TRAM linker: A safety-catch linker for the traceless release of acrylamides

Article abstract of DOI:10.1021/ol049711i

Equation presented. A novel safety-catch linker for the solid-phase synthesis of small-molecule libraries containing electrophilic reactive groups has been developed. Upon cleavage from solid support, the linker generates a Michael acceptor (an acrylamide

Full text of DOI:10.1021/ol049711i

ORGANIC
LETTERS
2004
Vol. 6, No. 12
1891-1894
TRAM Linker: A Safety-Catch Linker for
the Traceless Release of Acrylamides
,†,§
Christopher J. Ciolli,† Sean Kalagher,‡ and Peter J. Belshaw*
Departments of Chemistry and Biochemistry, UniVersity of Wisconsin-Madison,
1101 UniVersity AVenue, Madison, Wisconsin 53706
belshaw@chem.wisc.edu
Received February 17, 2004 (Revised Manuscript Received April 28, 2004)
ABSTRACT
A novel safety-catch linker for the solid-phase synthesis of small-molecule libraries containing electrophilic reactive groups has been developed.
Upon cleavage from solid support, the linker generates a Michael acceptor (an acrylamide) on each library member. Utilization of a two-resin
system in the final cleavage step provides crude products in high purity, allowing direct use in biological assays following filtration and
evaporation.
Small molecules that covalently modify proteins have long
been of interest as irreversible inhibitors and affinity labeling
agents.1 More recently, electrophilic small molecules have
gained promise as tools for proteomic profiling,2 in new
approaches for lead compound identification,3 selective target
inhibition/activation, and active site mapping in engineered
systems.4,5 To facilitate the preparation of electrophilic small
molecules, we have prepared a new linker for solid-phase
synthesis that releases a Michael acceptor (here an acryl-
amide) upon cleavage from the resin. We chose acrylamides
as electrophiles since they have been shown to be effective
for affinity alkylation of cysteines both in vitro5,6 and in vivo7
yet are unreactive toward common cellular nucleophiles (e.g.,
glutathione).
Recently, several reports describing the introduction of
potential Michael acceptors onto compounds upon release
from solid support have appeared. Vinyl sulfones have been
introduced onto peptides during cleavage from Kenner’s
safety-catch linker.8 Several groups have utilized oxidative
eliminations of selenium or sulfones for the synthesis of
acrylamides,9 R,â-unsaturated esters and ketones,10 R,â-
unsaturated aldehydes,11 â-dicarbonyl enones,12 cyclobu-
tenones,13 cyclopent-2-enones,14 and R,â-unsaturated γ-bu-
tyrolactones.15 Cyclic acrylamides have also been generated
via a ring-closing metathesis cleavage.16 While many of the
aforementioned examples produce compounds in high yields,
(7) Fry, D. W.; Bridges, A. J.; Denny, W. A.; Doherty, A.; Greis, K. D.;
Hicks, J. L.; Hook, K. E.; Keller, P. R.; Leopold, W. R.; Loo, J. A.;
McNamara, D. J.; Nelson, J. M.; Sherwood, V.; Smaill, J. B.; Trumpp-
kallmeyer, S.; Dobrusin, E. M. Proc. Nat. Acad. Sci. U.S.A. 1998, 95,
12022-12027.
* To whom correspondence should be addressed.
† Department of Chemistry.
‡ Current Address: Boston University School of Medicine, 715 Albany
St., Boston, MA 02118.
(8) Overkleeft, H. S.; Bos, P. R.; Hekking, B. G.; Gordon, E. J.; Ploegh,
H. L.; Kessler, B. M. Tetrahedron Lett. 2000, 41, 6005-6009.
(9) Sheng, S.-R.; Wang, X.-C.; Liu, X.-L.; Song, C.-S. Synth. Commun.
2003, 33, 2867-2872.
§ Department of Biochemistry.
(1) Baker, B. R. J. Pharm. Sci. 1964, 53, 347-364.
(2) (a) Jessani, N.; Cravatt, B. F. Curr. Opin. Chem. Biol. 2004, 8, 54-
59. (b) Jeffery, D. A.; Bogyo, M. Curr. Opin. Biotechnol. 2003, 14, 87-
95.
(10) Sheng, S.-R.; Liu, X.-L.; Wang, X.-C. J. Chin. Chem. Soc. 2003,
50, 279-281.
(11) Sheng, S.-R.; Wu, L.-L.; Huang, X. J. Chem. Res., Synop. 2003,
258-259.
(3) Erlanson, D. A.; Braisted, A. C.; Raphael, D. R.; Randal, M.; Stroud,
R. M.; Gordon, E. M.; Wells, J. A. Proc. Nat. Acad. Sci. U.S.A. 2000, 97,
9367-9372.
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553.
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Sci. 2001, 22, 170-173.
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2002, 4, 741-744.
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(15) (a) Fujita, K.; Watanabe, K.; Oishi, A.; Ikeda, Y.; Taguchi, Y. Synlett
1999, 1760-1762. (b) Huang, X.; Sheng, S.-R. J. Comb. Chem. 2003, 5,
273-277.
(5) Levitsky, K.; Ciolli, C. J.; Belshaw, P. J. Org. Lett. 2003, 5, 693-
696.
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U.S.A. 2001, 98, 8480-8484.
10.1021/ol049711i CCC: $27.50 © 2004 American Chemical Society
Published on Web 05/11/2004

each method requires an extractive workup and/or chroma-
tography following cleavage from a solid phase. In this paper,
we demonstrate a cleavage strategy that directly yields
acrylamides in high purity (>95%) following simple filtration
and evaporation allowing direct use of released compounds
in biological screens.17 The linker was designed such that
the Michael acceptor is generated from the linker attachment
site, maximizing the potential for scaffold diversification.
Scheme 1. Synthesis of the TRAM Linker
Our linker cleavage strategy is shown in Figure 1. During
synthesis of a library, the safety-catch exists as a protected
secondary amine. Upon completion of library synthesis, the
secondary amine is deprotected and alkylated to give the
quaternary ammonium salt. Library members are then
cleaved from resin with a volatile base, generating the
acrylamide electrophile. The cleavage chemistry of our
safety-catch linker for the traceless release of acrylamides
(TRAM linker) is similar to the REM resins,18 generating a
Michael acceptor via a Hofmann elimination. While the REM
resins release tertiary amines from the resin, the TRAM
linker releases the Michael acceptor.
length was shown to be sufficient for exo-mechanism affinity
alkylation of cysteines.5 This spacer could possibly be
substituted as required for other applications. Alkylation of
the sulfonamide nitrogen of 3 with ethyl bromoacetate
yielded the protected linker 4. Hydrolysis of the ethyl ester
4 gave linker 5, suitable for loading onto solid supports.
Although the sulfonamide moiety present in compound 3 is
necessary for the alkylation step, it should be possible to
substitute the secondary amine protecting group of 4 to
accommodate other library chemistries.
For optimization of our linker cleavage strategy (Figure
1, Scheme 2), we prepared model derivatives 9a and 10a of
the TRAM linker to readily evaluate conditions for linker
quaternization and cleavage. We incorporated the naphthyl-
acetamide moiety into our model derivative to facilitate
quantitation of experimental yields by HPLC-UV.
Optimization of the cleavage sequence began with the final
cleavage step. We used a volatile tertiary amine base (DIEA)
along with an acid scavenger resin21 to allow the direct
isolation of TRAM-derived acrylamides free from contami-
nating tertiary ammonium salts produced in the cleavage
reaction. Four variables were considered: equivalents of
DIEA, solvent, and both the identity and equivalents of the
supported acid scavenger. For our experiments varying DIEA
and solvent, we used 10 equiv of silica-TBD (1,5,7-
triazabicyclo[4.4.0]dec-5-ene) as an acid scavenger in the
cleavage reaction. Simultaneous variation of equivalents of
DIEA and solvent (Table 1) indicated that 2 equiv of DIEA
in DMF were the optimal conditions.
We next turned our attention to the supported acid
scavenger. Two previous reports21 describe the use of two-
resin systems for directly isolating REM resin products in
high purity. We chose to evaluate the previously reported
acid scavenger resins as well as a silica-bound derivative in
our cleavage reaction (Table 2). Use of 10 equiv of silica-
TBD gave the highest yields, although polystyrene-TBD
(PS-TBD) was comparable. When assessing the purity of
Figure 1. Cleavage strategy for the TRAM linker.
For ease of synthesis, we chose the ortho-nitrobenzene
sulfonyl (oNBS)19 group for protection of the secondary
amine. Synthesis of the linker (Scheme 1) began with oNBS
protection of â-alanine followed by EDCI-mediated coupling
with N-Boc-ethylenediamine (2)20 to yield sulfonamide 3.
We selected an ethylenediamine spacer because this linker
(16) Veerman, J. J. N.; Van Maarseveen, J. H.; Visser, G. M.; Kruse, C.
G.; Schoemaker, H. E.; Hiemstra, H.; Rutjes, F. P. J. T. Eur. J. Org. Chem.
1998, 2583-2589.
(17) Zaragoza, F. Angew. Chem., Int. Ed. 2000, 39, 2077-2079.
(18) (a) Morphy, J. R.; Rankovic, Z.; Rees, D. C. Tetrahedron Lett. 1996,
37, 3209-3212. (b) Heinonen, P.; Lonnberg, H. Tetrahedron Lett 1997,
38, 8569-8572. (c) Kroll, F. E. K.; Morphy, R.; Rees, D.; Gani, D.
Tetrahedron Lett. 1997, 38, 8573-8576. (d) Plater, M. J.; Murdoch, A.
M.; Morphy, J. R.; Rankovic, Z.; Rees, D. C. J. Comb. Chem. 2000, 2,
508-512.
(20) Pons, J.-F.; Fauchere, J.-L.; Lamaty, F.; Molla, A.; Lazaro, R. Eur.
J. Org. Chem. 1998, 853-859.
(21) (a) Ouyang, X.; Armstrong, R. W.; Murphy, M. M. J. Org. Chem.
1998, 63, 1027-1032. (b) Alhambra, C.; Castro, J.; Chiara, J. L.; Fernandez,
E.; Fernandez-Mayoralas, A.; Fiandor, J. M.; Garcia-Ochoa, S.; Martin-
Ortega, M. D. Tetrahedron Lett. 2001, 42, 6675-6678.
(19) Fukuyama, T.; Jow, C.-K.; Cheung, M. Tetrahedron Lett. 1995, 36,
6373-6374.
1892
Org. Lett., Vol. 6, No. 12, 2004

Scheme 2. Solid-Phase Synthesis of Acrylamides
Table 2. Evaluation of Solid-supported Acid Scavengers in
Cleavage Reaction
a Molar equivalents of scavenger amine relative to loading of resin 6.
b Percent yield determined via HPLC-UV using (()-γ-(2-naphthyl)-γ-
butyrolactone as an internal standard (see Supporting Information).
systems. Both DMSO and NMP23 have been shown to be
superior to DMF as solvents for REM resin quaternization;
however, a side-by-side comparison has not been reported.
We therefore compared cleavage yields in these two solvents
with varying equivalents of MeI/2,6-lutidine (Table 3). These
Table 3. Evaluation of Solvent and MeI/2,6-Lutidine in
Quaternary Amine Formation
yield (%)b
equiva of
MeI/2,6-lutidine
DMSO
NMP
10
20
40
59
59
63
68
69
71
a Molar equivalents relative to loading of resin 6. b Percent yield
determined via HPLC-UV using (()-γ-(2-naphthyl)-γ-butyrolactone as an
internal standard (see Supporting Information).
experiments indicated that 40 equiv MeI/2,6-lutidine in NMP
gave the highest yields for quaternization, although yields
with 10 and 20 equiv of MeI/2,6-lutidine were comparable
to those using 40 equiv. It is noteworthy that the inclusion
of 2,6-lutidine in the quaternization reaction roughly doubled
the final cleavage yields under otherwise identical experi-
mental conditions (data not shown). Previous reports of
similar transformations24 may similarly benefit by including
this acid scavenger. Although NMP gave the highest yields
in the quaternization reaction, colored impurities generated
in this reaction contaminated the isolated products. On the
basis of these observations, we chose DMSO with 40 equiv
of MeI/2,6-lutidine as our optimal condition, giving the
highest purities with minor decreases in yield.
1
isolated products by H NMR, we found batch-dependent
impurities arising from the silica-TBD scavenger contami-
nated our products and thus chose 10 equiv of PS-TBD as
our optimal scavenger.
Previous investigations of the quaternization reaction had
found that 20 equiv of alkylating reagent in DMSO22 were
optimal for amine quaternization in the related REM resin
Table 1. Evaluation of Base and Solvent in Cleavage Reaction
yield (%)b
equiva of
Having determined optimal quaternization and cleavage
conditions25 in our model system, we next sought to
demonstrate the applicability of the TRAM linker for a
variety of substrates (Scheme 2). Loading of Fmoc-Gly-OH
DIEA
DCM
DMF
DMSO
THF
1
2
5
57
58
58
59
62
65
65
64
43
45
47
44
53
56
55
57
10
(22) Cameron, K. S.; Morphy, J. R.; Rankovic, Z.; York, M. J. Comb.
Chem. 2002, 4, 199-203.
a Molar equivalents relative to loading of resin 6. b Percent yield
determined via HPLC-UV using (()-γ-(2-naphthyl)-γ-butyrolactone as an
internal standard (see Supporting Information).
(23) Yamamoto, Y.; Tanabe, K.; Okonogi, T. Chem. Lett. 1999, 103-
104.
(24) Wu, T. Y. H.; Schultz, P. G. Org. Lett. 2002, 4, 4033-4036.
Org. Lett., Vol. 6, No. 12, 2004
1893

onto aminomethyl polystyrene resin and capping with acetic
anhydride/pyridine afforded resin 6. Fmoc quantitation of 6
established an experimental loading level for all subsequent
experiments. Removal of the Fmoc protecting group and
coupling with linker 5 yielded resin 7. Following removal
of the Boc protecting group, a variety of carboxylic acids
were coupled onto the linker-functionalized resin to give 8a-
h. The oNBS protecting groups of 8a-h were removed with
the potassium salt of thiophenol to give secondary amine
resins 9a-h. Quaternization of the secondary amine was
achieved with 40 equiv of MeI/2,6-lutidine in DMSO to
afford quaternary ammonium salt resins 10a-h. Last, the
product acrylamides were cleaved from solid support with
2 equiv of DIEA and 10 equiv of PS-TBD scavenger resin
in DMF to yield compounds 11a-h.
All eight TRAM-derived acrylamides were isolated in
good yields (Table 4) after filtration and evaporation. More
importantly, acrylamides 11a-h were all isolated in greater
than 95% purity as judged by 1H NMR. Common protecting
groups such as Boc (11e,h), Cbz (11f,g), and tert-butyl ester
(11g) were stable to the linker cleavage chemistries.
In summary, we have developed a general linkage strategy
for the introduction of acrylamide electrophiles onto small-
molecule library members. Our strategy should be generaliz-
able to other Michael acceptors such as acrylates, enones,
and R,â-unsaturated sulfones/sulfonamides. This linker should
facilitate the synthesis and identification of small molecules
that covalently modify protein targets via proximity-acceler-
ated alkylation.5 Such compounds may find utility in
Table 4. Yields and Purities of TRAM-derived Acrylamides
model compound
isolated yield (%)
purity (%)a
11a
11b
11c
11d
11e
11f
11g
11h
63
64
69
69
73
64
67
70
>95
>95
>95
>95
>95
>95
>95
>95
1
a Determined via integration of H NMR spectra.
proteomic profiling, as isoform-selective modulators of
protein function in engineered systems, and possibly in
disrupting protein-protein interactions.
Acknowledgment. We gratefully acknowledge the NIH
(GM065406), Burroughs Wellcome Fund, and the W.M.
Keck Foundation for financial support of this work and NSF
(CHE-9208463) and NIH (RR08389) for support of NMR
facilities. C.J.C. was an NIH Chemistry-Biology Interface
Trainee (GM08505). We thank Silicycle for the generous
gift of silica-TBD, Melissa Boersma for technical assistance,
and Yi He for insightful discussions.
Supporting Information Available: Experimental pro-
cedures and structural characterization for all compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(25) Optimal protocol: alkylation with 40 equiv of MeI/2,6-lutidine in
DMSO and cleavage with 2 equiv of DIEA in DMF in the presence of 10
equiv of PS-TBD scavenger.
OL049711I
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Org. Lett., Vol. 6, No. 12, 2004