Orthogonally Protected Diaminoterephthalate Scaffolds: Installation of Two Functional Units at the Chromophore

Article abstract of DOI:10.1021/acs.joc.8b00347

The 2,5-diaminoterephthalate structural motif is a powerful chromophore with remarkable fluorescence properties. Containing two carboxylate and two amino functions, it defines a colored molecular scaffold which allows for orthogonal functionalization with different functional units. Therefore, different applications in life sciences and materials science could be addressed. In this study, the two amino functions were alkylated by reductive amination with side chains carrying amino (orthogonally protected as Boc or Alloc) and carboxylate functions (orthogonally protected as tBu or allyl ester). After sequential deprotections, functional units were introduced by amidation reactions. As three examples, the chromophore was coupled with retinoic acid and fullerene C₆₀ in order to obtain a triad for studying photoinduced electron transfer processes. Furthermore, cyclooctyne and azide moieties were introduced as functional units, allowing for ligation by click reactions. These two clickable groups were applied in combination with maleimide units which are reactive toward thiol residues. The latter dyes define so-called turn on probes, since the fluorescence quantum yields increased by one order of magnitude upon reaction with the molecular target.

Full text of DOI:10.1021/acs.joc.8b00347

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Orthogonally Protected Diaminoterephthalate Scaffolds
—Installation of Two Functional Units at the Chromophore
Leon Buschbeck, and Jens Christoffers
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b00347 • Publication Date (Web): 13 Mar 2018
Downloaded from http://pubs.acs.org on March 15, 2018
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Orthogonally Protected Diaminoterephthalate Scaffolds – Installation of
Two Functional Units at the Chromophore
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Leon Buschbeck and Jens Christoffers*
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Dedicated to Professor Jürgen Rullkötter on the occasion of his 70th birthday
Institut für Chemie, Carl von Ossietzky Universität Oldenburg, 26111 Oldenburg, Germany
ABSTRACT:
FU1
PG1
NH
NH
CO2Et
CO2Et
EtO2C
EtO2C
FU2
NHPG2
HN
The 2,5ꢀdiaminoterephthalate structural motif is a powerful chromophore with remarkable
fluorescence properties. Containing two carboxylate and two amino functions it defines a coꢀ
lored molecular scaffold which allows for orthogonal functionalization with different functiꢀ
onal units. Therefore, different applications in Life Sciences and Materials Science could be
addressed. In this study, the two amino functions were alkylated by reductive amination with
side chains carrying amino (orthogonally protected as Boc or Alloc) and carboxylate funcꢀ
tions (orthogonally protected as tBu or allyl ester). After sequential deprotections, functional
units were introduced by amidation reactions. As three examples, the chromophore was coupꢀ
led to retinoic acid and fullerene C60 in order to obtain a triad for studying photoinduced elecꢀ
tron transfer processes. Furthermore, cyclooctyne and azide moieties were introduced as funcꢀ
tional units allowing for ligation by Click reactions. These two clickable groups were applied
in combination with a maleimide units which are reactive towards thiol residues. The latter
dyes define so called "turn on" probes, since the fluorescence quantum yields increased by an
order of magnitude upon reaction with the molecular target.
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INTRODUCTION
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Fluorescence dyes are versatile tools for analytical applications in the Life Sciences1 as well
as Materials Science.2 Particularly useful are chromophores holding a reactive functional unit,
thus, allowing for the covalent ligation of biomolecules like proteins or other targets.3 As
functional units, for example, the maleimide moiety is suited for the conjugate addition of a
thiol, e.g. a cysteine residue on the surface of a protein.4 Furthermore, the copperꢀcatalyzed
Huisgen 1,3ꢀdipolar cycloaddition5 of an alkyne with an organo azide (soꢀcalled Click reacꢀ
tion)6 became very popular in the recent years for binding biomolecules to dyes.7 A newer deꢀ
velopment in this area is the use of highly reactive cyclooctyne derivatives that allow for a
copperꢀfree Click reaction.8
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Diaminoterephthalates (DATs) are bright and colorful dyes which exhibit powerful fluoresꢀ
cence properties.9 The DAT motif defines a structurally relative simple chromophore, which
is in contrast to other classes of dyes, so far underrated in the literature.10 These compounds
are readily accessed by conversion of succinyl succinates with primary amines,11 allowing a
flexible introduction of side chains with various functional units. Actually, the chromophore
defines a molecular scaffold,12 which can be orthogonally equipped with different functional
units,13 thus, tailored functional materials can be prepared for addressing different applicaꢀ
tions of Life Sciences14 and Materials Science.15
We have recently reported on the synthesis of bifunctional dye 1 holding a maleimide and
cyclooctyne functional unit (Scheme 1).16 We have used this probe 1 for studying proteinꢀproꢀ
tein interactions. Anyhow, there are two significant problems associated with the synthesis of
compound 1 from building block 2. One carboxylate function is released from compound 2 by
debenzylation (catalytic hydrogenation) which is then followed by introduction of the first
functional unit by amidation. The first problem arises during the saponification of the second
ester group because the respective basic reactions conditions are not fully compatible with the
functional unit already installed. The second problem is the coupling of the two functional
units by amidation of the aromatic carboxylate functions, which proceeded not always with
satisfying results because the the electron donating amino groups at the chromophore deplete
the electrophilicity of the carbonyl groups.
MeHN
O
NHMe
CO2Bn
H
N
O
N
O
H
H
N
O
N
MeO2C
O
NHMe
NHMe
O
1
2
Scheme 1.
Compound 1 as an example of the first generation of a bifunctionalized DAT
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and its synthetic precursor 2.
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In order to overcome the aboveꢀmentioned drawbacks during synthesis of probe 1, this maꢀ
nuscript suggests the next generation of bifunctionalized DATs to be prepared from the three
intermediate compounds 3, 4, and 5 (Scheme 2). The two key advantages of the new synthetic
concept are the following: (1) The functional units are sterically and electronically decoupled
from the chromophore by introduction of alkyl spacers, and (2) the compounds hold either
two orthogonally protected amino groups (compound 3), two carboxylic acids (compound 5),
or a mixed amineꢀcarboxylic acid scaffold (compound 4), which is an enrichment of synthetic
options compared to diprotected dicarboxylic acid 2. Actually, this new concept allows for the
first time to install two different functional units at the nitrogen atoms of the core structure.
We are planning to introduce the spacer units by reductive aminations of aldehydes 6, 7, 9,
and 10 with the aromatic amine 8. Compounds 6 and 9 are protected amines, compounds 7
and 10 protected carboxylic acids with either acid cleavable groups (BocNH, 6 and tBuO2C,
7) or groups cleavable by palladium catalyzed allylic substitutions (NHAlloc, 9 and CO2Allyl,
10).
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CO Bu
2t
HN
NHBoc
CO2Et
HN
NHBoc
CO2Et
HN
CO2Et
EtO2C
EtO2C
EtO2C
HN
NHAlloc
HN
HN
CO2Allyl
CO2Allyl
5
9
3
4
NHBoc
CO2Et
NHAlloc
CHO
OHC
BocHN
6
7
+
+
EtO2C
BuO C
CHO
t
NH2
OHC
CO2Allyl
10
2
8
Scheme 2.
Concept for the second generation of functional dyes with the orthogonally
protected DATs 3, 4, and 5 as chromophore building blocks and respective starting materials
6–10 for reductive aminations.
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RESULTS AND DISCUSSION
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The preparation of literature known aminopropanals 617 and 918 proceeded straightforward in
two steps starting from aminopropanol 11 (Scheme 3). Standard protocols were applied for
Bocꢀ19 and Allocꢀprotections20 and the subsequent oxidation was accomplished according to
the Swernꢀprocedure.17 After chromatographic purification, compounds 6 and 9 were obtained
in 82% and 59% yields over two steps, respectively. Compound 6 slowly decomposes at amꢀ
bient temperature, but is stable for at least a week at –35°C.
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(a), (b)
CHO
= Boc, 82%),
H2N
OH
HN
PG
11
6 (
9 (
PG
PG
= Alloc, 59%)
Scheme 3.
Synthesis of protected aminopropanals 6 and 9 (yields over two steps); reaꢀ
gents and conditions: (a) for 6: Boc2O, CH2Cl2, 23°C, 18 h, 99%; for 9: AllocCl, Na2CO3,
MeCN–H2O, 23°C, 20 h, 96%; (b) (COCl)2, DMSO, NEt3, CH2Cl2, –78°C→23°C, 3 h, 83%
(for 6), 61% (for 9).
The synthesis of formyl acetic esters from formylꢀMeldrums acid 12 was actually reported in
the literature,21 however, it turned out to require substantial practice when we tried to reproꢀ
duce the synthesis of tertꢀbutyl and allylic esters 7 and 10. First of all, starting material 1222
was obtained in two steps from Meldrums acid in 68% yield. Compound 12 was then converꢀ
ted with tBuOH and allylic alcohol in toluene to furnish a crude mixture (Scheme 4), from
which the products 721, 23 and 1024 were obtained by Kugelrohr distillation in vacuum (31%
and 65% yield, resp.). Both compounds are stable at –35°C at least for a week. Both
decompose at ambient temperature, tertꢀbutyl ester 7 faster than allyl ester 10. In this context,
we would like to point out, that a longer linker moiety, which is easier to prepare, e.g. a 3ꢀ
formyl propionic ester as a C4ꢀunit was initially investigated, which however lead in
subsequent operations to intramolecular amidations under formation of γꢀbutyrolactams.
O
O
+ ROH
RO2C
CHO
O
O
toluene
80°C, 1.5 h
OH
7 (R = Bu, 31%),
t
(R = Allyl, 65%)
10
12
Scheme 4.
Synthesis of aldehydes 7 and 10 from formyl Meldrums acid 12.
Synthesis of orthogonally protected building blocks 3, 4, and 5 started with DAT 13, which
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was obtained according to Sinnreich11 from diethyl succinyl succinate25 and then was monoꢀ
Boc protected (90% of product 8,26 Scheme 5). After some experimentation, two complemenꢀ
tary procedures turned out to be optimal for the reductive aminations: For protected aminoꢀ
propanals 6 and 9, NaBH(OAc)3 with or without AcOH27 gave better results than NaBH3CN
with ZnCl2.28 For protected formylacetates 7 and 10, the choice was opposite. Accordingly,
compounds 14 and 15 were obtained under different reactions conditions with 83% and 89%
yield, respectively. The Bocꢀdeprotection proceeded for both compounds under acidic conꢀ
ditions, although the allyl ester 17 (89% yield) showed slightly lower stability under these
conditions than allyl carbamate 16 (99% yield). Finally, the second reductive amination was
accomplished, again for aminopropanal 6 with NaBH(OAc)3 (without AcOH, but with longer
reaction times; in the presence of AcOH, the Boc group turned out to be not completely
stable) and tertꢀbutyl ester 7 with NaBH3CN with ZnCl2. Whereas the yields with aminoproꢀ
panal 6 were good if no additional AcOH was used (70% of product 3 and 76% of product 4),
the yield for the diester 5 was lower (51%). Anyhow, the overall procedures turned out to be
robust and compounds 3–5 were accessed on reasonable scales, with 52% (for 3), 54% (for 4)
and 36% (for 5) yields over four steps, respectively. In addition to compound 4, one might
suggest a second "mixed" case with an acid cleavable carboxylate protection and an amino
protection cleavable by palladium catalysis. However, such a constitution would provide no
added value.
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NH2
CO2Et
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EtO2C
NH2
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Boc2O
CH2Cl2, 23°C, 2 d
NHBoc
NHBoc
CO2Et
CO2Et
(a) or (b)
R2
CHO
EtO2C
EtO2C
NH2
HN
R2
14 (R2 = CH2NHAlloc, 83%),
15 (R2 = CO2Allyl, 89%)
(90%)
8
TFA, CH2Cl2
23°C, 18 h
R1
HN
NH2
CO2Et
CO2Et
(c) or (d)
R1
CHO
EtO2C
EtO2C
HN
HN
R2
R2
3 (R1 = CH2NHBoc, R2 = CH2NHAlloc, 70%), 16 (R2 = CH2NHAlloc, 99%),
4 (R1 = CH2NHBoc, R2 = CO2Allyl, 76%),
17 (R2 = CO2Allyl, 89%)
5 (R1 = CO Bu, R2 = CO Allyl, 51%),
2t
2
Scheme 5.
Synthesis of orthogonally protected building blocks 3, 4, and 5; reagents and
conditions, for 14: (a) aldehyde 9, R2 = CH2NHAlloc, NaBH(OAc)3, AcOH, CH2Cl2, 23°C, 1
h; for 15: (b) aldehyde 10, R2 = CO2Allyl, NaBH3CN, ZnCl2, CH2Cl2, 0°C→23°C, 2 d; for 3
and 4: (c) aldehyde 6, R1 = CH2NHBoc, NaBH(OAc)3, CH2Cl2, 0°C→23°C, 18 h; for 5: (d)
aldehyde 7, R2 = CO2tBu, NaBH3CN, ZnCl2, CH2Cl2, 0°C→23°C, 2 d.
In order to mimic natural photosynthesis and to understand fundamental photoinduced elecꢀ
tron and energy transfer processes, many photosynthetic models have been constructed and
investigated in the past few decades.29 Among the energy and electron accepting moieties, fulꢀ
lerene (C60) has become the most promising one due to its unique physical and chemical proꢀ
perties. For example, it can be easily ligated to dyes and other larger molecular entities by
cycloaddition reactions. Several dyeꢀfullerene conjugates, so called dyads15b,30 or triads,31
have been prepared in the past years. Herein we projected the preparation of the triad 20 by
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consecutive coupling of diprotected diamine 3 with retinoic acid and fullerene C60. The instalꢀ
lation of a C60 unit is conveniently achieved by 1,3ꢀdipolar cycloaddition with an azomethine
ylide prepared in situ from an aromatic aldehyde and Nꢀmethylglycine (sarcosine) (soꢀcalled
Pratoꢀreaction).32 For this purpose, the allyl carbamate 3 was first deprotected in a palladium
catalyzed allylic substitution reaction with morpholine as a scavenger of the allylic cation
(Scheme 6).33 The primary amine 18 was obtained in quantitative yields and further coupled
with paraꢀformyl benzoic acid using COMUꢀDIPEA34 {COMU = 1ꢀ[1ꢀ(cyanoꢀ2ꢀethoxyꢀ2ꢀ
oxoethylideneaminooxy)dimethylaminomorpholino]uranium hexafluorophosphate, DIPEA =
ethyldiisopropylamine} to yield the aromatic aldehyde 19 in 50% yield (as mentioned in the
introduction, the coupling of benzoic acid derivatives sometimes gives not fully satisfying
results). Next, the cycloaddition with C60 and N-methylglycine was accomplished and the
chromophore was linked by a N-methyl pyrrolidine unit to the fullerene (95% of product 21).
Bocꢀdeprotection was straightforward with TFA in CH2Cl2, and finally, retinoic acid was
coupled with using now the HATUꢀDIPEA35 [HATU = Oꢀ(7ꢀazabenzotriazolꢀ1ꢀyl)ꢀN,N,N′,N′ꢀ
tetramethyluronium hexafluorophosphate] protocol furnishing the target compound 20 (75%).
The yield over five steps was from compound 3 was 32%. The major problem associated with
this sequence was the extraordinary limited solubility of compounds 20–22 with a C60 moiety
and their pronounced tendency to stick irreversibly to glass surfaces, which caused loss of
material at every operational step. Therefore, it was important to carry out subsequent
reactions always directly after isolation of the product of the previous step. Moreover, due to
limited solubility and high molecular weight, signalꢀtoꢀnoise ratios of the carbon NMR
spectra a unsatisfying; some signals could only be identified by crossꢀpeaks of respective
1H,13Cꢀcorrelations. Signals in the proton spectra also appear very broad with unsatisfactory
resolution, presumably due to isomerization of the CꢀC double bonds of the retinoate and
strong associations of the molecules in solution. Anyhow, satisfactory HRMS data from comꢀ
pounds 20–22 were obtained in the MALDI and ESI spectra. As expected, the fluorescence of
the DAT scaffold was quenched by introduction of the fullerene moiety. The quantum yield
decreased from Φ = 0.02 (compound 19) to Φ = 0.001 in compounds 20–22. The photoꢀ
physics of this compound is currently under investigation in cooperation with a group speꢀ
cialized in femtosecond spectroscopy.
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HN
NHBoc
CO2Et
HN
NHBoc
HN
HN
NHBoc
CO2Et
+ HO2C
CHO
CO2Et
cat. [Pd(PPh3)4]
CHO
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morpholine
CH2Cl2, 23°C, 1.5 h
COMU, DIPEA
CH2Cl2, 23°C, 17 h
EtO2C
EtO2C
EtO2C
H
N
HN
NHAlloc
HN
NH2
O
3
18 (quant.)
19 (50%)
+ C60
+ MeHN
toluene
110°C, 17 h
CO2H
O
+ retinoic acid
HATU, DIPEA
N
NH
RHN
NH
H
CO2Et
CO2Et
CH2Cl2,
23°C, 18 h
N
N
Me
EtO2C
EtO2C
H
H
N
Me
HN
N
HN
O
O
20 (75%)
21 (R = Boc, 95%)
TFA, CH2Cl2
23°C, 18 h
22 (R = H, 91%)
Scheme 6.
Five step synthesis of a retinoic acidꢀDATꢀC60 triad 20 from building block 3.
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As a second target compound 28, we aimed at the preparation of an analog of compound 1
with the functional units cyclooctyne and maleimide now bound at one 3ꢀaminopropylamino
and one to 2ꢀcarboxyethylamino moiety of scaffold 4 (Scheme 7). For this purpose, the allyl
ester of compound 4 was deprotected by palladium catalyzed allylic substitution with pyrroliꢀ
dine as nucleophile36 to quantitatively furnish the acid 23, which was subsequently amidated
with maleimide building block 24, which was accessed according to a literature procedure.37
Compound 25 was obtained in 87% yield. The tertꢀbutyl carbamate was then cleaved with
TFA in CH2Cl2 under release of the primary amino function, which was actually reactive toꢀ
wards the maleimide group. For this reason, it was quantitatively isolated as the trifluoroaceꢀ
tate salt 26. Finally, the reaction of N-hydroxysuccinimidyl active carbonate of cyclooctyne
27, which was commercially available, in the presence of base gave to target structure 28 in
78% yield. The yield over four steps from compound 4 was 68%. Fortunately, and in contrast
to fullerene derivatives 20–22, all products in Scheme 7 are wellꢀbehaved organic comꢀ
pounds, which could easily be purified by column chromatography and spectroscopically
characterized without problems. As known for other maleimide functional dyes,14 the fluoꢀ
rescence of compound 28 is quenched by the maleimde moiety (quantum yield Φ = 0.005).
Upon reaction with a thiol, the emission was restored. As a model study to prove the "turn on"
effect of fluorescence and to show its feasibility in a copperꢀfree Click reaction, compound 28
was reacted with BnSH and BnN3 as model substrates. Compound 29 was isolated in 73%
yield and it showed efficient fluorescence (Φ = 0.11).
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BocNH
NH
4
5
CO2Et
6
7
EtO2C
HN
4 (R = Allyl)
8
9
CO2R
cat. [Pd(PPh3)4]
pyrrolidine
CH2Cl2, 23°C, 1.5 h
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(R = H, quant.)
23
O
CF3CO2 H3N
HATU, DIPEA
CH2Cl2, 23°C, 17 h
N
O
24
RHN
NH
CO2Et
EtO2C
O
H
N
N
HN
O
O
25 (R = Boc, 87%)
TFA, CH2Cl2
23°C, 18 h
(R = H CF CO H, quant.)
26
—
3
2
O
DIPEA, CH2Cl2,
23°C, 16 h
O
OSucc
O
27
O
N
NH
H
CO2Et
EtO2C
O
H
HN
28 (78%)
N
N
O
O
+ BnN3, BnSH
O
MeCN, 23°C, 20 h
N
N
O
N
NH
N
H
CO2Et
Bn
EtO2C
O
H
N
N
HN
29 (73%)
SBn
O
O
Scheme 7.
Preparation of fluorescence probe 28 with cyclooctyne and maleimide funcꢀ
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tional units and its "turn on" by reaction with model azide and thiol BnN3 and BnSH.
4
5
6
7
8
9
Finally in the last one of three case studies, dicarboxylic acid scaffold 5 was twice functionaliꢀ
zed with organo azide and maleimide in the following sequence (Scheme 8): First of all, the
allyl ester 5 was again deprotected (product 30 in quantitative yield) and amidated with the
maleimide building block 24 to give compound 31 in 83%. The tertꢀbutyl ester 31 was then
cleaved with TFA (product 32 with quantitative yield) and the carboxylic acid 32 amidated a
second time with amino azide reagent 33 (prepared according to a literature procedure)16 to
furnish target structure 34 in 39% yield. The yield over four steps from compound 5 was 32%.
Similar to the above case, compound 34 (Φ = 0.02) was also a "turn on" probe: Upon reaction
with BnSH, compound 35 was isolated in 41% yield and it showed efficient fluorescence (Φ =
0.15).
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13
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17
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20
21
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24
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29
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BuO C
t
2
NH
4
CO2Et
5
6
7
EtO2C
8
9
HN
CO2R
cat. [Pd(PPh3)4]
pyrrolidine
5 (R = Allyl)
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17
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28
29
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31
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60
CH2Cl2, 23°C, 1.5 h
30 (R = H, quant.)
O
CF3CO2 H3N
HATU, DIPEA
CH2Cl2, 23°C, 18 h
N
O
24
RO2C
NH
CO2Et
EtO2C
O
H
HN
N
N
O
O
31 (R = Bu, 83%)
t
TFA, CH2Cl2
23°C, 18 h
32 (R = H, quant.)
COMU, DIPEA
DMF, 23°C, 4.5 h
N3
NH2 — HCl
33
O
N3
N
H
NH
CO2Et
EtO2C
O
N
H
HN
34 (39%)
N
O
O
CH2Cl2, 23°C, 17 h
O
+ BnSH, NEt3
N3
N
H
NH
CO2Et
EtO2C
O
H
N
HN
35 (41%)
N
SBn
O
O
Scheme 8.
Preparation of bifunctional probe 34 with azide and maleimide functional units
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from dicarboxylic acid precursor 5; "turn on" of fluorescence upon reaction with BnSH.
4
5
6
7
8
9
Spectroscopy. All compounds with diaminoterephthalate chromophore are yellow, orange or
red, fluorescent materials (Table 1). As expected for a typical pushꢀpull aromatic system, the
absorption and emission wavelengths are sensitively dependent on the electron accepting or
donating nature of the substituents at both nitrogen atoms bound to the aromatic ring. The
prototypic compound 13 carries two NH2 groups and shows absorption and emission at 432
nm and 532 nm, resp. (Table 1, entry 1, Stokes shift 100 nm). When introducing a single alkyl
residue at only one nitrogen (compounds 16 and 17 in entries 5 and 6), both, absorption and
emission receive a bathochromic shift of ca. 20 nm. With a second alkyl residue at the other
nitrogen, both bands were shifted further towards the red region by ca. 20 nm. Most products
in this study (compounds 3–5 and 18–35, entries 7–24) represent this substitution pattern.
They show absorption at 466–478 nm and emission band at 564–571 nm, thus, the Stokes
shift is in the range of 92–106 nm. On the other hand, lowering electron density by
introduction of a carbamate group resulted in hypsochromic shifts: Compound 8 (entry 2)
absorbs at 408 nm and emits at 487 nm. This effect is at least partly compensated by
introduction of an alkyl group at the remaining NH2 function of compound 8: Products 14 and
15 showed absorption at 426–430 nm and emission at 505–511 nm (entries 3 and 4). It is
furthermore evident, that compounds with a NꢀBoc group show a Stokes shift of about 80 nm,
whereas all others have the Stokes shift around 100 nm.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
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40
41
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45
46
47
48
49
50
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52
53
54
55
56
57
58
59
60
The quantum yields range between 0.02 and 0.40, apart from compounds 20–21 (entries 12–
14), 25–28 (entries 16–18) and 31 (entry 21), where the fluorescence intensity is quenched by
the fullerene or maleimide moiety, resp. The "turnꢀon" effect of fluorescence by conjugated
addition of a thiol to the maleimide moiety is clearly visible for compounds 29 and 35 (entries
19 and 24), where it is higher than for the maleimide precursors 28 and 34.
Compound 20 with two chromophors, retinoate (RA) and diaminoterephthalate (DAT), shows
two absorption bands (335 nm at RA and 474 nm at DAT, entry 14), but only one emission
band at 554 nm (from DAT). If irradiated at 335 nm, this compound 20 shows emission also
at 554 nm due to a FRET process from RA to DAT moiety.
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Table 1.
Spectroscopic properties of diaminoterepthalates; solvent CH2Cl2.
4
5
Entry
Compound
λmax / nm
lg (ε / dm3 mol–1 cm–1)
λem / nm[a]
Φ[b]
6
7
8
9
1
432
408
430
426
455
452
478
474
472
469
475
475
448[d]
475, 335
469
474
466
473
473
466
470
466
469
469
3.75
3.87
3.86
3.78
3.77
3.85
3.75
3.80
3.99
3.70
3.71
3.87
532
487
511
505
553
551
570
567
564
571
570
567
552
554
571
571
568
566
570
571
567
572
568
567
0.18
0.21
0.34
0.40
0.13
0.10
0.10
0.10
0.07
0.05
0.02
0.001
13
2
8
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
14
4
15
5
16
6
17
7
3
8
4
9
5
18[c]
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
19
21
22[c]
[d]
[d]
–
–
3.49, 4.65
3.68
0.002
0.05
20
23[c]
25
26[c]
3.67
0.005
0.01
3.70
3.66
0.005
0.11
28
3.71
29
30[c]
3.80
0.05
3.66
0.007
0.02
31
32[c]
34
3.25
3.59
0.02
3.38
0.15
35
[a] Excitation at λmax of the absorption band. [b] Quantum yields were determined according
to the Parker Rees method38 using rhodamine B in EtOH as standard [λmax = 544 nm, lg(ε /
dm3 mol–1 cm–1) = 3.23, λem = 569 nm, Φ = 0.46].39 [c] Solvent MeOH. [d] Could not be
determined with certainty, because the absorption it is located as a shoulder in a very broad
UVꢀabsoption of the C60 unit.
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CONCLUSION
4
5
6
7
8
9
Diaminoterepthalates are bright and colorful dyes showing fluorescence with high quantum
yields. The chromophore defines a molecular scaffold allowing a flexible introduction of side
chains with various functional units. Herein we reported on the orthogonal functionalization at
the two amino functions with different effector groups, thus, tailored functional materials
could be prepared for addressing different applications of Life Sciences and Materials Sciꢀ
ence. In particular, the scaffold was equipped with protected amino and carboxylate functions
in three different manners: (1) with two protected propylamino functions (compound 3, NꢀBoc
and NꢀAlloc), (2) with two protected carboxyethyl groups (compound 5, tBu and allyl esters)
and (3) the mixed case with amino and carboxy functionalization (compound 4, NꢀBoc and
allyl ester). In all three cases, the two protective groups were orthogonally cleavable either
with Brønsted acid (NꢀBoc and tBu ester) or palladium catalysis (NꢀAlloc and allyl ester).
Starting from monoꢀNꢀBoc diethyl diaminoterephthalate 8, side chains were introduced by reꢀ
ductive amination of the two amino groups with carbaldehydes, which carried the above menꢀ
tioned orthogonally protected amino and carboxylate functionalities. The sequences gave the
intermediate compounds 3, 4, and 5 in 58%, 60% and 40% yields, respectively, over three
steps. As functional units, retinoic acid and fullerene C60 (both for studying electron transfer
processes), cyclooctyne and azide (for Click reactions) and maleimide (for ligation to proteins
by conjugate addition of cysteine residues) were chosen.
10
11
12
13
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17
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20
21
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24
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31
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58
59
60
First of all, the NꢀBoc and NꢀAlloc protected compound 3 was elaborated towards a molecular
triad equipped with an electron donating (retinoic acid) and accepting (fullerene C60) moiety
in order to obtain a molecular model 20 (in 32% yield over five steps) for studying photoinꢀ
duced electron transfer processes. As a second example, a cyclooctyneꢀmaleimide reactive
dye 28 was prepared from the NꢀBoc allyl ester 4 (68% over four steps). This compound was
designed to be conjugated by copperꢀfree alkyneꢀazide Click reaction as well as conjugated
addition of a thiol group to the maleimide moiety and could therefore serve as a fluorescent
crossꢀlinker for two proteins. A special feature of this compound 28 is the "turn on" effect of
fluorescence, since the fluorescence quantum yield was increased by an order of magnitude
upon chemical reaction with the molecular target; this feature was proven by reaction of comꢀ
pound 28 with benzyl azide and benzyl mercaptan as two model substrates. Finally, the
scaffold 5 with tBu and allyl ester functions was transformed to a reactive dye 34 (32% over
four steps) with azide and maleimide moieties again for Click reaction and conjugated adꢀ
dition with a thiol. Just as in the above case, a "turn on" effect by reaction with benzyl
mercaptan was observed.
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5
EXPERIMENTAL SECTION
6
7
8
9
General: Preparative column chromatography was carried out using Merck SiO2 (35–70 µm,
type 60 A) with hexanes (bp. 40–60°C), tertꢀbutyl methyl ether (MTBE), EtOAc or MeOH as
1
eluents. TLC was performed on aluminum plates coated with SiO2 F254. Hꢀ and 13CꢀNMR
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spectra were recorded on 500 MHz and 300 MHz instruments. Multiplicities of carbon signals
were determined with DEPT experiments. HRMS spectra were obtained with an EI spectroꢀ
meter (EI and CI) with sector field analyzer, an ESI spectrometer (pos. mode) with QꢀTOF
analyzer or in one case with a MALDI spectrometer. IR spectra were recorded on a spectroꢀ
meter equipped with a diamond ATR unit. UV/Vis spectra were recorded with a Shimadzu
UVꢀ1800, fluorescence spectra with a Shimadzu RFꢀ5301PC spectrometer. The following
compounds were prepared according to literature procedures: 6,17 7,21 9,18 10,24 12,22 13,11
24,37 and 33.16 All other starting materials were commercially available. Cyclooctyne derivaꢀ
tive 27 was purchased from Synaffix BV (Oss, Netherlands).
tert-Butyl Formylacetate (7). Under exclusion of air and moisture, a solution of formyl
Meldrum’s acid (12) (15.1 mmol, 2.60 g, 1.0 eq) and tert-butyl alcohol (18.1 mmol, 1.34 g,
1.2 eq) in abs. toluene (25 mL) was stirred at 80°C for 1.5 h. All volatile materials were
removed under reduced pressure at ambient temperature and the residue was purified by
Kugelrohr destillation (70°C, 10 mbar) to yield compound 7 (667 mg, 4.63 mmol, 31%) as a
colorless liquid. According to NMR spectroscopy, the compound is a mixture of an aldehyde
and an enol tautomer (aldehyde/enol = 0.65/0.35).
1H NMR (500 MHz, CDCl3), aldehyde: δ = 1.50 (s, 9H), 3.30 (d, J = 2.6 Hz, 2H), 9.79 (t, J =
2.6 Hz, 1H) ppm; enol: δ = 1.50 (s, 9H), 4.89 (d, J = 6.0 Hz, 1H), 7.06 (dd, J = 12.7 Hz, J =
6.1 Hz, 1H), 11.55 (d, J = 12.6 Hz, 1H) ppm. 13C{1H} NMR (125 MHz, CDCl3), aldehyde: δ
= 28.2 (3 CH3), 50.0 (CH2), 82.7 (C), 163.2 (C), 195.7 (CH) ppm; enol: δ = 28.4 (3 CH3), 81.4
(C), 95.0 (CH), 163.1 (CH), 172.2 (C) ppm. IR (ATR): λ–1 = 2980 (w), 1723 (m), 1657 (w),
1153 (m), 632 (s) cm–1. HRMS (ESI) m/z: [M + Na+] calcd. 167.0684 for C7H12O3Na; found
167.0690. C7H12O3 (144.17 g mol–1).
Allyl Formylacetate (10). Under exclusion of air and moisture, a solution of formyl
Meldrum’s acid (12) (10.7 mmol, 1.84 g, 1.0 eq) and allylic alcohol (12.9 mmol, 747 mg, 1.2
eq) in abs. toluene (24 mL) was stirred at 80°C for 1.5 h. All volatile materials were removed
under reduced pressure at ambient temperature and the residue was purified by Kugelrohr
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destillation (80°C, 3.2 mbar) to yield compound 10 (896 mg, 6.99 mmol, 65%) as a colorless
liquid. According to NMR spectroscopy, the compound is a mixture of an aldehyde and two
enol tautomers (aldehyde/enol1/enol2 = 0.57/0.26/0.17).
1H NMR (500 MHz, CDCl3), aldehyde: δ = 3.43 (d, J = 2.4 Hz, 2H), 4.68 (dt, J = 5.9 Hz, J =
1.3 Hz, 2H), 5.40–5.25 (m, 2H), 5.99–5.89 (m, 1H), 9.82 (t, J = 2.4 Hz, 1H) ppm; enol 1: δ =
4.65 (dt, J = 5.7 Hz, J = 1.4 Hz, 2H), 5.12 (d, J = 6.0 Hz, 1H), 5.40–5.25 (m, 2H), 5.99–5.89
(m, 1H), 7.13 (dd, J = 12.6 Hz, J = 6.0 Hz, 1H), 11.33 (d, J = 12.9 Hz, 1H) ppm; enol 2: δ =
4.78 (dt, J = 5.9 Hz, J = 1.3 Hz, 2H), 5.40–5.25 (m, 2H), 5.99–5.89 (m, 1H), 8.53 (s, 1H),
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13
12.66 (br.s, 1H), 14.49 (br.s, 1H) ppm. C{1H} NMR (125 MHz, CDCl3), aldehyde: δ = 48.6
(CH2), 66.2 (CH2), 119.3 (CH2), 131.5 (CH), 166.6 (C), 194.9 (CH) ppm; enol 1: δ = 65.0
(CH2), 93.3 (CH), 118.7 (CH2), 132.0 (CH), 164.1 (CH), 172.5 (C) ppm; enol 2: δ = 66.7
(CH2), 97.0 (CH), 120.0 (CH2), 131.0 (CH), 171.8 (C), 176.8 (CH) ppm. IR (ATR): λ–1
=
1718 (vs), 1182 (vs), 988 (s), 932 (s) cm–1. HRMS (CI) m/z: [M + H+] calcd. 129.0546 (for
C6H9O3; found 129.0550. C6H8O3 (128.13 g mol–1).
Diethyl 2,5-Diamino-N-(tert-butyloxycarbonyl)terephthalate (8). A solution of Boc2O
(4.83 mmol, 1.05 g, 1.0 eq) in CH2Cl2 (10 mL) was added dropwise to a cooled solution (iceꢀ
water bath) of diethyl 2,5ꢀdiaminoterephthalate (13) (4.83 mmol, 1.22 g, 1.0 eq) in CH2Cl2
(15 mL). The mixture was stirred for 2 d at ambient temperature and then poured into water
(50 mL). The layers were separated and the aqueous layer was extracted with CH2Cl2 (3 x 50
mL). The combined organic layers were dried over MgSO4, filtered and the solvent was evaꢀ
porated. The residue was chromatographed (SiO2, hexanes/EtOAc 5:1 with 1 vol% NEt3, Rf =
0.22) to yield compound 8 (1.53 g, 4.33 mmol, 90%) as a bright yellow solid, m.p. 121–
123°C.
1H NMR (500 MHz, CDCl3): δ = 1.38 (t, J = 7.1 Hz, 3H), 1.39 (t, J = 7.1 Hz, 3H), 1.51 (s,
9H), 4.34 (q, J = 7.1 Hz, 2H), 4.35 (q, J = 7.1 Hz, 2H), 5.49 (br.s, 2H), 7.32 (s, 1H), 8.77 (s,
1H), 9.52 (br.s, 1H) ppm. 13C{1H} NMR (125 MHz, CDCl3): δ = 14.3 (CH3), 14.5 (CH3), 28.5
(3 CH3), 61.1 (CH2), 61.7 (CH2), 80.1 (C), 116.1 (C), 118.7 (CH), 120.9 (C), 121.9 (CH),
130.8 (C), 144.3 (C), 153.2 (C), 167.2 (C), 167.6 (C) ppm. IR (ATR): λ–1 = 1690 (s), 1566 (s),
1525 (s), 1422 (m), 1367 (m), 1266 (m), 1207 (vs), 1154 (vs), 1101 (s), 792 (m) cm–1. MS
(EI, 70 eV), m/z (%): 352 (11) [M+], 296 (22), 252 (100), 233 (13), 224 (23), 206 (18), 205
(11), 178 (26), 133 (13), 132 (21), 131 (11), 57 (66), 41 (16). HRMS (EI) m/z: [M+] calcd.
352.1629 for C17H24N2O6; found 352.1629. UV/Vis (CH2Cl2): λmax (lg ε) = 408 nm (3.87);
fluorescence (CH2Cl2): λem = 487 nm, λex = 408 nm, Φ = 0.21. C17H24N2O6 (352.34 g mol–1).
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9
Diethyl 2-[3-(Allyloxycarbonylamino)propylamino]-5-(tert-butoxycarbonylamino)tere-
phthalate (14). Under exclusion of air and moisture, a solution of compound 9 (4.26 mmol,
670 mg, 1.5 eq) in abs. CH2Cl2 (10 mL) was added to a solution of compound 8 (2.84 mmol,
1.00 g, 1.0 eq) in abs. CH2Cl2 (10 mL). The mixture was stirred for 15 min at ambient tempeꢀ
rature. Subsequently, AcOH (4.26 mmol, 256 mg, 1.5 eq) and NaBH(OAc)3 (4.26 mmol, 903
mg, 1.5 eq) were added and the resulting mixture was stirred for 1 h at ambient temperature
and then poured into saturated aqueous NaHCO3ꢀsolution (50 mL). After extraction with
CH2Cl2 (3 x 50 mL), the combined organic layers were dried over MgSO4, filtered and the
solvent was removed under reduced pressure. The residue was chromatographed (SiO2, hexꢀ
anes/EtOAc 4:1, Rf = 0.25) to yield compound 14 (1.16 g, 2.34 mmol, 83%) as a yellow solid,
m.p. 128–130°C.
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28
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1H NMR (500 MHz, CDCl3): δ = 1.40 (t, J = 7.1 Hz, 3H), 1.42 (t, J = 7.1 Hz, 3H), 1.52 (s,
9H), 1.90 (quint, J = 6.8 Hz, 2H), 3.24–3.27 (m, 2H), 3.34 (q, J = 6.5 Hz, 2H), 4.35 (q, J = 7.0
Hz, 2H), 4.39 (q, J = 7.0 Hz, 2H), 4.56 (d, J = 5.0 Hz, 2H), 4.88 (br.s, 1H), 5.20 (dq J = 10.4
Hz, J = 1.3 Hz, 1H). 5.29 (dq, J = 17.2 Hz, J = 1.5 Hz, 1H), 5.91 (ddt, J = 17.1 Hz, J = 10.9
13
Hz, J = 5.7 Hz, 1H), 7.26 (s, 1H), 7.35 (br.s, 1H), 8.81 (s, 1H), 9.47 (br.s, 1H) ppm. C{1H}
NMR (125 MHz, CDCl3): δ = 14.4 (CH3), 14.5 (CH3), 28.5 (3 CH3), 29.6 (CH2), 39.1 (CH2),
40.6 (CH2), 61.1 (CH2), 61.8 (CH2), 65.7 (CH2), 80.1 (C), 112.9 (CH), 115.3 (C), 117.8
(CH2), 121.4 (CH), 122.9 (C), 129.6 (C), 133.0 (CH), 145.3 (C), 153.3 (C), 156.5 (C), 167.5
(C), 168.1 (C) ppm. IR (ATR): λ–1 = 1702 (s), 1677 (s), 1534 (s), 1237 (s), 1211 (vs), 1154 (s)
cm–1. MS (EI, 70 eV), m/z (%): 493 (17) [M+], 437 (54), 393 (85), 379 (40), 335 (100), 291
(33), 265 (61), 245 (75), 219 (89), 57 (32). HRMS (EI) m/z: [M+] calcd. 493.2419 for
C24H35N3O8; found 493.2413. UV/Vis (CH2Cl2): λmax (lg ε) = 430 nm (3.86); fluorescence
(CH2Cl2): λem = 511 nm, λex = 430 nm, Φ = 0.34. C24H35N3O8 (493.56 g mol–1).
Diethyl 2-[2-(Allyloxycarbonyl)ethylamino]-5-(tert-butoxycarbonylamino)terephthalate
(15). A solution of compound 10 (0.43 mmol, 55 mg, 1.5 eq) in CH2Cl2 (1 mL) was added unꢀ
der cooling (iceꢀwater bath) to a solution of compound 8 (0.28 mmol, 100 mg, 1.0 eq) in
DCM (2 mL). The mixture was stirred for 1 h at 0°C. Then ZnCl2 (0.14 mmol, 19 mg, 0.5 eq)
and NaBH3CN (0.43 mmol, 27 mg, 1.5 eq) were added and the resulting mixture was stirred
for 2 d at ambient temperature. It was then poured into water (30 mL) and extracted with
CH2Cl2 (3 x 30 mL). The combined organic layers were dried over MgSO4, filtered and the
solvent was removed under reduced pressure. The residue was chromatographed (SiO2, hexꢀ
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anes/EtOAc 6:1, Rf = 0.46) to yield compound 15 (116 mg, 0.25 mmol, 89%) as a yellow soꢀ
lid, m.p. 123–125°C.
1H NMR (500 MHz, CDCl3): δ = 1.39 (t, J = 7.1 Hz, 3H), 1.41 (t, J = 7.1 Hz, 3H), 1.51 (s,
9H), 2.70 (t, J = 6.8 Hz, 2H), 3.55 (q, J = 6.7 Hz, 2H), 4.35 (q, J = 7.1 Hz, 2H), 4.39 (q, J =
7.1 Hz, 2H), 4.62 (d, J = 5.8 Hz, 2H), 5.23 (dq, J = 10.4 Hz, J = 1.3 Hz, 1H), 5.31 (dq, J =
17.2 Hz, J = 1.4 Hz, 1H), 5.91 (ddt, J = 16.2 Hz, J = 10.5 Hz, J = 5.8 Hz, 1H), 7.30 (s, 1H),
7.50 (t, J = 5.6 Hz, 1H), 8.83 (s, 1H), 9.50 (br.s, 1H) ppm. 13C{1H} NMR (125 MHz, CDCl3):
δ = 14.3 (CH3), 14.4 (CH3), 28.5 (3 CH3), 34.2 (CH2), 38.9 (CH2), 61.1 (CH2), 61.7 (CH2),
65.5 (CH2), 80.1 (C), 112.8 (CH), 115.7 (C), 118.5 (CH2), 121.2 (C), 122.9 (CH), 129.9 (C),
132.1 (CH), 144.8 (C), 153.2 (C), 167.4 (C), 167.9 (C), 171.5 (C) ppm. IR (ATR): λ–1 = 1716
(s), 1687 (s), 1546 (s), 1243 (s), 1223 (vs), 1176 (s), 1155 (s), 1102 (s) cm–1. MS (EI, 70 eV),
m/z (%): 464 (17) [M+], 408 (40), 365 (26), 364 (100), 363 (17), 265 (25), 263 (18), 245 (16),
219 (59), 57 (56), 41 (41). HRMS (EI) m/z: [M+] calcd. 464.2153 for C23H32N2O8; found
464.2155. UV/Vis (CH2Cl2): λmax (lg ε) = 426 nm (3.78); fluorescence (CH2Cl2): λem = 505
nm, λex = 426 nm, Φ = 0.40. C23H32N2O8 (464.52 g mol–1).
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Diethyl 2-[3-(Allyloxycarbonylamino)propylamino]-5-aminoterephthalate (16). TFA (25
mL) was added dropwise to a cooled (iceꢀwater bath) solution of compound 14 (3.28 mmol,
1.62 g) in CH2Cl2 (25 mL). The mixture was stirred for 18 h at ambient temperature. All volaꢀ
tile materials were then removed under reduced pressure. The residue was suspended in aqueꢀ
ous KOH solution (10%, 30 mL), the mixture was diluted with water (20 mL) and extracted
with CH2Cl2 (3 x 50 mL). The combined organic layers were dried over MgSO4, filtered and
the solvent was evaporated. The residue was chromatographed (SiO2, hexanes/EtOAc 2:1
with 1 vol% NEt3, Rf = 0.20) to yield compound 16 (1.28 g, 3.25 mmol, 99%) as an orange
solid, m.p. 135–136°C.
1H NMR (500 MHz, CDCl3): δ = 1.37 (t, J = 7.1 Hz, 3H), 1.39 (t J = 7.1 Hz, 3H), 1.88 (quint,
J = 6.7 Hz, 2H), 3.21 (t, J = 6.6 Hz, 2H), 3.33 (q, J = 6.6 Hz, 2H), 4.30 (q, J = 7.1 Hz, 2H),
4.36 (q, J = 7.1 Hz, 2H), 4.55 (d, J = 5.2 Hz, 2H), 4.93 (br.s, 1H), 5.04 (br.s, 2H), 5.19 (dq, J
= 10.5 Hz, J = 1.3 Hz, 1H), 5.29 (dq, J = 17.1 Hz, J = 1.5 Hz, 1H), 5.91 (ddt, J = 16.2 Hz, J =
10.9 Hz, J = 5.6 Hz, 1H), 6.81 (br.s, 1H), 7.19 (s, 1H), 7.34 (s, 1H) ppm. 13C{1H} NMR (125
MHz, CDCl3): δ = 14.4 (CH3), 14.5 (CH3), 29.7 (CH2), 39.2 (CH2), 41.1 (CH2), 60.9 (CH2),
61.0 (CH2), 65.6 (CH2), 113.2 (CH), 117.0 (C), 117.7 (CH2), 118.1 (C), 119.9 (CH), 133.1
(CH), 139.5 (C), 142.4 (C), 156.4 (C), 167.6 (C), 167.8 (C) ppm. IR (ATR): λ–1 = 1692 (s),
1677 (s), 1203 (vs), 1102 (s) cm–1. MS (EI, 70 eV), m/z (%): 393 (36) [M+], 335 (36), 265
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(31), 259 (20), 219 (100), 191 (23), 165 (17), 73 (45), 66 (13), 57 (17). HRMS (EI) m/z: [M+]
calcd. 393.1894 for C19H27N3O6; found 393.1900. UV/Vis (CH2Cl2): λmax (lg ε) = 455 nm
(3.77); fluorescence (CH2Cl2): λem = 553 nm, λex = 455 nm, Φ = 0.13. C19H27N3O6 (393.44 g
mol–1).
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Diethyl 2-[2-(Allyloxycarbonyl)ethylamino]-5-aminoterephthalate (17). TFA (6 mL) was
added dropwise to a cooled (iceꢀwater bath) solution of compound 15 (0.95 mmol, 441 mg) in
CH2Cl2 (6 mL). The mixture was stirred for 16 h at ambient temperature. All volatile materiꢀ
als were then removed under reduced pressure. The residue was suspended in aqueous KOH
solution (10%, 10 mL), the mixture was diluted with water (15 mL) and extracted with
CH2Cl2 (3 x 25 mL). The combined organic layers were dried over MgSO4, filtered and the
solvent was evaporated. The residue was chromatographed (SiO2, hexanes/EtOAc 4:1 with 1
vol% NEt3, Rf = 0.24) to yield compound 17 (308 mg, 0.85 mmol, 89%) as an orange solid,
m.p. 96–98°C.
1H NMR (500 MHz, CDCl3): δ = 1.37 (t, J = 7.1 Hz, 3H), 1.39 (t, J = 7.1 Hz, 3H), 2.69 (t, J =
6.8 Hz, 2H), 3.51 (t, J = 6.1 Hz, 2H), 4.31 (q, J = 7.1 Hz, 2H), 4.35 (q, J = 7.1 Hz, 2H), 4.61
(dt, J = 5.6 Hz, J = 1.3 Hz, 2H), 5.06 (br.s, 2H), 5.22 (dq, J = 10.4 Hz, J = 1.2 Hz, 1H), 5.31
(dq, J = 17.2 Hz, J = 1.4 Hz, 1H), 5.92 (ddt, J = 17.2 Hz, J = 10.5 Hz, J = 5.8 Hz, 1H), 6.89
(br.s, 1H), 7.24 (s, 1H), 7.34 (s, 1H) ppm. 13C{1H} NMR (125 MHz, CDCl3): δ = 14.4 (CH3),
14.5 (CH3), 34.3 (CH2), 39.4 (CH2), 60.7 (CH2), 60.9 (CH2), 65.4 (CH2), 113.2 (CH), 117.4
(C), 117.9 (C), 118.4 (CH2), 120.0 (CH), 132.2 (CH), 139.7 (C), 141.7 (C), 167.57 (C),
167.58 (C), 171.8 (C) ppm. IR (ATR): λ–1 = 1687 (s), 1209 (vs), 1102 (s), 632 (s) cm–1. MS
(EI, 70 eV), m/z (%): 365 (16), 364 (77) [M+], 265 (27), 220 (12), 219 (100), 191 (24), 173
(13), 132 (13), 41 (19). HRMS (EI) m/z: [M+] calcd. 364.1629 for C18H24N2O6; found
364.1632. UV/Vis (CH2Cl2): λmax (lg ε) = 452 nm (3.85); fluorescence (CH2Cl2): λem = 551
nm, λex = 452 nm, Φ = 0.10. C18H24N2O6 (364.40 g mol–1).
Diethyl
2-[3-(Allyloxycarbonylamino)propylamino]-5-[3-(tert-butoxycarbonylamino)-
propylamino]terephthalate (3). The transformation was performed in two parallel batches,
which were executed as follows: Under exclusion of air and moisture, a solution of compound
6 (0.38 mmol, 66 mg, 1.5 eq) in abs. CH2Cl2 (3 mL) was added to a cooled (iceꢀwater bath)
solution of compound 16 (0.25 mmol, 100 mg, 1.0 eq) in abs. CH2Cl2 (3 mL). The mixture
was stirred for 15 min at 0°C. Subsequently NaBH(OAc)3 (0.38 mmol, 81 mg, 1.5 eq) was adꢀ
ded and the resulting mixture was stirred for 18 h at ambient temperature. The two batches
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were poured together into saturated aqueous NaHCO3ꢀsolution (30 mL). After extraction with
CH2Cl2 (3 x 30 mL), the combined organic layers were dried over MgSO4, filtered and the
solvent was evaporated. The residue was chromatographed (SiO2, hexanes/EtOAc 4:1, Rf =
0.23) to yield compound 3 (193 mg, 0.35 mmol, 70%) as a red solid, m.p. 133–134°C.
1H NMR (500 MHz, CDCl3): δ = 1.39 (t, J = 7.1 Hz, 6 Hz), 1.43 (s, 9 H), 1.86 (quint, J = 6.7
Hz, 2H), 1.88 (quint, J = 6.7 Hz, 2H), 3.22 (q, J = 7.0 Hz, 4H), 3.26 (q, J = 6.1 Hz, 2H), 3.33
(q, J = 6.5 Hz, 2H), 4.34 (q, J = 7.1 Hz, 4H), 4.55 (d, J = 4.5 Hz, 2H), 4.67 (br.s, 1H), 4.91
(br.s, 1H), 5.19 (dq, J = 10.4 Hz, J = 1.4 Hz, 1H), 5.29 (dq, J = 17.2 Hz, J = 1.6 Hz, 1H), 5.91
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(ddt, J = 17.0 Hz, J = 10.6 Hz, J = 5.6 Hz, 1H), 6.79 (br.s, 2H), 7.28 (s, 2H) ppm. C{1H}
NMR (125 MHz, CDCl3): δ = 14.5 (2 CH3), 28.5 (3 CH3), 29.7 (CH2), 29.9 (CH2), 38.7
(CH2), 39.2 (CH2), 41.3 (CH2), 41.4 (CH2), 61.0 (2 CH2), 65.6 (CH2), 79.3 (C), 87.4 (C),
114.3 (2 CH), 117.2 (C), 117.3 (C), 117.7 (CH2), 133.1 (CH), 141.2 (C), 141.3 (C), 156.1 (C),
156.5 (C), 168.0 (C) ppm. IR (ATR): λ–1 = 1675 (s), 1548 (s), 1194 (vs), 1164 (s), 1110 (s),
1088 (s) cm–1. MS (EI, 70 eV), m/z (%): 550 (100) [M+], 494 (92), 476 (14), 450 (44), 406
(10), 360 (14), 302 (32), 276 (9), 219 (9), 142 (9), 57 (13). HRMS (EI) m/z: [M+] calcd.
550.2997 for C27H42N4O8; found 550.3004. UV/Vis (CH2Cl2): λmax (lg ε) = 478 nm (3.75);
fluorescence (CH2Cl2): λem = 570 nm, λex = 478 nm, Φ = 0.10. C27H42N4O8 (550.65 g mol–1).
Diethyl 2-[2-(Allyloxycarbonyl)ethylamino]-5-[3-(tert-butoxycarbonylamino)propylami-
no]terephthalate (4). Under exclusion of air and moisture, a solution of compound 6 (0.49
mmol, 86 mg, 1.5 eq) in abs. CH2Cl2 (3 mL) was added to a cooled (iceꢀwater bath) solution
of compound 17 (0.33 mmol, 119 mg, 1.0 eq) in abs. CH2Cl2 (3 mL). The mixture was stirred
for 15 min at 0°C. Subsequently, NaBH(OAc)3 (0.49 mmol, 104 mg, 1.5 eq) was added and
the resulting mixture was stirred for 18 h at ambient temperature. It was then poured into satuꢀ
rated aqueous NaHCO3ꢀsolution (15 mL). After extraction with CH2Cl2 (3 x 15 mL), the comꢀ
bined organic layers were dried over MgSO4, filtered and the solvent was evaporated. The
residue was chromatographed (SiO2, hexanes/EtOAc 4:1, Rf = 0.46) to yield compound 4
(131 mg, 0.25 mmol, 76%) as a red solid, m.p. 94–96°C.
1H NMR (500 MHz, CDCl3): δ = 1.37 (t, J = 7.1 Hz, 6H), 1.42 (s, 9H), 1.84 (quint, J = 6.7
Hz, 2H), 2.68 (t, J = 6.8 Hz, 2H), 3.20 (t, J = 6.7 Hz, 2H), 3.25 (q, J = 6.1 Hz, 2H), 3.50 (t, J
= 6.8 Hz, 2H), 4.33 (q, J = 7.1 Hz, 4H), 4.60 (d, J = 5.7 Hz, 2H), 4.69 (br.s, 1H), 5.21 (dd, J =
10.4 Hz, J = 1.1 Hz, 1H), 5.30 (dd, J = 17.2 Hz, J = 1.3 Hz, 1H), 5.91 (ddt, J = 17.1 Hz, J =
10.6 Hz, J = 5.7 Hz, 1H), 6.86 (br.s, 2H), 7.27 (s, 1H), 7.31 (s, 1H) ppm. 13C{1H} NMR (125
MHz, CDCl3): δ = 14.4 (2 CH3), 28.5 (3 CH3), 29.9 (CH2), 34.4 (CH2), 38.7 (CH2), 39.5
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(CH2), 41.2 (CH2), 60.86 (CH2), 60.90 (CH2), 65.4 (CH2), 79.2 (C), 114.25 (CH), 114.27
(CH), 116.9 (C), 117.7 (C), 118.3 (CH2), 132.2 (CH), 140.5 (C), 141.6 (C), 156.1 (C), 167.8
(C), 168.0 (C), 171.7 (C) ppm. IR (ATR): λ–1 = 1674 (s), 1514 (s), 1196 (s), 1164 (vs), 1106
(s), 1086 (s), 787 (s) cm–1. MS (EI, 70 eV), m/z (%): 521 (100) [M+], 465 (97), 448 (10), 421
(16), 377 (9), 331 (22), 57 (14). HRMS (EI) m/z: [M+] calcd. 521.2732 for C26H39N3O8; found
521.2729. UV/Vis (CH2Cl2): λmax (lg ε) = 474 nm (3.80); fluorescence (CH2Cl2): λem = 567
nm, λex = 474 nm, Φ = 0.10. C26H39N3O8 (521.61 g mol–1).
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Diethyl 2-[2-(Allyloxycarbonyl)ethylamino]-5-[2-(tert-butoxycarbonyl)ethylamino]tere-
phthalate (5). A solution of compound 7 (0.74 mmol, 106 mg, 1.5 eq) in CH2Cl2 (3 mL) was
added under cooling (iceꢀwater bath) to a solution of compound 17 (0.49 mmol, 186 mg, 1.0
eq) in CH2Cl2 (2 mL). After stirring for 15 min at 0°C ZnCl2 (0.25 mmol, 33 mg, 0.5 eq) was
added and it was stirred for 15 min at 0°C. Subsequently, NaBH3CN (0.74 mmol, 46 mg, 1.5
eq) was added and the resulting mixture was stirred for 2 d at ambient temperature. It was
then poured into water (10 mL) and extracted with CH2Cl2 (3 x 30 mL). The combined orꢀ
ganic layers were dried over MgSO4, filtered and the solvent was removed under reduced
pressure. The residue was chromatographed (SiO2, hexanes/EtOAc 4:1, Rf = 0.33) to yield
compound 5 (122 mg, 0.25 mmol, 51%) as a red solid, m.p. 59–61°C.
1H NMR (500 MHz, CDCl3): δ = 1.361 (t, J = 7.1 Hz, 3H), 1.363 (t, J = 7.1 Hz, 3H), 1.44 (s,
9H), 2.55 (t, J = 6.8 Hz, 2H), 2.67 (t, J = 6.8 Hz, 2H), 3.44 (t, J = 6.6 Hz, 2H), 3.50 (t, J = 6.7
Hz, 2H), 4.32 (q, J = 7.1 Hz, 4H), 4.59 (dt, J = 5.7 Hz, J = 1.3 Hz, 2H), 5.20 (dq, J = 10.5 Hz,
J = 1.3 Hz, 1H), 5.29 (dq, J = 17.2 Hz, J = 1.5 Hz, 1H), 5.90 (ddt, J = 17.0 Hz, J = 10.6 Hz, J
= 5.7 Hz, 1H), 6.94 (br.s, 2H), 7.30 (s, 2H) ppm. 13C{1H} NMR (125 MHz, CDCl3): δ = 14.4
(2 CH3), 28.1 (3 CH3), 34.3 (CH2), 35.6 (CH2), 39.4 (CH2), 39.6 (CH2), 60.8 (2 CH2), 65.4
(CH2), 80.8 (C), 114.2 (CH), 114.3 (CH), 117.3 (C), 117.5 (C), 118.3 (CH2), 132.2 (CH),
140.7 (C), 141.0 (C), 167.7 (C), 167.8 (C), 171.4 (C), 171.7 (C) ppm. IR (ATR): λ–1 = 1727
(s), 1676 (s), 1528 (s), 1206 (vs), 1154 (vs), 1118 (s), 1107 (s), 1094 (s), 1018 (s), 788 (s) cm–
1. MS (EI, 70 eV), m/z (%): 493 (13), 492 (44) [M+], 437 (22), 436 (100), 377 (14), 331 (21),
291 (27), 245 (10), 231 (13), 199 (9), 173 (10), 57 (22). HRMS (EI) m/z: [M+] calcd.
492.2466 for C25H36N2O8; found 492.2472. UV/Vis (CH2Cl2): λmax (lg ε) = 472 nm (3.99);
fluorescence (CH2Cl2): λem = 564 nm, λex = 472 nm, Φ = 0.07. C25H36N2O8 (492.57 g mol–1).
Diethyl 2-[3-(Aminopropyl)amino]-5-[3-(tert-butoxycarbonylamino)propylamino]tere-
phthalate (18). Morpholine (1.35 mmol, 118 mg, 5.0 eq) was added to a solution of comꢀ
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pound 3 (0.27 mmol, 146 mg, 1.0 eq) in abs. CH2Cl2 (4 mL). After degassing the mixture,
Pd(PPh3)4 (13 µmol, 15 mg, 0.05 eq) was added under a nitrogen atmosphere and the mixture
was stirred for 1.5 h at ambient temperature. Charcoal (spatula tip, ca. 2 mg) was added and
the mixture was stirred for additional 5 min. After filtration, all volatile materials were evaꢀ
porated. The residue was chromatographed [SiO2, EtOAc → EtOAc/MeOH 6:1 with 1 vol%
NEt3, Rf(EtOAc/MeOH 6:1) = 0.15] to yield compound 18 (125 mg, 0.27 mmol, quant.) as a
red solid, m.p. 160–161°C.
1H NMR (500 MHz, CD3OD): δ = 1.38 (t, J = 7.1 Hz, 6H), 1.43 (s, 9H), 1.80 (quint, J = 6.7
Hz, 2H), 2.03 (quint, J = 6.8 Hz, 2H), 3.07–3.10 (m, 2H), 3.16 (t, J = 6.4 Hz, 2H), 3.18 (t, J =
6.6 Hz, 2H), 3.28–3.31 (m, 2H), 4.34 (q, J = 7.1 Hz, 4H), 7.30 (s, 1H), 7.32 (s, 1H) ppm.
13C{1H} NMR (125 MHz, CD3OD): δ = 14.7 (2 CH3), 28.3 (CH2), 28.8 (3 CH3), 30.7 (CH2),
39.0 (CH2), 39.2 (CH2), 41.6 (CH2), 42.1 (CH2), 61.96 (CH2), 61.99 (CH2), 79.9 (C), 115.3
(CH), 115.4 (CH), 118.1 (C), 118.8 (C), 141.7 (C), 142.7 (C), 158.5 (C), 168.9 (C), 169.0 (C)
ppm. IR (ATR): λ–1 = 1674 (s), 1199 (vs), 1176 (s) cm–1. MS (EI, 70 eV), m/z (%): 466 (100)
[M+], 410 (71), 393 (9), 366 (19), 322 (14), 276 (15), 219 (9), 59 (9). HRMS (EI) m/z: [M+]
calcd. 466.2786 for C23H38N4O6; found 466.2789. UV/Vis (MeOH): λmax (lg ε) = 469 nm
(3.70); fluorescence (MeOH): λem = 571 nm, λex = 469 nm, Φ = 0.05. C23H38N4O6 (466.58 g
mol–1).
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Diethyl 2-[3-(tert-butoxycarbonylamino)propylamino]-5-[3-(4-formylbenzamido)propyl-
amino]terephthalate (19). COMU (0.27 mmol, 116 mg, 1.2 eq) was added to a solution of 4ꢀ
carboxybenzaldehyde (0.27 mmol, 41 mg, 1.2 eq) and DIPEA (0.27 mmol, 35 mg, 1.2 eq) in
CH2Cl2 (2 mL). After stirring the mixture for 30 min at ambient temperature, a solution of
compound 18 (0.23 mmol, 105 mg, 1.0 eq) and DIPEA (23 mmol, 30 mg, 1.0 eq) was added
and the mixture was stirred for 17 h at ambient temperature. The mixture was then poured into
water (25 mL) and extracted with CH2Cl2 (3 x 25 mL). The combined organic layers were
dried over MgSO4, filtered and all volatile materials were evaporated. The residue was chroꢀ
matographed (SiO2, hexanes/EtOAc 1:1, Rf = 0.28) to yield compound 19 (67 mg, 0.11 mmol,
50%) as a red solid, m.p. 125–127°C.
1H NMR (500 MHz, CDCl3): δ = 1.36 (t, J = 7.1 Hz, 3H), 1.37 (t, J = 7.1 Hz, 3H), 1.42 (s,
9H), 1.85 (quint, J = 6.6 Hz, 2H), 2.02 (quint, J = 6.2 Hz, 2H), 3.20 (t, J = 6.7 Hz, 2H), 3.26
(q, J = 6.1 Hz, 2H), 3.31 (t, J = 6.1 Hz, 2H), 3.64 (q, J = 6.1 Hz, 2H), 4.30 (q, J = 7.2 Hz, 2H),
4.31 (q, J = 7.2 Hz, 2H), 4.72 (br.s, 1H), 6.80 (br.s, 2H), 6.91–6.94 (m, 1H), 7.26 (s, 1H), 7.30
(s, 1H), 7.85–7.89 (m, 4H), 10.03 (s, 1H) ppm. 13C{1H} NMR (125 MHz, CDCl3): δ = 14.4 (2
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CH3), 28.5 (3 CH3), 28.9 (CH2), 29.9 (CH2), 38.7 (CH2), 39.2 (CH2), 41.2 (CH2), 42.2 (CH2),
61.03 (CH2), 61.04 (CH2), 79.3 (C), 114.2 (CH), 114.4 (CH), 116.9 (C), 117.5 (C), 127.8 (2
CH), 129.8 (2 CH), 138.1 (C), 139.9 (C), 140.9 (C), 141.7 (C), 156.1 (C), 166.5 (C), 167.9
(C), 168.1 (C), 191.7 (CH) ppm. IR (ATR): λ–1 = 1673 (s), 1524 (s), 1203 (vs), 1171 (s), 1111
(s) cm–1. MS (EI, 70 eV), m/z (%): 598 (52) [M+], 542 (40), 524 (100), 480 (16), 374 (12),
362 (16), 328 (22), 316 (36), 302 (55), 190 (16), 133 (36), 59 (14). HRMS (EI) m/z: [M+]
calcd. 598.2997 for C31H42N4O8; found 598.3011. UV/Vis (CH2Cl2): λmax (lg ε) = 475 nm
(3.71); fluorescence (CH2Cl2): λem = 570 nm, λex = 475 nm, Φ = 0.02. C31H42N4O8 (598.70 g
mol–1).
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Diethyl
2-[3-(tert-butoxycarbonylamino)propylamino]-5-{3-[4-(1-methyl-2,3,4,5-tetra-
hydro[60]fullero[1',2':3,4]pyrrol-2-yl)benzamido]propylamino}terephthalate (21). Under
exclusion of air and moisture, fullereneꢀC60 (127 µmol, 84 mg, 2.0 eq) and sarcosine (580
µmol, 52 mg, 10.0 eq) were added to a solution of compound 19 (58 µmol, 35 mg, 1.0 eq) in
abs. toluene (100 mL). The mixture was heated to reflux for 17 h. Subsequently, the solvent
was evaporated and the residue was chromatographed [SiO2, hexanes/EtOAc 1:1 → EtOAc,
Rf = 0.50 (hexanes/EtOAc 1:1)]. For further purification, it was recrystallized
(CH2Cl2/hexanes 5:1, 200 mL) to yield compound 21 (75 mg, 557 µmol, 95%) as a brownishꢀ
black solid, m.p. > 300°C.
1H NMR (500 MHz, CDCl3): δ = 1.37 (t, J = 7.1 Hz, 3H), 1.39 (t, J = 7.1 Hz, 3H), 1.44 (s,
9H), 1.86 (quint, J = 6.6 Hz, 2H), 2.01 (quint, J = 6.4 Hz, 2H), 2.79 (s, 3H), 3.21 (t, J = 6.1
Hz, 2H), 3.25–3.31 (m, 4H), 3.59–6.64 (m, 2H), 4.27 (d, J = 9.5 Hz, 1H), 4.32 (q, J = 7.2 Hz,
2H), 4.34 (q, J = 7.1 Hz, 2H), 4.66 (br.s, 1H), 4.97 (s, 1H), 4.99 (d, J = 9.5 Hz, 1H), 6.60
(br.s, 1H), 7.29 (s, 1H), 7.34 (s, 1H), 7.83–7.94 (m, 4H) ppm; signals for two NHꢀprotons are
not observed. 13C{1H} NMR (125 MHz, CDCl3): δ = 14.52 (CH3), 14.54 (CH3), 28.6 (3 CH3),
29.2 (CH2), 29.8 (CH2), 38.7 (CH2), 38.8 (CH2), 40.1 (CH3), 41.4 (CH2), 42.3 (CH2), 61.0
(CH2), 61.1 (CH2), 69.2 (C), 70.2 (CH2), 79.4 (C), 83.3 (CH), 114.4 (CH), 114.9 (CH), 117.1
(C), 117.7 (C), 127.5 (2 CH), 129.6 (2 CH), 134.8 (C), 135.8 (C), 136.1 (C), 136.6 (C), 137.1
(C), 139.7 (C), 140.1 (C), 140.3 (C), 140.4 (C), 140.8 (C), 141.7 (C), 141.8 (C), 141.97 (C),
142.00 (C), 142.12 (C), 142.16 (C), 142.19 (C), 142.24 (C), 142.27 (C), 142.31 (C), 142.4 (2
C), 142.68 (C), 142.73 (2 C), 142.9 (C), 143.15 (C), 143.24 (2 C), 143.3 (C), 144.5 (C), 144.6
(C), 144.7 (C), 144.9 (C), 145.3 (C), 145.37 (C), 145.42 (2 C), 145.49 (2 C), 145.54 (C),
145.66 (C), 145.69 (2 C), 145.9 (C), 146.0 (C), 146.09 (C), 146.10 (C), 146.26 (C), 146.30
(C), 146.33 (C), 146.37 (C), 146.41 (C), 146.46 (C), 146.52 (C), 146.7 (C), 147.5 (2 C), 152.9
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(C), 153.1 (C), 154.0 (C), 156.1 (C), 156.2 (C), 167.4 (C), 167.9 (C), 168.1 (C) ppm. The
signal of one quaternary bridgehead carbon atom is hidden by the CDCl3 triplet, but it can be
3
detected at ca. 77 ppm by a J(1H,13C) crosspeak in the HMBC spectrum to the pyrrolidine
proton at 4.97 ppm. IR (ATR): λ–1 = 1682 (s), 1531 (s), 1213 (s), 1192 (vs), 1167 (s), 1092 (s)
cm–1. HRMS (ESI) m/z: [M + H+] calcd. 1346.3548 for C93H48N5O7; found 1346.3539.
UV/Vis (CH2Cl2): λmax (lg ε) = 475 nm (3.87); fluorescence (CH2Cl2): λem = 567 nm, λex =
475 nm, Φ = 0.001. C93H47N5O7 (1346.43 g cm–1).
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Diethyl
2-[3-(aminopropyl)amino]-5-{3-[4-(1-methyl-2,3,4,5-tetrahydro[60]fullero-
[1',2':3,4]pyrrol-2-yl)benzamido]propylamino}terephthalate (22). TFA (5 mL) was added
dropwise to a cooled (iceꢀwater bath) solution of compound 21 (39 µmol, 52 mg) in CH2Cl2
(5 mL). The mixture was stirred for 18 h at ambient temperature. All volatile materials were
then removed under reduced pressure. The residue was suspended in a mixture of aqueous
KOH solution (10%, 20 mL) and CH2Cl2 (25 mL). The undissolved material was dissolved by
first stirring the mixture at 40°C for 5 min in then using an ultrasonic bath (ambient temperaꢀ
ture, 5 min). The layers were separated and the aqueous layer was extracted with CH2Cl2 (2 x
25 mL). The combined organic layers were dried over a small amount (400 mg) of MgSO4,
filtered and the solvent was evaporated to yield compound 21 (44 mg, 35 µmol, 91%) as a
brownishꢀblack solid, m.p. > 300°C.
1H NMR (300 MHz, CDCl3): δ = 1.38 (q, J = 7.1 Hz, 6H), 1.83 (quint, J = 6.8 Hz, 2H), 1.97–
2.04 (m, 2H), 2.79 (s, 3H), 2.87 (t, J = 7.0 Hz, 2H), 3.22–3.32 (m, 4H), 3.62 (q, J = 6.1 Hz,
2H), 4.26–4.37 (m, 5H), 4.97 (s, 1H), 4.99 (d, J = 9.4 Hz, 1H), 6.55 (br.s, 1H), 7.00 (br.s, 2H),
13
7.31 (s, 2H), 7.81–7.90 (m, 4H) ppm. C{1H} NMR (125 MHz, CDCl3): δ = 14.5 (2 CH3),
29.3 (CH2), 33.3 (CH2), 38.8 (CH2), 40.1 (CH2), 40.2 (CH3), 41.4 (CH2), 42.0 (CH2), 60.95
(CH2), 61.03 (CH2), 69.2 (C), 70.2 (CH2), 83.3 (CH), 114.3 (CH), 114.4 (CH), 116.8 (C),
117.5 (C), 127.5 (2 CH), 129.6 (CH), 129.7 (CH), 134.8 (C), 135.1 (C), 136.1 (C), 136.6 (C),
137.0 (C), 139.7 (C), 140.0 (C), 140.3 (C), 140.8 (C), 140.9 (C), 141.7 (C), 141.8 (2 C),
141.96 (C), 141.98 (C), 142.15 (C), 142.17 (C), 142.23 (C), 142.26 (C), 142.29 (C), 142.4 (2
C), 142.68 (C), 142.72 (2 C), 142.84 (C), 143.1 (C), 143.2 (2 C), 143.3 (C), 144.48 (C),
144.54 (C), 144.7 (C), 144.8 (C), 145.32 (C), 145.35 (C), 145.41 (2 C), 145.48 (2 C), 145.53
(C), 145.65 (C), 145.68 (2 C), 145.9 (C), 146.0 (C), 146.1 (2 C), 146.25 (C), 146.29 (C),
146.31 (C), 146.36 (C), 146.40 (C), 146.45 (C), 146.51 (C), 146.7 (C), 147.5 (2 C), 152.9 (C),
153.1 (C), 154.0 (C), 156.2 (C), 167.4 (C), 168.03 (C), 168.14 (C) ppm. The signal of one
quaternary bridgehead carbon atom is hidden by the CDCl3 triplet, but it can be detected at ca.
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77 ppm by a J(1H,13C) crosspeak in the HMBC spectrum to the pyrrolidine proton at 4.97
ppm. IR (ATR): λ–1 = 1668 (s), 1158 (s), 1131 (vs) cm–1. HRMS (ESI) m/z: [M + H+] calcd.
1246.3024 for C88H40N5O5; 1246.3024 found. UV/Vis (MeOH): λmax = 448 nm; fluorescence
(MeOH): λem = 552 nm, λex = 448 nm. Since the absorption band is located as a shoulder in a
very broad UVꢀabsorption of the C60 unit, the absorption coefficient ε and the calculated
quantum yield Φ could not be determined with certainty. C88H39N5O5 (1246.31 g mol–1).
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Diethyl 2-[3-(N-retinoylamino)propylamino]-5-{3-[4-(1-methyl-2,3,4,5-tetrahydro[60]ful-
lero[1’,2’:3,4]pyrrol-2-yl)benzamido]propylamino}terephthalate (20). HATU (36 µmol,
14 mg, 2.5 eq) was added to a solution of retinoic acid (36 µmol, 13 mg, 2.5 eq) and DIPEA
(49 µmol, 6.3 mg, 3.5 eq) in CH2Cl2 (5 mL). The mixture was stirred for 1.5 h at ambient
temperature and was then added to suspension of compound 22 (14 µmol, 18 mg, 1.0 eq) in
CH2Cl2 (5 mL). The mixture was stirred for 18 h at ambient temperature. Subsequently, the
mixture was diluted with CH2Cl2 (40 mL) and washed with saturated aqueous NaHCO3 soluꢀ
tion (4 x 50 mL). The organic layer was dried over MgSO4, filtered and the solvent was evaꢀ
porated. The residue was chromatographed (SiO2, hexanes/EtOAc 1:1 → EtOAc →
CH2Cl2/MeOH 20:1) to yield compound 20 in two fractions [(1): Rf(CH2Cl2/MeOH 20:1) =
0.77, 6.5 µmol, 10.0 mg, 47%; (2): Rf(CH2Cl2/MeOH 20:1) = 0.46, 3.9 µmol, 6.0 mg, 28%]
as a brownish black solid, m.p. 220°C (dec.).
Due to the high molecular mass, the low solubility and isomerization of the double bonds of
the retinoate residue, it was not possible to get satisfactory NMRꢀspectra. IR (ATR): λ–1
=
3374 (br), 1713 (s), 1693 (s), 1660 (s), 1651 (s), 1097 (vs), 1017 (s) cm–1. MS (MALDI,
DCTB), m/z (%): 1527 (100) [M+]. HRMS (ESI) m/z: [M + H+] calcd 1528.5008 for
C108H66N5O6; found 1528.4966. UV/Vis (CH2Cl2): λmax (lg ε) = 475 nm (3.49; DAT), 335 nm
(4.65; retinoate); fluorescence (CH2Cl2): λem = 554 nm, λex = 475 nm, Φ = 0.002. C108H65N5O6
(1528.74 g mol–1).
Diethyl
2-[3-(tert-butoxycarbonylamino)propylamino]-5-(2-carboxyethylamino)tere-
phthalate (23). Under exclusion of air and moisture, pyrrolidine (1.0 mmol, 71 mg, 5.0 eq)
was added to a solution of compound 4 (200 ꢁmol, 106 mg, 1.0 eq), in abs. CH2Cl2 (7 mL).
After degassing the mixture, Pd(PPh3)4 (10 µmol, 12 mg, 0.05 eq) was added under an nitroꢀ
gen atmosphere and the mixture was stirred for 1.5 h at ambient temperature. Charcoal (spatuꢀ
la tip, ca. 2 mg) was added and the mixture was stirred for additional 5 min. After filtration,
all volatile materials were evaporated. The residue was chromatographed [SiO2, EtOAc →
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EtOAc/MeOH 6:1, Rf(EtOAc/MeOH 6:1) = 0.38] to yield compound 23 (102 mg, 0.21 mmol,
quant.) as a red solid, m.p. 135–137°C.
1H NMR (500 MHz, CDCl3): δ = 1.377 (t, J = 7.1 Hz, 3H), 1.382 (t, J = 7.1 Hz, 3H), 1.44 (s,
9H), 1.85 (quint, J = 6.7 Hz, 2H), 2.69 (t, J = 6.7 Hz, 2H), 3.21 (t, J = 6.7 Hz, 2H), 3.24–3.28
(m, 2H), 3.50 (t, J = 6.4 Hz, 2H), 4.33 (q, J = 7.1 Hz, 2H), 4.34 (q, J = 7.1 Hz, 2H), 4.70 (br.s,
1H), 6.31 (br.s, 2H), 7.28 (s, 1H), 7.33 (s, 1H) ppm, the signal for the carboxylate H is not
observed. 13C{1H} NMR (125 MHz, CDCl3): δ = 14.5 (2 CH3), 28.5 (3 CH3), 29.9 (CH2),
34.3 (CH2), 38.8 (CH2), 39.6 (CH2), 41.3 (CH2), 60.96 (CH2), 61.04 (CH2), 79.4 (C), 114.4
(CH), 114.6 (CH), 116.9 (C), 118.1 (C), 140.4 (C), 141.8 (C), 156.2 (C), 167.9 (C), 168.0 (C),
176.6 (C) ppm. IR (ATR): λ–1 = 1674 (s), 1196 (vs), 1226 (s), 1109 (s), 817 (s) cm–1. MS (EI,
70 eV), m/z (%): 481 (89) [M+], 425 (100), 408 (11), 381 (16), 337 (18), 305 (8), 291 (42),
145 (6), 219 (8), 57 (15). HRMS (EI) m/z: [M+] calcd. 481.2419 for C23H35N3O8; found
481.2417. UV/Vis (MeOH): λmax (lg ε) = 469 nm (3.68); fluorescence (MeOH): λem = 571 nm,
λex = 469 nm, Φ = 0.05. C23H35N3O8 (481.55 g mol–1).
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Diethyl 2-[3-(tert-butoxycarbonylamino)propylamino]-5-{3-[2-(2,5-dihydro-1H-pyrrol-1-
yl)ethylamino]-3-oxopropylamino}terephthalate (25). HATU (0.24 mmol, 91 mg, 1.2 eq),
DIPEA (0.44 mmol, 57 mg, 2.2 eq) and maleimide building block 24 (0.24 mmol, 61 mg, 1.2
eq) were added to a suspension of compound 23 (0.20 mmol, 96 mg, 1.0 eq) in CH2Cl2 (9
mL). The mixture was stirred for 17 h at ambient temperature. Subsequently, it was diluted
with CH2Cl2 (15 mL) and washed with saturated aqueous NaHCO3 solution (4 x 25 mL). The
organic layer was dried over MgSO4, filtered and all volatile materials were evaporated. The
residue was chromatographed (SiO2, EtOAc/hexanes 2:1, Rf = 0.19) to yield compound 25
(105 mg, 017 mmol, 87%) as a red solid, m.p. 111–112°C.
1H NMR (500 MHz, CDCl3): δ = 1.38 (t, J = 7.1 Hz, 3H), 1.39 (t, J = 7.1 Hz, 3H), 1.43 (s,
9H), 1.85 (quint, J = 6.7 Hz, 2H), 2.47 (t, J = 6.5 Hz, 2H), 3.20 (t, J = 6.0 Hz, 2H), 3.26 (q, J
= 6.5 Hz, 2H), 3.43–3.47 (m, 4H), 3.66–3.68 (m, 2H), 4.33 (q, J = 7.1 Hz, 2H), 4.34 (q, J =
7.1 Hz, 2H), 4.65 (br.s, 1H), 6.20 (t, J = 5.1 Hz, 1H), 6.64 (s, 2H), 6.80 (br.s, 2H), 7.26 (s,
13
1H), 7.32 (s, 1H) ppm. C{1H} NMR (125 MHz, CDCl3): δ = 14.5 (2 CH3), 28.5 (3 CH3),
29.9 (CH2), 36.4 (CH2), 37.7 (CH2), 38.7 (CH2), 38.9 (CH2), 40.2 (CH2), 41.3 (CH2), 61.0 (2
CH), 79.3 (C), 114.2 (CH), 114.7 (CH), 116.9 (C), 118.0 (C), 134.2 (2 CH), 140.6 (C), 141.8
(C), 156.1 (C), 167.9 (C), 168.0 (C), 171.0 (2 C), 171.8 (C) ppm. IR (ATR): λ–1 = 1693 (s),
1677 (s), 1662 (s), 1528 (s), 1202 (vs), 1217 (vs), 1173 (s), 1105 (s), cm–1. MS (EI, 70 eV),
m/z (%): 603 (20) [M+], 547 (21), 529 (11), 503 (13), 413 (8), 316 (8), 302 (14), 245 (8), 231
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(10), 124 (9), 110 (16), 82 (7), 59 (90). HRMS (EI) m/z: [M+] calcd. 603.2899 for
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+
C29H41N5O9 ; found 603.2902. UV/Vis (CH2Cl2): λmax (lg ε) = 474 nm (3.67); fluorescence
(CH2Cl2): λem = 571 nm, λex = 474 nm, Φ = 0.005. C29H41N5O9 (603.67 g mol–1).
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Diethyl 2-(3-ammoniopropylamino)-5-{3-[2-(2,5-dihydro-1H-pyrrol-1-yl)ethylamino]-3-
oxopropylamino}terephthalate trifluoroacetate (26). TFA (5 mL) was added dropwise to a
cooled (iceꢀwater bath) solution of compound 25 (260 ꢁmol, 157 mg) in CH2Cl2 (5 mL). The
mixture was stirred for 16 h at ambient temperature. MTBE (10 mL) and hexanes (10 mL)
were added. The red precipitate was isolated by filtration and washed with cold MTBE (40
mL). It was redissolved in MeOH (15 mL), filtered and the solvent was evaporated to yield
compound 26 (176 mg, 28 mmol, quant.) as a red resin.
1H NMR (500 MHz, CD3OD): δ = 1.40 (t, J = 7.1 Hz, 3H), 1.41 (t, J = 7.1 Hz, 3H), 2.13
(quint, J = 7.3 Hz, 2H), 2.59 (t, J = 6.6 Hz, 2H), 3.12 (t, J = 7.3 Hz, 2H), 3.34–3.37 (m, 2H),
3.46 (t, J = 6.6 Hz, 2H), 3.57 (t, J = 5.5 Hz, 2H), 3.62 (t, J = 5.7 Hz, 2H), 4.41 (q, J = 7.0 Hz,
2H), 4.42 (q, J = 7.0 Hz, 2H), 6.78 (s, 2H), 7.68 (s, 1H), 7.80 (s, 1H) ppm. 13C{1H} NMR
(125 MHz, CD3OD): δ = 14.4 (2 CH3), 27.1 (CH2), 34.2 (CH2), 38.1 (CH2), 38.3 (CH2), 39.0
(CH2), 44.4 (CH2), 45.3 (CH2), 63.0 (CH2), 63.2 (CH2), 117.7 (q, J = 292 Hz, CF3), 120.1
(CH), 122.4 (CH), 122.5 (2 C), 135.4 (2 CH), 136.0 (C), 140.5 (C), 162.2 (q, J = 35 Hz, C),
167.2 (C), 167.5 (C), 172.5 (2 C), 173.1 (C) ppm. IR (ATR): λ–1 = 1674 (s), 1200 (s), 1174
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(vs), 1142 (vs), 1106 (s) cm–1. HRMS (ESI) m/z: [M – CF3CO2 ] calcd. 504.2453 (for
C24H34N5O7; found 504.2449. UV/Vis (MeOH): λmax (lg ε) = 466 nm (3.70); fluorescence
(MeOH): λem = 568 nm, λex = 466 nm, Φ = 0.01. C26H34F3N5O9 (617.58 g mol–1).
Diethyl
2-{3-[(1R,8S,9s)-(bicyclo[6.1.0]-4-nonyne-9-yl)methoxycarbonylamino]propyl-
amino}-5-{3-[2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethylamino]-3-oxopropylamino}-
terephthalate (28). DIPEA (21 mmol, 27 mg, 2.5 eq) was added to a suspension of comꢀ
pound 26 (83 µmol, 51 mg, 1.0 eq) and cyclooctyne active carbonate 27 (66 µmol, 19 mg, 0.8
eq) in CH2Cl2 (2 mL). The mixture was stirred for 16 h at ambient temperature. Subsequently,
it was diluted with CH2Cl2 (20 mL) and the organic layer was washed with aqueous saturated
NaHCO3 solution (4 x 25 mL). It was dried over MgSO4, filtered and all volatile materials
were evaporated. The residue was chromatographed (SiO2, EtOAc/hexanes 2:1, Rf = 0.24) to
yield compound 28 (35 mg, 51 µmol, 78%) as a red solid, m.p. 101–102°C.
1H NMR (500 MHz, CDCl3): δ = 0.90–0.94 (m, 2H), 1.31–1.35 (m, 1H), 1.380 (t, J = 7.1 Hz,
3H), 1.383 (t, J = 7.1 Hz, 3H), 1.52–1.60 (m, 2H), 1.88 (quint, J = 6.6 Hz, 2H), 2.17–2.29 (m,
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6H), 2.48 (t, J = 6.5 Hz, 2H), 3.22 (t, J = 6.6 Hz, 2H), 3.32 (q, J = 6.2 Hz, 2H), 3.42–3.47 (m,
4H), 3.66–3.68 (m, 2H), 4.13–4.18 (m, 2H), 4.325 (q, J = 7.1 Hz, 2H), 4.334 (q, J = 7.1 Hz,
2H), 4.85 (br.s, 1H), 6.20 (t, J = 4.8 Hz, 1H), 6.65 (s, 2H), 6.83 (br.s, 2H), 7.26 (s, 1H), 7.32
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(s, 1H) ppm. C{1H} NMR (125 MHz, CDCl3): δ = 14.5 (2 CH3), 17.9 (CH), 20.2 (2 CH),
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21.5 (2 CH2), 29.2 (2 CH2), 29.7 (CH2), 36.4 (CH2), 37.6 (CH2), 38.9 (CH2), 39.1 (CH2), 40.2
(CH2), 41.1 (CH2), 60.99 (CH2), 61.03 (CH2), 62.8 (CH2), 98.9 (2 C), 114.1 (CH), 114.7
(CH), 116.9 (C), 117.9 (C), 134.2 (2 CH), 140.6 (C), 141.7 (C), 156.9 (C), 167.8 (C), 168.0
(C), 171.0 (2 C), 171.8 (C) ppm. IR (ATR): λ–1 1694 (s), 1678 (s), 1662 (s), 1528 (s), 1202
(vs), 1181 (vs), 1105 (s) cm–1. HRMS (ESI) m/z: [M + H+] calcd. 680.3290 for C35H46N5O9;
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found 680.3301. UV/Vis (CH2Cl2): λmax (lg ε) = 473 nm (3.66); fluorescence (CH2Cl2): λem
566 nm, λex = 473 nm, Φ = 0.005. C35H45N5O9 (679.77 g mol–1).
=
Diethyl
2-{3-[((5aR*,6S*,6aS*)-1-benzyl-1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]-
cycloocta[1,2-d][1,2,3]triazol-6-yl)methoxycarbonylamino]propylamino}-5-{3-[2-(3-ben-
zylthio-2,5-dioxopyrrolidin-1-yl)ethylamino]-3-oxopropylamino}terephthalate (29). A
solution of BnSH (0.47 mmol, 63 mg, 10 eq) and BnN3 (0.47 mmol, 63 mg, 10 eq) in MeCN
(2 mL) was added dropwise to a solution of compound 28 (47 µmol, 32 mg, 1.0 eq) in MeCN
(3 mL). The mixture was stirred for 20 h at ambient temperature. Subsequently, NEt3 (0.71
mmol, 71 mg, 15 eq) was added and the mixture was stirred for additional 3 h at ambient temꢀ
perature. All volatile materials were evaporated and the residue was chromatographed [SiO2,
CH2Cl2/MeOH 40:1, Rf(CH2Cl2/MeOH 20:1) = 0.50] to yield compound 29 (32 mg, 34 µmol,
73%) as a red solid, m.p. 55–57°C.
1H NMR (500 MHz, CDCl3): δ = 0.75–0.83 (m, 1H), 0.93–1.00 (m, 1H), 1.10–1.17 (m, 1H),
1.36 (t, J = 7.1 Hz, 3H), 1.37 (t, J = 7.1 Hz, 3H), 1.41–1.58 (m, 2H), 1.85 (quint, J = 6.4 Hz,
2H), 1.91–1.97 (m, 1H), 2.13–2.20 (m, 1H), 2.37 (dd, J = 18.6 Hz, J = 3.8 Hz, 1H), 2.48 (t, J
= 6.5 Hz, 2H), 2.50–2.54 (m, 1H), 2.72–2.78 (m, 1H), 2.87–2.91 (m, 1H), 2.96 (dd, J = 18.7
Hz, J = 9.2 Hz, 1H), 3.05–3.10 (m, 1H), 3.20 (t, J = 6.6 Hz, 2H), 3.30 (q, J = 6.1 Hz, 2H),
3.40–3.46 (m, 3H), 3.48–3.54 (m, 2H), 3.61–3.71 (m, 2H), 3.82 (d, J = 13.5 Hz, 1H), 4.02–
4.11 (m, 2H), 4.14 (d, 13.5 Hz, 1H), 4.30 (q, J = 7.2 Hz, 2H), 4.32 (q, J = 7.2 Hz, 2H), 4.85
(br.s, 1H), 5.41–5.49 (m, 2H), 6.33 (br.s, 1H), 6.91 (br.s, 2H), 7.07 (s, 1H), 7.08 (s, 1H),
13
7.22–7.35 (m, 10H) ppm. C{1H} NMR (125 MHz, CDCl3): δ = 14.46 (CH3), 14.48 (CH3),
17.6 (CH), 19.3 (CH), 19.5 (CH), 21.6 (CH2), 22.3 (CH2), 23.3 (CH2), 26.1 (CH2), 29.7
(CH2), 35.5 (CH2), 36.1 (CH2), 36.2 (CH2), 37.7 (CH), 38.2 (CH2), 38.9 (CH2), 39.1 (CH2),
40.3 (CH2), 41.2 (CH2), 52.1 (CH2), 61.0 (CH2), 61.1 (CH2), 62.7 (CH2), 114.3 (CH), 114.9
29
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