Nondestructive, colorimetric monitoring of amines and thiols on a solid support

Article abstract of DOI:10.1021/ol901154e

A new, nondestructive, highly sensitive method for colorimetric monitoring of primary amines, secondary amines, and thiols on a solid support was developed. The resin used in this method is simply regenerated for the repetition of the reaction or an ensui

Full text of DOI:10.1021/ol901154e

ORGANIC
LETTERS
2009
Vol. 11, No. 15
3438-3441
Nondestructive, Colorimetric Monitoring
of Amines and Thiols on a Solid
Support
Seh-Jin Yang, Xi Zhe Tian, and Injae Shin*
Department of Chemistry, Yonsei UniVersity, Seoul 120-749, Korea
injae@yonsei.ac.kr
Received June 10, 2009
ABSTRACT
A new, nondestructive, highly sensitive method for colorimetric monitoring of primary amines, secondary amines, and thiols on a solid support
was developed. The resin used in this method is simply regenerated for the repetition of the reaction or an ensuing reaction. By using this
new method, several peptides containing secondary amide linkages and C-terminal hydrazide groups were prepared in high purities and
yields.
Since the seminal technology of solid-phase peptide synthesis
(SPPS) was developed by Merrifield in 1963,1 solid-phase
synthesis (SPS) has become the leading edge technology for
the preparation of various substances such as peptides/
proteins, unnatural biopolymers, oligonucleotides, carbohy-
drates, and small molecules.2 Solid support-based chemistry
has several practical advantages over conventional solution-
based chemistry, including the fact that (1) SPS does not
require implementation of extensive workup procedures and
chromatographic purifications and (2) large excesses of
reagents can be used to drive reactions to completion.
However, the rapid, reliable monitoring of the progress of
on-resin reactions remains a significant problem.
Over the past decade, much effort has been devoted to
the development of detection methods for solid-phase reac-
tions. One technique involves HPLC analysis of synthetic
intermediates that are cleaved from the solid support.
Although facile, this method requires relatively large amounts
of samples that have to be discarded after analysis. Single-
bead FT-IR analysis can also be used to monitor on-resin
reactions. However, this method is restricted to analysis of
a limited set of substances containing IR discernible func-
tional groups, and it does not enable the reuse of analyzed
samples.3 Nondestructive, NMR spectroscopic detection
methods have been developed to analyze the progress of
(1) Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149–2154.
(2) (a) Caruthers, M. H. Science 1985, 230, 281–5. (b) Schno¨lzer, M.;
Kent, S. B. Science 1992, 256, 221–225. (c) Danishefsky, S. J.; McClure,
K. F.; Randolph, J. T.; Ruggeri, R. B. Science 1993, 260, 1307–1309. (d)
Cho, C. Y.; Moran, E. J.; Cherry, S. R.; Stephans, J. C.; Fodor, S. P.; Adams,
C. L.; Sundaram, A.; Jacobs, J. W.; Schultz, P. G. Science 1993, 261, 1303–
1305. (e) Bunin, B. A.; Ellman, J. A. J. Am. Chem. Soc. 1992, 114, 10997–
10998.
(3) Yan, B.; Sun, Q.; Wareing, J. R.; Jewell, C. F. J. Org. Chem. 1996,
61, 8765–8770.
10.1021/ol901154e CCC: $40.75
Published on Web 07/07/2009
 2009 American Chemical Society

Scheme 1. Synthesis of 1 and 2 Used for the Nondestructive
Colorimetric Resin Test
Figure 1. Strategy for a nondestructive colorimetric resin test: resin-
bound amines and thiols are monitored by reacting with an activated
ester of a reagent, and after the resin test a dye moiety is removed
by treatment with Bu4NF to regenerate the resin via a 1,6-
elimination process.
solid-phase reactions.4 Although the NMR techniques have
the advantage that samples can be reused after analysis, they
are not suitable for rapid monitoring of functional group
transformations. Overall, chromatographic and spectroscopic
methods for the detection of on-resin reactions are neither
simple nor convenient, and they require relatively long times
for analysis.
As part of efforts to develop simple and rapid methods
for monitoring functional groups on the resin-supported
molecules without the need for specialized instrumentation,
colorimetric monitoring tools have been explored.5 These
methods are based on the technique of staining resins that
contain specific functional groups. As a result, the progress
of a solid-phase reaction can be readily monitored by
examining the level of color of the stained resin using eye
or microscopic detection. For example, the Kaiser ninhydrin
test, widely used for SPPS, has been employed to determine
the presence of a primary aliphatic amine on a resin.6
However, this test is limited to primary aliphatic amines,
and it sometimes leads to false positives due to the harsh
reaction conditions that are used (1-5 min, 100 °C). The
TNBS (2,4,6-trinitrobenzenesulfonic acid) test has also been
used to detect on-resin amines, but this method also gives
positive results with any resin-bound base.7
is present in cysteine and other small molecules that are
frequently modified with other functional groups. Polymer-
supported substances possessing thiol groups can be moni-
tored by using the NF31 procedure.9 Other functional groups,
such as halogens, alcohols, carboxylic acids, and carbonyl
groups can be monitored on solid supports by using colo-
rimetric methods.5
Although colorimetric resin tests have been successfully
employed to monitor the progress of on-resin reactions, most
of these methods are limited by the fact that samples can
not be reused for the next reactions after resin tests, and as
a consequence, the need for repetitive colorimetric resin tests
causes a large sample loss. Therefore, the development of a
rapid, simple, and nondestructive method to detect the
progress of on-resin reactions remains as a significant
challenge. Below, we describe the results of a study that has
led to the first, resin-reusable, colorimetric method for
polymer-bound primary amine, secondary amine, and thiol
detection.
The new, nondestructive, colorimetric resin test involves
treatment of solid-supported amines and thiols with a dye
containing an activated ester group. This leads to the rapid
formation of intensely colored beads. Subsequent removal
of a colored moiety from solid support after the resin test is
performed regenerates the resin for the repetition of the
reaction (in case of incomplete reactions) or a subsequent
reaction. The molecular system used for this process consists
of three parts, including a dye for color detection, an activated
To improve these old techniques, several colorimetric resin
tests, including chloroanil and NF31 tests, have been
developed to analyze the extent of completeness of reactions
carried out on a polymeric matrix.8 The detection of resin-
bound thiol groups is also important since this functionality
(4) (a) Seeberger, P. H.; Beebe, X.; Sukenick, G. D.; Pochapsky, S.;
Danishefsky, S. Angew. Chem., Int. Ed. 1997, 36, 491–493. (b) Kanemitsu,
T.; Wong, C.-H.; Kanie, O. J. Am. Chem. Soc. 2002, 124, 3591–3599. (c)
Mogemark, M.; Elofsson, M.; Kihlberg, J. Org. Lett. 2001, 3, 1463–1466.
(5) Gaggini, F.; Porcheddu, A.; Reginato, G.; Rodriquez, M.; Taddei,
M. J. Comb. Chem. 2004, 6, 805–810.
(6) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal.
Biochem. 1970, 34, 595–598.
(8) (a) Marik, J.; Song, A.; Lam, K. S. Tetrahedron Lett. 2003, 44, 4319–
4320. (b) Madder, A.; Farcy, N.; Hosten, N. G. C.; De Muynck, H.; De
Clercq, P. J.; Barry, J.; Davis, A. P. Eur. J. Org. Chem. 1999, 2787–2791.
(9) Caroen, J.; Van der Eycken, J. Tetrahedron Lett. 2009, 50, 41–44.
(7) Hancock, W. S.; Battersby, J. E. Anal. Biochem. 1976, 71, 260–
264.
Org. Lett., Vol. 11, No. 15, 2009
3439

Figure 3. Regeneration of amines and thiols after the color test.
Bottom: microscopic images of the resin after reactions of the
colored resin with 0.2 M Bu4NF for 5 min twice at room
temperature.
were observed from reactions of 1 with on-resin amines and
thiols in the presence of a mixture of DBU (3 equiv) and
DMAP (0.2 equiv) for 7 min. However, 2 reacts with
secondary amines slower than 1. Thus, compound 1 was
selected for further colorimetric tests.
Figure 2. Colorimetric monitoring of polymer-bound amines and
thiols using 1 and 2 (NEM: N-ethylmorpholine; DBU: 1,8-
diazabyclo[5,4,0]undec-7-ene; dye: disperse red 1). Bottom: mi-
croscopic images of the resin after reactions of resin-bound amines
and thiols (1 equiv) with 1 or 2 (3 equiv) for the given time in the
presence of a mixture of DBU (3 equiv) and DMAP (0.2 equiv).
The results of concentration optimization experiments
showed that reactions of polymer-bound amines and thiols
with 50 mM 1, 50 mM DBU, and 10 mM DMAP for 7 min
at room temperature gave efficient color tests. Since high-
temperature reactions may result in decomposition of resin-
bound compounds, making regeneration of compounds after
color tests problematic, the resin test was performed at room
temperature.
To examine the sensitivity of this new colorimetric
method, the resin-bound amine and thiol containing dipep-
tides (10 µmol) shown in Figure 2 were coupled to 0.9 equiv
of Fmoc-Leu. After washing, the resin was treated with 1
(50 mM) and 50 mM DBU-10 mM DMAP for 7 min at
room temperature to monitor unreacted amine and thiol
groups. The resin was clearly stained by using this procedure,
indicating that the minimum detection sensitivity is ca. 1
µmol/g of resin.11
Since colorimetric tests of amines and thiols using 1
worked well, we next explored the regeneration of free
amines and thiols after performing the color tests. The
colored resin, obtained from reactions of resin-bound amines
and thiols with 1, was treated with 0.2 M Bu4NF in THF for
1-13 min or twice for 5 min at room temperature. These
treatments led to the complete removal of the dye moiety
from the amine and thiol groups on the resin (Figure 3). On
the basis of these results, double cleavage conditions
ester for reaction with resin-bound amines and thiols, and a
TBS blocked 4-hydroxymandelic acid group to initiate
removal of a dye moiety after the color test (Figure 1).10
The route for synthesis of 1 and 2, substances used for
the nondestructive colorimetric resin test, was initiated by
reaction of disperse red 1 with 3 (Scheme 1). After removal
of the Boc group of the product, the liberated amine group
was coupled to TBS-protected 4-hydroxymandelic acid (5)
under HBTU-DIEA conditions to produce 6. The hydroxyl
group in 6 was reacted with p-nitrophenyl chloroformate or
1,1′-carbonyldiimidazole to give an activated ester 1 or 2,
respectively. Substances 1 and 2 can be stored as solids and
in DMF solutions at room temperature for several weeks.
Initially, we examined whether 1 or 2 is a better reagent
for colorimetric monitoring of ploymer-bound amines and
thiols. Primary amines, secondary amines, and thiols ap-
pended to dipeptides (1 equiv) on a solid support were
independently treated with 1 and 2 (3 equiv) in the presence
of a single base (3 equiv of NEM, DIEA, DBU, or DMAP)
or a mixture of DBU and DMAP for various time periods
(1-13 min) at room temperature (Figure 2). After thorough
washing of the treated resin, the level of bead coloration was
determined using microscopy. Strong, positive responses
(10) Lee, M.-R.; Baek, K.-H.; Jin, H. J.; Jung, Y.-G.; Shin, I. Angew.
Chem., Int. Ed. 2004, 43, 1675–1678.
(11) See Supporting Information.
3440
Org. Lett., Vol. 11, No. 15, 2009

Figure 5. Sequence of peptides synthesized using the nondestructive
colorimetric resin test.
This finding demonstrates the practicability of this nonde-
structive resin test (Figure 4).
Finally, the new colorimetric resin test was applied for
the preparation of several peptides. First, three pentapeptides
containing the secondary amide linkage were assembled on
the Rink amide resin (Figure 5a-c). In the event that
incomplete peptide bond formation takes place during SPPS,
the tested resin was regenerated by treatment with 0.2 M
Bu4NF. The recovered resin was then combined with the
untested resin for the repetition of the coupling reaction. By
following this procedure, we were able to prepare three
pentapeptides containing secondary amide linkage in high
purities and yields.11 We were also able to efficiently prepare
relatively pure samples of five peptides containing C-terminal
hydrazide groups by using this method (Figure 5d-h).11
In conclusion, we have developed the first, simple,
efficient, nondestructive colorimetric resin test to monitor
primary amines, secondary amines, and thiols on solid
supports. The solid-supported functional groups can be easily
detected by this method, and the tested resin can be simply
regenerated for the repetition of the same reactions or
subsequent reactions. It is expected that this new technique
will be applicable in monitoring other nucleophilic functional
groups, such as alcohols and hydrazides, since the reagent
used in this procedure contains an activated ester.
Figure 4. Use of regenerated resins for the next reaction. Bottom:
HPLC profiles for (a) tripeptides obtained by using regenerated
resins and (b) tripeptides obtained from normal SPPS.
(treatment of the colored resin twice with 0.2 M Bu4NF for
5 min) were used in further studies. Importantly, the purity
of peptides obtained from regenerated resin was quite similar
to that obtained without employing the resin test, showing
that the resin was successfully recovered.
In practical applications of this new testing method, the
resin regenerated after the color test should be used for the
next reaction or the repetition of the same reaction. To
examine this capability, three polymer-bound dipeptides were
first reacted with 1 to stain the resin, and then the dye moiety
was removed by the treatment with 0.2 M Bu4NF. The
resulting polymer-bound dipeptides were coupled to Fmoc-
Leu to produce polymer-bound tripeptides. HPLC analysis
shows that the purity of tripeptides obtained from imple-
mentation of this procedure was quite similar to that of
tripeptides obtained without intervention of the resin test.
Acknowledgment. This work was supported by grants
from the NRL and WCU (R32-2008-000-10217-0) programs
(KOSEF/MEST).
Supporting Information Available: Experimental pro-
cedure, spectral data, and HPLC chromatograms. This
material is available free of charge via the Internet at
http://pubs.acs.org.
OL901154E
Org. Lett., Vol. 11, No. 15, 2009
3441