Building addressable libraries: Site-selective formation of an N-acyliminium ion intermediate

Article abstract of DOI:10.1021/ol8007827

(Chemical Equation Presented) A strategy for site-selectively generating reactive N-acyliminium ion intermediates on a microelectrode array has been developed. The route capitalizes on the use of an electroauxiliary for building a methoxylated amino acid

Full text of DOI:10.1021/ol8007827

ORGANIC
LETTERS
2008
Vol. 10, No. 12
2501-2504
Building Addressable Libraries:
Site-Selective Formation of an
N-Acyliminium Ion Intermediate
David Kesselring,† Karl Maurer,‡ and Kevin D. Moeller*,†
Department of Chemistry, Washington UniVersity, St. Louis, Missouri 63130, and
CombiMatrix Corporation, 6500 Harbor Heights Parkway, Suite 301, Mukilteo,
Washington 98275
moeller@wustl.edu
Received April 5, 2008
ABSTRACT
A strategy for site-selectively generating reactive N-acyliminium ion intermediates on a microelectrode array has been developed. The route
capitalizes on the use of an electroauxiliary for building a methoxylated amino acid substrate, and then the electrochemical generation and
solution phase confinement of acid in order to form the N-acyliminium ion. Keys to this strategy were the stability of an N-r-methoxyalkyl
amide to basic reaction conditions and the generality of the electrogenerated acid conditions for conducting microelectrode array reactions
in a site-selective fashion.
Chip-based molecular arrays are important tools for probing
interactions between libraries of potential ligands and
biological receptors.1,2 Of particular interest are addressable
microelectrode arrays that have the potential to monitor
binding events between a molecular library and a target
receptor in “real-time”.3 In order to take advantage of
microelectrode arrays for this purpose, we must first have
the synthetic tools needed for building the unique members
of a molecular library by unique, individually addressable
microelectrodes in the array.4,5 For this reason, our group
has been developing methods for conducting site-selective
syntheses on microelectrode arrays.5,6 In this effort, the
microelectrode array is coated with a porous polymer and
† Washington University.
‡ CombiMatrix Corporation.
(4) For a description of the chips used here, see: Dill, K.; Montgomery,
D. D.; Wang, W.; Tsai, J. C. Anal. Chim. Acta 2001, 444, 69. For alternative
addressable electrode arrays, see: (a) Sullivan, M. G.; Utomo, H.; Fagan,
P. J.; Ward, M. D. Anal. Chem. 1999, 71, 4369. (b) Zhang, S.; Zhao, H.;
John, R. Anal. Chim. Acta 2000, 421, 175. (c) Hintsche, R.; Albers, J.;
(1) For reviews, see: (a) Lipshutz, R. J.; Fodor, S. P. A.; Gingeras, T. R.;
Lockhart, D. J. Nat. Genet. 1999, 21, 20. (b) Pirrung, M. Chem. ReV. 1997,
97, 473
.
(2) For more recent examples, see: (a) Webb, S. M.; Miller, A. L.;
Johnson, B. H.; Fofanov, Y.; Li, T.; Wood, T. G.; Thompson, E. B. J.
Steroid Biochem. Mol. Biol. 2003, 85, 183. (b) Shih, S.-R.; Wang, Y.-W.;
Chen, G.-W.; Chang, L.-Y.; Lin, T.-Y.; Tseng, M.-C.; Chiang, C.; Tsao,
K.-C.; Huang, C. G.; Shio, M.-R.; Tai, T.-H.; Wang, S.-H.; Kuo, T.-L.;
Bernt, H.; Eder, A. Electroanal. 2000, 12, 660
.
(5) For Pd(II) reactions see ref 3 as well as: (a) Tesfu, E.; Maurer, K.;
Ragsdale, S. R.; Moeller, K. D. J. Am. Chem. Soc. 2004, 126, 6212–6213.
(b) Tesfu, E.; Maurer, K.; McShae, A.; Moeller, K. D. J. Am. Chem. Soc.
Liu, W.-T. J. Virol. Methods 2003, 111, 55
(3) Tesfu, E.; Roth, K.; Maurer, K.; Moeller, K. D. Org. Lett. 2006, 8,
709.
.
2006, 128, 70.
(6) Tian, J.; Maurer, K.; Tesfu, E.; Moeller, K. D. J. Am. Chem. Soc.
2005, 127, 1392-
10.1021/ol8007827 CCC: $40.75
Published on Web 05/21/2008
2008 American Chemical Society

then substrates for building the library are covalently attached
to the polymer proximal to the electrodes. Site-selective
chemical reactions are then conducted to further develop the
substrates. These site-selective reactions are essentially
competition experiments between the electrochemical gen-
eration of a chemical reagent or catalyst and the solution
phase destruction of the reagent or catalyst before it can
migrate away from the electrodes employed for its formation.
Hence, each new microelectrode array reaction requires two
new developments; an electrochemical method for generating
a reagent or catalyst and a solution phase “confining-agent”
that destroys the reagent or catalyst generated.
generating N-acyliminium ions on a microelectrode array
would represent an important addition to this effort. We
report herein a synthetic strategy for accomplishing this task.
In principle, site-selective N-acyliminium ion generation
can be achieved by using the electrode to generate either an
acid for catalyzing the departure of a leaving group from a
suitable precursor or an oxidant for converting an amide with
an silyl-electroauxiliary into the N-acyliminium ion (Scheme
2).11,12 For the first, a confining agent would be needed to
Scheme 2
Because reactive N-acyliminium ion intermediates have
proven very useful for building alkaloids (Scheme 1, eq 1)7
Scheme 1
scavenge acid generated at selected electrodes in the micro-
electrode array. For the second, a confining agent would be
needed to scavenge an oxidant generated at the electrodes.
While both approaches are attractive, the recent demonstra-
tion that t-Boc groups can be deprotected in a site-selective
fashion using acid on a microelectrode array13 led us to start
our investigation by examining the acid-catalyzed approach.
Our twin goals were to both develop an effective route for
site-selectively producing N-acyliminium ions on a micro-
electrode array and demonstrate the generality of the
electrochemical generation of acid/ confining-agent strategy
developed for the t-Boc deprotection reaction. Can reaction
conditions worked out for the site-selective generation of a
chemical reagent on a microelectrode array be extended to
new applications?
The study began with the synthesis of a suitable test
substrate for use on the microelectrode array (Scheme 3).
To this end, phenylalanine methyl ester was alkylated with
trimethylsilylmethyl chloride and the resulting product acyl-
and constrained peptidomimetics (Scheme 1, eq 2),8–10 and
because they are potentially useful for building peptide-based
bioconjugates (Scheme 1, eq 3), a method for site-selectively
(10) For examples using electrochemistry as a tool to functionalize amino
acids and generate N-acyliminium ions, see: (a) Beal, L. M.; Liu, B.; Chu,
W.; Moeller, K. D. Tetrahedron 2000, 56, 10113. and citations therein. (b)
Fobian, Y. M.; d’Avignon, D. A.; Moeller, K. D. Bioorg. Med. Chem. Lett.
1996, 6, 315. (c) Fobian, Y. M.; Moeller, K. D. Methods Mol. Med. 1999,
23 (Peptidomimetic Protocols) , 259. (d) Tong, Y.; Fobian, Y. M.; Wu,
M.; Boyd, N. D.; Moeller, K. D. J. Org. Chem. 2000, 65, 2484. (e) Cornille,
F.; Fobian, Y. M.; Slomczynska, U.; Beusen, D. D.; Marshall, G. R.;
Moeller, K. D. Tetrahedron Lett. 1994, 35, 6989. (f) Cornille, F.;
Slomczynska, U.; Smythe, M. L.; Beusen, D. D.; Moeller, K. D.; Marshall,
G. R. J. Am. Chem. Soc. 1995, 117, 909. (g) Slomczynska, U.; Chalmers,
D. K.; Cornille, F.; Smythe, M. L.; Beusen, D. D.; Moeller, K. D.; Marshall,
G. R. J. Org. Chem. 1996, 61, 1198. (h) Simpson, J. C.; Ho, C.; Shands,
E. F. B.; Gershengorn, M. C.; Marshall, G. R.; Moeller, K. D. Bioorg. Med.
Chem. 2002, 10, 291. (i) Li, W.; Hanau, C. E.; d’Avignon, A.; Moeller,
K. D. J. Org. Chem. 1995, 60, 8155.
(7) For an extensive review of N-acyliminium ions in synthesi, see:
Maryanoff, B. E.; Zhang, H.-C.; Cohen, J. H.; Turchi, I. J.; Maryanoff, C.
A Chem. ReV. 2004, 104, 1431.
(8) For reviews concerning the use of lactam based peptidomimetics,
see: (a) Cluzeau, J.; Lubell, W. D. Biopolymers 2005, 80, 98. (b) I-lalab,
L.; Gosselin, F.; Lubell, W. D. Biopolymers 2000, 55, 101. (c) Hanessian,
S.; McNaughton-Smith, G.; Lombart, H.-G.; Lubell, W. D. Tetrahedron
1997, 53, 12789. For additional lead references, see: (d) Polyak, F.; Lubell,
W. D. J. Org. Chem. 1998, 63, 5937. (e) Curran, T. P.; Marcaurell, L. A.;
O’Sullivan, K. M. Org. Lett. 1999, 1, 1225. (f) Gosselin, F.; Lubell, W. D.
J. Org. Chem. 2000, 65, 2163. (g) Polyak, F.; Lubell, W. D. J. Org. Chem.
2001, 66, 1171. (h) Feng, Z.; Lubell, W. D. J. Org. Chem. 2001, 66, 1181
.
(9) For synthetic routes to peptidomimetics, see: (a) Scott, W. L.; Alsina,
J.; Kennedy, J. H.; O’Donnell, M. J. Org. Lett. 2004, 6, 1629. (b) Palomo,
C.; Aizpurua, J. M.; Benito; A.; Miranda, J. I.; Fratila, R. M.; Matute, C.;
Domercq, M.; Gago, F.; Martin- Santamaria, S.; Linden, A. J. Am. Chem.
Soc. 2003, 125, 16243. (c) Colombo, L.; Di Giacomo, M.; Vinci, V.;
Colombo, M.; Manzoni, L.; Scolastico, C. Tetrahedron 2003, 59, 4501.
(d) Dolbeare, K.; Pontoriero, G. F.; Gupta, S. K.; Mishra, R. K.; Johnson,
R. L. J. Med. Chem. 2003, 46, 727. (e) Khalil, E. M.; Pradhan, A.; Ojala,
W. H.; Gleason, W. B.; Mishra, R. K.; Johnson, R. L. J. Med. Chem. 1999,
42, 2977. (f) Aube, J. AdV. Amino Acid Mimetics Peptidomimetics 1997, 1,
193. (g) Tong, Y.; Olczak, J.; Zabrocki, J.; Gershengorn, M. C.; Marshall,
G. R.; Moeller, K. D. Tetrahedron 2000, 56, 9791. (h) Duan, S.; Moeller,
K. D. Tetrahedron 2001, 57, 6407. (i) Liu, B.; Brandt, J. D.; Moeller, K. D.
(11) For the generation of N-acyliminium ions using a silyl group as an
electroauxiliary, see: (a) Yoshida, J.; Isoe, S. Tetrahedron Lett. 1987, 28,
6621. (b) Suga, S.; Watanabe, M.; Yoshida, J. J. Am. Chem. Soc. 2002,
124, 14825. For electroauxiliary as a general concept, see: (c) Yoshida, J.;
Nishiwaki, K. J. Chem. Soc., Dalton Trans. 1998, 2589
.
(12) For applications to the synthesis of peptide derivatives, see: Sun,
H.; Martin, C.; Kesselring, D.; Keller, R.; Moeller, K. D. J. Am. Chem.
Soc. 2006, 128, 13761
.
(13) For the use of acid on the chips, see: (a) Rossi, F. M. ; Montgomery,
D. D. ,PCT Int. Appl. (2000), 52 pp. CODEN: PIXXD2 WO 0053625 A2
20000914. (b) Maurer, K.; McShea, A.; Strathmann, M.; Dill, K. J. Comb.
Chem. 2005, 7, 637.
Tetrahedron 2003, 59, 8515
.
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Org. Lett., Vol. 10, No. 12, 2008

The placement of substrate on the microelectrode array
was done so that the substrate was located proximal to each
of the electrodes in the array. In this case, there was little
choice in this matter. In sharp contrast to previous studies
using this same electrogenerated-base catalyzed coupling,14
the activated amino acid substrate used here could not be
placed onto the microelectrode array in a site-selective
fashion. This suggests that placement of the amino acid
substrate on the microelectrode array proceeds through a
different mechanism than the earlier activated ester reactions,
perhaps involving formation of a ketene intermediate that
would react with alcohols in the absence of a base catalyst.
Such a reaction would not be confined to electrodes used to
generate the catalyst. While this observation leads to obvious
concerns about the stereochemistry of the substrate and the
need for a more gentle approach to placing amino acid
derivatives onto a microelectrode array, it did not interfere
with our plan to investigate the potential for site-selectively
generating N-acyliminium ions on the array.
Scheme 3
ated to form 2. The silyl group in 2 was then oxidatively
exchanged for a methoxy group using a constant current
electrolysis in an undivided cell.12 The use of the silyl
electroauxiliary-based approach to 3 was essential for making
the methoxylated amino acid building block in that it avoided
any elimination problems associated with incorporating the
leaving group into the amino acid substrate prior to forming
the amide while aiding the anodic oxidation reaction.12
The methoxylated amide 3 was converted into an activated
ester and placed onto a microelectrode array as outlined in
Scheme 4. Key to this sequence was the compatibility of
For site-selective N-acyliminium ion formation, the sub-
strate modified microelectrode array was inserted into a
solution of diphenylhydrazine and 4-pyrenebutanol in aceto-
nitrile/DMF containing tetrabutylammonium hexafluoro-
phosphate electrolyte (Scheme 5). Selected electrodes in the
Scheme 5
Scheme 4
array were then used as anodes to oxidize the diphenyl-
hydrazine and generate an acid in a manner analogous to
the previously reported t-Boc deprotection.14 The acid was
confined to the region of the microelectrode array surround-
ing the selected electrodes with the use of excess phenyl-
hydrazine. The excess phenylhydrazine served as a base to
neutralize any acid that migrated away from the selected
electrodes. In the region of the array proximal to the active
electrodes, the generation of acid occurred at a rate fast
enough to overwhelm the base and lead to the formation of
an N-acyliminium ion, an event that was detected by an
exchange of the methoxy group on the carbon alpha to the
amide nitrogen with 4-pyrene butanol. For this transforma-
tion, the selected electrodes were cycled; on at a potential
of + 3 V for 0.5 s and off for 1.0 s for 400 cycles. The
longer off cycle is necessary to allow for substrate diffusion
and maintain high current flow. Longer reaction times, up
the R-methoxy alkylamide with saponification of the methyl
ester to deprotect the C-terminus of the amino acid derivative,
conversion of the resulting acid into an activated ester, and
placement of the substrate onto the agarose polymer coating
the surface of the microelectrode array using the electro-
generated base conditions (reduction of vitamin B12 in
methanol/DMF). In the microelectrode array reaction, the
electrodes in the array were cycled on at a potential of -2.4
V for 0.1 s and off for 0.1 s. Three hundred such cycles
were used.5,6
(14) Unpublished results with Melissae Stuart.
Org. Lett., Vol. 10, No. 12, 2008
2503

to 1000 cycles (500s on) did not seem to improve the
intensity of the fluorescence from the surface of the array
above the microelectrodes utilized. Key to the success of
this reaction appears to be adequate swelling the polymer
coating the array. The presence of a measurable current flow
proved to be a useful monitor of polymer swelling since an
unswelled polymer coating effectively quenched the current
flow. When dichloromethane was used as a solvent for the
reaction and a checkerboard pattern of electrodes selected,
the current flow for the reaction was <10 µA and no reaction
was observed. The use of acetonitrile as solvent improved
both swelling of the polymer and the current flow, especially
when a small amount of DMF was employed as a cosolvent.
In addition, the reactions benefited from soaking the chip in
ethanol for a minute before beginning the reaction. When
these conditions were employed, a current of >200 µA could
be consistently obtained when activating a checkerboard
pattern of microelectrodes on the array.
Figure 1. Site-selective reaction. (a) Fluorescence image of the
checkerboard pattern. (b) Confinement around a single electrode
in the array.
ion based syntheses on the microelectrode arrays, but also
shows the generality of the strategy for electrochemically
generating and confining acid to preselected sites on a
microelectrode array. Efforts to develop oxidative strategies
for N-acyliminium ion formation and to take advantage of
the reactive intermediates generated for making substituted
peptides are underway.
Following the reaction, the microelectrode array was
washed by dipping the chip into vials of ethanol, water, and
then ethanol. The arrayed pattern was viewed using a
fluorescence microscope (Figure 1a). The pattern observed
clearly shows the checkerboard pattern of electrodes that
were used as anodes. A closer view shows the level of
confinement that was obtained with this reaction (Figure 1b).
The success of this experiment demonstrates that, like the
Pd(II) reactions studied earlier,5 the conditions for acid
generation and confinement on the microelectrode array are
generally applicable.
Acknowledgment. We thank the ACS Petroleum Research
Fund (42276-AC1) and the National Science Foundation
(CHE-9023698) for their generous support of this work. We
also gratefully acknowledge the Washington University High
Resolution NMR facility, partially supported by NIH grants
RR02004, RR05018, and RR07155, and the Washington
University Mass Spectrometry Resource Center, partially
supported by NIHRR00954, for their assistance.
In conclusion, a synthetic strategy for generating N-
acyliminium ion intermediates site-selectively on a micro-
electrode array has been developed. The reaction uses an
electrochemically generated acid to trigger formation of the
reactive intermediate and excess phenylhydrazine as a
confining agent for preventing the migration of acid away
from the selected electrodes. The success of the chemistry
not only demonstrates the potential for doing N-acyliminium
Supporting Information Available: Experimental pro-
cedures and NMR spectra for obtained compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL8007827
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Org. Lett., Vol. 10, No. 12, 2008