Microwave-Assisted Synthesis of 2,2′-Azopyridine-Labeled Amines, Amino Acids, and Peptides

Article abstract of DOI:10.1055/s-0035-1560523

A microwave-assisted procedure for labeling amines, amino acids, and peptides with 2,2′-azopyridines (2,2′-AzPy) is described using the corresponding 2,2′-azopyridine-diacylbenzotriazoles. The efficiency of the procedure is proven by the generation of thr

Full text of DOI:10.1055/s-0035-1560523

SYNTHESIS
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© Georg Thieme Verlag Stuttgart · New York
2016, 48, 365–378
paper
365
I. Avan
Paper
Synthesis
Microwave-Assisted Synthesis of 2,2′-Azopyridine-Labeled Amines,
Amino Acids, and Peptides
Ilker Avan*
HO2C
O
O
H
O
Department of Chemistry, Faculty of Science, Anadolu Universi-
ty, 26470 Eskişehir, Turkey
iavan@anadolu.edu.tr
N
H
H
N
H
N
N
N
2,2'-AzPy
H
N
O
H
O
O
CO2H
Dedicated to the memory of Alan Roy Katritzky (1928–2014)
N
N
N
N
N
N
N
N
N
N
N
N
2,2'-AzPy-4,4'-di(Gly-Leu-Phe) 2,2'-AzPy-5,5'-di(Gly-Leu-Phe) 2,2'-AzPy-6,6'-di(Gly-Leu-Phe)
Received: 21.07.2015
Accepted after revision: 16.10.2015
Published online: 27.10.2015
effective light-driven trans-to-cis isomerization.5 Azoben-
zene derivatives are used in a variety of light-driven biopro-
cess including protein and DNA folding,6 enzymatic,7 and
ion-transfer activity through channels.8
DOI: 10.1055/s-0035-1560523; Art ID: ss-2015-t0447-op
Abstract A microwave-assisted procedure for labeling amines, amino
acids, and peptides with 2,2′-azopyridines (2,2′-AzPy) is described us-
ing the corresponding 2,2′-azopyridine-diacylbenzotriazoles. The effi-
ciency of the procedure is proven by the generation of three constitu-
tionally isomeric sets of 2,2′-AzPy-X,X′-labeled amino conjugates
(where X = 4, 5, 6) including amines, amino acids, and peptides. Micro-
wave-assisted synthesis conditions provide good to excellent yields in
less than 15 minutes with retention of original chirality. A trans-to-cis
isomerization of the 2,2′-azopyridine-labeled amino conjugate upon la-
ser irradiation at 325 nm is visualized with UV and NMR spectroscopy.
Azopyridine (AzPy) is also an azoaromatic compound
that is fashioned by two pyridyl group attached to N=N
bond and show similar trans-cis isomerization. Azopyri-
dines have attracted considerable attention in the field of
coordination chemistry due to their high propensity to co-
ordinate metallic fragments through electron pairs of pyr-
idyl and azo nitrogen atoms. Structural identity, gas sorp-
tion ability, thermal stability, and magnetic property of
AzPy-metal complexes are of current interest.9,10 The earli-
est examples concerning photoresponsive AzPy compounds
are reported by Shinkai et al. who described the synthesis
of 2,2′-AzPy-bridged crown ether, which enables extraction
of substantial amounts of heavy metal ions; Cu2+, Ni2+, Co2+,
and Hg2+.11 Since the trans-isomer is vertically positioned
over the crown ether ring, it builds better coordination with
metal ions, and thus, extracts greater amounts of metal
ions than the cis-isomer does. Later on, the same group re-
ported a thiacrown ether attached 2,2′-Azopyridines.12 In
this case, the cis-isomer formed by photoirradiation shows
better binding affinity to Cu2+ and Hg2+ than the trans-
form.12 Consequently, when thiacrown ether is used as an
ion carrier on a membrane, Cu2+ transport rate is enhanced
by UV irradiation. Recently, Bardaji et al.10 reported the syn-
thesis of photosensitive 2,2′- and 4,4′-Azpy gold(I) and sil-
ver(I) complexes, which undergo trans-to-cis isomerization
in solution. Azo-metal coordination of an Ag-2,2′-AzPy
complex, having a confirmed structure in solid state via X-
ray, is simultaneously broken by UV irradiation during pho-
toisomerization in solution.
Key words azopyridine, microwave synthesis, N-acylbenzotriazole, ac-
ylation, peptides, photoisomerization, cis–trans isomerization
Peptides and proteins are essential biomolecules that
have diverse functions such as hormones, neurotransmit-
ters, and neuromodulators in living systems. Their inherent
interaction with biological systems makes them suitable
candidates for therapeutic and biological use.1 Peptides
have long been studied and prepared as drugs, immuno-
gens, hormones, vaccines, and peptidic probes.2 In fact, the
secrets of biofunction of peptides are mostly hidden in their
constitutions and well-defined three-dimensional struc-
tures. Introduction of shape or size labile modules into pep-
tide chains, which response to external stimuli such as
light, heat, pH, or physicochemical change, might affect
folding pattern, and thus allow a temporal control on pep-
tide’s bioactivity.3 Based on aforementioned approach,
many smart-modules and polymeric fragments are devised
and utilized for the manipulation of peptide-protein func-
tion.3,4 Among them, azoaromatic compounds (especially
azobenzene) are the most prominent moieties due to their
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Despite the coordination ability over other azoaromat-
ics and stimuli-responsive behavior of azopyridines, they
were not reported as labeling agent for amino acids and
peptides. Here, a general microwave assisted procedure is
presented for labeling amines, amino acids, and peptides
with AzPy in good to excellent yields in less than 15 min-
utes. The efficiency of the procedure is shown by the syn-
thesis of AzPy-labeled peptides without causing detectable
racemization. Preparations of AzPy precursors, microwave-
assisted synthesis conditions for AzPy labeling, and prelimi-
nary results on light-driven trans-cis isomerization are dis-
cussed. The methodology presented here expands labeling
toolbox for amino acids-peptides, and might be used for fu-
ture light-driven bioapplications.
Table 1 2,2′-AzPy-Dicarboxylates 2a–c and 2,2′-AzPy-Diacylbenzotri-
azoles 3a–c
Entry Acyl position 2, Yield (%) 2, Mp (°C)
3, Yield (%) 3, Mp (°C)
1
2
3
4,4′
5,5′
6,6′
2a, 44
2b, 44
2c, 41
>340
3a, 89
3b, 62
3c, 62
255–260
238–242
236–238
323–326
252–254
few decades, Katritzky and co-workers showed synthetic
utilities of N-acylbenzotriazoles as stable and easy to han-
dle acylation reagents for the preparation of tetra-, penta-,
hexa- and cyclic heptapeptides in solution phase,20,21 ‘diffi-
cult’ sequence peptides on solid phase,22 and peptidomi-
metics.23,24–26 Recently, Katritzky et al. reported the prepa-
ration of azobenzene-labeled amino acids, amines, nucleo-
sides, terpenes, sugar, and steroids through activated
benzotriazole intermediates under conventional meth-
ods.17–19 Benzotriazole-activated intermediates were pre-
pared under reflux condition for 5 hours in THF from the
corresponding azobenzene-carboxylic acids by reacting 1-
(methylsulfonyl)-1H-benzotriazole. Furthermore, benzotri-
azole-activated azobenzene carboxylic acids were reacted
with free amino acids and amines under reflux conditions
or at room temperature for 1–48 hours to give azo-dye-
labeled amino acids and amines in 74–100% yields.17 The
treatment of benzotriazole-activated carboxylic acids with
terpenes, sugar, and steroids at room temperature for 12–
36 hours gave azobenzene-labeled adjuncts in 45–82%
yields.18 Azobenzene-labeled nucleosides were obtained in
30–79% yields after 24-hour reaction of benzotriazole-acti-
vated intermediates with corresponding nucleosides at
room temperature. Herein, microwave-assisted reaction
conditions enable the coupling of 2,2′-AzPy-diacylbenzotri-
azoles 3a–c with amino conjugates in less than 15 minutes
to give 2,2′-AzPy-labeled amines, amino acids, and peptides
in good to excellent yields (40–91%) with retention of chi-
rality.
Preparation of 2,2′-AzPy-Dicarboxylic Acids 2a–c
Up to date, several methods have been employed for the
synthesis of azo compounds including (i) oxidation of aro-
matic amines and (ii) hydrazo derivatives; reduction of aro-
matic (iii), nitro, and (iv) azoxy compounds; (v) coupling of
primary arylamines with nitroso compounds; and (vi) cou-
pling of aryldiazonium salts.13 Synthetic methods have been
selected based on starting material availability and substit-
uent dependent reaction efficacy. Herein, symmetric 2,2′-
AzPy-dicarboxylic acids 2a–c were obtained by the oxida-
tion of 2-aminopyridine carboxylic acids 1a–c as previously
applied by Shinkai et al.11 using aqueous sodium hypochlo-
rite (10–15%), an inexpensive reagent, in moderate yields of
41–44% (Scheme 1, Table 1).
CO2H
N
HO2C
N
N
N
CO2H
CO2H
H2N
H2N
H2N
N
N
N
2a, 44%
N
1a
1b
1c
NaClO (10–15%)
NaOH (10%), H2O
N
CO2H
N
2b, 44%
5 °C, 3 h
N
N
HO2C
CO2H
N
Active intermediates, 2,2′-AzPy-diacylbenzotriazoles
(2,2′-AzPy-diBt) 3a–c, were prepared from 2,2′-AzPy-dicar-
boxylic acids 2a–c in 15 minutes by treating with 1-(meth-
ylsulfonyl)-1H-benzotriazole in anhydrous dimethylforma-
mide under microwave irradiation with 30 W irradiation
power at 70 °C in the presence of triethylamine (Scheme 2,
Table 1).
N
CO2H
2c, 41%
N
CO2H
Scheme 1 Synthesis of 2,2′-AzPy-dicarboxylates 2a–c
Preparation of 2,2′-AzPy-Diacylbenzotriazoles 3a–c
2,2′-AzPy-diacylbenzotriazoles 3a–c are readily stable
solid compounds at room temperature with high melting
points (>200 °C). However, NMR data are not available,
since they are not well soluble in organic solvents. Addition
of trifluoroacetic acid (TFA) to NMR tube improves solubili-
ty, nevertheless they decompose over time. The structures
In the published methods, azoaromatic carboxylic acids
were linked to biomoieties by using coupling reagents such
as (i) DCC, EDCI, HBTU, HATU, CDI;14 (ii) acid chlorides;11,15
and (iii) other carboxylic acid activators including N-hy-
droxysuccinimide ester,16 and benzotriazole.17–19 In the last
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Synthesis
O
N
N
N
N
Bt
Bt
Bt
N
HO2C
N
N
CO2H
CO2H
3a, 89%
N
N
O
O
N
2a
O
O
S
N
Me
Bt
N
N
N
N
N
3b, 62%
Et3N, DMF
MW (30 W, 70 °C)
15 min
Bt
N
O
Bt
2b
HO2C
CO2H
O
N
N
N
N
N
3c, 61%
N
N
O
2c
CO2H
Bt
Scheme 2 Preparation of 2,2′-AzPy-diacylbenzotriazoles 3a–c
of intermediates 3a–c are confirmed by elemental analysis
and further reactions for the preparation of 2,2′-AzPy-la-
beled amines, amino acids, and peptides.
Table 2 2,2′-AzPy-Diacylamines 5aa–cc
Entry Acyl position Reactant Amine
5, Yield (%) Mp (°C)
1
2
4,4′
4,4′
3a
3a
ethylamine
5aa, 75
5bb, 87
231–232
260–261
Preparation of 2,2′-AzPy-Diacylamines 5aa–cc
cyclohexyl-
amine
Reaction of 2,2′-AzPy-diacylbenzotriazoles 3a–c with
primary amines 4 in anhydrous DMF under microwave ex-
posure with 30 W irradiation power for 5–10 minutes at
70 °C afforded the corresponding amide derivatives 5aa–cc
(67–88%) (Scheme 3 and Table 2). While the reactions of al-
iphatic amines took 5 minutes, the coupling reaction of aro-
matic amine for the preparation of 5cc took 10 minutes be-
cause of its lower reactivity.
3
4
5,5′
5,5′
3b
3b
ethylamine
5ba, 70
5bb, 88
262–264
286–288
cyclohexyl-
amine
5
6
6,6′
6,6′
3c
3c
ethylamine
5ca, 67
5cb, 82
208–210
188–190
cyclohexyl-
amine
7
6,6′
3c
4-methoxy-
aniline
5cc, 69
234–235
O
N
H
N
R
R
N
O
N
N
H
N
N
Bt
Bt
N
O
Bt
N
5aa, R: Et, 75%
N
O
O
5ab, R: c-Hexyl, 87%
3a
O
R
R
H2N
N
N
N
H
4
N
N
N
N
3b
N
H
N
5ba, R: Et, 70%
5bb, R: c-Hexyl, 88%
Bt
N
N
Et3N, DMF
MW (30 W, 70 °C)
5–10 min
R
O
N
Bt
O
O
O
H
N
R
N
N
N
3c
N
O
N
N
R
5ca, R: Et, 67%
5cb, R: c-Hexyl, 82%
5cc, R: 4-MeOC6H4, 69%
Bt
N
H
O
Scheme 3 Preparation of 2,2′-AzPy-diacylamines 5aa–cc
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Preparation of 2,2′-AzPy-Labeled Amino Acids 7aa–cc
Table 3 2,2′-AzPy-Labeled Amino Acids 7aa–cc
N-Acylbenzotriazoles are advantageous peptide cou-
pling reagents that allow the use of unprotected amino
acids in aqueous media without causing racemization.21 In
initial experiments, the reaction of 2,2′-AzPy-diacylben-
zotriazoles 3a and 3b with free amino acids 6a–c were not
completed after stirring for 24 hours at room temperature,
while the reactions of 3c with amino acids were completed
after 6 hours at room temperature. When the reaction was
heated up to 50 °C by conventional heating, the reaction of
3a resulted after 3 hours at 50 °C with the formation of
product 7ab and some starting material 2a as an insepara-
ble mixture. Therefore, the reaction temperature was ele-
vated to 70 °C. At this temperature, the reactions of 3a with
amino acids were completed after 30 minutes, but yielded
the formation of some small amount of starting material
2a. When the conventional heating was replaced by micro-
wave irradiation in a modified procedure, the reaction time
of 3a was shortened to 15 minutes providing pure products
7ab without formation of starting material [see Supporting
Information (SI), pages S2–S3]. Microwave-assisted cou-
pling reactions of 2,2′-AzPy-diacylbenzotriazoles 3a–c with
free amino acids 6a–c in a mixture of water, acetonitrile,
and triethylamine with 20 W microwave irradiation power
for 5–15 minutes at 70 °C afforded 2,2′-AzPy-labeled amino
acids 7aa–cc and (7cb + 7cb′) (65–91%) (Scheme 4, Table 3).
While the complete conversion of 3c took 5 minutes, cou-
pling reaction of 3b took 10 minutes and reactions with 3a
took 15 minutes because of their lower reactivity. The end
of reactions could not be determined by TLC analysis since
the active intermediates, 2,2′-AzPy-diacylbenzotriazoles 3,
were not soluble in common organic solvents. The coupling
reactions were carried out until the disappearance of all
colored solid intermediates 3 in the reaction vessel.
Entry Acyl
posi-
Reac-
tant
Amino Reaction 7, Yield (%)
Mp (°C)
acid
time
(min)
tion
1
2
3
4
5
6
7
8
9
10
4,4′
4,4′
4,4′
5,5′
5,5′
5,5′
6,6′
6,6′
6,6′
6,6′
3a
3a
3a
3b
3b
3b
3c
3c
3c
3c
L-Ala
15
15
15
10
10
10
5
7aa, 85
7ab, 73
7ac, 88
205–206
199–201
210–212
224–225
91–92
L-Val
L-Phe
L-Ala
L-Val
L-Phe
L-Ala
L-Val
DL-Val
L-Phe
7ba, 65
7bb, 74
7bc, 84
7ca, 91
203–205
240–241
195–197
193–196
205–207
5
7cb, 69
7cb + 7cb′, 82
7cc, 82
5
5
Chiral integrity of compounds 7aa–cc was supported by
the NMR spectroscopy. Compound 7cb, valine analogue of
2,2′-AzPy-6,6′-dicarboxylate, showed duplication of signals
in the 1H and 13C NMR spectra (Figure 1). For example,
methyl protons of isopropyl group provided two separate
doublets at 0.98 ppm (J = 5.1 Hz) and 0.96 ppm (J = 5.1 Hz),
and they appeared again as two separate doublets at 0.72
ppm (J = 6.8 Hz) and 0.69 ppm (J = 6.8 Hz). For this particu-
lar compound, those minor repetitive signals were scruti-
nized to figure out whether they originate from impurities
or possible in situ racemization. Considering racemization
of acidic proton on alpha carbon for duplication of signals,
the racemic mixture (7cb + 7cb′) of this compound was
prepared from DL-valine and 3c. The racemic mixture (7cb +
7cb′) also provided similar duplicate signals within the
same integration ratio (20:1) and appear as chirally pure
R
O
N
O
H
HO
N
N
N
H
N
OH
O
N
O
N
O
R
R
N
Bt
Bt
7aa, R: Me, 85%
7ab, R: i-Pr,73%
7ac, R: Ph, 88%
Bt
N
N
3a
O
O
O
H2N
CO2H
OH
N
N
H
R
N
6a–c
N
O
O
N
N
H
N
N
7ba, R: Me, 65%
7bb, R: i-Pr,74%
7bc, R: Ph, 84%
N
Et3N
HO
3b
Bt
N
MeCN–H2O
MW
(20 W, 70 °C)
5–15 min
R
O
O
N
Bt
O
O
O
H
N
OH
N
R
N
N
N
N
R
N
HO
N
O
7ca, R: Me, 91%
7cb, R: i-Pr, 69%
7cb+7cb', R: i-Pr, 82%
7cc, R: Ph, 82%
3c
N
H
O
O
Bt
Scheme 4 Preparation of 2,2′-AzPy-labeled amino acids 7aa–cc
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compound 7cb in the 1H NMR spectrum, and each repeating
signals form their own racemate splitting patterns (see SI,
page S31). For instance, while shifted signals of isopropyl
group of 7cb provided two separate doublets at 0.72 ppm
and 0.69 ppm, its racemic mixture (7cb + 7cb′) gave multi-
ple signals in the range of 0.80–0.67 ppm due to further
splitting of parent signals. The 13C NMR spectrum of race-
mic mixture (7cb + 7cb′) was almost identical to the 13C
NMR spectrum of chirally pure compound 7cb, except the
mixture of 7cb and 7cb′ provided two separated signals at
116.4 and 116.3 ppm, whereas 7cb gave one signal at 116.4
ppm. Attempts for chiral separation of 7cb and 7cb′ were
not successful via analytical HPLC using Chirobiotic T col-
adjuncts 7ab, 7bb, 7cb in DMSO were measured to deter-
mine λmax values (Figure 2, A). In particular, azopyridine
compounds showed quite similar characteristic of azoben-
zene compounds in their UV/Vis spectra having a high-in-
tensity π–π* band in UV region and a low-intensity n–π*
band in visible region (Table 4).27 Those λmax values in the
UV region closely fit for irradiation with He-Cd laser at 325
nm. When a 60 μM solution of 7cb in DMSO was exposed to
laser irradiation at 325 nm for 2 minutes, UV/Vis spectra of
7cb changed by a decrease at π–π* band and an increase at
n–π* band upon the trans-to-cis photoisomerization (Fig-
ure 2, B). An NMR tube containing a solution of compound
7cb in DMSO-d6 was exposed to laser irradiation at 325 nm
for 10 minutes to monitor the possible changes in NMR
spectra. Intensities of minor NMR signals contributed by
cis-isomer were increased from a ratio of 12:1.6 to 12:8
upon laser irradiation, because trans-7cb was transformed
to cis-7cb isomer (Figure 1, A). The 1H NMR chemical shifts
of cis-7cb were shifted to upfield and coupling constants of
methyl protons of 7cb were amplified from 5.1 Hz to 6.8 Hz.
1
umn. Based on those findings, these minor signals on H
NMR of 7cb were not formed because of racemization. Ap-
parently, trans-to-cis isomerization over azo bond (N=N)
was another possibility. More stable trans-isomer of 7cb
could form the cis-isomer via a conformational change by
absorbing UV light at wavelength of its maximum absor-
bance (λmax). UV/Vis spectra of 30 μM of 2,2′-AzPy-valine
Figure 1 (A) 1H NMR and (B) 13C NMR spectra change upon trans-to-cis photoisomerization of 7cb via laser irradiation at 325 nm for 10 minutes (blue
spectra were obtained after 10 minutes laser exposure of NMR tube).
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Figure 2 (A) UV/Vis spectra of 30 μM 2,2′-AzPy-valine conjunctions 7ab (blue), 7bb (red), and 7cb (black) in DMSO. (B) UV/Vis spectral changes upon
photoisomerization of 60 μM 7cb in DMSO before (black) and after (green) 2 minutes laser irradiation at 325 nm.
Table 4 UV-Vis Bands of 2,2′-AzPy-Labeled Valine Compounds
2,2′-AzPy-valine compound
Acyl position
UV bands
λmax (nm)
322
Vis bands
λmax (nm)
460
εmax (M–1·cm–1
)
εmax (M–1 cm–1
)
7ab
7bb
7cb
4,4′
5,5′
6,6′
13 833
333
433
367
329
17 133
470
319
12 667
460
These results indicate that the electron density of afore-
mentioned molecule was increased while its molecular vol-
ume was decreased, since pyridines were cis-oriented over
the azo bond, and amino acid arms of cis-7cb are much
closer to themselves than before. The 13C NMR spectrum of
7cb also presented the duplicate signals due to photoisom-
erization upon laser irradiation (Figure 1, B). When the
NMR tube containing an enriched amount of cis-7cb isomer
was left under sunlight for 30 minutes, the initial ratio lev-
els (12:1.8) of isomers in the mixture was set again by re-
verse cis-to-trans isomerization. 1H NMR spectrum con-
taining only trans-7cb was obtained, when the NMR sample
was prepared avoiding sunlight. For this sample, the photo-
dynamic equilibrium between trans- and cis-isomers of 7cb
was established in 15 minutes under sunlight (see SI, page
S30).
nals of subgroups to monitor racemization during reactions
via NMR spectroscopy (in order to eliminate possible over-
laps or interferences to individual NMR signals). Reactions
of 2,2′-AzPy-diacylbenzotriazoles 3a–c with free dipeptide-
amide 8 in anhydrous DMF under microwave with 20 W ir-
radiation power in 10 minutes at 70 °C afforded 2,2′-AzPy-
labeled dipeptide-amides 9a–c (61–76%) (Scheme 5 and
Table 5). 2,2′-AzPy-6,6′-diacylbenzotriazole 3c was treated
with free dipeptides 10a, 10b, and free tripeptide 11a in a
mixture of H2O–MeCN–DMF in the presence of triethyl-
amine under microwave irradiation (20 W, 70 °C) for 10
minutes to afford 2,2′-AzPy-6,6′-diacyl-labeled di- and
tripeptides 12a, 12b, and 13 (72–89%) (Scheme 6 and Table
5). The reactions of 2,2′-AzPy-4,4′-diacylbenzotriazole 3a
and 2,2′-AzPy-5,5′-diacylbenzotriazole 3b with free dipep-
tides 10a, 10b, and free tripeptide 11a using various reac-
tion conditions provided a mixture of coupling products
and starting materials, 2,2′-AzPy-4,4′-dicarboxylic acid 2a
and 2,2′-AzPy-5,5′-dicarboxylic acid 2b; however, those
mixtures in low quantities could not be separated. There-
fore, active intermediates 3a and 3b were allowed to react
with a free tripeptide ester 11b under microwave condi-
tions (20 W, 70 °C) for 10 minutes in anhydrous DMF to give
2,2′-AzPy-diacyltripeptide methyl esters 14a and 15a,
which further underwent deprotection by LiOH in DMF–
MeOH–H2O to give the unprotected 2,2′-AzPy-diacyltripep-
Preparation of 2,2′-AzPy-Labeled Di- and Tripeptides
Synthetic utilities of 2,2′-AzPy-diacylbenzotriazoles in
coupling reactions were extended by reacting them with
small peptides. Free di- and tripeptides were obtained by
following previously published method for depsipep-
tides25,26 (see SI, page S4). Amino acid types and sequences
in a small peptide were selected by considering NMR sig-
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O
O
N
O
H
N
H
N
H
N
N
N
H
N
H
N
N
H
O
O
N
O
O
O
O
9a, 61%
O
H
N
H
H2N
·HCl
N
N
N
N
H
N
H
N
H
N
O
8
O
H
N
H
N
3a, 3b, 3c
N
Et3N, DMF
MW (20 W, 70 °C), 10 min
9b, 70%
N
H
O
O
O
H
N
H
N
N
H
N
N
N
O
O
H
N
O
O
9c, 76%
N
N
H
N
H
O
Scheme 5 Preparation of 2,2′-AzPy-labeled dipeptide-amides 9a–c
R2
O
H
N
HO2C
N
N
N
O
R1
·HCl
H2N
O
R2
H
H
R1
O
N
N
N
CO2H
N
N
CO2H
12a, R1: H, R2: i-Bu, 72%
12b, R1: Me, R2: Bn, 83%
H
R2
H
O
R1
10a, 10b
3c
·HCl
H2N
O
H
N
O
N
H
H
H
N
N
O
CO2H
11a
N
N
N
O
O
CO2H
H
H
N
Et3N, MeCN–DMF–H2O
MW (20W, 70 °C), 10 min
CO2H O
O
N
N
N
N
13, 89%
H
H
O
Scheme 6 Preparation of 2,2′-AzPy-6,6′-diacyldipeptides 12a, 12b and -tripeptide 13
RO2C
O
O
N
O
H
H
H
N
N
N
N
N
N
H
N
N
H
H
O
O
N
O
CO2R
·HCl
H2N
O
H
14a, R: Me, 65%
14b, R: H, 69%
(i)
N
N
H
11b
O
CO2Me
3a, 3b
DIEA, DMF
MW (20 W, 70 °C), 10 min
O
O
CO2R
H
N
N
N
H
N
H
N
O
O
N
H
H
N
15a, R: Me, 59%
15b, R: H, 41%
N
N
(i)
N
H
RO2C
O
O
(i) LiOH, DMF, MeOH, H2O
Scheme 7 Preparation of 2,2′-AzPy-4,4′-diacyltripeptides 14a, 14b and 2,2′-AzPy-5,5′-diacyltripeptides 15a, 15b
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, 365–378

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tilled over CaH2. THF was dried and distilled over metallic Na in the
presence of benzophenone. CH2Cl2 and EtOAc were dried and distilled
over CaO. The trans-to-cis photoisomerization was carried out by us-
ing a Kimmon He-Cd laser system (325 nm, 50 mW). Starting materi-
als, aminopyridine carboxylic acids 1a, 1b, and 1c were prepared by
following reported methods.28 Primary amines 4 and unprotected
amino acids 6 were purchased from commercial sources. 1-(Methyl-
sulfonyl)-1H-benzotriazole (BtSO2Me), Boc-Gly (16a), Boc-Ala (16b),
Boc-Gly-Bt (17a), and Boc-Ala-Bt (17b) were prepared by previously
reported methods.24,26,29 Unprotected dipeptides 10a and 10b were
characterized by 1H and 13C NMR spectroscopy and used for the next
coupling step without further purification. The structures of 2,2′-
AzPy-diacylbenzotriazoles 3a, 3b, and 3c were confirmed by elemen-
tal analysis, IR spectroscopy, and following reactions. NMR data of
these compounds are not available because of their insufficient solu-
bility in common NMR solvents.
Table 5 2,2′-AzPy-Labeled Di- and Tripeptides 9a–c, 12a, 12b, 13,
14a, 14b, 15a, and 15b Prepared
Entry Acyl position Reac-
tant
Product, yield Peptide part
(%)
Mp (°C)
1
2
3
4
5
6
7
4,4′
5,5′
6,6′
6,6′
6,6′
6,6′
4,4′
3a
3b
3c
3c
3c
3c
3a
9a, 61
9b, 70
9c, 76
Gly-Leu-NHi-Pr 242–244
Gly-Leu-NHi-Pr 212–216
Gly-Leu-NHi-Pr 244–246
12a, 72
12b, 83
13, 89
14a, 65
Gly-Leu
113–115
134–138
212–214
176–180
Ala-Phe
Gly-Leu-Phe
Gly-Leu-Phe-
OMe
8
9
4,4′
5,5′
3a
3b
14b,a 69
15a, 59
Gly-Leu-Phe
138–142
196–200
2,2′-AzPy-Dicarboxylic Acids (2,2′-AzPy-DiCAs) 2a–c; General Pro-
cedure
Gly-Leu-Phe-
OMe
A solution of 6-aminopyridinecarboxylic acid 1a–c (1 g, 7.25 mmol)
in aq 10% NaOH (5 mL) was added dropwise in 15 min to a solution of
aq 10–15% NaOCl (15 mL) at 0 °C. After the mixture had been stirred
for 1 h at 0 °C, additional aq 10–15% NaOCl (10 mL) was added to the
reaction mixture. After further stirring (3 h at 5 °C), the resulting or-
ange precipitate was filtered through a sintered glass filter. The re-
maining aqueous filtrate was treated with NaOH pellets (~5 g) to form
more precipitate, which was filtered later. The collected solid portions
were dissolved in aq 1 M NaOH solution (200 mL) to give a red solu-
tion. The red solution was heated to 50 °C and treated with active
charcoal. The solution was filtered through a pad of Celite (2 cm). The
solution was cooled to 10 °C, then acidified with concd HCl to pH 3–4.
A pale yellow precipitate, which was formed upon acidification was
collected by filtration, washed with cold H2O, and dried under re-
duced pressure to give 2,2′-AzPy-dicarboxylic acids 2a–c as micro-
crystals (Table 1).
10
5,5′
3b
15b,a 41
Gly-Leu-Phe
180–184
a Hydrolysis product.
tides 14b and 15b (Scheme 7, Table 5). Chiral integrity of
2,2′-AzPy-labeled peptides was confirmed by the NMR
spectroscopic data. Notably, there was no duplication of
peaks in the 1H and 13C NMR spectra of 9a–9c, 12a, 12b, 13,
14a, 14b, 15a, and 15b.
In conclusion, microwave-assisted syntheses of novel
2,2′-AzPy-labeled amines 5aa–cc, amino acids 7aa–cc, and
peptides 9a–c, 12a–c, 13, 14a,b, and 15a,b were achieved
by treating 2,2′-AzPy-diacylbenzotriazoles with primary
amines, unprotected amino acids, and peptides. All amino
acid and peptide derivatives were prepared in less than 15
minutes under microwave reaction conditions in good to
excellent yields and without any detectable racemization.
Trans-to-cis isomerization of 2,2′-AzPy-labeled amino acid
conjugate was brought about by laser exposure at 325 nm
and monitored using UV and NMR spectroscopy. More im-
portantly, these newly prepared photoactive 2,2′-AzPy de-
rivatives could be useful tools for remotely modulating the
folding pattern of a peptide and thus allowing a temporal
control of their bioactivity.
(E)-2,2′-(Diazene-1,2-diyl)diisonicotinic Acid (2,2′-AzPy-4,4′-diCA,
2a)
Yield: 0.43 g (44%); orange microcrystals; mp >340 °C.
1H NMR (500 MHz, 0.1 M K2CO3 in D2O): δ = 8.72 (d, J = 4.9 Hz, 2 H),
8.20 (s, 2 H), 7.92 (dd, J = 4.9, 1.3 Hz, 2 H).
13C NMR (125 MHz, 0.1 M K2CO3 in D2O): δ = 174.7, 168.6, 152.5,
151.1, 128.9, 119.1.
Anal. Calcd for C12H8N4O4: C, 52.95; H, 2.96; N, 20.58. Found: C, 52.49;
H, 2.96; N, 20.44.
(E)-6,6′-(Diazene-1,2-diyl)dinicotinic Acid (2,2′-AzPy-5,5′-diCA,
2b)
Melting points were determined with Mettler Toledo MP90 apparatus
and are uncorrected. 1H (500 MHz) and 13C (125 MHz) NMR spectra
were recorded on a Bruker Biospin 500 MHz spectrometer and 1H
(400 MHz) and 13C (100 MHz) NMR spectra were recorded on a Agi-
lent DD2 400 MHz spectrometer in CDCl3, DMSO-d6, D2O, or TFA-d1
with TMS as internal standard. NMR spectra were analyzed using
MestRe Nova 5.2 software. Elemental analyses were performed on a
Vario EL III CHNOS elemental analyzer. HRMS analyses were mea-
sured on a Waters SYNAPT MS-TOF or Agilent TOF system. Micro-
wave-assisted amino acid and peptide coupling reactions were per-
formed by using a single-mode cavity CEM Discover microwave syn-
thesizer with a continuous irradiation at 2450 MHz and equipped
with an infrared temperature control system. DMF was dried and dis-
Yield: 0.43 g (44%); pale red-orange microcrystals; mp 323–326 °C.
1H NMR (500 MHz, 0.1 M K2CO3 in D2O): δ = 8.96 (s, 2 H), 8.40 (d,
J = 8.2 Hz, 2 H), 7.92 (d, J = 8.2 Hz, 2 H).
13C NMR (125 MHz, 0.1 M K2CO3 in D2O): δ = 174.6, 166.4, 152.6,
143.1, 137.7, 120.1.
Anal. Calcd for C12H8N4O4: C, 52.95; H, 2.96; N, 20.58. Found: C, 52.46;
H, 2.99; N, 20.41.
(E)-6,6′-(Diazene-1,2-diyl)dipicolinic Acid (2,2′-AzPy-6,6′-diCA, 2c)
Yield: 0.40 g (41%); orange microcrystals; mp 252–254 °C (Lit.11 mp
143.3–144.5 °C).
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1H NMR (500 MHz, DMSO-d6): δ = 13.62 (br s, 2 H), 8.34–8.27 (m, 4
H), 8.04 (dd, J = 6.7 Hz, 2.0 Hz, 2 H).
(E)-2,2′-(Diazene-1,2-diyl)bis(N-ethylisonicotinamide) (2,2′-AzPy-
4,4′-diEtA, 5aa)
13C NMR (125 MHz, DMSO-d6): δ = 165.5, 162.0, 148.5, 140.6, 127.2,
Yield: 51 mg (75%); pale orange microcrystals; mp 231–232 °C.
1H NMR (500 MHz, CDCl3 + TFA): δ = 9.11 (d, J = 5.6 Hz, 2 H), 8.84 (d,
J = 1.1 Hz, 2 H), 8.60 (dd, J = 5.6, 1.4 Hz, 2 H), 3.62 (q, J = 7.3 Hz, 4 H),
1.33 (t, J = 7.3 Hz, 6 H).
116.5.
Anal. Calcd for C12H8N4O4: C, 52.95; H, 2.96; N, 20.58. Found: C, 52.71;
H, 3.00; N, 20.44.
13C NMR (125 MHz, CDCl3 + TFA): δ = 164.0, 156.5, 150.6, 146.6, 129.6,
2,2′-AzPy-Diacylbenzotriazoles (2,2′-AzPy-DiacylBt) 3a–c; General
115.8, 37.0, 13.5.
Procedure
HRMS [ESI(+)-TOF]: m/z [M + H]+ calcd for C16H18N6O2: 327.1569;
Et3N (0.22 g, 2.2 mmol) was added to a suspension of 2,2′-AzPy-dicar-
boxylic acid 2a–c (0.275 g, 1 mmol) in anhydrous DMF (4 mL) in a
dried 10 mL heavy-walled Pyrex tube containing a long stir bar.
BtSO2Me (0.43 g, 2.2 mmol) was added to this solution and exposed to
microwave irradiation (30 W) for 15 min at 70 °C with vigorous stir-
ring and simultaneous air cooling. After completion of the reaction,
the mixture was cooled to r.t. and poured onto crushed ice-water
mixture (~20 g). The precipitate formed was filtered and washed with
aq 10% Na2CO3 (3 × 5 mL), H2O, and Et2O. The solid was dried to yield
2,2′-AzPy-diacylbenzotriazoles 3a–c as microcrystals (Table 1).
found: 327.1571.
Anal. Calcd for C16H18N6O2: C, 58.88; H, 5.56; N, 25.75. Found: C,
58.75; H, 5.64; N, 26.16.
(E)-2,2′-(Diazene-1,2-diyl)bis(N-cyclohexylisonicotinamide) (2,2′-
AzPy-4,4′-dicHA, 5ab)
Yield: 79 mg (87%); pale orange microcrystals; mp 260–261 °C.
1H NMR (500 MHz, DMSO-d6): δ = 9.10 (d, J = 5.6 Hz, 2 H), 8.78 (d,
J = 1.1 Hz, 2 H), 8.58 (dd, J = 5.6, 1.4 Hz, 2 H), 4.05–3.95 (m, 2 H), 2.08–
2.00 (m, 4 H), 1.90–1.80 (m, 4 H), 1.76–1.68 (m, 2 H), 1.50–1.30 (m, 8
H), 1.30–1.18 (m, 2 H).
(E)-[Diazene-1,2-diylbis(pyridine-2,4-diyl)]bis[(1H-ben-
zo[d][1,2,3]triazol-1-yl)methanone] (2,2′-AzPy-4,4′-diBt, 3a)
13C NMR (125 MHz, CDCl3 + TFA): δ = 163.1, 156.6, 150.5, 146.5, 129.4,
Yield: 0.42 g (89%); pale red-brown microcrystals; mp 255–260 °C
(dec.).
115.6, 51.8, 32.8 (2 C), 25.2, 24.9 (2 C).
Anal. Calcd for C24H30N6O2: C, 66.34; H, 6.96; N, 19.34. Found: C,
66.31; H, 7.26; N, 19.73.
Anal. Calcd for C24H14N10O2: C, 60.76; H, 2.97; N, 29.52. Found: C,
60.38; H, 2.98; N, 29.08.
(E)-6,6′-(Diazene-1,2-diyl)bis(N-ethylnicotinamide) (2,2′-AzPy-
5,5′-diEtA, 5ba)
(E)-[Diazene-1,2-diylbis(pyridine-6,3-diyl)]bis[(1H-ben-
zo[d][1,2,3]triazol-1-yl)methanone] (2,2′-AzPy-5,5′-diBt, 3b)
Yield: 48 mg (70%); pale red microcrystals; mp 262–264 °C.
Yield: 0.29 g (62%); pale red-brown microcrystals; mp 238–242 °C
(dec.).
1H NMR (500 MHz, CDCl3 + TFA): δ = 9.50 (s, 2 H), 9.12 (d, J = 7.8 Hz, 2
H), 8.59 (d, J = 8.0 Hz, 2 H), 3.63 (q, J = 7.3 Hz, 4 H), 1.34 (t, J = 7.3 Hz, 6
Anal. Calcd for C24H14N10O2: C, 60.76; H, 2.97; N, 29.52. Found: C,
60.17; H, 3.04; N, 29.14.
H).
13C NMR (125 MHz, CDCl3 + TFA): δ = 164.0, 156.7, 146.8, 144.5, 136.4,
121.6, 37.2, 13.7.
(E)-[Diazene-1,2-diylbis(pyridine-6,2-diyl)]bis[(1H-ben-
zo[d][1,2,3]triazol-1-yl)methanone] (2,2′-AzPy-6,6′-diBt, 3c)
Anal. Calcd for C16H18N6O2: C, 58.88; H, 5.56; N, 25.75. Found: C,
58.71; H, 5.99; N, 25.37.
Yield: 0.29 g (62%); pale red-orange microcrystals; mp 236–238 °C
(dec.).
(E)-6,6′-(Diazene-1,2-diyl)bis(N-cyclohexylnicotinamide) (2,2′-
AzPy-5,5′-dicHA, 5bb)
Anal. Calcd for C24H14N10O2: C, 60.76; H, 2.97; N, 29.52. Found: C,
60.40; H, 3.06; N, 29.24.
Yield: 80 mg (88%); orange microcrystals; mp 286–288 °C.
1H NMR (500 MHz, CDCl3 + TFA): δ = 9.43 (d, J = 18 Hz, 2 H), 9.04 (dd,
J = 8.4, 2.0 Hz, 2 H), 8.52 (d, J = 8.4 Hz, 2 H), 4.06–3.96 (m, 2 H), 2.10–
2.00 (m, 4 H), 1.90–1.80 (m, 4 H), 1.76–1.68 (m, 2 H), 1.50–1.36 (m, 8
H), 1.30–1.16 (m, 2 H).
13C NMR (125 MHz, CDCl3 + TFA): δ = 163.0, 156.9, 145.9, 144.6, 136.0,
120.6, 51.6, 32.3 (2 C), 25.2, 24.9 (2 C).
2,2′-AzPy-Diacylamines 5aa–cc; General Procedure
Et3N (0.11 g, 1.1 mmol) was added to a mixture of 2,2′-AzPy-diacyl-
benzotriazole 3a–c (0.10 g, 0.21 mmol) and primary amine hydro-
chloride salt 4 (0.84 mmol) in anhydrous DMF (2.5 mL) in a dried 10
mL heavy-walled Pyrex tube containing a long stir bar. The reaction
mixture was exposed to microwave irradiation (30 W, 70 °C) for 5 min
with vigorous stirring and simultaneous air cooling. After completion
of the reaction, the mixture was cooled to r.t. and poured onto
crushed ice-water mixture (~20 g). The precipitate formed was fil-
tered and washed with aq 10% Na2CO3 (3 × 5 mL), aq 1 N HCl (3 × 5
mL), H2O, and Et2O, respectively. The solid was dried under vacuum to
yield 2,2′-AzPy-diacylamines 5aa–cc as microcrystals. Et3N was not
used in the case of cyclohexylamine (1.26 mmol). In the preparation
of 5cc, 4-methoxyaniline (0.104 g, 0.84 mmol) and Et3N (0.43 g, 0.42
mmol) were used and MW reaction took 10 min (if a precipitate did
not form upon acidification, the solution was saturated by adding sol-
id NaCl and cooled for 2–3 h) (Table 2).
HRMS [ESI(+)-TOF]: m/z [M + Na]+ calcd for C16H18N6O2Na: 457.2322;
found: 457.2343.
(E)-6,6′-(Diazene-1,2-diyl)bis(N-ethylpicolinamide) (2,2′-AzPy-
6,6′-diEtA, 5ca)
Yield: 46 mg (67%); pale orange-yellow microcrystals; mp 208–
210 °C.
1H NMR (500 MHz, CDCl3): δ = 8.44 (d, J = 7.5 Hz, 2 H), 8.18–8.10 (m, 4
H), 8.01 (d, J = 7.8 Hz, 2 H), 3.64–3.52 (m, 4 H), 1.32 (t, J = 7.3 Hz, 6 H).
13C NMR (125 MHz, CDCl3): δ = 163.2, 161,2, 150.4, 140.0, 124.9,
116.4, 34.5, 14.9.
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HRMS [ESI(+)-TOF]: m/z [M + H]+ calcd for C16H18N6O2: 327.1569;
13C NMR (125 MHz, DMSO-d6): δ = 173.6, 163.7, 163.0, 150.4, 143.8,
found: 327.1570.
124.2, 111.1, 48.3, 16.7.
HRMS [ESI(+)-TOF]: m/z [M + H]+ calcd for C18H18N6O6: 415.1361;
found: 415.1358.
(E)-6,6′-(Diazene-1,2-diyl)bis(N-cyclohexylpicolinamide) (2,2′-
AzPy-6,6′-dicHA, 5cb)
Yield: 75 mg (82%); pale orange microcrystals; mp 188–190 °C.
(2S,2′S)-2,2′-({2,2′-[(E)-Diazene-1,2-diyl]bis(isonicoti-
noyl)}bis(azanediyl))bis(3-methylbutanoic Acid) (2,2′-AzPy-4,4′-
diVal, 7ab)
1H NMR (500 MHz, CDCl3): δ = 8.44 (dd, J = 7.6 Hz, 0.8 Hz, 2 H), 8.11
(t, J = 7.8 Hz, 2 H), 8.04–7.96 (m, 4 H), 4.08–3.96 (m, 2 H), 2.12–2.04
(m, 4 H), 1.86–1.76 (m, 4 H), 1.73–1.64 (m, 2 H), 1.52–1.32 (m, 8 H),
1.28–1.16 (m, 2 H).
13C NMR (125 MHz, CDCl3): δ = 162.4, 161.3, 150.6, 125.0, 115.9, 48.7,
33.1 (2c), 25.6, 25.1 (2C).
Yield: 110 mg (73%); pale orange microcrystals; mp 199–201 °C.
1H NMR (500 MHz, DMSO-d6): δ = 12.78 (br s, 2 H), 9.08 (d, J = 8.1 Hz,
2 H), 8.94 (d, J = 4.9 Hz, 2 H), 8.24 (s, 2 H), 8.06 (d, J = 4.9 Hz, 2 H), 4.36
(t, J = 7.2 Hz, 2 H), 2.26–2.16 (m, 2 H), 1.00 (d, J = 6.4 Hz, 6 H), 0.98 (d,
J = 6.4 Hz, 6 H).
HRMS [ESI(+)-TOF]: m/z [M + H]+ calcd for C24H30N6O2: 435.2508;
found: 435.2504.
13C NMR (125 MHz, DMSO-d6): δ = 172.6, 164.6, 162.8, 150.2, 144.0,
124.4, 111.5, 58.5, 16.7, 29.41, 19.16, 18.63.
HRMS [ESI(+)-TOF]: m/z [M + H]+ calcd for C22H26N6O6: 471.1992;
found: 471.1992.
(E)-6,6′-(Diazene-1,2-diyl)bis[N-(4-methoxyphenyl)picolinamide]
(2,2′-AzPy-6,6′-diMeOPhA, 5cc)
Yield: 70 mg (69%); orange microcrystals; mp 234–235 °C.
1H NMR (500 MHz, CDCl3): δ = 9.92 (s, 2 H), 8.54 (d, J = 7.5 Hz, 2 H),
8.19 (t, J = 7.8 Hz, 2 H), 8.08 (d, J = 7.8 Hz, 2 H), 7.75 (d, J = 8.8 Hz, 4 H),
6.95 (d, J = 8.8 Hz, 4 H), 3.83 (s, 6 H).
(2S,2′S)-2,2′-[{2,2′-[(E)-Diazene-1,2-diyl]bis(isonicoti-
noyl)}bis(azanediyl)]bis(3-phenylpropanoic Acid) (2,2′-AzPy-4,4′-
diPhe, 7ac)
Yield: 160 mg (88%); pale brown microcrystals; mp 210–212 °C.
13C NMR (125 MHz, CDCl3): δ = 161.1, 160.9, 150.4, 140.3, 130.7,
125.1, 121.8 (2 C), 116.6, 144.3 (2 C), 55.5.
1H NMR (500 MHz, DMSO-d6): δ = 12.93 (br s, 2 H), 9.31 (d, J = 8.2 Hz,
2 H), 8.94 (d, J = 5.0 Hz, 2 H), 8.16 (s, 2 H), 7.96 (dd, J = 5.0, 1.4 Hz, 2
H), 7.35–7.10 (m, 8 H), 7.22–7.14 (m, 2 H), 4.76–4.66 (m, 2 H), 3.22
(dd, J = 13.9, 4.5 Hz, 2 H), 3.08 (dd, J = 13.9, 10.8 Hz, 2 H).
Anal. Calcd for C26H22N6O2: C, 64.72; H, 4.60; N, 17.42. Found: C,
64.36; H, 4.62; N, 17.43.
13C NMR (125 MHz, DMSO-d6): δ = 172.5, 163.9, 162.9, 150.4, 143.7,
137.8, 128.9 (2 C), 128.1 (2 C), 126.4, 124.0, 111.1, 54.2, 36.1.
HRMS [ESI(+)-TOF]: m/z [M + H]+ calcd for C30H26N6O6: 567.1992;
2,2′-AzPy-Diacylamino Acids 7aa–cc and 7cb + 7cb′; General Proce-
dure
Et3N (0.16 g, 1.58 mmol) was added to a suspension of amino acid 6a–
c (1.58 mmol) in H2O (1.5 mL) in a 10 mL heavy-walled Pyrex tube
containing a long stir bar. MeCN (3.5 mL) was added to the amino acid
solution and stirred for 5 min at r.t. 2,2′-AzPy-diacylbenzotriazole 3
(0.15 g, 0.32 mmol) was added to this white suspension. The mixture
was exposed to microwave irradiation (20 W, 70 °C) for the specified
reaction time (15 min for 3a, 10 min for 3b, and 5 min for 3a) with
vigorous stirring and simultaneous air cooling. The reactions were
carried out until the disappearance of all colored solid starting mate-
rials 3 in the reaction vessel. After completion of the reaction, the
mixture was cooled to r.t. and poured onto crushed ice-water mixture
(~10 g). The mixture was acidified with aq 2 N HCl to pH 3–4. The
precipitate formed was filtered and washed with aq 1 N HCl (3 × 5
mL), H2O, and Et2O, respectively (if a precipitate did not form upon
acidification, the solution was saturated by adding solid NaCl and
cooled for 2–3 h). Compound 7aa and 7ba were quite water soluble,
so the compound in aqueous part (at pH 3–4) was recovered by ex-
tracting with EtOAc (3 × 10 mL). The combined organic phases were
washed with brine (3 mL), dried (MgSO4), and evaporated under re-
duced pressure. The solid obtained was washed with Et2O and dried
under vacuum to yield 2,2′-AzPy-diacylamino acids (Table 3).
found: 567.2002.
(2S,2′S)-2,2′-[{6,6′-[(E)-Diazene-1,2-diyl)bis(nicoti-
noyl)}bis(azanediyl)]dipropionic Acid (2,2′-AzPy-5,5′-diAla, 7ba)
Yield: 86 mg (65%); pale red-brown microcrystals; mp 224–225 °C.
1H NMR (500 MHz, DMSO-d6): δ = 12.69 (br s, 2 H), 9.22 (d, J = 1.8 Hz,
2 H), 9.12 (d, J = 7.1 Hz, 2 H), 8.54 (dd, J = 8.4, 2.1 Hz, 2 H), 7.93 (d,
J = 8.3 Hz, 2 H), 4.54–4.44 (m, 2 H), 1.45 (d, J = 7.3 Hz, 6 H).
13C NMR (125 MHz, DMSO-d6): δ = 173.7, 163.8, 163.5, 148.8, 138.4,
131.4, 113.5, 48.2, 16.7.
HRMS [ESI(+)-TOF]: m/z [M + H]+ calcd for C18H18N6O6: 415.1361;
found: 415.1366.
Anal. Calcd for C18H18N6O6: C, 52.17; H, 4.38; N, 20.28. Found: C,
51.71; H, 4.02; N, 20.11.
(2S,2′S)-2,2′-({6,6′-[(E)-Diazene-1,2-diyl]bis(nicoti-
noyl)}bis(azanediyl))bis(3-methylbutanoic Acid) (2,2′-AzPy-5,5′-
diVal, 7bb)
Yield: 112 mg (74%); pale red-brown microcrystals; mp 91–92 °C.
(2S,2′S)-2,2′-({2,2′-[(E)-Diazene-1,2-diyl]bis(isonicoti-
noyl)}bis(azanediyl))dipropionic Acid (2,2′-AzPy-4,4′-diAla, 7aa)
1H NMR (500 MHZ, DMSO-d6): δ = 12.79 (br s, 2 H), 9.20 (d, J = 1.6 Hz,
2 H), 8.92 (d, J = 8.1 Hz, 2 H), 8.54 (dd, J = 8.3, 1.9 Hz, 2 H), 7.92 (d,
J = 8.3 Hz, 2 H), 4.37 (t, J = 7.4 Hz, 2 H), 2.30–2.18 (m, 2 H), 1.02 (d,
J = 6.9 Hz, 6 H), 1.01 (d, J = 6.9 Hz, 6 H).
13C NMR (125 MHZ, DMSO-d6): δ = 172.6, 164.7, 163.5, 148.9, 138.6,
131.7, 113.4, 58.4, 29.5, 19.2, 18.6.
Yield: 112 mg (85%); pale red microcrystals; mp 205–206 °C.
1H NMR (500 MHz, DMSO-d6): δ = 12.74 (br s, 2 H), 9.28 (d, J = 7.0 Hz,
2 H), 8.96 (d, J = 4.9 Hz, 2 H), 8.27 (s, 2 H), 8.08 (d, J = 4.4 Hz, 2 H),
4.54–4.44 (m, 2 H), 1.44 (d, J = 7.23 Hz, 6 H).
HRMS [ESI(+)-TOF]: m/z [M + H]+ calcd for C22H26N6O6: 471.1992;
found: 471.1984.
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(2S,2′S)-2,2′-({6,6′-[(E)-Diazene-1,2-diyl)bis(nicoti-
noyl)}bis(azanediyl))bis(3-phenylpropanoic Acid) (2,2′-AzPy-5,5′-
diPhe, 7bc)
13C NMR (125 MHz, DMSO-d6): δ = 172.4, 162.8, 161.0, 149.2, 141.2,
124.7, 116.4, 116.3, 57.2, 30.3, 19.0, 17.8.
HRMS [ESI(+)-TOF]: m/z [M + H]+ calcd for C22H26N6O6: 471.1992;
Yield: 152 mg (84%); orange microcrystals; mp 203–205 °C.
found: 471.1997.
1H NMR (500 MHz, DMSO-d6): δ = 12.90 (br s, 2 H), 9.18 (d, J = 8.1 Hz,
2 H), 9.10 (d, J = 1.9 Hz, 2 H), 8.42 (dd, J = 8.3, 2.1 Hz, 2 H), 7.89 (d,
J = 8.3 Hz, 2 H), 7.38–7.26 (m, 8 H), 7.24–7.17 (m, 2 H), 4.76–4.66 (m,
2 H), 3.25 (dd, J = 13.8, 4.5 Hz, 2 H), 3.08 (dd, J = 13.8, 10.8 Hz, 2 H).
(2S,2′S)-2,2′-({6,6'-[(E)-Diazene-1,2-diyl]bis(picoli-
noyl)}bis(azanediyl))bis(3-phenylpropanoic Acid) (2,2′-AzPy-6,6′-
diPhe, 7cc)
13C NMR (125 MHz, DMSO-d6): δ = 172.6, 164.0, 163.5, 148.6, 138.3,
Yield: 148 mg (82%); pale orange microcrystals; mp 205–207 °C.
1H NMR (500 MHz, DMSO-d6): δ = 13.07 (br s, 2 H), 8.84 (d, J = 8.2 Hz,
2 H), 8.34–8.22 (m, 4 H), 7.98 (d, J = 7.7 Hz, 2 H), 7.28–7.22 (m, 8 H),
7.22–7.14 (m, 2 H), 4.86–4.76 (m, 2 H), 3.29–3.24 (m, 4 H).
137.8, 131.4, 129.0 (2 C), 128.2 (2 C), 126.4, 113.6, 54.2, 36.3.
Anal. Calcd for C30H26N6O6: C, 63.60; H, 4.63; N, 14.83. Found: C,
63.27; H, 4.69; N, 14.87.
13C NMR (125 MHz, DMSO-d6): δ = 172.4, 162.8, 161.2, 149.4, 141.1,
(2S,2′S)-2,2'-({6,6'-[(E)-Diazene-1,2-diyl]bis(picoli-
137.4, 129.1 (2 C), 128.2 (2 C), 126.5, 124.7, 116.1, 53.4, 36.3.
noyl)}bis(azanediyl))dipropionic Acid (2,2′-AzPy-6,6′-diAla, 7ca)
Anal. Calcd for C30H26N6O6: C, 63.60; H, 4.63; N, 14.83. Found: C,
63.12; H, 4.35; N, 14.82.
Yield: 120 mg (91%); pale orange microcrystals; mp 240–241 °C.
1H NMR (500 MHz, DMSO-d6): δ = 12.86 (br s, 2 H), 8.93 (d, J = 7.6 Hz,
2 H), 8.38–8.28 (m, 4 H), 8.09 (d, J = 7.6 Hz, 2 H), 4.62–4.52 (m, 2 H),
1.48 (d, J = 7.2 Hz, 6 H).
13C NMR (125 MHz, DMSO-d6): δ = 173.6, 162.6, 161.1, 149.6, 141.0,
124.7, 115.9, 47.8, 17.2.
2,2′-AzPy-Diacyldipeptide-Amides 9a–c; General Procedure
2,2′-AzPy-diacylbenzotriazole 3 (0.050 g, 0.10 mmol) was added to a
mixture of free dipeptide-amide hydrochloride salt 8 (0.135 g, 0.5
mmol) and Et3N (0.10 g, 1.0 mmol) in anhydrous DMF (2.0 mL) in a
dried 10 mL heavy-walled Pyrex tube containing a long stir bar. The
reaction mixture was exposed to microwave irradiation (20 W, 70 °C)
for 10 min with vigorous stirring and simultaneous air cooling. After
completion of the reaction, the mixture was cooled to r.t. and poured
onto crushed ice-water mixture (~5 g). The mixture was acidified
with aq 1 N HCl (3 mL). The precipitate formed was filtered and
washed with aq 10% Na2CO3 (3 × 5 mL), aq 1 N HCl (3 × 5 mL), H2O and
Et2O, respectively. The solid product was dried under vacuum to yield
2,2′-AzPy-diacyldipeptide-amides 9a–c (Table 5).
HRMS [ESI(+)-TOF]: m/z [M + H]+ calcd for C18H18N6O6: 415.1361;
found: 415.1366.
(2S,2′S)-2,2′-({6,6′-[(E)-Diazene-1,2-diyl]bis(picoli-
noyl)}bis(azanediyl))bis(3-methylbutanoic Acid) (2,2′-AzPy-6,6′-
diVal, 7cb)
Yield: 104 mg (69%); pale orange microcrystals; mp 195–197 °C.
E-Isomer
1H NMR (500 MHz, DMSO-d6): δ = 13.11 (br s, 2 H), 8.57 (d, J = 8.8 Hz,
2 H), 8.38–8.28 (m, 4 H), 8.08 (dd, J = 7.4, 0.9 Hz, 2 H), 4.49 (dd, J = 8.8,
5.3 Hz, 2 H), 2.34–2.24 (m, 2 H), 0.98 (d, J = 5.1 Hz, 6 H), 0.96 (d, J = 5.1
Hz, 6 H).
2,2′-[(E)-Diazene-1,2-diyl]bis[N-(2-{[(S)-1-(isopropylamino)-4-
methyl-1-oxopentan-2-yl]amino}-2-oxoethyl)isonicotinamide]
[2,2′-AzPy-4,4′-di(Gly-Leu-NHPr-i), 9a]
Yield: 42 mg (61%); pale pink-orange microcrystals; mp 242–244 °C.
13C NMR (125 MHz, DMSO-d6): δ = 172.4, 162.8, 161.0, 149.2, 141.2,
124.7, 116.4, 57.2, 30.3, 19.0, 17.8.
1H NMR (400 MHz, TFA-d1): δ = 9.58 (d, J = 5.7 Hz, 2 H), 9.46 (d, J = 1.4
Hz, 2 H), 9.58 (dd, J = 5.7, 1.5 Hz, 2 H), 5.10–5.02 (m, 2 H), 4.96–4.82
(m, 4 H), 4.56–4.44 (m, 2 H), 2.20–1.96 (m, 6 H), 1.60 (d, J = 3.7 Hz, 6
H), 1.58 (d, J = 3.7 Hz, 6 H), 1.32 (d, J = 5.8 Hz, 6 H), 1.28 (d, J = 5.8 Hz,
6 H).
13C NMR (100 MHz, TFA-d1): δ = 176.2, 173.6, 167.0, 157.2, 152.9,
148.2, 131.5, 121.9, 55.7, 46.7, 45.6, 42.4, 26.9, 23.2, 22.4, 22.3.
Z-Isomer
NMR spectra of samples containing a mixture of both isomers (E and
Z) were recorded after laser irradiation at 325 nm of the NMR tube for
10 min (see SI, p S28–S29).
1H NMR (500 MHz, DMSO-d6): δ = 13.11 (br s, 2 H), 8.20 (t, J = 7.8 Hz,
2 H), 8.00 (d, J = 7.9, 2 H), 7.86 (d, J = 8.6 Hz, 2 H), 7.27 (d, J = 9.1 Hz, 2
H), 4.18 (dd, J = 9.0, 5.4 Hz, 2 H), 2.34–2.24 (m, 2 H), 0.72 (d, J = 6.8 Hz,
6 H), 0.69 (d, J = 6.8 Hz, 6 H).
HRMS [ESI(+)-TOF]: m/z [M + H]+ calcd for C34H50N10O6: 695.3988;
found: 695.3986.
13C NMR (125 MHz, DMSO-d6): δ = 172.0, 162.0, 161.9, 146.9, 140.8,
122.1, 121.0, 56.6, 30.1, 18.8, 17.6.
6,6′-[(E)-Diazene-1,2-diyl]bis[N-(2-{[(S)-1-(isopropylamino)-4-
methyl-1-oxopentan-2-yl]amino}-2-oxoethyl)nicotinamide] [2,2′-
AzPy-5,5′-di(Gly-Leu-NHPr-i), 9b]
Anal. Calcd for C22H26N6O6: C, 56.16; H, 5.57; N, 17.86. Found: C,
55.79; H, 5.58; N, 17.74.
Yield: 49 mg (70%); pale brown microcrystals; mp 212–216 °C.
1H NMR (400 MHz, TFA-d1): δ = 9.99 (s, 2 H), 9.62 (d, J = 8.4 Hz, 2 H),
9.07 (d, J = 8.3 Hz, 2 H), 5.10–5.00 (m, 2 H), 4.96–4.80 (m, 4 H), 4.56–
4.44 (m, 2 H), 2.18–1.96 (m, 6 H), 1.58 (d, J = 3.0 Hz, 6 H), 1.57 (d,
J = 3.2 Hz, 6 H), 1.32 (d, J = 5.6 Hz, 6 H), 1.28 (d, J = 5.6 Hz, 6 H).
13C NMR (100 MHz, TFA-d1): δ = 176.2, 173.8, 166.8, 157.9, 149.0,
147.6, 137.5, 124.4, 55.8, 46.8, 45.6, 42.5, 26.9, 23.2, 22.5, 22.4.
(E)-2,2′-{[6,6′-(Diazene-1,2-diyl)bis(picolinoyl)]bis(azanedi-
yl)}bis(3-methylbutanoic Acid) [2,2′-AzPy-6,6′-di(DL)Val, (7cb +
7cb′)]
Yield: 123 mg (82%); pale orange microcrystals; mp 193–196 °C.
1H NMR (500 MHz, DMSO-d6): δ = 13.11 (br s, 2 H), 8.66–8.54 (m, 2
H), 8.38–8.28 (m, 4 H), 8.14–8.06 (m, 2 H), 4.54–4.44 (m, 2 H), 2.36–
2.24 (m, 2 H), 1.08–0.90 (m, 12 H).
HRMS [ESI(+)-TOF]: m/z [M + H]+ calcd for C34H50N10O6: 695.3988;
found: 695.3998.
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6,6′-[(E)-Diazene-1,2-diyl]bis[N-(2-{[(S)-1-(isopropylamino)-4-
methyl-1-oxopentan-2-yl]amino}-2-oxoethyl)picolinamide] [2,2′-
AzPy-6,6′-di(Gly-Leu-NHPr-i), 9c]
(2S,2′S)-2,2′-[{(2S,2′S)-2,2′-[{2,2′-[{6,6′-[(E)-Diazene-1,2-di-
yl]bis(picolinoyl)}bis(azanediyl)]bis(acetyl)}bis(azanediyl)]bis(4-
methylpentanoyl)}bis(azanediyl)]bis(3-phenylpropanoic Acid)
[2,2′-AzPy-6,6′-di(Gly-Leu-Phe), 13]
Yield: 53 mg (76%); pale orange microcrystals; mp 244–246 °C.
Yield: 81 mg (89%); pale orange microcrystals; mp 212–214 °C.
1H NMR (400 MHz, TFA-d1): δ = 9.05–8.95 (m, 4 H), 8.92–8.84 (m, 2
H), 5.08–5.00 (m, 2 H), 4.94–4.80 (m, 4 H), 4.52–4.42 (m, 2 H), 2.12–
1.94 (m, 6 H), 1.56 (d, J = 2.6 Hz, 6 H), 1.54 (d, J = 2.6 Hz, 6 H), 1.28 (d,
J = 6.0 Hz, 6 H), 1.24 (d, J = 5.8 Hz, 6 H).
13C NMR (100 MHz, TFA-d1): δ = 176.3, 174.0, 165.9, 159.9, 148.4,
147.6, 130.2, 123.6, 55.7, 46.9, 45.4, 42.4, 26.9, 23.2, 22.4 (2 C).
1H NMR (500 MHz, DMSO-d6): δ = 12.64 (br s, 2 H), 8.96 (t, J = 5.5 Hz,
2 H), 8.36–8.26 (m, 4 H), 8.24 (d, J = 7.7 Hz, 2 H), 8.13 (d, J = 8.3 Hz, 2
H), 8.02 (d, J = 7.4 Hz, 2 H), 7.28–7.18 (m, 8 H), 7.18–7.14 (m, 2 H),
4.44–4.36 (m, 4 H), 4.06–3.96 (m, 4 H), 3.04 (dd, J = 13.9, 5.2 Hz, 2 H),
2.92 (dd, J = 13.7, 8.9 Hz, 2 H), 1.64–1.54 (m, 2 H), 1.46–1.36 (m, 4 H),
0.86 (d, J = 6.4 Hz, 6 H), 0.86 (d, J = 6.4 Hz, 6 H).
13C NMR (125 MHz, DMSO-d6): δ = 172.6, 171.7, 167.9, 163.1, 161.1,
149.7, 140.1, 137.4, 129.0 (2 C), 128.0 (2 C), 126.2, 124.6, 115.9, 53.3,
50.6, 42.2, 40.9, 36.4, 23.9, 23.0, 21.6.
HRMS [ESI(+)-TOF]: m/z [M + H]+ calcd for C34H50N10O6: 695.3988;
found: 695.4009.
2,2′-AzPy-6,6′-Di(di- and tripeptides) 12a, 12b, and 13; General
Procedure
HRMS [ESI(+)-TOF]: m/z [M + H]+ calcd for C46H54N10O10: 907.4097;
found: 907.4124.
Et3N (0.10 g, 1.00 mmol) was added to a suspension of the appropri-
ate free peptide hydrogen chloride salt 10a,b, 11a (0.50 mmol) in H2O
(0.5 mL) in a 10 mL heavy-walled Pyrex tube containing a long stir
bar. MeCN (1.5 mL) and DMF (0.2 mL) was added to the peptide solu-
tion and stirred for 2 min at r.t. 2,2′-AzPy-6,6′-diacylbenzotriazole 3c
(0.05 g, 0.10 mmol) was added to this solution. The mixture was ex-
posed to microwave irradiation (20 W, 70 °C) for 10 min with vigor-
ous stirring and simultaneous air cooling. After completion of the re-
action, the mixture was cooled to r.t. and poured onto crushed ice-
water mixture (~10 g). The mixture was acidified with aq 2 N HCl to
pH 3–4. The precipitate formed was filtered and washed with aq 1 N
HCl (3 × 5 mL), H2O, and Et2O, respectively (if a precipitate did not
form upon acidification, the solution was saturated by adding solid
NaCl and cooled for 2–3 h). The solid product was dried under vacu-
um to yield 2,2′-AzPy-6,6′-diacylpeptides (Table 5).
2,2′-AzPy-Di(tripeptides) 14a, 14b, 15a, and 15b
2,2′-AzPy-diacyltripeptide-esters 14a and 15a were prepared by fol-
lowing the same general procedure as described for 2,2′-AzPy-dia-
cyldipeptide-amides 9a–c (Table 5).
Dimethyl 2,2′-[{(2S,2′S)-2,2′-[{2,2′-[{2,2′-[(E)-Diazene-1,2-di-
yl]bis(isonicotinoyl)}bis(azanediyl)]bis(acetyl)}bis(azanedi-
yl)]bis(4-methylpentanoyl)}bis(azanediyl)](2S,2′S)-bis(3-
phenylpropanoate) [2,2′-AzPy-4,4′-di(Gly-Leu-Phe-OMe), 14a]
Yield: 61 mg (65%); brown microcrystals; mp 176–180 °C.
1H NMR (400 MHz, DMSO-d6): δ = 9.38–9.28 (m, 2 H), 8.96 (d, J = 4.4
Hz, 2 H), 8.41 (d, J = 7.0 Hz, 2 H), 8.25 (s, 2 H), 8.13 (d, J = 7.9 Hz, 2 H),
8.06 (d, J = 4.1 Hz, 2 H), 7.36–7.10 (m, 10 H), 4.56–4.34 (m, 4 H), 4.04–
3.90 (m, 4 H), 3.55 (s, 6 H), 3.10–2.90 (m, 4 H), 1.70–1.50 (m, 2 H),
1.50–1.34 (m, 4 H), 0.88 (d, J = 6.2 Hz, 6 H), 0.85 (d, J = 6.2 Hz, 6 H).
13C NMR (100 MHz, DMSO-d6): δ = 171.9, 171.6, 167.9, 164.0, 162.9,
150.4, 143.8, 137.0, 129.0 (2 C), 128.1 (2 C), 126.4, 124.0, 111.0, 53.4,
51.6, 50.4, 42.3, 40.8, 36.3, 23.9, 22.9, 21.6.
(2S,2′S)-2,2′-[{2,2′-[{6,6′-[(E)-Diazene-1,2-diyl]bis(picoli-
noyl)}bis(azanediyl)]bis(acetyl)}bis(azanediyl)]bis(4-methylpen-
tanoic Acid) [2,2′-AzPy-6,6′-di(Gly-Leu), 12a]
Yield: 44 mg (72%); pale orange microcrystals; mp 113–115 °C.
1H NMR (500 MHz, DMSO-d6): δ = 12.61 (br s, 2 H), 8.94 (t, J = 5.6 Hz,
2 H), 8.36–8.26 (m, 6 H), 8.02 (d, J = 7.4 Hz, 2 H), 4.32–4.26 (m, 2 H),
4.04 (m, 4 H), 1.74–1.60 (m, 2 H), 1.60–1.48 (m, 2 H), 0.90 (d, J = 6.5
Hz, 6 H), 0.86 (d, J = 6.5 Hz, 6 H).
HRMS [ESI(+)-TOF]: m/z [M + H]+ calcd for C48H58N10O10: 935.4410;
found: 935.4445.
(2S,2′S)-2,2′-[{(2S,2′S)-2,2′-[{2,2′-[{2,2′-[(E)-Diazene-1,2-di-
yl]bis(isonicotinoyl)}bis(azanediyl)]bis(acetyl)}bis(azanedi-
yl)]bis(4-methylpentanoyl)}bis(azanediyl)]bis(3-phenylpropanoic
Acid) [2,2′-AzPy-4,4′-di(Gly-Leu-Phe), 14b)]
13C NMR (125 MHz, DMSO-d6): δ = 173.8, 168.3, 163.1, 161.1, 149.7,
141.0, 124,6 116,0 50.2, 41.9, 40.1, 24.2, 22.7, 21.3.
HRMS [ESI(+)-TOF]: m/z [M + H]+ calcd for C28H36N8O8: 613.2729;
found: 613.2728.
LiOH (8 mg, 0.33 mmol) was added to a suspension of 14a (30 mg,
0.032 mmol) in DMF–MeOH–H2O ((3:1:1), 2 mL) in a test tube at
10 °C. After stirring the reaction mixture for 2 h at 10 °C, ice-cold wa-
ter (5 mL) was added to this mixture and acidified with aq 1 N HCl
until pH 3–4. The precipitate formed was collected by suction filtra-
tion and washed with cold H2O and Et2O. The solid product was dried
under vacuum to give 14b as pale brown microcrystals; yield: 20 mg
(69%); mp 138–142 °C.
1H NMR (400 MHz, DMSO-d6): δ = 12.68 (br s, 2 H), 9.38–9.28 (m, 2
H), 8.96 (d, J = 4.9 Hz, 2 H), 8.32–8.20 (m, 4 H), 8.13 (d, J = 8.4 Hz, 2 H),
8.06 (d, J = 4.2 Hz, 2 H), 7.32–7.14 (m, 10 H), 4.46–4.34 (m, 4 H), 4.04–
3.88 (m, 4 H), 3.05 (dd, J = 13.8, 5.2 Hz, 2 H), 2.92 (dd, J = 13.8, 9.0 Hz,
2 H), 1.66–1.54 (m, 2 H), 1.50–1.36 (m, 4 H), 0.86 (d, J = 6.4 Hz, 6 H),
0.84 (d, J = 6.4 Hz, 6 H).
(2S,2′S)-2,2′-[{(2S,2′S)-2,2′-[{6,6′-[(E)-Diazene-1,2-diyl]bis(picoli-
noyl)}bis(azanediyl)]bis(propanoyl)}bis(azanediyl)]bis(3-phenyl-
propanoic Acid) [2,2′-AzPy-6,6′-di(Ala-Phe), 12b]
Yield: 59 mg (83%); pale orange microcrystals; mp 134–138 °C.
1H NMR (500 MHz, DMSO-d6): δ = 12.79 (br s, 2 H), 8.70 (d, J = 7.9 Hz,
2 H), 8.48 (d, J = 8.0 Hz, 2 H) 8.36–8.26 (m, 4 H), 8.04 (dd, J = 7.3, 0.9
Hz, 2 H), 7.28–7.18 (m, 8 H), 7.18–7.12 (m, 2 H), 4.64–4.56 (m, 2 H),
4.52–4.42 (m, 2 H), 3.09 (dd, J = 13.8, 5.0 Hz, 2 H), 2.82 (dd, J = 13.8,
9.3 Hz, 2 H), 1.32 (d, J = 6.9 Hz, 6 H).
13C NMR (125 MHz, DMSO-d6): δ = 172.5, 171.5, 162.0, 161.0, 149.4,
141.0, 137.3, 129.0 (2C), 128.3 (2C), 126.3, 124.6, 115.8, 53.4, 48.1,
36.5, 18.7.
13C NMR (100 MHz, DMSO-d6): δ = 172.6, 171.8, 167.9, 164.0, 162.9,
150.4, 143.9, 137.4, 129.0 (2C), 128.0 (2C), 126.3, 124.1, 111.0, 53.3,
50.6, 42.3, 40.9, 36.4, 23.9, 23.0, 21.6.
HRMS [ESI(+)-TOF]: m/z [M + H]+ calcd for C36H36N8O8: 709.2729;
found: 709.2739.
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HRMS [ESI(+)-TOF]: m/z [M + H]+ calcd for C46H54N10O10: 907.4097;
found: 907.4135.
Supporting Information
Supporting information for this article is available online at
http://dx.doi.org/10.1055/s-0035-1560523.
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Dimethyl 2,2′-[{(2S,2′S)-2,2′-[{2,2′-[{6,6′-[(E)-Diazene-1,2-di-
yl]bis(nicotinoyl)}bis(azanediyl)]bis(acetyl)}bis(azanediyl)]bis(4-
methylpentanoyl)}bis(azanediyl)](2S,2′S)-bis(3-phenylpropa-
noate) [2,2′-AzPy-5,5′-di(Gly-Leu-Phe-OMe), 15a]
References
Yield: 55 mg (59%); pale red microcrystals; mp 196–200 °C.
(1) (a) Albericio, F.; Kruger, H. G. Future Med. Chem. 2012, 4, 1527.
(b) Antosova, Z.; Mackova, M.; Kral, V.; Macek, T. Trends Biotech-
nol. 2009, 27, 628. (c) Mok, W. W. K.; Li, Y. F. Curr. Pharm. Des.
2014, 20, 771. (d) Lien, S.; Lowman, H. B. Trends Biotechnol.
2003, 21, 556.
(2) (a) Vlieghe, P.; Lisowski, V.; Martinez, J.; Khrestchatisky, M.
Drug Discov. Today 2010, 15, 40. (b) Lee, S.; Xie, J.; Chen, X. Bio-
chemistry 2010, 49, 1364.
(3) (a) Brieke, C.; Rohrbach, F.; Gottschalk, A.; Mayer, G.; Heckel, A.
Angew. Chem. Int. Ed. 2012, 51, 8446. (b) Gautier, A.; Gauron, C.;
Volovitch, M.; Bensimon, D.; Jullien, L.; Vriz, S. Nat. Chem. Biol.
2014, 10, 533. (c) Young, D. D.; Deiters, A. Org. Biomol. Chem.
2007, 5, 999. (d) Pieroni, O.; Fissi, A.; Angelini, N.; Lenci, F. Acc.
Chem. Res. 2001, 34, 9.
1H NMR (400 MHz, DMSO-d6): δ = 9.28–9.16 (m, 4 H), 8.52 (dd, J = 8.3,
1.9 Hz, 2 H), 8.42 (d, J = 7.4 Hz, 2 H), 8.13 (d, J = 8.3 Hz, 2 H), 7.93 (d,
J = 8.3 Hz, 2 H), 7.32–7.18 (m, 10 H), 4.52–4.36 (m, 4 H), 4.04–3.92 (m,
4 H), 3.56 (s, 6 H), 3.04 (dd, J = 13.7, 5.9 Hz, 2 H), 2.97 (dd, J = 13.7, 8.7
Hz, 2 H), 1.66–1.54 (m, 2 H), 1.48–1.38 (m, 4 H), 0.88 (d, J = 6.5 Hz, 6
H), 0.85 (d, J = 6.5 Hz, 6 H).
13C NMR (100 MHz, DMSO-d6): δ = 172.0, 171.6, 168.1, 164.2, 163.4,
148.7, 138.3, 137.0, 131.5, 128.9 (2 C), 128.1 (2 C), 126.4, 113.6, 53.5,
51.6, 50.5, 42.4, 40.8, 36.3, 23.9, 22.9, 21.6.
HRMS [ESI(+)-TOF]: m/z [M + H]+ calcd for C48H58N10O10: 935.4410;
found: 935.4438.
(2S,2′S)-2,2′-[{(2S,2′S)-2,2′-[{2,2′-[{6,6′-[(E)-Diazene-1,2-di-
yl]bis(nicotinoyl)}bis(azanediyl)]bis(acetyl)}bis(azanediyl)]bis(4-
methylpentanoyl)}bis(azanediyl)]bis(3-phenylpropanoic Acid)
[2,2′-AzPy-5,5′-di(Gly-Leu-Phe), 15b]
(4) (a) Riggsbee, C. W.; Deiters, A. Trends Biotechnol. 2010, 28, 468.
(b) Yu, M.; Tang, T.; Takasu, A.; Higuchi, M. Polym. J. 2014, 46, 52.
(c) Pochan, D. J.; Schneider, J. P.; Kretsinger, J.; Ozbas, B.;
Rajagopal, K.; Haines, L. J. Am. Chem. Soc. 2003, 125, 11802.
(5) (a) Beharry, A. A.; Woolley, G. A. Chem. Soc. Rev. 2011, 40, 4422.
(b) Renner, C.; Moroder, L. ChemBioChem 2006, 7, 868.
(6) (a) Schrader, T. E.; Schreier, W. J.; Cordes, T.; Koller, F. O.;
Babitzki, G.; Denschlag, R.; Renner, C.; Löweneck, M.; Dong, S.-
L.; Moroder, L.; Tavan, P.; Zinth, W. Proc. Natl. Acad. Sci. U.S.A.
2007, 104, 15729. (b) Guerrero, L.; Smart, O. S.; Weston, C. J.;
Burns, D. C.; Woolley, G. A.; Allemann, R. K. Angew. Chem. Int. Ed.
2005, 44, 7778. (c) Guerrero, L.; Smart, O. S.; Woolley, G. A.;
Allemann, R. K. J. Am. Chem. Soc. 2005, 127, 15624. (d) Woolley,
G. A.; Jaikaran, A. S. I.; Berezovski, M.; Calarco, J. P.; Krylov, S. N.;
Smart, O. S.; Kumita, J. R. Biochemistry 2006, 45, 6075. (e) Dong,
S.-L.; Loeweneck, M.; Schrader, T. E.; Schreier, W. J.; Zinth, W.;
Moroder, L.; Renner, C. Chem. Eur. J. 2006, 12, 1114. (f) Samanta,
S.; Qin, C.; Lough, A. J.; Woolley, G. A. Angew. Chem. Int. Ed. 2012,
51, 6452.
LiOH (8 mg, 0.33 mmol) was added to a suspension of 15a (25 mg,
0.027 mmol) in DMF–MeOH–H2O (3:1:1, 2 mL) in a test tube at 10 °C.
After stirring the reaction mixture for 2 h at 10 °C, ice-cold H2O (5 mL)
was added to this mixture and acidified with aq 1 N HCl until pH 3–4.
The precipitate formed was collected by suction filtration and washed
with cold H2O and Et2O. The solid product was dried under vacuum to
give 15b as red-brown microcrystals; yield: 10 mg (41%); mp 180–
184 °C.
1H NMR (400 MHz, DMSO-d6): δ = 12.68 (br s, 2 H), 9.26–9.16 (m, 4
H), 8.51 (dd, J = 8.3, 1.9 Hz, 2 H), 8.24 (d, J = 7.7 Hz, 2 H), 8.12 (d, J = 8.6
Hz, 2 H), 7.93 (d, J = 8.3 Hz, 2 H), 7.32–7.18 (m, 10 H), 4.46–4.36 (m, 4
H), 4.02–3.94 (m, 4 H), 3.06 (dd, J = 13.8, 5.4 Hz, 2 H), 2.94 (dd,
J = 13.8, 8.9 Hz, 2 H), 1.66–1.54 (m, 2 H), 1.50–1.36 (m, 4 H), 0.88 (d,
J = 6.5 Hz, 6 H), 0.84 (d, J = 6.5 Hz, 6 H).
13C NMR (100 MHz, DMSO-d6): δ = 172.6, 171.8, 168.0, 164.1, 163.4,
148.7, 138.3, 137.4, 131.5, 129.0 (2 C), 128.1 (2 C), 126.3, 113.6, 53.5,
50.6, 42.3, 40.9, 36.4, 23.9, 23.0, 21.6.
HRMS [ESI(+)-TOF]: m/z [M + H]+ calcd for C46H54N10O10: 907.4097;
found: 907.4132.
(7) (a) Kim, Y.; Phillips, J. A.; Liu, H.; Kang, H.; Tan, W. Proc. Natl.
Acad. Sci. U.S.A. 2009, 106, 6489. (b) Westmark, P. R.; Kelly, J. P.;
Smith, B. D. J. Am. Chem. Soc. 1993, 115, 3416. (c) Nakayama, K.;
Endo, M.; Majima, T. Chem. Commun. 2004, 2386. (d) Willner, I.;
Rubin, S.; Riklin, A. J. Am. Chem. Soc. 1991, 113, 3321.
(8) (a) Banghart, M.; Borges, K.; Isacoff, E.; Trauner, D.; Kramer, R.
H. Nat. Neurosci. 2004, 7, 1381. (b) Mourot, A.; Kienzler, M. A.;
Banghart, M. R.; Fehrentz, T.; Huber, F. M. E.; Stein, M.; Kramer,
R. H.; Trauner, D. ACS Chem. Neurosci. 2011, 2, 536.
Acknowledgment
(9) (a) Bhattacharya, B.; Dey, R.; Pachfule, P.; Banerjee, R.; Ghoshal,
D. Cryst. Growth Des. 2013, 13, 731. (b) Kanoo, P.; Ghosh, A. C.;
Cyriac, S. T.; Maji, T. K. Chem. Eur. J. 2012, 18, 237. (c) Gai, Y.-L.;
Jiang, F.-L.; Xiong, K.-C.; Chen, L.; Yuan, D.-Q.; Zhang, L.-J.; Zhou,
K.; Hong, M.-C. Cryst. Growth Des. 2012, 12, 2079. (d) Wang, J.;
Zhang, Y.; Liu, X.-Q.; Xiao, J.; Zhou, H.; Yuan, A.-H. Micropor.
Mesopor. Mat. 2012, 159, 100. (e) Kaim, W. Coord. Chem. Rev.
2001, 219–221, 463.
This work was supported by Anadolu University Scientific Research
Projects Commission (Project No: 1306F157 and 1404F193). The au-
thor thanks all faculty and staff members of Anadolu University Me-
dicinal Plants, Drugs and Scientific Research Center for 500 MHz NMR
studies. Thanks are also due to all faculty and students of Nanoboyut
Research Laboratory for He-Cd laser system. The author also acknowl-
edges Anadolu University Scientific Research Projects Commission
(Project No. 1306F110) for purchasing 400 MHz NMR spectrometer
for the chemistry department and Dr. B. Inci, Dr B. Gülbakan, and Dr
H. Berber for helpful discussion and manuscript reading.
(10) Bardaji, M.; Barrio, M.; Espinet, P. Dalton Trans. 2011, 40, 2570.
(11) Shinkai, S.; Kouno, T.; Kusano, Y.; Manabe, O. J. Chem. Soc.,
Perkin Trans. 1 1982, 2741.
(12) Shinkai, S.; Honda, Y.; Ueda, K.; Manabe, O. Bull. Chem. Soc. Jpn.
1984, 57, 2144.
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, 365–378

378
I. Avan
Paper
Synthesis
(13) (a) Hamon, F.; Djedaini-Pilard, F.; Barbot, F.; Len, C. Tetrahedron
2009, 65, 10105. (b) da Silva, D. B.; Samadi, A.; Infantes, L.;
Carreiras, M. D.; Marco-Contelles, J. Tetrahedron Lett. 2010, 51,
6278.
(21) Panda, S. S.; Hall, C. D.; Scriven, E.; Katritzky, A. R. Aldrichimica
Acta 2013, 46, 43.
(22) (a) Katritzky, A. R.; Haase, D. N.; Johnson, J. V.; Chung, A. J. Org.
Chem. 2009, 74, 2028. (b) Katritzky, A. R.; Khashab, N. M.;
Yoshioka, M.; Haase, D. N.; Wilson, K. R.; Johnson, J. V.; Chung,
A.; Haskell-Luevano, C. Chem. Biol. Drug Des. 2007, 70, 465.
(23) (a) Avan, I.; Hall, C. D.; Katritzky, A. R. Chem. Soc. Rev. 2014, 43,
3575. (b) Abo-Dya, N. E.; Biswas, S.; Basak, A.; Avan, I.; Alamry,
K. A.; Katritzky, A. R. J. Org. Chem. 2013, 78, 3541. (c) Panda, S.
S.; El-Nachef, C.; Bajaj, K.; Katritzky, A. R. Eur. J. Org. Chem. 2013,
4156.
(24) Katritzky, A. R.; Avan, I.; Tala, S. R. J. Org. Chem. 2009, 74, 8690.
(25) Avan, I.; Tala, S. R.; Steel, P. J.; Katritzky, A. R. J. Org. Chem. 2011,
76, 4884.
(26) Biswas, S.; Avan, I.; Basak, A. K.; Abo-Dya, N. E.; Asiri, A.;
Katritzky, A. R. Amino Acids 2013, 45, 159.
(27) Bandara, H. M. D.; Burdette, S. C. Chem. Soc. Rev. 2012, 41, 1809.
(28) (a) Schmuck, C.; Machon, U. Chem. Eur. J. 2005, 11, 1109.
(b) Ferrari, G.; Marcon, E. Farmaco Ed. Sci. 1958, 13, 485.
(29) (a) Vitale, R.; Ottonello, G.; Petracca, R.; Bertozzi, S. M.;
Ponzano, S.; Armirotti, A.; Berteotti, A.; Dionisi, M.; Cavalli, A.;
Piomelli, D.; Bandiera, T.; Bertozzi, F. ChemMedChem 2014, 9,
323. (b) Katritzky, A. R.; He, H.-Y.; Suzuki, K. J. Org. Chem. 2000,
65, 8210. (c) Hansen, F. K.; Ha, K.; Todadze, E.; Lillicotch, A.;
Frey, A.; Katritzky, A. R. Org. Biomol. Chem. 2011, 9, 7162.
(14) (a) Asanuma, H.; Shirasuka, K.; Takarada, T.; Kashida, H.;
Komiyama, M. J. Am. Chem. Soc. 2003, 125, 2217. (b) Behrendt,
R.; Schenk, M.; Musiol, H.-J.; Moroder, L. J. Pept. Sci. 1999, 5, 519.
(c) Harvey, A. J.; Abell, A. D. Bioorg. Med. Chem. Lett. 2001, 11,
2441. (d) Goulet-Hanssens, A.; Lai Wing Sun, K.; Kennedy, T. E.;
Barrett, C. J. Biomacromolecules 2012, 13, 2958. (e) Li, H.; Qin,
Q.; Qiao, L.; Shi, X.; Xu, G. Chem. Commun. 2015, 51, 11321.
(f) Bergbreiter, D. E.; Osburn, P. L.; Li, C. Org. Lett. 2002, 4, 737.
(15) (a) Sano, M.; Amaike, M.; Kamino, A.; Shinkai, S. Langmuir 2001,
17, 4367. (b) Srinivas, O.; Mitra, N.; Surolia, A.; Jayaraman, N.
Glycobiology 2005, 15, 861.
(16) (a) Rose, T. M.; Prestwich, G. D. Org. Lett. 2006, 8, 2575. (b) Jiang,
Y. L.; McGoldrick, C. A.; Yin, D.; Zhao, J.; Patel, V.; Brannon, M. F.;
Lightner, J. W.; Krishnan, K.; Stone, W. L. Bioorg. Med. Chem. Lett.
2012, 22, 3632.
(17) Katritzky, A. R.; Chen, Q.-Y.; Tala, S. R. Org. Biomol. Chem. 2008,
6, 2400.
(18) Katritzky, A. R.; Tala, S. R.; Abo-Dya, N. E.; Abdel-Samii, Z. K. Syn-
thesis 2009, 1708.
(19) Katritzky, A. R.; Khelashvili, L.; Kovacs, J.; Shanab, K. Chem. Biol.
Drug Des. 2009, 73, 396.
(20) (a) Abdelmajeid, A.; Tala, S. R.; Amine, M. S.; Katritzky, A. R. Syn-
thesis 2011, 2995. (b) El-Khatib, M.; Elagawany, M.; Caliskan, E.;
Davis, E. F.; Faidallah, H. M.; El-Feky, S. A.; Katritzky, A. R. Chem.
Commun. 2013, 49, 2631.
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2016, 48, 365–378