Vinyl sulfone: A versatile function for simple bioconjugation and immobilization

Article abstract of DOI:10.1039/b920576d

The easy functionalization of tags and solid supports with the vinyl sulfone function is a valuable tool in omic sciences that allows their coupling with the amine and thiol groups present in the proteogenic residues of proteins, in mild and green conditions compatible with their biological function.

Full text of DOI:10.1039/b920576d

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Vinyl sulfone: a versatile function for simple bioconjugation and
immobilization†
Julia Morales-Sanfrutos, Javier Lopez-Jaramillo, Mariano Ortega-Mun˜oz, Alicia Megia-Fernandez,
Francisco Perez-Balderas, Fernando Hernandez-Mateo and Francisco Santoyo-Gonzalez*
Received 2nd October 2009, Accepted 3rd November 2009
First published as an Advance Article on the web 11th December 2009
DOI: 10.1039/b920576d
The easy functionalization of tags and solid supports with the vinyl sulfone function is a valuable tool
in omic sciences that allows their coupling with the amine and thiol groups present in the proteogenic
residues of proteins, in mild and green conditions compatible with their biological function.
under mild conditions, the second least abundant amino acid
Introduction
in proteins with a frequency of 1.36%.2 The water stability of
The covalent coupling of two biomolecules to each other (bio-
the vinyl sulfone function, the lack of by-products, the almost
conjugation) or to a solid support (immobilization) is one of the
cornerstones of omic sciences.1 Thus, labeling with biophysical
probes and biotinylation of proteins are standard techniques
quantitative yields of the reaction with thiols and the stability of
the thioether linkage formed are appealing characteristics of this
reaction that in practice, however, has been used almost exclusively
nowadays, while tethering of biomolecules to solid supports is
for PEGylation of proteins using end-functionalized vinyl-sulfone
PEG derivatives.8 In contrast with the facile functionalization
of cysteine by reaction with vinyl sulfones, the reaction of this
function with lysine residues has been reported8a,9 to be slow
and incomplete, occurring only at high pH values (pH >9.3),
conditions not always compatible with the biological function of
proteins.
Taking into account our previous results on the good reactivity
of primary amines with vinyl sulfone sugar derivatives,10 we
decided to explore the feasibility of using the vinyl sulfone function
in bioconjugation under mild conditions. We report here on the
capabilities of the vinyl sulfone function for the covalent coupling
of proteins to detection labels, other biomolecules and solid
supports by exploiting the Michael-type addition of this function
to the amine-containing residues, in conditions that preserve
the functionality and biological integrity of those biological
macromolecules.
exploited in the fabrication of arrays and in the immobilization
of enzymes. For proteins, the straightforward and probably most
widely used methodologies take advantage of the reactivity of
the naturally occurring functional groups present in proteogenic
amino acids towards labeling reagents or supports, commonly by
means of a nucleophile-to-electrophile attack. Lysine is by far
the most targeted residue because of its predominant presence
(the 11th most frequent residue),2 the reactivity of the e-amine
group of its side chain, its minor relevance from a biological
point of view, and its accessibility at the surface of those
biomolecules. Amine-reactive labels are usually acylating agents
such as sulfonyl chlorides, isothiocyanates and succinimidyl esters.
However, they are not exempt from drawbacks. Sulfonyl chlorides
are highly reactive but also unstable in water,3 especially at the
high pH required for the reaction with aliphatic amines, and
they can also react with phenols (tyrosine), aliphatic alcohols
(serine, threonine), thiols (cysteine) and imidazoles (histidine).
Isothiocyanates are stable in water, although their reactivity is
only moderate, and the degradation of the resulting thiourea
has been reported.4 Succinimidyl esters are the best suited amine
reagent for derivatization, and the less reactive but more soluble
sulfosuccinimidyl esters have been used to overcome their poor
water solubility.5 For these reasons, the chemical modification
of complex biomolecules, such as proteins, by simple procedures
under physiological conditions remains a challenge.
Results and discussions
Synthesis of vinyl sulfone-derivatized reagents
To study the value of the vinyl sulfones as a viable new tool in
the arsenal of bioconjugation strategies, we introduced the vinyl
sulfone function to a set of commonly used synthetic tags with a
biotechnological interest in bioconjugation such as rhodamine B
and dansyl (fluorescent tags), biotin (affinity tag), and ferrocene
(electrochemical tag),11 as well as on glucose, chosen as an example
of a simple biomolecule (Scheme 1).
The vinyl sulfone-derivatization of the fluorescent tags and
biotin was carried out by reaction of a scaffold bearing both a vinyl
sulfone group and an amine group with commercial rhodamine
B and biotin, previously transformed into the corresponding acid
chloride derivatives, and dansyl chloride. The synthesis of such
an amine–vinyl sulfone scaffold was approached by reaction of a
bis-vinyl sulfone with a primary amine. To avoid the reported
intramolecular process in the reaction of divinyl sulfone with
In this context, the Michael-type addition of vinyl sulfones6
is an attractive methodology for protein conjugation.7 Up to
the present, vinyl sulfone chemistry has been demonstrated to
be suitable for the selective modification of cysteine residues
Departamento de Q. Orga´nica, Facultad de Ciencia, Instituto de Biotecnolo-
gia, Universidad de Granada, Granada, 18071 SPAIN. E-mail: fsantoyo@
ugr.es; Fax: (+34)-958243186; Tel: (+34)-958248087
† Electronic supplementary information (ESI) available: 1H and 13C-NMR
spectra for compounds 1–5, 8, 9 and 11, and some further details on the
characterization of the reactions of vinyl sulfone derivatives with proteins.
See DOI: 10.1039/b920576d
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Scheme 1 Synthesis of vinyl sulfone-functionalized tags and sugars.
primary amines leading to 1,4-thiazane-1,1-dioxide derivatives,12
a longer bis-vinyl sulfone (1) was synthesized by reaction of
divinyl sulfone with ethylene glycol. However, the reaction of 1
with ethanolamine or propylamine did not lead to the desired
monoaddition products and, to overcome this difficulty, the use of
a steric hindrance amine was envisioned as a feasible alternative.
By this route, the desired amine–vinyl sulfone scaffold 2 was
obtained in 57% yield by reaction of 1 with sec-butylamine in
Cl2CH2 : isopropanol. Treatment of the amine–vinyl sulfone 2 with
rhodamine B acid chloride and dansyl chloride gave the fluorescent
labels 3 and 4, in 54% and 74% yield, respectively. The reactions
were carried out in anhydrous Cl2CH2 and anhydrous acetonitrile,
respectively, because the former overcame the solubility problems
of rhodamine B acid chloride, and the latter led to shorter
reaction times and higher yields. The same procedure was used
for the coupling of biotin acid chloride in anhydrous THF in the
presence of Et3N, leading to the biotinylation reagent 5 in 63%
yield.
On the other hand, the vinyl sulfone-functionalized glucose
derivative 9 was obtained by oxidation and elimination of the
2-chloroethyl-1-thio-b-D-glucopyranoside 8, which was easily ac-
cessible from per-O-acetylated-b-D-glucose by successive treat-
ment with thiourea and 1-bromo-2-chloroethane, and subsequent
de-O-acetylation. Finally, vinyl sulfone ferrocene 11 was easily
obtained by reaction of methanol ferrocene 10 with divinyl sulfone
(Scheme 1).
are affected by the surrounding residues. The degree to which a
protein is labeled is also dependent on the conjugation process,
and labeling reactions are influenced by both the molar ratio of
the reactants and the activity of the labeling reagent. Considering
these facts, and in order to put to the test our hypothesis
about the bioconjugation capabilities of the synthesized vinyl
sulfone reagents, a pool of four commercial proteins (avidin,
Concanavalin A (ConA), lysozyme and BSA), comprising both
acidic and alkaline isoelectric points, monomeric and oligomeric
structures, different numbers of potential Michael donors and
the presence/absence of free thiol groups, and post-translational
modifications were selected (Table 1).
The evaluation of the vinyl sulfone-derivatization of tags to label
proteins in mild conditions was carried out in the first instance on
fluorescent labels 3 and 4 that were conjugated with ConA and
avidin, respectively, to facilitate the monitoring of the reaction
(Scheme 2). The conjugation process was carried out at pH 8,
37 ◦C, and with different stoichiometries and reaction times (3, 8
and 24 h). After analysis of conjugates 12 and 13 by SDS-PAGE,
the results showed that ConA is labeled very efficiently within
3 h, with no appreciable differences detected between 8 and 24 h
of reaction, while avidin is poorly labeled within 3 h and longer
reaction times yield more intense fluorescence (Fig. 1).
In spite of the number of potential nucleophiles (20 for ConA
versus 10 for avidin), the molar excess of the labeling reagent
was depleted by the latter but not by the former. A closer
analysis reveals that higher stoichiometries do not yield more
intense fluorescence, but they provoke the precipitation of ConA
(ESI, Fig. S1†), probably due to the hydrophobicity associated
with the modification of the hydrophilic nature of the lysine
residues, and in the case of avidin, even less intense fluorescence,
whose over-labeling causes quenching (ESI, Fig. S2†). These
results demonstrate that the protein itself influences the extent
of the coupling, and that the labeling of proteins with vinyl
Bioconjugation of proteins by vinyl sulfone-functionalized reagents
The vinyl sulfone-derivatization described above yields activated
electrophilic olefin reagents that may react with nucleophiles by
a Michael-type addition. In mild conditions, amine and thiol
groups may deprotonate and act as Michael donors, but for
the particular case of proteins, their accessibility and pKa values
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Table 1 Main features of the pool of model proteins
Nucleophilic residues (per monomer)
Quaternary Molecular
structure
Isoelectric Post-translational
Entry Protein
weight/kDaa pointa
modifications
Lys
His
Cys (free)
S–S bonds
1
2
3
4
ConA
Avidin
Lysozyme Monomer
BSA Monomer
Tetramer
Tetramer
4 ¥ 26.6
4 ¥ 17
14.3
5.3
9.5
11
None
Glycosylation
None
12
9
6
6
1
1
17
None
None
None
1
None
1
4
17
66.5
5.6
None
59
a Estimated from the sequence, except for lysozyme whose isoelectric point was taken from Alderton et al.13
Scheme 2 Applications of vinyl sulfone-functionalized tags and sugars in bioconjugation of proteins.
Fig. 1 SDS-PAGE of the reaction of ConA with 3 (red) and avidin with
4 (green) for 3, 8 and 24 h (from left to right) (conjugates 12 and 13,
respectively).
Fig. 2 Identification of residues present in the HEW lysozyme that react
with vinyl sulfone glucose 5. 2mFo-DFc maps contoured at 1.2 s (blue) or
5 s (red) showing the electron density that defines the sugars bonded to
Lys96 (right) and His15 (left).
sulfone-derivatized reagents is feasible regardless of the
isoelectric point, number of potential nucleophiles or
glycosylation.14
Influence of the reaction conditions on the bioconjugation of vinyl
sulfone-functionalized reagents
Having demonstrated the conjugation of vinyl sulfone-
functionalized reagents with proteins lacking free Cys under
mild conditions, we focused on the identification of the amino
acid residues that react with the vinyl sulfone function. For
this purpose, HEW lysozyme was glycosylated by reaction with
glucopyranosyl vinyl sulfone 9 in phosphate buffer pH 7.7 at
room temperature. The resulting conjugate 15 (Scheme 2) was
We next addressed the influence of the reaction conditions on
the coupling of vinyl sulfone-functionalized reagents to model
proteins. First, HEW lysozyme was labeled with vinyl sulfone
rhodamine B 3 at pH ranging from 5 to 8, and both at room
temperature and 30 ◦C (conjugate 14, Scheme 2). The resulting
labeled proteins were analyzed by MALDI-TOF (Fig. 3), which
showed that the reaction takes place even in acidic media, and that
slight variations of pH or temperature exert a clear direct effect
on the number of labels (3) coupled to the HEW lysozyme, i.e.
the number of fluorescent tags increases with pH or temperature.
Second, it was demonstrated that the stoichiometry, in terms of
the vinyl sulfone groups : protein reactive groups (i.e. Lys, His
and Cys residues) ratio, also plays a role. Thus, the extent of the
˚
crystallized by hanging drop, and the structure solved at 1.6 A
by X-ray diffraction (PDB entry 2B5Z). The electron density map
clearly revealed that both Hys and Lys residues act as Michael
donors in the reaction with 9.15 These results prove that, contrary
to the generally accepted view, Lys residues not only react with
a vinyl sulfone-containing reagent at a pH compatible with the
biological nature of the sample, but that they may also yield a
double addition process (Fig. 2).
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Fig. 3 MALDI-TOF spectra of the HEW lysozyme labeled with vinyl sulfone rhodamine B 3 at rt (left) and 30 ◦C (right), and pH 5, 6, 7 and 8 (from
top to bottom). Molecular weight of lysozyme is 14 290 and that is increased by 796.4 Da per molecule of rhodamine B 3 coupled: A HEW + 1Rh;
B HEW + 2Rh; C HEW + 3Rh; D HEW + 4Rh; E HEW + 5Rh; F HEW + 6Rh.
Table 2 Influence of the stoichiometry on the labeling of BSA with vinyl
sulfone ferrocene 11
and is considered as essentially non-reversible and unaffected by
denaturing agents. Thus, after denaturation of the sample in mild
conditions, the SDS-PAGE showed a fluorescent signal at a high
molecular weight that corresponded to BSA–biotin–avidin–dansyl
complexes (Fig. 4, left). In order to further assess the identity
of these complexes, a new electrophoresis was carried out with
samples being denatured in standard conditions (Fig. 4, right).
The gel demonstrated that the fluorescence at high molecular
weights did not correspond to BSA since its molecular weight
is higher, and it disappeared when more stringent denaturation
conditions were employed. These results demonstrate that the
fluorescent high molecular weight band consists of BSA–biotin–
avidin–dansyl complexes, and that the coupling of vinyl sulfone-
derivatized biotin and dansyl yield fully functional species.
BSA : 11/mola
BSA reactive groups : 11
Fe per BSA/mol
1 : 10
1 : 50
1 : 100
1 : 0.13
1 : 0.65
1 : 1.30
9.28
25.03
28.75
a Estimated from the determination of Fe present in the sample resulting
from the coupling.16
labeling of BSA with the vinyl sulfone ferrocene electrochemical
tag 11, at pH 8.5 and room temperature, that yielded conjugated
16 (Scheme 2), showed that at an 11 : BSA ratio as unfavourable as
0.13 : 1 (i.e. 10 mols of 11 per mol of BSA) yielded between 9 and
10 labels per molecule of BSA. As the 11 : BSA ratio increased,
the extent of labeling also increased to reach a maximum that
corresponded to less than 30 labels (see Table 2). A closer analysis
of these results based on the existence in BSA of a single free Cys
and 77 reactive groups further demonstrates the good reactivity of
amine groups present in proteins towards vinyl sulfones, and the
existence of reactive groups that do not react.
Functionality of the vinyl sulfone-labeled model proteins
Although fluorescent labeling is an important application, macro-
molecule labeling is also aimed at promoting/detecting the
interaction between the labeled molecule and others. In this
context, the avidin–biotin technology17 plays a central role, and the
introduction of the avidin–biotin complex into a given system is a
widely used approach that serves to mediate between a recognition
system (e.g. antibody–antigen or lectin–carbohydrate) and a
reporter group (e.g. enzymatic activity or fluorescence). To explore
this range of applications with the new vinyl sulfone tags, BSA was
labeled with vinyl sulfone-derivatized biotin 5 and the resulting
conjugate 17 combined with the dansyl-labeled avidin conjugate
13 reported above (Scheme 2).14 The functionality of both the
biotinylated BSA and the fluorescently labeled avidin was analyzed
by SDS-PAGE, since the avidin–biotin interaction is one of the
strongest non-covalent biological interactions (Ka = 1015 M-1),
Fig. 4 SDS-PAGE, in mild (left) and standard (right) denaturing
conditions, of the complex BSA–biotin–avidin–dansyl at BSA–biotin
(17) : avidin–danysl (13) stoichiometries of 4 : 4 (lane 1) and 4 : 1 (lane 2).
Lane 3 is BSA pre-stained with 3.
Considering that avidin is a tetramer, a 4 : 4 stoichiometry
of dansyl–avidin : biotin–BSA should yield a full occupancy of
the four biotin binding sites, and a concomitant more intense
fluorescence than that for a 4 : 1 stoichiometry. As expected, this
higher intensity was observed. However, in both cases, single
species were detected by Coomassie because either they did not
interact, or they did not withstand the mild denaturing conditions
(ESI, Fig. S3†).
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Vinyl sulfone-derivatized rhodamine B as a pre-stain reagent for
electrophoresis
During the electrophoresis performed in the labeling assays
described above, it was noticed that the bright purple color of
the vinyl sulfone-derivatized rhodamine B 3 was transferred to
the labeled protein and made it visible to the naked eye. This
feature suggests a potential application in SDS-PAGE as a pre-
stain dye that, besides yielding fluorescence for sensitive detec-
tion/quantification, allows monitoring of the protein migration
during the electrophoretic separation. In fact, the use of Remazol
dyes, which at alkaline pH are converted to their vinyl sulfone
derivatives, was proposed as a pre-stain reagent for visualization
of proteins in SDS–polyacrylamide gels.18
Rhodamine–vinyl sulfone (3) was put to the test, bearing in
mind that a pre-stain dye may be attractive if (i) the protocol of
pre-staining is straightforward and general, (ii) the detection limit
is good enough, (iii) the migration pattern remains unaltered,
and (iv) the pre-staining is compatible with conventional post-
electrophoresis stains. The study was carried out on the model
monomeric proteins BSA and lysozyme (Table 1). BSA, with an
isoelectric point of 5.6 and 77 potential nucleophilic residues,
among them 1 free Cys, is a good model for an acidic protein,
while lysozyme, with an alkaline isoelectric point of 11 and only
7 potential nucleophilic residues, represents an extreme case of
a protein with low reactivity expected towards vinyl sulfone.
The results led to the conclusion that a procedure as simple
as incubation of the sample with the vinyl sulfone-derivatized
rhodamine B 3 at 100 ◦C for 5 min, in HEPES pH 8.8, yielded the
detection of roughly 1 mg by the purple color (Fig. 5) and 125 ng
by fluorescence in a conventional transilluminator (Fig. 6). The
subsequent staining of the sample revealed that the pre-staining
is fully compatible with a post-electrophoresis silver stain, and
electrophoretic mobility is not altered (Fig. 6). Improvement in the
sensitivity of the detection may be achieved by exciting the sample
at a more suitable wavelength (ESI, Fig. S4†) and recording on a
CCD camera.14
Fig. 6 SDS-PAGE of pre-stained samples. Fluorescence (left) of BSA
(lanes 1 and 3) and lysozyme (lanes 2 and 4) pre-stained with vinyl sulfone
rhodamine 3, and post-electrophoresis silver staining (right). The amount
of protein per lane was 250 ng (lanes 1 and 2) and 125 ng (lanes 3 to 6).
Control non-pre-stained samples were in lanes 5 and 6.
decoupling pre-staining from denaturation, since the latter is
carried out in a mixture containing amine and thiol groups that
react with the vinyl sulfone groups. However, the penalty of the
decoupling is an affordable short incubation of 10 min at no risk,
since pre-staining with vinyl sulfone-derivatized rhodamine B 3
does not interfere with a conventional post-electrophoresis silver
stain (Fig. 6).
Immobilization of proteins by vinyl sulfone silica
Another explored area of application of the vinyl sulfone group’s
reactivity towards biomolecules was immobilization onto solid
supports. The preservation of the biological functionality of
the biomolecules once immobilized is a requisite of primary
importance. In this context, we chose silica as a solid support based
on the appealing characteristics of silica-based hybrid materials.20
Our approach was based on the vinyl sulfone-functionalization
of silica that was easily performed by silanization of activated
commercial silica with [3-(methylamine)propyl]-trimethoxysilane
and subsequent reaction with divinyl sulfone (Scheme 3).21
The capability of the resulting vinyl sulfone silica 19 to immo-
bilize biomolecules while preserving their functionality was then
evaluated using invertase (Scheme 3). Invertase is a model enzyme
that catalyzes the hydrolysis of sucrose to fructose and glucose,
a process of interest to the food industry. The immobilization on
vinyl sulfone silica 19 was carried out in batch, at both room
temperature and 4 ◦C, by simple addition of the functionalized
silica to a solution of the enzyme in a standard phosphate buffer
at pH 7.5. The results show that the reaction takes place even at
4
◦C, although with lower yield, as expected from the influence
of the temperature on the reaction, but the enzymatic activity is
preserved, as the specific activity of the enzyme immobilized at
4 ◦C to hydrolyze sucrose is about 2.5 times larger (Table 3).21
Fig. 5 Sensitivity of pre-staining BSA (lanes 1 to 4) and lysozyme (lanes 5
to 8) with vinyl sulfone rhodamine (3). The amount of pre-stained protein
loaded was 5 mg (lanes 1 and 5), 2.5 mg (lanes 2 and 6), 1.25 mg (lanes 3
and 7) and 0.625 mg (lanes 4 and 8).
Table 3 Immobilization of invertase onto vinyl sulfone silica 19 and
functionality of the immobilized enzyme 20 to hydrolyze sucrose
Further experiments demonstrated that shorter incuba-
tion times were insufficient, and that longer times or a larger molar
excess of vinyl sulfone-derivatized rhodamine B 3 yielded stronger
fluorescence at the cost of retarding the mobility of the proteins, a
common drawback described for other lysine-modifying agents.19
At this point, it is important to remark on the importance of
Immobilized
invertase/mg
invertase per gram 19 immobilized invertase
Specific activity of immobilized
invertase/arbitrary units per mg
Temperature
rt
100.8
50.2
1.40
3.44
4 ◦
C
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Scheme 3 Synthesis of vinyl sulfone silica 19 and immobilization of proteins.
Elmer 141 polarimeter at room temperature. IR spectra were
recorded on a Satellite Mattson FTIR. 1H and 13C NMR spectra
were recorded at room temperature on a Varian Direct Drive
(300, 400 and 500 MHz) spectrometer. Chemical shifts are given
in ppm and referenced to internal CDCl3. J values are given in
Hz. FAB mass spectra were recorded on a Fissons VG Autospec-
Q spectrometer, using m-nitrobenzyl alcohol or thioglycerol as
matrix. MALDI-TOF and NALDI-TOF mass spectra were
recorded on an Autoflex Brucker spectrometer using HCCA
and NaI, respectively, as matrix. Divinylsulfone, rhodamine B,
dansyl chloride, biotin, ferrocene methanol, lysozyme, ConA,
avidin, BSA, Saccharomyces cerevisiae, and sec-butylamine were
purchased from commercial suppliers.
Conclusions
In conclusion, the Michael-type addition of amine and thiol
groups naturally occuring in biomolecules to vinyl sulfone is a
methodology wide in scope. The novelty of the methodology
presented herein relies on the ability of a protein to react with vinyl
sulfone-functionalized reagents via the amine groups present in the
side chain of their Lys and His residues, in mild conditions that
preserve the biological functionality of proteins. The approach
is a general strategy for bioconjugation and immobilization of
proteins. The results described herein demonstrate the feasibility
of the vinyl sulfone-derivatization to tackle the fluorescent labeling
of proteins and their biotinylation, two important tools in omic
sciences, and also the capability of vinyl sulfone silica to act as
a universal support to immobilize proteins while preserving their
functionality. Moreover, the vinyl sulfone-derivatized rhodamine
B is a suitable reagent for the routine labeling of proteins prior
to electrophoretic separation, the pre-staining being compatible
with a later standard silver or Coomassie Brilliant Blue staining.
Although the protein itself influences the extent of the coupling,
the reactions take place regardless of their isoelectric point, num-
ber of potential nucleophiles, or the presence of post-translational
modifications or free Cys residues, and the reaction is simple (it
involves the simple combination of both species), efficient (it takes
place even at 4 ◦C) and “green” as it requires benign solvent (i.e.
aqueous media), leading to stable linkages with an absence of by-
products. In addition, it can be anticipated that in the context
of life sciences, the scope of the vinyl sulfone function will also
involve its use as a practical convergent approach for protein post-
translational modifications that will expand the actual repertory
of methodologies, as demonstrated with the glycosylation of
lysozyme
Synthesis of vinyl sulfone tags and reagents
Synthesis of bis-vinyl sulfone 1. t-BuOK (119 mg, 1.1 mmol)
was added to a solution of divinyl sulfone (DVS) (1.6 mL, 16 mmol)
and ethylene glycol (330 mg, 5.3 mmol) in THF (100 mL). The
reaction mixture was stirred at rt for 30 min. Removal of the
solvent under reduced pressure yielded a crude that was purified
by column chromatography (AcOEt–hexane 2 : 1 → 3 : 1) giving 1
(805 mg, 51%) as a syrup. nmax(film)/cm-1: 1608, 1472, 1382, 1310,
1
and 1121; H-NMR (CDCl3, 400 MHz): d 6.76 (dd, 2 H, J 16.6
and 9.8 Hz), 6.41 (d, 2H, J 16.6 Hz), 6.10 (d, 2 H, J 9.8 Hz), 3.90
(t, 4 H, J 5.8 Hz), 3.63 (s, 4 H), 3.25 (t, 4 H, J 5.6 Hz); 13C-NMR
(CDCl3, 75 MHz): d 137.9, 129.1, 70.3, 64.7, 55.0. HRMS (m/z)
(FAB+) calcd. for C10H18O6S2Na [M + Na]+: 321.0442; found:
321.0442.
Synthesis of amino vinyl sulfone 2. To a solution of 1 (1.0 g,
3.3 mmol) in Cl2CH2–2-propanol (2 : 1, 45 mL) was added sec-
butylamine (227 mL, 2.2 mmol). The reaction mixture was kept
at rt for 6 h. Evaporation of the solvent yielded a crude that was
purified by column chromatography (AcOEt → AcOEt–MeOH
10 : 1) giving 2 (472 mg, 57%) as a syrup. nmax(film)/cm-1: 2961,
Experimental
1
2922, 2872, 1604, 1459, 1378, 1311, 1287, and 1121; H-NMR
General experimental procedures
(CDCl3, 400 MHz): d 6.71 (dd, 1 H, J 16.6 and 10 Hz), 6.34 (d, 1
H, J 16.6 Hz), 6.06 (d, 1 H, J 10 Hz), 3.84 (m, 4 H), 3.58 (s, 4 H),
3.29 (t, 2 H, J 5.3 Hz), 3.21 (m, 4 H), 3.06 (m, 2 H), 2.53 (m, 1 H),
2.00 (br s, 1 H), 1.42 (m, 1 H), 1.27 (m, 1 H), 0.98 (d, 3 H, J 6.3 Hz),
0.83 (t, 3 H, J 7.4 Hz); 13C-NMR (CDCl3, 75 MHz): d 137.9, 129.1,
70.4, 70.3, 64.8, 64.6, 55.3, 54.9, 54.6, 54.4, 40.3, 29.4, 19.6, 10.1.
HRMS (MALDI-TOF) calcd. for C14H29O6NS2Na [M + Na]+:
394.1334; found: 394.1334.
TLC was performed on Merck Silica Gel 60 F254 aluminium
sheets. Reagents used for developing plates include potassium
permanganate (1% w/v), ninhydrin (0.3% w/v) in ethanol and
UV light when applicable. Flash column chromatography was
performed on Silica Gel, Merck, (230–400 mesh, ASTM). Melting
points were measured on a Gallenkamp melting point apparatus
and are uncorrected. Optical rotations were recorded on a Perkin-
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Synthesis of vinyl sulfone rhodamine B 3. A solution of
rhodamine B (340 mg, 0.71 mmol) in Cl2SO (7 mL) was kept at
rt overnight. The reaction mixture was evaporated under vacuum
and co-evaporated with anhydrous toluene (3 ¥ 15 mL) to give
rhodamine B acid chloride. The crude acid chloride was dissolved
in anhydrous CH2Cl2 (10 mL) and added dropwise to a solution of
2 (240 mg, 0.65 mmol) and Et3N (184 mL, 1.29 mmol) in anhydrous
CH2Cl2 (15 mL). The reaction mixture was kept at rt for 10 min.
Evaporation of the solvent yielded a crude that was purified by
column chromatography (CH2Cl2–MeOH 30 : 1 → 10 : 1) giving
3 as a foam solid (278 mg, 54%). nmax(film)/cm-1: 1640, 1589,
1466, 1413, 1339, 1275, 1180, 1128;1H-NMR (Cl3CD, 500 MHz):
d 7.71-7.55 (several m, 3 H), 7.39-7.23 (several m, 3 H), 7.03 (d,
1 H, J 9.4 Hz), 6.89 (d, 1 H, J 10 Hz), 6.82 (br s, 2 H), 6.75 (dd,
1 H, J 16.6 and 9.9 Hz), 6.33 (d, 1 H, J 16.6 Hz), 6.07 (d, 1 H,
J 9.9 Hz), 3.87-3.57 (several m, 21 H), 3.23 (t, 2 H, J 5.7 Hz),
3.13 (t, 2 H, J 5.3 Hz), 1.43-1.26 (several m, 14 H), 0.89-0.67
(several m, 6 H); 13C-NMR (Cl3CD, 125 MHz): d 169.5, 157.8,
157.6, 155.7, 155.0, 137.9, 136.1, 132.6, 130.5, 130.4, 129.8, 129.5,
128.9, 128.2, 114.6, 113.7, 113.6, 113.5, 96.6, 96.5, 70.3, 70.2, 67.1,
64.5, 64.2, 56.7, 54.8, 53.7, 52.5, 46.3, 46.2, 34.6, 18.4,13.7, 12.7,
11.2; HRMS (m/z) (MALDI-TOF) calcd. for C42H58N3O8S2 [M]+:
796.366; found: 796.366.
43.8, 38.2, 37.0, 32.6, 32.5, 31.5, 29.1, 22.4, 22.3, 14.3. HRMS
(MALDI-TOF) calcd. for C24H43N3O8S3Na [M + Na]+: 620.2109;
found: 620.2110.
Synthesis of 2-chloroethyl 2,3,4,6-tetra-O-acetyl-1-thio-b-D-
glucopyranoside (7). To a solution of 1,2,3,4,6-penta-O-acetyl-
b-D-glucopyranose 623 (11.7 g, 30 mmol) and thiourea (2.53 g,
33 mmol) in dry acetonitrile (70 mL) was added BF3·Et2O (8 mL,
63 mmol). The reaction mixture was refluxed for 3 h. After cooling
1-bromo-2-chloroethane (5.9 mL, 69 mmol) and Et3N (13.5 mL,
90 mmol) were added and the reaction mixture was magnetically
stirred at rt for 12 h. Evaporation under vacuum of the solvent
gave a crude that was dissolved in CH2Cl2 (200 mL) and washed
with water (100 mL). The organic phase was dried (Na2SO4).
Evaporation of the solvent yielded a crude that was purified by
column chromatography (CH2Cl2 → EtOAc–hexane 1 : 1) giving
7 as a solid (10.6 g, 83%). M.p. 100–102 ◦C (lit22 97–98 ◦C); [a]D
◦
◦
1
-30 (c 1, chloroform) [lit22 -38 (c 0.7, chloroform)]; H NMR
(CDCl3, 300 MHz): d 5.24 (t, 1H, J = 9.3 Hz), 5.07 (t, 1H, J =
9.7 Hz), 5.03 (t, 1H, J = 9.8 Hz), 4.55 (d, 1H, J = 10.0 Hz), 4.30-
4.10 (m, 2H), 3.80-3.60 (m, 3H), 3.09 (ddd, 1H, J = 14.0, 9.8 and
6.1 Hz), 2.90 (ddd, 1H, J = 14.1, 9.4 and 6.6 Hz), 2.10, 2.06, 2.04,
2.01 (4 s, 12H); 13C NMR (CDCl3, 75 MHz): d 169.4, 83.9, 76.1,
76.7, 69.8, 68.3, 62.1, 43.4, 32.7, 20.7, 20.6; HRMS (m/z) (FAB+)
calcd. for C16H23ClO9S + Na: 449.065; found 449.063.
Synthesis of vinyl sulfone dansyl 4. To a solution of dansyl
chloride (130 mg, 0.48 mmol) in anhydrous acetonitrile (15 mL)
were added 2 (150 mg, 0.40 mmol) and Et3N (115 mL, 0.8 mmol).
The reaction mixture was kept at rt for 2.5 d. Evaporation
of the solvent yielded a crude that was purified by column
chromatography (AcOEt–hexanes 1 : 1 → 3 : 1) giving 4 as a syrup
(182 mg, 74%). nmax(film)/cm-1: 2929, 2871, 1569, 1456, 1387, 1311,
Synthesis of 2-chloroethyl-1-thio-b-D-glucopyranoside (8). To
a solution of 7 (3 g, 7.03 mmol) in methanol (60 mL) was added
Et3N (15 mL). The reaction was kept at 40 ◦C for 8 h. Evaporation
under vacuum yielded a crude product that was purified by column
chromatography (AcOEt–MeOH 9 : 1) yielding 8 (1.56 g, 86%)
as a solid. M.p. 88–90 ◦C; [a]D -42◦ (c 1, methanol); H NMR
1
1
and 1132 cm-1; H-NMR (CDCl3, 400 MHz): d 8.55 (d, 1 H, J
(CD3OD, 300 MHz): d 4.42 (d, 1H, J = 9.7 Hz), 3.86 (dd, 1H, J =
12.0 and 1.2 Hz), 3.80-3.60 (m, 5H), 3.18 (t, 1H, J = 8.7 Hz),
3.10 (ddd, 1H, J = 14.0, 9.6 and 6.0 Hz), 2.96 (ddd, 1H, J =
13.9, 9.6 and 6.2 Hz); 13C NMR (CD3OD, 75 MHz): d 87.4, 82.1,
79.6, 74.4, 71.5, 62.9, 44.8, 33.7; HRMS (m/z) (FAB+) calcd. for
C8H15ClO5S + Na: 281.0226; found: 281.0222.
8.6 Hz), 8.28 (d, 1 H, J 8.6 Hz), 8.24 (d, 1 H, J 7.4 Hz), 7.53 (m, 2
H), 7.17 (d, 1 H, J 7.6 Hz), 6.73 (dd, 1 H, J 16.6 and 10 Hz), 6.37
(d, 1 H, J 16.6 Hz), 6.05 (d, 1 H, J 10 Hz), 3.91 (m, 4 H), 3.80-3.46
(several m, 9H), 3.27 (t, 2 H, J 5.9 Hz), 3.22 (m, 2 H), 2.87 (s, 6
H), 1.38 (m, 2 H), 0.98 (d, 3 H, J 6.6 Hz), 0.65 (t, 3 H, J 7.3 Hz);
13C-NMR (CDCl3, 75 MHz): d 138.0, 134.9, 130.9, 130.7, 130.3,
130.2, 129.1, 128.5, 123.4, 119.5, 115.5, 70.8, 70.4, 64.9, 64.8, 56.0,
55.9, 55.0, 54.5, 45.6 ¥ 2, 35.7, 28.4, 18.8, 11.3. HRMS (m/z)
(NALDI-TOF) calcd. for C26H40O8N2S3Na [M + Na]+: 627.1844;
found: 627.1840.
Synthesis of 2-ethenyl 1-thio-b-D-glucopyranoside-S,S-dioxide
(9). Compound 8 (1.56 g, 6.02 mmol) was dissolved in a mixture
of AcOH–H2O2 (2 : 1, 45 mL) and kept in the dark for 2 d.
Lyophilization gave a crude that was dissolved in acetone (60 mL).
K2CO3 (2.4 g, 17.5 mmol) was then added and the resulting
suspension was refluxed for 8 h. Evaporation of the solvent was
followed by purification by column chromatography (AcOEt–
MeOH 10 : 1) yielding a compound that was lyophilized giving
9 (0.5 g, 37%) as a syrup. [a]D -8.9◦ (c 1, water), [a]436 = -16◦ (c
Synthesis of vinyl sulfone biotin 5. A solution of biotin (120 mg,
0.49 mmol) in Cl2SO (5 mL) was kept at rt for 1 h. The reaction
mixture was then evaporated under vacuum and co-evaporated
with toluene (3 ¥ 15 mL) to give biotin acid chloride. The crude
acid chloride was dissolved in anhydrous THF (15 mL) and added
dropwise to a solution of 2 (150 mg, 0.40 mmol) and Et3N (114 mL,
0.80 mmol) in anhydrous THF (10 mL). The new reaction mixture
was kept at rt for 10 min. Evaporation of the solvent yielded a crude
that was purified by column chromatography (AcOEt–MeOH
10 : 1 → 5 : 1) giving 5 as a syrup (152 mg, 63%). nmax(film)/cm-1:
1
1, water); H NMR (MeOH-d4, 300 MHz): d 6.97 (dd, 1H, J =
16.7 and 10.0 Hz), 6.43 (d, 1H, J = 16.7 Hz), 6.30 (d, 1H, J =
10.0 Hz), 4.78 (s, 3H), 4.34 (d, 1H, J = 9.5 Hz), 3.86 (dd, 1H, J =
12.5 and 2 Hz), 3.69 (t, 1H, J = 9.1 Hz), 3.68 (dd, 1H, J = 12.6
and 5.3 Hz), 3.45 (t, 1H, J = 8.8 Hz), 3.40 (ddd, 1H, J = 9.6,
5.5 and 2.1 Hz), 3.34 (s, 1H), 3.31 (t, 1H, J = 9.3 Hz); 13C NMR
(MeOH-d4, 75 MHz): d 136.2, 132.4, 92.6, 82.8, 78.8, 71.0, 70.5,
62.4; HRMS (m/z) (FAB+) calcd. for C8H14O7S + Na: 277.0358;
found: 277.0356.
1
3354, 3258, 2923, 2870, 1696, 1622, 1457, 1287, and 1121; H-
NMR (CD3OD, 400 MHz): d 6.93 (m, 1 H), 6.34 (m, 1 H), 6.18
(m, 1 H), 4.50 (m, 1 H), 4.33 (m, 1 H), 3.91 (m, 4 H), 3.66 (m, 4
H), 3.52 (m, 2 H), 3.42-3.24 (m, 8 H), 2.94 (m, 1 H), 2.72 (d, 1H,
J 12.7 Hz), 2.46 (m, 2H), 1.76-1.46 (several m, 8H), 1.26 (m, 3 H),
0.90 (m, 3 H); 13C-NMR (CD3OD, 75 MHz): d 178.5, 168.8, 142.3,
132.5, 74.2, 74.1, 68.5, 68.4, 66.1, 64.4, 59.7, 59.1, 58.5, 57.4, 57.1,
Synthesis of vinyl sulfone ferrocene (11). To a deoxygenated
solution of ferrocene methanol 10 (400 mg, 1.85 mmol) in
anhydrous THF (30 mL) were added NaH (89 mg, 3.71 mmol) and
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DVS (0.46 mL, 4.635 mmol). The reaction mixture was kept at rt
for 1 h. AcOH was added to destroy the NaH excess. Evaporation
of the solvent under vacuum yielded a crude that was purified by
column chromatography (hexane–AcOEt 7 : 1) giving 11 (509 mg,
83%) as an orange solid. M.p. 67.0 oC; IR (nmax) (film): 3099, 2921,
2857, 1638, 1313, 1128, 1099, 816, 754 cm-1; 1H NMR (400 MHz,
CDCl3): d 6.67 (dd, 1H, J = 16.6 and 9.9 Hz); 6.36 (d, 1H, J =
16.7 Hz); 6.03 (d, 1H, J = 9.9 Hz); 4.29-4.16 (m, 11H); 3.82 (t,
2H, J = 5.0 Hz); 3.18 (t, 2H, J = 5.2 Hz); 13C NMR (100 MHz,
CDCl3): d 137.8, 128.7, 82.3, 69.7, 69.5, 68.8, 68.6, 63.2, 55.1;
HRMS (FAB+) calcd. for C15H18FeO3S + H: 335.0404; found:
335.0403.
Labeling of HEW lysozyme with vinyl sulfone rhodamine B (3)
at different pH and temperatures (conjugate 14). The labeling of
11.2 nmols of HEW lysozyme with 288 nmols of vinyl sulfone
rhodamine B (3) was carried out by incubation at rt or 30 ◦C for
64 h in 160 mM buffer pH 5 and 6 (acetate buffer), or 7 and 8
(HEPES buffer). The samples were then dialyzed against distilled
water for 24 h and analyzed by MALDI-TOF (Fig. 3).
Labelling of BSA with ferrocene vinylsulfone 11 (conjugate 16).
To three aliquots (2 mL) of a 2 mg mL-1 solution of BSA in 50 mM
HEPES buffer at pH 8.5 were added the ferrocene vinyl sulfone
derivative 11 (0.2, 1.0 and 2.0 mg) dissolved in DMSO (0.1 mL)
to get a 1 : 10, 1 : 50 and 1 : 100 BSA : 11 molar ratio, respectively.
The samples were kept at rt for 24 h. Centrifugation was followed
by dialysis against an 85 mM phosphate buffer at pH 7.0. BSA
concentration was determined by Bradford’s method24 and Fe
content was spectrophotometrically determined.16
Synthesis of 3-(methylamino)-propyl silica (18). A suspension
of commercial silica gel (Merck 70-230 mesh, ASTM) (5 g),
previously activated by thermal treatment (120 ◦C under vacuum
for 24 h), in toluene (25 mL) containing 3-[(methylamino)-propyl]-
trimethoxysilane (1.250 g) was heated under reflux for 2 h. After
partial evaporation of the solvent under vacuum to approximately
80% of the original volume, the reaction mixture was refluxed
for an additional hour. The resulting functionalized silica 18 was
filtered, washed with toluene and dried under vacuum at 50 ◦C.
Biotinylation of BSA (conjugate 17) and interaction with dansyl–
avidin (conjugate 13). BSA (75 nmols) and vinyl sulfone-
derivatized biotin 5 (370 nmols) were incubated in 600 mL of
50 mM HEPES pH 8.0 at 37 ◦C for 24 h. The resulting conjugate
17 was dialyzed first against 100 mM HEPES pH 8, supplemented
with ethanolamine (2 ¥ 100 mL) to block the excess of 5,
and then against 100 mM HEPES pH 8 (7 ¥ 100 mL) for 4
d. The concentration of protein was determined by Bradford’s
method.24 The formation of BSA–biotin–avidin–dansyl complexes
was achieved by the incubation in 33 mM HEPES pH 8.5 at rt for
30 min of 0.15 nmols of tetrameric avidin with either 0.15 or
6 nmol of biotin–BSA to yield 1 : 1 and 1 : 4 dansyl–tetrameric
avidin : biotin–BSA complexes respectively. SDS-PAGE loading
buffer was added and the samples were denatured in mild
conditions by heating at 100 ◦C for 2 min.
Synthesis of vinyl sulfone-functionalized silica (19). To a sus-
pension of the amine functionalized silica 18 (1.53 g) in THF–
isopropanol (1 : 2, 10 mL) was added DVS (0.640 mL). The mixture
was kept at rt for 16 h. The resulting vinyl sulfone–silica 19 was
filtered, washed successively with MeOH and CH2Cl2 and dried
under vacuum at 50 ◦C.
Protein labeling and bioconjugation assays
Labeling of ConA and avidin with vinyl sulfone-derivatized
rhodamine B (3) and dansyl (4) (conjugates 12 and 13). ConA
(195 nmol) and avidin (140 nmol) were incubated with the
vinyl sulfone-derivatized fluorescent labeling agents 3 (578 nmol,
1 : 4 stoichiometry, and 1154 nmol, 1 : 8 stoichiometry) and 4
(990 nmol, 1 : 5 stoichiometry, and 1980 nmol, 1 : 5 and 1 : 10
stoichiometry), respectively, in 50 mM HEPES pH 8 at 37 ◦C
for 24 h. Samples were dialyzed first against 100 mM HEPES
pH 8 supplemented with ethanolamine (2 ¥ 100 mL) to block
the excess of vinyl sulfone-derivatized reagents and then against
100 mM HEPES pH 8 (7 ¥ 100 mL) for 4 d. The extent of labeling
was monitored at 3, 8 and 24 h by taking aliquots of 50 mL and
blocking by addition of 50 mL of SDS-PAGE loading buffer prior
denaturing at 100 ◦C for 4 min. The samples were not dialyzed.
Labeling of BSA and lysozyme with vinyl sulfone-derivatized
rhodamine B 3 as a pre-stain dye. BSA and lysozyme (33 mg),
and 3 (3.6 mmol) were incubated in 222 ml of 75 mM HEPES
pH 8.8 at 100 ◦C for 5 min prior mixing with the SDS-PAGE
loading buffer and denaturing at 100 ◦C for 4 min.
Electrophoresis of the labeled proteins. Samples analyzed by
SDS-PAGE25 in a 12% polyacrylamide gel (12% T : 1.3% C) in
a MiniProtean 3 (BioRad). The fluorescence was detected by
exciting at 365 nm with a conventional UV transilluminator
(Genesys Inst.) and the gels were stained by either Coomassie
Brilliant Blue26 or silver stain.27
Immobilization assays
Immobilization assays of invertase with bioconjugable vinyl
sulfone silica (19) and enzymatic activity determination. Vinyl
sulfone silica 19 (0.5 g) was suspended in 5 mL of a 47 mg mL-1
solution of invertase from Saccharomyces cerevisiae in 244 mM
phosphate buffer pH 7.5 and then 2.88 mL of water. The samples
were prepared in duplicate and incubated at either 4 ◦C or rt.
The reaction was allowed to proceed for 30 h (until there was
no variation in A280 between two consecutive measurements of
the protein in solution), and then the samples were washed with
100 mM phosphate buffer at pH 7.5 (6 ¥ 5 mL) and 2 M NaCl in
100 mM phosphate buffer pH 6.5 (2 ¥ 5 mL + 2 ¥ 10 mL). The
enzymatic activity of the immobilized invertase was monitored by
analysis of the optical rotation with a Perkin-Elmer polarimeter.
Glycosylation of commercial HEW lysozyme with glucopyranosyl
vinyl sulfone 9 (conjugate 15). The reaction was carried out by
mixing and stirring 500 mL of 50 mg mL-1 HEW lysozyme in
90 mM phosphate buffer pH 7.7 and 10% (v/v) 2-propanol with
10.5 mg glucopyranosyl vinyl sulfone 9 for 72 h at rt. To remove
the excess of 9 and stop the reaction, the mixture was transferred
to a 3500 kDa cutoff membrane (Spectrum) and dialyzed against a
total volume of 1500 mL of 20 mM acetate buffer at pH 4.5 in three
steps. The reaction was monitored every 24 h by native PAGE run
with a BioRad apparatus with reversed polarity and stained with
Coomassie Brilliant Blue. For the crystallization procedure and
the determination of the tridimensional structure see the ESI.†
674 | Org. Biomol. Chem., 2010, 8, 667–675
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The immobilized enzyme was incubated with a 3% (w/v) solution
of sucrose in 100 mM phosphate buffer pH 7.5 and the optical
rotation was measured 5 times within 17 h. The values were fitted
by linear regression (coefficient of determination >0.9), the slope
multiplied by 1000 being the enzymatic activity in arbitrary units.
Results are summarized in Table 3.
9 M. S. Masri and M. Friedman, J. Protein Chem., 1988, 7, 49–54.
10 F. Perez-Balderas, Ph.D. Dissertation, Universidad de Granada, Spain,
2005.
11 D. R. van Staveren and N. Metzler-Nolte, Chem. Rev., 2004, 104, 5931–
5986.
12 (a) W. E. Lawson and E. E. Reid, J. Am. Chem. Soc., 1925, 47, 2821–
2836; (b) A. H. Ford-Moore, J. Chem. Soc., 1949, 2433–2440.
13 G. Alderton, W. Ward and H. Febold, J. Biol. Chem., 1945, 157, 43–58.
14 F. Santoyo-Gonzalez, F. Hernandez-Mateo, F. Lopez-Jaramillo, J.
Ortega-Mun˜oz and J. Morales-Sanfrutos, WO Patent 200910664,
2009.
Acknowledgements
15 F. J. Lo´pez-Jaramillo, F. Pe´rez-Banderas, F. Herna´ndez-Mateo and F.
Santoyo-Gonza´lez, F., Acta Crystallogr., Sect. F: Struct. Biol. Cryst.
Commun., 2005, 61, 435–438.
16 A. Badia, N. H. H. Thai, A. M. English, S. R. Mikkelsen and R. T.
Patterson, Anal. Chim. Acta, 1992, 262, 87–90.
17 (a) M. Wilchek and E. A. Bayer, Anal. Biochem., 1988, 171,
1–32; (b) Methods Enzymol., vol. 184, Avidin-Biotin Technology, ed.
M. Wilchek and E. A. Bayer, Academic Press, New York, 1990;
(c) M. D. Savage, G. Mattson, S. Desai, G. W. Nielander, S. Morgensen
and E. J. Conklin, Avidin-Biotin Chemistry: A Handbook, Pierce
Chemical Company, USA, 1992.
Financial Support was provided by Direccio´n General de In-
vestigacio´n Cient´ıfica y Te´cnica (DGICYT) (CTQ2008-01754)
and Junta de Andaluc´ıa (P07-FQM-02899). J. M.-S. thanks the
University of Granada for a research contract (Programa Puente).
A. M.-F. thanks the Spanish Ministerio de Educacion for a
research fellowship (FPU).
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