Development of carbamate-tethered coumarins as phototriggers for caged nicotinamide

Article abstract of DOI:10.1016/j.bmcl.2013.09.067

The syntheses of 7-diethylaminocoumarin- or modified DEACM-nicotinamide and 6-bromo-7-methoxycoumarin- or BMCM-nicotinamide have been accomplished by reaction of nicotinoyl isocyanate with the corresponding coumarin allylic alcohol derivatives. The resulting compounds contain an N-acyl O-alkyl carbamate as a new type of linkage for the caging of nicotinamide with a coumarin phototrigger, which undergoes cleavage upon photolysis. Our design of specific caged-nicotinamides was based upon NBO and TD-FT calculations to predict absorption wavelengths and photocleavage potential. This work provides a potentially general method for the caging of amides with coumarin photolabile protecting groups.

Full text of DOI:10.1016/j.bmcl.2013.09.067

Bioorganic & Medicinal Chemistry Letters 23 (2013) 6321–6324
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of carbamate-tethered coumarins as phototriggers
for caged nicotinamide
Pauline Bourbon a, Qian Peng a, Guillermo Ferraudi a,b, Cynthia Stauffacher c, Olaf Wiest a, Paul Helquist a,
⇑
a Department of Chemistry and Biochemistry, University of Notre Dame, 251 Nieuwland Science Hall, Notre Dame, IN 46556, USA
b Notre Dame Radiation Research Laboratory, Notre Dame, IN 46556, USA
c Department of Biological Sciences, 927 S. Martin Jischke Street, West Lafayette, IN 47907, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The syntheses of 7-diethylaminocoumarin- or modified DEACM-nicotinamide and 6-bromo-7-methoxy-
coumarin- or BMCM-nicotinamide have been accomplished by reaction of nicotinoyl isocyanate with the
corresponding coumarin allylic alcohol derivatives. The resulting compounds contain an N-acyl O-alkyl
carbamate as a new type of linkage for the caging of nicotinamide with a coumarin phototrigger, which
undergoes cleavage upon photolysis. Our design of specific caged-nicotinamides was based upon NBO
and TD-FT calculations to predict absorption wavelengths and photocleavage potential. This work pro-
vides a potentially general method for the caging of amides with coumarin photolabile protecting groups.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 13 August 2013
Revised 18 September 2013
Accepted 23 September 2013
Available online 30 September 2013
Keywords:
Caged compound
Nicotinamide
Coumarin
Photocleavage
For the purpose of studying biochemical processes, controlled
release of bioactive molecules is frequently required. This need is
often met in solution, crystals, or cells by cleavage of photoactivat-
able protecting groups. These protected biomolecules are known as
caged compounds.1–4 Upon irradiation with light, photolysis occurs
irreversibly, providing the reactive molecule for which subsequent
biochemical reactions can be monitored by any of several tech-
niques. Of utmost importance for this process is the choice of the
caging group, which should completely inhibit the normal function
of the bioactive molecule. The photocleavage should be achieved
with useful conversions rates, which can be achieved even for
quantum yields of 2% or less if high-intensity lasers, standard
lamps or LEDs are used. However, the use of high-powered lasers
or non-laser based illumination techniques in biological systems
is only feasible at wavelengths at which absorptions by other spe-
cies that may be present do not lead to competing processes. This
requirement suggests the need for wavelengths above ca. 350 nm,
well outside the absorption window of most interfering cellular
components.
derivatives is that the absorption maximum (kmax) of the nitroben-
zyl group occurs at ca. 260 nm, which overlaps with the absorption
of cellular tryptophan residues at ca. 270 nm.8,9 To avoid this prob-
lem, these investigators developed various substituted nitrobenzyl
cages having much longer wavelengths of absorption (>300 nm)
but poorer quantum yields than the parent nitrobenzyl derivatives.
As an alternative, 7-diethylaminocoumarin (DEACM) and 6-bro-
mo-7-hydroxycoumarin (BHC) groups have been shown to have
kmax values in the cellular transparent region.10,11 Coumarin-caged
compounds that release alcohols,12 carboxylic acids,13 amines,13
diols,14 ketones15 and aldehydes15 upon irradiation are known.
Previously reported caged compounds bearing a carbonate linkage
can undergo cleavage releasing CO2 as a basis for caging of com-
pounds having an oxygen atom as a point of attachment.12,13 In
contrast, the use of an N-acyl O-alkyl carbamate linkage would in
principle permit amide nitrogen atoms to be the point of
attachment.
In a previous study,16 we stated our objective of pursuing this
further need. Our aim was to develop coumarin-based phototrig-
gers for nicotinamide as a model for NAD+ that can be cleaved
readily at higher wavelengths (>350 nm). We synthesized BHC-nic-
otinamide (1) and DEACM-nicotinamide (2), but we found that
they did not undergo the desired C–N bond cleavage at their kmax
values. In comparison, BHC-acetate17,18 (3) was found by others
to undergo cleavage of its corresponding C–O bond (Fig. 1).
We explained these results by Natural Bond Orbital (NBO) cal-
culations, which indicate in a quantitative fashion the expectation
that the C–N antibonding orbital r⁄ in 1 and 2 is higher in energy
Nicotinamide adenine dinucleotide (NAD+) is an important
cofactor for many enzymes catalyzing biological redox reactions.
In comparison with other caged cofactors such as ATP,5 GTP,6
and cAMP,7 caged NAD+ or NADP+ derivatives have not been widely
developed. Salerno et al. and Cohen et al. have reported N-nitro-
benzyl caged derivatives of NAD+, but a major problem with these
⇑
Corresponding author.
E-mail address: phelquis@nd.edu (P. Helquist).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.09.067

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P. Bourbon et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6321–6324
DCE with nicotinamide (6) followed by increasing the temperature
H
N
H
N
to 60 °C over one hour and then addition of DEACM-OH 8 resulted
in the formation of modified DEACM-nicotinamide 4 in 20% yield
(Scheme 1). The substrate 8 was obtained in one step from com-
mercially available 7-dimethylamino-4-methylcoumarin.20
The synthesis of 6-bromo-7-methoxycoumarin-nicotinamide
(BMCM-nicotinamide, 5) started from the chloromethylcoumarin
9, which was converted into the alcohol 10 in 87% yield
(Scheme 2).18 The phenolic hydroxy group was etherified using
methyl iodide and potassium carbonate to provide 11 in 35%
yield.10 Coupling with nicotinoyl isocyanate (7) furnished BMCM-
nicotinamide 5 in 38% yield.
The kmax of the newly synthesized modified DEACM-nicotin-
amide 4 in methanol is 379 nm, which is in good agreement with
the value of 382 nm predicted by the TD-DFT calculations, and
has an extinction coefficient of 14,027 MÀ1 cmÀ1. Continuous pho-
tolysis of 4 using a high pressure Xe–Hg lamp (Schoeffel, model
LPS255HR) followed by a Bausch & Lomb monochromator, was
N
N
O
Br
O
O
O
O
Br
O
O
HO
O
Et2N
O
HO
O
2
3
1
DEACM-nicotinamide,
E(σ*C-N)= 0.325 Ha
BHC-acetate,
E(σ*C-O)= 0.230 Ha
BHC-nicotinamide,
E(σ*C-N)= 0.323 Ha
Figure 1. Structure of 1, 2 and 3.
than the C–O antibonding orbital r⁄ of 3. On the other hand, we
envisioned that successful cleavage may be more likely for com-
pounds bearing an N-acyl O-alkyl carbamate linkage between the
nicotinamide and the coumarin as depicted in the previously unex-
plored derivatives 4 and 5 (Fig. 2). Initial calculations indicated
that if these compounds could be obtained, they would have rele-
vant bond energies more comparable to the photocleavable acetate
3 than the recalcitrant amides 1 and 2. In this Letter, we now report
that this prediction has been successfully borne out in practice. We
describe herein that we have been able to synthesize the desired
compounds and that they provide an alternative to the previously
reported nitrobenzyl-caged nicotinamide derivatives.
Prior to investing efforts in the synthesis of the envisioned
caged candidates 4 and 5, we chose to pursue additional computa-
tional studies to predict their utility as photocleavable nicotin-
amide derivatives. The M06L/6-311+G(d,p) level of theory was
used to predict the absorption wavelengths and estimate the ease
of cleavage based on the energies of the bond orbitals as described
previously.16 The calculations (see Table S1 in the Supplementary
data) show that the linkage in compounds 4 and 5 has a lower en-
ergy C–O r⁄ antibonding orbital and that these compounds are
therefore predicted to be better candidates compared to 1 and 2
in which the C–N bonds have higher energy r⁄ antibonding orbi-
tals. The predicted kmax values for 4 and 5 were 382 and 332 nm,
respectively, based upon TD-DFT calculations with the CPCM sol-
vation model.
With these positive indications of suitable photophysical char-
acteristics, we designed a synthetic route to produce the desired
N-acylated carbamates 4 and 5 based upon reaction of an amide
with oxalyl chloride to form the corresponding acyl isocyanate, fol-
lowed by reaction of this intermediate with a coumarin bearing a
free alcohol. This overall conversion proved to be straightforward
with the use of benzamide as a simple substrate, but upon
switching to nicotinamide, the task proved to be more difficult
due to a side reaction producing an undesired salt as a precipitate.
This observation is consistent with a report in 2005 that 2-chloro-
4,6-dimethylnicotinamide underwent rapid reaction with oxalyl
chloride to give the corresponding nicotinamideÁHCl salt.19 We
believe that in our reaction this side reaction is also happening.
To minimize salt formation, the previous workers conducted the
reaction under dilute conditions whereby the nicotinamide was
added slowly to a solution of oxalyl chloride. We have found sim-
ilarly that treatment of a cold, dilute solution of oxalyl chloride in
done for 15 min at a concentration of 50 lM in methanol at its
kmax, and the photolytic reaction was followed by LC/MS at differ-
ent time intervals (Fig. 3).
In the HPLC trace, the peak at tR 5.35 min corresponded to mod-
ified DEACM-nicotinamide 4, which steadily diminished during the
course of the photolysis. In conjunction with the disappearance of
4, a peak at tR 6.35 min started to appear. The m/z of this compound
was 262.15, which corresponds to the coumarin methyl ether 12.
The solvent methanol could participate in the reaction. To confirm
this hypothesis, the photolysis was repeated in trideuterated
methanol. The product showed an increase of m/z by three mass
units, which corresponds to incorporation of the three deuterium
atoms (Scheme 3, Fig. 4) suggesting a cationic pathway for the
cleavage of this new N-acylated carbamate linkage in contrast to
O
O
(COCl)2
NH
2
N
C
O
N
N
DCE,
0 to 60 °C
6
7
OH
H
Et2N
O
O
O
N
N
8
O
O
4
Et N
O
O
2
Scheme 1. Synthesis of modified DEACM nicotinamide 4.
Cl
OH
Br
Br
i)
ii)
HO
O
O
O
HO
O
O
10
H
N
H
N
9
N
O
N
H
N
O
OH
O
N
O
O
O
Br
O
O
O
Br
Br
MeO
O
O
O
iii)
Et2N
O
MeO
O
MeO
O
O
Modified DEACM-nicotinamide, 4
11
BMCM-nicotinamide, 5
E(σ*C-O)= 0.215 Ha
5
E(σ*C-O)= 0.217 Ha
Scheme 2. Synthesis of BMCM-nicotinamide 5. Reagents and condions: (i) H2O,
reflux; (ii) MeI, K2CO3, DMF; (iii) 7, DCE.
Figure 2. New type of caged nicotinamide.

P. Bourbon et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6321–6324
6323
Table 1
Properties of 4 and 5
kmax (nm)
e
(MÀ1 cmÀ1
)
I0 (E.minÀ1
)
U
4
5
379
330
14027
11442
1.075 Â 10À3
5.63 Â 10À3
5.06 Â 10À3
0.010
0.016a
0.012b
a
At 330 nm.
At 350 nm.
b
the more commonly proposed radical pathway for nitrobenzyl
groups.21
In addition, the appearance of a peak in the extracted ion chro-
matogram (EIC) at m/z 123.05 (data not shown) corresponds to for-
mation of nicotinamide (6) during the photolysis. Taken together,
these data showed that modified DEACM-caged nicotinamide 4
undergoes cleavage at 380 nm, releasing the coumarin methyl
ether 12 and nicotinamide 6 (Scheme 4).
Figure 3. HPLC traces during the photolysis of 4 at different times (0, 1, 2, 3, 7, 10
and 15 min, respectively), (k of irradiation 380 nm).
From the plot of the concentration of 4 as a function of the pho-
tolysis time, (dC/dt), the rate of disappearance of 4 was determined
to be 9 Â 10À6 mol minÀ1 (Supplementary data). The quantum
yield / of the photolysis of 4 was determined to be 0.010 using
the relationship / = (dC/dt)/I0(1À10ÀA), where I0 is the irradiation
intensity determined either by potassium reineckate actinometry
or bromopentaamminecobalt2+ actinometry (Table 1).22
For BMCM-nicotinamide 5, the kmax was determined to be
330 nm, again in excellent agreement with the TD-DFT prediction
H
D
O
N
N
O
D
O
O
O
D
O
hν, CD3OD
Et N
O
2
Et N
O
2
of 332 nm, with an
e
of 11,442 MÀ1 cmÀ1. The photolysis of 5
4
12 d3
was done at both 330 nm and 350 nm over 20 min in a solution
of 10% DMSO in methanol during which the EIC peak for 5
decreased over time with a quantum yield in the range of
Scheme 3. Photolysis of 4 in CD3OD.
Figure 4. Mass spectrum of the product from photolysis of 4 in CD3OD at 380 nm.
H
O
N
N
O
O
R1
R2
O
O
O
R1
R2
NH2
hν
O
N
O
O
6
1
2
1
2
12
13
4
, R = H, R = NEt2
, R = Br, R = OMe
Modified DEACM-nicotinamide, , R = H, R = NEt2
BMCM-nicotinamide, , R = Br, R = OMe
1
2
1
2
5
Scheme 4. Photolysis reaction of 4 and 5.

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P. Bourbon et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6321–6324
0.012–0.016. Concomitant with this decrease of the starting mate-
rial, the EIC peaks for both the coumarin methyl ether 13 and nic-
otinamide (6) increased.
the Purdue Center for Cancer Research. P.H. thanks the Research
Council of Sweden for the award of the Tage Erlander Guest Profes-
sorship at Gothenburg University and Stockholm University.
In summary, a key finding of this work is that the performance
of phototriggers for caged compounds can be predicted by use of
appropriate computational methods. Their kmax absorption values
can be calculated with good accuracy, and their potential for
photocleavage can also be assessed in advance of obtaining the
compounds by synthesis. As an outcome of using this approach,
modified DEACM-nicotinamide (4) and BMCM-nicotinamide (5)
bearing N-acyl O-alkyl carbamate linkages between the nicotin-
amide and the coumarins have been designed with the aid of
NBO and TD-DFT calculations and have been synthesized experi-
mentally. These compounds undergo cleavage upon photolysis in
methanol, releasing nicotinamide (6) and the coumarins as methyl
ethers. Based upon these observations, the BMCM group in partic-
ular is a candidate as a phototrigger for biologically relevant
amides with absorption wavelengths and extinction coefficients
appropriate for study of many cellular pathways. Although the
quantum yields for photocleavage of the new caged nicotinamides
4 and 5 are low, they are adequate for applications using high-
power lasers such as time-resolved Laue crystallography that
forms the basis of our ongoing uses of caged compounds to study
enzyme mechanisms in the solid state,23 and they form the basis
for further use of the computational methods described in this
work.
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
09.067.
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The authors thank Dr. William Boggess and Dr. Michelle Joyce in
the University of Notre Dame Mass Spectrometry & Proteomics
Facility for their assistance in performing LC/MS measurements
as well as the Notre Dame Center for Research Computing and
the TeraGrid (TG-CHE090124) for the use of computing resources.
P.B. gratefully acknowledges support from the Notre Dame Center
for Rare and Neglected Diseases. C.V.S. gratefully acknowledges
support of this research through NSF grant MCB-0444247 and