Synthesis of C-linked immobilized analogs of aloisine A by 'click' chemistry

Article abstract of DOI:10.1016/j.tetlet.2008.11.051

An efficient approach for the immobilization of a series of analogs of aloisine A, an in vitro inhibitor of protein kinases, to polymeric supports via a [3+2] cycloaddition reaction is reported.

Full text of DOI:10.1016/j.tetlet.2008.11.051

Tetrahedron Letters 50 (2009) 741–744
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Tetrahedron Letters
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Synthesis of C-linked immobilized analogs of aloisine A by ‘click’ chemistry
*
Rose Haddoub, David Gueyrard, Peter G. Goekjian
Université de Lyon, CNRS, UMR 5246, ICBMS, Laboratoire Chimie Organique 2—Glycochimie, Université Lyon 1, Bât. 308—CPE Lyon, 43,
Bd du 11 Novembre 1918, F-69622 Villeurbanne Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
An efficient approach for the immobilization of a series of analogs of aloisine A, an in vitro inhibitor of
protein kinases, to polymeric supports via a [3+2] cycloaddition reaction is reported.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 17 September 2008
Revised 11 November 2008
Accepted 14 November 2008
Available online 20 November 2008
Keywords:
Protein kinase inhibitor
Immobilization
[3+2] Cycloaddition
Affinity chromatography
Structure-activity relationship studies
Cyclin-dependent kinases (CDKs) belong to the large family of
protein kinases that catalyze the phosphorylation of proteins at a
Ser/Thr residue. CDKs are involved in numerous biological pro-
cesses such as cellular differentiation, transcription, apoptosis,
and cell cycle control.1,2 Furthermore, CDKs have been linked to
several diseases, for example, cancer, neurodegenerative diseases,
and diabetes.3 Due to their key roles, tremendous efforts have been
engaged to identify potential inhibitors of CDKs.4 The selectivity of
kinase inhibitors is usually addressed by testing inhibitors on a
panel of purified kinases.5 However, even the most sophisticated
panels cannot cover all kinases of the human genome, of which
more than 800 have been postulated. Furthermore, other non-ki-
nase targets need to be considered, either for detrimental or for
synergistic effects.6 Alternative approaches have therefore been
developed to assess the selectivity of kinase inhibitors. Of these,
affinity chromatography7 of cellular extracts on immobilized
inhibitors has previously been applied successfully to purvalanol,8
paullone,9 indirubin,10 and roscovitine.11
N
N
7
4'
OH
N
H
3'
Figure 1. Aloisine A.
the molecule have to be considered. In our previous work, we have
synthesized aloisine conjugates bearing an extended tri(ethylene-
glycol) (TEG) linker either on the butyl chain or in the 40-hydroxyl
position to assess its selectivity by affinity chromatography.14 In
this Letter, we report the synthesis of aloisine conjugates bearing
a TEG chain in the C-30 position. Furthermore, the synthetic strat-
egy developed, based on the well-documented Cu(I)-catalyzed
Huisgen 1,3-dipolar cycloaddition (so-called ‘click’ reaction),15,16
also allows for the direct immobilization of a series of aloisine ana-
logs onto beads.
The synthesis of pyrrolo[2,3-b]pyrazine cores typically involves
the condensation of a benzonitrile derivative with a lithiated
alkyl pyrazine.17 Introduction of the triethylene glycol linker
can be performed either on the pyrrolo[2,3-b]pyrazine or, in
most cases, on its benzonitrile or pyrazine precursor. However,
numerous problems were observed using the combination of a
basic and hygroscopic pyrazine core and a chelating, hydrophilic
TEG linker,14 which we sought to overcome by incorporating the
chain at a very late stage. Our strategy thus relies on the prep-
aration of the 30-alkynyl aloisine 2 as key intermediate, allowing
subsequent conjugation with the azido-functionalized TEG linker
318 (Fig. 2).
Aloisine
A (7-n-butyl-6-(4-hydroxyphenyl)-5H-pyrrolo [2,3-
b]pyrazine, Fig. 1) is a potent inhibitor of CDKs, highly selective
for CDK1/cyclinB, CDK2/cyclin A-E, CDK5/p25, and GSK-3 (Glyco-
gen synthase kinase-3) based on in vitro assays.12,13 A thorough
investigation of the selectivity of aloisine is of interest for a better
understanding of its cellular and physiological action, as well as the
optimization of its pharmacological properties. However, to take
into account the potential perturbations arising from introduction
of a linker tail necessary for immobilization, various positions of
* Corresponding author. Tel.: +33 4 72448183; fax: +33 4 78898914.
E-mail address: P.goekjian@univ-Lyon1.fr (P.G. Goekjian).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.11.051

742
R. Haddoub et al. / Tetrahedron Letters 50 (2009) 741–744
previous SAR studies on aloisine,12 we thus decided to explore
some structural modifications, such as replacement of the 40-hy-
droxyl for a chlorine (aloisine B), a methoxy, or a tetrahydropyran-
oxy, as well as the suppression of the butyl chain at C-7. To this
aim, a small ‘proof of concept’ library of 30-alkynyl aloisines was
prepared according to Figure 4. 4-methoxybenzonitrile (10) was
brominated by ortho-lithiation/bromination in the presence of LiT-
N
N
N
N
OH
OTHP
R
N3
Ts
3
+
O
N
R
N
H
N
N
3
2
N
O
H2N
26
O
MP and ZnCl2 to give an inseparable 15:85 mixture of 2- and
1
3-bromo-4-methoxybenzonitrile regioisomers 12a and 12b. Sono-
gashira coupling with (trimethylsilyl)acetylene on the mixture
afforded, after purification, the desired regioisomer 14b in a 47%
yield over two steps, along with a small amount of the 2-ethynyl
regiosiomer 14a. The same strategy applied to the 4-chlorobenzo-
nitrile (11) gave the 2-bromo-4-chorobenzonitrile regioisomer 13a
as the major product (ratio 80:20). Sonogashira coupling with (tri-
methylsilyl)acetylene yielded an unseparable mixture of 15a and
15b. The condensation step was performed using either methyl
or pentylpyrazine. As before, reaction of the 3-alkynyl-4-methoxy-
benzonitrile 14b in the presence of 2 equivalents of LDA followed
by removal of the silyl protecting group afforded the desired pyr-
rolo[2,3-b]pyrazine cores 18 and 19 in 66% and 82% yields, respec-
tively. Surprisingly, all attempts at condensation with the 2-
alkynyl isomer 14a failed, presumably due the steric hindrance of
the acetylene group. Attempted condensation of the mixture of the
4-chlorobenzonitrile acetylenes 15a and 15b with pentylpyrazine
under the same conditions yielded a complex mixture, of which
only the de-halogenated pyrrolo[2,3-b]pyrazine 20 could be iden-
tified as a product.
Having in hand the acetylene-substituted pyrrolo[2,3-b]pyra-
zine compounds 2, 18, 19, and 21 (the latter obtained from 2 by
removal of the THP), we next turned our attention to their
immobilization onto beads. In order to immobilize the compounds,
commercially available 1,4-bis(2:3-epoxypropoxy)butane-deriva-
tized agarose beads (Epoxy-Agarose, Aldrich) were reacted with
sodium azide in water while maintaining the pH below 9 to pro-
vide the azido functionalized gel 22. A corresponding azido-substi-
tuted gel 23 was prepared based on LCC-ReactospheresÒ beads, an
amino-functionalized polymethyl methacrylate polymer with
highly polar surface, which have the advantage of being resistant
to a wide range of chemical conditions, in the event that further
derivatization of the analogs on the gel would be desired. The alky-
nyl-aloisines 2, 18, 19, and 21 were then immobilized under click
coupling conditions24,25 in the presence of CuI and DIPEA in aceto-
nitrile to afford the corresponding immobilized aloisines 24–28
(Table 1).27 The efficiency of the reaction was monitored qualita-
tively by visualization of the characteristic aloisine fluorescence
on the beads under a UV lamp.
Figure 2. Strategy for the introduction of a TEG linker at C-30 position.
The synthesis of 1 is depicted in Figure 3. The commercially
available 3-bromo-4-hydroxybenzonitrile was protected as
4
the THP ether by reaction with DHP in the presence of PPTS in
dichloromethane to afford 5 in 57% yield. 5 was then reacted un-
der Sonogashira conditions with (trimethylsilyl)acetylene to give
the 3-alkynylbenzonitrile 6 in 77% yield.19 Compound 6 was re-
acted with pentylpyrazine in the presence of 2 equiv of LDA, to
provide a mixture of the expected 7 and its desilylated product
2.20,21 Treatment of this mixture with TBAF allowed obtention of
the acetylene 2 in 80% yield over two steps. The key cycloaddi-
tion step of 2 with the azido chain 3 was performed using CuI
and DIPEA in acetonitrile at room temperature and afforded
the triazole 8 in 85% yield.22 Nucleophilic substitution of the to-
syl with sodium azide gave 9 (77%), which upon cleavage of the
40-OTHP with p-TSA, and reduction of the azide gave the amino-
terminated linked aloisine 1 in 64% yield. This route proved to
be considerably more efficient than those in which the TEG
chain was incorporated at an early stage.
In view of the encouraging results obtained by this approach,
we then explored the possibility of using the cycloaddition reac-
tion directly on solid-phase to immobilize the molecule onto
beads. Indeed, several examples have shown the potential of the
copper-catalyzed ‘click’ reaction on solid support under a broad
range of conditions.23–25 Such an approach would only require
incorporating a relatively inert alkyne functionality on the parent
molecule, and would thus be sufficiently efficient to allow the
preparation of libraries of immobilized inhibitors, and thus to per-
form simultaneous structure-activity relationships on the com-
plete proteome of the cell by affinity chromatography. Based on
N
NC
OTHP
i
NC
OH
iii
OTHP
R
N
H
N
X
Br
5 : X = Br
6 : X = CCSiMe3
7 : R = SiMe3
2 : R = H
ii
iv
4
i
NC
R1
NC
R1
NC
R1
+
X
X
10 : R1= OMe
N
N
a
b
N
N
11 : R1= Cl
v
vii-viii
OH
OTHP
12 : R1= OMe, X=Br
13 : R1= Cl, X=Br
14 : R1= OMe, X= CCSiMe3
15 : R1= Cl, X=CCSiMe3
N
H
N
ii
H
N
N
N
N
N
N
R2
O
H2N
O
X
N
N
iii
iv
O
R2
O
14b or 15b
+
R1
H
N
H
N
N
1
8 : X = OTs
9 : X = N3
vi
18 : R1= OMe, R2=H
19 : R1= OMe, R2=C4H9
20 : R1= H, R2=C4H9
16 : R2=H
17 : R2=C4H9
Figure 3. Synthesis of aloisine A analog 1 bearing a linker at the C-30 position.
Reagents and conditions: (i) DHP, PPTS, CH2Cl2 (57%) (ii) PPh3, PdCl2(PPh3)2,
trimethylsilyl)acetylene, TEA, CuI, THF (77%); (iii) LDA, n-pentylpyrazine, THF,
ꢀ40 °C; (iv) TBAF, THF (80%); (v) 3, DIPEA, MeCN, CuI, rt (85%); (vi) NaN3, DMF,
70 °C (77%); (vii) p-TSA, MeOH (84%); (viii) H2, 10%Pd/C, MeOH, rt (64%).
Figure 4. Synthesis of 30-alkynylaloisines. Reagents and conditions: (i) LiTMP,
ZnCl2, Br2, ꢀ70 °C; (ii) PPh3, PdCl2(PPh3)2, (trimethylsilyl)acetylene, TEA, CuI, THF
(14a, 8% 14b, 47%); (iii) LDA, THF, ꢀ40 °C; (iv) TBAF, THF (18, 66%; 1 82%).

R. Haddoub et al. / Tetrahedron Letters 50 (2009) 741–744
743
Table 1
References and notes
Immobilization of the alkynyl aloisines
R2
R2
1. Dhavan, R.; Tsai, L. H. Nat. Rev. Mol. Cell Biol. 2001, 2, 749–759.
2. Morgan, D. Annu. Rev. Cell Dev. Biol. 1997, 13, 261–291.
N
N
N
N3
R1
R1
i
Linker
+
3. See for example: Cohen, P. Nat. Rev. Drug Discovery 2002, 1, 309–315;
Malumbres, M.; Barbacid, M. Nat. Rev. Cancer 2001, 1, 222–231.
4. Fischer, P. M. Curr. Opin. Drug Discovery Dev. 2001, 4, 623–634.
5. Bain, J.; Plater, L.; Elliott, M.; Shapiro, N.; Hastie, J.; McLauchlan, H.; Klevernic,
I.; Arthur, J. S. C.; Alessi, D.; Cohen, P. Biochem. J. 2007, 408, 297–315.
6. See for example: Trapp, J.; Jochum, A.; Meier, R.; Saunders, L.; Marshall, B.;
Kunick, C.; Verdin, E.; Goekjian, P.; Sippl, W.; Jung, M. J. Med. Chem. 2006, 49,
7307–7316.
7. Knockaert, M.; Greengard, P.; Meijer, L. TRENDS Pharmacol. Sci. 2002, 23, 417–
425; Knockaert, M.; Gray, N.; Damiens, E.; Chang, Y.-T.; Grellier, P., et al Chem.
Biol. 2000, 7, 411–422.
8. Knockaert, M.; Gray, N.; Damiens, E.; Chang, Y. T.; Grellier, P.; Grant, K.;
Fergusson, D.; Mottram, J.; Soete, M.; Dubremetz, J. F.; LeRoch, K.; Doerig, C.;
Schultz, P. G.; Meijer, L. Chem. Biol. 2000, 7, 411–422.
N
R
N
H
N
N
N
22 : Agarose
N
H
23 : LCC-Reactospheres
Linker
i : DIPEA, CuI, MeCN
Alkyne-Aloisine
R1
R2
Azido-Gel
Product
18
19
2
21
21
OMe
OMe
OTHP
OH
H
22
22
22
22
23
24
25
26
27
28
C4H9
C4H9
C4H9
C4H9
OH
9. Knockaert, M.; Wieking, K.; Schmitt, S.; Leost, M.; Mottram, J.; Kunick, C.;
Meijer, L. J. Biol. Chem. 2002, 277, 25493–25501.
10. Meijer, L.; Skaltsounis, A. L.; Magiatis, P.; Polychronopoulos, P.; Knockaert, M.;
Leost, M.; Ryan, X. P.; Vonica, C. A.; Brivanlou, A.; Dajani, R.; Crovace, C.;
Tarricone, C.; Musacchio, A.; Roe, S. M.; Pearl, L.; Greengard, P. Chem. Biol. 2003,
10, 1255–1266.
11. Bach, S.; Knockaert, M.; Reinhardt, J.; Lozach, O.; Schmitt, S.; Baratte, B. J. Biol.
Chem. 2005, 280, 31208–31219.
12. Mettey, Y.; Gompel, M.; Thomas, V.; Garnier, M.; Leost, M.; Ceballos-Picot, I.,
et al J. Med. Chem. 2003, 46, 222–236.
13. Mapelli, M.; Massimiliano, L.; Crovace, C.; Seeliger, M.; Tsai, L. H.; Meijer, L.;
Musacchio, A. J. Med. Chem. 2005, 48, 671–679.
14. Corbel, C.; Haddoub, R.; Guiffant, D.; Lozach, O.; Gueyrard, D.; Lemoine, J.;
Ratin, M.; Meijer, L.; Bach, S.; Goekjian, P.G., unpublished results.
15. Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40, 2004–
2021.
16. Kolb, H. C.; Sharpless, K. B. Drug Discovery Today 2003, 8, 1128–1137.
17. Vierfond, J. M.; Mettey, Y.; Mascrier-Demagny, L.; Miocque, M. Tetrahedron Lett.
1981, 22, 1219–1222.
The affinity chromatography assays were performed on porcine
brain extracts using the different gels (see Supplementary data
Fig. S1). While a detailed biological discussion of the results will be
reported in due course,14 a qualitative analysis of the results permits
a few relevant observations: comparing the results using the com-
pound 1 immobilized on cyanogen bromide-activated agarose with
those of the gel 27 having essentially the same structure, although
the capacity of the latter is lower (consistent with the lower degree
of functionalization of the commercial epoxy sepharosecomparedto
cyanogen-bromide activated agarose), the same target proteins are
retained, and are not retained by the ethanolamine control. This
demonstrates that the aloisine analogs were indeed immobilized
by click chemistry to the azido-substituted Epoxy-Agarose gel. The
reactosphere gel (28), although having the advantage of greater
chemical stability, appears to bind the same target proteins, but also
leads to more extensive non-specific binding. Comparison of the re-
sults obtained with the different gels (see Supplementary data
Fig. S2) shows that it is in fact possible to obtain qualitative struc-
ture-activity relationships against the entire proteome by affinity
chromatography. Although we have used a very limited library as
a proof of concept, the synthesis of the precursors and the immobi-
lization and affinity chromatography protocols are sufficiently sim-
ple to be applied to larger focused libraries.
In conclusion, we have described the preparation of immobi-
lized forms of aloisine for selectivity screening by affinity chroma-
tography, based on the incorporation of an acetylene group,
followed by late stage immobilization, either in solution or in the
solid state. This method represents an advantageous solution to
the problem of incorporating hydrophilic and chelating polyethyl-
ene glycol chains that are often incompatible with the synthesis of
the original compounds. This approach is efficient enough for
immobilizing small libraries for selectivity optimization against
complete cell extracts.
18. Eduardo, F. M.; Correa, J.; Irene, R. M.; Ricardo, R. Macromolecules 2006, 39,
2113–2120.
19. Typical procedure for the Sonogashira reaction: Synthesis of compound 6: PPh3
(61 mg, 0.23 mmol), PdCl2(PPh3)2 (378 mg, 0.54 mmol), and 1.1 g of compound
5 (3.9 mmol., prepared by THP protection of the 4-hydroxybenzonitrile) were
dried for 30 min in a schlenk tube. 10 mL of THF, 2.7 mL of TEA (19.5 mmol),
and 1.1 mL of (trimethylsilyl)acetylene (7.8 mmol) were added to the mixture.
After 30 min, CuI (0.05 equiv) were added to the solution. The reaction mixture
was stirred at rt under an atmosphere of argon for 12 h. The solvent was
removed under vacuum and the solid was filtered off. The crude product was
purified by flash chromatography over silica gel and was eluted with PE/EA
(85/15). Yield 900 mg (77%). 1H NMR (CDCl3): d (ppm) 7.68 (1H, d, J = 2.1 Hz);
7.50 (1H, dd, J = 8.7 Hz, J = 2.2 Hz); 7.12 (1H, d, J = 8.7 Hz); 5.60 (1H, s); 3.80
(1H, dt, J = 2.6 Hz, J = 11.0 Hz); 3.60 (1H, m); 2.05–1.60 (6H, m); 0.25 (9H, s).
HRMS calcd for C17H21NO2Si: 300.1420; found: 300.1420.
20. Davis, M.; Wakefield, B.; Wardell, J. Tetrahedron 1992, 48, 939–952.
21. Typical procedure: Synthesis of compound 7: n-pentylpyrazine (175 mg,
1.16 mmol) was added to a freshly prepared solution of LDA (2.32 mmol in
4 ml of THF-hexane) at ꢀ40 °C under argon. After 30 min, compound
6
(180 mg, 0.58 mmol) in THF (1 mL) was added, and the solution was stirred for
30 min at ꢀ40 °C and for 18 h at rt, then hydrolyzed with a saturated aqueous
solution of NH4Cl. After extraction with ethyl acetate, the organic layer was
dried over Na2SO4 and concentrated under vacuum. The crude product was
purified by flash chromatography over silica gel and was eluted with EA/PE (2/
8) to yield a mixture of compound 7 and 2, from which a pure fraction of 7 was
characterized. 1H NMR: (300 MHz, CDCl3): d (ppm) 12.60 (1H, s); 8.40 (1H, d,
J = 2.8 Hz); 8.20 (1H, d, J = 2.7 Hz); 7.90 (1H, d, J = 2.2 Hz); 7.68 (1H, dd,
J = 8.8 Hz, J = 2.4 Hz); 7.25 (1H, d, J = 8.8 Hz); 5.68 (1H, br s); 4.00 (1H, m); 3.68
(1H, m); 2.99 (2H, t, J = 7.7 Hz); 2.10–1.60 (8H, m); 1,40 (2H, m); 0,90 (3H, t,
J = 7.3 Hz); 0.25 (9H, s). HRMS calcd for C26H33N3O2Si: 448.2420; found:
448.2423.
Acknowledgments
We thank Pr. Laurent Meijer, Dr. Stéphane Bach, Pr. Jérôme
Lemoine, and Miss Caroline Corbel for the biological and protein
studies shown in the supplementary materials. Financial support
from the European Union (Contract No. LSHB-CT-2004-503467)
and a partial Ph.D. scholarship from 3Ds Lebanon (RH) are grate-
fully acknowledged. We also thank LCC Engineering & Trading
GmbH, CH-2246 Egerkingen, Switzerland for providing a sample
of LCC-Reactospheres beads.
22. Typical procedure for the click reaction: Synthesis of compound 8. To a stirred
solution of compound 2 (320 mg, 0.85 mmol) in MeCN (10 mL) were added
280 mg of compound 3 (0.85 mmol). After 20 min at rt, 445 lL of DIPEA
(2.55 mmol) and a catalytic amount of CuI (ꢁ0.05 equiv) were added, the
resulting mixture was stirred at room temperature for 14 h and the reaction
was stopped by adding 2 mL of water. The resulting solution was extracted
with ethyl acetate, the organic phase washed with NaCl, dried over Na2SO4, and
concentrated under vacuum. The crude product was purified by flash
chromatography over silica gel and was eluted with EA/PE (95/5). Yield
520 mg (86%). 1H NMR: (300 MHz, CDCl3): d (ppm) 11.10 (1H, br s); 8.72 (1H, d,
J = 2.1 Hz); 8.40–8.30 (3H); 8.22 (1H, d, J = 2.9 Hz); 7.72 (2H, d, J = 7.8 Hz); 7.65
(1H, dd, J = 8.6 Hz, J = 2.1 Hz); 7.42 (1H, d, J = 8.9 Hz); 7.25 (2H, d, J = 7.8 Hz);
5.60 (1H, br s); 4.62 (2H, t, J = 4.9 Hz); 4.20 (2H, t, J = 5.0 Hz); 3.90 (2H, t,
J = 5.1 Hz); 3.83 (1H, m); 3.70 (1H, m); 3.62–3.58 (6H, m); 3.05 (2H, t,
J = 7.7 Hz) 2.43 (3H, s, CH3Ts); 2.00–1.65 (8H, m); 1.45 (2H, tq, J = 7.5 Hz); 0.88
(3H, t, J = 7.4 Hz). HRMS calcd for C36H44N6O7S: 705.3070; found: 705.3068.
23. Lober, S.; Gmeiner, P. Tetrahedron 2004, 60, 8699–8702.
Supplementary data
Affinity chromatography results with the immobilized aloisines.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2008.11.051.

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R. Haddoub et al. / Tetrahedron Letters 50 (2009) 741–744
24. Punna, S.; Kaltgrad, E.; Finn, M. G. Bioconjugate Chem. 2005, 16, 1536–1541.
25. Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057–3064.
26. Menzel, K.; Fischer, E.; DiMichele, L.; Frantz, D.; Nelson, T.; Kress, M. J. Org.
Chem. 2006, 71, 2188–2191.
27. (a) Azido substituted Agarose: Commercial Epoxy-Agarose (Aldrich 1 g, 20–
40 mol/mL) was suspended in aqueous sodium azide (6 mL, 10 equiv) and
stirred by rotation at 50 °C, while maintaining the pH below 9 by addition of
aliquots of saturated aqueous solution of NH4Cl. After 8 h, the resin was filtered
and washed with water. (b) Azido substituted Reactospheres: The azido
diglycolic acid PEG n7, MW 554.6 (500 mg, LCC) is attached on the amino
PMMA LCC-ReactospheresÒ A310 resin (2.5 g) in DMF (4 mL) by adding EDCI
(180 mg, 2 equiv) and HOBt (400 mg, 3 equiv). The mixture is allowed to react
overnight at room temperature. The resin is then filtered and washed with
MeOH/DMF, MeOH, DCM, and DMF. The synthesis step is repeated twice.(c)
Typical conditions for the on-gel click reaction: The azido substituted agarose
(1 mL) was suspended in acetonitrile (3 mL) containing 2 (0.03 mmol), DIPEA
(5 equiv to the alkyne), and a catalytic amount of CuI (ꢁ0.05 equiv). The
mixture was stirred by rotation overnight. The resin was filtered and washed
thoroughly with MeOH, DMF, CH2Cl2, and 0.2 M EDTA to provide 26 as a white,
UV-fluorescent gel, indicating the presence of immobilized 2. Further IR
measurements indicated the disappearance of the azido group and the affinity
chromatography results provided in the supplementary material demonstrate
the presence of the immobilized aloisines.