (vs), 1577.7 (s), 1505.5 (w), 1435.3 (w), 1403.9 (m), temperature, collected by filtration (the NMP phase can be used
1333.0 (vs), 1247.7 (m), 1167.2 (m), 1124.4 (w), 1104.9 (w), 1004.2 for further labelling of peptides), washed with water and methanol,
(w), 982.1 (w), 889.7 (w), 851.5 (w), 808.1 (m), 780.7 (m), 747.7 separated from molecular sieves by fractionated sedimentation
(m), 723.8 (w), 665.6 (w), 623.1 (w) cm–1. UV/Vis (CHCl3): λmax from methanol and dried in air to give a red powder. The red color-
(Erel.) = 459.8 (0.22), 490.8 (0.60), 527.6 (1.00) nm; fluorescence
ation could not be removed, neither with water nor with organic
(CHCl3): λmax (Irel.) = 535.5 (1.00), 578.8 (0.52), 627.5 (0.12) nm; solvents such as chloroform. Method 2: Compound 1c (18.0 mg,
fluorescence quantum yield {CHCl3, λexc = 490 nm, E490nm
=
26.1 μmol) was allowed to react as was described in Method 1 at
50 °C for 30 min to give a reddish-orange material. The coloration
could not be removed, neither with water nor with organic solvents
such as chloroform. Method 3: Compound 1c (10.0 mg, 14.5 μmol)
was dissolved in DMSO (10 mL) at 50 °C and a microspatulum of
0.0202 cm–1, reference: 2,9-bis(1-hexylheptyl)anthra[2,1,9-
def:6,5,10-dЈeЈfЈ]diisoquinoline-1,3,8,10-tetraone with Φ = 1.00}: Φ
= 1.00. MS (DEI+, 70 eV): m/z (%) = 886 (15) [M]+, 703 (100),
[M – C13H26]+, 374 (21) [M – C34H43N2O2]+, 346 (27) [M –
C
3 5 H4 7 N2 O3 ]+ , 314 (30) [M – C3 7 H3 5 N2 O4 ]+ , 137 (81) dicyclocarbodiimide (DCC) was added with stirring to a mixture
[C7H7NO2]+, 120 (90) [C7H4O2]+, 92 (23) [C6H6N]+. HMRS: calcd.
for C58H51N3O6 [M]+ 885.378; found 885.379.
of bovine catalase (18.0 mg) and molecular sieves (4 Å; 300 mg) in
DMSO and stirred at 50 °C for 5 h and further treated as was de-
scribed for Method 1. A dark-red material was obtained the color-
ation of which could not be removed, neither with water nor with
organic solvents such as chloroform. Method 4: A microspatulum
of 1c and bovine catalase were dispersed in NMP (5 mL) at 50 °C
for 1 h giving uncoloured flakes of catalase. A microspatulum of
DCC was added (red colouration within a few minutes). The mix-
ture was further stirred at 50 °C for 1 h and further treated as was
described for Method 1. A reddish-orange material was obtained
the colouration of which could not be removed, neither with water
nor with organic solvents such as chloroform.
2-[(1-{4-[9-(1-Hexylheptyl)-1,3,8,10-tetraoxo-3,8,9,10-tetrahydro-
1H-anthra[2,1,9-def:6,5,10-dЈeЈfЈ]diisoquinolin-2-ylmethyl]phenyl}-
methylidene)amino]-3-phenylpropionic Acid (12d). Method 1: 1c
(30.0 mg, 43.4 μmol) was dissolved in N-methylpyrrolidone (NMP,
20 mL) at 110 °C, treated with MgSO4 (400 mg) and with l-phenyl-
alanine (25.0 mg, 151 μmol) in NMP (10 mL, dissolved at 110 °C),
stirred at 110 °C for 5 h and at room temperature for 12 h, sepa-
rated from the solid by filtration, shaken with concentrated brine
(100 mL) and extracted with toluene (100 mL). The combined or-
ganic phases were three times shaken with concentrated brine
(100 mL each), dried with MgSO4, concentrated in vacuo and puri-
fied by column chromatography (silica gel; toluene/ethanol, 10:1)
(second fraction). Yield: about 25%. Rf (silica gel; CHCl3/ethanol,
Fluorescence Labelling of Catalase with 4Ј-[9-(1-Hexylheptyl)-
1,3,8,10-tetraoxo-3,8,9,10-tetrahydro-1H-anthra[2,1,9-def:6,5,10-
dЈeЈfЈ]diisoquinolin-2-ylmethyl]biphenyl-4-carbaldehyde
(1e):
A
l
microspatulum of 1e was allowed to react as was described for
Method 2. A reddish-orange material was obtained the colouration
of which could not be removed, neither with water nor with organic
solvents such as chloroform.
40:1) = 0.03; Rf (silica gel; CHCl3/ethanol, 10:1) = 0.51. H NMR
3
(200 MHz, CDCl3, 25 °C, TMS): δ = 0.86 [t, J(H,H) = 7.0 Hz, 6
H, 2ϫCH3], 1.18–1.38 (m, CH2, 16 H), 1.38 (m, 1.80–2.30 (m, 4
H, β-CH2), 3.89 (m, 1 H, CH), 4.46 (m, 2 H, CH2), 5.10–5.28 (m,
1 H, α-CH), 5.41 (s, 2 H, NCH2), 6.70–7.34 (m, 5 H, CHaryl), 7.53–
7.83 (m, 4 H, CHaryl), 8.24 (s, 1 H, CHN), 8.59–8.78 (m, 8 H,
CHperylene) ppm. UV/Vis (CHCl3): λmax (Erel.) = 459.2 (0.22), 490.4
(0.61), 527.2 (1.00) nm; fluorescence (CHCl3): λmax (Irel.) = 536.0
(1.00), 579.2 (0.53), 629.0 (0.13) nm; fluorescence quantum yield
{CHCl3, λexc = 490 nm, E490nm = 0.0295 cm–l, reference: 2,9-bis(1-
hexylheptyl)anthra[2,1,9-def:6,5,10-dЈeЈfЈ]diisoquinoline-1,3,8,10-
tetraone with Φ = 1.00}: Φ = 1.00. Method 2: 1c (30.0 mg,
43.4 μmol) was dissolved in dimethyl sulfoxide (DMSO, 20 mL) at
110 °C, treated with MgSO4 (400 mg) and with l-phenylalanine in
DMSO (25 mg in 10 mL DMSO), dissolved at 110 °C, stirred at
110 °C for 5 h and at room temperature for a further 12 h, sepa-
rated from the solid by filtration, shaken with concentrated brine
(100 mL) and extracted with toluene (100 mL). The combined or-
ganic phases were three times shaken with concentrated brine
(100 mL each), dried with MgSO4, concentrated in vacuo (reddish-
violet powder) and purified by column chromatography (silica gel;
toluene/ethanol, 10:1) (second fraction). Yield: ca. 25%.
Supporting Information (see footnote on the first page of this arti-
cle): Synthesis and spectroscopic characterization of 3, 4, 5, 6, 9,
10 and 11.
Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft
and the Fonds der Chemischen Industrie.
[1] C. Bräuchle, G. Seisenberger, T. Endreß, M. U. Ried, H.
Büning, M. Hallek, ChemPhysChem 2002, 3, 299–303.
[2] H. Schiff, Justus Liebigs Ann. Chem. 1864, 131, 118.
[3] K. Moonen, C. V. Stevens, Synthesis 2005, 20, 3603–3612.
[4] H. Zollinger, Color Chemistry, Synthesis Properties, and Appli-
cations of Organic Dyes and Pigments, 3rd ed., Wiley-VCH,
Zürich, 2003.
[5] W. Herbst, K. Hunger, Industrielle Organische Pigmente. Her-
stellung, Eigenschaften, Anwendung, 2nd ed., VCH, Weinheim,
1995.
[6] J. Fabian, H. Hartmann, Light Absorption of Organic Color-
ants, Springer Verlag, Berlin, 1980.
[7] H. R. Schweizer, Künstliche Organische Farbstoffe und ihre Zwi-
schenprodukte, 1st ed., Springer, Berlin, 1964, p. 385.
[8] H. Langhals, J. Karolin, L. B.-Å. Johansson, J. Chem. Soc.,
Faraday Trans. 1998, 94, 2919–2922.
2-[(1-{4Ј-[9-(1-Hexylheptyl)-1,3,8,10-tetraoxo-3,8,9,10-tetrahydro-
1H-anthra[2,1,9-def:6,5,10-dЈeЈfЈ]diisoquinolin-2-ylmethyl]biphenyl-
4-yl}methylidene]amino}-3-phenylpropionic Acid (13d): 1e (30.0 mg,
39.1 μmol) in NMP (20 mL) and l-phenylalanine (22.6 mg,
137 μmol) in NMP (10 mL) were allowed to react as was described
for the preparation of 12d (reddish-violet powder).
[9] H. Langhals, S. Demmig, Ger. Offen. DE 4007618.0, 1990
[Chem. Abstr. 1992, 116, P117172n].
[10] H. Langhals, Helv. Chim. Acta 2005, 88, 1309–1343.
Fluorescence Labelling of Catalase with 4-[9-(1-Hexylheptyl)-
1,3,8,10-tetraoxo-3,8,9,10-tetrahydro-1H-anthra[2,1,9-def:6,5,10-
Eur. J. Org. Chem. 2007, 4328–4336
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4335

H. Langhals, T. Becherer, J. Lindner, A. Obermeier
FULL PAPER
[11] H. Langhals, Heterocycles 1995, 40, 477–500.
[12] S. Demmig, H. Langhals, Chem. Ber. 1988, 121, 225–230.
[13] H. Langhals, S. Demmig, T. Potrawa, J. Prakt. Chem. 1991,
333, 733–748.
[17] H. Kaiser, J. Lindner, H. Langhals, Chem. Ber. 1991, 124, 529–
535.
[18] N. Miyaura, A. Suzuki, Chem. Rev. 1995, 95, 2457–2483.
[19] H. Kaiser, J. Lindner, H. Langhals, Chem. Ber. 1991, 124, 529–
535.
[14] P. D. R. Rose, A. Williams, J. Chem. Soc., Perkin Trans. 2 2002,
1589–1595.
[20] H. Langhals, Chem. Ber. 1985, 118, 4641–4645.
[15] R. Kumar, A. K. Chakraborti, Tetrahedron Lett. 2005, 46,
8319–8323.
Received: March 15, 2007
Published Online: July 6, 2007
[16] O. Ouari, F. Chalier, R. Bonaly, B. Pucci, P. Tordo, J. Chem.
Soc., Perkin Trans. 2 1998, 2299–2307.
4336
www.eurjoc.org
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2007, 4328–4336