Probing the limitations of the fluorous content for tag-mediated microarray formation

Article abstract of DOI:10.1039/c1cc16022b

The synthesis of a di-perfluorohexyl tag is reported for use in a fluorous-based carbohydrate microarray. A comparative microarray study with this di-perfluorohexyl tag and a mono-perfluorooctyl and mono-perfluorohexyl tag found the increased fluorous content conducive to better spot morphology and easier washing protocols without precluding reuse of the fluorous slide.

Full text of DOI:10.1039/c1cc16022b

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Probing the limitations of the fluorous content for tag-mediated
microarray formationw
Heather D. Edwards,a Sahana K. Nagappayyaa and Nicola L. B. Pohl*ab
Received 27th September 2011, Accepted 15th November 2011
DOI: 10.1039/c1cc16022b
The synthesis of a di-perfluorohexyl tag is reported for use in a
fluorous-based carbohydrate microarray. A comparative microarray
study with this di-perfluorohexyl tag and a mono-perfluorooctyl
and mono-perfluorohexyl tag found the increased fluorous
content conducive to better spot morphology and easier washing
protocols without precluding reuse of the fluorous slide.
Perfluorinated compounds (PFCs) have been extensively used
in the past decade in commercial, industrial, and research
studies, but now PFCs are becoming a major environmental
concern.1–7 The most environmentally persistent PFCs are
Scheme 1 Synthetic route to the mono-C6F13-allyl fluorous tag.
perfluorooctane sulfonate (PFOS) and perfluorooctanoate
(PFOA). Recent studies have found that the growing amounts
of PFOS and PFOA found in water correlate with the
bioaccumulation of PFCs in human and animals globally.1–7
However, unlike the longer PFCs, C6 and shorter perfluorous
chains are not environmentally persistent.8 Growing safety
and health issues with these 8-carbon-length fluorocarbons
threaten their continued bulk manufacturing and therefore
also the inexpensive use of this fluorous chain length in a
variety of applications in which environmental escape of the
PFCs is not a concern. However, such octylfluorous tags have
already proven useful not only for efficient separations using
fluorous solid phase extraction (FSPE)9 but also as a con-
venient fluorous handle for microarray formation.10 Fluorous
microarrays rely on non-covalent fluorous–fluorous inter-
actions between the fluorous tail linked to the molecule used
for screening and the fluorous coated glass slide.11,12 In
addition to their use for carbohydrates, such octylfluorous-
based microarrays have also been successful for screening
other small molecule–protein interactions.13,14 More recently,
biotin was adhered to a fluorous-coated glass slide and it was
demonstrated that C8F17-tagged molecules were better for
fluorous biotin–avidin microarrays than the C6F13-tagged
molecules in terms of spot intensity, size and spot morphology.15
Given that a shorter fluorous tag was likely not an option, we set
out to discover a fluorous tag that could form strong enough
non-covalent interactions for robust fluorous microarray studies
without reliance on the C8F17 motif.
First a route to the desired mono-C6F13-tag was designed
based on the synthesis of the known mono-C8F17-allyl tag for
direct comparison.16 We started the synthetic route with
perfluorohexyl iodide as an inexpensive precursor to obtain
our desired product 3 (Scheme 1). Radical addition of per-
fluorohexyl iodide to allyl alcohol followed by reduction of the
iodide provided perfluorohexyl alcohol 2. Alcohol 2 was then
mesylated for displacement by (Z)-1,4-but-2-endiol to provide
the desired allyl tag 3 in 28% overall yield from allyl alcohol.
As one hexylfluorous tag was insufficient for good spot
formation on a fluorous slide, we next designed a new tag
containing two C6F13-moieties for direct comparison with the
related mono-C8F17- and mono-C6F13-tagged carbohydrates
described above. Such a di-C6F13-tag would still need to allow
the attached sugar to orient away from the slide surface in
such a way that protein-binding could take place. As many
sugars come in the form of glycolipids, these structures were
seen as inspiration for the design of compound 7 with two
fluorous tails attached to a group that served as a UV-active
and removable linker (Scheme 2). Using ceric ammonium
nitrate17 the fluorous linker could be readily removed from the
di-C6F13-tagged peracetylated glucose. The desired di-C6F13-tag
synthesis then started with the known diester 4 made by addition
of two allyl groups to diethylmalonate followed by radical
addition of perfluorohexyl iodide to the resulting alkenes.18
The presence of the diesters precluded use of lithium aluminium
hydride to remove the iodides; therefore, microwave-assisted
conditions using zinc in acidic medium were developed for
iodide removal to produce, after base-mediated saponification,
diacid 5. The diacid was immediately subjected to heat-mediated
a Department of Chemistry, Iowa State University, Ames,
IA 50011-3111, USA
b Department of Chemical and Biological Engineering, Iowa State
University, Ames, IA 50011-3111, USA. E-mail: npohl@iastate.edu;
Fax: +1 515-294-0105; Tel: +1 515-294-2339
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c1cc16022b
c
510 Chem. Commun., 2012, 48, 510–512
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Scheme 2 Synthetic route to the di-C6F13 fluorous tag.
decarboxylation to produce monoacid 6.19,20 This monoacid was
reduced to the alcohol for a Mitsunobu reaction analogous to
those previously carried out with other fluorous alcohols21 to
produce the desired di-fluorous tag 7 in a 14% overall yield from
diester 4. The di-fluorous tag was found to be soluble to at least
1 M concentrations in the common solvents dichloromethane
and toluene at room temperature.
With the necessary fluorous tags 3 and 7 in hand, the next step
was their glycosylation with peracetylated trichloroacetimidate-
activated22 mannose, glucose, and rhamnose glycosyl donors.
After Zemplen deacylation and reduction of the alkene, nine
compounds (Fig. 1) were obtained to perform microarray experi-
ments with fluorescein isothiocyanate-labeled concanavalin A
(FITC-ConA). ConA is known to bind to terminally a-linked
D-mannose, whereas b-D-glucose and a-L-rhamnose are not
ligands for this plant lectin.23 Previously reported FSPE
protocols for the octylfluorous tagged monosaccharides were
used for the purification of our new fluorous-tagged mono-
saccharides.21 Interestingly, the same solvents could be used
for eluting the compounds with a single as for a double fluorous
hexyl moiety. The aromatic ring also does not override the
fluorous content in the FSPE protocol.
Fig. 1 The nine fluorous linked saccharides that were spotted at
250 mM concentration on the microarray slide and then screened for
binding to ConA-FITC. The slide was visualized using a fluorescent
scanner at 488 nm.
fluorous glass slide resulted in uneven spots; washing the slides
with a 1 : 1 dichloromethane : methanol solution before print-
ing solved this problem. The morphology of the spots was
found to also be affected by several physical factors like
temperature, humidity and drying time. We printed the slides
under three different humidity conditions (60%, 65% and
70% humidity) while maintaining the temperature at 22 1C
and observed that the slides printed at 70% humidity showed
the best spots with no donut effect.
In order to compare the performance of the three fluorous-tags
on the fluorous-coated glass slide, the carbohydrate microarray
study was set up as shown in Fig. 1. To make a 250 mM
concentration of the carbohydrates, the fluorous-tagged
monosaccharides were dissolved in methanol/DMSO/water
(2 : 6 : 2).24 These monosaccharides were then spotted multiply
in groups of nine onto a commercially available fluoroalkylsilane-
derivatized glass slide using a standard microarray spotting
robot.24,25 The slides were then incubated with a 200 mM
solution of FITC-ConA in phosphate-buffered saline (PBS)
for one hour. After incubation, the slide was washed with a
1Â PBS containing 1% BSA solution twice and then washed
once with distilled water.26 Next, the slide was scanned using a
General Scanning ProScan Array HT at 488 nm to visualize the
carbohydrate–ConA binding (Fig. 1). Finally, the data collected
from the scan were processed through ImaGenes 8.0 software to
obtain the intensities of each fluorous-tagged monosaccharide.
(For details of spotting and scanning see ESI.w)
We speculate that the higher water content in air helps the
hydrophilic carbohydrates to better orient on the glass slide to
create the non-covalent fluorous–fluorous interactions with
the fluorous molecules on the slide. Drying of the slide after
printing also had an effect on the spot morphology. Less donut
effect was observed when the slides were dried for a longer
period of time. After testing for various drying times, we
concluded that the slides should be kept in the humidity
chamber for 18 hours and then outside of it for 2 hours before
incubating.
After optimal spotting and drying conditions were found,
comparisons among the three different fluorous tags were
made. Fluorescence scans after various washing protocols
show that the di-C6F13-tag-containing sugars were robust
and could withstand more than two washings with 1Â PBS
containing 1% BSA. In many cases, the mono-C6F13-tag con-
taining sugars were being washed away when washed more than
Multiple spotting and scanning experiments revealed several
key experimental details. We found that spotting on a new
c
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Chem. Commun., 2012, 48, 510–512 511

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once with the PBS solution containing BSA, whereas the
intensity of the di-C6F13-tag containing sugars was the same
even after washing multiple times. There was only a slight
decrease in the intensity of the –C8F17 tag containing sugars
with multiple washings with PBS as shown by the scans. Fig. 1
shows all nine of the fluorous-linked monosaccharides spotted
on the same fluorous slide. As shown, only two of these fluorous-
linked monosaccharides are seen bound to the ConA-FITC.
The relatively weak non-covalent fluorous interaction of the
mono-C6F13-tag with the slide due to less fluorous content
precluded its visualization. Note also that the b-D-glucose and
a-L-rhamnose compounds did not bind at all, as expected.
From the slide we can see also that the di-C6F13-tag has a
larger spot size and is brighter than the mono-C8F17-tag.
Despite the apparent greater adhesion of the sugars attached
to the di-C6F13-tag to the slides, the fluorous slides could still
be washed and reused at least five times without significantly
increasing background noise.
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By software analysis of spot intensities, we can conclude that
the spot intensity of the sugars attached to the di-C6F13-tag is two
to four times more intense than that of the mono-C8F17-tag
and its binding ability is superior to both the mono-C8F17- and
-C6F13-tag. The synthesis of the di-C6F13-tag is relatively
simple and has high yielding steps which could be carried
out on a larger scale. Clearly, the standard mono-C8F17 tag
can be effectively and efficiently replaced by the di-C6F13-tag.
Given its comparable behaviour on fluorous silica gel, this new
tag could also possibly replace the octylfluorous tag in the
purification of compounds using FSPE in both manual and
automated syntheses.
N.L.B.P. acknowledges the Wilkinson Professorship in Inter-
disciplinary Engineering. This work was supported in part by
the U. S. National Science Foundation (CHE-0911123).
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Notes and references
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512 Chem. Commun., 2012, 48, 510–512
This journal is The Royal Society of Chemistry 2012