1,2,3-Triazoles as peptide bond isosteres: Synthesis and biological evaluation of cyclotetrapeptide mimics

Article abstract of DOI:10.1039/b616751a

Since the discovery of Cu^I-catalysed click chemistry, the field of peptidomimetics has expanded to include 1,4-connected 1,2,3-triazoles as useful peptide bond isosteres. Here, we report the synthesis of triazole-containing analogues of the nat

Full text of DOI:10.1039/b616751a

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www.rsc.org/obc | Organic & Biomolecular Chemistry
1,2,3-Triazoles as peptide bond isosteres: synthesis and biological evaluation
of cyclotetrapeptide mimics†
Victoria D. Bock,a Dave Speijer,b Henk Hiemstraa and Jan H. van Maarseveen*a
Received 15th November 2006, Accepted 30th January 2007
First published as an Advance Article on the web 6th February 2007
DOI: 10.1039/b616751a
Since the discovery of CuI-catalysed click chemistry, the field of peptidomimetics has expanded to
include 1,4-connected 1,2,3-triazoles as useful peptide bond isosteres. Here, we report the synthesis of
triazole-containing analogues of the naturally occurring tyrosinase inhibitor cyclo-[Pro-Val-Pro-Tyr]
and show that the analogues retain enzyme inhibitory activity, demonstrating the effectiveness of a
1,4-connected 1,2,3-triazole as a trans peptide bond isostere.
Introduction
In the pursuit of potential lead compounds that bind to thera-
peutically relevant targets, numerous classes of peptidomimetic
compounds have been developed to enhance the stability and
availability of short peptide fragments in vivo.1 Although the
use of heterocyclic moieties in peptidomimetics has been widely
reported,2 the application of 1,2,3-triazoles to this field has oc-
curred only recently, following the discovery of regioselective CuI-
catalysed click chemistry in 2002.3 1,2,3-Triazoles show particular
promise as amide bond isosteres, given their favourable pharma-
cophoric properties4 and facile synthesis from readily available5
azide- and alkyne-functionalised derivatives of chiral amino acids.
Recently, reports have surfaced describing the incorporation
of 1,2,3-triazoles into peptide nanotubes,6 b-turn mimics,7 pro-
tease inhibitors,4f ,8 cyclopeptide analogues9 and peptide chain
analogues.10 Although these results suggest that triazoles have
similar atom placement and electronic properties to those of a
transoid peptide bond,6a,b,11 no studies to date have analysed the
effects on binding affinity or enzyme inhibition resulting from
replacement of an amide bond in a natural product by a 1,2,3-
triazole.
Scheme 1
To directly assess these effects, we selected a series of three
triazole analogues to the naturally occurring cyclotetrapeptide
cyclo-[Pro-Tyr-Pro-Val] (1), a potent tyrosinase inhibitor isolated
from L. helveticus12 (Scheme 1). All reported attempts to synthesise
1 failed to yield the natural product due to the problematic ring
closure step,13 but we obtained cyclo-[(L)Pro-(L)Val-w(triazole)-
(L)Pro-(L)Tyr] (2) in good yield via CuI-catalysed click chemistry-
mediated macrocyclisation.9e Herein, we report the synthesis of
two other triazole analogues (3 and 4, Scheme 1) and compare the
inhibitory activity of the three peptidomimetic compounds to that
of the natural cyclotetrapeptide.
Results and discussion
Synthesis of triazole analogues 3 and 4 commenced with the prepa-
ration of the azido alkyne linear precursors via a modular, flexible
approach based on coupling reactions between amino acids and
their azido acid and amino alkyne counterparts. Construction
of N3-Tyr(OBn)-Pro-Val-Pro-C≡CH 14, the linear precursor to
cyclotetrapeptide analogue 3, required dipeptides 8 and 12, which
could be obtained via EDC–HOBt-mediated peptide coupling
between azido acid 6 and protected proline 7 and between proline
alkyne 109e and Boc-protected valine 11, respectively (Scheme 2).
Subsequent TFA-mediated tBu–Boc removal and block coupling
of the product dipeptide analogues gave linear precursor 14 in 62%
yield.
Synthesis of the linear precursor to cyclotetrapeptide analogue
4, which contains two triazole moieties in its backbone, began
with CuI-catalysed alkyne–azide coupling of proline alkyne 1514
with valine azido acid 16.5a Conveniently, this reaction required
no protection for the free carboxylic acid and proceeded without
difficulty to yield Boc-Pro-w(triazole)-Val 17 (Scheme 3). Peptide
aVan’t Hoff Institute for Molecular Sciences, University of Amster-
dam, Nieuwe Achtergracht 129, 1018 WS, Amsterdam, The Netherlands.
E-mail: jvm@science.uva.nl; Fax: +31 20 5255670; Tel: +31 20 5255671
bDepartment of Medical Biochemistry, Academic Medical Center, University
of Amsterdam, Meibergdreef 15, 1105 AZ, Amsterdam, The Netherlands
† Electronic supplementary information (ESI) available: Procedures and
spectroscopic data for compounds 5, 6, 8, 9, 12, 13, 18 and 19 and protocol
for the mushroom tyrosinase activity assay. See DOI: 10.1039/b616751a
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Scheme 2 Reagents and conditions: a) NaN3, Tf2O, CH2Cl2, H2O, 0 ◦C. b)
K2CO3, CuSO4, MeOH, H2O, rt. c) BnBr, K2CO3, 2 : 1 = CHCl3 : MeOH,
reflux.
Scheme 3
coupling of 17 with deprotected proline alkyne 10 gave Boc-Pro-
w(triazole)-Val-Pro alkyne 18 in 73% yield over two steps. TFA
deprotection and peptide coupling with tyrosine azido acid 6
provided N3-Tyr(OBn)-Pro-w(triazole)-Val-Pro alkyne 20 in 45%
overall yield for the two transformations due to difficulties in
driving the coupling reaction to completion. Fortunately, sufficient
amounts of linear precursor 20 were obtained to enable direct
progression to cyclisation reactions.
Based on the optimised CuI-catalysed click chemistry macrocy-
clisation conditions for cyclotetrapeptides,9e cyclisation of azido
alkynes 14 and 20 provided rapid access to the two additional
analogues of cyclo-[Pro-Val-Pro-Tyr] (Scheme 4). In the case
of the cyclisation of azido alkyne 14, purification of protected
cyclotetrapeptide analogue 21 proved difficult due to low solubil-
ity, so cyclisation yields of approximately 50% were the highest
obtainable.
Cyclisation of azido alkyne 20 proved more difficult. While the
cyclisations leading to analogues 2 and 3 provided clean products
following column chromatography, purification of the product
from cyclisation of azido alkyne 20 afforded a complex mixture
of compounds, forcing us to precipitate the cyclic product from
an acetonitrile–water mixture, substantially lowering the yield.
Overall, however, cyclotetrapeptide analogue 22 was obtained in
an isolated yield of 36%.
Scheme 4
Removal of the benzyl protecting groups in 21 and 22 via
catalytic hydrogenation yielded the desired analogues 3 and 4
in 91% and 90% yields, respectively (Scheme 4). Although the
cyclisation yields proved lower than expected from the previously
described optimised cyclisation conditions,9e our methodology still
provided an efficient route to the desired products.
With all the desired compounds in hand, the inhibitory effect of
cyclotetrapeptide analogues 2–4 on mushroom tyrosinase activity
was compared to that of the natural product12 via an in vitro
spectrophotometric assay (Table 1). Results indicate that all of the
triazole analogues not only retain inhibition activity, but in fact,
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Table 1 Biological activity of cyclotetrapeptide analogues compared with
spectrometer, coupled to a JEOL MS-MP9021D/UPD system
program. Samples were loaded in a matrix solution (3-nitrobenzyl
alcohol) on a stainless steel probe and bombarded with xenon
atoms with an energy of 3 keV. During the high resolution
FAB-MS measurements a resolving power of 10000 (10% valley
definition) was used. MALDI-TOF mass spectra were recorded
on a Micromass TofSpec 2EC (Micromass, Whyttenshawe, UK).
Samples (50 pmol) were dissolved in EtOAc, mixed with a
concentrated solution of DHB in EtOAc and spotted directly on
the stainless steel MALDI target. Silver nitrate was used for the
ionisation of the molecules. Analytical TLC chromatography was
performed on 250 lm silica gel 60 plates with 254 nm fluorescent
indicator. The mushroom tyrosinase (EC 1.14.18.1) used for the
bioassay was purchased from Sigma Chemical Co. (St. Louis, MO,
USA). Although mushroom tyrosinase differs somewhat from
other sources,16 this fungal source was used for the experiment
because it is readily available. Enzyme inhibition was determined
on a ULTROSPEC 2100 PRO measuring dopachrome formation
at 470 nm for two minutes. All samples were first dissolved in
DMSO and used at ten times dilution. Each experiment was
carried out in triplicate and averaged.
the natural product
Compound
Tyrosinase activity IC50/mMa
cyclo-[Pro-Tyr-Pro-Val]
Triazole analogue 2
Triazole analogue 3
Triazole analogue 4
1.5b
0.6
0.5
1.6
a Concentration for 50% reduction in tyrosinase activity from mushroom
tyrosinase extract. b Data taken from Ref. 12
cyclotetrapeptide analogues 2 and 3 both show an approximately
threefold increase in activity relative to the natural product. These
IC50 values put cyclotetrapeptide analogues 2 and 3 in the mid-
range of known naturally-occurring and synthetic inhibitors of
mushroom tyrosinase.15
Conclusions
These results provide the strongest evidence to date that 1,4-
disubstituted 1,2,3-triazoles can serve as transoid amide bond
mimics in natural compounds without compromising biological
activity. Although previous studies on which this work is based
have elegantly shown the similar hydrogen-bonding properties of
transoid amide bonds and 1,2,3-triazoles,6a,b,11 the introduction
of a triazole into a natural product to determine direct effects
on biological activity provides an additional assessment of the
effects of the triazole incorporation into peptide backbones. This
study clearly demonstrates that analogues of an inaccessible nat-
ural product are readily available via CuI-catalysed alkyne–azide
coupling and that these analogues show retention of biological
activity when compared with a parent cyclotetrapeptide.
(S)-1-((S)-2-Azido-3-(4-(benzyloxy)phenyl)propanoyl)-N-((S)-1-
((S)-2-ethynylpyrrolidin-1-yl)-3-methyl-1-oxobutan-2-
yl)pyrrolidine-2-carboxamide (14)
To a 25 cm3 round-bottomed flask equipped with a CaCO3 drying
tube and charged with Val-Pro alkyne 13 (0.2441 g, 0.714 mmol,
1.1 equiv.) in freshly distilled CH2Cl2 (2.5 cm3) was added DIPEA
(0.12 cm3, 0.714 mmol, 1.1 equiv.). After 10 min of stirring,
this solution was added to a 25 cm3 oven-dried flask equipped
with a CaCO3 drying tube and charged with N3-Tyr(OBn)-Pro
9 (0.2813 g, 0.647 mmol, 1 equiv.), EDC (0.124 g, 0.647 mmol,
1 equiv.), HOBt (0.087 g, 0.647 mmol, 1 equiv.) and freshly distilled
CH2Cl2 (2.5 cm3). After 16 h, this solution was diluted with CHCl3
(20 cm3) and washed with H2O (1 × 20 cm3), satd aq NaHCO3
(1 × 20 cm3), and 1 N HCl (aq) (1 × 20 cm3). The combined
organics were then dried over Na2SO4, filtered, and concentrated
in vacuo to yield a yellow solid. The product was purified via flash
chromatography (75% EtOAc–PE) to afford N3-Tyr(OBn)-Pro-
Val-Pro alkyne 14 (0.2293 g, 0.4018 mmol, 62%) as a white solid.
1H NMR (CDCl3, 400 MHz) d 7.41–7.26 (m, 5H), 7.18–6.87 (m,
4H), 5.02 (m, 2H), 4.97–4.44 (m, 3H), 4.00–2.91 (m, 8H), 2.38–1.94
(m, 10H), 0.99–0.86 (m, 6H) ppm. 13C NMR (CDCl3, 100 MHz) d
170.1, 169.9, 169.8, 169.7, 169.5, 169.3, 169.2, 168.9, 168.7, 168.5,
168.4, 168.2, 168.1, 167.9, 167.6, 156.9, 156.8, 135.9, 135.8, 135.7,
130.3, 129.2, 129.1, 127.8, 127.7, 127.6, 127.5, 127.2, 127.0, 126.9,
126.7, 126.5, 126.4, 126.3, 124.8, 114.2, 114.1, 114.0, 82.7, 82.0,
81.9, 81.7, 80.8, 71.7, 71.1, 69.2, 69.1, 68.9, 68.8, 60.7, 60.6, 60.2,
59.9, 59.7, 59.4, 59.3, 59.2, 58.6, 55.5, 55.0, 54.9, 54.8, 54.7, 54.6,
47.6, 47.2, 46.9, 46.8, 46.4, 46.2, 46.0, 45.8, 45.5, 45.4, 44.9, 36.2,
36.0, 35.5, 35.4, 35.3, 35.2, 34.9, 33.2, 33.1, 31.4, 31.3, 31.2, 30.9,
30.8, 30.7, 30.6, 30.3, 30.0, 29.9, 28.6, 27.5, 27.4, 27.2, 27.1, 26.8,
26.5, 24.0, 23.8, 23.7, 23.5, 21.9, 21.5, 21.4, 18.7, 18.6, 18.3, 18.2,
17.1, 17.0, 17.09, 16.9, 16.8, 16.7, 16.4, 16.2 ppm. IR 3301, 3218,
2966, 2873, 2243, 2106, 1684, 1650, 1632, 1510, 1429, 1379, 1339,
1303, 1234, 1179, 1107, 1015, 912 cm−1. [a]D20 = −64.3 (c 1.96 in
CHCl3).
Experimental
General notes
Oxygen- and moisture-sensitive reactions were carried out us-
ing standard Schlenk techniques under a nitrogen atmosphere.
Tetrahydrofuran and diethyl ether were freshly distilled from
sodium–benzophenone. Dry DMF and CH2Cl2 were freshly dis-
tilled from CaH2. All commercially available reagents were used as
received, unless indicated otherwise. NMR spectra were recorded
in Fourier transform mode on a Bruker ARX 400 (1H at 400 MHz,
13C at 100 MHz) magnetic resonance spectrophotometer. 1H NMR
spectra are reported as chemical shifts in parts per million (ppm)
downfield from a tetramethylsilane internal standard (0.00 ppm).
Spin multiplicity is described by the following abbreviations: s =
singlet, d = doublet, t = triplet, q = quartet, m = multiplet,
dd = doublet of doublets, and br = broad. Coupling constants
(J) are reported in Hertz (Hz). 13C NMR spectra are reported
as chemical shifts in ppm with the solvent resonance as the
internal standard (CDCl3: 77.07 ppm; MeOD: 49.00 ppm; MeCN-
d3: 118.26) and were recorded with complete heterodecoupling
as APT (attached proton test) spectra. Infrared spectra were
obtained from CDCl3 solutions on a Bruker IFS 28 Fourier
Transform spectrometer (FTIR) and are reported in wavenumbers
(cm−1). Fast atom bombardment (FAB) mass spectrometry was
carried out using a JEOL JMS SX/SX 102A four-sector mass
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(S)-2-(4-((S)-1-((S)-2-Azido-3-(4-(benzyloxy)phenyl)propanoyl)-
pyrrolidin-2-yl)-1H-1,2,3-triazol-1-yl)-1-((S)-2-ethynylpyrrolidin-
1-yl)-3-methylbutan-1-one (20)
(m, 1H, Tyr CaH), 5.33 (d, J = 6.9 Hz, 1H, Pro CaH), 5.01–4.98
(m, 2H, OCH2Ph), 4.23 (d, J = 8.1 Hz, 1H, Pro CaH), 4.01 (t, J =
9.0, 1H, Val CaH), 3.80–3.78 (m, 1H, one of two Pro NCH2), 3.69–
3.67 (m, 2H, Pro NCH2), 3.48–3.44 (m, 2H, one of two Pro NCH2
and one of two Tyr CaCH2), 3.35–3.32 (m, 1H, one of two Tyr
CaCH2), 2.46–2.41 (m, 1H, one of two Pro CaCH2), 2.31–1.78 (m,
8H, Val CaCH, Pro CaCH2CH2, Pro CaCH2), 0.85 (d, J = 6.6 Hz,
3H, one of two Val CaCH(CH3)2), 0.79 (d, J = 6.8 Hz, 3H, one of
two Val CaCH(CH3)2) ppm. 13C NMR (CDCl3, 100 MHz) d 169.4,
168.6, 165.5, 156.9, 149.7, 135.8, 129.9, 127.5, 126.9, 126.7, 126.1,
120.8, 113.7, 68.9, 62.7, 61.2, 56.6, 54.3, 47.3, 45.4, 36.4, 36.3, 32.6,
31.1, 31.0, 28.6, 20.7, 20.5, 18.0, 17.4 ppm. IR 3208, 2966, 2874,
1657, 1616, 1512, 1434, 1386, 1302, 1246, 1231, 1178, 1094, 1048,
1016, 913 cm−1. HMRS (FAB) Calculated for C32H39N6O4 (MH+):
571.3035; Found: 571.3033. [a]2D0 = −146.1 (c 0.95 in CHCl3).
To a 50 cm3 round-bottomed flask equipped with a CaCO3 drying
tube and charged with Pro-w(triazole)-Val-Pro alkyne TFA salt
19 (0.3254 g, 0.956 mmol, 1 equiv.) in freshly distilled CH2Cl2
(3 cm3) was added DIPEA (0.17 cm3, 0.956 mmol, 1 equiv.). After
10 min of stirring, this solution was added to a 50 cm3 oven-
dried flask equipped with a CaCO3 drying tube and charged with
tyrosine azido acid 6 (0.313 g, 1.051 mmol, 1.1 equiv.), EDC
(0.183 g, 0.956 mmol, 1 equiv.), HOBt (0.129 g, 0.956 mmol,
1 equiv.) and freshly distilled CH2Cl2 (7 cm3). After 16 h, this
red–orange solution was diluted with CHCl3 (50 cm3) and washed
with H2O (1 × 50 cm3), satd aq NaHCO3 (1 × 50 cm3), and
1 N HCl (aq) (1 × 50 cm3). The combined organics were then
dried over Na2SO4, filtered, and concentrated in vacuo to yield a
brown solid. The product was purified via flash chromatography
(33% EtOAc–PE) to afford N3-Tyr(OBn)-Pro-w(triazole)-Val-Pro
alkyne 20 (0.2552 g, 0.430 mmol, 45% over two steps) as a white
cyclo-[Pro-Tyr-w(triazole)-Pro-Val] 3
To a 50 cm3 round-bottomed flask charged with Bn-protected
cyclic peptide 21 (55.0 mg, 0.0964 mmol, 1 equiv.) in MeOH (1 cm3)
and CH2Cl2 (9 cm3) was added 10% Pd/C (27.5 mg). The resulting
mixture was subjected to a three-cycle of vacuum–H2 and was
stirred at rt under a H2 balloon for 16 h. The catalyst was then
removed by filtration through Celite, and the filtrate concentrated
in vacuo to afford a white solid. This solid was then dissolved in
a minimal volume of CH2Cl2 and titrated with EtOAc. The white
solid was collected by filtration, yielding cyclic peptide 3 (42 mg,
0.0877 mmol, 91% yield). 1H NMR (CDCl3–MeOD,17 400 MHz)
d 6.87–6.85 (m, 3H, triazole C5H, Tyr ArH), 6.63 (d, J = 8.4 Hz,
2H, Tyr ArH), 6.14–6.10 (m, 0.4H, amide NH), 5.68 (t, J = 5.9 Hz,
1H, Tyr CaH), 5.26 (d, J = 7.2 Hz, 1H, Pro CaH), 4.18 (d, J =
8.2 Hz, 1H, Pro CaH), 3.99–3.96 (m, 1H, Val CaH), 3.69–3.65 (m,
3H, Pro NCH2), 3.44–3.39 (m, 3H, Tyr CaCH2 and one of two
Pro NCH2), 2.42–2.39 (m, 1H, one of two Pro CaCH2), 2.13–1.77
(m, 8H, Val CaCH, Pro CaCH2, Pro CaCH2CH2), 0.81 (d, J =
6.5 Hz, 3H, one of two Val CaCH(CH3)2), 0.74 (d, J = 6.8 Hz, 3H,
one of two Val CaCH(CH3)2) ppm. 13C NMR (CDCl3–MeOD,17
100 MHz) d 170.3, 170.1, 170.0, 166.5, 130.8, 126.3, 122.5, 115.0,
63.6, 62.2, 57.2, 55.2, 48.3, 46.6, 37.0, 33.4, 32.0, 31.7, 21.5, 21.4,
19.0, 18.1 ppm. IR 3473, 2957, 2926, 1657, 1618, 1516, 1434, 1385,
1309, 1264, 1226, 1172, 1156, 1095 cm−1. HMRS (FAB) Calculated
for C25H33N6O4 (MH+): 481.2565; Found: 481.2563. [a]2D0 = −175.5
(c 0.74 in CHCl3).
1
solid. H NMR (CDCl3, 400 MHz) d 8.02–7.60 (m, 1H), 7.41–
7.28 (m, 5H), 7.13–6.76 (m, 4H), 5.38–4.66 (m, 3.6H), 4.29–4.26
(m, 0.4H), 3.94–2.79 (m, 8H), 2.64–1.66 (m, 10H), 1.09–0.98 (m,
3H), 0.74–0.66 (m, 3H) ppm. 13C NMR (CDCl3, 100 MHz) d
169.2, 169.1, 169.0, 167.9, 167.0, 166.6, 166.3, 166.2, 165.8, 157.9
157.8, 149.8, 149.4, 148.6, 148.1, 147.2, 137.0, 136.9, 136.8, 130.4,
130.2, 130.1, 128.6, 128.5, 128.4, 128.3, 128.2, 128.0 127.9, 127.7,
127.5, 127.4, 122.3, 121.5, 121.1, 120.9, 120.8, 120.4, 120.1, 119.9,
119.7, 119.3, 119.1, 115.1, 115.0, 114.9, 114.8, 83.3, 83.2, 83.1,
83.0, 82.3, 82.2, 81.4, 81.2, 74.0, 73.8, 73.2, 73.1, 73.0, 71.1,
70.9, 70.7, 70.6, 70.0, 69.9, 67.2, 67.1, 67.0, 61.2, 61.1, 61.0,
60.9, 60.8, 60.6, 60.4, 54.1, 53.9, 53.7, 53.6, 53.1, 52.9, 52.9, 52.8,
48.8, 48.7, 48.6, 48.5, 48.1, 47.9, 47.8, 47.0, 46.9, 46.8, 46.7, 46.6,
46.4, 46.3, 46.2, 37.5, 37.3, 36.5, 36.4, 35.6, 34.3, 34.0, 33.9, 33.7,
33.6, 33.5, 33.3, 33.2, 33.1, 32.8, 32.7, 32.2, 32.0, 31.9, 31.8, 31.7,
31.6, 30.7, 30.6, 30.4, 29.6, 24.9, 24.8, 24.7, 24.6, 24.2, 24.0, 22.8,
22.7, 22.1, 21.9, 21.8, 19.4, 19.3, 19.2, 19.0, 18.8, 18.7, 18.6, 18.5,
18.3, 18.2 ppm. IR 3300, 2968, 2876, 2246, 2102, 1654, 1610,
1583, 1510, 1427, 1390, 1299, 1238, 1177, 1153, 1109, 1045, 1016,
911, 810 cm−1. HMRS (FAB) Calculated for C33H39N8O3 (MH+):
595.3147; Found: 595.3145. [a]2D0 = +5.1 (c 0.95 in CHCl3).
Bn-protected cyclic peptide 21
To a 100 cm3 round-bottomed flask charged with alkyne azide 14
(0.033 g, 0.0578 mmol, 1 equiv.) in toluene (58 cm) was added DBU
(0.026 cm3, 0.1735 mmol, 3 equiv.). The solution was degassed with
argon for thirty minutes and then heated to reflux while flushing
with argon. At reflux, CuBr (1.7 mg, 0.0116 mmol, 0.2 equiv.)
was added, and the solution was stirred at reflux under argon for
16 h. The mixture was then cooled to rt and poured through a
2 inch pad of Celite. The Celite pad was washed with CH2Cl2
(3 × 25 cm3). The filtrate was concentrated in vacuo to provide a
blue–green oil. The product was purified via flash chromatography
(3% MeOH in CH2Cl2) to afford Bn-protected cyclic peptide 21
Bn-protected cyclic peptide 22
To a 100 cm3 round-bottomed flask charged with azido alkyne
20 (0.0269 g, 0.0452 mmol, 1 equiv.) in toluene (45 cm3) was
added DBU (0.020 ml3, 0.1356 mmol, 3 equiv.). The solution was
degassed with argon for thirty minutes and then heated to reflux
while flushing with argon. At reflux, CuBr (1.3 mg, 0.0091 mmol,
0.2 equiv.) was added, and the solution was stirred at reflux under
argon for 16 h. The mixture was then cooled to rt and poured
through a 2 in pad of Celite. The Celite pad was washed with
CH2Cl2 (3 × 20 cm3). The filtrate was concentrated in vacuo
to provide a brown solid. The product was purified via flash
chromatography (3% MeOH in CH2Cl2) to afford a white solid.
This solid was then washed with 50% MeCN–H2O (5 cm3) to yield
protected cyclotetrapeptide analogue 22 (9.6 mg, 0.0161 mmol,
1
(0.0184 g, 0.0326 mmol, 56% yield) as a white solid. H NMR
(CDCl3, 400 MHz) d 7.41–7.30 (m, 5H, OCH2Ph), 7.01 (d, J =
8.5 Hz, 2H, Tyr ArH), 6.88 (s, 1H, triazole C5H), 6.81 (d, J =
8.6 Hz, 2H, Tyr ArH), 5.72–5.68 (m, 0.67H, amide NH), 5.43–5.41
974 | Org. Biomol. Chem., 2007, 5, 971–975
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36% yield) as a white solid. 1H NMR (CDCl3, 400 MHz) d 7.37–
7.26 (m, 5H, OCH2Ph), 7.10 (d, J = 8.5 Hz, 2H, Tyr ArH), 6.95 (s,
1H, triazole C5H), 6.84 (d, J = 8.5 Hz, 2H, Tyr ArH), 6.45 (s, 1H,
triazole C5H), 5.56–5.53 (m, 1H, Tyr CaH), 5.26 (d, J = 7.2 Hz,
1H, Pro CaH), 5.06 (d, J = 7.2 Hz, 1H, Pro CaH), 4.99–4.97 (m,
3H, Val CaH and OCH2Ph), 3.73–3.31 (m, 6H, Pro NCH2 and
Tyr CaCH2), 2.53–2.50 (m, 1H, Val CaCH), 2.31–2.28 (m, 2H,
Pro CaCH2), 1.97–1.76 (m, 6H, Pro CaCH2 and Pro CaCH2CH2),
1.01 (d, J = 6.5 Hz, 3H, one of two Val CaCH(CH3)2), 0.70 (d,
J = 6.8 Hz, 3H, one of two Val CaCH(CH3)2) ppm. 13C NMR
(CDCl3, 100 MHz) d 181.1, 166.7, 166.2, 159.2, 150.4, 149.1, 138.2,
132.1, 129.9, 129.4, 129.3, 128.8, 120.5, 120.3, 116.2, 71.3, 69.0,
64.9, 56.6, 56.3, 48.4, 48.3, 39.4, 35.1, 35.0, 32.2, 31.0, 23.0, 22.0,
21.4, 19.6 ppm. IR 3136, 3100, 2960, 1653, 1539, 1510, 1432,
1369, 1345, 1306, 1262, 1221, 1175, 1019 cm−1. HMRS Calculated
for C33H39N8O3 (MH+): 595.3147 (FAB) Found: 595.3145; (ESI)
Found: 595.32. [a]2D0 = −35.9 (c 0.41 in CHCl3).
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H. Schiewe and H. Waldmann, Proc. Natl. Acad. Sci. U. S. A., 2006,
103, 10606–10611.
5 For the rapid synthesis of azido acids from amino acids, see: (a) J. T.
Lundquist, IV and J. C. Pelletier, Org. Lett., 2001, 3, 781–783; (b) For
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4503–4506.
7 (a) J. H. Billing and U. J. Nilsson, J. Org. Chem., 2005, 70, 4847–4850;
(b) K. Oh and Z. Guan, Chem. Commun., 2006, 3069–3071; (c) W. J.
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R. J. Fisher and T. R. Burke, Jr., Bioorg. Med. Chem. Lett., 2006, 16,
5265–5269.
8 M. Whiting, J. Muldoon, Y.-C. Lin, S. M. Silverman, W. Lindstom,
A. J. Olson, H. C. Kolb, M. G. Finn, K. B. Sharpless, J. H. Elder and
V. V. Fokin, Angew. Chem., Int. Ed., 2006, 45, 1435–1439.
9 (a) M. Roice, I. Johannsen and M. Meldal, QSAR Comb. Sci., 2004,
23, 662–673; (b) J. H. van Maarseveen, W. S. Horne and M. R. Ghadiri,
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17135; (b) V. Aucagne and D. A. Leigh, Org. Lett., 2006, 8, 4505–
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cyclo-[Val-w(triazole)-Pro-Tyr-w(triazole)-Pro] 4
To a 50 cm3 round-bottomed flask charged with Bn-protected
cyclotetrapeptide analogue 22 (33.2 mg, 0.0558 mmol, 1 equiv.)
in MeOH (0.5 cm) and CH2Cl2 (4.5 cm3) was added 10% Pd/C
(50.0 mg). The resulting mixture was subjected to a three-cycle of
vacuum–H2 and was stirred at rt under a H2 balloon for 16 h. The
catalyst was then removed by filtration through Celite, and the
filtrate concentrated in vacuo to afford cyclotetrapeptide analogue
4 (25.3 mg, 0.05022 mmol, 91% y◦ield) as a white solid. 1H NMR
(CDCl3–MeOD,16 400 MHz at 40 C) d 6.98 (s, 1H, triazole C5H),
6.95 (d, J = 8.4 Hz, 2H, Tyr ArH), 6.66 (d, J = 8.4 Hz, 2H, Tyr
ArH), 6.53 (s, 1H, triazole C5H), 5.54–5.51 (m, 1H, Tyr CaH), 5.22
(d, J = 7.2 Hz, 1H, Pro CaH), 5.06 (d, J = 7.1 Hz, 1H, Pro CaH),
5.00 (d, J = 8.9 Hz, 1H, Val CaH), 3.76–3.74 (m, 2H, Pro NCH2),
3.62–3.58 (m, 2H, Pro NCH2), 3.37–3.34 (m, 2H, Tyr CaCH2),
2.52–2.49 (m, 1H, Val CaCH), 2.28–2.25 (m, 2H, Pro CaCH2),
2.00–1.77 (m, 6H, Pro CaCH2 and Pro CaCH2CH2), 1.01 (d, J =
6.6 Hz, 3H, one of two Val CaCH(CH3)2), 0.70 (d, J = 6.8 Hz, 3H,
one of two Val CaCH(CH3)2) ppm. 13C NMR (CDCl3–MeOD,17
100 MHz at 40 ◦C) d 165.3, 165.0, 155.8, 148.7, 147.7, 130.3,
126.2, 119.4, 119.3, 115.2, 67.5, 63.5, 55.0, 54.8, 46.9, 46.8, 37.8,
33.5, 33.4, 30.6, 21.2, 20.4, 19.7, 17.9 ppm. IR 3106, 3059, 2962,
2925, 2875, 2244, 1731, 1633, 1538, 1511, 1432, 1379, 1344, 1299,
1260, 1177, 1086, 1021, 911, 860 cm−1. HMRS (FAB) Calculated
for C26H33N8O3 (MH+): 505.2677; Found: 505.2676. [a]2D0 = −87.7
(c 0.13 in DMF).
11 For a review, see: H. C. Kolb and K. B. Sharpless, Drug Discovery
Today, 2003, 8, 1128–1137.
12 H. Kawagishi, A. Somoto, J. Kuranari, A. Kimura and S. Chiba,
Tetrahedron Lett., 1993, 34, 3439–3440.
13 U. Schmidt and J. Langner, J. Pept. Res., 1997, 49, 67–73.
14 H. D. Dickson, S. C. Smith and K. W. Hinkle, Tetrahedron Lett., 2004,
45, 5597–5599.
15 For reviews of tyrosinse inhibition, see: (a) A. Rescigno, F. Sollai, B.
Pisu, A. Rinaldi and E. Sanjust, J. Enzyme Inhib. Med. Chem., 2002,
17, 207–218; (b) S.-Y. Seo, V. K. Sharma and N. Sharma, J. Agric. Food
Chem., 2003, 51, 2837–2853; (c) Y.-J. Kim and H. Uyama, Cell. Mol.
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Notes and references
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Giannis and T. Kolter, Angew. Chem., Int. Ed. Engl., 1993, 32, 1244–
1267.
2 S. Borg, G. Estennebouhtou, K. Luthman, I. Csoregh, W. Hesselink
and U. Hacksell, J. Org. Chem., 1995, 60, 3112–3120, and references
therein.
16 C. W. van Gelder, W. H. Flurkey and H. J. Wichers, Phytochemistry,
1997, 45, 1309–1323.
17 The product was dissolved in approximately 0.5 cm CDCl3 with several
drops of MeOD added to improve solubility.
3 (a) V. V. Rostovtsev, L. G. Green, V. V. Fokin and K. B. Sharpless,
Angew. Chem., Int. Ed., 2002, 41, 2596–2599; (b) C. W. Tornøe, C.
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