New photocleavable linker: α-Thioacetophenone-type linker

Article abstract of DOI:10.1016/j.bmcl.2014.04.104

Photocleavable linkers are advantageous over the common linkers because they could be cleaved without using reagents. A novel photocleavable linker with an α-thioacetophenone moiety has been developed. This linker, which can be cleaved upon irradiation at 365 nm via the Norrish type II reaction, is applicable to a protein affinity purification system, allowing target proteins to be effectively isolated. This novel linker would serve as an effective tool in chemical biology.

Full text of DOI:10.1016/j.bmcl.2014.04.104

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New photocleavable linker:
a-Thioacetophenone-type linker
Hirotaka Yonezawa a, Yoshitake Nishiyama a,b, Koji Takeo a, Takeshi Iwatsubo a,c, Taisuke Tomita a,
Satoshi Yokoshima b, Tohru Fukuyama b,
⇑
a Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
b Graduate School of Pharmaceutical Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan
c Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Photocleavable linkers are advantageous over the common linkers because they could be cleaved without
Received 14 March 2014
Revised 22 April 2014
Accepted 25 April 2014
Available online xxxx
using reagents. A novel photocleavable linker with an
a-thioacetophenone moiety has been developed.
This linker, which can be cleaved upon irradiation at 365 nm via the Norrish type II reaction, is applicable
to a protein affinity purification system, allowing target proteins to be effectively isolated. This novel
linker would serve as an effective tool in chemical biology.
Ó 2014 Published by Elsevier Ltd.
Keywords:
Cleavable linker
Affinity purification
Fluorescence spectroscopy
Photolysis
Norrish type II reaction
A cleavable linker is defined as a molecular spacer that can be
cleaved selectively under relatively mild conditions. Cleavable
linkers have been broadly used in the field of chemical biology
for drug delivery, proteomics, imaging, or DNA sequencing,1 espe-
cially those that can be cleaved by irradiation with light.2–11 In
general, photoreactions proceed in high selectivity and the photo-
reactive moieties are stable under biological conditions. One of the
distinct advantages of photocleavable linkers is that they could be
cleaved without using reagents, resulting not only in simple and
easy operations but also they could be used in a highly congested
environment where reagents cannot reach.
Perhaps the most popular photocleavable linker is an ortho-
nitrobenzyl-type linker (Scheme 1, i),3,4 which is very stable under
a variety of conditions and can be cleaved selectively and effec-
tively upon irradiation at 365 nm. However, photolysis of the
linker generates a harmful nitrosobenzene, which acts as an
electrophile, an oxidant, or a reductant, inducing undesired side
reactions.12 Another common photocleavable linker is a phenacyl
ester–type linker (Scheme 1, ii).5,6 While the products generated
in photolysis, acetophenone and carboxylic acid, may not be very
harmful, irradiation at 254 nm, a shorter wavelength, could dam-
age biomolecules such as proteins and nucleic acids.1
on the a-thioacetophenone structure, which undergoes a Norrish
type II reaction by irradiation at a longer wavelength, 365 nm,
to generate acetophenone and thioaldehyde as products
(Scheme 2).13–19 By taking advantage of this reaction, we designed
a core unit of
a-thioacetophenone-type linker 6, which has two
orthogonally protected amine moieties at the end so that a variety
of functional moieties can be introduced (Scheme 3).
Linker unit 6 was synthesized in a four-step sequence. The
Friedel–Crafts reaction of 120 with chloroacetyl chloride afforded
phenacyl chloride 2, while condensation of carboxylic acid 321 with
amine 422 and ensuing removal of the trityl group gave 5. Subse-
quently, the coupling between 2 and 5 provided the desired linker
unit 6.
These drawbacks of the known photocleavable linkers have
inspired us to develop a novel and effective linker. We focused
Scheme 1. Two examples of the photocleavable linkers. (i) ortho-Nitrobenzyl-type
linkers generate reactive nitrosobenzenes. (ii) Photolysis of phenacyl esters requires
short wavelength.
⇑
Corresponding author. Tel.: +81 527476817.
E-mail address: fukuyama@ps.nagoya-u.ac.jp (T. Fukuyama).
http://dx.doi.org/10.1016/j.bmcl.2014.04.104
0960-894X/Ó 2014 Published by Elsevier Ltd.
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2
H. Yonezawa et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
via successive deprotection and introduction of a biotin moiety
and a fluorescent group onto each amine. The efficiency of the
cleavage of the linker was estimated as follows (Supplementary
Table 1 and Fig. 1). First, fluorescent probe 7 was immobilized on
avidin beads in a PBS buffer (Fig. 1, i). The amount of the immobi-
lized fluorescent probe was calculated by measuring the fluores-
cent intensity of the supernatant which contained the unloaded
probe (Supplementary Table 1, a). After rinsing, the avidin beads
were irradiated at 365 nm (Fig. 1, ii), and the fluorescence intensity
of the supernatant, which reflects the amount of the cleaved probe,
was recorded over time (Supplementary Table 1, b). The amount of
the cleavage was calculated using 8 as a standard compound. The
cleavage increased over time and reached the plateau at 45% yield
after 1 h (Fig. 2).23
Scheme 2. Norrish type II reaction of a-thioacetophenones.
Since the effective cleavage of the a-thioacetophenone-type lin-
ker on avidin beads was confirmed, we then applied it to the pro-
tein affinity purification using the avidin–biotin interaction.1,24–28
We designed molecular probe 9, which has biotin as an affinity
tag at one end of the linker and a fluorophosphonate moiety at
the other (Fig. 3). FP-biotin 10, in which biotin and a fluorophosph-
onate unit are connected via an alkyl chain, is known to biotinylate
selectively serine hydrolases.29 Therefore, the probe with cleavable
linker 9 is likely to serve as a tool for affinity purification of serine
hydrolases. To test this possibility, affinity purification of trypsin as
a serine hydrolase was attempted with probe 9 (Fig. 3). Trypsin
was treated with 9 followed by avidin beads. The resultant avidin
beads with immobilized trypsin were irradiated at 365 nm. Grati-
fyingly, the linker was effectively cleaved and 61%30 of trypsin
was recovered from the avidin beads (Fig. 4). In contrast, without
ultraviolet light irradiation, nonspecific elution of trypsin was not
observed. Replacing 9 with normal FP-biotin 10 did not release
trypsin upon UV irradiation.
Scheme 3. Preparation of the linker unit. Reagents and conditions: (a) ClCH2COCl,
AlCl3, CH2Cl2, rt, 87%; (b) EDCI, CH2Cl2, rt, 76%; (c) Et3SiH, TFA, CH2Cl2, rt; (d) 2, Et3N,
CH2Cl2, rt, 60% (2 steps).
We next carried out a quantitative analysis of the cleavage of
the linker unit on avidin beads using fluorescence spectrometry
(Fig. 1). Fluorescent probe 7 was synthesized from linker unit 6
Figure 1. Schematic diagram of the efficiency of cleavage determined by fluorescent spectrometry. F = Fluorescent group, B = biotin, A = avidin. (i) Immobilization of
fluorescent probe 7 on avidin beads; (ii) elution of cleavage product 8 by irradiation.
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H. Yonezawa et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
will serve as a powerful tool that would find valuable applications
in the field of chemical biology.
Acknowledgments
This work was financially supported by JSPS KAKENHI Grant
Numbers 20002004, 24111511, 25221301, and Platform for Drug
Discovery, Informatics, and Structural Life Science (MEXT). Y.N.
and K.T. were supported by research fellowships from JSPS.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.04.
104.
Figure 2. Efficiency of the cleavage by irradiation. Probe 7 immobilized on avidin
beads was cleaved by irradiation at 365 nm. The ratio of the cleavage was
determined by fluorescence spectrometry. t: irradiation time; R: the ratio of the
cleavage.
References and notes
1. Leriche, G.; Chisholm, L.; Wagner, A. Bioorg. Med. Chem. 2012, 20, 571.
2. Bochet, C. G. J. Chem. Soc., Perkin Trans. 1 2002, 125.
3. Patchonik, A.; Amit, B.; Woodward, R. B. J. Am. Chem. Soc. 1970, 92, 6333.
4. Wang, Z.; Udeshi, N. D.; O’Malley, M.; Shabanowitz, J.; Hunt, D. F.; Hart, G. W.
Mol. Cell. Proteomics 2010, 9, 153.
5. Anderson, J. C.; Reese, C. B. Tetrahedron Lett. 1962, 1, 1.
6. For an application example, see: Orth, R.; Sieber, S. A. J. Org. Chem. 2009, 74,
8476.
7. Enders, D.; Rijksen, C.; Bremus-Köbberling, E.; Gillner, A.; Köbberling, J.
Tetrahedron Lett. 2004, 45, 2839.
8. Du, L.; Zhang, S.; Wang, Y. Tetrahedron Lett. 2005, 46, 3399.
9. Johnsson, R.; Lackey, J. G.; Bogojeski, J. J.; Damha, M. J. Bioorg. Med. Chem. Lett.
2011, 21, 3721.
10. Chumachenko, N.; Novikov, Y.; Shoemaker, R. K.; Copley, S. D. J. Org. Chem.
2011, 76, 9409.
11. Zhang, Y.; Tanimoto, H.; Nishiyama, Y.; Morimoto, T.; Kakiuchi, K. Synlett 2012,
367.
12. Mayer, G.; Heckel, A. Angew. Chem., Int. Ed. 2006, 45, 4900. Angew. Chem. 2006,
118, 5020.
Figure 3. Molecular design of photocleavable FP-biotin 9 and structure of FP-biotin
10.
13. Padwa, A.; Pashayan, D. J. Org. Chem. 1971, 36, 3550.
14. Caserio, M. C.; Lauer, W.; Novinson, T. J. Am. Chem. Soc. 1970, 92, 6082.
15. Vedejs, E.; Perry, D. A. J. Org. Chem. 1984, 49, 573.
16. Vedejs, E.; Perry, D. A.; Wilde, R. G. J. Am. Chem. Soc. 1986, 108, 2985.
17. Cheney, J.; Moores, C. J.; Raleigh, J. A.; Scott, A. I.; Young, D. W. J. Chem. Soc.,
Chem. Commun. 1974, 47.
18. Brandt, A.; Bassignani, L.; Re, L. Tetrahedron Lett. 1976, 17, 3975.
19. Vedejs, E.; Reid, J. G. J. Am. Chem. Soc. 1984, 106, 4617.
20. Joullié, M. M.; Day, A. R. J. Am. Chem. Soc. 1954, 76, 2990.
21. Kozikowski, A. P.; Chen, Y.; Gaysin, A.; Chen, B.; D’Annibale, M. A.; Suto, C. M.;
Langley, B. C. J. Med. Chem. 2007, 50, 3054.
22. Hurevich, M.; Tal-Gan, Y.; Klein, S.; Barda, Y.; Levitzki, A.; Gilon, C. J. Pept. Sci.
2010, 16, 178.
23. The amount of fluorescent probe remaining on avidin beads was measured by
boiling the beads after cleavage (Supplementary Table 1, c). The amount of
cleavage calculated using the amount of non-cleavage sufficiently matched
with the one shown in Fig. 2 (see also Supplementary Fig. 1).
24. Koopmans, T.; Dekker, F. J.; Martin, N. I. RSC Adv. 2012, 2, 2244.
25. Yang, Y.; Verhelst, S. H. Chem. Commun. 2013, 5366.
26. Tsuchiya, A.; Asanuma, M.; Hirai, G.; Oonuma, K.; Muddassar, M.; Nishizawa,
E.; Koyama, Y.; Otani, Y.; Zhang, K. Y. J.; Sodeoka, M. Mol. BioSyst. 2013, 9, 1026.
27. Zheng, T.; Jiang, H.; Wu, P. Bioconjugate Chem. 2013, 24, 859.
28. Yang, Y.; Hahne, H.; Kuster, B.; Verhels, S. H. L. Mol. Cell. Proteomics 2013, 12,
237.
Figure 4. Results of trypsin recovery (single experiment). Trypsin was treated with
9 or 10 followed by avidin beads, and the resultant beads were irradiated at 365 nm
for 45 min. The supernatants were analyzed by SDS–PAGE and Coomassie Brilliant
Blue staining.
In summary, we have developed a novel
a-thioacetophenone-
type linker that can be photocleaved at 365 nm, which would
cause a minimal effect on biomolecules such as proteins and
nucleic acids. Fluorescence spectrometry confirmed the effective
cleavage of the linker on avidin beads. To test the possibility for
protein affinity purification, this linker was successfully applied
to a FP-biotin system. We strongly believe that our novel linker
29. Liu, Y.; Patricelli, M. P.; Cravatt, B. F. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 14694.
30. The amount of cleavage was calculated using the value of absorbance of each
band (Supplementary Figs. 2 and 3).
Please cite this article in press as: Yonezawa, H.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.04.104