Caged Proline in Photoinitiated Organocatalysis

Article abstract of DOI:10.1021/acs.joc.9b00220

Organocatalysis is an emerging field, in which small metal-free organic structures catalyze a diversity of reactions with a remarkable stereoselectivity. The ability to selectively switch on such pathways upon demand has proven to be a valuable tool in biological systems. Light as a trigger provides the ultimate spatial and temporal control of activation. However, there have been limited examples of phototriggered catalytic systems. Herein, we describe the synthesis and application of a caged proline system that can initiate organocatalysis upon irradiation. The caged proline was generated using the highly efficient 4-carboxy-5,7-dinitroindolinyl (CDNI) photocleavable protecting group in a four-step synthesis. Advantages of this system include water solubility, biocompatibility, high quantum yield for catalyst release, and responsiveness to two-photon excitation. We showed the light-triggered catalysis of a crossed aldol reaction, a Mannich reaction, and a self-aldol condensation reaction. We also demonstrated light-initiated catalysis, leading to the formation of a biocide in situ, which resulted in the growth inhibition of E. coli, with as little as 3 min of irradiation. This technique can be broadly applied to other systems, by which the formation of active forms of drugs can be catalytically assembled remotely via two-photon irradiation.

Full text of DOI:10.1021/acs.joc.9b00220

Subscriber access provided by UNIV AUTONOMA DE COAHUILA UADEC
Article
Caged Proline in Photoinitiated Organocatalysis
Charitha Guruge, Saad Y. Rfaish, Chanel Byrd, Shukun
Yang, Anthony K. Starrett, Eric Guisbert, and Nasri Nesnas
J. Org. Chem., Just Accepted Manuscript • Publication Date (Web): 25 Mar 2019
Downloaded from http://pubs.acs.org on March 26, 2019
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 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 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.
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 32
The Journal of Organic Chemistry
1
2
3
4
Caged Proline in Photoinitiated Organocatalysis
5
6
7
Charitha Guruge, Saad Y. Rfaish, Chanel Byrd, Shukun Yang, Anthony K. Starrett, Eric Guisbert,
Nasri Nesnas*
8
9
Department of Biomedical and Chemical Engineering and Sciences, Florida Institute of Technology,
Melbourne, Florida 32901, United States
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
* Corresponding Author
Professor Nasri Nesnas, PhD
Department of Biomedical and Chemical Engineering and Sciences
Florida Institute of Technology, 150 West University Blvd., Melbourne, FL 32901, United States
E-mail: nesnas@fit.edu
Key words: organocatalysis, caged proline, photoinitiated, CDNI-Pro, cages, photoprotecting groups, proline
Caged Proline
Spent Cage
Proline
O
OH
O
OH
O
O
O
O2N
O2N
350 nm
OH
NH
N
N
H
Active catalyst
O
NH
Inactive catalyst
Graphical Abstract
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 2 of 32
1
2
3
4
5
6
7
8
9
ABSTRACT: Organocatalysis is an emerging field in which small metal-free organic
structures catalyze a diversity of reactions with remarkable stereoselectivity. The ability
to selectively switch on such pathways upon demand has proven to be a valuable tool in
biological systems. Light as a trigger provides the ultimate spatial and temporal control
of activation. However, there have been limited examples of photo triggered catalytic
systems. Herein, we describe the synthesis and application of a caged proline system
that can initiate organocatalysis upon irradiation. The caged proline was generated
using the highly efficient 4-carboxy-5,7-dinitroindolinyl (CDNI) photoprotecting group in
a 4-step synthesis. Advantages of this system include water solubility, biocompatibility,
high quantum yield for catalyst release, and responsiveness to two-photon excitation.
We showed the light triggered catalysis of a crossed aldol, a Mannich, and a self-aldol
condensation reaction. We also demonstrated light initiated catalysis leading to the
formation of a biocide in situ, which resulted in the growth inhibition of E. coli, with as
little as 3 minutes of irradiation. This technique can be broadly applied to other systems
by which the formation of active forms of drugs can be catalytically assembled remotely
via two-photon irradiation.
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
ACS Paragon Plus Environment

Page 3 of 32
The Journal of Organic Chemistry
1
2
3
4
INTRODUCTION
5
6
7
8
Catalysts that can be activated upon demand are useful tools for multiple applications,
especially in recent years alongside the growth of the field of bioorthogonal chemistry.1,2
There are multiple types of triggers that include: physical, chemical, biological, as well
as mechanical forces.3–6 However, triggering pathways using light has shown numerous
advantages primarily due to the ability to precisely control the location and time of
activation, generally referred to as spatio-temporal control.7 Organocatalysts have
emerged as biomimetic catalysts displaying advantages of being both greener as well
as biocompatible8–15 The simple amino acid, proline, has been shown by several groups
to be among the simplest and most efficient organocatalyst.16–30
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
The ability to externally control cellular systems is of extreme value. It can provide
ample opportunities for not only learning complex biological pathways but also enabling
their manipulation. Optical methods have dramatically enhanced biological research to
enable precise spatial and temporal control of specific processes.31–33 From the ability to
release small molecules upon demand across synaptic junctions to the field of
optogenetics,34–36 light triggered processes continue to emerge as crucial techniques to
study biological pathways37–39 and localize treatment of diseases.40,41 Molecules that are
photo-released upon demand are generally referred to as caged molecules or caged
agonists.42,43 Another term for the cage is PPG (Photo Protective Group).44–47
Herein, we report an efficient light initiated catalytic system that can catalyze reactions
upon irradiation to generate in situ products. We synthesized a protected form of
L-proline (proline or Pro) that we refer to as caged proline, and we report the kinetics of
its release using light (Figure 1). Proline has two functional groups: a secondary amine
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 4 of 32
1
2
3
4
5
6
7
8
9
and a carboxylic acid group. We have shown that both functional groups play a critical
role in catalysis, and here we report the caging of the carboxy end of proline using a 4-
step synthesis of 4-carboxymethoxy-5,7-dinitroindolinyl proline, 1 (CDNI-Pro). Among
photocleavable cages, CDNI has a notably high quantum yield, ϕ ≈ 0.5, and several
labs, including ours, have also shown its ability to respond to two-photon excitation at
720 nm.46,48 The two photon uncaging cross section for CDNI caged molecules is 0.06
GM/nm at 720 nm wavelength.37 Using CDNI-Pro, we demonstrated light initiated
catalysis of a cross aldol reaction, a Mannich reaction, and a self-aldol condensation
reaction that generates an antibacterial agent. Furthermore, we showed a biological
application by which light initiated catalysis leads to the formation of a biocide that
effectively inhibited further growth in Escherichia coli.
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
Photoinitiated Organocatalyst
Inactive catalyst
Active catalyst
O
OH
O
OH
O
O
4
O2N
O
5
350 nm
OH
NH
O2N
N
photo
uncaging
7
O
N
O2
N
H
NH
ON
Caged Proline
L-Proline
Pro
spent cage
1
CDNI-Pro
Figure 1. Photoinitiated release of catalytically active proline from CDNI-Pro under UV light.
ACS Paragon Plus Environment

Page 5 of 32
The Journal of Organic Chemistry
1
2
3
4
RESULTS AND DISCUSSION
5
6
7
8
Catalytic Importance of Proline’s Functional Groups
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
Since proline has two functional groups: the carboxylic acid and the secondary amine,
we hypothesized that protecting either of its functional groups would inhibit its catalytic
activity.
However, there are published mechanisms that demonstrate different
mechanistic pathways in proline catalysis,28,49 and these pathways vary in the role of
these two functional groups. Hence, we initially verified that adding the protection to
either group would eliminate catalytic activity in the asymmetric aldol reaction. Using
commercially available N-Boc-Pro and the methyl-L-prolinate (synthesized according to
standard procedures),50 we demonstrated that both functional units are critical toward
proline’s catalytic potential in the asymmetric aldol reaction.51,52 Other experimental as
well as computational mechanistic studies also showed the role of the two functional
groups and the ratio of the Mannich route to the aldol condensation vs. the aldol type
product.53
The classic cross aldol reaction between acetone (2) and 4-nitrobenzaldehyde (3) was
conducted in the presence of three versions of proline, displayed as three entries in
Table S1: 1. Pro, 2. C-protected-Pro, and 3. N-protected-Pro. Within the span of 4 h of
reaction time, we observed the formation of aldol product 4 only in the presence of
native proline, but not in the presence of the other two protected versions. This
highlights the need for both functional groups to be present to catalyze this reaction.
The lack of reactivity may, in part, be due to the bulk of the carboxy protection since
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 6 of 32
1
2
3
4
5
6
7
8
9
pyrrolidine (i.e. proline lacking the carboxy unit) has been shown to be an active catalyst
in other Schiff base promoted reactions such as the Stork enamine synthesis.53,54 Some
reactivity of the pyrrolidine unit has also been observed after 24 h reaction time.53 Table
S1 summarizes the control experiments conducted to highlight the critical role of both
the secondary amine as well as the carboxylic acid unit in these types of aldol reactions.
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
Synthesis of CDNI-Pro
Since protection of either functional group eliminates proline’s catalytic potential, we
sought to protect the carboxylic end of proline with a photoprotecting group (PPG) to
generate CDNI-Pro (1). Although we have shown that CDNI could also be used to
protect the amine functionality, our quantum yields of uncaging were far superior when
CDNI was attached to the carboxy moiety.48 A recent report discussed the protection of
the secondary amine with NVOC,55 however, here we resorted to the much higher
quantum yield PPG: CDNI, which can also respond to two-photon excitation.
Proline was coupled to the CDNI cage to form the amide bond in CDNI-Pro. The
protected carboxylic acid functionality rendered this caged proline catalytically inactive.
The CDNI cage is photocleavable under UV radiation of 350 nm wavelength,46,56,57 or
with two-photon fast pulse laser at 720 nm.46,48,58,59 Thus irradiation leads to the
uncaging of CDNI-Pro and the release of the carboxylic acid group, leading to the active
catalyst, proline (Figure 1). This process provided a method to trigger the activation of
proline, using an external source, where the precision of its spatial and temporal control
adds extreme value in its broader applications. This concept could be expanded to
ACS Paragon Plus Environment

Page 7 of 32
The Journal of Organic Chemistry
1
2
3
4
assemble or cross-link drugs in situ via photoactivation. Furthermore, other catalysts
5
6
can be conjugated to CDNI to enable their external activation for specific pathways.
7
8
9
Our synthesis of CDNI-Pro (scheme 1) relied on an improved synthetic procedure for
CDNI-Glu that we recently reported.48 Starting with 4-hydroxyindole, tert-butyl 2-
bromoacetate was used to form 6 in 80% yield, under mild basic conditions of K2CO3. A
subsequent reduction of the pyrrole ring of the indole with NaBH3CN broke down its
aromaticity. This frees the lone pair of electrons on the pyrrolidine moiety making this
secondary amine more reactive. The Boc protected proline, 8, was linked to compound
7 via EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) based coupling reaction to
form 9. The tert-butyl and Boc protection were removed with TFA (trifluoroacetic acid)
and followed by nitration using NO2BF4.48,60–62 The final product CDNI-Pro, 1, was
purified by HPLC using a reverse phase C18 column. The nitration step and all
subsequent steps were conducted in the dark under dim red light. The four steps to
prepare CDNI-Pro are relatively safe and the only necessary precaution is to avoid
moisture in the final stages, as it could lead to the formation of caustic nitric acid.
Furthermore, TFA must be handled with care and ample ventilation due its volatility and
toxic nature.
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
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 8 of 32
1
2
3
4
Scheme 1. Synthetic pathway for CDNI-Proa
5
6
7
8
9
O
OtBu
O
O
OtBu
O
O
OH
OH
N
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
Boc
8
a
b
c
N
N
N
H
H
H
5
6
7
OtBu
O
OH
O
O
O
O2N
d
N
N
O
O
NH
N
Boc
1
9
CDNI-Pro
aExperimental conditions: a) K2CO3, dry (CH3)2CO, rt, 18 h, 80%; b) NaBH3CN, dry
CH3COOH, rt, 2 h, 97%; c) N-(tert-butoxycarbonyl)-L-proline (8), EDC, DMAP, dry
CH2Cl2, rt, 18 h, 62%; d) (i) TFA, rt, 1 h, quantitative; (ii) TFA, NO2BF4, 0 °C, 1 h, 24%.
Photoinitiated Catalysis
The purified photo responsive catalyst, CDNI-Pro, was introduced to three different
reactions: aldol addition, Mannich reaction, and a self-aldol condensation reaction. All
three reactions were conducted in pairs: 1) a control reaction in the dark, as well as, 2)
the identical condition followed by irradiation (Figure 2). The reactions were irradiated
with UV light at a wavelength of 350 nm to uncage CDNI-Pro. A photochemical reactor
with an intensity of 60.97 Wm-2 was used as the light source. In the cross aldol reaction,
ACS Paragon Plus Environment

Page 9 of 32
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
acetone (2) was reacted with 4-nitrobenzaldehyde (3) in the presence of CDNI-Pro at rt
for 24 h.51 The control reaction that lacked light as a trigger did not yield any product,
while the irradiated reaction gave the aldol addition product, 4-hydroxy-4-(4-
nitrophenyl)butan-2-one, 4, in 65% yield. Similarly, a Mannich reaction27 was performed
with acetone (2), 4-nitrobenzaldehyde (3) and 4-methoxyaniline (10). The irradiated
reaction produced the expected product, 11, in 45% yield, while the analogous one in
the dark did not yield any product in the span of 4 h. The self-aldol condensation
reaction was conducted using butyraldehyde (12) as the only substrate in the presence
of the caged catalyst. Irradiation resulted in the catalysis of the self-aldol condensation
reaction of butyraldehyde affording 13 in 47% yield, while the control setup did not lead
to any product.57
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
It is interesting to note that the cross aldol and the self-aldol reactions formed different
types of products, namely, the aldol adduct and the enal product of the aldol
condensation, respectively. It is likely that the condensation product is driven by the
formation of a Mannich type reaction, in which the enamine adds to the iminium,
resulting from the condensation of proline with butyraldehyde, leading to a protonated β-
ammonium intermediate, which eliminates far easier than the corresponding β-hydroxy
in the cross aldol reaction.
In the latter reaction, the 4-nitrobenzaldehyde is
substantially more electrophilic than butyraldehyde, and thus may react with the proline-
generated enamine without the need of forming the electrophilic iminium species formed
in the self-aldol of butyraldehyde. The distribution of the two types of products resulting
from proline catalysis has been elegantly described by Gschwind,49 Bargeman,53 and
co-workers.
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 10 of 32
1
2
3
4
5
6
Caged CDNI-Pro
Uncaged CDNI-Pro
Reaction
UV 350 nm
(5 min)
In dark/ 24 h
7
8
cross aldol reaction
OH
O
O
9
O
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
20% CDNI-Pro
+
H
0%
O2N
DMSO, 25 °C
O2N
4
2
3
+
65%
Mannich reaction
OCH3
NH2
O
O
O
HN
20% CDNI-Pro
H
+
DMSO, 25 °C
0%
O2N
NO2
OCH3
11
45%
2
3
10
O
self-aldol condensation
H
O
20% CDNI-Pro
0%
H
DMSO, 25 °C
12
13
47%
Figure 2. Reactions catalyzed by CDNI-Pro in the dark and upon irradiation: Cross aldol
reaction between (CH3)2CO (2) and 4-nitrobenzaldehyde (3); Mannich reaction between
(CH3)2CO (2), 4-nitrobenzaldehyde (3) and 4-methoxyaniline (10); self-aldol
condensation reaction of butyraldehyde (12).
Spectroscopic Studies
When CDNI-Pro is irradiated with UV light, the ortho nitro aromatic system is predicted
to get excited to a singlet state which can intersystem cross to a triplet state leading to
the cleavage of the amide bond thus releasing proline.44,63,64 A rapid method to follow
ACS Paragon Plus Environment

Page 11 of 32
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
the photochemical progress of catalyst release can generally be accomplished via UV
measurements. The shift in UV absorbance during photolysis was studied and is
displayed in Figure S4, and described in SI. CDNI-Pro has a λmax of 330 nm and a
calculated molar attenuation coefficient of 6400 Lmol-1cm-1.
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
Although UV is generally the method of choice for following reactions involving
conjugated systems, the lack of a clear isosbestic point precludes precise kinetic
calculations. The starting CDNI cage carries two nitro groups at positions C-5 and C-7,
and upon irradiation, the nitro group at C-7 is converted to the less electron withdrawing
nitroso group (Figure 1). Therefore, since the starting cage and the byproduct of
uncaging both harbor the additional nitro group at C-5, the UV properties of the starting
cage and the spent cage product are not sufficiently distinct to result in a tangible shift in
absorption of reactant vs. product. We have previously observed this phenomenon with
CDNI-Glu but not with the mononitrated cage MNI-Glu which bears only one nitro group
at C-7 which gets converted to the nitroso in the spent cage.56
Kinetics of Catalyst Release by NMR
Since UV-vis spectra were not sufficiently distinct to enable kinetic assessment of
1
catalyst release, we monitored the release of proline via H NMR spectroscopy (Figure
3). This enabled a more accurate determination of the concentration of species
throughout photolysis. A specific amount of CDNI-Pro and maleic acid were dissolved
in 0.5 mL of D2O and transferred in to an NMR tube. The maleic acid was used as an
internal integration reference, and all the peak areas were calculated relative to the area
of the maleic acid peak at 6.39 ppm. This experiment was conducted in the dark with
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 12 of 32
1
2
3
4
5
6
the assistance of dim red light. The NMR tube was irradiated in the photochemical
reactor at a wavelength of 350 nm, with an intensity of 60.97 Wm-2, for 30 s intervals.
7
8
9
1
After each irradiation, H NMR spectra were acquired. One of the main advantages of
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
using NMR spectroscopy is the fact that the aromatic proton of the nitroindolinyl system
is a singlet and therefore simple to quantify with reliable accuracy. During the
photoinitiated release of proline, the aromatic hydrogen of CDNI’s benzene ring, shifts
upfield from 8.59 ppm (a) to 7.94 ppm (b). We monitored this peak shift and its peak
area diminution with respect to the maleic acid peak (internal standard). As a result of
photolysis, the indolinyl ring of CDNI gets oxidized to indole, thus forming a double bond
in the five membered ring which leads to the formation of two new aromatic peaks, c at
7.20 ppm and d at 6.74 ppm, that gradually became visible with increased photolysis
time across sequential 30s intervals of irradiation. The rate of the photocleavage
reaction for CDNI-Pro was thus determined using this experiment.
ACS Paragon Plus Environment

Page 13 of 32
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
1
Figure 3. H NMR (400 MHz, D2O) of selected spectra for progressive photolysis of
CDNI-Pro, concentration of 0.94 mM, with a wavelength of 350 nm at 60 s intervals, rt.
To obtain the photolysis rate of CDNI-Pro, the decreasing area of aromatic hydrogen,
Ha, was used to calculate the concentration of the starting cage. The mean of the
results of duplicate photolysis experiments were used to calculate the rate constant.
The photolysis of CDNI-Pro follows a first order rate law with a rate constant of 7.23 ×
10-3 s-1. The quantum yield, (ϕ), of CDNI-Pro was calculated to be 0.52, similar to
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 14 of 32
1
2
3
4
previously published CDNI based cages (see SI for details).37,44 The photo cross-
5
6
section, (εϕ), of CDNI-Pro was calculated to be 3328 Lmol-1cm-1.
7
8
9
Biological Application: In Situ Catalysis of the Formation of an Antibacterial
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
The photo activatable catalyst, CDNI-Pro, was also shown to be a valuable tool as a
bioorthogonal system under physiological conditions. The self-aldol condensation
reaction of 12, photoinitiated with CDNI-Pro, was used for the in situ generation of a
toxic product, (E)-2-ethylhex-2-enal (13), and then its effects on bacteria growth were
measured.65–67
(a)
3 min
+
CDNI-Pro
Butyraldehyde
(b)
(c)
(d)
Irradiator
350 nm
E coli
LB Media
In Dark
In Dark
2 h
Incubation at 37 °
C for 24 h
(e)
(g)
(f)
Colony count
In Dark
In Dark
In Dark
Figure 4. Graphical illustration of biological assay.
In this assay (summarized in Figure 4), E. coli bacteria (DH5α) were transferred to LB
media (Lysogenic broth) containing CDNI-Pro and butyraldehyde under dark conditions.
Then, the mixture was exposed to UV irradiation for 3 min and allowed to stir for another
2 h. Finally, the bacteria were plated onto LB-agar, incubated for 24 h, and the bacterial
colonies were counted. Five control reactions were done to verify the significance of
ACS Paragon Plus Environment

Page 15 of 32
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
each parameter, Table 1. The results showed that there were large amounts of E. coli
growing on all the plates, except for entry 2. Since nitrosoarenes are known to induce
cellular toxicities, we ran a control experiment to photorelease proline and the spent
nitrosoarene cage, and we observed that these cleaved products did not affect bacterial
growth (entry 3, Table 1). Hence, the coexistence of all three components: CDNI-Pro,
butyraldehyde, and UV irradiation is critical to the formation of 13, which inhibits
bacterial growth.
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
Table 1. Bioassay Studies: including all the control experiments revealing the need for
all three components of caged proline, substrate, and light to catalyze the formation of
13, which leads to the inhibition of bacterial growtha
entry
CDNI-Pro
butyraldehyde Irradiation
E. coli
growth
(2.63 × 10-2 M)
(2.63 × 10-2 M)
(350 nm)
1
2
3
4
5
Yes
Yes
Yes
Yes
No
aExperimental conditions: CDNI-Pro (1 eq), butyraldehyde (1 eq) was dissolved in LB
media with E. coli at pH 7 in RT. Each parameter was either X = missing or = present
in the reaction media. Entries 2, 3 and 5 were irradiated for 3 min in the photochemical
reactor at 350 nm with an intensity of 60.97 Wm-2.
As summarized in Table 1, CDNI-Pro was innocuous to E. coli, highlighting its
compatibility as a bioorthogonal tool. Proline was released only after irradiation, which
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 16 of 32
1
2
3
4
5
6
7
8
9
resulted in the catalysis of the self-aldol condensation of butyraldehyde (12) forming the
antibacterial 13. We further optimized the assay conditions by performing a series of
experiments with variations in the concentration of CDNI-Pro.
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
The assay was conducted according to the diagram shown in Figure 4. The steps
included: (a) the average colony count of E. coli was measured as 8.70×104 in 10 mL of
LB media, (b) the butyraldehyde, CDNI-Pro and E. coli were transferred in to the
sterilized Eppendorf tubes in dark, as per the ratios shown in Table 2. Fifteen sterilized
Eppendorf tubes were prepared in triplicate trials as outlined by entries 1-5 (Table 2).
Table 2. E. coli. growth relative to CDNI-Pro concentrationa
E coli
12
13
entry
CDNI-Pro
Butyraldehyde
LB
E. coli
(µL)
50
(µL)
0
40
(µL)
5
5
(µL)
45
5
1
2
50
3
4
5
30
20
10
5
5
5
15
25
35
50
50
50
aExperimental conditions: Samples were prepared with LB media, [CDNI-Pro] = 0.02 M
stock solution; [butyraldehyde] = 0.14 M stock solution; E. coli = 8.70×103 CFUmL-1.
Samples were prepared in sterile conditions in the dark, at rt.
ACS Paragon Plus Environment

Page 17 of 32
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
Subsequent steps included (Figure 4): (c) 350 nm UV irradiation of each trial for 3 min;
the reactor was cooled down to rt between each irradiation, (d) trials were incubated in
the dark for 2 h with gentle shaking. For the final dilution, 5 µL from each irradiated tube
were transferred to a new tube with 1 mL of LB media, (e) then, 300 µL from each tube
was transferred to the sterilized LB plates and plated with sterile glass beads, (f)
samples were incubated at 37 °C for 24 h, and colonies were counted for each plate
and averaged.
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
We observed a drastic reduction in E. coli growth when the CDNI-Pro concentration was
25% of butyraldehyde. The colony count was reduced from 3.54×109 CFU/mL (colony
forming unit per mL) to 3.45×107 CFU/mL. With increased CDNI-Pro concentration, E
coli growth was further inhibited. Ultimately, there were undetectable levels of E. coli
when the CDNI-Pro concentration was at stoichiometric amounts relative to the
substrate, Figure 5.
As a control, an experiment was performed with the same conditions as per entry 5 of
Table 2, but without E. coli. After following each step, the product was extracted using
dichloromethane from the LB media and confirmed by 1H NMR spectroscopy and DART
(Direct Analysis in Real Time) mass spectrometry to be aldol product, (E)-2-ethylhex-2-
enal (13), identical to the one obtained from the reaction presented in Figure 2.
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 18 of 32
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. Plot of E. coli colony count (CFU/mL) with respect to CDNI-Pro percentage.
CONCLUSION
In summary, we have demonstrated the photoinitiation of proline catalysis using light.
This spatiotemporal control of organocatalysis enhances its applicability in noninvasive
treatments, where CDNI-Pro and similarly inspired structures could be used as
bioorthogonal tools. We used a bioassay to show how light can be applied as an
external trigger to release an active catalyst that assembled a biocide in cells within 3
ACS Paragon Plus Environment

Page 19 of 32
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
minutes. This methodology could broaden the field of in situ drug synthesis for varying
applications including but not limited to cancer therapeutics.68–72 Furthermore, CDNI-Pro
could be used for applications that involve other roles for proline beyond aldol
catalysis.73,74 Future work aims to expand the irradiation wavelengths to enable catalyst
activation with visible to near IR regions for a more functionally versatile caged catalyst.
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 Experimental Methods
Unless otherwise mentioned all reagents were purchased in highly pure form from
Fisher or Sigma-Aldrich. The solvents used in the experiments were purchased from
VWR or Fisher. When required anhydrous methylene chloride (CH2Cl2) was obtained
from a solvent purification system, which uses an activated alumina column ((MBRAUN,
MB-SPS). Reaction vessels were flame dried (alternative vacuum and N2 flow three
times) using a Schlenk line. The reactions were done under Ar or N2 conditions. The
progress of reactions was monitored by TLC, carried out on EMD Millipore TLC Silica
Gel 60 F254 plates. Short/long wavelength UV light or stains (p-anisaldehyde), followed
by heating, were used to detect the progress of appropriate spots in TLC. Column
chromatography (using 60 Å silica gel from Fisher) was conducted to purify the products
from steps a to c (scheme 1). The product from step d, compound (1), was purified
using HPLC, Waters 2695 separation module with Waters 996 photodiode array
detector with an XSELECT C18 OBD prep column (10 mm × 25 mm). The purified
product was lyophilized via LABCONCO Freezone 4.5. From step d onwards, all
characterizations and experiments were conducted in the dark with the aid of a low
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 20 of 32
1
2
3
4
5
6
7
8
9
intensity red lamp. The purified products were stored at -20 °C temperature and
compound (1) was covered with an aluminum foil to avoid exposure to light. The
products were characterized by NMR spectroscopy (Bruker 400 MHz NMR), UV/Vis
spectroscopy (Agilent 8453 diode array UV−vis), high-resolution mass spectroscopy
(JEOL DART-AccuTOF mass spectrometer) and low-resolution LCMS (Agilent LC-MS
with ES+ and 6120 quadrupole). The HRMS for compounds 1, 11 and 13 was
conducted at University of Florida-Mass Spectrometry Research and Education Center
using Agilent 6220 Time-of-Flight spectrometer (funding from NIH S10 OD021758-
01A1). Solvents for NMR experiments were purchased from Cambridge Isotopes and
Acros (Fisher). Chemical shifts were calculated relative to the solvent residual peaks
(CDCl3 = 7.26 ppm, D2O = 4.79 ppm). The following abbreviations were used to
describe NMR peak multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m =
multiplet. For uncaging of CDNI-Pro, Rayonet photochemical reactor (RPR 100), with 16
UV lamps (wavelength of 350 nm), was used. The photo intensity of the reactor was
calculated as 60.97 Wm-2. The instrument was equipped with a fan to regulate the
temperature inside. After each irradiation (3-5 min), the instrument was cooled down to
room temperature. For the biological experiment E coli DH5α strain was used.
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
tert-butyl (S)-2-(4-(2-(tert-butoxy)-2-oxoethoxy)indoline-1-carbonyl)pyrrolidine-1-
carboxylate (9). Up to compound 7, the syntheses for steps a and b were done per our
previously published procedure.58 Then a 50 mL round bottomed flask was charged with
compound 7 (250.00 mg, 1.07 mmol), EDC (155.26 mg, 1.00 mmol) , DMAP (61.13 mg,
0.50 mmol), and 10 mL of dry CH2Cl2. The reaction mixture was allowed to stir for 15
min until complete solubility of all components. Subsequently, N-(tert-Butoxycarbonyl)-L-
ACS Paragon Plus Environment

Page 21 of 32
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
proline (N-Boc-proline ,8) (215.30 mg, 1.00 mmol) dissolved in 5 mL of dry CH2Cl2, was
slowly added to the reaction mixture. The reaction was allowed to stir for 18 h at rt
under Ar. After completion, the organic layer was extracted with CH2Cl2, neutralized with
NaHCO3, washed with brine, and dried over anhydrous Na2SO4. The crude product was
purified by column chromatography (2:1 hexane/ ethyl acetate) to isolate (1-tert-
butoxypyrrolidin-2-yl)-tert-butyl 2-(indolin-4-yloxy) acetate, compound 9, in 62% yield as
a brown oil, (230.00 mg, 0.66 mmol). 1H NMR (400 MHz, CDCl3) δ 7.90 - 7.86 (m, 1H),
7.16 - 7.08 (m, 1H), 6.43 (t, J = 8.8 Hz, 1H), 4.60 – 4.58 (m, 1H), 4.54 (s, 1H), 4.52 (s,
1H), 4.24–4.17 (m, 1H), 4.10-4.04 (m, 1H), 3.70-3.60 (m, 1H), 3.54-3.43 (m, 1H), 3.22 (t,
J = 8.4, 2H), 2.29–2.07 (m, 2H), 2.05-1.83 (m, 2H), 1.47 (s, 9H), 1.45 -1.33 (m, 9H).
13C{1H} NMR (101 MHz, CDCl3) δ 172.0 – 170.4 (m), 167.9, 155.1 – 153.0 (m), 144.7,
128.9, 118.9, 111.0, 106.9, 82.3, 79.7, 65.7, 60.4, 58.7, 48.1, 46.8, 30.4, 29.5, 28.9,
25.3, 24.0, 21.0, 14.1. HRMS (DART-AccuTOF) m/z [M+H]+ cacld for (without Boc
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
+
protection) C19H27N2O4 347.1966, found 347.1994.
(S)-2-((5,7-dinitro-1-prolylindolin-4-yl)oxy)acetic acid (1). A 25 mL round bottomed flask
was charged with compound 9 (125.0 mg, 0.20 mmol) and TFA (2 mL) and allowed to
stir for 1 h at rt. A TLC was taken (3:2 hexane/ethyl acetate) to determine the
completion of the reaction. After complete deprotection, the TLC spot for the product will
remain on the baseline. Subsequently, a nitrogen stream was applied to remove the
TFA. To the same flask, 2 mL of fresh TFA was added and cooled to 0 °C. Then,
nitronium tetrafluoroborate (NO2BF4) (106.0 mg, 0.80 mmol) was added under a N2 flow
and stirred in dark for 1.5 h. Finally, TFA was removed via a nitrogen stream followed
by vacuum. The product was then purified using reverse phase HPLC via isocratic
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 22 of 32
1
2
3
4
5
6
7
8
9
elution at 2 mL min-1 using a solvent system of 60% water and 40 % acetonitrile
acidified with 0.1% TFA, tR = 9 min, (Figure S1 in SI). Finally, product was lyophilized to
dryness, generating compound 1 in 24% yield as a light-yellow powder (18.25 mg, 0.05
mmol). 1H NMR (400 MHz, D2O) δ 8.58 (s, 1H), 4.96 (td, J = 6.8Hz, 2.4Hz, 1H), 4.77 (s,
2H), 4.52 (td, J = 10.4 Hz, 4Hz, 1H), 4.35 (q, J = 10Hz, 1H), 3.52-3.48 (m, 1H), 3.46 (t, J
= 5.2 Hz, 1H), 3.44-3.38 (m, 1H), 2.64 - 2.56(m, 1H), 2.27 – 2.20 (m, 2H), 2.18- 2.08 (m,
2H). 13C{1H} NMR (101 MHz, D2O) δ 172.0, 168.0, 151.2, 139.2, 138.4, 135.0, 132.3,
123.0, 69.3, 60.0, 50.7, 46.2, 28.1, 26.4, 23.7. LC−MS (ESI) 381.1 [M + H]+; HRMS
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
+
(ESI-TOF) m/z [M+H]+ calcd: C15H17N4O8 381.1041, found 381.1054 (MS spectrum can
be found in SI).
Photoinitiated organocatalytic reactions
Cross aldol reaction to form 4-hydroxy-4-(4-nitrophenyl)butan-2-one (4). The catalyst 1
(7.6 mg, 0.02 mmol) was stirred in 5 mL of DMSO:acetone (4:1) with 4-
nitrobenzaldehyde (15.12 mg, 0.1 mmol) in a 25 mL round-bottomed flask, in the dark at
rt. After 24 h, no reaction was observed. Then, the reaction mixture was irradiated in the
Rayonet photochemical reactor at 350 nm for a total irradiation time of 5 min.
Afterwards, the mixture was stirred for 4 h at rt. The organic layer was extracted with
ethyl acetate and dried over anhydrous Na2SO4, filtered, and concentrated under
vacuum. The crude product was purified through column chromatography (1:1
hexane/ethyl acetate) to give compound 4 in 65% yield, as a brown oil (13.60 mg, 0.065
mmol). 1H NMR (400 MHz, CDCl3) δ 8.22 (d, J = 7.9 Hz, 2H), 7.54 (d, J = 8.1 Hz, 2H),
5.34 – 5.20 (m, 1H), 3.56 (s, 1H), 2.85 (d, J = 7.5 Hz, 2H), 2.20 (d, J = 21.5 Hz, 3H).
ACS Paragon Plus Environment

Page 23 of 32
The Journal of Organic Chemistry
1
2
3
4
13C{1H} NMR (101 MHz, CDCl3) δ 208.3, 150.4, 147.4, 126.6, 123.5, 68.7, 51.8, 30.6.
5
6
+
HRMS (DART-AccuTOF) m/z [M+H]+ calcd for C10H12NO4 210.0761, found 210.0778.
7
8
9
Mannich reaction to form 4-((4-methoxyphenyl)amino)-4-(4-nitrophenyl)butan-2-one (11).
The catalyst 1 (76 mg, 0.2 mmol), was stirred in 8 mL of DMSO, p-anisidine (135 mg,
1.1 mmol), 4-nitrobenzaldehyde (151.12 mg, 1.0 mmol) and 2 mL of acetone in a 25 mL
round-bottomed flask in the dark at rt. After 24 h, no product could be observed.
Subsequently, the reaction mixture was irradiated in the Rayonet photochemical reactor
at 350 nm for a total irradiation time of 5 min and stirred at rt for 24 h. The organic layer
was extracted with ethyl acetate, dried over anhydrous Na2SO4, filtered, and then
concentrated under vacuum. The crude product was purified through column
chromatography (1:1 hexane/ethyl acetate) to give compound 11 in 40% yield, as dark
brown oil (141.45 mg, 0.45 mmol). 1H NMR (400 MHz, CDCl3) δ 8.17 (d, J = 8.6 Hz, 2H),
7.55 (d, J = 8.6 Hz, 2H), 6.69 (d, J = 8.9 Hz, 2H), 6.46 (d, J = 8.9 Hz, 2H), 4.85 (t, J =
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13
7.5 Hz, 1H), 4.28 (s, 1H), 3.69 (s, 3H), 2.95 (d, J = 6.6 Hz, 2H), 2.15 (s, 3H). C{1H}
NMR (101 MHz, CDCl3) δ 206.0, 153.0, 150.7, 140.2, 140.0, 129.1, 127.4, 124.2, 122.7,
116.44, 115.4, 114.8, 114.6, 55.9, 54.6, 50.8, 30.7. HRMS (ESI-TOF) m/z [M+H]+ calcd
+
for C17H19N2O4 315.1339, found 315.1346.
Self-aldol condensation to form (E)-2-ethylhex-2-enal (13). CDNI-Pro (1) (840 mg, 2.2
mmol) and butyraldehyde (1 mL, 11.10 mmol) were mixed in 5 mL of methanol in a 25
mL round-bottomed flask at rt for 24 h. No reaction product could be observed under
dark conditions. Then mixture was irradiated in the Rayonet photochemical reactor at
350 nm for a total irradiation time of 5 min. Subsequently, the reaction mixture was
stirred at rt for 4 h. The solvent was subsequently removed under vacuum. The crude
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 24 of 32
1
2
3
4
5
6
7
8
9
product was purified through column chromatography (4:1 hexane/ethyl acetate) to give
compound 13 in 47% yield, as a brown oil (658.76 mg, 5.22 mmol). 1H NMR (400 MHz,
CDCl3) δ 9.3 (s, J,1H), 6.34 (t, J = 7.4 Hz, 1H), 2.25 (q, J = 7.4 Hz, 2H), 2.19 (q, J = 7.5
Hz, 2H), 1.50 -1.45 (m, 2H), 0.90 - 0.85 (m, 1H). 13C{1H} NMR (101 MHz, CDCl3) δ
195.0, 154.5, 145.1, 30.7, 22.1, 17.2, 13.7, 13.2. HRMS (ESI-TOF) m/z [M+H]+ calcd for
C8H15O+ 127.1117, found 127.1121.
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
Generation of in-situ biocide (E)-2-ethylhex-2-enal to inhibit E. coli growth via
photo initiated self-aldol condensation
Preparation of minimal media with E. coli
Initially, 1 L of LB (Lysogeny broth) agar media was sterilized and poured to 40 plates,
which were stored at 0 °C. Two tubes of sterilized LB agar liquid media (30 mL for each)
were prepared. One of the two tubes were referred as the “Blank” and a minimum
amount of -150 °C stored DH5α E. coli was transferred to the other tube. The two tubes
were incubated at 30 °C with gentle shaking for 24 h. Then, 30 µL of liquid from the tube
with E. coil was transferred to the “Blank” and the new solution was incubated at 30 °C
with gentle shaking for 2 hours after which, OD (optical density) value was measured,
and dilutions were performed based on the colonies number. The average colony count
was calculated as 4.35×104.
Measuring E. coli growth with varying CDNI-Pro concentration
Five trials were conducted varying the CDNI-Pro concentration. Fifteen sterilized
Eppendorf tubes were prepared and grouped as triplicate trials. The solution for each
entry was prepared according to the details in Table S3 in SI. Stock solutions of 0.79 M
ACS Paragon Plus Environment

Page 25 of 32
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
CDNI-Pro (2.00 mg in 300 µL LB solution), 0.082 M butyraldehyde (12.96 µL in 1 mL LB
solution) and E. coli minimal media (with 4.35×104 colony count), were used to prepare
each trial sample. Samples were prepared in dark with an aid of a red dim light. All the
sample tubes were treated with UV light of 350 nm in the photoreactor for 3 min. The
photo reactor was cooled to rt before each irradiation. Then, all the tubes were
incubated in the dark for 2 h with gentle shaking. New sets of 15 sterilized Eppendorf
tubes were prepared with 1 mL of sterilized LB liquid media. Then, 5 µL from each trial
tube was transferred to the new tube for dilution. Finally, 300 µL from each reaction
mixture was transferred to a prepared LB plate, and 15 plates were incubated at 37 °C
for 24 h. Colonies were counted for each plate, and CFU values were calculated.
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
ASSOCIATED CONTENT
The supporting Information is available free of charge on the ACS Publication website
at: http://pubs.acs.org.
NMR spectra of CDNI-Pro (1) and all intermediates leading up to it; Kinetic data; LCMS
data, and Direct Analysis in Real Time (DART)-MS data, ESI HRMS for 1.
AUTHOR INFORMATION
Corresponding Author
*E-mail: nesnas@fit.edu
ORCID
Nasri Nesnas: 0000-0003-3511-940X
Eric Guisbert: 0000-0003-1901-5598
Charitha Guruge: 0000-0003-4405-2905
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 26 of 32
1
2
3
4
Author Contributions
5
6
7
8
C.G. and N.N. conceived this work, and C.G. developed and performed the synthesis of
all compounds reported; S.Y.R. and C.B. participated in the synthetic protocols and
purifications. A.K.S. conducted HPLC purifications of finals products. C.G. conducted
all UV, NMR, and kinetic measurements. S.Y., C.B., and E.G. performed the
bioorthogonal study in E. coli. N.N. directed the project, and C.G. and N.N. wrote the
manuscript. All authors contributed to and approved the final manuscript.
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
Funding
The study was support by NIH Award R15-GM112119-01A1.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors wish to thank NIH for their financial support for C.G. and supplies (R15-
GM112119-01A1). N.N. thanks Profs. Roberto Peverati and Rudolf J. Wehmschulte
(FIT) for helpful discussions and Mr. Alec Pabarue for research assistance. The
authors acknowledge NIH S10 OD021758-01A1 and the University of Florida for HRMS
of three compounds: 1, 11, and 13.
ACS Paragon Plus Environment

Page 27 of 32
The Journal of Organic Chemistry
1
2
3
4
REFERENCES
5
6
7
8
9
(1) Sletten, E. M.; Bertozzi, C. R. Bioorthogonal Chemistry: Fishing for Selectivity in a
Sea of Functionality. Angew. Chem., Int. Ed. 2009, 48, 6974–6998.
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
(2) Wang, F.; Zhang, Y.; Du, Z.; Ren, J.; Qu, X. Designed Heterogeneous Palladium
Catalysts for Reversible Light-Controlled Bioorthogonal Catalysis in Living Cells.
Nat. Commun. 2018, 9, 1–8.
(3) Nagaoka, K.; Eboshi, T.; Takeishi, Y.; Tasaki, R.; Honda, K.; Imamura, K.; Sato, K.
Carbon-Free H2 Production from Ammonia Triggered at Room Temperature with
an Acidic RuO2/g-Al2O3 Catalyst. Sci. Adv. 2017, 3, 1–8.
(4) Pizzolato, S. F.; Stacko, P.; Kistemaker, J. C. M.; van Leeuwen, T.; Otten, E.;
Feringa, B. L. Central-to-Helical-to-Axial-to-Central Transfer of Chirality with a
Photoresponsive Catalyst. J. Am. Chem. Soc. 2018, 140, 17278–17289.
(5) Blanco, V.; Leigh, D. A.; Marcos, V. Artificial Switchable Catalysts. Chem. Soc.
Rev. 2015, 44, 5341–5370.
(6) Hölzl-Hobmeier, A.; Bauer, A.; Silva, A. V.; Huber, S. M.; Bannwarth, C.; Bach, T.
Catalytic Deracemization of Chiral Allenes by Sensitized Excitation with Visible
Light. Nature 2018, 564, 240–243.
(7) Spatiotemporal Mechanisms of Life. Nat. Chem. Biol. 2007, 3, 593–593.
(8) List, B. Introduction: Organocatalysis. Chem. Rev. 2007, 107, 5413–5415.
(9) MacMillan, D. W. C. The Advent and Development of Organocatalysis. Nature
2008, 455, 304–308.
(10) Marchetti, L.; Levine, M. Biomimetic Catalysis. ACS Catal. 2011, 1, 1090–1118.
(11) Evans, C. S.; Davis, L. O. Recent Advances in Organocatalyzed Domino C–C
Bond-Forming Reactions. Molecules 2018, 23, 1–13.
(12) Song, J.; Chen, D. F.; Gong, L. Z. Recent Progress in Organocatalytic
Asymmetric Total Syntheses of Complex Indole Alkaloids. Natl. Sci. Rev. 2017, 4,
381–396.
(13) Bisogno, F. R.; López-Vidal, M. G.; de Gonzalo, G. Organocatalysis and
Biocatalysis Hand in Hand: Combining Catalysts in One-Pot Procedures. Adv.
Synth. Catal. 2017, 359, 2026–2049.
(14) Dalko, P. I.; Moisan, L. In the Golden Age of Organocatalysis. Angew. Chem., Int.
Ed. 2004, 43, 5138–5175.
(15) Hughes, D. L. Asymmetric Organocatalysis in Development - Highlights of the
Recent Patent Literature. Org. Process Res. Dev. 2018, 22, 574–584.
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 28 of 32
1
2
3
4
5
(16) Hajos, Z. G.; Parrish, D. R. Asymmetric Synthesis of Bicyclic Intermediates of
Natural Product Chemistry. J. Org. Chem. 1974, 39, 1615–1621.
6
7
8
(17) Jarvo, E. R.; Miller, S. J. Amino Acids and Peptides as Asymmetric
Organocatalysts. Tetrahedron 2000, 58, 2481–2495.
9
(18) Limbach, M. “Five at One Stroke”: Proline and Small Peptides in the
Stereoselective de Novo Synthesis and Enantiotopic Functionalization of
Carbohydrates. Chem. Biodivers. 2005, 2, 825–836.
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
(19) Tian, J.; Zhong, J.; Li, Y.; Ma, D. Organocatalytic and Scalable Synthesis of the
Anti-Influenza Drugs Zanamivir, Laninamivir, and CS-8958. Angew. Chem., Int.
Ed. 2014, 53, 13885–13888.
(20) Kotkar, S. P.; Sudalai, A. A Short Enantioselective Synthesis of the Antiepileptic
Agent, Levetiracetam Based on Proline-Catalyzed Asymmetric α-Aminooxylation.
Tetrahedron Lett. 2006, 47, 6813–6815.
(21) Tanner, J. J.; Fendt, S. M.; Becker, D. F. The Proline Cycle As a Potential Cancer
Therapy Target. Biochemistry 2018, 57, 3433–3444.
(22) Tafida, U. I.; Uzairu, A.; Abechi, S. E. Mechanism and Rate Constant of Proline-
Catalysed Asymmetric Aldol Reaction of Acetone and p-Nitrobenzaldehyde in
Solution Medium: Density-Functional Theory Computation. J. Adv. Res. 2018, 12,
11–19.
(23) Buchschacher, P.; Fürst, A.; Gutzwiller, J. (S)-8a-Methyl-3,4,8,8a-Tetrahydro-
1,6(2H, 7H)-Naphthalenedione. Org. Synth. 1985, 63, 37.
(24) Ahrendt, K. A.; Borths, C. J.; Macmillan, D. W. C. New Strategies for Organic
Catalysis: The First Highly Enantioselective Organocatalytic Diels-Alder Reaction.
J. Am. Chem. Soc. 2000, 122, 4243–4244.
(25) Notz, W.; Tanaka, F.; Barbas III, C. F. Enamine-Based Organocatalysis with
Proline and Diamines: The Development of Direct Catalytic Asymmetric Aldol,
Mannich, Michael, and Diels-Alder Reactions. Acc. Chem. Res. 2004, 37, 580–
591.
(26) List, B.; Lerner, R. A.; Barbas III, C. F. Enantioselective Aldol Cyclodehydrations
Catalyzed by Antibody 38C2. Org. Lett. 1999, 1, 59–62.
(27) List, B. Proline-Catalyzed Asymmetric Reactions. Tetrahedron 2002, 58, 5573–
5590.
(28) List, B.; Hoang, L.; Martin, H. J. New Mechanistic Studies on the Proline-
Catalyzed Aldol Reaction. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 5839–5842.
(29) Córdova, A. Direct Catalytic Asymmetric Cross-Aldol Reactions in Ionic Liquid
Media. Tetrahedron Lett. 2004, 45, 3949–3952.
ACS Paragon Plus Environment

Page 29 of 32
The Journal of Organic Chemistry
1
2
3
4
5
(30) Northrup, A.B.; MacMillan, D. W. C. Two-Step Synthesis of Carbohydrates by
Selective Aldol Reactions. Science 2004, 305, 1752–1755.
6
7
8
9
(31) Corrie, J. E. T. Photoremovable Protecting Groups Used for the Caging of
Biomolecules. In Dynamic Studies in Biology; Givens, M. G. R., Ed.; WILEY-VCH
Verlag GmbH & Co. KGaA: Weinheim, 2005; p 1.
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
(32) Mayer, G.; Heckel, A. Biologically Active Molecules with a “Light Switch” Angew.
Chem., Int. Ed. 2006, 45, 4900–4921.
(33) Hansen, M. J.; Velema, W. A.; Lerch, M. M.; Feringa, B. L. Wavelength-Selective
Cleavage of Photoprotecting Groups: Strategies and Applications in Dynamic
Systems. Chem. Soc. Rev. 2015, 44, 3358–3377.
(34) Zemelman, B. V.; Nesnas, N.; Lee, G. A.; Miesenböck, G. Photochemical Gating
of Heterologous Ion Channelsꢀ: Remote Control over Genetically Designated
Populations of Neurons. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 1352–1357.
(35) Zemelman, B. V.; Lee, G. A.; Ng, M.; Miesenböck, G. Selective Photostimulation
of Genetically ChARGed Neurons. Neuron 2002, 33, 15–22.
(36) Miesenböck, G. The Optogenetic Catechism. Science 2009, 326, 395–399.
(37) Ellis-Davies, G. C. R. Chemist and Biologist Talk to Each Other about Caged
Neurotransmitters. Beilstein J. Org. Chem. 2013, 9, 64–73.
(38) Kramer, R. H.; Mourot, A.; Adesnik, H. Optogenetic Pharmacology for Control of
Native Neuronal Signaling Proteins. Nat. Neurosci. 2013, 16, 816–823.
(39) Kramer, R. H.; Fortin, D. L.; Trauner, D. New Photochemical Tools for Controlling
Neuronal Activity. Curr. Opin. Neurobiol. 2009, 19, 544–552.
(40) Wegener, M.; Hansen, M. J.; Driessen, A. J. M.; Szymanski, W.; Feringa, B. L.
Photocontrol of Antibacterial Activity: Shifting from UV to Red Light Activation. J.
Am. Chem. Soc. 2017, 139, 17979–17986.
(41) Gorka, A. P.; Nani, R. R.; Schnermann, M. J. Harnessing Cyanine Reactivity for
Optical Imaging and Drug Delivery. Acc. Chem. Res. 2018, 51, 3226–3235.
(42) Papageorgiou, G.; Ogden, D.; Kelly, G.; Corrie, J. E. T. Synthetic and
Photochemical Studies of Substituted 1-Acyl-7-Nitroindolines. Photochem.
Photobiol. Sci. 2005, 4, 887–896.
(43) Papageorgiou, G.; Ogden, D. C.; Barth, A.; Corrie, J. E. T. Photorelease of
Carboxylic Acids from 1-Acyl-7-Nitroindolines in Aqueous Solution: Rapid and
Efficient Photorelease of L-Glutamate. J. Am. Chem. Soc. 1999, 121, 6503–6504.
(44) Klán, P.; Šolomek, T.; Bochet, C. G.; Blanc, A.; Givens, R.; Rubina, M.; Popik, V.;
Kostikov, A.; Wirz, J. Photoremovable Protecting Groups in Chemistry and
Biology: Reaction Mechanisms and Efficacy. Chem. Rev. 2013, 113, 119–191.
ACS Paragon Plus Environment