Aza-1,2,3-triazole-3-alanine synthesis via copper-catalyzed 1,3-dipolar cycloaddition on Aza-progargylglycine

Article abstract of DOI:10.1021/jo100957z

(Figure presented) The parallel synthesis of seven aza-1,2,3-triazole-3- alanine azapeptides of the Growth Hormone Releasing Peptide-6 (GHRP-6) was accomplished via a Cu-catalyzed azide-alkyne [3+2] cycloaddition on an aza-propargylglycine residue anchored on Rink amide resin. Circular dichroism spectroscopy in water demonstrated that azapeptides which possess an aza-1,2,3-triazole-3-alanine residue at the Trp⁴ position of the GHRP-6 sequence adopt β-turn conformations.

Full text of DOI:10.1021/jo100957z

pubs.acs.org/joc
recently a three-step procedure featuring regioselective alky-
Aza-1,2,3-triazole-3-alanine Synthesis via
Copper-Catalyzed 1,3-Dipolar Cycloaddition on
Aza-progargylglycine
lation and chemoselective deprotection of an azaglycine
semicarbazone to afford a variety of azapeptides directly
on solid support.5 With the idea that multiple combinatorial
libraries of azapeptides could be obtained via the making of
“libraries from libraries”,6 we have incorporated aza-pro-
pargylglycine moieties within a peptide to introduce a new
series of aza-1,2,3-triazole-3-alanine residues (Figure 1).
Growth Hormone Releasing Peptide-6 (His-D-Trp-Ala-
Trp-D-Phe-Lys-NH2, GHRP-6), a synthetic hexapeptide that
binds to two distinct receptors,7 was chosen as the target pep-
tide to develop this methodology. Incorporation of aza-Phe
for Trp4 conferred selectivity for the CD36 receptor,5 a target
for the development of treatments of angiogenesis-related
diseases. A combinatorial approach to incorporate novel hete-
roaryl alanine residues at the 4-position was deemed desirable
for improving binding affinity of the lead [AzaPhe4]GHRP-6
azapeptide.
Caroline Proulx and William D. Lubell*
Deꢀpartement de Chimie, Universiteꢀ de Montreꢀal, C.P. 6128,
ꢀ ꢀ
Succursale Centre-Ville, Montreal, Quebec H3C 3J7, Canada
william.lubell@umontreal.ca
Received May 17, 2010
The Cu-catalyzed azide-alkyne [3þ2] cycloaddition has
proven effective for making triazole peptidomimetics.8 Pro-
pargyl glycine, propargyl amides, and N-propargyl glycine
peptoids all have served as substrates for triazole formation
leading to macrocyclization,9,10 carbohydrate ligation,11
peptoid oligomer functionalization,12 as well as amide bond
and histidine isostere synthesis.8a,13 Precluding the solution-
phase synthesis of azido acids and propargyl glycine resi-
dues, and taking advantage of the propensity for aza-amino
acids to adopt β-turns, we describe now the solid-phase
synthesis of aza-1,2,3-triazole-3-alanine-containing azapep-
tides using aza-propargyl glycine residues anchored on Rink
amide resin.
The parallel synthesis of seven aza-1,2,3-triazole-3-ala-
nine azapeptides of the Growth Hormone Releasing
Peptide-6 (GHRP-6) was accomplished via a Cu-cata-
lyzed azide-alkyne [3þ2] cycloaddition on an aza-pro-
pargylglycine residue anchored on Rink amide resin.
Circular dichroism spectroscopy in water demonstrated
that azapeptides which possess an aza-1,2,3-triazole-3-
alanine residue at the Trp4 position of the GHRP-6
sequence adopt β-turn conformations.
[Aza-1,2,3-triazole-3-alanine4]GHRP-6 analogues were
synthesized by elaboration of the submonomer methodology
for aza-propargylglycine synthesis by using propargyl bro-
mide as electrophile5 to alkylate a semicarbazone residue on
solid phase. Subsequent 1,3-dipolar cycloaddition was ac-
complished with aryl iodides, sodium azide, and copper iodide
in a tandem aryl azide formation/cycloaddition reaction cas-
cade (Table 1, Figure 2).14 Seven triazoles were synthesized in
Azapeptides are peptidomimetics in which the R carbon of
one or more amino acids has been replaced with a nitrogen
atom.1 The longstanding interest in aza-amino acids as
peptide mimics stems from their increased stability,2 resist-
ance to proteases,3 and ability to induce conformational
rigidity, favoring the formation of β turns.4 Until recently,
the incorporation of side-chain diversity onto the aza-amino
acid residue was limited by the solution-phase synthesis of
protected N-alkyl hydrazine building blocks prior to their
activation and incorporation onto a growing peptide chain.
Toward the combinatorial synthesis of azapeptides, we described
(5) Sabatino, D.; Proulx, C.; Klocek, S.; Bourguet, C. B.; Boeglin, D.;
Ong, H.; Lubell, W. D. Org. Lett. 2009, 11, 3650.
€
(6) Houghten, R. A.; Blondelle, S. E.; Dooley, C. T.; Dorner, B.; Eichler,
J.; Ostresh, J. M. Mol. Diversity 1996, 2, 41.
(7) Demers, A.; McNicoll, N.; Febbraio, M.; Servant, M.; Marleau, S.;
Silverstein, R.; Ong, H. Biochem. J. 2004, 417.
(8) (a) Tornoe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67,
3057–3064. (b) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless,
K. B. Angew. Chem., Int. Ed. 2002, 41, 2596.
(9) Selected examples include: (a) Bock, V. D.; Speijer, D.; Hiemstra, H.;
van Maarseveen, J. H. Org. Biomol. Chem. 2007, 5, 971. (b) Turner, R. A.;
Oliver, A. G.; Lokey, R. S. Org. Lett. 2007, 9, 5011.
(1) Gante, J. Synthesis 1989, 405.
(10) Selected examples include: (a) Holub, J. M.; Jang, H.; Kirshenbaum,
K. Org. Lett. 2007, 9, 3275. (b) Jagasia, R.; Holub, J. M.; Bollinger, M.;
Kirshenbaum, K.; Finn, M. G. J. Org. Chem. 2009, 74, 2964.
(11) (a) Kuijpers, B. H. M.; Groothuys, S.; Keereweer, A. R.; Quaedflieg,
P. J. L. M.; Blaauw, R. H.; van Delft, F. L.; Rutjes, F. P. J. T. Org. Lett. 2004,
6, 3123. (b) Lee, D. J.; Mandal, K.; Harris, P. W. R.; Brimble, M. A.; Kent,
S. B. H. Org. Lett. 2009, 11, 5270.
(2) (a) Gassman, J. M. Bioorg. Med. Chem. Lett. 1996, 6, 1771. (b) Wipf,
P.; Adeyeye, C. M.; Rusnak, J. M.; Lazo, J. S. Bioorg. Med. Chem. 1996, 4,
1585.
(3) Dugave, C.; Demange, L. Lett. Pept. Sci. 2003, 10, 1.
(4) (a) Reynolds, C. H.; Hormann, R. E. J. Am. Chem. Soc. 1996, 118,
9395–9401. (b) Lee, H.-J.; Song, J.-W.; Choi, Y.-S.; Park, H.-M.; Lee, K.-B.
ꢀ
J. Am. Chem. Soc. 2002, 124, 11881–11893. (c) Andre, F.; Boussard, G.;
Bayeul, D.; Didierjean, C.; Aubry, A.; Marraud, M. J. Pept. Res. 1997, 49,
(12) Holub, J. M.; Jang, H.; Kirshenbaum, K. Org. Biomol. Chem. 2006,
4, 1497.
ꢀ
556–562. (d) Andre, F.; Vicherat, A.; Boussard, G.; Aubry, A.; Marraud, M.
J. Peptide Res. 1997, 50, 372–381.
(13) Gajewski, M.; Seaver, B.; Esslinger, C. S. Bioorg. Med. Chem. Lett.
2007, 17, 4163–4166.
DOI: 10.1021/jo100957z
r
Published on Web 06/28/2010
J. Org. Chem. 2010, 75, 5385–5387 5385
2010 American Chemical Society

Proulx and Lubell
SCHEME 1. Synthesis of [Aza-1,2,3-triazole-3-alani-
ne]GHRP-6 Analogues
JOCNote
FIGURE 1. 1,2,3-Triazole-3-alanine and aza-1,2,3-triazole-3-
alanine residues.
TABLE 1. LCMS Conversions of the Aryl Azide Formation/1,3-Di-
polar Cycloddition Step
entry
ArI
LCMS conversions at 214 nm (%)
a
b
c
d
e
f
iodobenzene
4-iodoanisole
3-iodoanisole
4-iodotoluene
4-fluoroiodobenzene
3-trifluoromethyliodobenzene
1-iodonaphthalene
61
82
58
80
69
79
62
g
peptide synthesis.15 Azapeptides 8a-g were obtained in
crude purities ranging from 52% to 69% and purified by
using reverse-phase HPLC to afford a product of >99%
purity in 5-11% overall yields based on initial resin loading
(Table 2).
Circular dichroism (CD) spectroscopy in water was used
to study the conformational bias of the [aza-1,2,3-triazole-3-
alanine4]GHRP-6 analogues and compared with the native
sequence (Figure 3). The CD curve of the native GHRP-6
sequence in water is characteristic of a random coil exhibiting
a negative maximum around 190 nm. On the other hand,
azapeptides 8a-g displayed CD signatures consistent with
β-turn conformations exhibiting distinct negative maxima at
230 and 190 nm, and a distinct positive maximum at 215 nm.
A similar curve shape was observed for [aza-Phe4]GHRP-6
indicating that such modification of the aza-residue side chain
does not alter the backbone conformation of the azapeptide in
water.
Seven new GHRP-6 azapeptides containing aza-1,2,3-
triazole-3-alanine residues were prepared employing aza-
propargylglycine in a copper-catalyzed tandem aryl azide
formation/1,3-dipolar cycloaddition approach on Rink
amide resin. Considering that aza-propargylglycine may
be effectively introduced anywhere in a peptide sequence
by submonomer solid-phase azapeptide synthesis, featur-
ing semicarbazone propargylation,5 this libraries-from-
libraries methodology should find general application
for modifying peptide structures, because solution phase
synthesis of the propargyl moiety and aryl azide are
avoided. The effectiveness of this reaction cascade com-
bined with the conformational rigidity imposed by the aza-
amino acid residue make this method particularly attrac-
tive for structure-activity studies of biologically active
peptides.
FIGURE 2. Representative LCMS traces of intermediate 5a
(upper) and peptide 8a (lower), using MeOH/H2O eluent (0-80%
MeOH) containing 0.1% formic acid.
this way and subsequently converted to GHRP-6 analogues
modified at the Trp4 position (Scheme 1).
˚
Addition of 4 A powdered molecular sieves to the resin
proved to be essential to afford the desired triazoles in good
conversions. Both electron-rich and electron-poor aryl iodides
furnished aza-1,2,3-triazole-3-alaninyl residues in good con-
versions (Table 1), with complete disappearance of starting
material as indicated by LCMS analysis after resin cleavage.
Copper scavenging was achieved by shaking the resin in a 3:1
DMF:0.1 N HCl mixture for 12 h. Selective removal of the
hydrazone to reveal the aza-amino acid residue was accom-
plished by using hydroxylamine in pyridine under previously
established conditions,5 and azapeptide elongation was com-
pleted according to standard Fmoc protocols for solid-phase
(14) A one-pot aryl azide formation/1,3-dipolar cycloaddition sequence
on solid support was reported in: Xu, W.-M.; Huang, X.; Tang, E. J. Comb.
Chem. 2005, 7, 726–733.
(15) Lubell, W. D.; Blankenship, J. W; Fridkin, G.; Kaul, R. Peptides.
In Science of Synthesis, 21.11, Chemistry of Amides; Thieme: Stuttgart,
Germany, 2005; p 713.
5386 J. Org. Chem. Vol. 75, No. 15, 2010

Proulx and Lubell
JOCNote
TABLE 2. Yields and Purities of GHRP-6 Azapeptides 8a-g
HRMS [M þ H]þ or [M þ Na]þ ions
peptide
crude puritya (%)
purityb (%)
yieldc (%)
m/z (calcd)
m/z (obsd)
8a
8b
8c
8d
8e
8f
63
52
59
66
65
69
52
>99
>99
>99
>99
>99
>99
>99
5
8
8
8
9
902.4532
932.4638
932.4638
916.4689
920.4438
970.4406
952.4689
902.4516
932.4617
932.4617
916.4657
920.4423
970.4375
952.4660
10
11
8g
aRP-HPLCpurity at 214nm of the crude peptide in MeOH/H2O eluent containing0.1% formic acid (FA). bRP-HPLCpurity at 214 nm of the purified
peptide in MeOH/H2O eluent containing 0.1% formic acid (FA). cYields after purification by RP-HPLC are based on resin loading.
with parafilm, and heated in a water bath with a sonicator at
80 °C for 24 h. The resin was filtered and washed with DMF (3 ꢀ
10 mL), DMF/0.1 N HCl (3:1, 3 ꢀ 10 mL), H2O (3 ꢀ 10 mL),
MeOH (3 ꢀ 10 mL), THF (3 ꢀ 10 mL), and DCM (3 ꢀ 10 mL).
The resin was suspended in a DMF/0.1 N HCl mixture for 12 h,
filtered, and subsequently washed with solvent as above. The
extent of reaction conversion was shown to be 61% by LCMS
analysis of the crude filtered solution, after cleavage of an aliquot
of resin using TFA/TES/H2O (95:2.5:2.5, v/v/v. Figure 2).
LCMS (0-80% MeOH, 35 min) retention time (RT) = 19.89 min;
LCMS (ESI) calcd for C32H38N9O3 [M þ H]þ 596.3, found
m/z 596.2.
Acknowledgment. This research was supported by the
Natural Sciences and Engineering Research Council of
FIGURE 3. Circular dichroism in water of azapeptides 8a-g and
native GHRP-6 (red).
ꢀ
Canada (NSERC) and the Fonds Quebecois de la Recherche
sur la Nature et les Technologies (FQRNT). C.P. is grateful
to NSERC and Boehringer Ingelheim for graduate student
Experimental Section
€ €
fellowships. The authors thank Dr. A. Furtos, K. Venne, and
Representative Triazole Synthesis (5a). In a 6.0 mL filtration
tube, equipped with a cap and stopcock under argon, swollen
semicarbazone resin 4 (200 mg, 0.128 mmol) in DMSO was trea-
ted sequentially with iodobenzene (15 μL, 5 equiv, 0.64 mmol),
CuI (122 mg, 0.64 mmol, 5 equiv), L-proline (74 mg, 0.64 mmol,
5 equiv), sodium azide (42 mg, 0.64 mmol, 5 equiv), triethyl-
ꢀ
M-C. Tang (Universite de Montreal) for assistance with
ꢀ
mass spectrometry.
Supporting Information Available: General Experimental
Methods, compound characterization data, and copies of
LCMS traces. This material is available free of charge via
the Internet at http://pubs.acs.org.
˚
amine (89 μL, 0.64 mmol, 5 equiv), and powdered 4 A molecular
sieves. The filtration tube was flushed with argon, capped, sealed
J. Org. Chem. Vol. 75, No. 15, 2010 5387