Design of a reversible biotin analog and applications in protein labeling, detection, and isolation

Article abstract of DOI:10.1039/c1cc12738a

To expand the applicability of the biotin-(strept)avidin system, a biotin analog with reversible binding under non-denaturing conditions has been designed, and its applications in protein labeling, detection, and isolation have been evaluated. The Royal S

Full text of DOI:10.1039/c1cc12738a

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Design of a reversible biotin analog and applications in protein labeling,
detection, and isolationw
Lai-Qiang Ying* and Bruce P. Branchaud
Received 10th May 2011, Accepted 10th June 2011
DOI: 10.1039/c1cc12738a
To expand the applicability of the biotin–(strept)avidin system, a
biotin analog with reversible binding under non-denaturing
conditions has been designed, and its applications in protein
labeling, detection, and isolation have been evaluated.
The interaction between biotin and avidin or streptavidin is
the strongest non-covalent interaction known in nature.1 The
Fig. 1 Illustrations of the biotin-binding pocket and key H-bonding
for many applications in biotechnology and nanotechnology.2–4
interactions between biotin and streptavidin.
biotin–(strept)avidin system has become a standard platform
One of the major limitations of the biotin–(strept)avidin system is
the almost irreversible binding under physiological conditions.5
To disrupt a biotin–(strept)avidin complex, it is usually necessary
to denature the avidin or streptavidin proteins using harsh
conditions.6
In order to make the binding of biotin with (strept)avidin
more easily reversible under mild conditions, much work has
been done to modify avidin or streptavidin by chemical
modification7 and genetic mutations.8 Another approach is
to design biotin analogs with reduced affinity to avidin and
streptavidin.9–11 The reported biotin analogs such as biotin
sulfone, 20-thiobiotin, and desthiobiotin provide partial
reversibility, but there is still a need for better biotin analogs
that permit truly reversible binding, both (1) fast and complete
Scheme 1 Synthesis of N30-ethyl biotin derivatives. (a) HCl, MeOH;
binding, and (2) fast and complete release, under mild,
DMTrCl, DMAP, pyridine; CH3CH2I, NaH, DMF; 80% HOAc; 1 M
physiological conditions.
NaOH, MeOH; TSTU, TEA, DMF; (b) 1,8-diaminotriethyleneglycol,
The crystal structures of biotin–avidin and biotin–streptavidin
DMF; (c) amino-dPEG4-acid, TEA, DMF, H2O; TSTU, TEA, DMF;
complexes provide valuable information for designing new
(d) H-Lys(Boc)-OBut, TEA, DMF; TFA, DCM; AF488-5TFP, TEA,
biotin analogs.12,13 The biotin-binding pocket of streptavidin,
DMF; (e) TSTU, DMAP, DMF; propargylamine, DMF.
shown in Fig. 1, displays a virtually perfect fit with biotin,
and the ureido-ring of biotin provides the most important
Scheme 1 shows the synthesis of the new biotin analog,
N30-ethyl biotin. Biotin 1 was converted to biotin methylester,
followed by selective protection with the 4,40-dimethoxytrityl
(DMTr) group at the N10 position, and alkylation at the
N30 position with iodoethane. The DMTr protecting group
was removed by 80% HOAc, and the methylester was hydrolyzed
to the free acid by NaOH, then was converted to succinimidylester
2 using O-(N-succinimidyl)-1,1,3,3-tetramethyluronium tetra-
fluoroborate (TSTU). The N30-ethyl biotin succinimidylester 2
was converted to 3 using 1,8-diaminotriethyleneglycol.
Compound 2 was also reacted with amino-dPEG4-acid, and
then converted to succinimidylester 4 with TSTU. Compound
4 was conjugated with H-Lys(Boc)-OBut, followed by deprotection
with TFA and labeling with Alexa Fluor 488 to give 5.
hydrogen bonding interactions at the streptavidin-binding
sites. By detailed analysis of the biotin-binding pocket of
streptavidin, the position of 30-NH of biotin has open space
available for modification. Thus, we decided to focus on the
modification at 30-NH of biotin to make lower affinity biotin
analogs by blocking the hydrogen bonding interaction
with Ser45.
Life Technologies, 29851 Willow Creek Road, Eugene, OR 97402,
USA. E-mail: lai-qiang.ying@lifetech.com; Fax: +1 541 335 0206;
Tel: +1 541 335 0158
w Electronic supplementary information (ESI) available: Details of
experiments for synthesis, characterization, capture and release assay,
kinetic measurement, and protein labeling, detection and isolation,
additional cell images. See DOI: 10.1039/c1cc12738a
c
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Chem. Commun., 2011, 47, 8593–8595 8593

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Table 1 The capture and release efficiency of biocytin and N30-ethyl
biotin with streptavidin
off-rate from streptavidin in PBS buffer (koff E 1.2 ꢀ 10ꢁ4 Sꢁ1).
The dissociation constant KD of N30-ethyl biotin is about
0.8 nM. In the presence of 2 mM biotin, the N30-ethyl biotin
Compound
Biocytin
Capture efficiency (%) Release efficiency (%)
displays fast off-rate (koff E 5.8 ꢀ 10ꢁ2
S
ꢁ1), thus leading to
96 ꢂ 2
o5
complete dissociation from streptavidin.
N30-Ethyl biotin 5 94 ꢂ 3
95 ꢂ 4
The reversibility of N30-ethyl biotin with streptavidin
provides valuable applications in protein labeling, detection,
and isolation. To evaluate the efficiency using N30-ethyl biotin,
the N30-ethyl biotin alkyne 6 was prepared for labeling and
isolation of azide-containing proteins via click chemistry.15
Recently, the bio-orthogonal labeling of glycoproteins in
cells incubated with azido sugars or alkynyl sugars has been
developed for imaging the localization, trafficking, and dynamics
of glycans.16–18 We have adapted the azido sugar labeling
system to demonstrate the ability of N30-ethyl biotin for
efficient labeling, detection, and isolation in a biological
system. The HeLa cells were first incubated with azido sugars
(Ac4GlcNAz, Ac4GalNAz, Ac4ManNAz) containing media
for 24 h, respectively. The fixed HeLa cells were labeled
with N30-ethyl biotin alkyne 6 in the presence of CuSO4,
tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), and
sodium ascorbate.19 The cells incubated with azido sugars
show a increased fluorescence signal when compared to control
cells that were not incubated with azido sugars, as shown in
Fig. 3 and ESIw, indicating the labeling of cells with N30-ethyl
biotin alkyne 6 via formation of a triazole linkage. The
enhancement of the fluorescence signal is only observed in
the presence of both catalyst Cu(I) and azido glycans (ESIw).
The Cu(I) chelating ligand, THPTA, facilitates the reaction
and protects protein damage,19 but is not necessary for labeling
of cells. These results demonstrate N30-ethyl biotin alkyne 6 is
an efficient reagent for labeling and detection in cells.
Compound 5 was converted to succinimidylester, then reacted
with propargylamine to afford 6.
In order to evaluate the binding affinity and reversibility of
N30-ethyl biotin with streptavidin, a simple fluorescent binding
and release assay was designed. Briefly, N30-ethyl biotin-
AF488 conjugate 5 was incubated with M-280 Streptavidin
Dynabeads in pH 7.4 PBS buffer. After 25 min incubation at
room temperature, the magnetic beads were separated from
the supernatant, which was used to measure the capture
efficiency, by measuring the fluorescent intensity of the
supernatant solution to determine the amount that was not
captured. Then, the N30-ethyl biotin 5 bound magnetic beads
were washed with PBS buffer to remove weak or non-specific
binding. The reversibility was tested by incubating the N30-ethyl
biotin 5 bound magnetic beads with 2 mM biotin in PBS buffer
at room temperature for 5 min, then the magnetic beads were
concentrated by a magnet, and the fluorescent intensity in
the solution was measured to determine the amount that was
released. As a control, the biocytin-AF488 conjugate was
also tested in the same assay format. As shown in Table 1,
the N30-ethyl biotin displays high binding efficiency and the
binding is fully reversible with a biotin competing reagent
under non-denaturing conditions.
The binding constant and on-and-off rate constant of
N30-ethyl biotin with streptavidin were tested using a label-
free ForteBio system.14 The N30-ethyl biotin 3 was covalently
immobilized to amine reactive sensor tips from ForteBio. To
generate the association curve, the N30-ethyl biotin labeled
biosensor tips were incubated with streptavidin at 25 1C. The
dissociation curve was determined by incubating the streptavidin
bound sensor tips in PBS buffer described above, as well
as in PBS buffer containing 2 mM biotin at 25 1C. As
shown in Fig. 2, the N30-ethyl biotin shows a fast on-rate with
In order to isolate azido sugar containing glycoproteins
from a cell mixture, the Jurkat cells were cultured in Ac4GlcNAz
supplemented media for 24 h. The cell lysate was prepared at
B3 mg mLꢁ1 in 0.5% SDS/PBS. N30-Ethyl biotin alkyne 6
and biotin alkyne 7 (Invitrogen) were used to perform parallel
protein enrichment experiments. Briefly, cell lysate was incubated
with alkyne 6 or 7 in the presence of CuSO4, THPTA, and
sodium ascorbate at room temperature. After click labeling,
the proteins were enriched with streptavidin agarose at room
streptavidin (kon E 1.5 ꢀ 105 Mꢁ1
S
ꢁ1), and a relative slow
Fig. 3 Fluorescence microscopy of HeLa cells labeled with N30-ethyl
biotin alkyne 6. Cells incubated with Ac4ManNAz supplemented
media (top) or unsupplemented media (bottom) were labeled with
N30-ethyl biotin alkyne 6 in the presence of Cu(I)/THPTA as a
catalyst. Fluorescence signal from AF488 colored green (left), the
nuclear stain, Hoechst 33342, colored blue (center), and the merged
images (right) are shown.
Fig. 2 Association and dissociation curves between N30-ethyl biotin
and streptavidin on the ForteBio system.
c
8594 Chem. Commun., 2011, 47, 8593–8595
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agarose via interaction of N30-ethyl biotin with streptavidin.
One major problem using biotin alkyne 7 is the isolated
proteins are contaminated with streptavidin (SA), which is
leached out from agarose beads by the harsh, denaturing
condition. These results demonstrate using N30-ethyl biotin
has great advantage in gentle release to isolate clean proteins
for downstream analysis.
In summary, we have developed a new biotin analog,
N30-ethyl biotin, with reversible binding affinity with streptavidin
(KD E 0.8 nM), and with excellent capture and release
efficiency with streptavidin under gentle non-denaturing
conditions. Applications in glycoprotein labeling, detection,
and isolation using N30-ethyl biotin alkyne 6 via click
chemistry have been demonstrated. This new biotin analog
should be useful to expand the new applications of standard
biotin–streptavidin technology.
Notes and references
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Fig. 4 Isolation of glycoproteins using click chemistry. Cell lysate
was incubated with N30-ethyl biotin alkyne 6 or biotin alkyne 7 in the
presence of Cu(I)/THPTA, captured by streptavidin agarose, washed,
and eluted with either 10 mM HCl or 1% SDS in H2O at 95 1C,
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temperature. After the washing step, the proteins labeled with
N30-ethyl biotin alkyne 6 were eluted out from streptavidin
agarose using 2 mM biotin or 10 mM HCl in H2O; the proteins
labeled with biotin alkyne 7 were eluted out by heating
protein-bound streptavidin agarose in 1% SDS in H2O at
95 1C for 5 min. As a control, cell lysate was also incubated
with alkyne 6 or 7 without the Cu(I) catalyst. Another control
is using untreated cell lysate to perform the same isolation
procedure. As shown in Fig. 4, both control experiments
without the Cu(I) catalyst, and without Ac4GlcNAz treatment,
show a very low background using N30-ethyl biotin alkyne 6,
indicating the azido containing glycoproteins were selectively
isolated by labeling with N30-ethyl biotin alkyne 6 via formation
of a triazole linkage, followed by capture with streptavidin
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 8593–8595 8595