Synthesis of crosslinking amino acids by click chemistry

Article abstract of DOI:10.1016/j.tet.2009.07.054

Crosslinking amino acids are naturally existing protein crosslinkers. Herein, we described the synthesis of several novel bis-amino acids constituted of serine, alanine, lysine, and tyrosine with click chemistry. The Huisgen 1,3-dipolar cycloadditions bet

Full text of DOI:10.1016/j.tet.2009.07.054

Tetrahedron 65 (2009) 7935–7941
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of crosslinking amino acids by click chemistry
,
,
Chun Li a b, Jie Tang b, Juan Xie a
*
a PPSM, Institut d’Alembert, ENS Cachan, CNRS, UniverSud, 61 av President Wilson, F-94230 Cachan, France
b Institute of Medicinal Chemistry, Department of Chemistry, East China Normal University, Shanghai 200062, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 May 2009
Received in revised form 20 July 2009
Accepted 23 July 2009
Available online 26 July 2009
Crosslinking amino acids are naturally existing protein crosslinkers. Herein, we described the synthesis of
several novel bis-amino acids constituted of serine, alanine, lysine, and tyrosine with click chemistry. The
Huisgen 1,3-dipolar cycloadditions between azido- and alkyne-functionalized amino acids can be easily
realized in the presence of catalytic amount of CuSO4 and Na ascorbate. In addition, fluorescent bis-
amino acid derivatives have also been prepared using anthracene, fluorescein, and benzothiadiazole as
fluorophores. With symmetrical bis-alkyne benzothiadiazole, it was possible to synthesize asymmetrical
derivative bearing two different amino acid moieties.
Keywords:
Bis-amino acids
Crosslinks
Ó 2009 Elsevier Ltd. All rights reserved.
Click chemistry
Fluorescence
1. Introduction
the biosciences and appear to be among the most powerful analytical
research tools. We have also synthesized fluorescent bis-amino acid
Crosslinking amino acids like histidinoalanine, lysinoalanine, and
pyridinoline are natural metabolism products identified from
a range of sources like milk products, human tissues including con-
nective tissue, bone, dentin, and eye cataracts, phosphoproteins of
bivalve molluscs as well as bicyclic dodecapeptides theonellamides.1
As covalent protein crosslinks, histidinoalanine and lysinoalanine
have been shown to increase the stability toward proteases and
modify the quaternary structure of proteins. They seem to play an
important role in mineral binding in bivalve molluscs and in ad-
vanced eye cataracts. On the other hand, deoxypyridinoline and
pyridinoline, two fluorescent crosslinks derived from the degrada-
tion of bone collagen, have been used as biochemical markers of
collagen turnover.2 Synthesis of these crosslinking amino acids has
therefore attracted increasing attentions.1,3 However, preparation of
bis-amino acids like histidinoalanine remained difficult because of
the problem of regioisomers and low yield.4 As a continuing program
on the use of click chemistry for bioorganic and analytical applica-
tions,5 we decided to synthesize new bis-amino acid derivatives of
serine, alanine, tyrosine, and lysine (compounds 1–10, Fig. 1) using
the Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction.6
Since the triazole ring has similar chemical properties as the imid-
azole ring and is resistant to hydrolysis, oxidation, reduction, or other
modes of cleavage, compounds 1–3 can be considered as mimetics of
histidinoalanine. Fluorescence methods have been widely used in
derivatives 5–10 based on anthracene, fluorescein, and benzothia-
diazole, which absorb at wavelengths longer than 320 nm, where
interference from the tryptophan units is negligible. These molecules
could then be used as multivalent organic probes for fluorescent
labeling of biomolecules.7
2. Results and discussion
In order to obtain various bis-amino acids, we have firstly syn-
thesized protected amino acids functionalized with azido or alkyne
functions. O-Propargyl Boc-Ser-OMe 11,8 3-azido Boc-Ala-OMe 12,9
3-azido Boc-Ala-OBn 13,10 O-propargyl Boc-Tyr-OMe 1411 have
been prepared according to the literature procedure. O-Propargyl
Boc-Ser-OBn 15 was obtained from Boc-Ser-OH by O-alkylation and
esterification reactions (Scheme 1). By a diazotransfer reaction with
imidazole-1-sulfonyl azide,12 Na-Cbz-Lys-OH has been transformed
into the corresponding azide 16 in 70% yield after esterification
with SOCl2 and MeOH.
Click reaction of azides 12 and 13 with alkynes 11, 14, and 15
underwent smoothly in the presence of catalytic amount of CuSO4
and sodium ascorbate in a mixture of CH2Cl2 and H2O (Table 1). Bis-
amino esters 1–4 were obtained in 55–86% yield. By saponification
or hydrogenolysis, these compounds can be easily transformed into
the carboxylic acids 17–20 for further derivatization (Scheme 2).
Synthesis of anthracene and fluorescein-based bis-amino acid
derivatives is depicted in the Scheme 3. Reaction of the alkyne 15
(2 equiv) with the known diazido anthracene 2113 (1 equiv) led to
the desired compound 5 in 60% yield. However, hydrogenolysis
* Corresponding author. Tel.: þ33 1 47405586; fax: þ33 1 47402454.
E-mail address: joanne.xie@ens-cachan.fr (J. Xie).
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.07.054

7936
C. Li et al. / Tetrahedron 65 (2009) 7935–7941
CO2H
NH2
CO2H
H2N
BocHN
CO2R1
OH
H2N
CO2H
NH
H2N
CO2H
N
N+
N
N
N
1 = R2 = Me
1
R
N
O
2 R1 = Me, R2 = Bn
3 R1 = Bn, R2 = Me
H2N
Histidinoalanine
BocHN CO2Me
CO2H
H2N
CO2H
H2N
CO2H
BocHN
CO2R2
Deoxypyridinoline
Lysinoalanine
CO2Bn
O
NHBoc
O
O
O
N
N
N
N
N
(CH2)n
N
N
N
N
CO2
RHN CO2Me
O
N
N
(CH2)n
CO2Me
6 n=1, R=Boc
7 n=4, R=Cbz
N
N
N
N
N
BocHN
CO2Me
5
RHN
4
NHBoc
CO2Bn
O
S
R1HN
MeO2C
NHR2
n = 1, R1 = R2 = Boc
n = 4, R1 = R2 = Cbz
=
8
=
N
m
m
m
(CH2)m
(CH2)n
9
=
N
N
N
CO2Me
1, n = 4, R1 = Boc, R2 = Cbz
10
N
N
N
Figure 1. Structure of natural and target crosslinking amino acid derivatives.
under different conditions failed to give the corresponding free
acid. Treatment of the azido alanine 12 or azido lysine 16 with the
bis-propargyl fluorescein 2214 furnished the fluorescent molecules
6 and 7 as a yellow solid in good yield (82% for 6 and 92% for 7).
Tentative saponification of 6 and 7 also hydrolyzed the ester bond
of the fluorescein moiety. We then decided to deprotect the amino
function of compounds 6 and 7 for further modification. Reaction of
6 with TFA led to the free amino derivative 23 in quantitative yield.
Hydrogenolysis or HBr treatment of 7 failed to give the corre-
sponding free acid.
1. NaH, BrCH2C CH
DMF
BocHN
CO2Bn
O
BocHN
CbzHN
CO2H
2. BnBr, K2CO3, DMF
56 %
OH
15
CO2H
1. SOCl2, MeOH
CbzHN
CO2Me
2. ImSO2N3, MeOH
K2CO3, CuSO4
70 %
(CH2)4NH2
(CH2)4N3
16
Benzothiadiazole fluorophore was easily accessible and pos-
sessed high fluorescence efficiency.15 Treatment of its bis-alkyne
Scheme 1. Synthesis of azido or propargyl amino esters 15, 16.
Table 1
Synthesis of bis-amino esters 1–4 via click chemistrya
Entry
1
Azide
CO2Me
Alkyne
CO2Me
Product
Yieldb%
55
CO2Me
NHBoc
N
N
BocHN
BocHN
BocHN
BocHN
BocHN
BocHN
MeO2C
BocHN
N
O
O
O
N3
O
12
12
13
11
15
11
1
CO2Bn
NHBoc
N
N
CO2Me
N3
CO2Bn
O
MeO2C
BocHN
N
2
3
70
86
2
CO2Me
NHBoc
N
N
CO2Bn
N3
CO2Me
O
BnO2C
BocHN
N
3
CO2Me
NHBoc
BocHN
CO2Me
BocHN
CO2Me
N3
4
86
N
N
N
MeO2C
BocHN
12
O
14
O
4
a
The reaction was conducted in the presence of 0.01–0.1 equiv of CuSO4 and 0.02–0.2 equiv of sodium ascorbate in a mixture of CH2Cl2/H2O (1/1) at rt for 12–20 h.
Isolated yield after column chromatography.
b

C. Li et al. / Tetrahedron 65 (2009) 7935–7941
7937
CO2H
prepared. Furthermore, their fluorescent derivatives have also been
prepared using azido or alkyne-functionalized anthracene, fluo-
rescein, and benzothiadiazole as fluorophores. These bis-amino
acids should be useful as protein crosslinks for various biological
applications. Fluorescent bis-amino acids could also be used for
biomolecules labeling or as fluorescent sensors.17 Work is in prog-
ress in our laboratory for potential applications of these molecules.
N
N
N
LiOH
HO2C
1
O
MeOH/H2O
96 %
NHBoc
BocHN
17
CO2H
N
N
N
10% Pd/C
MeO2C
BocHN
2
H2, MeOH
92 %
O
NHBoc
18
4. Experimental
4.1. General
CO2Me
NHBoc
CO2H
N
N
N
10% Pd/C
HO2C
3
H2, MeOH
86 %
O
BocHN
19
1Hand13CNMRspectrawererecordedonJeol400spectrometerin
CDCl3, DMSO-d6 or CD3OD solutions. Column chromatography was
performed on E. Merck Silica Gel 60 (230–400 mesh). Analytical thin-
layer chromatography was performed on E. Merck aluminum per-
colated plates of Silica Gel 60F-254 with detection by UV and nin-
hydrin. Optical rotations were measured using a Jasco P-2000
polarimeter. High-resolution mass spectra (HRMS) were recorded on
a MA1212 instrument using standard conditions (ESI, 70 eV).
NHBoc
LiOH
4
N
N
N
MeOH/H2O
90 %
HO2C
O
BocHN
20
Scheme 2. Synthesis of bis-amino acids 17–20.
N3
4.2. Preparation of O-propargyl Boc-Ser-OBn 15
15 (2 equiv), CuSO4, Na ascorbate
5
To an ice cold solution of Boc-Serine (0.380 g, 1.86 mmol) in
10 mL DMF was added slowly NaH (60%, 107 mg, 4.45 mmol). After
kept stirring for 30 min, propargyl bromide (0.23 mL, 2.61 mmol)
was then added. After 12 h at rt, brine (15 mL) was added. The re-
action mixture was extracted with EtOAc (2ꢀ10 mL). The water
layer was acidified to pH¼1–2 with 1 N KHSO4, then extracted with
EtOAc (3ꢀ15 mL). The combined organic layers were dried over
MgSO4, evaporated to give a liquid, which was dissolved in DMF
(10 mL). NaHCO3 (312 mg, 3.72 mmol) and BnBr (0.59 mL,
4.65 mmol) were added successively. After 12 h at rt, brine (15 mL)
was added. The reaction mixture was extracted with EtOAc
(3ꢀ15 mL). The combined organic layers were washed with brine
and dried over anhydrous MgSO4, then evaporated. Purification by
column chromatography (petroleum ether/EtOAc: 10/1) afforded
CH2Cl2/H2O, 60%
21
N3
O
O
O
12 (2 equiv), CuSO4, Na ascorbate
6
CH2Cl2/H2O, 82%
CO2
22
16 (2 equiv), CuSO4, Na ascorbate
7
22
CH2Cl2/H2O, 92%
O
O
O
the title compound as a colorless liquid (0.350 g, 56%). TLC: Rf¼0.6
25
ꢁ2.4 (c 0.3, CH2Cl2); 1H NMR
N
TFA
(petroleum ether/EtOAc¼4/1). [
a]
D
N
6
N
CH2Cl2
100%
(400 MHz, CDCl3):
d
7.35–7.36 (m, 5H, Ph), 5.39 (d, 1H, J¼8.3 Hz,
-
NH3+CF3CO2
CO2
MeO2C
NH), 5.27 (d, 1H, J¼12.4 Hz, CHPh), 5.15 (d, 1H, J¼12.4 Hz, CHPh),
4.49 (dt, 1H, J¼2.8, 8.7 Hz, CH), 4.10 (d, 2H, J¼2.3 Hz, CH2), 4.00 (dd,
1H, J¼3.2, 9.6 Hz, CH), 3.75 (dd, 1H, J¼3.2, 9.2 Hz, CH), 2.40 (t, 1H,
23
N
N
N
-
+
O2CF3C H3N
CO2Me
J¼2.3 Hz, C^CH). 13C NMR (100 MHz, CDCl3):
d 170.4, 155.6, 135.5
Scheme 3. Synthesis of anthracene and fluorescein-based bis-amino acid derivatives.
(Cq); 128.6, 128.4, 128.2 (Ph); 80.1 (Cq), 75.2 (CH^); 69.8, 67.3, 58.6
(CH2); 54.0 (CH), 28.4 (Boc). HRESIMS: calcd for C18H23NNaO5:
356.1474; found: m/z 356.1468.
derivative 2416 (1 equiv) with the azido alanine 12 or azido lysine
16 (2 equiv) in the presence of TBAF afforded, in one-pot,5b the bis-
triazole compounds 8 and 9 in 68–73% yield (Scheme 4). Reaction of
24 with 0.44 equiv of 16 led to a mixture of mono-click compounds
25 and 26. The TMS-protected compound 25 can be easily con-
verted into the deprotected alkyne 26 by TBAF in 86% total yield.
Further click reaction of 26 with the azide 12 led to the asymmet-
rical bis-amino ester 10 in 91% yield. This result is very interesting
since different azido derivatives can be linked to the benzothia-
diazole fluorophore through two consecutive click reactions.
Saponification of compounds 8–10 led to the corresponding free
acids 27–29 in good yield. The amino-protecting groups of 8 and 9
can also be easily removed with TFA (for Boc) or HBr in AcOH (for
Cbz), leading to the corresponding amino esters 30 and 31.
4.3. Preparation of 6-azido-2(S)-benzyloxycarbonylamino-
hexanoic acid methyl ester 16
To precooled (ice-salt bath) absolute MeOH (5 mL) was added
slowly SOCl2 (0.2 mL, 2.8 mmol), then Na-Cbz-Lys-OH (100 mg,
0.36 mmol) in one portion. The solution was allowed to warm to rt
slowly and stirred overnight. After evaporation, Na-Cbz-Lys-OMe
was obtained as clear oil, which was dissolved in dry MeOH (8 mL).
K2CO3 (248 mg, 1.8 mmol) and CuSO4$5H2O (23 mg, 0.09 mmol)
were added to the above solution, followed by ImSO2N3 (75 mg,
0.43 mmol) 10 min later. The reaction mixture was stirred at rt for
12 h. After addition of H2O (30 mL), the mixture was extracted with
CH2Cl2 (3ꢀ10 mL). The organic layers were combined, washed
with brine, dried over MgSO4, and evaporated under vacuum. The
residue was purified on silica gel with EtOAc/petroleum ether (1/4)
3. Conclusion
In summary, we have realized an efficient synthesis of new
crosslinking amino esters with click chemistry. Bis-amino acids
containing serine, alanine, tyrosine, and lysine can then be easily
as eluent to give 70 mg (70%) of 16 as a colorless liquid. TLC: Rf¼0.5
25
(petroleum ether/EtOAc¼4/1). [
a
]
þ109.2 (c 0.6, CH2Cl2); 1H
D
NMR (400 MHz, CDCl3):
d 7.36–7.37 (m, 5H, Ph), 5.31 (d, 1H,

7938
C. Li et al. / Tetrahedron 65 (2009) 7935–7941
S
N
N
12 (2 equiv), CuSO4, Na scorbate
8
TMS
TMS
TBAF, CH2Cl2/H2O, 68%
24
16 (2 equiv), CuSO4, Na scorbate
9
24
TBAF, CH2Cl2/H2O, 73%
CbzHN
N
CO2Me
(CH2)4
S
N
N
16 (0.44 equiv), CuSO4
Na ascorbate
24
R
N
TBAF, CH2Cl2/H2O
N
25 R = TMS
26 R = H
TBAF, 86%
12 (1 equiv), CuSO4
10
26
Na ascorbate , CH2Cl2/H2O, 91%
S
R1HN
NHR2
CO2H
N
N
LiOH
(CH2)m
(CH2)n
N
N
8 - 10
N
N
HO2C
MeOH/H2O
N
N
27 m = n = 1, R1 = R2 = Boc, 95%
28 m = n = 4, R1 = R2 = Cbz, 92%
29 m = 1, n = 4, R1 = Boc, R2 = Cbz, 90%
-
+
S
-
NH3+CF3CO2
O2CF3C H3N
TFA, CH2Cl2
100%
N
N
8
N
N
N
N
MeO2C
CO2Me
N
N
30
H2N
NH2
1. 33 % HBr
AcOH
S
(CH2)4
(CH2)4
N
N
9
MeO2C
CO2Me
2. NaHCO3
68%
N
N
N
N
N
N
31
Scheme 4. Synthesis of benzothiadiazole-based bis-amino acid derivatives.
J¼5.1 Hz, NH), 5.10 (s, 2H, CH2), 4.37–4.42 (m, 1H, CH), 3.72 (s, 3H,
3.93 (dd, 1H, J¼3.2, 9.6 Hz, CH), 3.79 (s, 3H, MeO), 3.75 (s, 3H,
MeO), 3.73 (dd, 1H, J¼3.2, 9.6 Hz, CH), 1.42 (s, 18H, 2ꢀt-Bu). 13C
OMe), 3.26 (t, 2H, J¼6.6 Hz, CH2N3), 1.39–1.99 (m, 6H, 3ꢀCH2). 13
C
NMR (100 MHz, CDCl3):
d
172.8, 155.9, 136.2 (Cq); 128.7, 128.3,
NMR (100 MHz, CDCl3): d 171.1, 169.5, 155.5, 155.2, 144.7 (Cq);
128.2 (Ph); 67.2 (PhCH2), 53.7 (CH), 52.6 (OMe), 51.2 (CH2N3), 32.4,
28.5, 22.5 (CH2). HRESIMS: calcd for C15H20N4NaO4: 343.1382;
found: m/z 343.1377.
123.8 (CH), 80.8 (Cq), 70.4, 64.8 (CH2), 53.9, 53.7 (MeO), 53.2, 52.5
(CH); 50.9 (CH2), 28.3 (CH3). HRESIMS: calcd for C21H35N5NaO9:
524.2333; found: m/z 524.2327.
4.4. General procedure for click reaction
4.4.2. 2(S)-tert-Butoxycarbonylamino-3-[4-(2(S)-tert-butoxy-
carbonylamino-2-methoxycarbonyl-ethoxymethyl)-1,2,3-triazol-1-
yl]-propionic acid benzyl ester 2. From 12 (80 mg, 0.33 mmol) and
15 (110 mg, 0.33 mmol), column chromatography (petroleum
To a solution of alkyne (1 mmol) and azide (1 mmol) in CH2Cl2
(10 mL) and water (10 mL) were added CuSO4$5H2O (0.01–
0.2 mmol) and sodium ascorbate (0.02–0.2 mmol) successively. The
reaction mixture was vigorously stirred at rt for 12–20 h. CH2Cl2
(30 mL) was then added. After separation of the organic layer, the
aqueous layer was extracted with CH2Cl2 (3ꢀ20 mL). The combined
organic layers were washed with brine (40 mL), dried over MgSO4,
evaporated and purified by column chromatography.
ether/EtOAc,1/1) afforded 2 as a colorless liquid (134 mg, 70%). TLC:
25
Rf¼0.2 (petroleum ether/EtOAc¼1/1). [
a
]
D
þ16.6 (c 0.5, CH2Cl2); 1H
NMR (400 MHz, CDCl3):
d
7.34 (s, 1H, CH), 7.33–7.34 (m, 5H, Ph),
5.38 (m, 2H, 2ꢀNH), 5.25 (d, 1H, J¼12.4 Hz, CHPh), 5.11 (d, 1H,
J¼12.4 Hz, CHPh), 4.78–4.81 (m, 1H, CH), 4.69–4.73 (m, 2H), 4.63 (d,
1H, J¼12.4 Hz, CH), 4.55 (d, 1H, J¼12.4 Hz, CH), 4.48 (td, 1H, J¼3.7,
9.2 Hz, CH), 3.92 (dd, 1H, J¼3.2, 9.2 Hz, CH), 3.76 (s, 3H, MeO), 3.73
(dd, 1H, J¼3.2, 9.6 Hz, CH), 1.44 (s, 18H, 2ꢀt-Bu). 13C NMR (100 MHz,
4.4.1. 2(S)-tert-Butoxycarbonylamino-3-[4-(2(S)-tert-butoxy-
carbonylamino-2-methoxycarbonyl-ethoxymethyl)-1,2,3-triazol-1-
yl]-propionic acid methyl ester 1. From 11 (84 mg, 0.33 mmol) and
12 (80 mg, 0.33 mmol), column chromatography (petroleum
CDCl3):
d 170.5, 169.5, 155.5, 155.2, 144.5, 135.5 (Cq); 128.6, 128.3,
128.1 (Ph), 123.8 (CH), 80.8 (Cq), 70.5, 67.2, 64.8 (CH2), 54.1 (MeO),
53.7, 53.1 (CH); 50.9 (CH2), 28.3 (CH3). HRESIMS: calcd for
C27H39N5NaO9: 600.2646; found: m/z 600.2640.
ether/EtOAc, 1/1,) afforded 1 as a colorless liquid (90 mg, 55%). TLC:
25
Rf¼0.2 (petroleum ether/EtOAc¼1/1). [
a
]
þ34.6 (c 0.5, CH2Cl2);
D
1H NMR (400 MHz, CDCl3):
d
7.47 (s, 1H, CH), 5.40 (d, 1H, J¼6.4 Hz,
4.4.3. 3-[4-(2(S)-Benzyloxycarbonyl-2-tert-butoxycarbonylamino-
ethoxymethyl)-1,2,3-triazol-1-yl]-2(S)-tert-butoxycarbonylamino-
propionic acid methyl ester 3. From 11 (187 mg, 0.583 mmol) and 13
(150 mg, 0.583 mmol), column chromatography (petroleum ether/
NH), 5.36 (d, 1H, J¼8.7 Hz, NH), 4.85 (dd, 1H, J¼5.0, 14.2 Hz, CH),
4.78 (dd, 1H, J¼4.1, 14.2 Hz, CH), 4.71–4.76 (m, 1H, CH), 4.65 (d, 1H,
J¼12.4 Hz, CH), 4.61 (d, 1H, J¼12.4 Hz, CH), 4.42–4.45 (m, 1H, CH),

C. Li et al. / Tetrahedron 65 (2009) 7935–7941
7939
EtOAc, 1/1) afforded 3 as a colorless liquid (280 mg, 86%). TLC:
129.0,124.8,124.7 (CH); 117.8,115.2 (Cq); 113.8,105.7,101.5 (CH), 80.7
(Cq), 62.4, 58.2 (CH2), 53.8, 53.5 (CH); 53.2 (OMe), 51.1, 51.0 (CH2),
28.3 (CH3). HRESIMS: calcd for C44H49N8O13: 897.3419; found: m/z
897.3414. Calcd for C44H48N8NaO13: 919.3239; found: m/z 919.3233.
25
Rf¼0.3 (petroleum ether/EtOAc¼1/1). [
a
]
þ10.8 (c 0.3, CH2Cl2); 1H
D
NMR (400 MHz, CDCl3):
d
7.33–7.35 (m, 5H, Ph), 7.25 (s, 1H, CH),
5.42 (d, 1H, J¼7.3 Hz, NH), 5.35 (d, 1H, J¼8.7 Hz, NH), 5.20 (d, 1H,
J¼12.4 Hz, CHPh), 5.15 (d, 1H, J¼12.4 Hz, CHPh), 4.82 (dd, 1H, J¼4.6,
14.2 Hz, CH), 4.76 (dd, 1H, J¼4.1, 14.2 Hz, CH), 4.66–4.71 (m, 1H),
4.58 (d, 1H, J¼12.4 Hz, CH), 4.52 (d, 1H, J¼12.4 Hz, CH), 4.41 (td, 1H,
J¼3.7, 8.7 Hz, CH), 3.88 (dd, 1H, J¼3.2, 9.6 Hz, CH), 3.71 (s, 3H, OMe),
3.70 (dd, 1H, J¼3.2, 9.2 Hz, CH), 1.41 (s, 18H, 2ꢀt-Bu). 13C NMR
4.4.7. 2-{6-[1-(5(S)-Benzyloxycarbonylamino-5-methoxycarbonyl-
pentyl)-1H-1,2,3-triazol-4-ylmethoxy]-3-oxo-3H-xanthen-9-yl}-
benzoic acid 1-(5(S)-benzyloxycarbonylamino-5-methoxycarbonyl-
pentyl)-1H-1,2,3-triazol-4-ylmethyl ester 7. From 22 (30 mg,
0.073 mmol) and 16 (45 mg, 0.145 mmol), compound 7 was
(100 MHz, CDCl3):
d 171.1, 168.9, 155.5, 155.2, 144.5, 134.8 (Cq);
128.9, 128.8 (Ph), 123.8 (CH), 80.8 (Cq), 70.4, 68.2, 64.7 (CH2), 54.0,
53.9 (CH); 52.6 (OMe), 50.9 (CH2), 28.3 (CH3). HRESIMS: calcd for
C27H39N5NaO9: 600.2645; found: m/z 600.2640.
obtained as a brown solid (69 mg, 92%). TLC: Rf¼0.2 (petroleum
25
ether/EtOAc¼1/2). [
a
]
ꢁ2.4 (c 0.5, CH2Cl2); 1H NMR (400 MHz,
D
CDCl3):
d 8.22–8.25 (m, 1H, H-Ar), 7.62–7.71 (m, 3H, H-Ar), 7.28–
7.35 (m, 11H, H-Ar), 7.11 (s, 1H, CH), 7.03 (s, 1H, CH), 6.85 (dd, 1H,
J¼2.8, 9.2 Hz, H-Ar), 6.76–6.80 (m, 2H, H-Ar), 6.49 (d, 1H, J¼9.6 Hz,
H-Ar), 6.39 (s, 1H, CH), 5.76 (d, 0.5H, J¼8.2 Hz, NH), 5.73 (d, 0.5H,
J¼8.2 Hz, NH), 5.41 and 5.43 (2d, 1H, J¼7.8 Hz, NH), 5.27 (s, 2H,
CH2), 5.01–5.12 (m, 6H, 3ꢀCH2), 4.18–4.33 (m, 6H, 2ꢀCH, 2ꢀCH2),
3.76 (s, 6H, 2ꢀOMe), 1.66–1.99 (m, 12H, 6ꢀCH2). 13C NMR
4.4.4. 2(S)-tert-Butoxycarbonylamino-3-{4-[4-(2(S)-tert-butoxy-
carbonylamino-2-methoxycarbonyl-ethyl)-phenoxymethyl]-1,2,3-
triazol-1-yl}-propionic acid methyl ester 4. From 12 (22 mg,
0.09 mmol) and 14 (30 mg, 0.09 mmol), column chromatography
(petroleum ether/EtOAc, 1/1) afforded 4 as a white crystal (45 mg,
25
86%). TLC: Rf¼0.4 (petroleum ether/EtOAc¼1/1). [
a
]
þ54.6 (c 0.5,
(100 MHz, CDCl3): d 172.8, 172.6, 165.3, 162.7, 156.1, 156.0, 154.1,
D
CH2Cl2); 1H NMR (400 MHz, CDCl3):
d
7.55 (s, 1H, CH), 7.01 (d, 2H,
150.1, 136.3, 136.2, 134.2 (Cq); 132.9, 131.5, 130.4 (CH); 130.2 (Cq),
129.9, 129.1, 128.6, 128.5, 128.3, 128.1, 123.3 (CH); 117.6, 115.1 (Cq);
113.9, 101.5 (CH); 67.1, 62.5, 58.5 (CH2); 53.6, 53.5 (CH); 52.6
(OMe), 50.2, 50.0 (CH2); 32.1, 31.8, 29.7, 22.5, 22.4, 22.2 (CH2).
J¼8.7 Hz, Tyr), 6.88 (d, 2H, J¼8.7 Hz, Tyr), 5.36 (d, 1H, J¼7.3 Hz,
NH), 5.16 (s, 2H, CH2), 4.94 (d, 1H, J¼7.8 Hz, NH), 4.80 (dd, 1H,
J¼4.6, 14.2 Hz, CH), 4.78 (dd, 1H, J¼4.6, 13.8 Hz, CH), 4.68–4.71 (m,
1H, CH), 4.50–4.55 (m, 1H, CH), 3.75 (s, 3H, OMe), 3.70 (s, 3H,
OMe), 3.02 (dd, 1H, J¼5.5, 14.2 Hz, CH), 2.99 (dd, 1H, J¼6.0, 13.8 Hz,
CH), 1.42 (s, 9H, t-Bu), 1.40 (s, 9H, t-Bu). 13C NMR (100 MHz, CDCl3):
HRESIMS: calcd for C56H57N8O13
1049.4046.
: 1049.4045; found: m/z
d
172.4, 169.5, 157.3, 155.1 (Cq); 130.4 (CH), 128.7 (Cq), 129.3 (CH),
4.4.8. 2(S)-tert-Butoxycarbonylamino-3-(4-{7-[1-(2(S)-tert-butoxy-
carbonylamino-2-methoxycarbonyl-ethyl)-1H-1,2,3-triazol-4-yl]-
2,1,3-benzothiadiazol-4-yl}-1,2,3-triazol-1-yl)-propionic acid methyl
ester 8. Compound 8 was prepared from 24 (11 mg, 0.032 mmol)
and 12 (17 mg, 0.064 mmol) according to the general procedure, in
the presence of nBu4NF$3H2O (40 mg, 0.128 mmol). After the
completion of the reaction, the crude mixture was diluted in
a mixture of 10 mL CH2Cl2/0.1 N EDTA solution (1/1) to remove the
copper complexes. Column chromatography (petroleum ether/
114.9 (CH), 80.8 (Cq), 62.1 (CH2), 54.6, 53.8 (CH); 53.2, 52.3 (OMe),
51.0, 37.5 (CH2), 28.4 (CH3). HRESIMS: calcd for C27H39N5NaO9:
600.2646; found: m/z 600.2647.
4.4.5. 3-(1-{10-[4-(2(S)-Benzyloxycarbonyl-2-tert-butoxy-
carbonylamino-ethoxymethyl)-1,2,3-triazol-1-ylmethyl]-anthracen-
9-ylmethyl}-1H-1,2,3-triazol-4-ylmethoxy)-2(S)-tert-butoxy-
carbonylamino-propionic acid benzyl ester 5. From diazido an-
thracene 21 (20 mg, 0.07 mmol) and 15 (47 mg, 0.14 mmol),
column chromatography (petroleum ether/EtOAc, 1/1) afforded 5
EtOAc, 1/1) afforded 8 as a yellow solid (15 mg, 68%). TLC: Rf¼0.2
25
(petroleum ether/EtOAc¼1/2). [
a
]
þ45.6 (c 0.5, CH2Cl2); 1H NMR
D
as a colorless liquid (40 mg, 60%). TLC: Rf¼0.1 (petroleum ether/
(400 MHz, CDCl3):
d
8.70 (s, 2H, 2ꢀCH), 8.58 (s, 2H, 2ꢀCH), 5.51 (d,
25
EtOAc¼1/2). [
a
]
ꢁ4.2 (c 0.5, CH2Cl2); 1H NMR (400 MHz, CDCl3):
2H, J¼7.3 Hz, 2ꢀNH), 4.98 (d, 4H, J¼3.6 Hz, 2ꢀCH2), 4.79 (m, 2H,
D
d
8.41 (dd, 4H, J¼3.2, 6.9 Hz, H-Ar), 7.66 (dd, 4H, J¼3.2, 6.9 Hz, H-
2ꢀCH), 3.83 (s, 6H, 2ꢀOMe), 1.46 (s, 18H, 2ꢀt-Bu). 13C NMR
Ar), 7.20–7.25 (m, 10H, Ph), 7.09 (s, 2H, 2ꢀCH), 6.52 (s, 4H, 2ꢀCH2),
5.28 (d, 2H, J¼8.7 Hz, 2ꢀNH), 5.02 (d, 2H, J¼12.4 Hz, 2ꢀCHPh), 4.95
(d, 2H, J¼12.4 Hz, 2ꢀCHPh), 4.36–4.45 (m, 6H, 2ꢀCH, 2ꢀCH2), 3.84
(dd, 2H, J¼3.2, 9.2 Hz, 2ꢀCH), 3.65 (dd, 2H, J¼3.2, 9.2 Hz, 2ꢀCH),
(100 MHz, CDCl3): d 169.1, 155.0, 142.9 (Cq); 125.1, 124.6 (CH);
122.4, 80.9 (Cq); 53.8 (CH), 53.2 (OMe), 51.1 (CH2), 28.3 (CH3).
HRESIMS: calcd for C28H36N10NaO8S: 695.2336; found: m/z
695.2331.
1.39 (s, 18H, 2ꢀt-Bu). 13C NMR (100 MHz, CDCl3):
d 170.4, 155.4,
144.7, 135.4, 130.7 (Cq); 128.5, 128.3, 127.7, 127.1, 124.2 (CH), 122.2
(CH), 80.1 (Cq), 70.5, 67.0, 64.7 (CH2); 54.1 (CH), 46.6 (CH2), 28.4
(CH3). HRESIMS: calcd for C52H58N8NaO10: 977.4174; found: m/z
977.4168.
4.4.9. 2(S)-Benzyloxycarbonylamino-6-(4-{7-[1-(5(S)-benzyloxy-
carbonylamino-5-methoxycarbonyl-pentyl)-1H-1,2,3-triazol-4-yl]-
2,1,3-benzothiadiazol-4-yl}-1,2,3-triazol-1-yl)-hexanoic acid methyl
ester 9. Compound 9 was prepared from 24 (33 mg, 0.1 mmol)
and 16 (60 mg, 0.2 mmol) in the presence of nBu4NF$3H2O
(567 mg, 1.8 mmol), followed by treatment as for compound 8.
Column chromatography (petroleum ether/EtOAc, 1/3) afforded 9
4.4.6. 2-{6-[1-(2(S)-tert-Butoxycarbonylamino-2-methoxycarbonyl-
ethyl)-1H-1,2,3-triazol-4-ylmethoxy]-3-oxo-3H-xanthen-9-yl}-benzoic
acid 1-(2(S)-tert-butoxycarbonylamino-2-methoxycarbonyl-ethyl)-1H-
1,2,3-triazol-4-ylmethyl ester 6. From 22 (41 mg, 0.1 mmol) and 12
(49 mg, 0.2 mmol), column chromatography (petroleum ether/
as a yellow solid (60 mg, 73%). TLC: Rf¼0.2 (petroleum ether/
25
EtOAc¼1/2). [
a
]
þ4.0 (c 0.3, CH2Cl2); 1H NMR (400 MHz, CDCl3):
D
d
8.68 (s, 2H, 2ꢀCH), 8.60 (s, 2H, 2ꢀCH), 7.26–7.32 (m, 10H, Ph),
EtOAc, 1/3) afforded 6 as a brown solid (74 mg, 82%). TLC: Rf¼0.3
5.37 (d, 2H, J¼8.2 Hz, 2ꢀNH), 5.04 (s, 4H, 2ꢀCH2), 4.43 (t, 4H,
J¼6.9 Hz, 2ꢀN–CH2), 4.35 (m, 2H, 2ꢀCH), 3.70 (s, 6H, 2ꢀOMe),
þ2.8 (c 0.5, CH2Cl2); 1H NMR
25
(petroleum ether/EtOAc¼1/2). [
a]
D
(400 MHz, CDCl3):
d
8.21 (d, 1H, J¼7.3 Hz, H-Ar), 7.62–7.71 (m, 3H, H-
1.38–2.08 (m, 12H, 6ꢀCH2). 13C NMR (100 MHz, CDCl3):
d 172.7,
Ar), 7.27 (d, 1H, J¼6.8 Hz, H-Ar), 7.18 (s, 1H, CH), 7.04 (d, 1H, J¼1.8 Hz,
H-Ar), 6.73–6.83 (m, 3H, H-Ar), 6.47 (d, 1H, J¼9.6 Hz, H-Ar), 6.38 (s,
1H, CH), 5.47–5.56 (m, 2H, 2ꢀNH), 5.29 (m, 2H, CH2), 5.03–5.12 (m,
2H, CH2), 4.84 (m, 2H, CH2), 4.67–4.76 (m, 6H, 2ꢀCH, 2ꢀCH2), 3.76 (s,
6H, 2ꢀOMe), 1.40 (s, 9H, t-Bu), 1.38 (s, 9H, t-Bu). 13C NMR (100 MHz,
155.9,152.3, 143.2, 136.2 (Cq); 128.6,128.3, 128.1, 126.1,123.8 (CH);
122.7 (Cq), 67.1 (CH2), 53.6 (CH), 52.6 (OMe), 50.1, 32.1, 29.8, 22.3
(CH2). HRESIMS: calcd for C40H44N10NaO8S: 847.2962; found: m/z
847.2957.
CDCl3):
d
185.6, 169.6, 169.5, 165.2, 162.6, 162.5, 158.8, 155.2, 154.1,
4.4.10. 6-[4-(7-Ethynyl-2,1,3-benzothiadiazol-4-yl)-1,2,3-triazol-
1-yl]-2(S)-benzyloxycarbonylamino-hexanoic acid benzyl ester
149.9, 142.8, 141.5, 141.4, 134.2 (Cq); 132.9, 131.4, 130.5, 130.3, 129.8,

7940
C. Li et al. / Tetrahedron 65 (2009) 7935–7941
26. Click reaction of 24 (53 mg, 0.16 mmol) with 16 (22 mg,
0.07 mmol) in the presence of nBu4NF$3H2O (202 mg,
0.64 mmol), followed by treatment as for compound 8 afforded
a mixture of 25 and 26, which was further treated with
(Cq); 130.3 (CH), 124.5 (CH), 114.1 (CH), 79.2, 78.7 (Cq), 61.1 (CH2),
57.1, 56.0 (CH), 52.1, 37.5 (CH2), 27.4 (CH3).
4.5.3. 2(S)-tert-Butoxycarbonylamino-3-(4-{7-[1-(2(S)-tert-butoxy-
carbonylamino-2-carboxy-ethyl)-1H-1,2,3-triazol-4-yl]-2,1,3-benzo-
thiadiazol-4-yl}-1,2,3-triazol-1-yl)-propionic acid 27. Saponification
t-Bu4NF$3H2O (200 mg in CH2Cl2) to give 26 (30 mg, 86%) as a red
25
oil. TLC: Rf¼0.3 (petroleum ether/EtOAc¼1/2). [
a
]
þ2.4 (c 0.2,
D
CH2Cl2); 1H NMR (400 MHz, CDCl3):
d
8.73 (s, 1H, CH), 8.51 (d, 1H,
of 8 (44 mg, mmol) afforded 27 as a yellow solid (39 mg, 95%). TLC:
25
J¼7.4 Hz, CH), 7.90 (d, 1H, J¼7.4 Hz, CH), 7.30–7.34 (m, 5H, Ph),
5.30 (d, 1H, J¼8.5 Hz, NH), 5.06 (s, 2H, CH2), 4.47 (t, 2H, J¼6.8 Hz,
N–CH2), 4.30–4.38 (m, 1H, CH), 3.71 (s, 3H, OMe), 3.62 (s, 1H,
CH^), 1.42–2.05 (m, 6H, 3ꢀCH2). 13C NMR (100 MHz, CDCl3):
Rf¼0.3 (CH2Cl2/MeOH¼3/1). [
a]
þ29.4 (c 0.5, MeOH); 1H NMR
D
(400 MHz, CD3OD):
d
8.99 (s, 2H, 2ꢀCH), 8.50 (s, 2H, 2ꢀCH), 5.06
(m, 2H, 2ꢀCH), 4.75 (m, 2H, 2ꢀCH), 4.52 (m, 2H, 2ꢀCH),1.32 (s,18H,
2ꢀt-Bu). 13C NMR (100 MHz, CD3OD):
d 172.6, 156.1, 151.9, 142.4
d
172.6, 155.9, 155.3, 151.4, 142.8, 136.2 (Cq); 134.5, 128.6, 128.3,
(Cq); 125.6, 125.0 (CH); 122.5, 79.3 (Cq); 55.3 (CH), 51.7 (CH2), 27.3
(CH3).
128.1 (CH); 125.1 (CH), 124.5 (Cq), 124.3 (CH), 114.7 (Cq); 84.0
(CH^), 67.2 (CH2), 53.5, 52.6 (CH); 50.2 (CH2), 32.2, 29.8, 22.2
(CH2). HRESIMS: calcd for C25H24N6NaO4S: 527.1477; found: m/z
527.1472.
4.5.4. 2(S)-Benzyloxycarbonylamino-6-(4-{7-[1-(5(S)-benzyloxy-
carbonylamino-5-carboxy-pentyl)-1H-1,2,3-triazol-4-yl]-2,1,3-ben-
zothiadiazol-4-yl}-1,2,3-triazol-1-yl)-hexanoic acid 28. Saponi-
4.4.11. 6-(4-{7-[1-(2(S)-tert-Butoxycarbonylamino-2-methoxy-
carbonyl-ethyl)-1H-1,2,3-triazol-4-yl]-2,1,3-benzothiadiazol-4-
yl}-1,2,3-triazol-1-yl)-2(S)-benzyloxycarbonylamino-hexanoic
acid benzyl ester 10. From 26 (15 mg, 0.03 mmol) and 12 (8 mg,
0.03 mmol), column chromatography (petroleum ether/EtOAc, 1/
fication of 9 (50 mg, 0.06 mmol) afforded 28 as a yellow solid
25
(44 mg, 92%). TLC: Rf¼0.3 (CH2Cl2/MeOH¼3/1). [
a
]
þ12.0 (c 0.3,
D
DMSO); 1H NMR (400 MHz, DMSO-d6):
d
8.93 (s, 2H, 2ꢀCH), 8.48 (s,
2H, 2ꢀCH), 7.18–7.28 (m, 10H, Ph), 6.74 (d, 2H, J¼6.4 Hz, 2ꢀNH),
4.86 (m, 4H, 2ꢀCH2), 4.43 (t, 4H, J¼6.7 Hz, 2ꢀN–CH2), 3.67 (m, 2H,
2ꢀCH), 1.25–1.85 (m, 12H, 6ꢀCH2). 13C NMR (100 MHz, DMSO-d6):
3) afforded compound 10 as a yellow solid (20 mg, 91%). TLC:
25
Rf¼0.2 (petroleum ether/EtOAc¼1/2). [
a
]
þ22.4 (c 0.5, CH2Cl2);
d 174.0, 156.0, 152.0, 142.2, 137.7 (Cq); 128.8, 128.2, 128.0, 125.8,
D
1H NMR (400 MHz, CDCl3):
d
8.69 (s, 2H, 2ꢀCH), 8.59 (s, 2H,
125.1 (CH); 122.8 (Cq), 65.6 (CH2), 55.4 (CH), 50.2, 32.0, 30.3, 22.8
(CH2).
2ꢀCH), 7.26–7.32 (m, 5H, Ph), 5.50 (d, 1H, J¼6.9 Hz, NH), 5.35 (d,
1H, J¼8.2 Hz, NH), 5.05 (s, 2H, CH2), 4.95 (m, 2H, CH2), 4.77 (m,
1H, CH), 4.44 (t, 2H, J¼6.9 Hz, CH2), 4.36 (m, 1H, CH), 3.82 (s, 3H,
OMe), 3.70 (s, 3H, OMe), 1.38–2.06 (m, 6H, 3ꢀCH2), 1.43 (s, 9H,
4.5.5. 6-(4-{7-[1-(2(S)-tert-Butoxycarbonylamino-2-carboxy-
ethyl)-1H-1,2,3-triazol-4-yl]-2,1,3-benzothiadiazol-4-yl}-1,2,3-tri-
azol-1-yl)-2(S)-benzyloxycarbonylamino-hexanoic acid 29. Saponi-
t-Bu). 13C NMR (100 MHz, CDCl3):
d 172.7, 169.6, 155.9, 155.2,
152.2, 143.2, 136.2 (Cq); 128.6, 128.3, 128.1, 126.2, 126.0, 125.2,
123.9 (CH); 122.8, 122.3, 80.8 (Cq); 67.1 (CH2), 53.9, 53.5 (CH);
53.2, 52.6 (OMe); 50.9, 50.1, 32.1, 29.8 (CH2), 28.3 (CH3), 22.3
(CH2). HRESIMS: calcd for C34H40N10NaO8S: 771.2649; found:
m/z 771.2644.
fication of 10 (12 mg, 0.02 mmol) afforded 29 as a yellow solid
25
(10 mg, 90%). TLC: Rf¼0.3 (CH2Cl2/MeOH¼3/1). [
a
]
þ10.1 (c 0.7,
D
MeOH); 1H NMR (400 MHz, CD3OD):
d
8.94 (s, H, CH), 8.91 (s, H,
CH), 8.41 (s, 2H, 2ꢀCH), 7.15–7.27 (m, 5H, Ph), 5.03 (dd, 1H, J¼3.6,
13.7 Hz, CH), 4.94 (s, 2H, CH2), 4.72 (dd, 1H, J¼6.9, 13.8 Hz, CH),
4.50 (t, 2H, J¼6.9 Hz, CH2), 4.41 (m, 1H, CH), 4.01 (m, 1H, CH), 1.41–
2.06 (m, 6H, 3ꢀCH2), 1.30 (s, 9H, t-Bu). 13C NMR (100 MHz, CD3OD):
4.5. General procedure for the saponification
d 178.0, 173.5, 156.8, 156.6, 152.0, 142.6, 136.9 (Cq); 128.0, 127.5,
127.2, 125.5, 125.2, 125.1, 125.0, 124.8 (CH); 122.6, 122.3, 79.1 (Cq);
66.0 (CH2), 56.1, 56.0 (CH); 52.3, 50.1, 32.2, 29.7 (CH2), 27.4 (CH3),
22.3 (CH2).
To a solution of methyl ester in MeOH (1 mL) and water (0.3 mL)
were added LiOH (1.2 equiv/ester). The reaction mixture was stirred
at rt for 12–24 h, then acidified with resine Hþ, filtered, evaporated
directly to get the free acid without purification.
4.6. General procedure for the hydrogenolysis of benzyl ester
4.5.1. 2(S)-tert-Butoxycarbonylamino-3-[4-(2(S)-tert-butoxy-
carbonylamino-2-carboxy-ethoxymethyl)-1,2,3-triazol-1-yl]-pro-
pionic acid 17. Saponification of 1 (28 mg, 0.06 mmol) afforded 17
Benzyl ester (0.55 mmol) in MeOH (15 mL) was hydrogenated
over 10% Pd–C (5 wt %) at rt for 12 h. After filtration, the filtrate was
evaporated to get the pure product.
as a white solid (26 mg, 96%). TLC: Rf¼0.1 (CH2Cl2/MeOH¼3/1).
25
[
a
]
þ8.3 (c 1.4, MeOH); 1H NMR (400 MHz, CD3OD):
d
7.85 (s, 1H,
4.6.1. 2(S)-tert-Butoxycarbonylamino-3-[4-(2(S)-tert-butoxy-
carbonylamino-2-methoxycarbonyl-ethoxymethyl)-1,2,3-triazol-1-
yl]-propionic acid 18. Hydrogenolysis of 2 (320 mg, 0.55 mmol)
D
CH), 4.84 (dd, 1H, J¼4.2, 14.2 Hz, CH), 4.60 (dd, 1H, J¼7.4, 12.8 Hz,
CH), 4.57 (s, 2H, CH2), 4.40 (s, 1H, CH), 4.38–4.42 (m, 1H, CH), 3.70
(m, 2H, CH2), 1.41 (s, 9H, t-Bu), 1.37 (s, 9H, t-Bu). 13C NMR
afforded 18 as a colorless oil (266 mg, 92%). TLC: Rf¼0.1 (CH2Cl2/
25
(100 MHz, CD3OD):
d
173.5, 172.9, 156.5, 156.0, 144.2 (Cq); 124.4
MeOH¼3/1). [
a
]
ꢁ9.2 (c 0.5, MeOH); 1H NMR (400 MHz, CD3OD):
D
(CH), 79.2, 79.2 (Cq), 70.3, 63.8 (CH2), 55.6, 54.6 (CH); 51.6 (CH2),
27.4 (CH3).
d
7.89 (s, 1H, CH), 4.80 (dd, 1H, J¼4.2, 13.8 Hz, CH), 4.66 (m, 1H, CH),
4.62 (m, 1H, CH), 4.58 (s, 2H, CH2), 4.16 (s, 1H, CH), 3.71 (s, 3H,
MeO), 3.70 (m, 2H, CH2), 1.40 (s, 9H, t-Bu), 1.37 (s, 9H, t-Bu). 13C
4.5.2. 2(S)-tert-Butoxycarbonylamino-3-{4-[4-(2(S)-tert-butoxy-
carbonylamino-2-carboxy-ethyl)-phenoxymethyl]-1,2,3-triazol-1-yl}-
propionic acid 20. Saponification of 4 (21 mg, 0.04 mmol) afforded
NMR (100 MHz, CD3OD): d 170.0, 156.4, 156.1, 144.5 (Cq); 124.6
(CH), 79.8, 79.0 (Cq), 70.9, 63.8 (CH2), 53.8 (CH); 51.9 (MeO), 50.1
(CH2), 27.4, 27.3 (CH3).
20 as a white solid (18 mg, 90%). TLC: Rf¼0.1 (CH2Cl2/MeOH¼3/1).
25
[
a]
þ25.3 (c 1.2, MeOH); 1H NMR (400 MHz, CD3OD):
d
7.92 (s, 1H,
4.6.2. 3-[4-(2(S)-tert-Butoxycarbonylamino-ethoxymethyl)-2-carb-
oxy-1,2,3-triazol-1-yl]-2(S)-tert-butoxycarbonylamino-propionic acid
methyl ester 19. Hydrogenolysis of 3 (180 mg, 0.31 mmol) afforded
D
CH), 7.12 (d, 2H, J¼8.2 Hz, Tyr), 6.86 (d, 2H, J¼8.2 Hz, Tyr), 5.07 (s,
2H, CH2), 4.87 (dd, 1H, J¼6.4, 13.7 Hz, CH), 4.58 (dd, 1H, J¼6.4,
13.7 Hz, CH), 4.33 (m, 1H, CH), 4.13 (m, 1H, CH), 3.05 (dd, 1H, J¼4.6,
13.8 Hz, CH), 2.80 (dd, 1H, J¼7.3, 13.3 Hz, CH), 1.37 (s, 9H, t-Bu), 1.36
19 as a colorless oil (130 mg, 86%). TLC: Rf¼0.1 (CH2Cl2/MeOH¼3/1).
25
[a
]
ꢁ6.9 (c 0.9, MeOH); 1H NMR (400 MHz, CD3OD):
d 7.88 (s, 1H,
D
(s, 9H, t-Bu). 13C NMR (100 MHz, CD3OD):
d
157.1, 156.1, 143.6, 130.8
CH), 4.84 (d, 1H, J¼4.1 Hz, CH), 4.65 (dd, 1H, J¼7.8, 13.8 Hz, CH), 4.50

C. Li et al. / Tetrahedron 65 (2009) 7935–7941
7941
(s, 3H, CH and CH2), 4.29 (t, 1H, J¼3.1 Hz, CH), 3.80 (dd, 1H, J¼4.6,
9.6 Hz, CH), 3.68 (s, 4H, CH and OMe),1.42 (s, 9H, t-Bu),1.31 (s, 9H, t-
solid NaHCO3, extracted with CH2Cl2 (3ꢀ10 mL). The combined
organic layers were washed with brine and dried over anhydrous
MgSO4, then evaporated. Purification by column chromatography
Bu). 13C NMR (100 MHz, CD3OD):
d 171.2, 156.6, 156.5, 144.0 (Cq);
124.5 (CH), 79.6, 79.5 (Cq); 69.5, 63.8 (CH2), 54.0 (CH); 51.6 (OMe),
50.8 (CH2), 27.3 (CH3).
(CH2Cl2/MeOH¼15/1) afforded yellow oil (10 mg, 68%). TLC: Rf¼0.1
25
(CH2Cl2/MeOH¼15/1). [
a]
ꢁ8.0 (c 0.1, DMSO); 1H NMR (400 MHz,
D
CDCl3):
d
8.73 (s, 2H, 2ꢀCH), 8.66 (s, 2H, 2ꢀCH), 4.48 (t, 4H,
4.7. 2-{6-[1-(2(S)-Amino-2-methoxycarbonyl-ethyl)-1H-1,2,3-
triazol-4-ylmethoxy]-3-oxo-3H-xanthen-9-yl}-benzoic acid 1-
(2(S)-amino-2-methoxycarbonyl-ethyl)-1H-1,2,3-triazol-4-
ylmethyl ester, compound with trifluoroacetic acid 23
J¼7.1 Hz, 2ꢀN–CH2), 3.70 (s, 6H, 2ꢀOMe), 3.43 (m, 2H, 2ꢀCH),1.46–
2.08 (m, 12H, 6ꢀCH2). 13C NMR (100 MHz, CDCl3):
d 176.4, 152.4,
143.2 (Cq); 126.2, 123.9 (CH); 122.7 (Cq), 54.2 (CH), 52.2 (OMe),
50.3, 34.2, 30.2, 22.8 (CH2). HRESIMS: calcd for C18H21N10O4S:
473.1468; found: m/z 473.1463.
To a solution of 6 (12 mg, 0.013 mmol) in CH2Cl2 (10 mL) was
added CF3CO2H (0.1 mL, 1.4 mmol). The reaction mixture was stir-
Acknowledgements
red at rt for 12 h, then evaporated to get red oil (12 mg, 100%). TLC:
25
Rf¼0.2 (CH2Cl2/MeOH¼3/1). [
a
]
þ5.0 (c 0.8, MeOH); 1H NMR
C.L. thanks the China Scholarship Council for a Doctorate
scholarship.
D
(400 MHz, CD3OD):
d
8.32 (dd, 1H, J¼1.4, 9.2 Hz, H-Ar), 8.29 (s, 1H,
H-Ar), 7.80–7.91 (m, 3H, H-Ar), 7.68 (s, 1H, CH), 7.45 (dd, 1H, J¼1.4,
7.8 Hz, H-Ar), 7.30–7.34 (m, 2H, H-Ar), 7.16–7.19 (m, 1H, H-Ar), 7.07
(s, 1H, CH), 6.96–6.99 (m, 1H, H-Ar), 5.53 (s, 2H, CH2), 5.00–5.08 (m,
4H, 2ꢀCH2), 4.91–4.97 (m, 3H, CH and CH2), 4.68–4.77 (m, 1H, CH),
References and notes
1. Taylor, C. M.; Wang, W. Tetrahedron 2007, 63, 9033–9047.
2. (a) James, I. T.; Walne, A. J.; Perrett, D. Ann. Clin. Biochem. 1996, 33, 397–400;
(b) Christenson, R. H. Clin. Biochem. 1997, 30, 573–593.
3.80 (s, 6H, 2ꢀOMe). 13C NMR (100 MHz, CD3OD):
d 180.0, 168.1,
161.4, 158.5, 143.8, 143.3, 134.5, 134.4 (Cq); 133.1, 132.4, 131.1, 130.9,
130.8, 130.2, 126.3, 125.9 (CH); 118.7, 118.6 (Cq); 117.7, 104.3, 102.5
(CH), 63.7, 58.8 (CH2), 54.3 (OMe), 53.6, 53.5 (CH); 48.6, 48.5 (CH2).
3. (a) Allevi, P.; Longo, A.; Anastasia, M. Chem. Commun. 1999, 559–560;
(b) Adamczyk, M.; Johnson, D. D.; Reddy, R. E. Tetrahedron: Asymmetry 2000, 11,
2289–2298; (c) Allevi, P.; Cribiu` , R.; Giannini, E.; Anastasia, M. Bioconjugate
Chem. 2005, 16, 1045–1048; (d) Allevi, P.; Femia, E. A.; Rota, P.; Costa, M. L.;
Anastasia, M. Tetrahedron: Asymmetry 2008, 19, 2470–2478.
4. (a) Patchornik, A.; Sokolovsky, M. J. J. Am. Chem. Soc. 1964, 86, 1860–1861; (b)
Tohdo, K.; Hamada, Y.; Shiori, T. Pept. Chem. 1992, 7–12; (c) Tohdo, K.; Hamada,
Y.; Shioiri, T. Synlett 1994, 247–249.
5. (a) David, O.; Maisonneuve, S.; Xie, J. Tetrahedron Lett. 2007, 48, 6527–6530;
(b) Maisonneuve, S.; Fang, J.; Xie, J. Tetrahedron 2007, 64, 8716–8720; (c) Lin,
L.; Shen, Q.; Chen, Q.-R.; Xie, J. Bioorg. Med. Chem. 2008, 16, 9757–9763;
(d) Cheng, K. G.; Liu, J.; Liu, X. F.; Li, H. L.; Sun, H. B.; Xie, J. Carbohydr. Res.
2009, 344, 841–850.
4.8. Preparation of 2(S)-amino-3-(4-{7-[1-(2(S)-amino-2-
methoxycarbonyl-ethyl)-1H-1,2,3-triazol-4-yl]-2,1,3-
benzothiadiazol-4-yl}-1,2,3-triazol-1-yl)-propionic acid
methyl ester, compound with trifluoroacetic acid 30
To a solution of 8 (26 mg, 0.04 mmol) in CH2Cl2 (10 mL) was
added CF3CO2H (0.1 mL, 1.4 mmol). The reaction mixture was stir-
6. (a) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40, 2004–
2021; (b) Tornoe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057–
3069.
red at rt for 12 h, then evaporated to get red oil (26 mg, 100%). TLC:
25
Rf¼0.4 (CH2Cl2/MeOH¼3/1). [
a
]
þ25.8 (c 0.5, MeOH); 1H NMR
D
7. Gonçalves, M. S. T. Chem. Rev. 2009, 109, 190–212.
(400 MHz, CD3OD):
d
9.05 (s, 2H, 2ꢀCH), 8.48 (s, 2H, 2ꢀCH), 5.10–
8. Wei, Z.-L.; Petukhov, P. A.; Bizik, F.; Teixeira, J. C.; Mercola, M.; Volpe, E. A.;
Glazer, R. I.; Willson, T. M.; Kozikowski, A. P. J. Am. Chem. Soc. 2004, 126, 16714–
16715.
9. Otsuka, M.; Kittaka, A.; Iimori, T.; Yamashita, H.; Kobayashi, S.; Ohno, M. Chem.
Pharm. Bull. 1985, 33, 509–514.
5.22 (m, 4H, 2ꢀCH2), 4.83 (m, 2H, 2ꢀCH), 3.91 (s, 6H, 2ꢀOMe). 13
C
NMR (100 MHz, CD3OD):
d 168.4, 153.3, 144.4 (Cq); 127.3, 126.8
(CH); 123.7 (Cq); 54.3 (OMe), 53.9 (CH2), 50.2 (CH). HRESIMS: calcd
10. Gajewski, M.; Seaver, B.; Esslinger, C. S. Bioorg. Med. Chem. Lett. 2007, 17, 4163–
4166.
for C24H33N10O4S: 557.2407; found: m/z 557.2402.
11. Bew, S. P.; Brimage, R. A.; L’Hermite, N.; Sharma, S. V. Org. Lett. 2007, 9, 3713–
3716.
4.9. Preparation of 2(S)-amino-6-(4-{7-[1-(5(S)-amino-5-
methoxycarbonyl-pentyl)-1H-1,2,3-triazol-4-yl]-2,1,3-
benzothiadiazol-4-yl}-1,2,3-triazol-1-yl)-hexanoic acid methyl
ester 31
12. Goddard-Borger, E. D.; Stick, R. V. Org. Lett. 2007, 9, 3797–3800.
13. Varazo, K.; Xie, F.; Gulledge, D.; Wang, Q. Tetrahedron Lett. 2008, 49, 5293–5296.
14. DaSilveira Neto, B. A.; Sant’Ana Lopes, A.; Ebeling, G.; Gonçalves, R. S.; Costa,
V. E. U.; Ouina, F. H.; Dupont, J. Tetrahedron 2005, 61, 10975–10982.
15. (a) Raimundo, J.-M.; Blanchard, P.; Brisset, H.; Akoudad, S.; Roncali, J. Chem.
Commun. 2000, 939–940; (b) Huang, J.; Li, C.; Xia, Y.-J.; Zhu, X.-H.; Peng, J.; Cao,
Y. J. Org. Chem. 2007, 72, 8580–8583.
To precooled (ice bath) AcOH (0.5 mL) was added 9 (22 mg,
0.03 mmol) and 33% HBr/AcOH (0.2 mL) successively, the solution
was stirred for 12 h, then poured into 10 mL water, neutralized with
16. Chen, L.; Hu, T.-S.; Zhu, J.; Wu, H.; Yao, Z.-J. Synlett 2006, 1225–1229.
17. Callan, J. F.; de Silva, A. P.; Magri, D. C. Tetrahedron 2005, 61, 8551–8588.