Traceless azido linker for the solid-phase synthesis of N H-1,2,3-triazoles via Cu-catalyzed azide - Alkyne cycloaddition reactions

Article abstract of DOI:10.1021/ol102209p

A broadly useful acid-labile traceless azido linker for the solid-phase synthesis of NH-1,2,3-triazoles is presented. A variety of alkynes were efficiently immobilized on a range of polymeric supports by Cu(I)-mediated azide - alkyne cycloadditions. Suppo

Full text of DOI:10.1021/ol102209p

ORGANIC
LETTERS
2010
Vol. 12, No. 23
5414-5417
Traceless Azido Linker for the
Solid-Phase Synthesis of
NH-1,2,3-Triazoles via Cu-Catalyzed
Azide-Alkyne Cycloaddition Reactions
A. Emil Cohrt, Jakob F. Jensen, and Thomas E. Nielsen*
Department of Chemistry, Technical UniVersity of Denmark,
DK-2800 Kgs. Lyngby, Denmark
ten@kemi.dtu.dk
Received September 15, 2010
ABSTRACT
A broadly useful acid-labile traceless azido linker for the solid-phase synthesis of NH-1,2,3-triazoles is presented. A variety of alkynes were
efficiently immobilized on a range of polymeric supports by Cu(I)-mediated azide-alkyne cycloadditions. Supported triazoles showed excellent
compatibility with subsequent peptide chemistry. Release of pure material (typically >95%) from the solid support was readily achieved by
treatment with aqueous TFA.
Since the introduction by Merrifield,1 solid-phase synthesis
has emerged as an increasingly important tool in modern
chemical biology and medicinal chemistry research.2 Being
undisputedly beneficial in polypeptide synthesis, the field has
developed significantly in recent years, where more emphasis
has been given to the parallel synthesis of heterocycles,3 such
as diketopiperazines, benzodiazepines, and triazoles, but also
to more complex ring systems, such as those made during
diversity- and biology-oriented synthesis efforts.4
One of recent years’ most significant accomplishments in
solid-phase synthesis was reported by Meldal and co-workers
on their discovery of the copper(I)-catalyzed azide-alkyne
cycloaddition (CuAAC) reaction.5 The reaction has been
proven extremely reliable and robust for solid-phase ap-
plications, e.g., as evidenced by the successful synthesis and
screening of a library containing over 400 000 peptidotria-
zoles6 and very recently for the synthesis of antibody-like
protein-capture agents.7
The NH-1,2,3-triazoles (N-unsubstituted 1,2,3-triazoles)
possess many interesting biological properties.8 However,
(4) (a) Feliu, L.; Vera-Luque, P.; Albericio, F. J. Comb. Chem. 2007,
9, 521–565. (b) Bunin, B. A.; Plunkett, M. J.; Ellman, J. A. Proc. Natl.
Acad. Sci. U.S.A. 1994, 91, 4708–4712. (c) Lambert, J. N.; Mitchell, J. P.;
Roberts, K. D. J. Chem. Soc., Perkin Trans. 1 2001, 5, 471–484. (d) Nielsen,
T. E.; Meldal, M. Curr. Opin. Drug DiscoVery DeV. 2009, 12, 798–810.
(e) Mitchell, J. M.; Shaw, J. T. Angew. Chem., Int. Ed. 2006, 45, 1722–
1726. (f) Ludolph, B.; Waldmann, H. Tetrahedron Lett. 2009, 50, 3148–
3150. (g) Izumi, M. J. Pestic. Sci. 2006, 31, 1–5. (h) Burke, M. D.;
Schreiber, S. L. Angew. Chem., Int. Ed. 2004, 43, 46–58. (i) Nielsen, T. E.;
Schreiber., S. L. Angew. Chem., Int. Ed. 2008, 47, 48–56.
(5) (a) Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002,
67, 3057–3064. (b) For the first solution-phase CuAAC reaction, consult:
Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew.
Chem., Int. Ed. 2002, 41, 2596–2599.
(1) Merrifield, B. R. J. Am. Chem. Soc. 1963, 85, 2149–2154.
(2) (a) Fru¨chtel, J. S.; Jung., G. Angew. Chem., Int. Ed. 1996, 35, 17–
42. (b) Organic Synthesis on Solid Phase; Do¨rwald, F. Z., Ed.; Wiley-
VHC: Weinheim, 2002. (c) Linker Strategies in Solid-Phase Organic
Synthesis; Scott, P., Ed.; Wiley-VHC: Weinheim, 2009. (d) The power of
functional resins in organic synthesis; Tulla-Puche, J., Albericio, F., Ed.;
Wiley-VCH: Weinheim, 2008.
(6) Tornøe, C. W.; Sanderson, S. J.; Motrram, J. C.; Coombs, G. H.;
Meldal, M. J. Comb. Chem. 2004, 6, 312–324.
(7) Agnew, H. D.; Rohde, R. D.; Millward, S. W.; Nag, A.; Yeo, W. S.;
Hein, J. E.; Pitram, S. M.; Tariq, A. A.; Burns, V. M.; Krom, R. J.; Fokin,
V. V.; Sharpless., K. B.; Heath, J. R. Angew. Chem., Int. Ed. 2009, 48,
4944–4948.
(3) Bra¨se, S.; Gil, C.; Knepper, K. Bioorg. Med. Chem. 2002, 10, 2415–
2437.
10.1021/ol102209p  2010 American Chemical Society
Published on Web 11/04/2010

Table 1. Representative Solid-Phase Cu(I)-Catalyzed Azide-Alkyne Cycloaddition Reactions
entry
Cu source
base
reducing agent
solvent
conversionb
1
CuSO4·H2O (0.16 equiv)
CuI (2 equiv)
CuI (2 equiv)
-
-
-
Na ascorbate (3.4 equiv)
-
-
Na ascorbate (5 equiv)
Na ascorbate (3 equiv)
Na ascorbate (1 equiv)
Na ascorbate (1 equiv)
CH2Cl2:MeOH (1:1)
DMF:piperidine (4:1)
DIPEA:pyridine:DMF (1:3:1)
DMF:piperidine (4:1)
DMF
56%
43%
38%
49%
77%
68%
>95%
2
3a
4
CuI (5 equiv)
DIPEA (10 equiv)
5
6
7
CuBr (2 equiv)
CuI (0.5 equiv)
CuI (1 equiv)
2,6-lutidine (10 equiv)
2,6-lutidine (2 equiv)
2,6-lutidine (2 equiv)
MeCN:DMSO:H2O (8:2:1)
NMP:H2O (4:1)
a 5 equiv of Fmoc-propargylamine was used. b Conversions were measured using RP-HPLC Fmoc quantification (254 nm).14
contrary to the formation of 1,4-disubstituted 1,2,3-triazoles,
the general access to NH-1,2,3-triazoles on solid support is
not described in the literature. Various methods are known
for the solution-phase synthesis of this compound class,
including the reaction of nitro-alkenes with sodium azide9
and the application of various azide donors, such as azi-
domethyl pivalate and carbamates10 and trimethylsilyl
azide.11 However, none of these methods have been reported
for use on solid support. The direct synthesis of NH-1,2,3-
triazoles from alkynes and inorganic azides is impractical
on solid support, and copper(I)-catalyzed variants hereof are
not easily optimized with sodium azide.
Intrigued by the potential of NH-1,2,3-triazoles, we
envisioned a strategy that uses an acid-labile azido linker
(2) compatible with common resins and reaction conditions
typical for combinatorial chemistry.12 The unique chemose-
lectivity of the CuAAC reaction would allow loading of the
resin with multifunctional alkyne building blocks without
resorting to an extensive protecting group scheme. The
synthesis of 2 commenced with the addition of excess
amounts of Grignard reagent derived from bromobenzene
to vanillin followed by alkylation of the phenol with ethyl-
4-bromobutyrate to yield 1 in 76% over two steps (Scheme
1).
2-methoxyphenoxy)propanoic acid linker (2) in 87% yield
(three steps). In conclusion, the linker 2 can be made in only
five steps with an overall yield of 66% on a +10 g scale,
notably without resorting to chromatographic purification in
any of the steps.
To investigate the ability of the linker to function as a
traceless linker in the formation of NH-1,2,3 triazoles,13
2
was readily coupled to the amino-functionalized PEGA800
(polyethylene glycol dimethyl acrylamide) resin using TBTU
(O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tet-
rafluoroborate). A range of procedures were attempted to find
a quantitative protocol for the solid-phase synthesis of 1,2,3-
triazoles (representative results are shown in Table 1). The
efficiency hereof was evaluated by measuring the conversion
of 3a to 4a via subsequent Fmoc quantification.14 Copper(I)
species generated in situ from CuSO4 using sodium ascorbate
in CH2Cl2/MeOH resulted in a moderate conversion of 56%
(entry 1), and changing the copper(I) source to CuI gave a
further decrease in conversion to 43% (entry 2). Increasing
the amount of alkyne from 2 equiv to 5 equiv and use of
(8) (a) Kume, M.; Kubota, T.; Kimura, Y.; Nakashimizu, H.; Motokawa,
K.; Nakano, M. J. Antibiot. 1993, 46, 177–192. (b) Cristalli, G.; Eleuteri,
A.; Volpini, R.; Vittori, S.; Camaioni, E.; Lupidi, G. J. Med. Chem. 1994,
37, 201–205. (c) Nimkar, S. K.; Mabic, S.; Anderson, A. H.; Palmer, S. L.;
Graham, T. H.; de Jonge, M.; Hazelwood, L.; Hislop, S. J.; Castagnoli, N.
J. Med. Chem. 1999, 42, 1828–1835. (d) Fray, M. J.; Bull, D. J.; Car, C. L.;
Gautier, E. C. L; Mowbray, C. E.; Stobie, A. J. Med. Chem. 2001, 44,
1951–1962. (e) Komeda, S.; Lutz, M.; Spek, A. L.; Yamanaka, Y.; Sato,
T.; Chikuma, M.; Reedijk, J. J. Am. Chem. Soc. 2002, 124, 4738–4746.
(9) Quiclet-Sire, B.; Zard, S. Z. Synthesis 2005, 19, 3319–3326.
(10) Loren, J. C.; Krasinski, A.; Fokin, V. V.; Sharpless., K. B. Synlett
2005, 18, 2847–2850.
Chlorination of 1 followed by azidation and hydrolysis of
the ester moiety afforded the 3-(4-(azido(phenyl)-methyl)-
Scheme 1. Synthesis of the Azido Linker
(11) Jin, T.; Kamijo, S.; Yamamoto, Y. Eur. J. Org. Chem. 2004, 18,
3789–3791.
(12) For previous work on NH-1,2,3-triazole synthesis on Wang resins,
see: Harju, K.; Vahermo, M.; Mutikainen, I.; Yli-Kauhaluoma, J. J. Comb.
Chem 2003, 5, 826–833.
(13) For a review on traceless linkers, see: Gil, C.; Bra¨se, S. Curr. Opin.
Chem. Biol. 2004, 8, 230–237.
(14) The conversion of 4a was measured by subjecting the formed Fmoc-
protected peptide to a 2% DBU (DMF) solution containing 0.042 nM
anthracene as internal standard for 30 min, followed by HPLC quantification
of the anthracene/dibenzofulvene ratio. See also: Freeman, C. E.; Howard,
A. G. Talanta 2005, 65, 574–577.
Org. Lett., Vol. 12, No. 23, 2010
5415

DIPEA (N,N-diisopropylethylamine) as a base gave an even
lower conversion (entry 3). On the other hand, increasing
the amount of copper to 5 equiv and addition of sodium
Table 2. Test of Common Solid Supports for the Synthesis of
NH-1,2,3-Triazole
ascorbate increased the conversion to 49% (entry 4).15
A
significant improvement in conversion to 77% was observed
by switching to 2,6-lutidine as base (entry 5). Encouraged
by this observation, we tried to decrease the amount of
catalyst to 0.5 equiv as well as to change the solvent to
prevent precipitation of Cu-salts, but conversion decreased
to 68% (entry 6). Realizing that a stoichiometric amount of
catalyst is needed, a full equivalent of CuI in NMP/H:O gave
the desired conversion of >95% (entry 7), and no precipita-
tion of Cu salts was observed.15,16
With an optimized CuAAC protocol at hand, we naturally
turned our attention to the cleavage conditions. To measure
the reactivity of immobilized 2, small batches of the Fmoc-
protected triazole linker derivative 4a were subjected to 95%
TFA(aq) for various lengths of time. After cleavage, the resin
was washed with appropriate amounts of DMF and CH2Cl2,
and residual Fmoc on the resin was quantified by standard
HPLC methods (vide supra).17 As seen in Figure 1, more
than 90% of 4a was released as the corresponding triazole
after 24 h.
support
4a-PEGA800
4b-polystyrene-OH
4c-polystyrene-NH2
4d-tentagel-OH
4e-tentagel-NH2
4f-Chemmatrix-NH2
4g-Wang-OH
puritya
yield
>95%
80%
94%
>95%
94%
94%
38%
71%
57%
54%
67%
85%
59%
26%
a Purity was determined by RP-HPLC (215 and 254 nm).
tive set of common functional groups, including free carboxyl
acid moieties (entries 7 and 8). The purity was generally
very high for ethynyl aryls, except for the pyridine derivative
(entry 10). The yields are in most cases modest but
acceptable. Crude aliphatic NH-1,2,3-triazoles were generally
less pure, albeit with higher yields, except for the cyclopropyl
derivative (entry 15). Aryl ethers, thioethers, and esters were
synthesized in high purities and acceptable yields (entries
16-18).
To test the versatility of the linker, the NH-1,2,3-triazole
5 was synthesized on seven commercially available and
commonly used hydroxy- or amino-functionalized solid
supports.18
The reliability of the linker in multistep peptide synthesis
was explored by synthesizing an NH-1,2,3-triazole derivative
of the opioid pentapeptide Leu-enkephalin (7) found in the
brain.19
Figure 1. Acid-mediated release of triazole from 4a-PEGA800 as a
function of time.
As seen in Table 2, the linker is well applicable with all
polymeric supports examined, except for the Wang resin that
appeared unstable under the reaction conditions. Confident
with the solid-phase strategy, a small collection of aryl- and
aliphatic NH-1,2,3-triazoles were synthesized.
The peptide was synthesized using a standard Fmoc
strategy, employing TBTU as the coupling reagent. The
amino acids were coupled to the linker after an initial click
reaction with Fmoc-propargylamine. After cleavage with
95% TFA(aq) for 24 h, the pentapeptide was obtained as
the TFA salt in 85% purity and 80% yield.20
As seen in Table 3, the solid-phase strategy is very robust
and applicable to a range of alkynes containing a representa-
A complementary method that quantitatively assembles
the azido linker on the solid support was also developed
(Scheme 2). The alkylated benzhydrol 1 was hydrolyzed by
LiOH and attached to the solid support as a TBTU activated
ester (Scheme 2). After attachment, the linker was swollen
(15) Zhang, Z.; Fan, E. Tetrahedron Lett. 2006, 47, 665–669.
(16) Bock, V. D.; Hiemstra, H.; van Maarseveen, J. H. Eur. J. Org.
Chem. 2006, 1, 51–68
.
(17) Bernatowicz, M. S.; Daniels, S. B.; Ko¨ster, H. Tetrahedron Lett.
1989, 30, 4645–4648.
(18) (a) The following solid supports were tested: PEGA800 (Polymer
Labs), Hydroxymethyl polystyrene (Polymer Labs), Aminomethyl Poly-
styrene (Polymer Labs), Hydroxy Tentagel (Novasyn), Amino Tentagel
(Fluka), Chemmatrix (Aldrich), Wang (Aldrich). (b) The linker 2 was
attached to the solid support using TBTU (3 equiv of 2, 4 equiv of NEM,
and 2.88 equiv of TBTU in DMF for 2 h), or MSNT coupling (3 equiv of
1, 2.25 equiv of MeIm, and 3 equiv of MSNT in CH2Cl2 1 h, repeated
once). Fmoc-Phe-OH and 2-naphthoic acid were attached using standard
TBTU coupling. Cleavage was mediated with 95% TFA(aq) for 24 h.
(19) (a) Hughes, J.; Smith, T. W.; Kosterlitz, H. W.; Fothergill, L. A.;
Morgan, B. A.; Morris, H. R. Nature 1975, 258, 577–579. (b) Wilczynska,
D.; Kosson, P.; Kwasiborskal, M.; Ejchart, A.; Olma, A. J. Pept. Sci. 2009,
15, 777–782.
(20) Determined by use of HPLC (215 and 254 nm) quantification and
1H NMR.
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Org. Lett., Vol. 12, No. 23, 2010

Table 3. Solid-Phase Synthesis of NH-1,2,3-Triazoles
a Purity was determined by RP-HPLC (215 and 254 nm).
In summary, we have developed an acid-labile traceless
azido linker for the synthesis of NH-1,2,3-triazoles on the
solid phase. The linker can be synthesized in solution in five
steps from readily available starting materials or directly on
the solid phase. The linker is compatible with most com-
monly used solid supports and remains intact throughout the
multipeptide synthesis. Products are ultimately released from
the solid support in excellent purity using 95% TFA(aq).
Scheme 2. Solid-Phase Assembly of the Azido Linker
Acknowledgment. We thank the Danish Council for
Independent Research, Natural Sciences, Technology and
Production Sciences, the Lundbeck Foundation, and the
Technical University of Denmark for financial support.
in concentrated MsCl in pyridine for 1 h. The mesylated resin
was then shaken at 65 °C with 10 equiv of sodium azide in
DMSO for 24 h. The NH-1,2,3-triazole 5 was synthesized
in 88% purity and 44% yield using the conditions noted in
Table 2.20
Supporting Information Available: Experimental pro-
cedures and full spectroscopic data for all new compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
The observed yield and purity were almost identical to
those obtained with linker 2.
OL102209P
Org. Lett., Vol. 12, No. 23, 2010
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