Synthesis and application of photoaffinity probe containing an intact isoprenoid chain

Article abstract of DOI:10.1016/j.bmcl.2009.06.037

Two novel chemical probes each carrying an intact isoprenoid chain, a biotin tag and a benzophenone moiety were synthesized. Photoaffinity labeling of the Saccharomyces cerevisiae cell lysate revealed that these probes could selectively trap some proteins

Full text of DOI:10.1016/j.bmcl.2009.06.037

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4824–4826
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and application of photoaffinity probe containing an intact
isoprenoid chain
Lingdong Li a,b, Wei Tang a,b, Zongbao Kent Zhao a,
*
a Dalian Institute of Chemical Physics, CAS, Dalian 116023, PR China
b Graduate School of Chinese Academy of Sciences, Beijing 100049, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Two novel chemical probes each carrying an intact isoprenoid chain, a biotin tag and a benzophenone
moiety were synthesized. Photoaffinity labeling of the Saccharomyces cerevisiae cell lysate revealed that
these probes could selectively trap some proteins, and proteins with molecular weight of ꢀ70 KDa
appeared as a major band upon Streptavidin blot analysis.
Received 7 April 2009
Revised 20 May 2009
Accepted 10 June 2009
Available online 13 June 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Isoprenoid chain
Chemical probe
Photoaffinity labeling
Streptavidin blot
Polyprenyl diphosphates (Fig. 1, n P 1), generally derived from
consecutive enzymatic condensation of dimethylallyl diphosphate
and its isomer isopentenyl diphosphate, are widely involved in con-
structing skeletons of many metabolites including terpenoids,
carotenoids, dilichols and sterols.1,2 Various prenyltransferases
and prenylcyclases participate in these transformations, resulting
in enormous structural and stereochemical diversities of metabo-
lites.3 Meanwhile, a wide variety of biologically relevant molecules,
identified.7b Those early results implied that molecular interac-
tions involving isoprenoid chain may be underestimated.
However, the benzophenone moiety is not a perfect structural
mimic of an isoprenoid unit. Early biochemical studies also showed
that those benzophenone-based compounds exhibited much
weaker competitive inhibition to PPTases,8d,e suggesting that bind-
ing affinity to those compounds was rather low. To further improve
the efficacy of the synthetic probe for profiling isoprenoid chain-
interacting proteins,9 it is essential to design probes with an intact
isoprenoid chain as the baiting unit instead of the benzophenone
modified prenyl moiety. For this reason, the benzophenone moiety
may be inserted between the isoprenoid chain and the biotin moi-
ety.10 We herein report the synthesis of two photoaffinity probes
contained an intact isoprenoid chain and preliminary photoaffinity
labeling results of S. cerevisiae proteome.
such as vitamin K2, coenzyme Q, a-tocotrienol, and flavonoid, are
decorated with intact isoprenoid chains,4 especially those from
medicinal plants.5 In addition, with assistance of protein pren-
yltransferases (PPTase), the isoprenoid chains can also be attached
to proteins, such as Ras protein, the prenylation of which is impli-
cated in various cellular processes concerningcancer and many met-
abolic diseases.6
Interaction between the isoprenoid chain of a molecule and the
target protein is believed to be of great functional significance.
However, it is difficult to address such interactions at an -omics le-
vel7 because the isoprenoid chain per se is chemically inactive, and
confers neither covalent linkage nor ionic interaction to the corre-
sponding biological partners. On the basis of PPTase inhibitor stud-
We initially tried to synthesize the probe as depicted in Scheme1.
In this strategy, pyrophosphonate 4 was planed by a coupling proce-
dure, and then the biotin tag was introduced by click chemistry.
Starting from commercially available 4,4-dihydroxy-benzophe-
none,11 the precursor phosphoric acid 1 was obtained in 3 steps.
We then prepared phosphonate 2 and 3 according to procedures de-
scribed by DeGraw8e and Patel,12 respectively. Treatment of 2 or 3
with trimethylsilyl bromide (TMSBr)13 gave the corresponding
phosphonic acid. However, CDI-mediated coupling14 of these phos-
phonic acids with 1 failed to furnish compound 4, regardless of our
reaction condition optimization. It seemed that the hydroxyalkyl-
benzophenone moiety was incompatible to those conditions.
A new synthetic strategy outlined in Scheme 2 was then devel-
oped. In this protocol, we first set up the intermediate 8 with the
ies,8 we recently reported two photoaffinity probes with
a
benzophenone moiety attached to the terminus of the isoprenoid
chain (Probe 1 and Probe 2, Fig. 1).7 Probe 2 was further employed
in a chemical proteomic studies of Saccharomyces cerevisiae prote-
ome, and 30 proteins with a variety of biological functions were
* Corresponding author. Tel./fax: +86 041 84379211.
E-mail address: zhaozb@dicp.ac.cn (Z.K. Zhao).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.06.037

L. Li et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4824–4826
4825
Biosynyhesis of terpenoid metabolites, etc.
O
P
O-
O
P
O-
Posttranslational modification of
proteins, such as Ras protein.
Modification of functional small biomolecules
such as flavonoid, Coenzyme Q, etc.
O
O
O-
n
n = 0, 1, 2, 3...
O
O-
P
O-
O
HN
O
O
O
NH
P
N
n
H
O
O
S
O
O
Linker
Reactive unit
Synthetic probe (n = 1, Probe 1; n = 2, Probe 2)
Biotin tag
Figure 1. Isoprenoid chains involved biological processes and synthetic prenyl-derived photoaffinity probes.
O
MeO
MeO
P
n
n
in 3 steps
2, n = 1; 3, n = 2
O
O
HO
HN
OH
N3
HO
HO
OH
OH
O
P
O
O
P
1
O
O
S
O
Cu(I)-catalyzed click reaction
NH
CDI
O
H
N
O-
O-
O
O
N3
P
O
P
O
O
n
O
O
4
Photophore
Baiting unit
Biotin tag
Scheme 1. Initial synthetic strategy to photoaffinity probes bearing intact isoprenoid chain.
biotin tag linked to a benzophenone moiety and an azide group,
followed by click chemistry with pyrophosphonate 11 or 12 with
an alkyne chemical handle to reunite the intact isoprenoid chain.
Dibromide 5 was prepared by treating 4,4-dihydroxybenzophe-
none with excess 1,2-dibromoethane.15 After nucleophilic dis-
placement reaction of 5 with sodium azide in hot DMF, diazide 6
was obtained in 80% isolated yield. The azide-amine 7 was ob-
tained upon reduction of 6 in the presence of sub-stoichiometric
amount of PPh3, which was further linked to biotin with EDC/HOBt
as the coupling regent to give the biotinylated photophore 8. It
should be noted that compound 8 may be a powerful building
block for other chemical proteomic studies. Phosphorylation of
but-3-yn-1-ol by Oza’s procedure16 produced phosphate 9, which
was deprotected with TMSBr13 to give phosphate 10 as a white
powder. Pyrophosphonate 11 or 12 was then furnished by a CDI-
promoted coupling14 of phosphate 10 with the corresponding
phosphonic acid from phosphonate 2 or 3. Purification on silica
gel column eluted with i-PrOH/NH3ÁH2O (4/1, vol/vol) afforded
O
O
O
c
a
H2N
N3
X
X
OH
HO
O
O
O
O
O
7
5, X = Br
b
O
HN NH
6, X = N3
d
H
N
N3
S
O
O
8
O
O-
O-
OR
OR
g
e
O
O
O
OH
P
P
P
MeO
MeO
n
9, R = Me
10, R = H
O
P
O
O
n
f
O
11, n = 1; 12, n = 2
2, n = 1; 3, n = 2
O
S
O
HN
NH
O-
O-
h
O
O
H
8 + 11/12
P
P
n
N
N
N
O
O
N
O
O
O
Baiting unit
Photophore
Biotin tag
n=1, Probe 3; n=2, Probe 4
Scheme 2. Synthesis of Probe 3 and Probe 4. Reagents and conditions: (a) BrCH2CH2Br, K2CO3, MEK, reflux, 41%; (b) NaN3, DMF, 80 °C, 80%; (c) PPh3, THF, H2O, rt, 60% (based
on consumed 6); (d) biotin, EDC/HOBt, DMF, rt, 52%; (e) CBr4, P(OMe)3, pyridine, 0 °C, 70%; (f) TMSBr/collidine, CH2Cl2, rt, 50%; (g) (1) 2/3, TMSBr, CH2Cl2, rt; (2) CDI, Bu3N,
DMF, rt; (3) phosphate 10, 56% in three steps for 11, and 61% for Probe 12; (h) CuSO4/Cu, CHCl3/MeOH/H2O (1:1:2), rt, 60% for Probe 3 and 62% for Probe 4.

4826
L. Li et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4824–4826
Figure 2. Photoaffinity labeling of the yeast proteome with Probe 3/Probe 4. Soluble protein extracts (2.0 mg/mL) and the synthetic probe (20 lM) were exposed to UV
irradiation at 360 nm for 1 h, separated by SDS–PAGE and visualized by Coomassie staining (A) and streptavidin blot (B). M: protein markers; lane 1: total proteins; lanes 2
and 3: total proteins with Probe 3 and Probe 4 but without UV irradiation; lanes 4 and 5: total proteins with Probe 3 and Probe 4 in the presence of UV irradiation.
compound 11 or 12 as a white powder, as indicated by their char-
acteristic 31P NMR data of two sets of doublet signals with coupling
constants around 26.0 Hz (see Supplementary data). Compound 11
or 12 was linked with excess 8 in a mixture of CHCl3/MeOH/H2O
(1/1/2, vol/vol/vol) in the presence of a catalytic amount of Cu2+
and an excess of copper powder.17 This click reaction proceeded
slowly at room temperature, and up to 28 h was required to reach
complete conversion of 11 or 12. Probe 3 and Probe 4 were ob-
tained as a white powder upon purification by silica gel chroma-
tography. Both probes were soluble in water, which is helpful for
subsequent proteomic research.
be revealing to similar researches. We are now integrating more
advanced analytical techniques to identify those labeled proteins
and results will be reported in due course.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.06.037.
References and notes
We next applied Probe 3 and Probe 4 to S. cerevisiae cell lysate
to test their efficacy for protein labeling. Preparation of yeast pro-
teome, photoreaction, SDS–PAGE and streptavidin blot analysis
were performed using the procedures described elsewhere.7 The
yeast proteome was obtained with reasonable good quality as pro-
teins with a wide molecular weight were present (Fig. 2A, lane 1).
Incubation of Probe 3 and Probe 4 with the proteome sample in the
absence or presence of UV irradiation at 360 nm almost had no dis-
cernible affects on protein mobility in the gel electrophoresis
experiment (Fig. 2A, lanes 2–5), suggesting that these probes as
well as UV irradiation did not drastically change the protein abun-
dance distribution. However, streptavidin blot analysis indicated
that there was a distinct band at ꢀ70 KDa together with other min-
or bands in the UV-irradiated samples in the presence of these syn-
thetic probes (Fig. 2B, lanes 4 and 5). In contrast, these bands were
not presented in the untreated proteome (Fig. 2B, lanes 1) or sam-
ples without exposure to UV light (Fig. 2B, lanes 2 and 3). These
data demonstrated clearly that Probe 3 and Probe 4 had a similar
ability of covalent labeling some proteins upon UV irradiation. Fur-
thermore, the band at ꢀ70 KDa in lane 5 (Fig. 2B) looked stronger
than that in lane 4, suggesting that Probe 4 may have higher affin-
ity to these target proteins. These differences also suggested that it
was the isoprenoid chain, other than the biotin or the photophore
moiety, that mediated much of the molecular interaction to these
labeled proteins.18 Lastly, it should bear in mind that these differ-
ences based on gel electrophoresis did not necessarily mean that
only a few proteins were identified.
1. Sacchettini, J. C.; Poulter, C. D. Science 1997, 277, 1788.
2. Skorupinska-Tudek, K.; Wojcik, J.; Swiezewska, E. Chem. Rec. 2008, 8, 33. and
references therein.
3. Liang, P. H.; Ko, T. P.; Wang, A. H. Eur. J. Biochem. 2002, 269, 3339. and
references therein.
4. Hofius, D.; Sonnewald, U. Trends Plant Sci. 2003, 8, 6. and references therein.
5. (a) Liu, L.; Liu, S.; Chen, X.; Guo, L.; Che, Y. Bioorg. Med. Chem. 2009, 17, 606; (b)
Hirata, T.; Fujii, M.; Akita, K.; Yanaka, N.; Ogawa, K.; Kuroyanagi, M.; Hongo, D.
Bioorg. Med. Chem. 2009, 17, 25. and references therein; (c) Mahabusarakam,
W.; Rattanaburi, S.; Phongpaichit, S.; Kanjana-Opas, A. Phytochem. Lett. 2008, 1,
211.
6. Gelb, M. H.; Scholten, J. D.; Sebolt-Leopold, J. S. Curr. Opin. Chem. Biol. 1998, 2,
40.
7. (a) Li, L.; Tang, W.; Liu, W.; Zhao, Z. Chin. J. Org. Chem. 2008, 28, 489; (b) Tian, R.;
Li, L.; Tang, W.; Liu, H.; Ye, M.; Zhao, Z. K.; Zou, H. Proteomics 2008, 8, 3094.
8. (a) Basso, A. D.; Kirschmeier, P.; Bishop, W. R. J. Lipid Res. 2006, 47, 15. and
references therein; (b) Gaon, I.; Turek, T. C.; Weller, V. A.; Edelstein, R. L.; Singh,
S. K.; Distefano, M. D. J. Org. Chem. 1996, 61, 7738; (c) Turek, T. C.; Gaon, I.;
Distefano, M. D. J. Org. Chem. 2001, 66, 3253; (d) Turek-Etienne, T. C.;
Strickland, C. L.; Distefano, M. D. Biochemistry 2003, 42, 3716; (e) DeGraw, A.
J.; Zhao, Z.; Strickland, C. L.; Taban, A. H.; Hsieh, J.; Jefferies, M.; Xie, W.;
Shintani, D. K.; McMahan, C. M.; Cornish, K.; Distefano, M. D. J. Org. Chem. 2007,
72, 4587.
9. Barglow, K. T.; Cravatt, B. F. Nat. Methods 2007, 4, 822.
10. (a) Morohashi, Y.; Kan, T.; Tominari, Y.; Fuwa, H.; Okamura, Y.; Watanabe, N.;
Sato, C.; Natsugari, H.; Fukuyama, T.; Iwatsubo, T.; Tomita, T. J. Biol. Chem. 2006,
281, 14670; (b) Salisbury, C. M.; Cravatt, B. F. Proc. Natl. Acad. Sci. USA 2007, 104,
1171.
11. Deseke, E.; Nakatani, Y.; Ourisson, G. Eur. J. Org. Chem. 1998, 243.
12. Patel, D. V.; Gordon, E. M.; Schmidt, R. J.; Weller, H. N.; Young, M. G.; Zahler, R.;
Barbacid, M.; Carboni, J. M.; Gullo-Brown, J. L.; Hunihan, L.; Ricca, C.; Robinson,
S.; Seizinger, B. R.; Tuomari, A. V.; Manne, V. J. Med. Chem. 1996, 38, 435.
13. Salomon, C. J.; Breuer, E. Tetrahedron Lett. 1995, 36, 6759.
14. Zhao, Y.; Thorson, J. S. J. Org. Chem. 1998, 63, 7568.
15. Hubbard, S. C.; Jones, P. B. Tetrahedron 2005, 61, 7425.
16. Oza, V. B.; Corcoran, R. C. J. Org. Chem. 1995, 60, 3680.
17. (a) Lee, L. V.; Mitchell, M. L.; Huang, S.-J.; Fokin, V. V.; Sharpless, K. B.; Wong, C.-
H. J. Am. Chem. Soc. 2003, 125, 9588; (b) Rostovtsev, V. V.; Green, L. G.; Fokin, V.
V.; Sharpless, K. B. Angew. Chem., Int. Ed. 2002, 41, 2596.
In conclusion, we successfully prepared the photoaffinity probe
carrying an intact isoprenoid chain, a biotin tag and a photoreac-
tive benzophenone moiety. SDS–PAGE and streptavidin blot analy-
sis indicated that these probes were able to label S. cerevisiae
proteome. The photoaffinity strategy employed in this work may
18. Taylor, J. S.; Reid, T. S.; Terry, K. L.; Casey, P. J.; Beese, L. S. EMBO J. 2003, 22,
5963.