Design, synthesis, and photochemistry of modular caging groups for photoreleasable nucleotides

Article abstract of DOI:10.1021/ol3029093

A modular approach to preparing caged nucleotides having additional properties has been achieved. The modular caging agent includes three components: an amine reactive NHS ester moiety, a photoactive Bhc group, and tosylhydrazone as a precursor of the diazomethyl group. Various amines including biotin and an Arg-Gly-Asp (RGD) peptide were introduced into the key intermediate via amide linkage. The Bio-Bhc-diazo thus synthesized enables the preparation of a photoreleasable siRNA with additional properties.

Full text of DOI:10.1021/ol3029093

ORGANIC
LETTERS
2012
Vol. 14, No. 24
6182–6185
Design, Synthesis, and Photochemistry
of Modular Caging Groups for
Photoreleasable Nucleotides
Toshiaki Furuta,* Kaori Manabe, Aoi Teraoka, Kanako Murakoshi, Ai Ohtsubo, and
Akinobu Suzuki
Department of Biomolecular Science, Faculty of Science, Toho University, 2-2-1
Miyama, Funabashi, 274-8510, Japan
furuta@biomol.sci.toho-u.ac.jp
Received October 22, 2012
ABSTRACT
A modular approach to preparing caged nucleotides having additional properties has been achieved. The modular caging agent includes three
components: an amine reactive NHS ester moiety, a photoactive Bhc group, and tosylhydrazone as a precursor of the diazomethyl group. Various
amines including biotin and an Arg-Gly-Asp (RGD) peptide were introduced into the key intermediate via amide linkage. The Bio-Bhc-diazo thus
synthesized enables the preparation of a photoreleasable siRNA with additional properties.
Caged compounds are synthetic molecules with biologi-
cal functions that are masked temporarily by covalently
introduced photoremovable protecting groups.1 Spot illu-
mination of an appropriately designed caged compound
enables control of fundamental biological processes with
high spatial and temporal resolution. Phosphate-containing
molecules are an important class of compounds for bio-
logical sciences. Examples are cyclic nucleotides, which are
known as second messengers, phosphorylated proteins
as intracellular signaling components, and DNAs and RNAs
as the carriers of genetic information. Most of the mole-
cules are thought to be activated or produced transiently in
a highly localized manner in physiologically relevant cir-
cumstances. Therefore, caged compounds of phosphate
containing molecules are anticipated for use in the study of
fundamental biological processes. Structural variations of
caging groups have increased in recent years. Nevertheless,
the protecting groups used to produce caged phosphates
are limited to a few examples: o-nitrobenzyl,2 p-hydroxy-
phenacyl (includes desyl group),3 and (coumarin-4-yl)-
methyl groups.4 We have developed and used brominated
(coumarin-4-yl)methyl groups such as the (6-bromo-7-
hydroxycoumarin-4-yl)methyl (Bhc) group as a caging group
for carboxylates,5 amines,5,6 phosphates,7 and alcohols.8
Photochemical and physical properties of the Bhc group
have proven to be favorable as a photocaging group. If an
additional functional unit such as a specific ligand of a protein
of interest and a signal peptide could be installed easily on the
Bhc-ring, then the usefulness of the Bhc-caged compounds
(4) (a) Furuta, T.; Torigai, H.; Osawa, T.; Iwamura, M. Chem. Lett.
1993, 1179. (b) Furuta, T.; Torigai, H.; Sugimoto, M.; Iwamura, M.
J. Org. Chem. 1995, 60, 3953. (c) Hagen, V.; Bendig, J.; Frings, S.;
Eckardt, T.; Helm, S.; Reuter, D.; Kaupp, U. B. Angew. Chem., Int. Ed.
2001, 40, 1045.
(5) Furuta, T.; Wang, S. S.; Dantzker, J. L.; Dore, T. M.; Bybee,
W. J.;Callaway, E. M.;Denk, W.;Tsien, R. Y.Proc. Natl. Acad. Sci. U.S.A.
1999, 96, 1193.
(6) (a) Furuta, T.; Watanabe, T.; Tanabe, S.; Sakyo, J.; Matsuba, C.
Org. Lett. 2007, 9, 4717. (b) Yamaguchi, S.; Chen, Y.; Nakajima, S.;
Furuta, T.; Nagamune, T. Chem. Commun. 2010, 46, 2244. (c) Mizukami,
S.; Hosoda, M.; Satake, T.; Okada, S.; Hori, Y.; Furuta, T.; Kikuchi, K.
J. Am. Chem. Soc. 2010, 132, 9524.
(7) (a) Ando, H.; Furuta, T.; Tsien, R. Y.; Okamoto, H. Nat. Genet.
2001, 28, 317. (b) Furuta, T.; Takeuchi, H.; Isozaki, M.; Takahashi, Y.;
Kanehara, M.; Sugimoto, M.; Watanabe, T.; Noguchi, K.; Dore, T. M.;
Kurahashi, T.; Iwamura, M.; Tsien, R. Y. ChemBioChem 2004, 5, 1119.
(c) Kawakami, T.; Cheng, H.; Hashiro, S.; Nomura, Y.; Tsukiji, S.;
Furuta, T.; Nagamune, T. ChemBioChem 2008, 9, 1583.
(1) For example, see: Lee, H. M.; Larson, D. R.; Lawrence, D. S.
ACS Chem. Biol. 2009, 4, 409.
(2) (a) Engels, J.; Schlaeger, E. J. J. Med. Chem. 1977, 20, 907.
(b) Kaplan, J. H.; Forbush, B., III; Hoffman, J. F. Biochemistry 1978,
17, 1929.
(3) (a) Givens, R. S.; Athey, P. S.; Kueper, L. W.; Matuszewski, B.;
Xue, J. Y. J. Am. Chem. Soc. 1992, 114, 8708. (b) Givens, R. S.; Park,
C.-H. Tetrahedron Lett. 1996, 37, 6259. (c) Park, C.-H.; Givens, R. S.
J. Am. Chem. Soc. 1997, 119, 2453.
r
10.1021/ol3029093
Published on Web 12/03/2012
2012 American Chemical Society

Scheme 1. Synthesis of the Key Building Block (1)
Figure 1. Modular caged compounds.
with free phosphate in the presence of other functional
groups, and (3) whether the synthesized phosphate esters
can retain their photochemical properties as caged phos-
phates. To address the first question, the key intermediate
1 was reacted with amine-containing molecules (Figure 2).
The NHS ester moiety of 1 reacted quantitatively with a
propargyl amine to produce the corresponding propargyl
amide. Because the tosylhydrazone moiety of the amide
was partly transformed into a diazomethyl group, the
crude product was subjected to reaction with triethyl-
amine to give the desired Bhc-diazo derivative propargyl-
Bhc-diazo (4) in 93% isolated yield. The product is an
unexpectedly stable diazomethane derivative and can be
stored at ambient temperature for more than a year. After
being used in caging nucleotides, the propargyl moiety in 4
can be a chemical handle for additional modification using
Cu-catalyzed Huisgen [3 þ 2] cyclization.13
would be expanded. We report a modular approach to
the synthesis of caged nucleotides having additional
functionality.
To synthesize caged nucleotides, photoremovable pro-
tecting groups can be used either as protecting groups of
functional groups in riboses,9 phosphates,4,7,10 and nucleo-
bases11 or as photocleavable linkers of hairpin-like struc-
tures.12 Because free phosphates are known to react with
diazomethane derivatives to give the corresponding esters,
the synthetic route of phosphate protection is expected to
be simpler and to provide easier application for preparing
caged compounds having complex structures. Therefore,
we designed new precursor molecules of modular caged
nucleotides in which an additional functional unit was in-
stalled easily. The key compound (1) comprises three com-
ponents: an amine reactive NHS ester moiety, a photo-
active Bhc group, and tosylhydrazone as a precursor of a
diazomethyl moiety (Figure 1). The precursor molecule
NHS-Bhc-hydrazone (1) was synthesized from the known
alcohol Bhc-CH2OH (2)5 in five steps (Scheme 1).
The following questions are addressed by this study: (1)
which functional units can be added to precursor molecule
1, (2) whether the precursor molecule can react selectively
Using the same reaction conditions, we synthesized
other Bhc-diazo derivatives from amine-containing com-
pounds including pentylamine-biotin (Bio-Bhc-diazo 5) as
a ligand of a specific protein and hydrophobic octylamine
(Oct-Bhc-diazo 6). An Arg-Gly-Asp (RGD) peptide, a
targeting moiety to cancer cells,14 was also introduced into
1 to yield the corresponding tosylhydrazone 7. As far as
chemical stability is concerned, we observed decomposi-
tion of 7 during storage as a DMF solution. The decom-
posed products included the 4-hydroxymethyl coumarin
derivative 8, which was generated from hydrolysis of the
4-formyloxymethyl coumarin 9. A possible explanation of
this is the following: during storage, a small amount of
the DMF molecules are hydrolyzed to produce diethyl-
amine and formic acid, diethylamine drives the formation
of the 4-diazomethyl derivative (RGD-Bhc-diazo) from 7,
and the resulting RGD-Bhc-diazo reacts with formic acid
to produce the formic acid ester 9. Those results raised the
next question of whether the coumarinyl diazomethanes
react selectively with phosphates in the presence of carbox-
ylates, amines, and alcohols: functional groups that are
common among biologically important molecules.
(8) Suzuki, A. Z.; Watanabe, T.; Kawamoto, M.; Nishiyama, K.;
Yamashita, H.; Ishii, M.; Iwamura, M.; Furuta, T. Org. Lett. 2003, 5, 4867.
(9) (a) Chaulk, S. G.; MacMillan, A. M. Nucleic Acids Res. 1998, 26,
3173. (b) Chaulk, S. G.; MacMillan, A. M. Nat. Protoc. 2007, 2, 1052.
(10) (a) Furuta, T.; Momotake, A.; Sugimoto, M.; Hatayama, M.;
Torigai, H.; Iwamura, M. Biochem. Biophys. Res. Commun. 1996, 228, 193.
(b) Furuta, T.; Iwamura, M. Methods Enzymol. 1998, 291, 50. (c) Monroe,
W. T.; McQuain, M. M.; Chang, M. S.; Alexander, J. S.; Haselton, F. R.
J. Biol. Chem. 1999, 274, 20895. (d) Shah, S.; Rangarajan, S.; Friedman,
S. H. Angew. Chem., Int. Ed. 2005, 44, 1328. (e) Nguyen, Q. N.; Chavli,
R. V.; Marques, J. T.; Conrad, P. G., Jr.; Wang, D.; He, W.; Belisle,
B. E.; Zhang, A.; Pastor, L. M.; Witney, F. R.; Morris, M.; Heitz, F.;
Divita, G.; Williams, B. R.; McMaster, G. K. Biochim. Biophys. Acta
2006, 1758, 394. (f) Casey, J. P.; Blidner, R. A.; Monroe, W. T. Mol.
Pharmaceutics 2009, 6, 669. (g) Shah, S.; Jain, P. K.; Kala, A.;
Karunakaran, D.; Friedman, S. H. Nucleic Acids Res. 2009, 37, 4508.
(11) (a) Alvarez, K.; Vasseur, J. J.; Beltran, T.; Imbach, J. L. J. Org.
Chem. 1999, 64, 6319. (b) Krock, L.; Heckel, A. Angew. Chem., Int. Ed.
2005, 44, 471. (c) Hobertner, C.; Silverman, S. K. Angew. Chem., Int. Ed.
2005, 44, 7305. (d) Lusic, H.; Young, D. D.; Lively, M. O.; Deiters Org.
Lett. 2007, 9, 1903. (e) Mikat, V.; Heckel, A. RNA 2007, 13, 2341. (f)
Kuzuya, A.; Okada, F.; Komiyama, M. Bioconjugate Chem. 2009, 20,
6319. (g) Deiters, A.; Garner, R. A.; Lusic, H.; Govan, J. M.; Dush, M.;
Nascone-Yoder, N. M.; Yoder, J. A. J. Am. Chem. Soc. 2010, 132,
15644. (h) Young, D. D.; Lively, M. O.; Deiters, A. J. Am. Chem. Soc.
2010, 132, 6183. (i) Govan, J. M.; Uprety, R.; Hemphill, J.; Lively,
M. O.; Deiters, A. ACS Chem. Biol. 2012, 7, 1247.
Consequently, two coumarinyl diazomethane deriva-
tives 4 and 6 were chosen to address the second question.
Most diazomethane derivatives are known to react with
carboxylic acids as well as phosphates withthe evolution of
nitrogen. As expected, the reaction of 4 with diethyl
phosphate produced the corresponding ester 10. However,
(12) (a) Shestopalov, I. A.; Sinha, S.; Chen, J. K. Nat. Chem. Biol.
2007, 3, 650. (b) Tang, X.; Maegawa, S.; Weinberg, E. S.; Dmochowski,
I. J. J. Am. Chem. Soc. 2007, 129, 11000. (c) Tang, X.; Swaminathan, J.;
Gewirtz, A. M.; Dmochowski, I. J. Nucleic Acids Res. 2008, 36, 559. (d)
Ouyang, X.; Shestopalov, I. A.; Sinha, S.; Zheng, G.; Pitt, C. L.; Li,
W.-H.; Olson, A. J.; Chen, J. K. J. Am. Chem. Soc. 2009, 131, 13255.
(13) (a) Tornoe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem.
2002, 67, 3057. (b) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.;
Sharpless, B. K. Angew. Chem., Int. Ed. 2002, 41, 2596. (c) El-Sagheer,
A. H.; Brown, T. Chem. Soc. Rev. 2010, 39, 1388.
(14) Arap, W.; Pasqualini, R.; Ruoslahti, E. Science 1998, 279, 377.
Org. Lett., Vol. 14, No. 24, 2012
6183

Figure 3. Reaction of the coumarinyl diazomethane derivatives
with phosphates.
Photochemical and physical properties of the phosphate
ester 10 were investigated to address the third question.
The compound 10 has two absorption maxima. The longer
maximum, a πꢀπ* absorption band that is responsible for
the photolysis reaction, lies at 328 nm, which is similar to
that of the 7-methoxy analogue of the Bhcmoc group
(Bmcmoc group).6a The photolysis reaction of 10 was
conducted at 350 nm under simulated physiological con-
ditions (10 mM K-MOPS buffer solution at pH 7.2).
Photolysis mixtures were analyzed periodically using
reversed-phase HPLC. The HPLC traces shown in Figure 5
indicate the almost quantitative production of an ex-
pected photobyproduct Propargyl-Bhc-CH2OH (12), and
thereby indicate the production of diethyl phosphate. The
photolytic consumption of 10 and the production of 12 can
be approximated by single exponential equations (Figure 6),
suggesting no unexpected secondary effects that interfere with
photolysis throughout the photolysis reaction. From the data,
the quantum yield (Φ350) for the formation of 12 and diethyl
phosphate was calculated as 0.29, which is comparable to
those of Bmcmoc-dC (Φ350 = 0.30)6a and Bhc-cAMP
(Φ350 = 0.10).7b In view of the application to caged nucleo-
tides, photolysis efficiency which can be expressed quantita-
tively from the product of molar absorptivity (ε) and photo-
lysis quantum yield (Φ) is expected to be the property of
practical importance because the value acts as an index for the
amount of light intensity required for the uncaging reaction.
The εΦ350 value for the formation of diethyl phosphate from
10 was 2200 Mꢀ1 cmꢀ1, which is almost twice as high as those
of other coumarinyl methyl caged phosphates.4,7,10a,10b
To demonstrate the utility of the compounds, Bio-Bhc-
diazo (5) was used to test whether the modular caging
agents are applicable to the synthesis of caged oligonucleo-
tides. As we described above, direct esterification of
Figure 2. Chemical structures of Bhc-diazo derivatives having
additional functionality.
no ester product was obtained in the reaction with acetic
acid even in the presence of copper(II) acetate which is
known to promote carbene formation from diazomethane
derivatives. The results suggest that compound 4 would
react with organic acids through a protonation derived
SN2 mechanism, not through the formation of carbenes
(Figure 3). Therefore, the reactivity of 4 with organic acids
must depend on the acidity of the acid components and
nucleophilicity of their conjugated bases. The typical pKa
values of phosphates are 2. Those of carboxylates are 4,
indicating that organic acids that are less acidic than
carboxylates are unable to react with the coumarinyl
diazomethanes in the absence of acid catalysts.15 Because
the acetate anion is the second smallest conjugated base of
carboxylic acids, most carboxylic acids cannot react with
the coumarinyl diazomethanes. As a consequence, we
deduced that the coumarinyl diazomethanes can be caging
agents of phosphates but not of carboxylates. Unlike the
case with the o-nitrophenyl diazomethane derivatives,10g
an unexpected nucleobase modification reaction must be
avoided with the coumarinyl diazomethanes when the com-
pounds are applied to the preparation of caged nucleotides.
To test the assumption, a mononucleotide 30,50-cyclic
adenosine mononucleotide (cAMP) was mixed with 6 in
DMSO (Figure 4). Although the product was formed in
trace quantities, the desired ester (11) was obtained as the
sole product. The remainder was recovered starting cAMP.
The free acid form of cAMP has two functional groups
other than the cyclic phosphate, a hydroxyl group on the
ribose ring and an amino group on the adenine base.
Thereby the coumarinyl diazomethane derivatives are use-
ful as a phosphate caging agent with high chemoselectivity.
Figure 4. Oct-Bhc caged cAMP (11).
Org. Lett., Vol. 14, No. 24, 2012
(15) (a) Ito, K.; Maruyama, J. Chem. Pharm. Bull. 1983, 31, 3014. (b)
Ito, K.; Maruyama, J. Chem. Pharm. Bull. 1986, 34, 390.
6184

Figure 7. Relative luminescence intensities of biotinylated siR-
NAs. Luminescence intensity of an untreated siRNA (indicated
as 0) was set to 1.
Figure 5. HPLC traces for the photolysis of 10 (measured at 330 nm).
Samples were analyzed after the specified irradiation time.
quantified in the reactions of the siRNA with different molar
ratios of 5 (Figure 7). The observed intensities of lumines-
cence, and therefore the quantities of the attached caging
groups, increased when the larger amount of 5 was used. The
synthesized siRNA derivatives were exposed to 350-nm UV
light and subjected to electrophoretic analysis. The lumines-
cence intensities of the band correspond to the biotin-labeled
siRNA decreased by 60% after 5-min irradiation, clearly
indicating that deprotection of the Bio-Bhc group can also
be monitored. The biotin moiety of the Bio-Bhc modified
nucleotides act not only as a detection tag of the attached
caging group described here but also as an affinity tag for
purification and a tag for introducing steric bulkiness.6b
In summary, compound 1, a precursor molecule of
modular caged nucleotides, was designed and synthesized
from commercially available 4-bromoresorcinol. Amine
containing functional units including biotin and an RGD
peptide were introduced into precursor 1. After introdu-
cing a functional unit, the tosylhydrazone moiety in 1 was
transformed into the diazomethyl group to produce the
desired coumarinyl diazomethane derivatives. The coumar-
inyl diazomethanes react chemoselectively with the free acid
form of phosphates to yield the corresponding esters in the
presence of other functional groups including carboxylates,
amines, and alcohols. Propargyl-Bhc-caged diethyl phos-
phate was photolyzed to produce the parent acid with high
photolysis efficiency (εΦ350 = 2200 Mꢀ1 cmꢀ1). Bio-
Bhc-diazo (5) was used in producing siRNA derivatives
having a biotinylated photocaging group. The attached
caging group was detected in both caging and uncaging
reactions using standard polyacrylamide gel electrophoresis.
Figure 6. Time course for photolysis of 10. Samples (10ꢀ5 M)
were irradiated at 350 nm (10 mJ/s) under simulated physiolo-
gical conditions (10 mM K-MOPS buffer at pH 7.2). Closed
circles: consumption of 10. Open circles: yield of 12. Solid lines:
least-squares curve fit to a simple decaying exponential for 10
and rising exponential for 12.
oligonucleotides is easy to carry out if an appropriately
designed precursor molecule is available. Using these
methods, biologists can prepare caged compounds of their
own oligonucleotides, for example, plasmid vectors iso-
lated from cultured E. coli,10c mRNAs prepared by in vitro
translation,7a,16 and siRNAs synthesized using automated
solid-phase synthesis.10dꢀg We investigated the reaction of
Bio-Bhc-diazo (5) and a 22-mer siRNA. The precursor
molecule 5 has biotin as an affinity tag and the Bhc-diazo
group as a caging agent for oligonucleotides. We tried
to use biotin as a monitoring tag of the caging reaction.
The reaction mixtures were subjected to polyacrylamide
gel electrophoresis. The siRNAs were visualized by SYBR
Gold staining, whereas the attached Bio-Bhc group was
monitored by chemiluminescence-based detection of the bio-
tin tag with an HRP-streptavidin conjugate. The fact that
the bands detected by chemiluminescence staining showed the
same migration as those by SYBR Gold staining implies
that the caging group is attached covalently to the siRNA
molecules. The luminescence intensities of each band were
Acknowledgment. This work was supported by MEXT
KAKENHI Grant Nos. 19021043, 20016024, 22121520,
22310141, and 24121724.
Supporting Information Available. Experimental pro-
cedures and spectroscopic data for all new compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(16) Ando, H.; Kobayashi, M.; Tsubokawa, T.; Uyemura, K.;
Furuta, T.; Okamoto, H. Dev. Biol. 2005, 287, 456.
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 24, 2012
6185