A photolabile linker for the mild and selective cleavage of enriched biomolecules from solid support

Article abstract of DOI:10.1021/jo901809k

(Chemical Equation Presented) Selective release of enriched biomolecules from solid support is a desirable goal in proteomic and metabolomic studies. Here we demonstrate that photocleavage of a light-sensitive phenacyl ester bond is a suitable alternative

Full text of DOI:10.1021/jo901809k

pubs.acs.org/joc
that attach covalently onto proteins and allow their capture
A Photolabile Linker for the Mild and Selective
Cleavage of Enriched Biomolecules from Solid
Support
and enrichment via incubation with avidin or streptavidin
beads. While this technology has become a standard proce-
dure for proteomics applications, several disadvantages have
limited the scope and applicability of this method including
harsh, denaturing conditions to disrupt the strong biotin-
avidin interaction, unspecific binding of high abundance
proteins and endogenously biotinylated proteins to the beads,
as well as contamination of the MS-probe with avidin mono-
mers during the release procedure. Previously, several cleava-
ble linkers have been applied in order to circumvent the
limitations of heat-induced avidin denaturation.6 Among
those are acid and disulfide cleavable linker systems which
also encountered problems due to nonselective release and
premature cleavage, respectively.6,7 A better degree of selec-
tivity has been achieved with either protease cleavable linkers
ordiazobenzene derivatives that arecleaved by mildtreatment
with sodium dithionite.7,8 However, although these methods
represent a huge improvement compared to the heat-induced
release, chemical compounds or enzymes still must be added
for a successful cleavage.
Ronald Orth and Stephan A. Sieber*
Center for Integrated Protein Science Munich CIPSM,
Department of Chemistry and Biochemistry, Ludwig-
Maximilians-Universita€t Mu€nchen, Butenandtstrasse 5-13,
81377 Munich, Germany
stephan.sieber@cup.uni-muenchen.de
Received August 21, 2009
Here we introduce a novel strategy to approach the goal of
a selective cleavage procedure requiring light to induce a
chemical bond breakage. We synthesized a photolabile
phenacyl ester group and incorporated it into a multifunc-
tionalized linker system for the enrichment and photoclea-
vage of small molecule metabolites (Figure 1).9,10 The first
functional element of this linker is an azide group that
enables the attachment of small biomolecules labeled with
an alkyne-containing probe via the copper-catalyzed Huis-
gen cycloaddition.11-13 Once the linker is attached to the
labeled biomolecules, a biotin moiety facilitates the binding
and enrichment on avidin beads. After enrichment, cleavage
of biomolecules is achieved by UV-irradiation breaking
the corresponding photolabile phenacyl ester group. Once
released, a linker bound fluorophore provides a very sensi-
tive visualization of labeled metabolites on HPLC. Since
irradiation at 254 nm is not suitable for proteins due to
expected photodamage, we decided to demonstrate the gen-
eral value of our method with small molecule metabolites as
an initial model system. Recently, Cravatt and Carlson
introduced a method named “metabolite enrichment by
tagging and proteolytic release” (METPR) in which meta-
bolites were captured by reactive groups that are immobi-
lized on solid support, released by proteolysis, and identified
Selective release of enriched biomolecules from solid sup-
port is a desirable goal in proteomic and metabolomic
studies. Here we demonstrate that photocleavage of a
light-sensitive phenacyl ester bond is a suitable alternative
cleavage strategy for the selective release of enriched
biomolecules form avidin beads circumventing the dis-
advantages of conventional heat denaturation procedures.
In the era of postgenomic research, characterization of the
full cellular complement such as proteins and metabolites has
become amajorgoal tounderstand the molecular principles of
human diseases. Rising up to this challenge several custo-
mized technologies in the field of chemistry, biology, and
analytical sciences have been developed and implemented.
Especially mass spectrometry (MS) has evolved as a key
technology in the identification and characterization of pro-
teins and metabolites in prokaryotic and eukaryotic cells.1-3
Although MS is a very sensitive method for the detection of
proteins and metabolites, in many cases metabolites and
proteins especially thoseof lowabundance needtobe enriched
on solid support in order to be detected. Several methods such
as isotope coded affinity tagging (ICAT)4 and activity based
protein profiling (ABPP)5 use biotinylated reactive groups
(6) van der Veken, P.; Dirksen, E. H.; Ruijter, E.; Elgersma, R. C.; Heck,
A. J.; Rijkers, D. T.; Slijper, M.; Liskamp, R. M. ChemBioChem 2005, 6,
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Proteomics 2007, 6, 1761.
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(10) Carrasco, M. R.; Fitzgerald, M. C.; Oda, Y.; Kent, S. B. H.
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Angew. Chem., Int. Ed. 2002, 41, 2596.
(2) Saghatelian, A.; Cravatt, B. F. Curr. Opin. Chem. Biol. 2005, 9, 62.
(3) Cravatt, B. F.; Simon, G. M.; Yates, J. R. 3rd Nature 2007, 450, 991.
(4) Tao, W. A.; Aebersold, R. Curr. Opin. Biotechnol. 2003, 14, 110.
(5) Evans, M. J.; Cravatt, B. F. Chem. Rev. 2006, 106, 3279.
(12) Huisgen, R. 1,3 Dipolar Cylcoaddition Chemistry: Wiley: New York,
1984.
(13) Speers, A. E.; Adam, G. C.; Cravatt, B. F. J. Am. Chem. Soc. 2003,
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8476 J. Org. Chem. 2009, 74, 8476–8479
Published on Web 10/12/2009
DOI: 10.1021/jo901809k
r
2009 American Chemical Society

Orth and Sieber
JOCNote
SCHEME 1. Synthesis of the Biotinylated Phenacyl Ester
Group
SCHEME 2. Synthesis of Functionalized Peptide Backbone
FIGURE 1. Structure of the photocleavable linker and principal
workflow of the metabolite capture, enrichment, and cleavage
procedure.
by MS out of complex sample mixtures.14,15 On the basis of
this approach, we synthesized a small N-hydroxy succini-
mide (NHS) reactive probe with an alkyne tag that can be
added to metabolomic extracts and that selectively reacts
with amine-containing compounds. Here we show that
probe-captured model metabolites can be attached to the
azide moiety of the photocleavable linker via click chemistry,
immobilized on avidin beads, and selectively released via UV
light.
The structure of the photocleavable linker is based on a
modular assembly starting with the synthesis of a photolabile
phenacyl ester group. Phenacyl esters are commonly used as
photolabile protecting groups in organic synthesis.16-19
Usually they function as terminal protecting groups, whereas
our approach compromises a 4,20-disubstituted phenacyl
ester moiety linking the different functional elements of the
photocleavable linker. This group is easily formed by a
nucleophilic reaction between biotin and p-azido-2-bromo-
acetophenone (Scheme 1). Subsequent click chemistry be-
tween the azide moiety on the phenacyl ester and hexynoic
acid introduces a free acid moiety that was coupled onto the
free amine of a peptidic linker backbone that is immobilized
on a rink amide resin. This immobilized peptide was built up
by standard solid phase peptide synthesis (SPPS) coupling
procedures and contains an azide-modified lysine and a
MTT-protected lysine (Scheme 2). Removal of the peptide
and the MTT group was achieved in one step via TFA
treatment after successful coupling of the phenacyl ester
building block 3. The resulting free amine was coupled to
N-hydroxy succinimide (NHS)-activated tetramethyl rhoda-
mine (TAMRA). HPLC purification yielded the pure multi-
functional photolabile linker.
To evaluate the optimal conditions of photocleavage, we
first tested the solubility of the photolinker and realized that
up to 60% (v/v) DMSO in PBS buffer was required in order
to dissolve the compound. For all subsequent irradiation
experiments 0.8 mM DTT was added as a photoscavanger
neutralizing free radicals formed during UV-irradiation.20
Although photocleavage of the photolabile linker 7 was
already induced by room light, the efficiency of this process
was too low in order to be of preparative value. We therefore
used a 15 W UV-hand lamp placed 20 cm above the sample
and irradiated compound 7 for 5 min at 254 nm, which led to
75((11)% of the expected cleavage product as determined
by HPLC analysis (Figure 2).
The identity of the cleaved product was confirmed by mass
spectrometry and revealed that the bond breakage occurred at
the C-O bond and thereby released biotin as previously
reported (Experimental Section).21 Cleavage conditions could
be further improved by reducing the distance of the lamp to
about 1 cm above the sample. This significantly accelerated
the duration of cleavage to 1 min and increased the yield
(89((2)%) (Figure 2). Compared to other cleavage proce-
dures mentioned above, our initialexperiments appeared to be
advantageous due to the rapid reaction time and easy hand-
ling. We therefore evaluated the potential value of this ap-
proach in the release of immobilized small biomolecules from
avidin beads. To test whether UV-induced cleavage works on
solid support, the photocleavable linker (5 nmol) was incu-
bated with avidin beads, washed, and irradiated with UV light
(15 W hand lamp) for 1 min. This reaction yielded 69((4)%
fluorescent product relative to a control linker (Figure 1 in the
Supporting Information) that is already used in established
protocols and cleaved by conventional heat denaturation
(Figure 2 in the Supporting Information).22,23 This result
(14) Carlson, E. E.; Cravatt, B. F. J. Am. Chem. Soc. 2007, 129, 15780.
(15) Carlson, E. E.; Cravatt, B. F. Nat. Methods 2007, 4, 429.
(16) Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic
Synthesis, 4th ed.; Wiley & Sons Inc.: New York, 2007.
(17) Sheehan, J. C.; Umezawa, K. J. Org. Chem. 1973, 38, 3771.
(18) Wang, S.-S. J. Org. Chem. 1976, 41, 3258.
(20) Rinnova, M.; Novakova, M.; Kasicka, V.; Jiracek, J. J. Pept. Sci.
2000, 6, 355.
(21) Literak, J.; Dostalova, A.; Klan, P. J. Org. Chem. 2006, 71, 713.
€
(22) Bottcher, T.; Sieber, S. A. Angew. Chem., Int. Ed. Engl. 2008, 47,
4600.
(23) Staub, I.; Sieber, S. A. J. Am. Chem. Soc. 2008, 130, 13400.
(19) Bochet, C. G. J. Chem. Soc. 2002, 125.
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Orth and Sieber
JOCNote
FIGURE 2. HPLC traces of the photolinker before (A) and after 1
(B) and 5 min (C) of irraditation.
indicates that the procedure is still preparative useful,
although the irradiation on solid support slightly reduces the
cleavage efficiency. In the next step, the efficiency of click
chemistry for the probe attachment was evaluated. Unexpect-
edly, the standard procedure for the cycloaddition via CuSO4
and TCEP did not work since it destroyed the photolinker
probably by a reduction of the light-sensitive C-O bond. We
therefore avoided the use of TCEP and used CuBr, which
yielded a quantitative reaction. With these optimized condi-
tions, the NHS probe was reacted with lysineand benzylamine
as a representative metabolite mimic. The resulting amides
were then attached to the linker via click chemistry. The
reaction mixtures were each incubated with avidin beads,
washed with PBS buffer, and irradiated for 7 min with a
350 W UV lamp at 254 nm, which yielded 68((6)% lysine
(Figure 3) and 70((16)% benzylamine (Figure 3 in the
Supporting Information) product relative to the heat dena-
turation with the control linker (Figure 4 in the Support-
ing Information). For these experiments, higher irradiation
energy was required for bond-breaking, which is due to the
presence of an additional photoactive triazole moiety. This
higher irradiation energy caused the formation of two pro-
ducts which are a result of the quenching reaction of the
radical intermediate with water to give the methyl ketone and
the R-hydroxyl ketone as confirmed by mass spectrometric
analysis (Supporting Information).24
FIGURE 3. HPLC trace and structure of the photolinker after
lysine capture via the metabolite reactive group and UV irradiation.
our photocleavable linker could be used in automated meta-
bolite/metabolom screening. In future studies, the photoacti-
vating group will be adjusted for the needs of protein
chemistry and detachment from the resin should take place
at wavelengths higher than 300 nm in order to avoid photo-
damage.
Experimental Section
Synthesis of Phenacyl Ester (2). To a solution of 40-azido-2-
bromoacetophenone (500 mg, 2.08 mmol) and Biotin (422.5 mg,
1.74 mmol) in 2.6 mL of dried DMSO was added freshly
triturated K2CO3 (312 mg, 2.26 mmol). The reaction mixture
was strirred for 48 h at 40 °C under argon atmosphere. The
phenacyl ester (2) (424.3 mg, 60%) was obtained as a yellow solid
by silica gel chromatography eluted with TCM/MeOH (4/1). MS
(MALDI) m/z calcd for C18H21N5O4S (M þ H)þ 404.13,
found:404.27 (M þ H)þ; 1H NMR (CDCl3/CD3OD (7/1),
600 MHz) δ 7.89 (d, J = 8.4 Hz, 2 H), 7.07 (d, J = 8.4 Hz,
2 H), 5.31 (d, J = 16.4, 1 H), 5.24 (d, J = 16.2, 1H), 4.50-4.46 (m,
1 H), 4.33-4.28 (m, 1 H), 3.16-3.11 (m, 1 H), 2.91-2.86 (m, J =
4.8, 1 H), 2.69 (d, J = 12.7 Hz, 1H), 2.52-2.41 (m, 2H),
1.76-1.69 (m, 3H), 1.67-1.60 (m, 1H), 1.55-1.44 (m, 2H); 13
C
NMR (CDCl3/CD3OD (7/1), 150 MHz) δ 191.1 (C), 173.1 (C),
163.9 (C), 145.9 (C), 130.4 (C), 129.7 (CH), 119.2 (CH), 65.7
(CH2), 61.6 (CH), 60.1 (CH), 55.2 (CH), 40.43 (CH2), 40.35
(CH2), 33.4 (CH2), 28.0 (CH2), 24.7 (CH2).
In general, these results show that our model system
validates the use of photocleavable linkers as an attractive
strategy for the enrichment and release of biomolecules from
solid support. Advantages of our method include rapid reac-
tion times, easy handling, and reduced contamination as
compared to established methods. Due to these advantages
Synthesis of the Triazole (3). To a solution of phenacyl ester (2)
(424.3 mg, 1.05 mmol) and hexynoic acid (127.29 μL, 1.27 mmol)
in 9.1 mL of dried DMSO were added 10.50 μmol of Cu(II)SO4
(2.62 mg dissolved in 1 mL of H2O) and 105.00 μmol of freshly
prepared sodium ascorbate (20.80 mg dissolved in 105.2 μL of
H2O). The reaction mixture was stirred for 48 h at room
(24) Chai, W.; Takeda, A.; Hara, M.; Ji, S.; Horiuchi, C. A. Tetrahedron
2005, 61, 2453.
8478 J. Org. Chem. Vol. 74, No. 21, 2009

Orth and Sieber
JOCNote
temperature under argon atmosphere. The triazole (3) (320 mg,
59%) was obtained as a yellow solid by silica gel chromatography
eluted with TCM/MeOH (4/1). MS (ESI) m/z calcd for C24H29-
N5O6S (M - H)- 514.174, found 514.176 (M - H)-; 1H NMR
(DMSO, 400 MHz) δ 12.04 (s, 1H), 8.73 (s, 1H), 8.13 (d,
J = 8.9 Hz, 2H), 8.06 (d, J = 8.9 Hz, 2H), 6.36 (d, J = 29.2
Hz, 2H), 5.48 (s, 2H), 4.30-4.25 (m, 1H), 4.14-4.09 (m, 1H),
3.11-3.05 (m, 1H), 2.72 (t, J = 7.6 Hz, 2H), 2.42 (t, J = 7.4 Hz,
2H), 2.30 (t, J = 7.2 Hz, 2H), 1.92-1.84 (m, 2H), 1.65-1.55 (m,
4H), 1.51-1.32 (m, 4H); 13C NMR (DMSO, 100 MHz) δ 192.4
(C), 174.6 (C), 172.8 (C), 163.1 (C), 148.4 (CH), 140.6 (C), 133.6
(CH), 130.2 (CH), 120.8 (CH), 120.0 (CH), 66.8 (CH2), 61.4
(CH), 59.6 (CH), 55.7 (CH), 40.8 (CH2), 33.4 (CH2), 33.4 (CH2),
28.4 (CH2), 28.3 (CH2), 24.9 (CH2), 24.8 (CH2), 24.5 (CH2).
Synthesis of Phenacyl Linker (7): Coupling to Resin Bound
Peptide, Purification, and TAMRA Coupling. A solution of
triazole (3) (11.1 mg, 21.6 μmol), TBTU (6.7 mg, 20.88 μmol),
HOBt (2.9 mg, 21,6 μmol), and DIPEA (12.2 μL, 70.2 μmol) was
stirred for 10 min at room temperature prior to addition of the
(2ꢀ) and dried for 1 h at room temperature. The pellet was dissolved
in 100 μL of dried DMF. DIPEA (3.8 μL, 21.6 μL) and a mixture of
(5)- and (6)-carboxy-TAMRA-NHS isomers (3.80 mg, 7.2 μmol)
were added. The reaction mixture was strirred for 16 h at room
temperature. The crude product was purified via HPLC (column:
XBridge BEH130 C18 5 μm (10 ꢀ 150 mm), linear gradient 0 to
100% CH3CN/H2O with 0.1% TFA in 60 min) to give phenacyl
linker (7) as a purple powder (1.4 mg, 18.52%). It was possible to
separate the two isomers and all studies were performed with a
single isomer. tR = 25.4 min (42.1% CH3CN/H2O); MS (ESI) m/z
calcd for C73H87N16O13S (M)þ 1427.635, found 1427.632 (M)þ.
Acknowledgment. We thank Prof. Dr. Thomas Carell and
his group for their generous support, the excellent working
environment, and many fruitful scientific discussions. We
gratefully acknowledge funding by the Emmy Noether Pro-
gram of the Deutsche Forschungsgemeinschaft (DFG), a
€
DFG grant (SFB 749), a stipend by the Romer-Stiftung, and
support by the Fonds der chemischen Industrie and by the
Center for Integrated Protein Science Munich CIPSM.
deprotected resin bound peptide (4) (10 mg resin, 5.3 μmol, BH
=
0.53 mmol/g). The suspension was shaken for 16 h at room
temperature. This coupling procedure was repeated three times to
give a negative Kaiser assay. After washing with DMF (3ꢀ) and
DCM (2ꢀ), the resin was treated with 100 μL of TFA/H2O (95/5)
for 2 h at room temperature. Ice cold Et2O (1 mL) was added and
the suspension was incubated for 45 min at -20 °C. After centri-
fugation (2 min, 17 000 g) the resulting pellet was washed with Et2O
Supporting Information Available: General experimental
methods, additional experimental procedures, compound char-
acterization data, copies of spectra, and supporting figures. This
material is available free of charge via the Internet at http://
pubs.acs.org.
J. Org. Chem. Vol. 74, No. 21, 2009 8479