An Optimized Glutamate Receptor Photoswitch with Sensitized Azobenzene Isomerization

Article abstract of DOI:10.1021/acs.joc.5b01402

A new azobenzene-based photoswitch, 2, has been designed to enable optical control of ionotropic glutamate receptors in neurons via sensitized two-photon excitation with NIR light. In order to develop an efficient and versatile synthetic route for this mo

Full text of DOI:10.1021/acs.joc.5b01402

Subscriber access provided by UNIV OF LETHBRIDGE
Article
An Optimized Glutamate Receptor Photoswitch
with Sensitized Azobenzene Isomerization
Marta Gascón-Moya, Arnau Pejoan, Mercè Izquierdo-Serra, Silvia Pittolo,
Gisela Cabrè, Jordi Hernando, Ramon Alibés, Pau Gorostiza, and Felix Busque
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.5b01402 • Publication Date (Web): 28 Sep 2015
Downloaded from http://pubs.acs.org on October 2, 2015
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
The Journal of Organic Chemistry is published by the American Chemical Society.
1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 34
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
An Optimized Glutamate Receptor Photoswitch with
Sensitized Azobenzene Isomerization
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
Marta Gascón-Moya,a Arnau Pejoan,a Mercè Izquierdo-Serra,b Silvia Pittolo,b Gisela Cabré,a Jordi
Hernando,a Ramon Alibés,a Pau Gorostiza,b,c,d * Félix Busqué a *
a Departament de Química, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain
b Institut de Bioenginyeria de Catalunya (IBEC), Barcelona, Spain
c Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina (CIBERꢀ
BBN), Zaragoza, Spain
d Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain
pau@icrea.cat, felix.busque@uab.es
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 2 of 34
Abstract
1
2
3
4
5
6
7
8
9
A new azobenzeneꢀbased photoswitch, 2, has been designed to enable optical control of ionotropic
glutamate receptors in neurons via sensitized twoꢀphoton excitation with NIR light. In order to develop
an efficient and versatile synthetic route for this molecule, a modular strategy is described which relies
on the use of a new linear fully protected glutamate derivative stable in basic media. The resulting
compound undergoes oneꢀphoton transꢀcis photoisomerization via two different mechanisms: direct
excitation of its azoaromatic unit, and irradiation of the pyrene sensitizer, a well known twoꢀphoton
sensitive chromophore. Moreover, 2 presents large thermal stability of its cis isomer, in contrast to other
twoꢀphoton responsive switches relying on the intrinsic nonꢀlinear optical properties of pushꢀpull
substituted azobenzenes. As a result, the molecular system developed herein is a very promising
candidate for evoking large photoinduced biological responses during the multiphoton operation of
neuronal glutamate receptors with NIR light, which require accumulation of the proteinꢀbound cis state
of the switch upon repeated illumination.
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
Introduction
Neuroscience is being revolutionized in the last years by the emergence of optogenetic1 and
optochemical tools,2 a series of techniques that allow the optical control of neural networks by means of
lightꢀsensitive proteins and photoresponsive small molecules. An important strategy in this field is the
use of photoswitchable tethered ligands (PTLs), in which a pharmacologically active moiety is
covalently attached to a neuronal receptor through a photoisomerizable tether.2,3 This enables the
activity of the resulting proteinꢀPTL construct to be modulated upon photoisomerization of the switch,
which brings the ligand closer to or away from the binding site and, as such, triggers a reversible lightꢀ
induced recognition event and the subsequent biological response. Owing to their synthetic accessibility,
excellent photochemical properties and large geometrical change between their photointerconvertible
trans and cis configurations, azobenzene derivatives have become the most common lightꢀresponsive
ACS Paragon Plus Environment

Page 3 of 34
The Journal of Organic Chemistry
building blocks for the preparation of PTLs4 and they have been exploited to optically control a number
of different neuronal receptors.2,3,5
1
2
3
4
5
6
7
8
Since glutamate is the major excitatory neurotransmitter in the central nervous system, glutamate
receptors play a central role in the regulation of neural activity6 and are an important target of
optochemical techniques. Much attention has therefore been focused in recent years on the development
of PTLs for the lightꢀinduced operation of these proteins, especially in the case of the ionotropic
glutamate receptors (iGluR) controlling the channelꢀregulated flow of ions through cell membrane.2,3
The most successful approach towards this goal is based on maleimideꢀazobenzeneꢀglutamate
photoswitches (MAG, Figure 1a),7 which were originally designed to enable optical control of the
kainateꢀtype ionotropic glutamate receptor GluK2. In MAG compounds, a lateral maleimide unit is used
to covalently bind the PTL to a cysteine residue genetically engineered into the ligand binding domain
of the receptor, while its photoswitchable azobenzene core enables lightꢀcontrol of the separation
distance between the glutamate moiety and the binding pocket. In particular, transꢀtoꢀcis isomerization
of the system approaches the ligand towards the binding site and induces glutamate recognition, which
leads to ion channel opening and, eventually, to the generation of a photoinduced neuronal excitatory
potential (Figure 1b).7 Afterward, irradiation or thermal relaxation of the metastable cis isomer allow
reverting back the process and recovering the initial closed state of the channel.7 For the first MAG
switch developed (Figure 1a), irradiation with UVꢀviolet (λtrans-cis ~ 340ꢀ400 nm) and blueꢀgreen light
9
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
(λcis-trans ~ 440ꢀ580 nm) were required to photochemically operate the tagged GluK2 receptors,7 while
spontaneous cisꢀtoꢀtrans isomerization occurred in the dark on the minute time scale (τcis= 17.7 min)7b
owing to the large thermal stability of the cis isomer resulting from its symmetric 4,4'ꢀdiamide
substitution pattern.4 Although some efforts have been devoted to shift MAG photoactivity to longer
wavelengths,8,9 the application of these compounds in vivo is severely compromised by the reduced
penetration depth10 and low axial resolution associated to the conventional oneꢀphoton excitation of
azobenzeneꢀbased switches with UVꢀvisible light. To overcome these drawbacks and take full
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 4 of 34
advantage of the lightꢀregulated activity of MAGꢀlabeled receptors, multiphoton excitation with nearꢀ
infrared (NIR) light would be highly desirable, since it provides subꢀmicrometric resolution in three
dimensions,11 deep penetration into tissue12 and patterned illumination.13,14 Unfortunately, the twoꢀ
photon absorption crossꢀsections of common azobenzene chromophores are rather low,15 which makes
multiphoton operation of MAG PTLs under NIR irradiation a challenge.
1
2
3
4
5
6
7
8
9
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
Figure 1. (a) Structure of a MAG molecular switch composed of maleimide (green), azobenzene (red)
and glutamate (blue) units.7 (b) Operation mode of a cysteineꢀtagged ionotropic glutamate receptor
functionalized with a photoswitchable tethered ligand. Reversible isomerization of the azoaromatic core
of the switch modulates the separation distance between the glutamate ligand and the binding site of the
receptor to induce channel opening (transꢀtoꢀcis) and closing (cisꢀtoꢀtrans). In this work, an additional
building block is introduced into the structure of the PTL, an antenna enabling twoꢀphoton sensitization
of azobenzene isomerization (MAGAꢀtype switch). (c) Structures of the MAGA photoswitched tethered
ligands 1 and 2, whose additional sensitizer unit is shown in magenta.
ACS Paragon Plus Environment

Page 5 of 34
The Journal of Organic Chemistry
To tackle this problem, we have recently introduced two different molecular strategies, which allowed
1
2
for the first time the twoꢀphoton optical control of lightꢀgated ionotropic glutamate receptors based on
cysteineꢀtagged GluK2 and MAG switches (LiGluRꢀMAG).16 On the one hand, the intrinsic twoꢀphoton
activity of MAG compounds was shown to significantly increase upon pushꢀpull substitution of their
azobenzene core, as subsequently demonstrated by Isacoff and coꢀworkers for analogous PTLs.17
However, a decrease of the thermal stability of the cis state of these systems was concomitantly
observed,16,17 which imposes a severe constraint on their photochemical behavior: the ligandꢀbound
state of the LiGluRꢀMAG tether dissociates as soon as illumination ceases, since the cis liganding
configuration of the switch backꢀisomerizes in the dark on the ms scale. While this might be
advantageous for certain applications requiring fast operation of ionotropic glutamate receptors,16 rapid
thermal relaxation of the switch prevents building up a large population of the open state of the ion
channel and, therefore, the obtaining of large photoresponse amplitudes. Alternatively, multiphoton
operation of MAGꢀtype PTLs can be attained by incorporating a photosensitizer unit to the structure of
the switch, which should absorb NIR light via a twoꢀphoton process and efficiently transfer its
electronic excitation energy to the azobenzene chromophore (Figure 1b). We named this new family of
compounds maleimideꢀazobenzeneꢀglutamateꢀantenna switches (MAGA),16 whose multiphoton
response could then be disentangled from the substitution pattern of the azoaromatic core. Actually, the
oneꢀ and twoꢀphoton sensitized isomerization of a MAGAꢀtype PTL was already demonstrated in our
previous work using a naphthalene antenna (1 in Figure 1c); however, this compound also suffered from
low cis thermal stability owing to the pushꢀpull substitution of the azobenzene core selected (τcis = 265
ms at 298 K) and, consequently, displayed reduced multiphoton activity when conjugated to LiGluR.16
In view of this, we pursue herein the preparation of a new MAGA photoswitch featuring both sensitized
isomerization using a twoꢀphoton absorber and long cis state lifetime in the dark. As such, this
compound could be exploited to optically trigger the LiGluR receptor via multiphoton absorption of
NIR light in neuroscience applications demanding large biological photoinduced signals (i.e.
3
4
5
6
7
8
9
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
accumulation of the open state of the channel upon repeated excitation).
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 6 of 34
Figure 1c shows the structure of 2, the novel MAGAꢀtype PTL targeted in this work. This compound
was derived on account of two main design concepts. First, the use of a symmetrically substituted
azobenzene chromophore displaying two similar Nꢀamido substituents at positions 4 and 4’, which
should ensure a long thermal lifetime of its cis state at room temperature according to the previous
results obtained for MAG7 and other related azoaromatic derivatives.18 Second, the selection of a proper
multiphoton antenna, which should (i) display large twoꢀphoton absorption under NIR irradiation, and
(ii) emit in the same spectral region where the azobenzene core absorbs, thus enabling efficient
sensitization of the switch via resonance energy transfer (RET). Since the 4,4'ꢀdiamidoazobenzene core
in 2 must present a large hypsochromic absorption shift with respect to the azoaromatic group of 1 (~70
nm),7,16,18 the naphtalene sensitizer of the latter compound did not fulfil the second of the requirements
of our present design and could not be used as antenna for the development of switch 2. Instead, a
monoꢀalkylated pyrene derivative was chosen as sensitizer. Although this type of dyes present moderate
twoꢀphoton absorption crossꢀsections,19 they are often used as twoꢀphoton fluorescent probes for
labeling cell membranes in confocal fluorescence microscopy studies18 and can be detected even down
to the singleꢀmolecule level under twoꢀphoton excitation.20 More importantly, their emission spectra
(λfl,max ~ 390 nm19) largely overlaps the absorption spectra of the π→π* transition of transꢀ4,4’ꢀ
1
2
3
4
5
6
7
8
9
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
diamidoazobenzenes (λabs,max ~ 370 nm18), a critical requirement for photosensitization of their transꢀtoꢀ
cis isomerization.
Results and Discussion
Synthesis of photoswitchable ligand 2
A convergent synthetic strategy was devised for the preparation of 2, which relies on the use of five
different building blocks (Scheme 1): the N,Nꢀorthogonally diprotected
Lꢀlysine 3; transꢀ4,4’ꢀ
diaminoazobenzene (4) as the symmetricallyꢀsubstituted azoaromatic core; the linear protected
derivative of glutamate 5; 2ꢀ(pyrenꢀ1ꢀyl)acetic acid (6) as twoꢀphoton sensitizer; and maleimide
ACS Paragon Plus Environment

Page 7 of 34
The Journal of Organic Chemistry
derivative 7. Two key features differentiate our approach from the synthetic procedures previously
reported for the synthesis of analogous MAG photoswitches.7ꢀ9 On the one hand, the use of 3 as a
central scaffold to which the distinct functional fragments of the target compound would be sequentially
tethered, thus aiming to develop a modular route toward MAGA PTLs (or any other type of
multifunctional ligand derived from the original maleimideꢀazobenzeneꢀglutamate scheme). On the
other hand, 5 was developed as an alternative glutamate precursor to typical cyclic pyroglutamate
groups, providing an optimized strategy to achieve the final photoswitchable ligand in terms of both
deprotection reaction conditions and intermediate manipulation.
1
2
3
4
5
6
7
8
9
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
Scheme 1. Retrosynthetic analysis proposed for the synthesis of new MAG compound 2
Since all the other building blocks of 2 were commercially available or synthetically accessible via
prior reported procedures, our initial task focused on the preparation of glutamate derivative 5. Although
this compound has been recently used by us for the synthesis of 1,16 we describe herein the obtaining of
5 for the first time. The synthetic sequence commenced with the protection of the carboxylic acid and
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 8 of 34
the nitrogen atom of commercial Lꢀpiroglutamic acid as the corresponding tertꢀbutyl ester and tertꢀbutyl
1
2
3
4
5
6
7
8
9
carbamate, respectively, to obtain known pyroglutamate 821 in a 53% overall yield (Scheme 2). Next,
this product was treated with LiHMDS in THF at – 78 ºC to form the corresponding enolate, which was
allowed to react with allyl bromide at low temperature to afford a 3:1 diastereoisomeric mixture of
already described allylated derivatives trans 9a and cis 9b.22 The major trans diastereoisomer 9a was
isolated after purification by column chromatography in a 36% yield and used in the following steps of
the synthesis. The relative configuration assignment of these two isomers was confirmed by comparison
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
1
with the described NMR data.22 Thus, in the H NMR spectrum of 9a Hꢀ2 resonates at an upper field
than that of 9b and shows constant couplings with Hꢀ3 protons quite different (9.6 and 1.6 Hz), whereas
the values of these constants are more similar in the cis isomer (9.3 and 5.7 Hz).
Scheme 2. Synthesis of key intermediate 9a
Subsequent lithium hydroxide ring opening of 9a proceeded smoothly providing acid 10 in 93% yield
(Scheme 3), which was transformed into the corresponding diꢀtertꢀbutyl ester derivative 11 in 56% yield
by treatment with tertꢀbutyl 2,2,2,ꢀtrichloroacetimidate in the presence of catalytic BF3. The synthesis
was continued with a crossꢀmetathesis reaction of 11 with crotonaldehyde using the HoveydaꢀGrubs
second generation catalyst to afford aldehyde 12 in 89% yield, which was oxidized to the corresponding
acid 13 in 77% yield following a Pinnick protocol with NaClO2 in the presence of 2ꢀmethylꢀ2ꢀbutene as
scavenger of the hypochlorous acid generated. Direct metathesis reaction of intermediate 11 with acrylic
ACS Paragon Plus Environment

Page 9 of 34
The Journal of Organic Chemistry
acid afforded derivative 13 in lower yield (52%) than that obtained with the two consecutive
transformations described above (69% overall yield for the two steps). Finally, standard hydrogenation
under palladium catalyst rendered almost quantitatively the target glutamate derivative 5.
1
2
3
4
5
6
7
8
9
Scheme 3. Synthesis of glutamate derivative 5
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
With glutamate derivative 5 in hand, we proceeded to prepare the final MAGA photoswitch 2
(Scheme 4) following the modular synthetic strategy outlined above and previously implemented in our
group for the obtaining of 1.16 Accordingly, the N,Nꢀorthogonally diprotected
Lꢀlysine 3 was tethered to
the symmetric azobenzene core 4 using the carbodiimide coupling reagent EDCI along with HOBt and
DIPEA as base, which led to a chromatographically easily separable mixture of starting material and
compound 14 in 85% yield (over consumed starting material). Removal of the tertꢀbutyl carbamate
protection of this compound by treatment with 37% HCl in MeOH for 1 hour furnished amine 15 in
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 10 of 34
98% yield. Longer reaction times led to partial hydrolysis of the amide moiety and should be strictly
avoided. Next, the glutamate derivative 5 was introduced into this intermediate using again EDCI under
standard coupling conditions, affording derivative 16 in 88% yield. The incorporation of the pyrene
sensitizer was then achieved by treatment of 16 with 20% piperidine solution in DMF to render the
corresponding reactive amine 17 and successive EDCIꢀpromoted amide formation with 6. In this way,
18 was obtained in 64% overall yield for the two steps. The subsequent reaction between this
intermediate and the freshly made acid chloride of maleimide derivative 723 afforded compound 19 in
74% yield, which features all the functional fragments of the final MAGA switch.
1
2
3
4
5
6
7
8
9
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
Scheme 4. Synthesis of the new MAGA compound 2
ACS Paragon Plus Environment

Page 11 of 34
The Journal of Organic Chemistry
The preparation and manipulation of such advanced intermediate was highly favored by the use of the
new linear glutamate derivative 5. In our hands, 5 turned to be a better glutamate precursor than the
cyclic pyroglutamate group normally employed in the synthesis of MAG compounds,7ꢀ9 which we
found to be troublesome due to: (i) the moderate stability to the basic conditions required for the ring
opening reaction of pyroglutamate of the maleimide fragment, which should therefore be introduced in a
later stage of the route; and (ii) the low solubility of the dicarboxylic acid generated from the ring
opening process of the pyroglutamate precursor,7ꢀ9 which compromised its manipulation and the
efficiency of posterior reactions (e.g. incorporation of the maleimide group). These inconveniences are
overcome by the use of the already opened derivative of glutamate 5 whose carboxylic acid groups are
protected as the corresponding tertꢀbutyl esters. Finally, removal of these moieties and of the additional
tertꢀbutyl carbamate protecting group of intermediate 19 took place in a single final step by treatment
with a 2:1 mixture of trifluoroacetic acid and CH2Cl2, thus providing the monotrifluoroacetate salt of
target ligand 2 in 79% yield. As expected, this compound was selectively isolated as its more stable
trans isomer (transꢀ2), according to spectroscopic data.
1
2
3
4
5
6
7
8
9
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
One-photon optical characterization of photoswitchable ligand 2
Aiming to assess the validity of our molecular design, the optical properties of compound 2 were
investigated to demonstrate both sensitization of the trans-cis isomerization of the ligand upon
photoexcitation of the pyrene antenna and the long thermal stability of the cis state of the switch at room
temperature. For sake of simplicity, this study was conducted under oneꢀphoton excitation with UVꢀvis
light. However, based on our previous work16 and the well known nonꢀlinear optical properties of
pyrene derivatives,19ꢀ20 the results obtained herein could be extrapolated to the photochemical behavior
expected for 2 upon twoꢀphoton absorption of NIR light.
Figure 2a shows the absorption spectrum of transꢀ2 in DMSO, which is compared to those recorded
for its photoactive building blocks: pyrene derivative 6 and transꢀ4,4’ꢀbis(acetylamino)azobenzene
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 12 of 34
(transꢀ20), a model azoaromatic dye obtained by bisꢀacylation of 4,4’ꢀdiaminoazobenzene using
standard amide formation conditions according to literature precedents.24 Clearly, the narrow pyrene
vibronic absorption peaks at λabs ∼ 315, 330 and 345 nm and the broad transꢀazoaromatic π → π*
1
2
3
4
5
6
7
8
9
(λabs,max = 380 nm) and n → π* (λabs > 450 nm) absorption bands are both observed in the spectrum of
transꢀ2. This does not only demonstrate the successful introduction of the two chromophoric units, but
also that they are not electronically coupled in the ground state of the ligand. In contrast, nonꢀnegligible
interactions should take place in the electronic excited state, as revealed by the fluorescence emission
spectra measured for transꢀ2 and 6 (Figure 2b). While strong emission was detected for the separated
pyrene derivative in DMSO with a rather high fluorescence quantum yield (Φfl,6 = 0.30), a ∼ 43ꢀfold
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
decrease was observed after incorporation into the ligand (Φfl,transꢀ2 = 0.007). A small fraction of this
reduction must be attributed to the competitive absorption of the pyrene and azobenzene moieties of
transꢀ2 at the excitation wavelength of the fluorescence quantum yield measurements (λexc= 320 nm).
Actually, according to the extinction coefficients measured for the reference compounds 6 and transꢀ20
ꢂꢃꢄ ꢅꢆ
(ꢀꢁꢂꢃꢄ ꢅꢆ ꢀꢇꢈꢉꢊꢋꢌꢍꢎ ~ 2), about one third of the excitation photons in these experiments were absorbed
⁄
by the nonꢀemissive azoaromatic core of transꢀ2, thus accounting for an apparent ~ 1.5ꢀfold decrease in
Φfl for the pyrene antenna. However, the major part of the fluorescence quantum yield reduction (~ 30ꢀ
fold) can be ascribed to an intrinsic decrement of the emission efficiency of this fluorophore upon
tethering to the azobenzene group in transꢀ2, thus uncovering additional nonꢀradiative relaxation
pathways of its electronic excited state. As such, this result is indicative of the occurrence of the RET
processes required for sensitization of the photoisomerization of the azobenzene moiety.
ACS Paragon Plus Environment

Page 13 of 34
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
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
Figure 2. (a) Normalized absorption spectra of transꢀ2, 6 and transꢀ20 in DMSO (c = 5 10ꢀ6 M). (b)
Fluorescence emission spectra of transꢀ2 (x10, λexc = 320 nm) and 6 (λexc = 305 nm) in DMSO (c = 1
10ꢀ6 M). Both emission spectra were normalized relative to the absorption at the excitation wavelength.
In spite of this, we first investigated transꢀtoꢀcis isomerization of 2 upon direct excitation of the
azoaromatic moiety. Figure 3 depicts the changes registered in the absorption spectra of transꢀ2 and
reference compound transꢀ20 in DMSO when irradiated at λexc = 365 nm, an excitation wavelength
365 nm
transꢀ20
where the pyrene moiety of the ligand shows minimal absorption (ε
/ε3665 nm = 13; see Figure 2a).
In both cases, a noteworthy decrease of the broad π → π* azobenzene band around 380 nm and a very
slight increase of the n → π* azobenzene band at λabs > 450 nm were observed. These are clear
signatures of the photoinduced conversion of transꢀ2 and transꢀ20 into their corresponding cis isomers,
4,7,16 which should lead to the formation of an equilibrium photostationary state (PSS). According to the
1H NMR data measured for DMSOꢀd6 solutions of these compounds under equivalent irradiation
conditions, the cis composition of the PSS mixtures generated was found to be 65% and 63% for 2 and
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 14 of 34
20, respectively. Since nearly identical values were also determined for the transꢀtoꢀcis
photoisomerization quantum yields of these azobenzene derivatives (Φtrans-cis,2 = Φtrans-cis,20 = 0.28), we
could unambiguously conclude that the introduction of a pyrene group into the structure of ligand 2 did
not modify the intrinsic photochemical behavior of its azoaromatic moiety when subjected to direct light
excitation. A similar conclusion was drawn when analyzing the photoinduced cisꢀtoꢀtrans isomerization
of 2 and 20 upon selective excitation of their azobenzene core. Thus, when illuminating with green light
(λexc = 532 nm) the trans/cis mixtures of these compounds obtained by irradiation at λexc = 365 nm, our
absorption measurements demonstrated that both cisꢀ2 and cisꢀ20 backꢀisomerized to their trans isomers
nearly quantitatively and at similar rates (Figure S3). In particular, PSS mixtures with 94% and 93%
content in transꢀ2 and transꢀ20 were obtained under these experimental conditions, respectively,
according to our spectral data.
1
2
3
4
5
6
7
8
9
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
Figure 3. Variation of the absorption spectra of transꢀ2 (c = 5 10ꢀ6 M) and transꢀ20 (c = 2.7 10ꢀ5 M) in
DMSO upon irradiation at λexc = 365 nm. The arrows in the figures indicate the spectral changes
observed, while the dashed lines correspond to the absorption spectra of the resulting photostationary
state mixtures.
Next, the photosensitized isomerization of transꢀ2 upon irradiation of the pyrene antenna was
explored. Unfortunately, selective excitation of this chromophore to prevent direct transꢀtoꢀcis
isomerization of 2 could not be achieved because the absorption spectrum of the pyrene photosensitizer
ACS Paragon Plus Environment

Page 15 of 34
The Journal of Organic Chemistry
selected fully overlaps with that of the azobenzene core (see Figure 2a). To minimize this effect and
evaluate the actual contribution of the sensitized pathway to the photoswitching of 2, two main actions
were taken. On the one hand, photosensitization was investigated at λexc = 347 nm, for which the largest
difference in absorption between the pyrene and azoaromatic fragments of transꢀ2 was determined
1
2
3
4
5
6
7
8
9
(ε6347 nm/ε3tr4a7nsnꢀm20 = 2). On the other hand, photoisomerization was simultaneously studied at λexc = 365
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
nm, which should selectively promote direct lightꢀinduced transꢀtoꢀcis conversion, as discussed above.
Figure 4 shows the relative photoisomerization efficiencies of transꢀ2 and antennaꢀless compound transꢀ
20 in DMSO solution at λexc = 347 and 365 nm, which are plotted as the photogenerated ratios of cisꢀ2
and cisꢀ20 determined at different irradiation times from absorption measurements. Noticeably, a
straight line around %cisꢀ2 / %cisꢀ20 =1 was obtained at λexc = 365 nm, which indicates that direct transꢀtoꢀ
cis photoisomerization proceeds with equivalent rates and efficiencies for both azoaromatic compounds.
This is indeed the result expected according to our previous experiments, where nearly identical PSS
compositions and Φtrans-cis values were measured upon selective excitation of the transꢀazobenzene
moiety of 2 and 20.
Figure 4. Photogenerated ratios of cisꢀ2 and cisꢀ20 (%cisꢀ2 / %cisꢀ20) measured upon continuous
irradiation of DMSO solutions of transꢀ2 and transꢀ20 (c = 5 10ꢀ6 M) at λexc = 347 and 365 nm.
However, a more complex time profile was obtained at λexc = 347 nm. At short irradiation times (t <
50 min), we found %cisꢀ2 / %cisꢀ20 to be larger than 1, which indicates that transꢀtoꢀcis photoisomerization
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 16 of 34
takes place faster for ligand 2. This pinpoints the occurrence of an additional lightꢀinduced
isomerization mechanism in this compound with respect to reference transꢀ20, which we ascribe to
photosensitization evolving through pyrene absorption and subsequent resonance energy transfer toward
the azobenzene core. Such RET process would therefore account for the fluorescence quenching
observed for the pyrene unit in the ligand (see Figure 2b) and, actually, we estimated it to take place
with high critical Förster radius (R0= 3.42 nm) and efficiency (ERET= 99.8%) according to the optical
properties of 6 and transꢀ20 and the separation distances between the antenna and azoaromatic groups in
transꢀ2 (Figure S1). Nevertheless, it must be noted that energy transfer from pyrene to the cisꢀ
azobenzene moiety of 2 is also to be expected given the nonꢀnegligible spectral overlap between these
groups and, indeed, only slightly lower R0 and ERET values were determined for this process with respect
to that in transꢀ2: R0= 2.47 nm and ERET= 96.2% (Figure S2). This suggests that the cisꢀtoꢀtrans
isomerization of this compound can also be photosensitized by the antenna once the concentration of
cisꢀ2 significantly increases in the sample. As such, the PSS generated at λexc = 347 nm would depend
on the intricate balance between direct and sensitized transꢀtoꢀcis and cisꢀtoꢀtrans photoisomerization
rates, which explains the different equilibrium mixtures obtained under long irradiation times at this
wavelength with respect to λexc = 365 nm. In spite of this, it is clear that addition of the pyrene group to
the ligand enhances the photoconversion efficiency of transꢀ2 (up to 60% according to the data at short
irradiation times), thus demonstrating that photosensitization with a twoꢀphoton absorber can also be
exploited to trigger MAGAꢀtype PTLs bearing a symmetricallyꢀsubstituted azobenzene core.
1
2
3
4
5
6
7
8
9
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
Once proven that target photoswitch 2 can operate under photosensitized isomerization, the thermal
stability of the resulting cis isomer was analyzed. With this aim, the spontaneous cisꢀtoꢀtrans
isomerization of this compound and reference 20 was studied in the dark and at room temperature by
absorption spectroscopy. Spectral changes were observed in this way that are consistent with cisꢀ2 and
cisꢀ20 backꢀisomerization ꢀꢀ namely, the recovery of the intense trans absorption around 380 nm and a
slight decrease of the n → π* band at λabs > 450 nm (Figure S4). By plotting the time dependence of this
ACS Paragon Plus Environment

Page 17 of 34
The Journal of Organic Chemistry
data, the thermal cisꢀtoꢀtrans conversion rates could be retrieved from monoexponential fits,17 as shown
in Figure 5. In particular, the following values were obtained: kcis-trans = 3.2 10ꢀ5 and 4.1 10ꢀ5 sꢀ1 for 2 and
20, respectively, which correspond to thermal lifetimes of their cis isomers at room temperature of 8.7 h
and 6.8 h. Clearly, very similar cisꢀtoꢀtrans isomerization kinetics were found for both compounds in
the dark. This indicates that, under equivalent experimental conditions, the thermal stability of cisꢀ
azobenzenes is mainly governed by the electronic effects imparted by the substituents introduced, as
already reported.4 As such, the equivalent 4,4'ꢀdiamide substituent pattern of 2 and 20 accounts for their
comparable and long τcis values regardless of the different nature of their actual substituents. In spite of
this, a minor though nonꢀnegligible size effect could be inferred from our measurements, the bulkier
amide groups introduced in 2 slightly slowing down its thermal cisꢀtoꢀtrans backꢀisomerization process
and leading to a little larger cis state lifetime. On the other hand, the large thermal stability encountered
for cisꢀ2 and cisꢀ20 is in agreement with the behavior previously reported for cisꢀMAG (τcis= 17.7 min
1
2
3
4
5
6
7
8
9
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
in aqueous buffer)7b, which also bears a 4,4'ꢀdiamidoazobenzene core. The differences in τcis observed
between these compounds must be attributed to the distinct media where the measurements were
conducted, since it is known that the cis lifetime of azobenzene derivatives largely decreases in polar
solvents.4a,25 In fact, when monitoring the thermal cisꢀtoꢀtrans isomerization kinetics of cisꢀ2 in a 1:1
DMSO:water mixture (Figure S5), we determined a much shorter τcis value (τcis= 2.0 h) than in pure
DMSO and closer to that found for cisꢀMAG in aqueous buffer. Finally, the large thermal cis state
stability obtained herein for 2 is in striking contrast with the short cis lifetime measured for the previous
MAGA ligand 1 (τcis= 67 ms in PBS:DMSO 4:1 and 265 ms when tethered to LiGluR16), which arose
from its pushꢀpull substitution pattern. This unequivocally demonstrates that our photosensitized
approach toward twoꢀphoton responsive azobenzeneꢀbased PTLs also enables the use of switches
displaying longꢀlived cis isomers and, consequently, can be applied to the generation of large lightꢀ
induced biological responses upon continuous irradiation.
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 18 of 34
1
2
3
4
5
6
7
8
9
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
Figure 5. Variation of the absorption at 382 nm of the photostationary state mixtures of 2 and 20 in the
dark at 25 ºC in DMSO, which reports on the recovery of the concentration of transꢀ2 and transꢀ20
upon thermal cisꢀtoꢀtrans isomerization. Points correspond to the experimental data, while dashed lines
were obtained from monoexponential fits.
Actually, a preliminary assessment of the twoꢀphoton activity of 2 can be made on the basis of the
oneꢀphoton experiments conducted herein, the twoꢀphoton absorption crossꢀsections reported in the
literature for pyrene26 and azobenzene17 chromophores and our previous work on fast relaxing MAGA
switch 1.16 Since the same excited state of the pyrene antenna would be built, the efficiency of the RET
process towards the azobenzene core of 2 should be the same under oneꢀ or twoꢀphoton excitation. As
such, the photosensitized twoꢀphoton response of this switch would mainly depend on the following
parameters: (a) the selectivity of the twoꢀphoton excitation of the pyrene antenna with NIR light; (b) the
lifetime of the cis isomer of the switch created under such conditions. As discussed above, the oneꢀ
photon excitation of the pyrene moiety of 2 is poorly selective and the maximum photosensitization
effect was found at λexc = 347 nm, where a 2:1 pyrene:azobenzene absorption ratio was estimated.
Noticeably, we expect this absorption difference to increase under NIR irradiation due to the larger twoꢀ
photon activity of the pyrene antenna, thus favoring the sensitized pathway for photoisomerization. In
fact, 5ꢀfold higher twoꢀphoton absorption crossꢀsections at 820 nm have been reported for
monosubstituted pyrene derivatives (σ2=55 GM; 1 GM= 10ꢀ50 cm4 s γꢀ1) with respect to 4,4’ꢀ
diamidoazobenzenes (σ2=10 GM).17 As a result, sensitization via the pyrene antenna should be the
ACS Paragon Plus Environment

Page 19 of 34
The Journal of Organic Chemistry
major pathway for the twoꢀphoton isomerization of 2. It must be noted that the intrinsic efficiency of
this process should be lower than for our previous MAGA switch 1 bearing a higher absorbing
naphthalene sensitizer (σ2∼200 GM at 780 nm).16,27 In spite of this, an overall large twoꢀphoton
response would be expected for 2 on account of the longer lifetime of its cis isomer. Actually, the fast
thermal backꢀisomerization of 1 resulted in a ∼10ꢀfold lower biological signal for this compound with
respect to MAG under continuous oneꢀphoton excitation,16 which indicated that a less cisꢀenriched
photostationary state could be prepared for this MAGA switch. Taking into account that 2 also presents
a longꢀlived cis state as MAG, a similar factor can be assumed between the oneꢀphoton responses of the
two MAGAꢀtype compounds. This should counterbalance the ∼4ꢀfold lower twoꢀphoton absorptivity of
the pyrene sensitizer with respect to the naphthalene antenna of 1 and eventually lead to larger
biological signals for the switch developed herein under NIR irradiation.
1
2
3
4
5
6
7
8
9
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
Conclusions
In summary, we report herein the synthesis and photochemical characterization of a new azobenzeneꢀ
based photoswitchable ligand designed to enable optical control of ionotropic glutamate receptors in
neurons via twoꢀphoton excitation of a pyrene sensitizing unit with NIR light. Aiming to develop an
efficient and versatile synthetic route for this and other related ligands, a modular strategy is described
which benefits from the use of a new linear full protected glutamate derivative stable in basic media. In
this way, target compound 2 could be prepared in 7 steps with 27% overall yield starting from a
commercial N,Nꢀorthogonally diprotected Lꢀlysine derivative as central scaffold, to which its different
functional fragments were sequentially introduced: the azoaromatic switch, the glutamate moiety, the
pyrene sensitizer and the anchoring maleimide group. As originally planned, 2 presents oneꢀphoton
photoisomerization either upon selective irradiation of its azobenzene core, or via photoexcitation of the
pyrene antenna. In addition, by using a symmetrically substituted azoaromatic group, the thermal
lifetime of its cis isomer could be enhanced in a factor >105 with respect to the analogous twoꢀphoton
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 20 of 34
responsive ligands previously reported, which rely on the intrinsic nonꢀlinear optical properties of pushꢀ
pull azobenzenes. This is an essential requirement for enabling accumulation of the proteinꢀbound cis
state of the switch upon repeated illumination of tagged glutamate receptors, which makes compound 2
a promising candidate for evoking large photoinduced biological responses under multiphoton operation
of these systems with NIR light.
1
2
3
4
5
6
7
8
9
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
Experimental Section
General Methods: Commercially available reagents were used as received. The solvents were dried by
distillation over the appropriate drying agents. All reactions were performed avoiding moisture by
standard procedures and under nitrogen atmosphere. Flash column chromatography was performed
using silica gel (230ꢀ400 mesh). 1H NMR and 13C NMR spectra were recorded at 250 and 62.5 MHz or
360 and 90 MHz or 500 and 125 MHz. NMR signals were assigned with the help of DEPT, COSY,
HMBC, HMQC and NOESY experiments. Proton chemical shifts are reported in ppm (δ) (CDCl3, δ
7.26; DMSOꢀd6, δ 2.50 or methanolꢀd4, δ 3.31). Carbon chemical shifts are reported in ppm (δ)
(CDCl3, δ 77.2; DMSOꢀd6, δ 39.5 or methanolꢀd4, δ 49.0). Melting points were determined on hot
stage and are uncorrected. High resolution mass spectra (HRMS) were recorded in a micrOTOFQ
spectrometer using ESIMS (QTOF). Optical rotations were measured at 22 ± 2 ˚C.
Photochemical characterization: All spectroscopic and photochemical experiments were carried out in
HPLC or spectroscopy quality solvents and in Arꢀdegassed samples. Steadyꢀstate UVꢀvis absorption
measurements were recorded on a UVꢀvisible (190ꢀ1100 nm) Spectrophotometer. Fluorescence
emission spectra were measured in a fluorescence spectrometer using high energy pulsed Xenon source
for excitation. All the emission spectra registered were corrected by the wavelength dependence of the
spectral response of the detection system. Fluorescence quantum yields were determined using the
standard method28 and relative to pyrene in ethanol (Φpyrene = 0.32).29 The same excitation wavelength
was used for both the sample of interest and the standard (λexc = 305 (6) or 320 nm (transꢀ2)) and
ACS Paragon Plus Environment

Page 21 of 34
The Journal of Organic Chemistry
solutions with absorbance values below 0.05 were measured to prevent innerꢀfilter effects. The
fluorescence quantum yield of the target compound (Φsample) was determined by applying the following
equation:28
1
2
3
4
5
6
7
8
9
ꢟ
ꢊ
ꢔ
ꢝꢞꢋ
ꢛꢚꢜ
ꢕꢖꢗꢘꢙꢚ
ꢕꢖꢗꢘꢙꢚ
Φꢋꢉꢏꢐꢑꢒ = Φꢈꢒꢓ
(1)
ꢟ
ꢔ
ꢛꢚꢜ
ꢝꢞꢋ
ꢕꢖꢗꢘꢙꢚ
ꢊ
ꢛꢚꢜ
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
In this equation, Φref is the fluorescence quantum yield of the standard, Fsample and Fref are the integrated
emission intensities measured for the sample and the standard, Abssample and Absref are the optical
densities of the sample and the standard at the excitation wavelength, and nsample and nref are the
refractive indices of the solvents used. Photoisomerization of 2 and 20 was investigated by: (i) steadyꢀ
state UVꢀvis absorption spectroscopy for transꢀtoꢀcis and cisꢀtoꢀtrans photoisomerization and slow cisꢀ
1
toꢀtrans thermal backꢀisomerization processes; (ii) H NMR for the elucidation of the transꢀtoꢀcis
photostationary state mixtures. Photoisomerization quantum yields were determined using the
methodology reported in ref. 30 and azobenzene as reference compound (Φtransꢀcis= 0.15 in
acetonitrile31). Different excitation sources were used in the photochemical experiments depending on
the spectral requirements: a UV lamp equipped with two 4W tubes emitting at 365 nm, a Xe lamp
coupled to a spectrograph, the third harmonic of the Nd:YAG laser (λexc = 355 nm) or a cw laser diode
at λexc = 532 nm (ZꢀLaser). All absorption, fluorescence and photochemical measurements of 2, 6 and
pyrene were carried at low enough concentrations as to prevent aggregation processes driven by πꢀ
stacking of pyrene units.
(2R)-2-{(2S)-3-(tert-Butoxy)-2-[(tert-butoxycarbonyl)amino]-3-oxopropyl}-4-pentenoic acid, 10. To
an iceꢀcooled solution of 9a (1.80 g, 5.53 mmol) in a mixture 5:2 of THF and water (75 mL), 1 M LiOH
(8.0 mL, 8.00 mmol) was added. After stirring for 1 h at this temperature, the mixture was acidified to
pH 2 with 1 M HCl and extracted with EtOAc (3x50 mL). The combined organic extracts were dried
over anhydrous MgSO4 and the solvent was removed under vacuum. The residue was purified by
column chromatography (hexanes/EtOAc, 3:1) to furnish 10 (1.78 g, 5.18 mmol, 93% yield) as a
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 22 of 34
yellowish oil: [α]D ꢀ 0.8 (c 1.47, CH2Cl2); 1H NMR (360 MHz, CDCl3) δ 5.74 (m, 1H, Hꢀ4), 5.39 (br
20
1
2
3
4
i
i
d, JNH,2 = 8.5 Hz, 1H, NH), 5.08 (m, 2H, Hꢀ5), 4.22 (m, 1H, Hꢀ2 ), 2.49 (m, 2H, Hꢀ2, Hꢀ3), 2.31ꢀ2.04
13
(m, 2H, Hꢀ3, Hꢀ1i), 1.66 (m, 1H, Hꢀ1i), 1.46 (s, 9H, C(CH3)3), 1.45 (s, 9H, C(CH3)3); C NMR (90
5
6
7
8
9
MHz, CDCl3) δ 178.6 (Cꢀ1), 171.3 (Cꢀ3i), 156.3 (CO, carbamate), 134.8 (Cꢀ4), 117.8 (Cꢀ5), 82.7
(C(CH3)3), 80.7 (C(CH3)3), 52.7 (Cꢀ2i), 41.8 (Cꢀ2), 36.4 (Cꢀ3), 35.2 (Cꢀ1i), 28.4 (C(CH3)3), 28.1
(C(CH3)3); IR (ATR) 2978, 1707, 1367, 1247, 1150 cmꢀ1; HRMS (ESI+) calcd. for [C17H29NO6+Na]:
366.1887; found: 366.1893.
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
Di(tert-butyl) (2R,4S)-2-allyl-4-[(tert-butoxycarbonyl)amino]pentanedioate, 11. To a solution of
compound 10 (3.50 g, 7.28 mmol) in CH2Cl2 (90 mL), tertꢀbutyl 2,2,2ꢀtrichloroacetimidate (2.6 mL,
14.6 mmol) and BF3·OEt2 (370 µL, 2.96 mmol) were added. Then, a saturated aqueous solution of
NaHCO3 (20 mL) was added and the product was extracted with CH2Cl2 (3x50 mL). The combined
organic extracts were dried over anhydrous MgSO4 and the solvent was removed under vacuum. The
resulting yellow oil was purified by column chromatography (hexanes/EtOAc, 3:1) to give 11 (1.64 g,
20
4.10 mmol, 56%) as a yellow solid: mp 86ꢀ89 ºC (from hexanes/EtOAc); [α]D + 10.7 (c 0.59,
CH2Cl2); 1H NMR (250 MHz, CDCl3) δ 5.70 (ddt, J2 ,3
= 17.0 Hz, J2 ,3 = 10.1 Hz, J2 ,1 = 6.8 Hz,
i
i
i
i
i i
trans
cis
1H, Hꢀ2i), 5.09 (m, 3H, 2xHꢀ3i, NH), 4.22 (m, 1H, Hꢀ4), 2.49ꢀ2.26 (m, 2H, Hꢀ1i, Hꢀ2), 2.26ꢀ2.03 (m,
2H, Hꢀ1i, Hꢀ3), 1.65 (m, 1H, Hꢀ3), 1.44 (m, 27H, 3xC(CH3)3); 13C NMR (62.5 MHz, CDCl3) δ
173.9/171.7 (Cꢀ1/Cꢀ5), 155.3 (CO, carbamate), 135.0 (Cꢀ2i), 117.2 (Cꢀ3i), 81.8 (C(CH3)3), 80.7
(C(CH3)3), 79.5 (C(CH3)3), 52.7 (Cꢀ4), 42.3 (Cꢀ2), 36.8 (Cꢀ1i), 34.1 (Cꢀ3), 28.3 (C(CH3)3), 28.1
(C(CH3)3), 28.0 (C(CH3)3); IR (ATR) 3382, 2979, 1742, 1703, 1526, 1366, 1229, 1150 cmꢀ1; HRMS
(ESI+) calcd. for [C21H37NO6+Na]: 422.2513; found: 422.2520.
Di(tert-butyl) (2S,4R)-2-[(tert-butoxycarbonyl)amino]-4-[(2E)-4-oxo-2-butenyl]pentanedioate, 12.
To a boiling solution of (4R)ꢀ11 (2.30 g, 5.75 mmol) and crotonaldehyde (2.4 mL, 28.8 mmol) in dry
CH2Cl2 (36 mL), HoveydaꢀGrubbs II catalyst (56 mg, 89 ꢀmol) was added in 3 portions. The mixture
was heated to reflux for 2.5 h, when TLC analysis (hexanes/EtOAc, 2:1) showed no starting material.
ACS Paragon Plus Environment

Page 23 of 34
The Journal of Organic Chemistry
The reaction mixture was filtered off through a pad of silica gel and concentrated. The crude was
1
2
3
purified by column chromatography (hexanes/Et2O, from 3:1 to 1:1) to provide 12 (2.19 g, 5.12 mmol,
4
5
6
20
1
89% yield) as a yellow oil: [α]D + 88.4 (c 0.48, CH2Cl2); H NMR (250 MHz, CDCl3) δ 9.46 (d,
7
8
i
i
i
i
i
i
i i
JCHO,3 = 7.8 Hz, 1H, CHO), 6.73 (dt, J2 ,3 = 15.7 Hz, J2 ,1 = 6.7 Hz, 1H, Hꢀ2 ), 6.09 (dd, J3 ,2 = 15.7 Hz,
9
J3 ,CHO = 7.8 Hz, 1H, Hꢀ3i), 5.03 (d, JNH,2 = 7.9 Hz, 1H, NH), 4.20 (m, 1H, Hꢀ2), 2.68ꢀ2.39 (m, 3H, Hꢀ4,
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
i
2xHꢀ1i), 2.15 (ddd, Jgem = 12.7 Hz, J3,2 = 8.6 Hz, J3,4 = 4.8 Hz, 1H, Hꢀ3), 1.64 (ddd, Jgem = 12.7 Hz, J3,2
= 8.6 Hz, J3,4 = 3.7 Hz, 1H, Hꢀ3), 1.44 (m, 27H, 3xC(CH3)3); 13C NMR (90 MHz, CDCl3) δ 193.6
(CHO), 173.1/171.4 (Cꢀ1/Cꢀ5), 155.3 (CO, carbamate), 154.4 (Cꢀ2i), 134.7 (Cꢀ3i), 82.4 (C(CH3)3), 81.7
(C(CH3)3), 79.9 (C(CH3)3), 52.5 (Cꢀ2), 41.6 (Cꢀ4), 35.4 (Cꢀ1i), 34.8 (Cꢀ3), 28.4 (C(CH3)3), 28.2
(C(CH3)3), 28.1 (C(CH3)3); IR (ATR) 2977, 1699, 1392, 1367, 1251, 1150 cmꢀ1; HRMS (ESI+) calcd.
for [C22H37NO7+Na]: 450.2462; found: 450.2460.
(2E,5R,7S)-8-tert-Butoxy-5-(tert-butoxycarbonyl)-7-[(tert-butoxycarbonyl)amino]-8-oxo-2-octenoic
acid, 13. To an iceꢀcooled solution of the aldehyde 12 (2.50 g, 5.85 mmol) in a 5:1 mixture of t-BuOH
and water (240 mL), 2ꢀmethylꢀ2ꢀbutene (6.2 mL, 58.5 mmol), NaH2PO4·2H2O (4.60 g, 29.2 mmol) and
NaClO2 (3.17 g, 35.1 mmol) were successively added. The resulting mixture was stirred at 0 ºC for 1 h
and warmed up to rt. The reaction mixture was stirred overnight. Then, water (30 mL) and a saturated
aqueous solution of Na2CO3 were added until pH 8. The mixture was extracted with EtOAc (3x50 mL),
dried over anhydrous MgSO4 and concentrated under vacuum. Purification by column chromatography
20
(hexanes/EtOAc/CH3COOH, 4:1:0.1) gave 13 (2.00 g, 4.51 mmol, 77% yield) as a yellow oil: [α]D
+
59.8 (c 1.16, CH2Cl2); 1H NMR (360 MHz, MeOHꢀd4) δ 6.86 (dt, J3,2 = 15.1 Hz, J3,4 = 7.3 Hz, 1H, Hꢀ3),
5.84 (d, J2,3 = 15.1 Hz, 1H, Hꢀ2), 4.06 (dd, J7,6 = 10.6 Hz, J7,6 = 3.8 Hz, 1H, Hꢀ7), 2.56 (m, 1H, Hꢀ5),
13
2.41 (m, 2H, Hꢀ4), 2.08 (m, 1H, Hꢀ6), 1.65 (m, 1H, Hꢀ6), 1.45 (m, 27H, 3xC(CH3)3); C NMR (62.5
MHz, MeOHꢀd4) δ 174.5/172.8/169.0 (Cꢀ1/Cꢀ8/CO, ester), 157.3 (CO, carbamate), 146.7 (Cꢀ3), 124.4
(Cꢀ2), 82.4 (C(CH3)3), 82.1 (C(CH3)3), 80.2 (C(CH3)3), 53.6 (Cꢀ7), 42.9 (Cꢀ5), 35.9 (Cꢀ4), 34.4 (Cꢀ6),
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 24 of 34
28.8 (C(CH3)3), 28.4 (C(CH3)3), 28.3 (C(CH3)3); IR (ATR) 2977, 2932, 1703, 1366, 1247, 1147 cmꢀ1;
HRMS (ESI+) calcd. for [C22H37NO8+Na]: 466.2411; found: 466.2417.
1
2
3
4
5
6
7
8
(5R,7S)-8-tert-Butoxy-5-(tert-butoxycarbonyl)-7-[(tert-butoxycarbonyl)amino]-8-oxooctanoic acid,
5. To a solution of olefine 13 (1.50 g, 3.38 mmol) in EtOAc (60 mL), 10% Pd/C (0.15 g) was added.
The resulting suspension was stirred in a H2 atmosphere for 16 h. Then, it was filtered through Celite®
and the solvent was evaporated under vacuum. The resulting oil was purified by column
chromatography (hexanes/EtOAc/CH3COOH, 5:4:1) to afford a colourless oil identified as 5 (1.50 g,
9
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
1
3.36 mmol, quantitative yield): [α]D20 + 66 (c 0.30, CH2Cl2); H NMR (360 MHz, MeOHꢀd4) δ 6.83 (d,
JNH,7 = 8.4 Hz, 1H, NH), 3.98 (m, 1H, Hꢀ7), 2.44 (m, 1H, Hꢀ5), 2.29 (td, J2,3 = 6.8 Hz, J2,4 = 3.7 Hz, 2H,
13
Hꢀ2), 2.03 (m, 1H, Hꢀ6), 1.60 (m, 5H, 2xHꢀ3, 2xHꢀ4, Hꢀ6), 1.46 (m, 27H, 3xC(CH3)3); C NMR (90
MHz, MeOHꢀd4) δ 176.8/175.9/173.3 (Cꢀ1/Cꢀ8/CO, ester), 157.8 (CO, carbamate), 82.5 (C(CH3)3), 82.0
(C(CH3)3), 80.3 (C(CH3)3), 54.1 (Cꢀ7), 43.9 (Cꢀ5), 35.0 (Cꢀ6), 34.5 (Cꢀ2), 33.4 (Cꢀ4), 28.8 (C(CH3)3),
28.4 (C(CH3)3), 28.3 (C(CH3)3), 23.6 (Cꢀ1); IR (ATR) 2976, 1709, 1392, 1366, 1248, 1146 cmꢀ1; HRMS
(ESI+) calcd. for [C22H39NO8+Na]: 468.2568; found: 468.2570.
9H-Fluoren-9-ylmethyl
N-[(5S)-6-{4-[(E)-2-(4-aminophenyl)-1-diazenyl]aniline}-5-[(tert-
butoxycarbonyl)amino]-6-oxohexyl]carbamate, 14. To a stirred solution of 4ꢀ[(E)ꢀ(4ꢀaminophenyl)ꢀ
1ꢀdiazenyl]aniline (4) (1.00 g, 4.71 mmol) in dry THF (100 mL) under N2 atmosphere, a solution of 3
(2.43 g, 5.19 mmol), EDCI (1.17 g, 6.10 mmol), HOBt (955 mg, 7.07 mmol), DIPEA (3.3 mL, 18.8
mmol) in dry THF (100 mL) was added. The reaction mixture was stirred overnight at rt. Then, the
mixture was diluted with EtOAc (200 mL) and washed with water (2x100 mL). The organic layer was
dried over anhydrous MgSO4 and concentrated under vacuum. The residue was purified by column
chromatography (from CH2Cl2 to CH2Cl2/EtOAc, 4:1) to deliver amide 14 (1.62 g, 2.45 mmol, 85%
yield considering starting material consumed) as an orange solid and starting material (388 mg, 1.83
mmol): mp 94ꢀ97 ºC (from CH2Cl2/EtOAc); [α]D20 ꢀ 9.70 (c 1.01, CHCl3); 1H NMR (250 MHz, CDCl3)
δ 8.73 (br s, 1H, Cꢀ1iiiNH), 7.77 (m, 6H, Hꢀ4, Hꢀ5, Hꢀ3iii, Hꢀ5iii, Hꢀ2iv, Hꢀ6iv), 7.65 (d, J2
= J6
=
iii iii
iii iii
,5
,3
ACS Paragon Plus Environment

Page 25 of 34
The Journal of Organic Chemistry
8.0 Hz, 2H, Hꢀ2iii, Hꢀ6iii), 7.58 (d, J1,2 = J8,7 = 7.2 Hz, 2H, Hꢀ1, Hꢀ8), 7.38 (t, J3,2 = J3,4 = J6,7 = J6,5 = 7.2
Hz, 2H, Hꢀ3, Hꢀ6), 7.29 (td, J2,3 = J2,1 = J7,8 = J7,6 = 7.2 Hz, J2,4 = J7,5 = 1.2 Hz, 2H, Hꢀ2, Hꢀ7), 6.71 (d,
1
2
3
4
5
6
iv
iv
ii
iv iv
iv iv
ii
ii
ii
ii
J3
= J5
= 8.8 Hz, 2H, Hꢀ3 , Hꢀ5 ), 5.34 (d, JCꢀ5 NH,5 = 7.5 Hz, 1H, Cꢀ5 NH), 4.95 (t, JCꢀ1 NH,1 =
,2
,6
7
8
9
6.0 Hz, 1H, Cꢀ1iiNH), 4.41 (d, J1 ,9 = 6.9 Hz, 2H, Hꢀ1i), 4.20 (m, 2H, Hꢀ5ii, Hꢀ9), 4.06 (br s, 2H, NH2),
3.19 (m, 2H, Hꢀ1ii), 1.95 (m, 1H, Hꢀ4ii), 1.72 (m, 1H, Hꢀ4ii), 1.62ꢀ1.36 (m, 13H, 2xHꢀ2ii, 2xHꢀ3ii,
C(CH3)3); 13C NMR (100.6 MHz, CDCl3) δ 170.8 (Cꢀ6ii), 156.9 (CO, carbamate), 156.5 (CO,
carbamate), 149.5 (Cꢀ4iii, Cꢀ4iv), 145.7 (Cꢀ1iv), 144.0 (Cꢀ9a, Cꢀ8a), 141.4 (Cꢀ4a, Cꢀ4b), 139.4 (Cꢀ1iii),
127.8 (Cꢀ3, Cꢀ6), 127.2 (Cꢀ2, Cꢀ7), 125.1 (Cꢀ1, Cꢀ8, Cꢀ2iv, Cꢀ6iv), 123.4 (Cꢀ3iii, Cꢀ5iii), 120.1 (Cꢀ4, Cꢀ6,
Cꢀ2iii, Cꢀ6iii), 114.8 (Cꢀ3iv, Cꢀ5iv), 80.7 (C(CH3)3), 66.7 (Cꢀ1i), 55.2 (Cꢀ5ii), 47.4 (Cꢀ9), 40.3 (Cꢀ1ii), 31.4
(Cꢀ4ii), 29.5 (Cꢀ2ii), 28.4 (C(CH3)3), 22.7 (Cꢀ3ii); IR (ATR) 3323, 2926, 2856, 1687, 1665, 1596, 1504,
1245 cmꢀ1; HRMS (ESI+) calcd. for [C38H42N6O5+Na]: 685.3109; found: 685.3112.
i
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
9H-Fluoren-9-ylmethyl
N-((5S)-5-amino-6-{4-[(E)-2-(4-aminophenyl)-1-diazenyl]anilino}-6-
oxohexyl)carbamate, 15. To a solution of intermediate 14 (1.07 g, 1.62 mmol) in MeOH (100 mL),
37% HCl (21 mL, 0.22 mol) was added. The reaction mixture was stirred for 1 h at room temperature
until TLC (EtOAc) showed no presence of starting material. Then, the mixture was concentrated under
vacuum, diluted with EtOAc (20 mL) and neutralized with a saturated aqueous solution of NaHCO3.
Purification by column chromatography (from EtOAc to EtOAc/MeOH, 95:5) gave 15 (890 mg, 1.58
20
1
mmol, 98% yield) as an orange solid: mp 78ꢀ82 ºC (from EtOAc); [α]D ꢀ25.6 (c 1.19, CHCl3); H
NMR (400 MHz, CDCl3) δ 9.69 (br s, 1H, Cꢀ1iiiNH), 7.84 (d, J3
= J5
= 8.7 Hz, 2H, Hꢀ3 , Hꢀ
iii
iii iii
iii iii
,6
,2
5iii), 7.75 (m, 6H, Hꢀ4, Hꢀ5, Hꢀ2iii, Hꢀ6iii, Hꢀ2iv, Hꢀ5iv), 7.59 (d, J1,2 = J8,7 = 7.4 Hz, 2H, Hꢀ1, Hꢀ8), 7.39
(t, J3,2 = J3,4 = J6,7 = J6,5 = 7.4 Hz, 2H, Hꢀ3, Hꢀ6), 7.31 (td, J2,3 = J2,1 = J7,8 = J7,6 = 7.4 Hz, J2,4 = J7,5 = 0.9
iv
iv
iv iv
iv iv
ii
ii
Hz, 2H, Hꢀ2, Hꢀ7), 6.73 (d, J3
= J5
= 8.7 Hz, 2H, Hꢀ3 , Hꢀ5 ), 4.90 (t, JCꢀ1 NH,1 = 5.4 Hz, 1H,
,2
,6
Cꢀ1 NH), 4.40 (d, J1 ,9 = 6.9 Hz, 2H, Hꢀ1i), 4.21 (t, J9,1 = 6.5 Hz, 1H, Hꢀ9), 3.49 (m, 1H, Hꢀ5 ), 3.21 (m,
2H, Hꢀ1ii), 1.96 (m, 1H, Hꢀ4ii), 1.68ꢀ1.29 (m, 5H, 2xHꢀ2ii, 2xHꢀ3ii, Hꢀ4ii); 13C NMR (100.6 MHz,
CDCl3) δ 173.5 (Cꢀ6ii), 156.7 (CO, carbamate), 149.5/149.4 (Cꢀ4iii/Cꢀ4iv), 145.7 (Cꢀ1iv), 144.1 (Cꢀ9a, Cꢀ
ii
ii
i
i
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 26 of 34
8a), 141.4 (Cꢀ4a, Cꢀ4b), 139.4 (Cꢀ1iii), 127.8 (Cꢀ3, Cꢀ6), 127.2 (Cꢀ2, Cꢀ7), 125.1 (Cꢀ1, Cꢀ8, Cꢀ2iv, Cꢀ
6iv), 123.5 (Cꢀ3iii, Cꢀ5iii), 120.1 (Cꢀ4, Cꢀ6), 119.6 (Cꢀ2iii, Cꢀ6iii), 114.8 (Cꢀ3iv, Cꢀ5iv), 66.7 (Cꢀ1i), 55.5 (Cꢀ
5ii), 47.4 (Cꢀ9), 40.7 (Cꢀ1ii), 34.5 (Cꢀ4ii), 29.8 (Cꢀ2ii), 23.0 (Cꢀ3ii); IR (ATR) 3335, 2923, 1687, 1596,
1508, 1249, 1138 cmꢀ1; HRMS (ESI+) calcd. for [C33H34N6O3+H]: 563.2765; found: 563.2769.
Di(tert-butyl) (2R,4S)-2-{4-[((1S)-1-{4-[(E)-(4-aminophenyl)-1-diazenyl]anilino} carbonyl-5-{[(9H-
fluoren-9-ylmethoxy)carbonyl]amino}pentyl)amino]-4-oxobutyl}-4-[(tert-
1
2
3
4
5
6
7
8
9
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
butoxycarbonyl)amino]pentanedioate, 16. To a stirred solution of 15 (232 mg, 0.34 mmol) in dry
THF (18 mL) under N2 atmosphere, a solution of 5 (169 mg, 0.38 mmol), EDCI (86 mg, 0.45 mmol),
HOBt (70 mg, 0.52 mmol), DIPEA (240 ꢀL, 1.40 mmol) in dry THF (20 mL) was added. The reaction
mixture was stirred overnight at rt. Then, the mixture was diluted with EtOAc (40 mL) and washed with
water (2x40 mL). The organic layer was dried over anhydrous MgSO4 and concentrated under vacuum.
The residue was purified by column chromatography (hexanes/EtOAc, 1:9) to afford amide 16 (335 mg,
20
0.30 mmol, 88% yield) as an orange solid: mp 98ꢀ105 ºC (from hexanes/EtOAc); [α]D ꢀ 22.5 (c 1.18,
CHCl3); 1H NMR (400 MHz, CDCl3) δ 8.91 (br s, 1H, Cꢀ1vNH), 7.79 (m, 6H, Hꢀ4iv, Hꢀ5iv, Hꢀ3v, Hꢀ5v,
v
v
iv
Hꢀ2vi, Hꢀ6vi), 7.65 (d, J2 ,3 = J6 ,5 = 8.7 Hz, 2H, Hꢀ2 , Hꢀ6 ), 7.57 (d, J1
= J8 ,7 = 7.4 Hz, 2H, Hꢀ1 ,
v
v
v
v
iv iv
,2
i iv
Hꢀ8iv), 7.38 (t, J3
= J3
= J6
= J6
= 7.4 Hz, 2H, Hꢀ3iv, Hꢀ6iv), 7.31 (t, J2
= J2
=
iv iv
iv iv
iv iv
iv iv
iv iv
iv iv
,1
,2
,4
,5
,7
,3
vi
vi
iv iv
iv
i
vi vi
vi vi
,6
J7
= J7
= 7.4 Hz, 2H, Hꢀ2iv, Hꢀ7iv), 6.73 (d, J3
= J5
= 8.7 Hz, 2H, Hꢀ3 , Hꢀ5 ), 6.48 (s,
,6
,8
,2
ii
ii
1H, Cꢀ1iiNH), 5.10 (t, JCꢀ5 NH,5 = 5.4 Hz, 1H, Cꢀ5 NH), 4.56 (m, 1H, Hꢀ1 ), 4.38 (d, J1
= 6.9 Hz,
ii
ii
iii iv
,9
2H, Hꢀ1iii), 4.20 (m, 2H, Hꢀ4, Hꢀ9iv), 4.05 (br s, 2H, NH2), 3.22 (m, 2H, Hꢀ5ii), 2.34 (m, 1H, Hꢀ2), 2.24
(m, 2H, Hꢀ3i), 2.13ꢀ1.94 (m, 3H, Hꢀ3, Hꢀ1i, Hꢀ2ii), 1.86ꢀ1.37 (m, 7H, Hꢀ3, Hꢀ1i, 2xHꢀ2i, Hꢀ2ii, 2xHꢀ4ii),
13
1.44 (m, 29H, 3xC(CH3)3, 2xHꢀ3ii); C NMR (100.6 MHz, CDCl3) δ 174.5/173.8/171.9/170.1 (Cꢀ1/Cꢀ
5/Cꢀ4i/CO, amide), 157.0 (CO, carbamate), 155.5 (CO, carbamate), 149.5 (Cꢀ4v/Cꢀ4vi), 145.7 (Cꢀ1vi),
144.1 (Cꢀ9aiv, Cꢀ8aiv), 141.4 (Cꢀ4aiv, Cꢀ4biv), 139.5 (Cꢀ1v), 127.8 (Cꢀ3iv, Cꢀ6iv), 127.2 (Cꢀ2iv, Cꢀ7iv),
125.2/125.1 (Cꢀ1iv, Cꢀ8iv/Cꢀ2vi, Cꢀ6vi), 123.4 (Cꢀ3v, Cꢀ5v), 120.1 (Cꢀ4iv, Cꢀ5iv/Cꢀ2v, Cꢀ6v), 114.8 (Cꢀ3vi,
Cꢀ5vi), 82.1 (C(CH3)3), 81.1 (C(CH3)3), 79.8 (C(CH3)3), 66.8 (Cꢀ1iii), 54.0 (Cꢀ1ii), 52.8 (Cꢀ4), 47.4 (Cꢀ
ACS Paragon Plus Environment

Page 27 of 34
The Journal of Organic Chemistry
9iv), 42.4 (Cꢀ2), 40.5 (Cꢀ5ii), 36.1 (Cꢀ3i), 34.9 (Cꢀ3), 32.1 (Cꢀ1i), 30.7 (Cꢀ2ii), 29.8 (Cꢀ4ii), 28.5
(C(CH3)3), 28.2 (C(CH3)3), 28.1 (C(CH3)3), 23.2 (Cꢀ2i), 22.6 (Cꢀ3ii); IR (ATR) 3300, 2976, 2931, 1691,
1598, 1530, 1366, 1248, 1148 cmꢀ1; HRMS (ESI+) calcd. for [C55H71N7O10+H]: 990.5335; found:
990.5347.
1
2
3
4
5
6
7
8
9
Di(tert-butyl) (2R,4S)-2-(4-{(1S)-5-amino-1-({4-[(E)-(4-aminophenyl)-1-diazenyl] anilino}carbonyl)
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
pentyl]amino}-4-oxobutyl)-4-[(tert-butoxycarbonyl)amino]pentanedioate, 17.
A
commercially
available solution of 20% piperidine in DMF (18 mL) was added to compound 16 (775 mg, 0.78 mmol).
After 1 h of stirring at rt, TLC analysis (EtOAc/MeOH, 95:5) showed no presence of starting material.
The mixture was diluted with water (18 mL) and washed with EtOAc (3x30 mL). The combined organic
extracts were dried over anhydrous MgSO4 and concentrated under vacuum. The resulting solid was
purified by column chromatography (from EtOAc to EtOAc/MeOH/NH3, 9:2:1) to deliver 17 (459 mg,
20
0.60 mmol, 76% yield) as an orange solid: mp 89ꢀ96 ºC (from EtOAc); [α]D ꢀ 31.1 (c 0.88, CHCl3);
1H NMR (400 MHz, CDCl3) δ 9.56 (br s, 1H, Cꢀ1iiiNH), 7.81 (m, 4H, Hꢀ3iii, Hꢀ5iii, Hꢀ2iv, Hꢀ6iv), 7.70
iii
iii
iv
iv
iii iii
iii iii
iv iv
iv iv
= J5
,6
(d, J2
= J6
= 7.7 Hz, 2H, Hꢀ2 , Hꢀ6 ), 6.74 (d, J3
= 8.4 Hz, 2H, Hꢀ3 , Hꢀ5 ), 6.68
,3
,5
,2
(m, 1H, Cꢀ1iiNH), 5.21 (m, 1H, Cꢀ4NH), 4.63 (m, 1H, Hꢀ1ii), 4.19 (m, 1H, Hꢀ4), 4.06 (br s, 2H, Cꢀ
4ivNH2), 2.82 (m, 2H, Hꢀ5ii), 2.58ꢀ2.31 (m, 3H, Hꢀ2, Cꢀ5iiNH2), 2.27 (m, 2H, Hꢀ3i), 2.20ꢀ1.90 (m, 3H,
Hꢀ3, Hꢀ1i, Hꢀ2ii), 1.86ꢀ1.56 (m, 7H, Hꢀ3, Hꢀ1i, 2xHꢀ2i, Hꢀ2ii, 2xHꢀ4ii), 1.44 (m, 29H, 3xC(CH3)3, 2xHꢀ
13
3ii); C NMR (100.6 MHz, CDCl3) δ 174.5/173.5/171.8/170.3 (Cꢀ1/Cꢀ5/Cꢀ4i/CO, amide), 155.4 (CO,
carbamate), 149.5 (Cꢀ4iii, Cꢀ4iv), 145.7 (Cꢀ1iv), 139.7 (Cꢀ1iii), 125.1 (Cꢀ2iv, Cꢀ6iv), 123.4 (Cꢀ3iii, Cꢀ5iii),
120.1 (Cꢀ2iii, Cꢀ6iii), 114.8 (Cꢀ3iv, Cꢀ5iv), 82.7 (C(CH3)3), 81.1 (C(CH3)3), 79.7 (C(CH3)3), 53.9 (Cꢀ1ii),
52.8 (Cꢀ4), 42.4 (Cꢀ2), 41.2 (Cꢀ5ii), 36.1 (Cꢀ3i), 34.8 (Cꢀ3), 32.1 (Cꢀ1i/Cꢀ2ii), 31.6 (Cꢀ4ii), 28.5
(C(CH3)3), 28.2 (C(CH3)3), 28.1 (C(CH3)3), 23.2 (Cꢀ2i), 22.7 (Cꢀ3ii); IR (ATR) 3343, 2977, 2933, 1701,
1598, 1506, 1368, 1249, 1150 cmꢀ1; HRMS (ESI+) calcd. for [C40H61N7O8+H]: 768.4654; found:
768.4650.
Di(tert-butyl) (2R,4S)-2-(4-{(1S)-1-({4-[(E)-(4-aminophenyl)-1-diazenyl]anilino} carbonyl)-5-[(2-
pyrenylacetyl)amino]pentyl}amino)-4-oxobutyl]-4-[(tert-butoxycarbonyl)amino]pentanedioate, 18.
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 28 of 34
To a stirred solution of 17 (410 mg, 0.53 mmol) in dry THF (20 mL) under N2 atmosphere, a solution of
1ꢀpyreneacetic acid (6) (167 mg, 0.64 mmol), EDCI (133 mg, 0.69 mmol), DIPEA (465 ꢀL, 2.67 mmol)
in dry THF (30 mL) was added. The reaction mixture was stirred overnight at rt. Then, the mixture was
diluted with EtOAc (50 mL) and washed with water (2x20 mL). The organic layer was dried over
anhydrous MgSO4 and concentrated under vacuum. The residue was purified by column
chromatography (EtOAc) and triturated with diethyl ether (2x10 mL) to furnish an orange solid
identified as 18 (458 mg, 0.45 mmol, 84% yield): mp 157ꢀ161 ºC (from EtOAc); [α]D20 + 5.30 (c 0.96,
CHCl3); 1H NMR (400 MHz, DMSOꢀd6) δ 10.3 (s, 1H, Cꢀ1ivNH), 8.37 (d, J = 9.3 Hz, 1H, Hꢀpyr), 8.29ꢀ
8.17 (m, 5H, Cꢀ1iiNH, 4xHꢀpyr), 8.12 (s, 2H, Hꢀpyr), 8.08 (m, 1H, Cꢀ5iiNH), 8.05 (t, J = 7.6 Hz, 1H, Hꢀ
1
2
3
4
5
6
7
8
9
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
iv
iv
iv iv
,3
iv iv
= 9.1 Hz, 2H, Hꢀ2 , Hꢀ6 ), 7.71 (d, J3
,6
iv iv
,2
pyr), 7.99 (d, J = 7.9 Hz, 1H, Hꢀpyr), 7.76 (d, J2
= J5
iv
iv
v
v
iv iv
v
v
v
v
= J5
= 9.1 Hz, 2H, Hꢀ3 , Hꢀ5 ), 7.63 (d, J2 ,3 = J6 ,5 = 8.8 Hz, 2H, Hꢀ2 , Hꢀ6 ), 7.10 (d, JCꢀ4NH,4
=
,6
v
v
v
v
v v
8.2 Hz, 1H, Cꢀ4NH), 6.66 (d, J3 ,2 = J5 ,6 = 8.8 Hz, 2H, Hꢀ3 , Hꢀ5 ), 6.02 (s, 2H, NH2), 4.40 (m, 1H,
Hꢀ1ii), 4.17 (s, 2H, Hꢀ2iii), 3.76 (m, 1H, Hꢀ4), 3.09 (dd, J5 ,4 = 12.6 Hz, J5 ,Cꢀ5 NH = 6.7 Hz, 2H, Hꢀ5ii),
2.33 (m, 1H, Hꢀ2), 2.12 (m, 2H, Hꢀ3i), 1.85ꢀ1.51 (m, 6H, 2xHꢀ3, 2xHꢀ1i, 2xHꢀ2ii), 1.52ꢀ1.25 (m, 33H,
3xC(CH3)3, 2xHꢀ2i, 2xHꢀ3ii, 2xHꢀ4ii); 13C NMR (100.6 MHz, DMSOꢀd6) δ 173.8/172.1/171.7/171.2 (Cꢀ
1/Cꢀ5/Cꢀ4i/CO, amide), 169.9 (Cꢀ1iii), 155.5 (CO, carbamate), 152.4 (Cꢀ4v), 148.1 (Cꢀ4iv), 142.8 (Cꢀ1v),
140.2 (Cꢀ1iv), 131.1 (Cꢀpyr), 130.8 (Cꢀpyr), 130.4 (Cꢀpyr), 129.7 (Cꢀpyr), 129.0 (Cꢀpyr), 128.6 (CHꢀ
pyr), 127.4 (CHꢀpyr), 127.2 (CHꢀpyr), 126.8 (CHꢀpyr), 126.1 (CHꢀpyr), 125.1 (Cꢀ2v, Cꢀ6v), 124.9 (CHꢀ
pyr), 124.8 (CHꢀpyr), 124.7 (CHꢀpyr), 124.1 (Cꢀpyr), 124.0 (CHꢀpyr), 123.9 (Cꢀpyr), 122.5 (Cꢀ3iv, Cꢀ
5iv), 119.5 (Cꢀ2iv, Cꢀ6iv), 113.4 (Cꢀ3v, Cꢀ5v), 80.3 (C(CH3)3), 79.7 (C(CH3)3), 78.0 (C(CH3)3), 53.4 (Cꢀ
1ii), 52.5 (Cꢀ4), 42.2 (Cꢀ2), 40.1 (Cꢀ1iii), 38.6 (Cꢀ5ii), 34.8 (Cꢀ3i), 33.0 (Cꢀ3), 32.0 (Cꢀ1i), 31.6 (Cꢀ2ii),
28.8 (Cꢀ4ii), 28.2 (C(CH3)3), 27.9 (C(CH3)3), 27.6 (C(CH3)3), 23.0 (Cꢀ3ii, Cꢀ2i); IR (ATR) 3277, 2975,
2935, 1720, 1638, 1599, 1531, 1506, 1368, 1251, 1151 cmꢀ1; HRMS (ESI+) calcd. for [C58H71N7O9+H]:
1010.5386; found: 1010.5377.
ii ii
ii
ii
ACS Paragon Plus Environment

Page 29 of 34
The Journal of Organic Chemistry
Di(tert-butyl) (2S,4R)-2-[(tert-butoxycarbonyl)amino]-4-[4-({(1S)-1-({4-[(E)-(4-{[2-(2,5-dioxo-2,5-
dihydro-1H-1-pyrrolyl)-acetyl]amino}phenyl)-1-diazenyl]anilino}carbonyl)-5-[(2-
1
2
3
4
5
6
7
8
pyrenylacetyl)amino]pentyl}amino)-4-oxobutyl]pentanedioate, 19. To a stirred solution of 7 (120
mg, 0.77 mmol) and oxalyl chloride 2.0 M in THF (402 ꢀL, 0.80 mmol) in dry CH2Cl2 (12 mL) was
added one drop of DMF. After stirring for 2 h at rt, the mixture was concentrated. The resulting acid
chloride was taken up in dry THF (5 mL) and slowly added to an iceꢀcooled solution of 18 (200 mg,
0.20 mmol), DIPEA (207 ꢀL, 1.19 mmol) in dry THF (25 mL). After stirring for 10 min at this
temperature, the mixture was warmed up to rt and stirred for additional 4 h. Then, it was diluted with
EtOAc (30 mL) and washed with water (3x10 mL). The organic extract was dried over anhydrous
9
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
MgSO4, concentrated under vacuum and purified by column chromatography (EtOAc/MeOH, 9:1) to
20
provide 19 (168 mg, 0.15 mmol, 74% yield) as an orange solid: mp 187ꢀ198 ºC (from EtOAc); [α]D
+
1
68.5 (c 0.70, DMSO); H NMR (400 MHz, DMSOꢀd6) δ 10.7 (s, 1H, Cꢀ4vNH), 10.3 (s, 1H, Cꢀ1ivNH),
8.37 (d, J = 9.3 Hz, 1H, Hꢀpyr), 8.29ꢀ8.17 (m, 5H, Cꢀ1iiNH, 4xHꢀpyr), 8.14 (s, 2H, Hꢀpyr), 8.09 (m, 1H,
Cꢀ5iiNH), 8.07 (t, J = 7.8 Hz, 1H, Hꢀpyr), 7.98 (d, J = 7.8 Hz, 1H, Hꢀpyr), 7.84 (m, 6H, Hꢀ2iv, Hꢀ6iv, Hꢀ
v
v
vii
vii
3iv, Hꢀ5iv, Hꢀ3v, Hꢀ5v), 7.75 (d, J2 ,3 = J6 ,5 = 8.9 Hz, 2H, Hꢀ2 , Hꢀ6 ), 7.16 (s, 2H, Hꢀ3 , Hꢀ4 ), 7.09
v
v
v v
(d, JCꢀ2NH,2 = 8.0 Hz, 1H, Cꢀ2NH), 4.38 (m, 1H, Hꢀ1ii), 4.33 (s, 2H, Hꢀ2vi), 4.16 (s, 2H, Hꢀ2iii), 3.76 (m,
1H, Hꢀ2), 3.08 (dd, J5 ,4 = 12.6 Hz, J5 ,Cꢀ5 NH = 6.7 Hz, 2H, Hꢀ5ii), 2.31 (m, 1H, Hꢀ4), 2.11 (m, 2H, Hꢀ
3i), 1.85ꢀ1.51 (m, 6H, 2xHꢀ3, 2xHꢀ1i, 2xHꢀ2ii), 1.51ꢀ1.24 (m, 33H, 3xC(CH3)3, 2xHꢀ2i, 2xHꢀ3ii, 2xHꢀ
4ii); 13C NMR (100.6 MHz, DMSOꢀd6) δ 172.8/171.1/170.7/170.5/169.7 (Cꢀ1/Cꢀ5/Cꢀ4i/Cꢀ2vii, Cꢀ5vii/CO,
amide), 168.9 (Cꢀ1iii), 164.3 (Cꢀ1vi), 154.5 (CO, carbamate), 146.9/146.7 (Cꢀ4iv/Cꢀ1v), 140.7/140.1 (Cꢀ
1iv/Cꢀ4v), 134.0 (Cꢀ3vii, Cꢀ4vii), 130.1 (Cꢀpyr), 129.8 (Cꢀpyr), 129.4 (Cꢀpyr), 128.7 (Cꢀpyr), 128.0 (Cꢀ
pyr), 127.6 (CHꢀpyr), 126.4 (CHꢀpyr), 126.2 (CHꢀpyr), 125.8 (CHꢀpyr), 125.1 (CHꢀpyr), 124.1 (CHꢀ
pyr), 123.9 (CHꢀpyr), 123.7 (CHꢀpyr), 123.1 (Cꢀpyr), 123.0 (CHꢀpyr), 122.9 (Cꢀpyr), 122.5/122.4 (Cꢀ2v,
Cꢀ6v/Cꢀ3iv, Cꢀ5iv), 118.5 (Cꢀ2iv, Cꢀ6iv, Cꢀ3v, Cꢀ5v), 79.3 (C(CH3)3), 78.7 (C(CH3)3), 77.0 (C(CH3)3), 52.4
(Cꢀ1ii), 51.6 (Cꢀ2), 41.2 (Cꢀ4), 39.5 (Cꢀ2iii), 37.6 (Cꢀ5ii), 33.8 (Cꢀ3i), 32.0 (Cꢀ3), 31.0 (Cꢀ1i), 30.6 (Cꢀ2ii),
ii ii
ii
ii
ACS Paragon Plus Environment