Photoaffinity labeling on magnetic microspheres (PALMm) methodology for topographic mapping: Preparation of PALMm reagents and demonstration of biochemical relevance

Article abstract of DOI:10.1039/b303425a

Photoaffinity labeling (PAL) is a technique widely used for identifying the binding-site within proteins. Although the classic method is both versatile and powerful, it suffers significant disadvantages, such as the need to radiolabel the PAL ligand, and the need to conduct highly complicated separations of both the labeled protein and the labeled peptides derived from it. Here, we propose a novel and universal methodology - Photo-Affinity Labeling on Magnetic microspheres (PALMm) designed to simplify and shorten the PAL protocol. In this context, we describe the preparation of PALMm reagents and the evaluation of their biochemical relevance regarding two ATP-binding enzymes: hexokinase and apyrase.

Full text of DOI:10.1039/b303425a

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Photoaffinity labeling on magnetic microspheres (PALMm)
methodology for topographic mapping: preparation of PALMm
reagents and demonstration of biochemical relevance
Efrat Halbfinger,a Karine Gorochesky,a Sébastien A. Lévesque,b Adrien R. Beaudoin,b
Larisa Sheihet,c Shlomo Margelc and Bilha Fischer*a
a Department of Chemistry, Gonda-Goldschmied Medical Research Center, Bar-Ilan University,
Ramat-Gan 52900, Israel
b Biology Sciences, University of Sherbrooke J1K 2R1, Canada
c Department of Chemistry, Bar-Ilan University, Ramat-Gan 52900, Israel
Received 28th March 2003, Accepted 5th June 2003
First published as an Advance Article on the web 22nd July 2003
Photoaffinity labeling (PAL) is a technique widely used for identifying the binding-site within proteins. Although the
classic method is both versatile and powerful, it suffers significant disadvantages, such as the need to radiolabel the
PAL ligand, and the need to conduct highly complicated separations of both the labeled protein and the labeled
peptides derived from it. Here, we propose a novel and universal methodology—Photo-Affinity Labeling on
Magnetic microspheres (PALMm) designed to simplify and shorten the PAL protocol. In this context, we describe
the preparation of PALMm reagents and the evaluation of their biochemical relevance regarding two ATP-binding
enzymes: hexokinase and apyrase.
acids in the binding site. The photoreactive group should be
chemically stable, with the longest possible wavelength of
Introduction
The specific recognition between a protein and a bioactive lig-
and, occurs at a defined binding-site within the protein. The
identification of a protein’s binding-site, namely, the elucida-
tion of the amino acid residues constituting the binding-pocket,
is essential for understanding the protein function. Further-
more, this knowledge forms the basis for the design of
new inhibitors/ligands for the regulation of enzyme/receptor
activity.
A commonly used technique for identifying the binding/cata-
lytic-site within proteins is topographic mapping.1 Topographic
labeling of a protein is defined as a labeling pattern that
involves the full environment of a ligand within a binding-site.
This method is suitable for proteins whose primary sequence
is known. Topographic mapping is especially valuable when
studying proteins for which X-ray crystal-structures are
extremely difficult to obtain (e.g. membrane proteins). For
topographic mapping, a photoaffinity labeling (PAL) protocol
is usually applied.1
PAL protocol requires the use of a photoactiveable, but
chemically inert, ligand analogue that is recognized by the pro-
tein’s binding site.1 For the detection of the ligand throughout
the PAL protocol, the ligand is usually marked radioactively.
Upon irradiation, the photreactive ligand, incorporated into
the protein, transforms into a highly reactive species (nitrene or
carbene) that inserts into neighboring covalent bonds. Identifi-
cation of the resulting photo-crosslinked product provides
structural information on the protein’s binding site.
irradiation in order to avoid damage to the protein (>280 nm).
Azides, diazirines, and diazonium salts used for photoaffinity
labeling are usually fluoroaromatic derivatives in order to
increase their chemical stability.1b–3 Although highly reactive
species are formed, non-specific labeling is usually limited, due
to efficient quenching of the photogenerated species by the
solvent.1a
The versatile and powerful PAL method has been applied in
numerous structural studies. For instance, the PAL method
provided a detailed picture of ion channels,4 transporter pro-
teins,5 and nucleic acid–protein interactions.1e Photoaffinity
labeling was used to probe RNA–RNA contacts,6 and protein–
protein interactions, for studying signal transduction.7 The
PAL method was also harnessed for drug discovery and
development.1d,f
A critical step in the PAL method is the synthesis of the PAL
ligand. The additional synthetic steps, required for the intro-
duction of photosensitive groups and radiolabels to the ligand,
complicate this technique. Furthermore, the PAL protocol
involves two highly complicated separations: separation of the
photo-crosslinked protein from the hardly distinguishable free
protein; and, separation of the peptides marked by the photo-
affinity label from the multitude of non-labeled peptides. HPLC
separation of the tryptic digest results in a crowded chromato-
gram with overlapping peaks.8,9 The identification of the
HPLC peaks containing the labeled peptides is yet another
complication.8a,b
For this purpose, the photo-crosslinked protein is separated
from the free protein and then subjected to enzymatic degrad-
ation. Subsequently, the resulting labeled protein fragments are
separated from the multitude of peptides. Finally, the labeled
peptides are analyzed by Edman degradation or mass spectro-
scopic methods to identify labeled amino acids that constitute
the protein’s binding-site.
To obtain as complete mapping as possible, the PAL reagent
is required to have a dynamic interaction, allowing the probe to
adopt different positions in the binding site. Furthermore,
photoprobes with the highest possible photoreactivity, are
required to enable reaction with a maximum number of amino
Thus, the commonly used photoaffinity labeling remains a
labor-intensive method from the synthesis of the PAL reagent
to sequencing the labeled peptides.
Several improved PAL methodologies have been reported.
For instance, the replacement of a radioactive marker by a
fluorescent one made the classic PAL method easier to imple-
ment. Fluorescent marking is achieved by either binding the
ligand to a photoactiveable, fluorescent reagent,10 or by extend-
ing the ligand bearing a photoactiveable part to form a fluor-
escent molecule.11 Nevertheless, the use of a fluorescent marker
still did not solve the problem of complicated purifications,
required in the PAL protocol. However, conjugating the ligand
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
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to a biotin marker helped to avoid radioactive labeling, and to
simplify the purification steps.9,12 Yet, this method does not
completely exclude the use of HPLC.9 Furthermore, this
method requires an additional step of chemiluminescent detec-
tion of the labeled peptides,9 or alternatively, the addition of a
fluorescent marker.12b Thus, the limitations of the various PAL
methods justify the continued search for improved topographic
mapping techniques.
Here, we propose a novel and universal PAL methodology—
Photoaffinity Labeling on Magnetic microspheres, denoted as
PALMm. This method is designed to overcome the above-
mentioned limitations of the PAL technique. We describe the
preparation of PALMm reagents 1 and 2 and the evaluation
of their biochemical relevance regarding two ATP-binding
enzymes: hexokinase and apyrase. The application of the novel
PALMm reagents 1 and 2 for the topographic mapping of these
enzymes will be reported in due course.
MS/MS analysis of the peptides. 8. Correlation of mass spec-
troscopic results with biochemical and site-directed-mutagene-
sis data for the final topographic mapping.
The proposed PALMm methodology is expected to shorten
considerably the labor and time required to perform PAL
studies. One of the primary expected advantages of the
PALMm methodology is the cessation of the need to conduct
radiolabeling of the PAL reagent. In addition, the PALMm
methodology is expected to extremely simplify isolation steps
(steps 3, 5, Fig. 1). Separation of the cross-linked protein from
the free protein is made possible by ‘fishing’ it out magnetically.
Furthermore, the proposed methodology prevents the need
of complicated separations on HPLC of the multicomponent
mixture of peptides obtained after enzymatic degradation of
the crosslinked protein. In our methodology, HPLC separation
would not be required due to the limited number of labeled-
peptides expected to be ‘fished’ out magnetically (steps 5, 6),
and the further use of the ESI MS/MS technique (step 7).
Results and discussion
Selection of a scaffold for PALMm reagents
Description of the PALMm methodology
The above-mentioned PALMm methodology is expected to
serve as a universal means for topographic mapping. Here, we
describe the compatibility of PALMm reagents 1 and 2 for the
topographic mapping of several ATP-binding proteins. Speci-
fically, we focused on hexokinase, whose binding-site is well
characterized,13–17 and on apyrase whose catalytic-site has not
yet been deciphered. Hexokinase is targeted as a model for
proving the feasibility of the PALMm method. Once the known
ATP-binding-site of hexokinase is reproduced by the PALMm
method, we can then apply this method for elucidating the
unknown catalytic-site of apyrase.
As shown below, the elucidation of this catalytic-site is a
prerequisite for the design of novel drugs regulating nucleotide
signaling. Specifically, the major effects of ATP-receptors
(including P2Y-R and P2X-R subtypes)18,20 on most of the
physiological systems18b,21–25 are modulated by metabolic
enzymes.26 Among the key enzymes involved in the regulation
of extracellular nucleotide concentrations in the vicinity of
ATP-receptors, apyrases (NTPDases) play a major role in
hydrolyzing tri- and/or di-phosphate-nucleosides to mono-
phosphate-nucleosides.27
The PALMm protocol, outlined in Fig. 1, is based on the classic
PAL method, yet, it presents several significant improvements.
These improvements are made possible by the use of PALMm
reagents (e.g. 1 and 2). Specifically, this PALMm method
includes the following steps. 1. Incubation of the protein with a
photoactiveable PALMm ligand bound to magnetic micro-
spheres. 2. Irradiation of the ligand–protein complex at a
specific wavelength that produces a highly reactive ligand
species (e.g. carbene), which inserts into a covalent bond of a
near-by amino acid residue. 3. Magnetic separation of the
photo-cross-linked protein from the free protein. 4. Enzymatic
digestion of the labeled protein. 5. Magnetic separation
of microsphere-linked peptides from the tryptic digest. 6.
Detachment of peptides from the magnetic microspheres by
reduction of a disulfide bond. 7. Identification of labeled amino
acid residues that constitute the protein’s binding-site by ESI
The first member of this family, NTPDase1, was found,
purified, and characterized by Lebel et al.28 Its kinetic proper-
ties, substrate specificity, and influence of certain inhibitors
were later described.29,30 Sequence analysis and directed muta-
genesis on apyrase conserved regions (ACR) have indicated that
two ACRs (I and IV) play an essential role in catalytic activity
of NTPDases.31,32 Yet, substrate specificity may also depend on
other regions of the protein because the NTPDases which share
the ACR, exhibit different rates of catalysis with tri- and di-
nucleosides. In this respect, one cannot exclude that sequences
in the vicinity of these conserved regions have a role to play in
substrate specificity.33 Therefore, it is essential to define the
topographic configuration of the active-site. This step is a pre-
requisite in the design of potent and specific inhibitors of these
key enzymes that regulate nucleotide signaling.34
For the topographic mapping of NTPDase (apyrase) we
designed suitable PALMm reagents based on our previously
developed inhibitors of NTPDase.35
Earlier, we identified 8-substituted ATP derivatives 3a–c
(Scheme 2) as very poor P2Y-R ligands. Therefore, these ana-
logues served as an attractive scaffold for the development of
specific NTPDase inhibitors.36 On the basis of the promising
hydrolytic stability of derivatives 3a–c regarding NTPDase,35
a
new series of 8-thioether ATP analogues, 3d–g, was explored.35
Among these analogues, 8-BuS-ATP was found as a competi-
tive inhibitor with the lowest Ki value, 10 µM; where 8-CH2-
tBuS-ATP, 8-cycloheptylS-ATP and 8-hexylS-ATP analogues
produced a mixed type of inhibition. 8-BuS-ATP, 3a, was found
Fig. 1 A proposed protocol for topographic mapping by Photo-
Affinity Labeling on Magnetic microspheres—PALMm.
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Scheme 1 Structures of nucleotide-based PALMm reagents.
moiety (diazirine) (in 5); D) a magnetic bead (6); E) a cleavable
spacer between the aromatic linker and the magnetic bead (7).
A
Inhibitor. A specific inhibitor is required for targeting the
PAL moiety to the enzyme’s catalytic-site. This inhibitor should
have affinity towards the target protein, yet not high affinity, in
order to enable maximal labeling of the binding-site. Since we
aim to map the unknown catalytic-site of apyrase, we chose
an apyrase (NTPDase) inhibitor, 3a, which we previously
developed,35 as a scaffold for the construction of the related
PALMm reagents 1 and 2. PALMm reagent 1 was first evalu-
ated as both a substrate and inhibitor of hexokinase. This
enzyme serves here as a structurally known model for proving
the feasibility of the PALMm method.
B
Photoaffinity label. The photoaffinity label of choice
should be chemically stable during the preparation of the
PALMm reagent. Furthermore, the wavelength of irradiation
of the PAL moiety should be as long as possible, far from that
absorbed by proteins, in order to avoid damage to the enzyme.
The photoaffinity label of choice was 3-(trifluoromethyl)-3-
phenyldiazirine3a,37 (in 5), which is chemically stable in mild
acidic or basic environments,38 and is irradiated at a non-
destructive wavelength (ca. 360 nm).
Scheme 2 Structures of NTPDase inhibitors.
C
linker. The diazirine PAL moiety has to be linked to the
inhibitor scaffold, 4, and to the magnetic bead, 6, through a
chemically and enzymatically stable bond. Thus, the linking
groups of choice are amine and thioether, which are stable
under the synthesis conditions and the PALMm protocol con-
ditions (Fig. 1). The linker between the diazirine and inhibitor
precursor 4, should be as short as possible to enable maximum
proximity of the PAL moiety to the inhibitor, and therefore
ensure labeling of the catalytic-site. The linker of choice is m-
xylyl (in 5), where the distance between the inhibitor precursor
4 and the diazirine PAL moiety is only four atoms long (e.g. in
product 13, Scheme 5).
both to be the most effective non-hydrolyzable competitive
inhibitor, and also a specific inhibitor having no interaction
with P2X-receptors,35 and P2Y1-receptors.36
Based on these findings, we selected derivative 3a as an
attractive scaffold for the preparation of PALMm analogues 1
and 2, targeting NTPDase and possibly other ATP-binding
proteins.
Design of PALMm reagents
To implement the PALMm protocol, we propose special
reagents, e.g. 1 and 2 (Scheme 1). These reagents are composed
of five moieties (Scheme 3): A) inhibitor precursor (4); B) a
compact tri-functional aromatic linker (in 5); C) a small PAL
D
Cleavable spacer. A cleavable spacer, 7, is required to
enable the detachment of the inhibitor-linked-peptide from the
Scheme 3 General scheme for the preparation of a PALMm reagent.
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functionalized magnetic microsphere (step 6, Fig. 1). This
cleavage should be performed under mild conditions that would
not harm any functions of the inhibitor or the bound peptide.
For this purpose, cystamine is used as a cleavable spacer.
Cystamine is attached via one amino end to the linker, 5, and
via a second amino end to the magnetic microsphere, 6 (Scheme
3). The peptide-bound inhibitor can be detached from the bead
by dithiotriethol or tris(2-carboxyethyl)phosphine39 reduction
of the cystamine’s disulfide bond.
E
Magnetic microsphere. The proposed magnetic micro-
spheres, 6, consist of polystyrene microspheres of a narrow size
distribution, ca. 6 micron diameter. These particles are coated
with magnetite (Fe3O4), and then with silica40a bearing vinyl
sulfone functions.40b–d
Assembly of PALMm reagents 1 and 2
PAL reagent 5, is attached via one methylene bromide group, to
a precursor of apyrase inhibitor, 4, and via the other methylene
bromide, to a cystamine cleavable spacer, 7 (Scheme 3). The
resulting nucleoside is then phosphorylated at the 5Ј position of
the sugar, and finally the cystamine moiety, is added through its
free amine, to the vinyl sulfone derivatized magnetic bead, 6.
The detailed assembly of PALMm reagents is described in
Schemes 4–6.
Scheme 5 Conditions: a. 1. NaOH, 2. 5a, 4 h, 57%; b. H2N(CH2)2-
SS(CH2)2NHtBoc, 14, 42%.
aneous loss of the purple ninhydrin-colored spot of 11 from the
TLC plate, and the appearance of a typical diazirine absorption
at 360 nm. NBS, was found to be a mild diaziridine oxidizing
agent, as compared to other reagents used for this purpose such
as: AgNO3, Ag2O, I2, CrO3, NaIO4, and RuO2.43 This diazirdine
oxidation occurs probably via a radical mechanism leading to
the formation of HBr and succinimide, in addition to diazirine.
Optimized conditions of the reaction, avoiding the formation
of multi-brominated products, involved the addition of three
equivalents of NBS. Under these conditions, the desired PAL
reagent 5a was obtained in 25% yield. This product was accom-
panied with the mono-brominated product, 5b, obtained in
51% yield, which was then recycled with 1 eq NBS to form 5a in
64% yield.
PAL reagent 5a was linked to a precursor of apyrase inhibi-
tor, 4, via a thioether bond (Scheme 5). Selective S-monoalkyl-
ation of nucleoside 12 by 5a was achieved by first preparing the
sodium thiolate salt 4,35 and then adding it to a highly dilute
solution of 5a at room temperature. The resulting nucleoside,
13, obtained in 57% yield, was then coupled to the cleavable
spacer cystamine. To avoid the di-alkylation of cystamine by 13
and the phosphorylation of the cystamine’s free amine in the
next synthetic step (Scheme 6), cystamine 7, was mono-
protected by t-Boc group.44 This group is stable under condi-
tions of the alkylation step forming 15 and the phosphorylation
steps leading to 16 and 17 (Scheme 6). Nucleoside 13 was, there-
fore, treated with 5 eq mono-protected tBoc-cystamine, 14, in
the presence of a basic resin (Dowex MWA-1) in DMF to pro-
vide product 15 in 42% yield. The last synthetic step before
coupling the reagent to the magnetic microspheres, included the
preparation of nucleotides 16 and 17 by phosphorylation of the
5Ј-position of nucleoside 15. Under the conditions of one-pot
5Ј-triphosphorylation, involving the addition of POCl3 in
Scheme
4
Synthesis of PAL moiety bearing suitable linkers.
Conditions: a. 1. BuLi, 2. CF3CO2Me, 92%; b. HONH2, 97%; c. TsCl;
d. NH3 (g); e. 3 eq NBS, (PhCOO)2, 25% (5a); 51% (5b); f. 1 eq NBS,
64% (5a).
5-Bromo-1,3-xylene, 8, served as a scaffold for the PAL moi-
ety generation (Scheme 4). The brominated position was elab-
orated by BuLi and methyl trifluoroacetate to form a ketone 9.
Three equivalents of BuLi were required to obtain product 9 in
92% yield. Ketone 9 was converted to diaziridine 11 in three
steps in 97% overall yield.41 Next, each of the benzylic positions
was mono-brominated by NBS in methyl formate in the pres-
ence of dibenzoyl peroxide under light (either sunlight or fluor-
escent lamp).42 Under these conditions, the diaziridine function
in 11 was also oxidized to the corresponding diazirine to form
the desired PAL reagent, 5a. This was indicated by instant-
Scheme 6 Conditions: a. 1. POCl3, PO(OMe)3, 31%; b. 1. 10 eq CDI, 2. P2O7(Bu3NHϩ)4, 20%; c. TFA, quantitative yield; d. pH 8.6, 6, high dilution.
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PO(OMe)3 at
0
ЊC followed by bis(tributylammonium)-
quantitative yield. With constant nitrogen gas flow and a short
pyrophosphate and then hydrolysis,45 only the 5Ј-mono-
phosphate product 16 was obtained in low yield. All attempts
to modify the phosphorylation conditions for obtaining nucleo-
side 5Ј-triphosphate 17 failed.
reaction time, the decomposition of the triphosphate chain was
almost completely prevented. In the same way, the t-Boc pro-
tecting group was removed from the homologue 16 to provide
nucleotide 18.
Since our goal was the preparation of PALMm reagents tar-
geting ATP-binding proteins we attempted the preparation of
17 by a convergent synthesis starting from a precursor 20, bear-
ing already the triphosphate moiety at 5Ј-position to which
compound 22, that includes the linker, PAL, and spacer
moieties, will be attached (Scheme 7).
Pre-PALMm reagents 18 and 19, with free amine side-
chain, were coupled to the magnetic microspheres 6 via
a Michael addition to vinyl sulfone groups on the bead at
pH 8.6. As described below, this was performed under
high dilution, to provide the desired PALMm reagents 1
and 2.
8-SNa-ATP, 21b, was prepared in 48% yield from 8-Br-ATP,
20, in a 2 M NaSH solution, pH 8,46 followed by treatment of
21a with NaOH, pH 11. The product was identified by spraying
the TLC plate with Ellman’s reagent, dithiobis(nitrobenzoic
acid), and sodium sulfite,47 thus forming an indicative yellow
spot. For the preparation of compound 22, PAL reagent 5a was
treated with monoprotected tBoc-cystamine, 14, in the presence
of DOWEX-MWA-1 (basic resin). This reaction required an
excess of 5a and high dilution to avoid the reaction of tBoc-
cystamine with both benzylic positions of 5a. However, product
22 was obtained in a minute amount accompanied by a
complex mixture of products.
Characterization of magnetic microspheres 6
Magnetic microspheres, 6, consisting of polystyrene micro-
spheres (ca. 6 micron diameter), coated with Fe3O4 and then
with amine group bearing silica,40a were functionalized with
divinyl sulfone (DVS). This provided active olefin functions for
coupling with amines 18 and 19.40b–d The number of vinyl sul-
fone functions (free binding sites) on the magnetic microspheres
was quantified as follows.
An excess of mercaptoethanol in NaHCO3 solution (pH 8.6)
was added to magnetic microspheres, 6. After overnight reac-
tion, that produced 23 (Scheme 8A), the microspheres were
removed with a magnet and washed. The filtrate was analyzed
for its mercaptoethanol content with an excess of 4,4Ј-dipyridyl
disulfide, 4-PDS, 24 (Scheme 8B). 4-PDS reacts with thiols and
produces 4-thiopyridone, 4-TP, 25.49 Product 25 has a typical
absorption at 323 nm, far enough from that of 24 (247 nm),
thus minimizing the overlap of the UV bands of starting
material and product. This minimal overlap enabled the
determination of mercaptoethanol concentration in the filtrate
based on 4-TP’s absorbance and ε (13251 cmϪ1 MϪ1 at 323 nm
in 0.1 M sodium bicarbonate buffer, pH 8.6). The number
of moles of reacted mercaptoethanol provided the number
of free binding-sites on microspheres 6 (7.7 µeq per 1 mg
microspheres).
Eventually, the preparation of pre-PALMm reagent 17 was
achieved in two steps (Scheme 6). The first step included mono-
phosphorylation of nucleoside 15 with POCl3 in PO(OMe)3 to
obtain 16 in 31% yield. The second step included di-phos-
phorylation of nucleoside-5Ј-monophosphate 16, to produce
the triphosphate homologue 17, using an activating agent 1,1Ј-
carbonylbis(imidazole) (CDI), and bis(tetrabutylammonium)-
pyrophosphate.48 The 5Ј-phosphorimidazolidate intermediate,
formed from 16 and CDI, facilitates the nucleophilic attack of
the pyrophosphate salt.48 The desired nucleotide 17, obtained in
1
20% yield, was characterized by H and 31P NMR, UV, and
FAB spectra.
Next, the t-Boc protecting group was carefully removed in
neat TFA, for 3 min, to produce the desired product 19 in a
Scheme 7 Conditions: a. 1. NaSH, RT, overnight, 2. NaOH, pH 11, 48%; b. H2N(CH2)2SS(CH2)2NHtBoc, 14, 80 ЊC 3 h, 2 eq DOWEX-MWA-1,
acetonitrile; c. 1. DOWEX-MWA-1, DMF, 2. TFA, 3 min.
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Scheme 8 Conditions: a. HO(CH2)2SH, RT, overnight, 0.1 M NaHCO3 pH 8.6; b. 0.1 M NaHCO3 pH 8.6, 5 min.
Table 1 Percentage of phosphoryl group transfer by hexokinase in the
presence of ATP and/or nucleotide analogues
hexokinase activity to about 76%. 8-BuS-AMP, 3h, however,
did not inhibit the reaction of ATP at all.
Once the affinity of the model compound 3a to hexokinase
had been demonstrated, we set to evaluate PALMm reagent 2,
as either a co-substrate or inhibitor of hexokinase. First, the
bead-bound nucleotide 2 at 890 µM concentration, was sus-
pended in the solution containing the assay’s enzymes (hexo-
kinase and glucose-6-phosphate dehydrogenase) and reagent
(β-NADP),50 and the enzymatic reaction was monitored by UV
for 20 min (Fig. 2). During the first five minutes of the assay, in
addition to the absorbance of β-NADPH, the absorbance of
magnetic microspheres, 6, which are scattered in the solution, is
also observed (Fig. 2). The elevation of the spectrum’s baseline
is due to the black opaque color of the particles (Fig. 3). The
Nucleotide analogue
Relative % of hexokinase activity
ATP
8-BuS-ATP, 3a
PALMm reagent 2
8-BuS-ATP, 3a ϩ ATP
8-BuS-AMP, 3h ϩ ATP
PALMm reagent 2 ϩ ATP
a Evaluation of 8-BuS-ATP as a co-substrate compared to ATP.
Assays were performed for 5 min at 25 ЊC with 0.025 units protein and
8-BuS-ATP, 3a, or ATP (370 µM of each) in the incubation medium.
Reagent 2 was assayed at 890 µM concentration. Assays were per-
formed in duplicate (SEM 0.013). b Evaluation of 8-BuS-ATP, 3a, and
8-BuS-AMP, 3h, as inhibitors of phosphoryl group transfer from ATP.
Assays were performed for 5 min at 25 ЊC with 0.025 units protein,
8-BuS-ATP or 8-BuS-AMP and ATP (370 µM of each). Assays were
performed in duplicate (3a, SEM 0.0085) or triplicate (3h, SEM 0.039).
Reagent 2 was assayed at 500 µM concentration.
100a
58a
23a
76 b
100b
100b
Coupling PALMm reagents 18 and 19 to magnetic microspheres
6
Pre-PALMm reagents 18 and 19 were then coupled overnight to
magnetic microspheres 6 in bicarbonate buffer, pH 8.3. The
products, PALMm reagents 1 and 2 (Scheme 1), were sub-
sequently separated from the solution by a strong magnet. The
yield of the coupling reaction in both cases was ca. 70% based
on the amounts of 18 and 19 remaining in the solution, as
determined through their absorbance at 285 nm (ε 20955 MϪ1
cmϪ1).
Fig. 2 Evaluation of PALMm reagent 2 as a hexokinase co-substrate.
The reaction of hexokinase is monitored by the change of β-NADPH
absorbance at 340 nm. The elevated absorbance during the first minutes
is due to the scattered magnetic microspheres (Fig. 3). After 5 min,
microspheres 2 sank down and a linear increase of β-NADPH
absorbance was measured. For calculating the enzyme activity we used
absorbance data from the 5th min onwards.
Evaluation of PALMm reagent 2 as a substrate/inhibitor of
hexokinase
The compatibility of reagent 2 for a PALMm protocol was
evaluated by first studying its affinity to the primary target
enzyme, hexokinase. The reproduction of the known binding-
site of hexokinase13–17 by the PALMm protocol, with reagent 2,
will validate this method and will enable its application for
structurally unknown proteins (e.g. NTPDase/apyrase) as final
targets.
PALMm reagents 1 and 2 were designed and synthesized
based on the inhibitor of the final target, NTPDase, namely,
based on 8-BuS-ATP 3a.35 Therefore, for proving the affinity of
reagent 2 to hexokinase, we initially had to determine the ability
of the model compound, 8-BuS-ATP, 3a, to bind to hexokinase,
as either a co-substrate or an inhibitor.
8-BuS-ATP, 3a, was first evaluated as a co-substrate for hexo-
kinase,50 and the percentage of phosphoryl group transfer
to glucose was compared to that of ATP (Table 1). Next,
8-BuS-ATP and 8-BuS-AMP were evaluated as inhibitors of
hexokinase, at a concentration of 370 µM for each (Table 1).
8-BuS-ATP was found to be a moderate substrate for hexo-
kinase, producing about 58% activity compared to that
of ATP. The inhibitory effect of 3a was modest, reducing
Fig. 3 Magnetic microspheres 6 elevate the baseline of the UV spec-
trum. A) ATP solution. B) ATP solution and 1 mg of microspheres 6.
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absorbance of β-NADPH alone is measured ca. five minutes
after the beginning of the assay, when the suspended magnetic
microspheres sink. Calculation of percentage of phosphoryl
group transfer uses absorbance data from the fifth minute
onwards.
PALMm reagent 2 was found to be a weak co-substrate of
hexokinase with only 23% phosphoryl-group transfer, as com-
pared to ATP at the same concentration. PALMm reagent 2
was then evaluated as a potential hexokinase inhibitor, in the
presence of an equimolar amount of ATP (500 µM), and the
enzyme’s activity was compared to that with ATP alone.
PALMm reagent 2 had practically no inhibitory effect.
Evaluation of PALMm reagent 1 as an inhibitor of apyrase
After demonstrating the affinity of PALMm reagent 2 to the
primary target enzyme, hexokinase, we aimed to evaluate the
affinity of reagents 1 and 2 towards the final target: apyrase.
This goal was achieved in three sequential and dependent
steps. First, a model compound, 8-BuS-AMP, 3h, was tested as
an inhibitor of NTPDase1, with ATP as the substrate. In the
second step, pre-PALMm reagent 16, which is water-soluble
and still not bead-bound, was used as a model compound for
PALMm reagent 1 and was evaluated as an NTPDase1 inhibi-
tor. In the third step, after demonstrating the affinity of 16 to
NTPDase, the corresponding bead-bound reagent 1 was evalu-
ated as a potato-apyrase inhibitor. Potato-apyrase was chosen
as a model to characterize the NTPDase(s) active site, since it
contains all five conserved regions (ACR I to V), possesses simi-
lar activity to NTPDase1, and has 20.3% to 28.7% identity to
known mammal NTPDases. Yet, practically, potato-apyrase is
much easier to study because, unlike NTPDase1, it is water-
soluble and stable (loss of less than 10% activity after two
weeks at 4 ЊC).51
The activity of the enzyme was measured based on the strong
absorbance at 630 nm of the complex of malachite green with
Pi released upon hydrolysis of ATP by NTPDase1 or
apyrase.52,53
Thus, in the first step it was found that the activity of
the enzyme decreased to 18% at 100 µM concentration of
8-BuS-AMP (Fig. 4A). This significant inhibition prompted us
to explore the inhibitory potential of pre-PALMm reagent 16 as
a soluble model of PALMm reagent 1. Pre-PALMm reagent 16
at 100 µM indeed decreased the activity of NTPDase, yet, only
to 68% of that of ATP (Fig. 4B).
Fig. 4 Influence of 8-BuS-AMP and pre-PALMm reagent 16 on
NTPDase1 ATPase activity. Assays were performed for 7 min at 37 ЊC
in the presence of 1.0 µg of protein and different concentrations (1 to
100 µM) of 8-BuS-AMP and pre-PALMm reagent 16 in the incubation
medium. Reactions were started by adding 100 µM of ATP. A) A
concentration dependent inhibition of NTPDase1 ATPase activity is
observed for 8-BuS-AMP up to (82.2
compound 16 there is a weak inhibition (32.4
4.7%) at 100 µM. B) For
2.4% at 100 µM).
Results are the mean of triplicate samples. The maximum deviation
from the mean was within 10%.
The construction of the PALMm reagent on the scaffold of
8-BuS-ATP raised the following questions. Will the binding-site
of apyrase/hexokinase accommodate ATP analogues bearing a
large substituent at C8 (e.g. analogues 16–19)? Will the mag-
netic microsphere 6, which is ca. 100-fold larger than the
protein, interfere with nucleotide binding to the protein?
For assessing the biological relevance of PALMm reagents 1
and 2, we evaluated them as either substrates or inhibitors of
hexokinase and apyrase.
PALMm reagent 2 was identified as a weak hexokinase sub-
strate demonstrating 23% phosphoryl group transfer compared
to that of ATP. Likewise, PALMm reagent 1 was identified as a
weak inhibitor of potato-apyrase, reducing the percentage of
hydrolysis of ATP to 83%. The affinity of these reagents to the
corresponding enzymes is low compared to their precursors.
For instance, 8-BuS-ATP is a moderate substrate of hexokinase
with 58% enzyme activity compared to ATP, while reagent 2 is a
weak substrate (23%). Likewise, 8-BuS-AMP is an NTPDase1
inhibitor reducing the activity of the enzyme to 18% compared
to ATP; while pre-PALMm reagent 16, a soluble model of
reagent 1, and reagent 1 decreased the activity only to 68% and
83%, respectively.
Next, the bead bound reagent 1, at 1260 µM concentration,
was evaluated as a potato-apyrase inhibitor with an equimolar
concentration of ATP as the substrate. PALMm reagent 1 was
found to be a poor inhibitor of apyrase that decreases the
hydrolysis rate of ATP to 83%. Namely, reagent 1 demonstrates
a weak interaction with apyrase.
Summary
The essence of the PALMm method, proposed here to
overcome the severe limitations of the classic PAL protocol, is
the utilization of a PALMm reagent (e.g. 1 or 2), which is a
trifunctional affinity probe bound to a magnetic microsphere.
Specifically, we prepared here PALMm reagents targeting
adenine-nucleotide binding proteins.
Several adenine nucleotides bearing a PAL moiety have been
previously prepared for incorporation in DNA, yet they bear
the PAL moiety on the adenine’s N6-amine,54 the phosphate
backbone,55 or on the ribose.1e
The reduction in substrate/inhibitor nature of the PALMm
reagents compared to their precursors may be due to either the
bulkiness of the substitution at C8, or hindrance of the mag-
netic particle. However, such hindrance is less likely because
the 17-atoms long flexible spacer between the inhibitor and the
microsphere (from the aromatic ring on the inhibitor to the
silica surface of the microsphere) is expected to enable free
approach of the protein to the particle-bound-inhibitor.
Here, we considered that keeping free N6-position, phos-
phate, and ribose moieties, is essential for the molecular recog-
nition of the ATP-analogue by the protein. Therefore, we
attached the functionalized PAL moiety at the C8-position of
the nucleotide. Furthermore, we based this decision on the pro-
nounced inhibitory effects of 8-BuS-AMP and 8-BuS-ATP,
shown in this and previous studies.35
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It is known that the reagents used for PAL studies should not
bind to the protein too strongly. This is necessary in order to
enable their binding to as many amino acid residues as possible
to obtain as complete as possible structural information on the
binding-site.1 Therefore, PALMm reagents 1 and 2, found to be
a weak inhibitor and substrate, respectively, are suitable for the
PALMm protocol.
The PALMm approach is universal and may be in principle
applied to the topographic mapping of any protein, provided
the PALMm reagent is based on the appropriate inhibitor/
ligand scaffold. Here, we demonstrated the biochemical rele-
vance of 8-substituted-adenosine-nucleotide PALMm reagents
1 and 2 regarding two ATP-binding proteins—hexokinase
and apyrase. The application of reagents 1 and 2 for the topo-
graphic mapping of these enzymes will be published in due
course.
for 3 h. The cooling bath was then removed and the mixture was
hydrolyzed with saturated aqueous NH4Cl. The organic phase
was washed five times with aqueous NH4Cl, three times with
water, and dried over Na2SO4. The solvent was evaporated and
the crude material was purified on an alumina gel (neutral)
column eluted with petroleum ether till the impurity was
removed, then the eluent was gradually changed to chloroform.
The product was obtained as yellow oil in 91% yield (0.58 g). 1H
NMR (CDCl3, 200 MHz) δ 7.70–7.64 (m, 2H, Ar), 7.36–7.30
(m, 1H, Ar), 2.40 (q, J = 0.5 Hz, 6H, CH3); 13C NMR (CDCl3,
2
200 MHz) δ 180.8 (q, JCF = 35 Hz, COCF3), 138.9 (s, 2C,
Ar), 137.3 (d, Ar), 130.1 (s, Ar), 127.8 (d, 2C, Ar), 116.8 (q,
1JCF = 291 Hz, CF3), 21.2 (q, 2C, CH3); 19F NMR (CDCl3, 200
MHz) δ Ϫ72.3 (s); MS (CI/CH4) m/z 203 MHϩ; high-resolution
MS calcd for C10H10F3O 203.0684, found 203.0714.
1-(3,5-Dimethylphenyl)-2,2,2-trifluoroethanone oxime (10a)
Experimental
A mixture of hydroxylamine hydrochloride (206 mg, 2.97
mmol) and NaOH (119 mg, 2.97 mmol) in absolute ethanol
(10 mL) was heated under reflux. A solution of 5-(trifluoro-
acetyl)-m-xylene 9 (200 mg, 0.99 mmol) in absolute ethanol
(1.5 mL) was added. After 22 h the solvent was evaporated
under vacuum and the residue was partitioned between ether
and water. The organic layer was washed three times with 0.01
M aqueous HCl, three times with water, and dried over Na2SO4.
The solvent was removed to give the product as a cream solid
General
1H spectra were measured at 200, 300, or 600 MHz on Bruker
AC-200, DPX-300, or DMX-600 spectrometers. 13C NMR
spectra were measured at 75.4 MHz on Bruker AC-200, DPX-
300, or DMX-600 spectrometers. The chemical shifts are
reported in ppm relative to tetramethylsilane (TMS) as an
internal standard. 19F NMR spectra were measured on Bruker
AC-200, using CFCl3 as an external reference. Nucleotides were
also characterized by 31P NMR in D2O using 85% H3PO4 as an
external reference on a Bruker AC-200 spectrometer. Carbon
assignments in nucleosides are based on hetero-nuclear multiple
quantum correlation (HMQC) and hetero-nuclear multiple
bond correlation (HMBC) experiments. COSY (correlated
spectroscopy), HMQC, HMBC and NOESY (nuclear Over-
hauser effect spectroscopy) experiments were recorded on a
Bruker DMX-600 spectrometer. Mass spectra were recorded on
an AutoSpec-E-FISION VG high-resolution mass spectro-
meter. Nucleotides were characterized by FAB (fast atom bom-
bardment) and high-resolution FAB using glycerol matrix
under FAB negative conditions on an AutoSpec-E-FISION VG
high-resolution mass spectrometer. Separation of the newly
synthesized nucleotides was achieved on LC (Isco UA-6)
using DEAE A-25 Sephadex (HCO3-form) anion exchanger as
described below. Final purification was performed on an HPLC
(Merck-Hitachi) system using a semi-preparative LiChroCART
LiChrospher 60 RP-select B column (1 × 25 cm; Merck KGaA)
and a linear gradient of 0.1 M triethylammonium acetate buffer
(TEAA, pH 7.5) and acetonitrile at 6 mL minϪ1 flow rate. The
purity of the new nucleotides was evaluated on the same semi-
preparative column, at 6 mL minϪ1, in two different solvent
systems. Solvent system I was 0.1 M TEAA–CH3CN, 100 : 0 to
50 : 50 in 20 min. Solvent system II was 0.1 M TEAA–CH3OH,
90 : 10 to 10 : 90 in 20 min. For the enzymatic assays a UV-2041
PC spectrophotometer (Shimadzu Co.) equipped with a Helma
quartz cell type 9Q 10 was used.
1
(mp 97–98 ЊC) in 98% yield (213 mg). H NMR (CDCl3, 200
MHz) δ 9.04 (br s, 1H, NOH), 7.11 (br s, 3H, Ar), 2.36 (s, 3H,
CH3), 2.35 (s, 3H, CH3); 13C NMR (CDCl3, 200 MHz) δ 148.1
(q, 2JCF = 32 Hz, COCF3), 138.3 (s, 2C, Ar), 132.3 (d, Ar), 126.1
1
(d, 2C, Ar), 125. 87 (s, Ar), 120.6 (q, JCF = 275 Hz, CF3),
21.2 (q, 2C, CH3); 19F NMR (CDCl3, 200 MHz) δ Ϫ68.0 (s);
MS (CI/CH4) m/z 218 MHϩ; high-resolution MS calcd for
C10H11F3NO 218.0793, found 218.0800.
N-(4-Toluenesulfonyl)-1-(3,5-dimethylphenyl)-2,2,2-trifluoro-
ethanone oxime (10b)
Oxime 10a (192 mg, 0.88 mmol) dissolved in 3 mL of dry pyrid-
ine was heated under reflux with p-toluenesulfonyl chloride (253
mg, 1.5 eq). After 3 h the solvent was removed under vacuum,
and the residue was purified on an alumina (neutral) column
(petroleum ether : chloroform 3 : 1). The product was obtained as
1
a cream solid (mp 98–99 ЊC) in 88% yield (288 mg). H NMR
(CDCl3, 200 MHz) δ 7.88 and 7.38 (AAЈXXЈ, 2H each, Tos), 7.13
(br s, 1H, Ar), 6.95 (br s, 2H, Ar), 2.47 (s, 3H, Tos), 2.34 (s, 3H,
CH3), 2.33 (s, 3H, CH3); 13C NMR (CDCl3, 300 MHz) δ 154.6 (q,
2JCF = 33 Hz, COCF3), 146.1 (s, Tos), 138.6 (s, 2C, Ar), 133.3 (d,
Ar), 131.3 (s), 129.9 (d, 2C, Tos), 129.3 (d, 2C, Tos), 125.7 (d, 2C,
Ar), 124.5 (s), 119.6 (q, 1JCF = 278 Hz, CF3), 21.8 (q, Tos), 21.3 (q,
2C, CH3); MS (CI/CH4) m/z 372 MHϩ; high-resolution MS calcd
for C17H17F3NO3S 372.0881, found 372.0858.
3-(3,5-Dimethylphenyl)-3-trifluoromethyldiaziridine (11)
A solution of 10b (0.464 g, 1.25 mmol) in dry ether (5.5 mL), in
a flame dried two necked flask, was cooled to Ϫ60 ЊC. Gaseous
ammonia was liquified at Ϫ78 ЊC in a graded cylinder con-
nected to the two neck flask. When the required amount of
ammonia was accumulated (8 mL), the cooling bath was
removed and gaseous ammonia was let into the reaction flask at
Ϫ60 ЊC. The mixture was stirred for 8 h. The cooling bath was
removed and ammonia was allowed to evaporate overnight.
The residue was diluted with ether, washed with water, dried
over Na2SO4, and the solvent was removed to give the product
as a yellowish oil in 97% yield (0.261 g). The product was puri-
fied on a silica gel column and eluted with petroleum ether :
chloroform 2 : 1. 1H NMR (CDCl3, 200 MHz) δ 7.20 (br s, 2H,
Ar), 7.05 (br s, 1H, Ar), 2.75 (br s, 1H, NH), 2.32 (s, 6H, CH3),
2.21 (br s, 1H, NH); 13C NMR (CDCl3, 300 MHz) δ 138.4 (s,
Reagents
Ammonium sulfate, ATP, bovine serum albumin (BSA), cal-
cium chloride, imidazole, malachite green, MES, tetramisole,
thioglycollate and Tris-base were purchased from Sigma Chem-
ical Co. (St. Louis, MO, USA). Bradford reagent was obtained
from Bio-Rad Laboratories (Mississauga, Ont., Canada).
1-(3,5-Dimethylphenyl)-2,2,2-trifluoroethanone (9)
To a solution of 5-bromo-m-xylene (0.37 mL, 3.16 mmol) in dry
ether (10 mL), n-butyllithium (1.6 M, 4 mL, 2 eq) was added at
Ϫ30 ЊC under dry argon. The reaction mixture was allowed to
warm up to 0 ЊC within 2.5 h and cooled down again to Ϫ50 ЊC.
A solution of methyl trifluoroacetate (0.5 mL, 1.5 eq) in dry
ether (0.5 mL) was added at Ϫ50 ЊC and the mixture was stirred
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 8 2 1 – 2 8 3 2
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2C, Ar), 131.7 (d, Ar), 131.5 (s, Ar), 125.7 (d, 2C, Ar), 123.5
mmol) in DMF (5 mL). The reaction was stirred under nitrogen
1
2
(q, JCF = 278 Hz, CF3), 58.0 (q, JCF = 36 Hz, C-diaziridine),
21.1 (q, 2C, CH3); 19F NMR (CDCl3, 200 MHz) δ Ϫ76.7 (s);
MS (CI/CH4) m/z 217 MHϩ; high-resolution MS calcd for
C10H12F3N2 217.0953, found 217.0948.
at 60 ЊC for 6 h. Dowex-MWA-1 was removed by filtration. The
solvent was evaporated under vacuum and the residue was co-
evaporated repeatedly with EtOH. The product was purified on
a silica gel column eluted with chloroform : methanol 15 : 1.
After evaporation and drying for two days under vacuum the
product was obtained in 48% yield (73 mg). 1H NMR (CD3OD,
200 MHz) δ 8.08 (s, 1H, H-2), 7.57 (br s, 1H, Ar), 7.20 (br s, 1H,
Ar), 7.11 (br s, 1H, Ar), 5.94 (d, J = 7 Hz, 1H, H-1Ј), 4.95 (dd,
J = 7, 5 Hz, 1H, H-2Ј), 4.64 and 4.45 (Abq, J = 8 Hz, 2H,
CH2S), 4.32 (dd, J = 5, 1.5 Hz, 1H, H-3Ј), 4.19–4.09 (m, 1H,
H-4Ј), 3.86 (dd, J = 12, 2 Hz, 1H, H-5Ј), 3.75 (s, 2H,
Ar-CH2NH), 3.69 (dd, J = 12, 3 Hz, 1H, H-5Ј), 3.30 (t, J = 7 Hz,
2H, CH2NH), 2.79 (br s, 4H, CH2SSCH2), 2.71 (t, J = 7 Hz, 2H,
CH2NHCO), 1.42 (s, 9H, Boc); 13C NMR (CD3OD, 300 MHz)
δ 158.3 (s, Boc), 156.3 (s, C-6), 152.5 (d, C-2), 151.5 (s, C-4),
150.1 (s, C-8), 142.5 (s, Ar), 140.2 (s, Ar), 132.1 (d, Ar), 130.5 (s,
Ar), 127.2 (d, Ar), 126.8 (d, Ar), 123.5 (q, 1JCF = 275 Hz, CF3),
121.4 (s, C-5), 91.4 (d, C-1Ј), 88.9 (s, C-4Ј), 80.2 (s, Boc), 74.2 (d,
C-2Ј), 73.1 (d, C-3Ј), 64.1 (t, C-5Ј), 53.3 (d), 48.1 (d), 40.7 (d),
39.1 (d), 38.7 (d), 37.7 (d), 29.4 (q, 2JCF = 40 Hz, C-diazirine).
3-[3,5-Bis(bromomethyl)phenyl]-3-trifluoromethyl-3H-diazirine
(5a)
Compound 11 (0.247 g, 1.14 mmol) was dissolved in methyl for-
mate (10 mL). N-Bromosuccinimide (NBS) (611 mg, 3 eq) and
dibenzoyl peroxide (55 mg, 0.2 eq) were added. The reaction was
heated under reflux overnight under the light of four fluorescent
PL 11W lamps (or under sunlight for 8 h). TLC on a silica gel
plate, using petroleum ether : chloroform 2 : 1, indicated the
disappearance of the starting material (Rf = 0 pink spot with
ninhydrin spray) and the formation of two products: 3-(3-methyl-
5-bromomethylphenyl)-3-trifluoromethyl-3H-diazirine (5b) (Rf
0.54) and 3-[3,5-bis(bromomethyl)phenyl]-3-trifluoromethyl-3H-
diazirine (5a) as a minor product (Rf 0.43) in a ratio of 2 : 1,
respectively. Both products were not colored with ninhydrin
spray. The desired product was purified on a silica gel column
eluted with petroleum ether. Compound 5a was obtained in 24%
1
N-(2-{2-[3-[6-Amino-9-(3,4-dihydroxy-5-phosphonooxymethyl-
tetrahydrofuran-2-yl)-9H-purin-8-ylsulfanylmethyl]-5-
(3-trifluoromethyl-3H-diazirin-3-yl)benzylamino]ethyl-
disulfanyl}ethyl)carbamic acid tert-butyl ester (16)
yield as colorless oil. H NMR (CDCl3, 200 MHz) δ 7.48 (t, J =
1.5 Hz, 1H, Ar), 7.13 (br s, 2H, Ar), 4.44 (s, 4H, CH2Br); 13C
NMR (CDCl3, 300 MHz) δ 140.0 (s, 2C, Ar), 130.9 (d, Ar), 130.5
(s, Ar), 126.9 (d, 2CH, Ar), 121.9 (q, 1JCF = 275 Hz, CF3), 31.5 (t,
2
2CH2Br), 28.2 (q, JCF = 40 Hz, C-diazirine); UV λmax (CHCl3)/
A solution of nucleoside 15 (30 mg, 0.039 mmol) in dry tri-
methyl phosphate (1 mL) was added to a flame-dried flask
under nitrogen. The solution was cooled to 0 ЊC, then proton
sponge (13 mg, 2 eq) was added. After 20 min, distilled phos-
phorous oxychloride (8.4 µL, 3 eq) was added dropwise and a
purple clear solution was formed. Stirring was continued for 2 h
at 0 ЊC. TLC (1-propanol–28% NH4OH–H2O (11 : 2 : 7)), indi-
cated the disappearance of starting material and formation of a
polar product (Rf 0.39). 0.2 M TEAB solution (10 mL) was
added. Stirring continued for 45 min and then the solution was
freeze-dried. The residue was separated on HPLC applying a
linear gradient of TEAA–CH3CN 100 : 0 to 50 : 50 in 20 min
followed by isocratic elution for 5 min (TEAA–CH3CN, 50 : 50,
6 mL minϪ1) tR 19.48 min. The product was obtained as a white
solid in 31% yield (12 mg). 1H NMR (D2O, 200 MHz) δ 8.10 (s,
1H, H-2), 7.61 (br s, 1H, Ar), 7.26 (br s, 1H, Ar), 7.12 (br s, 1H,
Ar), 6.09 (d, J = 6 Hz, 1H, H-1Ј), (H-2Ј is hidden by the water
peak), 4.55–4.32 (m, 2H, CH2S), 4.31–3.87 (m, 6H, H-3Ј, H-4Ј,
H-5Ј & Ar-CH2-NH), 3.30–3.07 (m, 4H, CH2NH and CH2-
nm 353. 5b obtained in 51% yield as colorless oil. 1H NMR
(CDCl3, 200 MHz) δ 7.26 (br s, 1H, Ar), 6.99 (br s, 1H, Ar), 6.93
(br s, 1H, Ar), 4.41 (s, 2H, CH2Br), 2.35 (s, 3H, CH3); 13C NMR
(CDCl3, 300 MHz) δ 139.6 (s, Ar), 138.6 (s, Ar), 131.1 (d, Ar),
129.7 (s, Ar), 127.1 (d, Ar), 124.1 (d, Ar), 122.0 (q, 1JCF = 275 Hz,
CF3), 32.3 (t, CH2Br), 28.2 (q, 2JCF = 40 Hz, C-diazirine), 21.3 (q,
CH3); UV λmax (CHCl3)/nm 353. Compound 5b was recycled and
brominated again. The reaction was carried out on 0.85 mmol of
5b by the above procedure using only 1 eq of NBS. Product 5a
was obtained in 64% yield (202 mg).
5-{6-Amino-8-[3-bromomethyl-5-(3-trifluoromethyl-3H-diazirin-
3-yl)benzylsulfanyl]purin-9-yl}-2-hydroxymethyltetrahydro-
furan-3,4-diol (13)
Adenosine 8-thiolate sodium salt (0.14 mmol) was dissolved in
dry DMF (10 mL) and compound 5a (109 mg, 2 eq) dissolved
in DMF (2 mL) was added. The solution was stirred under
nitrogen at room temperature for 4 h. The solvent was evapor-
ated under vacuum and the residue was co-evaporated repeat-
edly with EtOH until it turned into a yellow solid. The product
was purified on a silica gel column eluted with chloroform :
methanol 15 : 1. After evaporation and drying for two days in
vacuo the product was obtained in 60% yield (50 mg). 1H NMR
(CD3OD, 200 MHz) δ 8.08 (s, 1H, H-2), 7.60 (br s, 1H, Ar), 7.21
(br s, 1H, Ar), 7.16 (br s, 1H, Ar), 5.96 (d, J = 7 Hz, 1H, H-1Ј),
4.98 (dd, J = 7, 5 Hz, 1H, H-2Ј), 4.60 and 4.44 (Abq, J = 12 Hz,
2H, CH2S), 4.51 (s, 2H, CH2Br), 4.33 (dd, J = 5, 1.5 Hz, 1H,
H-3Ј), 4.19–4.13 (m, 1H, H-4Ј), 3.87 (dd, J = 12.5, 2 Hz, 1H,
H-5Ј), 3.71 (dd, J = 12.5, 2.5 Hz, 1H, H-5Ј); 13C NMR (CD3OD,
300 MHz) δ 156.2 (s, C-6), 152.4 (d, C-2), 151.4 (s, C-4), 149.8
(s, C-8), 141.29 (s, Ar), 140.5 (s, Ar), 132.6 (d, Ar), 130.7 (s, Ar),
128.0 (d, Ar), 127.2 (d, Ar), 123.3 (q, 1JCF = 275 Hz, CF3), 121.3
(s, C-5), 91.3 (d, C-1Ј), 88.8 (s, C-4Ј), 74.1 (d, C-2Ј), 73.0 (d,
NHCO), 2.74–2.38 (m, 4H, CH2SSCH2), 1.35 (s, 9H, Boc); 31
P
NMR (D2O, 200 MHz, pH 5) δ 4.2 (s); FAB (negative) 839
(M2Ϫ), 841 (M2Ϫ ϩ 2Hϩ). High-resolution FAB calc for
C29H39N9O9F3PS 841.1722, found 841.1670; tR 19.21 min (90%
purity) using solvent system I, 21.60 min (92% purity) using
solvent system II.
N-(2-{2-[3-[6-Amino-9-(3,4-dihydroxy-5-(phosphonooxy-
phosphonooxyphosphonooxymethyl)tetrahydrofuran-2-yl)-9H-
purin-8-ylsulfanylmethyl]-5-(3-trifluoromethyl-3H-diazirin-3-yl)-
benzylamino]ethyldisulfanyl}ethyl)carbamic acid tert-butyl ester
(17)
CDI (22 mg, 0.13 mmol) was added to a flame-dried flask under
nitrogen containing nucleotide 16 (22 mg, 0.022 mmol) in dry
DMF (1 mL). The solution was stirred under nitrogen at room
temperature for 4 h. TLC (isopropanol–28% NH4OH–H2O
(6 : 3 : 1), indicated the disappearance of starting material. Dry
methanol (8 µL) was then added to the reaction mixture. After
5 min 0.1 M (Bu3NH)4P2O7 in DMF (110 mL), prepared from
Na2H4P2O7 with 4 eq of Bu3N in 0 ЊC, was added at once and
the mixture was stirred under nitrogen at room temperature for
3 h. TLC (isopropanol–28% NH4OH–H2O (6 : 3 : 1), indicated
the disappearance of starting material and formation of prod-
uct 17 (Rf 0.46). The solvent was evaporated under vacuum.
2
C-3Ј), 64.0 (t, C-5Ј), 37.5 (d), 32.4 (d), 29.3 (q, JCF = 40 Hz,
C-diazirine).
N-(2-{2-[3-[6-Amino-9-(3,4-dihydroxy-5-hydroxymethyltetra-
hydrofuran-2-yl)-9H-purin-8-ylsulfanylmethyl]-5-(3-trifluoro-
methyl-3H-diazirin-3-yl)benzylamino]ethyldisulfanyl}ethyl)-
carbamic acid tert-butyl ester (15)
N-(tert-Butyloxycarbonyl)cystamine44 (250 mg, 5 eq) and
Dowex-MWA-1 (57 mg) were added to 13 (117 mg, 0.198
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The crude residue was separated on HPLC applying a linear
gradient of TEAA–CH3CN 100 : 0 to 50 : 50 in 20 min followed
by isocratic elution for 5 min (TEAA–CH3CN, 50 : 50, 6 mL
minϪ1) tR 17.41 min. The product was obtained as a white solid
in 19% yield (4.6 mg). 1H NMR (D2O, 600 MHz) δ 8.13 (s, 1H,
H-2), 7.32 (br s, 1H, Ar), 7.27 (br s, 1H, Ar), 7.09 (br s, 1H, Ar),
6.16 (d, J = 6 Hz, 1H, H-1Ј), 5.17 (t, J = 6 Hz, 1H, H-2Ј), 5.09–
4.96 (m, 1H, H-3Ј), 4.65–4.45 (m, 2H, CH2S), 4.44–4.25 (m,
5H, H-4Ј, H-5Ј and Ar-CH2-NH), 3.56–2.98 (m, 4H, CH2NH
and CH2NHCO), 2.68–2.41 (m, 4H, CH2SSCH2), 1.35 (s, 9H,
Boc); 31P NMR (D2O, 200 MHz, pH 8) δ Ϫ9.1 (d), Ϫ10.7 (d),
Ϫ22.0 (t).
ically stirred solution was then preset to 73 ЊC. Nitrogen was
bubbled through the solution for ca. 15 min to exclude air, and
then a blanket of nitrogen was maintained over the solution
during the polymerization period. A deaerated solution con-
taining the initiator benzoyl peroxide (1.5 g, 0.6% w/v of total
solution) and styrene (37.5 mL, 16% w/v of total solution) was
then added to the reaction flask. The polymerization reaction
continued for 24 h and was then stopped by cooling. The micro-
spheres formed were washed by extensive centrifugation cycles
with EtOH and then with water. The particles were then dried
by lyophilization.
2) Magnetic polystyrene microspheres were formed by
seeded polymerization of iron salts on the former polystyrene
microspheres according to the literature.40b In a typical experi-
ment, polystyrene microspheres (2.3 µm, 1 g) were added to a
flask containing 20 mL distilled water. The formed mixture was
sonicated for a few minutes and then mechanically stirred at ca.
200 rpm. The temperature was then preset to 60 ЊC. Nitrogen
was bubbled through the suspension during the coating process
to exclude air. 0.1 mL of iron chloride tetrahydrate aqueous
solution (1.2 mmol in 10 mL H2O) and 0.1 mL of NaNO2
aqueous solution (0.02 mmol in 10 mL H2O) were then succes-
sively introduced into the reaction flask. Thereafter, NaOH
aqueous solution (0.5 mmol in 10 mL H2O) was added until a
pH of ca. 10 was reached. This procedure was repeated four
times. During this coating process the microspheres became
brown–black colored. The suspension was then cooled to room
temperature. The formed magnetic polystyrene microspheres
were then washed extensively in water with a magnet and dried
in a vacuum oven.
3) Silica microspheres of substantially smaller size (30–
40 nm) were coated on the magnetic polystyrene microspheres
by seeded polymerization of tetraethoxysilane on the surface of
these particles, according to the Stober method.40b,57 In a typical
experiment, magnetic polystyrene microspheres (2.3 µm, 1 g)
were added to a flask containing EtOH (93.6 mL) and distilled
water (1.9 mL), and the mixture was sonicated to disperse
the particles. Ammonium hydroxide (1.3 mL) and Si(OEt)4
(3.2 mL) were then added, and the resulting suspension was
shaken at room temperature for 12 h. The resulting magnetic
polystyrene microspheres coated with silica nanoparticles were
freed from free silica particles (30–40 nm diameter) by repeated
centrifugation cycles. The magnetic polystyrene–silica micro-
spheres were then dried in a vacuum oven.
2-{2-[3-[6-Amino-9-(3,4-dihydroxy-5-(phosphonooxyphosphono-
oxyphosphonooxymethyl)tetrahydrofuran-2-yl)-9H-purin-8-
ylsulfanylmethyl]-5-(3-trifluoromethyl-3H-diazirin-3-yl)benzyl-
amino]ethyldisulfanyl}ethylamine (19)
Nucleotide 17 (4.6 mg, 0.0042 mmol) was dissolved in neat TFA
(2 mL) and the solution was stirred at room temperature for
3 min. Then, TFA was evaporated by air flow for an additional
3 min. Product 19 was purified on HPLC applying a linear
gradient of TEAA–CH3CN 100 : 0 to 50 : 50 in 20 min followed
then by an isocratic elution for 5 min (TEAA–CH3CN, 50 : 50)
at 6 mL minϪ1, tR 13.98 min. The product was obtained as a
white solid, in a quantitative yield (4.3 mg). 1H NMR (D2O, 600
MHz) δ 8.20 (s, 1H, H-2), 7.35 (br s, 1H, Ar), 7.32 (br s, 1H,
Ar), 7.12 (br s, 1H, Ar), 6.20 (d, J = 6 Hz, 1H, H-1Ј), 5.17–5.26
(m, 1H, H-2Ј), 5.14–5.09 (m, 1H, H-3Ј), 4.66–4.52 (m, 2H,
CH2S), 4.51–4.24 (m, 5H, H-4Ј, H-5Ј and Ar-CH2-NH), 3.53–
3.02 (m, 4H, CH2NH and CH2NHCO), 2.85–2.70 (m, 2H,
CH2SSCH2), 2.67–2.5 (m, 2H, CH2SSCH2); 31P NMR (D2O,
200 MHz, pH 8) δ Ϫ7.6 (d), Ϫ10.7 (d), Ϫ21.0 (t); UV λmax
(H2O)/nm 285, 353; tR 13.10 min (92% purity) using solvent
system I, 14.03 min (93% purity) using solvent system II. FAB
(negative, glycerol) 923 (M2Ϫ Ϫ 2Hϩ ϩ 2Naϩ).
Product 18 was obtained in the same way in 100% yield start-
ing from 3.9 mg (0.0037 mmol) nucleotide 16. 1H NMR (D2O,
200 MHz) δ 8.19 (s, 1H, H-2), 7.61 (br s, 1H, Ar), 7.23 (br s, 1H,
Ar), 7.21 (br s, 1H, Ar), 6.01 (d, J = 6 Hz, 1H, H-1Ј), 4.87 (t,
J = 6, 1H, H-2Ј), 4.52–4.38 (m, 2H, CH2S), 4.29–4.04 (m, 6H,
H-3Ј, H-4Ј, H-5Ј and Ar-CH2-NH), 3.31–3.09 (m, 4H, CH2NH
and CH2NHCO), 2.86–2.74 (m, 4H, CH2SSCH2); 31P NMR
(D2O, 200 MHz, pH 8) δ 1.1 (s); UV λmax (H2O)/nm 285, 353;
FAB (negative, glycerol) 741 (M Ϫ H)Ϫ, 740 (M Ϫ 2H)Ϫ.
4) Magnetic polystyrene–silica microspheres containing
amine groups were prepared by reacting these particles with
Si(OEt)3(CH2)3NH2, according to the literature.58 In a typical
experiment, Si(OEt)3(CH2)3NH2 (8 mL) was added into a flask
containing 1 g of monodispersed silica coated microspheres of
2.3 µm average diameter dispersed in 100 mL buffer acetate,
0.1 M at pH 5.5. The suspension was stirred at 60 ЊC for 18 h.
Thereafter, the amino derivatized microspheres were washed
intensively by several centrifugation cycles with buffer acetate
and distilled water, respectively and dried in a vacuum oven.
5) The amino derivatized microspheres (10 mg) were shaken
in 0.1 M NaHCO3, pH 8.5, 1 mL, and treated with divinyl
sulfone (1 mg), at room temperature for 4 h. The particles were
collected with a magnet, washed several times, and freeze dried.
The diameter of microspheres 6 was 5.5 15% micron.
Coupling nucleotides 18 or 19 to magnetic microspheres
Nucleotide 19 (1.9 mg, 0.0017 mmol) was dissolved in 0.1 M
ammonium bicarbonate buffer, pH 8.3 (1 mL), and magnetic
microspheres, 6 (1 mg), were added. The reaction mixture was
shaken at room temperature overnight. Magnetic microspheres
were separated from the solution by a strong magnet and
washed with 0.1 M ammonium bicarbonate buffer (4 mL). The
amount of the remaining nucleotide 19 was established based
on the absorption of the filtrate at 285 nm. Finally, the number
of moles of 19 connected to the magnetic microspheres was
calculated. PALMm product 2 was obtained in 70% yield.
PALMm product 1 was obtained in the same way in 74%
yield starting from 12 mg (0.0019 mmol) compound 18.
Synthesis of functional magnetic polystyrene–silica hybrid
Characterization of the magnetic microspheres 6
microspheres 6
Mercaptoethanol (11.5 µL, 0.16 mmol) was dissolved in 0.1 M
sodium bicarbonate buffer (1.5 mL), pH 8.5, and magnetic
microspheres 6 (10 mg) were added. The reaction mixture was
shaken at room temperature overnight. The magnetic micro-
spheres were filtered and 4.6 µL of the filtrate was diluted in
0.1 M sodium bicarbonate buffer (1.5 mL) and added to the
solution of 4,4Ј-dipyridyl disulfide (4-PDS) (0.66 mg, 0.003
mmol) in 0.1 M sodium bicarbonate buffer, pH 8.5 (1.5 mL).
1) Monodispersed polystyrene microspheres were prepared
according to the literature.56 In a typical experiment, poly-
styrene microspheres of 4.5 µm median size diameter (SD 0.07
µm) were formed by introducing into a reaction flask (1 L) a
solution containing PVP, MW 360000 (1.25 g, 0.5% w/v of
total solution) dissolved in a mixture of EtOH (150 mL) and
2-methoxyethanol (62.5 mL). The temperature of the mechan-
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 2 8 2 1 – 2 8 3 2
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The reaction was stirred at room temperature for 5 min and the
absorbance of the final solution was measured at 321 nm.
Finally, the number of free binding sites on the microspheres
was calculated.
Idaho) as described before.61 Briefly, potatoes (5 kg) were
peeled, diced and homogenized in an ice cold solution of thio-
glycollate (50 mM) corresponding to half the volume of potato
(2.5 L per 5 Kg). This homogenate was agitated for 15 min,
filtered through 8 layers of cheesecloth and the turbid extract
was centrifuged 10 min at 1000g to eliminate a solid white pre-
cipitate, then centrifuged a second time 15 min at 13 000g to
eliminate cell debris. Two consecutive salting-outs in 40% and
70% of ammonium sulfate saturation were performed, the pH
was adjusted to pH 4.0. The extract was filtered through 3–4
layers of cheesecloth, then centrifuged 15 min at 13 000g. The
final pellet was re-suspended in 2% of initial potato volume.
The enriched fraction obtained (≈110 mg protein) was applied
to an affinity column (5.8 ml of Cibacron blue F3G-A, BioRad
Inc., Hercules, CA, USA) previously rinsed with at least 5 vol-
umes of washing buffer (MES 100 mM, NaCl 0.5 M, pH 6.0).
The fraction was put three times on the column for maximum
adsorption, then washed 5 times with washing buffer. The
enzyme was eluted by a linear gradient (0.5–4 M) of NaCl. 1
mL sample were collected and fractions containing activity
were pooled and concentrated using Centriprep30 (AMICON).
The concentrate was centrifuged 15 min at 13 000g then applied
to a Sephadex 75 HR 10/30 column (10 × 300 mm), previously
rinsed with 4 volumes of elution buffer (MES 100 mM, pH 6.0).
2 mL was injected in the injection loop of an ÄKTAexplorer
(Amersham Pharmacia Biotech, Baie dЈUrfé, Qc, Canada) and
1 mL samples were collected. Fractions containing more then
20% of total activity were pooled.
Evaluation of 8-BuS-ATP as a hexokinase substrate
The assay was conducted according to the Sigma quality con-
trol test procedure. Hexokinase used for this assay is from
Saccharomyces cerevisiae (Sigma Co). Fresh 8-BuS-ATP solu-
tion (19 mM) was used. 8-BuS-ATP concentration in the assay
solution was 370 µM. The assay was performed in duplicate
(SEM 0.013).
Evaluation of 8-BuS-ATP and 8-BuS-AMP as hexokinase
inhibitors
Freshly prepared 8-BuS-ATP, 8-BuS-AMP, and ATP solu-
tions, each at 19 mM concentration, were used. The resulting
activity of the enzyme was compared to that with ATP alone.
8-BuS-ATP, 8-BuS-AMP, and ATP concentrations in the assay
solution were 370 µM for each.
Evaluation of PALM reagent 2 as a hexokinase substrate or
inhibitor
A freshly prepared solution of PALMm reagent 2 was used.
Reagents were added according to the standard assay, and the
final solution was immediately mixed by inversion and the
increase in absorbance, at 340 nm, was recorded for 20 min.
The assay was done in duplicate. Calculations of enzyme activ-
ity used absorbance data from the fifth minute onwards, after
the magnetic microspheres had completely sank down.
PALMm reagent 2 at 890 µM reduced hexokinase activity to
23% compared to ATP.
Acknowledgements
This work was supported in part by the Marcus Center for
Medicinal Chemistry.
In the same way, PALMm reagent 2 was evaluated as hexo-
kinase inhibitor at 1.26 mM concentration, in the presence of
ATP (1.26 mM). The resulting activity of the enzyme was com-
pared to that with ATP alone (1.26 mM). Reagent 2 had no
inhibitory effect.
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Enzyme activity was measured by the release of inorganic
phosphate with the malachite green colorimetric assay.52 Activ-
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1 mL of the following incubation medium: 8 mM CaCl2, 5 mM
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