Chemoenzymatic synthesis of triazole-linked glycopeptides

Article abstract of DOI:10.1055/s-2006-942509

Triazole-linked glycopeptides are prepared by C-terminal elongation of glycoamino acids with proteinogenic amino acids following a chemical or enzymatic coupling protocol. Two orthogonal routes for a chemoenzymatic strategy were explored, involving a click-reaction before amide bond formation or in reverse order. It was found that enzymatic peptide coupling under the influence of alcalase proceeds cleanly and in high yields, while the resulting dipeptides can be efficiently clicked to acetylene- or azide-containing sugars. Georg Thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-2006-942509

3146
SPECIAL TOPIC
Chemoenzymatic Synthesis of Triazole-Linked Glycopeptides
Synthesis of
T
t
riazole
a
-Linked Gly
n
copeptides Groothuys,a Brian H. M. Kuijpers,a Peter J. L. M. Quaedflieg,b Harlof C. P. F. Roelen,b Roel W. Wiertz,b
Richard H. Blaauw,c Floris L. van Delft,a Floris P. J. T. Rutjes*a
a
Institute for Molecules and Materials, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands
Fax +31(24)3653393; E-mail: F.Rutjes@science.ru.nl
DSM Research, Life Sciences-Advanced Synthesis, Catalysis and Development, P.O. Box 18, 6160 MD Geleen, The Netherlands
Chiralix B.V., Toernooiveld 100, 6525 EC Nijmegen, The Netherlands
b
c
Received 29 March 2006
a number of specific advantages over chemical peptide
synthesis: (a) enzymatic peptide coupling reactions are
free from racemization, (b) side chain protection can
mostly be omitted, (c) the coupling reagent (enzyme) can
Abstract: Triazole-linked glycopeptides are prepared by C-termi-
nal elongation of glycoamino acids with proteinogenic amino acids
following a chemical or enzymatic coupling protocol. Two orthog-
onal routes for a chemoenzymatic strategy were explored, involving
a click-reaction before amide bond formation or in reverse order. It be recycled efficiently, (d) the process is environmentally
was found that enzymatic peptide coupling under the influence of
alcalase proceeds cleanly and in high yields, while the resulting
dipeptides can be efficiently clicked to acetylene- or azide-contain-
synthesis is posed by incorporation of proline and/or non-
ing sugars.
friendly, and (e) the reaction proceeds under mild condi-
tions. The greatest challenge in chemoenzymatic peptide
proteinogenic amino acids such as D-, b- and unnatural
amino acids.
Key words: glycopeptides, azides, alkynes, click chemistry, enzy-
matic coupling
We wish to report here that under the influence of alcalase
as the biocatalyst, N-protected acetylenic and azidoamino
acid methyl esters act as suitable acyl donors for conden-
sation with a proteinogenic amino acid amide. Alcalase-
catalyzed condensation was also applied to our triazole-
Since the development of the Cu(I)-catalyzed variant of
the Huisgen [3+2] cycloaddition,1 there has been a tre-
mendous explosion of interest in the preparation of
linked glycoamino acids and compared with a traditional
[1,2,3]-triazoles from acetylenes and organic azides.2,3
chemical peptide synthesis protocol. The inverse strategy,
The mild conditions for cycloaddition (inter alia aqueous
solvents), the generally high yields and the wide function-
i.e. enzymatic coupling before click-reaction was found to
be optimal, giving the desired triazole-linked glycodipep-
al group compatibility have led to broad application of the
tides in high yield and purity.
so-called ‘click-reaction’ to organic substrates including
In a typical enzymatic peptide synthesis protocol,9 an N-
(unprotected) biopolymers such as proteins4 and carbohy-
drates.5,6 Of particular interest in this respect is the poten-
tial of a triazole to function as an amide isostere, a
presumption that was put forward at an early occasion and
that appears to be corroborated by several reports in recent
literature.7 As part of a program to obtain chemically and
metabolically stable isosteres of N-glycopeptides, we
have also applied Cu(I)-catalyzed Huisgen addition as re-
cently described in the straightforward assembly of an ar-
ray of triazolyl glycoamino acids from a wide range of
carbohydrate and amino acid building blocks.5 In particu-
lar, it was rewarding to find that the combination of azi-
dosugars with acetylenic amino acids proceeded equally
well as the inverse situation employing acetylenic sugars
and azidoamino acids.5,8
,
protected amino acid (bearing a free carboxy group) is
condensed with a C-protected amino acid. Since the reac-
tion is equilibrium-controlled, the thermodynamically
more stable starting materials (or hydrolysis products) are
usually favored unless the desired condensation product
precipitates from the solution. An alternative kinetically
controlled approach involves C-activation of the N-pro-
tected amino acid. This is potentially more favorable since
the yield is now predominantly determined by enzymatic
properties such as substrate specificity and the ratio of
synthesis versus hydrolysis.9 The most suitable enzymes
for enzymatic peptide synthesis include the majority of
the commercially available serine, metallo-, and aspartate
endo- and exoproteases.11 Our synthetic efforts have fo-
cused on the condensation of a neutral and bulky N-pro-
tected unnatural amino acid [e.g. azidoornitine methyl
ester (1a) and propargylglycine methyl ester (1b),
Table 1] with a proteinogenic C-protected amino acid. To
attain such a goal, subtilisine or carboxypeptidase Y
seemed suitable candidates.10 Based on the work of Chen
et al.,11 we focused our attention on the cheap and indus-
trially available alcalase, a proteolytic enzyme mixture
produced by Bacillus licheniformis (containing subtilisine
Carlsberg as the major enzyme component), with high ac-
tivity and stability in alcoholic solvents.
We have now extended our research into triazole-linked
glycopeptides through elongation with proteinogenic ami-
no acids via both conventional solution-phase chemical
peptide synthesis and chemoenzymatic procedures.
Chemoenzymatic peptide synthesis has received increas-
ing interest over the last decade,9,10 and is characterized by
SYNTHESIS 2006, No. 18, pp 3146–3152
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x.
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x
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0
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Advanced online publication: 25.07.2006
DOI: 10.1055/s-2006-942509; Art ID: C03706SS
© Georg Thieme Verlag Stuttgart · New York

SPECIAL TOPIC
Synthesis of Triazole-Linked Glycopeptides
3147
Table 1 Chemical and Enzymatic Synthesis of Dipeptides
chemical
coupling
(EDCI)a
or
enzymatic
coupling
(alcalase)b
R1
R1
O
H
Cbz
OMe
Cbz
N
N
H
N
NH2
H
R2
O
O
1a R1
=
(CH2)3N3
2a–c (R2 = Bn)
3a–c (R2 = H)
b R1 =
CH2C CH
(CH2)3
N
OBn
c R1
=
BnO
OBn
OBn
O
N
N
Entry
Substrate
Chemical yield
(%)a
Enzymatic yield
(%)b
Dipeptide
1
2
3
4
5
6
1a
1b
1c
1a
1b
1c
Phe-NH2
Phe-NH2
Phe-NH2
Gly-NH2
Gly-NH2
Gly-NH2
69
55
86
80
60
82
77
79
40c
93
80
10
2a
2b
2c
3a
3b
3c
a Reaction conditions: 1. NaOH, THF; 2. Phe-NH2 or Gly-NH2, EDCI, HOAt, DIPEA, DMF.
b Reaction conditions: Phe-NH2 or Gly-NH2, alcalase, t-BuOH, DMF.
c Conversion of acyl donor as determined by HPLC.
In order to determine whether 1a–c are suitable substrates the dipeptides 2a and 3a in hand, we first determined
for alcalase, first hydrolysis was investigated in a 1:1 whether the C-terminal amide functionality would be dis-
mixture of tert-butyl alcohol and 1 M aqueous KHCO3. It turbing in a Cu(I)-catalyzed [3+2] cycloaddition. To this
was observed that both 1a and 1b were completely hydro- end, a range of conditions was applied to a model system
lyzed after five minutes, but the hydrolysis of the triazole- involving a mixture of 2a and phenylacetylene (Table 2).
linked glycoamino acid methyl ester 1c was much slower Unfortunately, the reaction with standard Cu(OAc)2/Na-
(complete conversion only after 24 h with a five-fold ex- ascorbate combination in a mixture of tert-butyl alcohol
cess of alcalase). Possibly, the bulky glycosidic side- and water, conditions that served our goal so well earlier,
chain of 1c is not well accommodated in the active site of failed in this case (Table 2, entry 1). The poor solubility of
the enzyme.
the substrates in t-BuOH–H2O led us to switch to a THF–
H2O mixture, but also in this case no reaction took place
(Table 2, entry 3). Presumably in these cases complex-
ation of copper(I), added in substoichiometric amount, to
the free amide functionality occurred, thereby catalyzing
oxidation to Cu(II), as suggested by an observed color
change from yellow to blue. Unfortunately, also with an
excess of a copper(I) species, in the presence of a variety
of tertiary amines in acetonitrile or THF, resulted in only
minute formation of the desired product (Table 2, entries
5, 7 and 9). A more successful strategy was found by ad-
dition of tris(benzyltriazolylmethyl)amine (TBTA), a
ligand reported by Fokin et al.12 to protect copper from ox-
idation and disproportionation, while enhancing its cata-
lytic activity. Indeed, addition of TBTA to any of the
previously applied conditions led to a dramatic improve-
ment in yield as well as a significant decrease in reaction
time. In particular, the use of a CuI/TBTA combination in
the presence of Et3N in acetonitrile resulted in a conver-
sion of more than 90% of 2a into the desired triazolylpep-
tide 4 (Table 2, entry 8).
In the next step, alcalase-promoted condensation of 1a
and 1b as the acyl donor with phenylalanine amide as ac-
ceptor was explored under anhydrous conditions
(Table 1) and we were delighted to find that dipeptides 2a
and 2b were formed in high yields (Table 1, entries 1 and
2). Similarly, dipeptides 3a and 3b were formed equally
successfully by alcalase-mediated coupling of 1a and 1b
to glycine amide (Table 1, entries 4 and 5) respectively. It
was found that not only the isolated yields compared fa-
vorably to chemical coupling with EDCI, but the resulting
dipeptides were formed with exclusive diastereoselectivi-
ty, i.e. with no detectable racemization. Not unexpectedly,
under similar conditions the condensation of phenylala-
nine or glycine amide with our glycotriazolylamino acid
methyl ester 1c proceeded extremely slow (40% conver-
sion of 1c after 8 days) and did not lead to a satisfactory
yield of the desired dipeptides.
Based on this observation, we were stimulated to investi-
gate whether the inverse mode of actions, i.e. initial pep-
tide coupling followed by Huisgen condensation with the
carbohydrate moiety, would be more successful. Having
Synthesis 2006, No. 18, 3146–3152 © Thieme Stuttgart · New York

3148
S. Groothuys et al.
SPECIAL TOPIC
Table 2 Optimization of the [3+2] Cycloaddition of Dipeptide 2a with Phenylacetylene
N
N
Ph
N3
N
Ph
O
O
H
N
H
N
[Cu]I, 35 °C
24 h
HN
Cbz
HN
Cbz
NH2
NH2
O
O
Ph
Ph
2a
4
Entry
1
Cu(I) source
Base
–
Ligand
Solvent
Conversiona (%)
0
Cu(OAc)2
–
t-BuOH–H2O (1:1)
Na-ascorbateb
2
3
4
5
6
Cu(OAc)2
–
TBTAc
–
t-BuOH–H2O (1:1)
THF–H2O (1:1)
THF–H2O (1:1)
MeCN
~50
0
Na-ascorbateb
Cu(OAc)2
–
Na-ascorbateb
Cu(OAc)2
–
TBTAc
–
~40
~30
~70
Na-ascorbateb
CuId
CuId
DIPEAd
2,6-lutidined
DIPEAd
TBTAd
MeCN
2,6-lutidined
7
8
CuId
Et3Nd
Et3Nd
–
–
MeCN
MeCN
THF
0
>90
0
CuId
TBTAd
PMDETAe
TBTAe
9
CuBre
CuBre
10
–
THF
~70
a Conversion determined by TLC and LC-MS based on consumption of 2a.
b 0.2 equiv of Cu(OAc)2 and 0.4 equiv of Na-ascorbate.
c 0.2 equiv.
d 2 equiv.
e 1 equiv.
Having established suitable conditions for the cycloaddi- in excellent yields upon Huisgen condensation of N-Cbz-
tion of dipeptide 2a with phenylacetylene, the optimiza- protected azidopropyl-containing dipeptides 2a and 3a
tion of the catalyst loading was investigated by with acetylenic C-glycosides 5 and 7,13b irrespective of the
condensation of 2a with glucose-derived acetylene 513a carbohydrate O-protective groups (acetyl in 5 and benzyl
(Table 3). It was found that reduction of the amount of in 7). Deprotection of the acetyl protective groups of 6 us-
catalyst from two equivalent to one gave a 100% conver- ing K2CO3/MeOH, proceeded smoothly to give the unpro-
sion within 16 hours (Table 3, entry 1). With 50 mol% of tected triazole-linked glycodipeptide 9 (Table 4).
copper, complete conversion was also attained within 24
hours (Table 3, entry 2) which was also the case with 20%
copper (Table 3, entry 3). Further reduction of catalyst to
Finally, the inverse mode of addition, i.e. condensation of
azidosugar 1013c with the acetylenic dipeptides 2b or 3b
was also investigated. Much to our satisfaction, the de-
10 mol% failed to give complete conversion even after 48
sired glycodipeptides were formed uneventfully
hours (Table 3, entry 4), whereas under the action of 1
(Scheme 1) and in excellent yield.
mol% catalyst no conversion was observed at all (Table 3,
In conclusion, a chemoenzymatic coupling strategy was
entry 5).
successfully applied to the preparation of triazole-linked
glycodipeptides. In particular, alcalase-induced conden-
Since the reduction of the copper(I) catalyst loading be-
low 20 mol% was found to be unsatisfactory for complete
sation of an acetylenic or azidoamino acid methyl ester
conversion to the product, all subsequent couplings were
with an amino acid amide, followed by Cu(I)-catalyzed
performed under identical conditions involving 20 mol%
conversion to a triazole-linked glycopeptide proceeded
CuI, TBTA and Et3N in acetonitrile. As summarized in
with high conversion and good yield. Although the in-
verse approach, i.e. applying triazole-linked glycoamino
Table 4, glycodipeptides 2c, 3c, 6, and 8 were all obtained
Synthesis 2006, No. 18, 3146–3152 © Thieme Stuttgart · New York

SPECIAL TOPIC
Synthesis of Triazole-Linked Glycopeptides
3149
AcO
Table 3 Optimization of Catalyst Loading
N
O
N
AcO
AcO
AcO
N
N
O
N
N
AcO
AcO
AcO
O
OAc
AcO
2b
or
3b
H
O
Cbz
N
+
OAc
N3
AcO
N
H
NH2
CuI, TBTA, Et3N
MeCN, 40 °C
AcO
R2
OAc
O
O
O
10
11 R2 = H (99%)
12 R2 = Bn (92%)
H
AcO
2a
+
Cbz
N
AcO
N
NH2
CuI, TBTA, Et3N
MeCN, 40 °C
H
OAc
O
Scheme 1 Click-reaction of dipeptides 2b and 3b with glucosyl
azide 10.
Ph
5
6
Entry
Loadinga
100 mol%
50 mol%
20 mol%
10 mol%
1 mol%
Concentrationb Time
Conversionc
100%
100%
100%
50%
1
2
3
4
5
1.1 mM
1.8 mM
12 mM
3.7 mM
5 mM
16 h
24 h
24 h
48 h
48 h
1H NMR spectra were recorded on a Varian Inova 400 (400 MHz)
spectrometer and 13C NMR spectra on a Bruker DMX300 (75 MHz)
or Bruker DPX200 (50 MHz) spectrometer. Chemical shifts (d) are
given in ppm, downfield from tetramethylsilane. IR spectra were re-
corded on an ATI Mattson, Genesis series, or a Bruker Tensor 27
FTIR spectrometer. High-resolution mass spectra were recorded on
a JEOL AccuTOF (ESI), or a MAT900 (EI, CI, and ESI). All reac-
tions were performed under N2 unless otherwise stated.
0%
a Loading of CuI, TBTA, and Et3N.
b Concentration of substrates.
Before use in the coupling reactions, L-Phe-NH2 was dried by co-
evaporation with DMF (2 × 5 mL per mmol) and Gly-NH2·HCl was
first neutralized by treatment with an excess of t-BuNH2, subse-
quently isolated as solid (Gly-NH2) by repeated co-evaporation of
the volatiles with EtOH and further dried by co-evaporation with
DMF (2 × 5 mL per mmol). t-BuOH was dried by distillation under
reduced pressure and kept under N2. Alcalase was obtained from
Novo Industrial (Denmark) as a brown aqueous liquid. For the hy-
drolysis experiments this enzyme solution was used as such. For the
(anhyd) peptide coupling reactions the water in the alcalase solu-
tion was removed using the method of Chen et al.11 As a matter of
fact, the aqueous alcalase (0.5 mL) and absolute EtOH (1.0 mL)
were mixed in an Eppendorf cup and the resulting suspension was
agitated on a Vortex mixer for 5 min and centrifuged (3000 rpm) for
10 min to spin down the enzyme. The supernatant was decanted and
the enzyme resuspended in absolute EtOH (1.0 mL), agitated for 5
min on a Vortex mixer and spun down by centrifugation. This pro-
cedure was repeated once with absolute EtOH (1.0 mL) and finally
once with anhyd t-BuOH (1.0 mL). The resulting enzyme was re-
suspended in t-BuOH (1.0 mL) for use in the enzymatic peptide
coupling reactions. All other commercially available reagents were
used as received. HPLC analyses were performed with an Inertsil
RP-18 column (25 × 0.46 cm) (RP-18, 5 mm). TfN3 was prepared as
described by Wong et al.14
c Conversion determined by HPLC.
Table 4 Click Reaction of Dipeptides 2a and 3a with Glucosyl
Acetylenes 5 and 7 and Deprotection of 6
RO
N
O
N
N
RO
RO
OR
RO
RO
O
2a
or
3a
O
H
Cbz
N
+
RO
N
NH2
20 mol% CuI,
TBTA, Et3N
MeCN, 40 °C, 16 h
H
OR
R2
O
5 R = Ac
or
2c, 3c, 8
6
9 (R = H)
K2CO3,
MeOH
(95%)
7 R = Bn
Entry
Dipeptide
2aa
Carbohydrate
5 (R = Ac)
7 (R = Bn)
5 (R = Ac)
7 (R = Bn)
Product
Yield (%)
1
2
3
4
6
88
87
62c
96
Chemical Peptide Coupling; General Procedure
2aa
2c
8
To a solution of 1 (2.5 mmol) in THF (12.5 mL) at 0 °C, was added
aq 0.25 N NaOH solution (20.0 mL, 5.0 mmol). After 1 h at this
temperature, the mixture was warmed to r.t. and stirring was contin-
ued. The extent of conversion was monitored by TLC analysis in
EtOAc–heptane (1:1). When the methyl ester of 1 was completely
hydrolyzed, the mixture was acidified with 1.0 N aq HCl until pH
2–3. The mixture was extracted with EtOAc (3 × 25 mL). The or-
ganic extracts were combined, dried (Na2SO4) and concentrated in
vacuo, giving a yellow oil, corresponding to the free carboxylic acid
(2.5 mmol). Part (1.0 mmol) of this amino acid was dried by co-
evaporation with DMF (2 × 5 mL) and dissolved in DMF (2.5 mL).
To the clear cooled (0–5 °C) solution, was added DIPEA (0.35 mL,
2.1 mmol), followed by EDCI (211 mg, 1.1 mmol) and HOAt (1-
hydroxy-7-azabenzotriazole; 150 mg, 1.1 mmol) and the mixture
was stirred for at least 0.5 h at 0–5 °C. Subsequently, a solution of
Phe-NH2 or Gly-NH2 (1.1 mmol) in DMF (2.5 mL) was added and
the mixture was stirred for 1 h at 0–5 °C. The mixture was warmed
to r.t. and stirred overnight. TLC analysis (EtOAc) showed com-
3ab
3ab
3c
a 2a: R2 = Bn.
b 3a: R2 = H.
c Conversion ca. 70%.
acid donors was less successful, we are currently explor-
ing alternative methods for this approach, for example, the
use of no or less bulky carbohydrate protective groups, by
using a more activated ester, or by the identification of a
more active biocatalyst.
Synthesis 2006, No. 18, 3146–3152 © Thieme Stuttgart · New York

3150
S. Groothuys et al.
SPECIAL TOPIC
plete conversion of the carboxylic acid starting material. The mix-
ture was partitioned between EtOAc (75 mL) and H2O (15 mL) to
which a few drops of 1 N aq HCl were added. The aqueous layer
was extracted with EtOAc (40 mL) and the combined organic phas-
es were washed with 0.5 N aq HCl (40 mL), 1 M aq KHCO3 (40 mL)
and brine (40 mL), and subsequently dried (Na2SO4) and concen-
trated in vacuo.
trated in vacuo. The product was suspended in dioxane (1 mL) and
2 N aq NaOH (0.35 mL) was added slowly at 0 °C. To the resulting
solution was added NaHCO3 (80.2 mg, 0.720 mmol) and subse-
quently Cbz-OSu (181 mg, 0.720 mmol) and the mixture was stirred
for 18 h at r.t. The mixture was taken up in H2O (10 mL) and EtOAc
(10 mL) and the layers were separated. The aqueous phase was ex-
tracted with EtOAc (2 × 10 mL) and the combined organic layers
were washed with aq sat. NaHCO3 (15 mL), aq sat. NH4Cl and
brine, dried (Na2SO4) and concentrated in vacuo. Purification by
column chromatography (heptane–EtOAc, 3:1) yielded 1b (162 mg,
88%) as a colorless oil, which solidified upon storage at –20 °C; Rf
0.60 (1:1 EtOAc–heptane).
Enzymatic Peptide Coupling; General Procedure
The N-Cbz-protected amino acid methyl ester 1 (1.0 mmol in the
case of 1a and 1b, 0.5 mmol in the case of 1c) was dried by co-evap-
oration with DMF (2 × 5 mL) and dissolved in t-BuOH (4.0 mL).
After adding a solution of Phe-NH2 or Gly-NH2 (1.5 mmol) in DMF
(2.0 mL), the mixture was stirred at 35 °C. Subsequently, the alca-
lase suspension in t-BuOH (1 mL) was added and the mixture was
stirred at 35 °C. Samples were taken at regular time intervals and
analyzed by HPLC. After almost complete conversion of 1, the mix-
ture was concentrated in vacuo to remove most of the volatiles. The
residue was taken up in EtOAc (50 mL) and H2O (20 mL), to which
a few drops of 1 N aq HCl were added. The aqueous layer was ex-
tracted with EtOAc (30 mL) and the combined organic phases were
washed with 1 M aq KHCO3 (40 mL), 1 N aq HCl (40 mL) and brine
(40 mL), dried (Na2SO4) and concentrated in vacuo. Analytically
pure samples were obtained by recrystallization.
IR (film): 3369, 3295, 3032, 2958, 1714, 1511 cm–1.
1H NMR (400 MHz, CDCl3): d = 7.35–7.28 (m, 5 H), 5.58 (br d, 1
H), 5.11 (s, 2 H), 4.55–4.50 (m, 1 H), 3.77 (s, 3 H), 2.80–2.70 (m, 2
H), 2.01 (t, J = 2.6 Hz, 1 H).
13C NMR (50 MHz, CDCl3): d = 170.6, 155.4, 135.9, 128.3, 128.0,
127.9, 78.1, 71.7, 66.9, 52.6, 52.2, 22.5.
HRMS (CI): m/z calcd for C14H16NO4 (M + H): 262.1079; found:
262.1092.
Cbz-L-Orn{4-(2,3,4,6-tetra-O-benzyl-b-D-glucosyl)[1,2,3]tri-
azol-1-yl}-OMe (1c)
To a solution of Bn4Glc-acetylene13b (7.50 g, 13.7 mmol) and Cbz-
L-Orn(N3)-OMe (4.20 g, 13.7 mmol) in t-BuOH (100 mL) was add-
ed a mixture of Cu(OAc)2 (0.550 g, 2.75 mmol) and sodium ascor-
bate (1.1 g, 5.5 mmol) in H2O (100 mL), and the reaction was stirred
overnight at r.t.. After the addition of H2O (150 mL), the mixture
was extracted with CH2Cl2 (2 × 150 mL). The combined organic
layers were washed with H2O (100 mL), dried (Na2SO4) and con-
centrated in vacuo. Purification by gradient flash chromatography
(EtOAc–heptane) yielded the clicked product 1c (10.2 g, 87%) as a
white solid; Rf 0.22 (1:1 EtOAc–heptane).
Optimized Click-Reaction; General Procedure
To a solution of the dipeptide (25 mmol) and the carbohydrate (25
mmol)13 in MeCN (0.5 mL) were added solutions of Et3N in MeCN
(10 mM, 0.50 mL, 0.20 equiv) and TBTA in MeCN (10 mM, 0.50
mL, 0.20 equiv). N2 was bubbled through the solution for 5 min. A
solution of CuI in MeCN (10 mM, 0.50 mL, 0.20 equiv) was added
and the mixture was stirred at 40 °C for 16 h. Brine (2 mL) was add-
ed and the mixture was extracted with EtOAc (2 × 4 mL). The com-
bined organic layers were dried (MgSO4) and concentrated in
vacuo. The product was purified by gradient flash chromatography
(MeOH–CH2Cl2).
IR (film): 3321, 3028, 2950, 2863, 2249, 1951, 1722, 1515 cm–1.
1H NMR (400 MHz, CDCl3): d = 7.41 (s, 1 H), 7.35–6.98 (m, 25 H),
5.30 (br d, J = 8.0 Hz, 1 H), 5.13–5.07 (m, 2 H), 4.94 (d, J = 10.9
Hz, 1 H), 4.90 (d, J = 10.9 Hz, 1 H), 4.85 (d, J = 10.5 Hz, 1 H),
4.64–4.49 (m, 5 H), 4.44–4.37 (m, 1 H), 4.35–4.27 (m, 3 H), 3.92 (t,
J = 9.3 Hz, 1 H), 3.81 (t, J = 8.9 Hz, 1 H), 3.74–3.60 (m, 4 H), 3.69
(s, 3 H), 1.92–1.62 (m, 4 H).
13C NMR (50 MHz, CDCl3): d = 172.0, 155.7, 145.4, 138.3, 137.8,
137.7, 135.9, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.7,
127.6, 127.5, 127.4, 122.6, 86.6, 81.5, 79.1, 77.9, 75.3, 74.8, 74.5,
73.8, 73.1, 68.9, 66.8, 52.9, 52.3, 49.2, 29.2, 25.9.
Cbz-L-Orn(N3)-OMe (1a)
To a solution Cbz-L-Orn-OH (15 g, 56 mmol), K2CO3 (16.5 g, 84.5
mmol), and CuSO4·5H2O (143 mg, 0.570 mmol) in H2O (160 mL)
and MeOH (320 mL), was added freshly prepared TfN3 in CH2Cl2
( 0.41 M, 270 mL) and the reaction was stirred overnight at r.t. The
mixture was concentrated in vacuo, acidified to pH 2 with 2 M aq
HCl, and extracted with EtOAc (2 × 200 mL). The combined organ-
ic layers were dried (MgSO4) and concentrated in vacuo to yield
crude Cbz-Orn(N3)-OH. The crude intermediate was subsequently
dissolved in MeOH (150 mL) and SOCl2 (4.1 mL, 56 mmol) was
added. After 4 h of reflux, and evaporation of the MeOH in vacuo,
the residue was taken up in EtOAc (300 mL), and the EtOAc layer
was washed with H2O (2 × 150 mL) and brine (100 mL), dried
(MgSO4) and concentrated in vacuo to yield 1a (15.6 g, 91%) as a
colorless oil; Rf 0.53 (1:1 EtOAc–heptane).
HRMS (ESI): m/z calcd for C50H54N4O9 + Na (M + Na): 877.3788;
found: 877.3805.
Cbz-L-Orn(N3)-L-Phe-NH2 (2a)
Chemical coupling; yield: 302 mg (69%). Enzymatic coupling;
yield: 339 mg (77%); Rf 0.29 (2:1 EtOAc–heptane).
IR (film): 3304, 2470, 2098, 1679, 1640 cm–1.
1H NMR (400 MHz, CDCl3/MeOD): d = 7.38–7.17 (m, 10 H), 5.10
(d, J = 12.3 Hz, 1 H), 5.06 (d, J = 12.3 Hz, 1 H), 4.63 (dd, J = 5.9,
8.4 Hz, 1 H), 4.04 (dd, J = 5.7, 8.0 Hz, 1 H), 3.25–3.16 (m, 3 H),
2.93 (dd, J = 5.5, 8.6 Hz, 1 H), 1.74–1.44 (m, 4 H).
IR (film): 3317, 3070, 3032, 2963, 2872, 2090, 1365, 1528 cm–1.
1H NMR (400 MHz, CDCl3): d = 7.37–7.32 (m, 5 H), 5.32 (br d,
J = 7.4 Hz, 1 H), 5.11 (s, 2 H), 4.42–4.38 (m, 1 H), 3.76 (s, 3 H),
3.31 (br s, 2 H), 1.98–1.58 (m, 4 H).
13C NMR (50 MHz, CDCl3): d = 172.4, 155.8, 135.8, 128.2, 127.9,
127.7, 66.8, 53.3, 53.1, 52.2, 50.4, 29.3, 24.5.
HRMS (ESI): m/z calcd for C22H26N6O4 + Na (M + Na): 461.19132;
found: 461.19216.
HRMS (CI): m/z calcd for C14H19N4O4 (M + H): 307.1406; found:
307.1407.
Cbz-L-propargylglycine-L-Phe-NH2 (2b)
Chemical coupling; yield: 536 mg (55%). Enzymatic coupling;
yield: 312 mg (79%); Rf 0.25 (2:1 EtOAc–heptane).
IR (film): 3351, 2474, 2220, 2064, 1671, 1632 cm–1.
Cbz-L-Propargylglycine-OMe (1b)
To a suspension of L-propargylglycine15 (80 mg, 0.71 mmol) in
MeOH (1.5 mL) was added slowly SOCl2 (114 mL, 1.56 mmol) at
0 °C. The mixture was refluxed for 2 h, cooled to r.t. and concen-
Synthesis 2006, No. 18, 3146–3152 © Thieme Stuttgart · New York

SPECIAL TOPIC
Synthesis of Triazole-Linked Glycopeptides
3151
1H NMR (400 MHz, CDCl3/MeOD): d = 7.39–7.20 (m, 10 H), 5.10
(s, 2 H), 4.65–4.62 (m, 1 H), 4.28–4.25 (m, 1 H), 3.20–3.15 (m, 1
H), 3.00–2.95 (m, 1 H), 2.65–2.54 (m, 2 H), 2.12 (br s, 1 H).
13C NMR (50 MHz, CDCl3/MeOD): d = 173.0, 172.5, 157.1, 145.6,
138.3, 137.8, 137.7, 137.6, 136.2, 128.3, 128.3, 128.3, 128.2, 128.0,
127.9, 127.8, 127.8, 127.7, 127.7, 127.6, 127.6, 127.5, 123.6, 86.7,
81.7, 79.1, 78.0, 75.5, 75.0, 74.6, 73.8, 73.3, 68.8, 66.9, 54.5, 42.0,
28.5, 26.3.
HRMS (ESI): m/z calcd for C22H23N3O4 + Na (M + Na): 416.15863;
found: 416.15939.
HRMS (ESI): m/z calcd for C51H56N6O9 + Na (M + Na): 919.40065;
Cbz-L-Orn{4-(2,3,4,6-tetra-O-benzyl-b-D-glucosyl)[1,2,3]tri-
azol-1-yl}-L-Phe-NH2 (2c)
Chemical coupling; yield: 418 mg (86%). Click-reaction dipeptide
2c; yield: 21 mg (87%); Rf 0.20 (2:1 EtOAc–heptane).
found: 919.39922.
Cbz-L-Orn{4-(2,3,4,6-tetra-O-acetyl-b-D-glucosyl)[1,2,3]tri-
azol-1-yl}-L-Phe-NH2 (6)
Yield: 18 mg (88%); Rf 0.42 (EtOAc).
IR (KBr): 3307, 2955, 1753, 1675, 1533 cm–1.
IR (film): 3369, 3282, 2945, 2828, 1696, 1645 cm–1.
1H NMR (400 MHz, CDCl3/MeOD): d = 7.71 (s, 1 H), 7.33–6.93
(m, 30 H), 5.06 (d, J = 12.2 Hz, 1 H), 5.03 (d, J = 12.2 Hz, 1 H),
4.89 (s, 2 H), 4.83 (d, J = 10.9 Hz, 1 H), 4.62–4.48 (m, 5 H), 4.44
(d, J = 11.9 Hz, 1 H), 4.25 (t, J = 7.0 Hz, 2 H), 4.24 (d, J = 10.9 Hz),
4.03 (dd, J = 5.8, 8.0 Hz, 1 H), 3.85–3.76 (m, 2 H), 3.73–3.66 (m, 3
H), 3.62–3.58 (m, 1 H), 3.16 (dd, J = 5.8, 14.0 Hz, 1 H), 2.90 (dd,
J = 8.4, 14.0 Hz, 1 H), 1.78–1.48 (m, 4 H).
13C NMR (75 MHz, CDCl3/MeOD): d = 174.4, 172.4, 157.3, 146.1,
138.8, 138.3, 138.2, 138.1, 137.0, 136.5, 129.5, 128.8, 128.8, 128.6,
128.5, 128.4, 128.3, 128.2, 128.1, 128.1, 128.0, 127.2, 123.8, 87.2,
82.2, 79.6, 78.4, 76.0, 75.4, 75.1, 74.3, 73.8, 69.3, 67.4, 54.8, 54.3,
50.0, 37.9, 29.2, 26.3.
1H NMR (400 MHz, CDCl3/MeOD): d = 7.71 (s, 1 H), 7.36–7.16
(m, 10 H), 5.37 (t, J = 9.4 Hz, 1 H), 5.23 (t, J = 9.7 Hz, 1 H), 5.19
(t, J = 9.7 Hz, 1 H), 5.09 (d, J = 12.1 Hz, 1 H), 5.05 (d, J = 12.1 Hz,
1 H), 4.75 (d, J = 10.0 Hz, 1 H), 4.63 (dd, J = 8.1, 14.3 Hz, 1 H),
4.37–4.21 (m, 3 H), 4.14 (dd, J = 2.0, 12.5 Hz, 1 H), 4.12–4.05 (m,
1 H), 3.91 (ddd, J = 2.0, 4.8, 12.4 Hz, 1 H), 3.17 (dd, J = 6.3 14.4
Hz, 1 H), 2.94 (dd, J = 8.2, 13.8 Hz, 1 H), 2.07 (s, 3 H), 2.06 (s, 3
H), 2.02 (s, 3 H), 1.88 (s, 3 H), 1.84–1.74 (m, 2 H), 1.68–1.59 (m, 1
H), 1.54–1.43 (m, 1 H).
13C NMR (50 MHz, CDCl3/MeOD): d = 173.5, 171.5, 170.9, 170.2,
169.9, 169.7, 143.9, 136.3, 135.7, 128.9, 128.2, 128.2, 128.0, 127.7,
126.6, 122.6, 75.8, 73.6, 72.8, 71.4, 68.1, 66.9, 61.9, 54.0, 53.5,
49.5, 37.2, 25.7, 20.3, 20.2, 20.0.
HRMS (ESI): m/z calcd for C58H62N6O9 + Na (M + Na):
1009.44760; found: 1009.44631.
HRMS (ESI): m/z calcd for C38H46N6O13 + Na (M + Na):
Cbz-L-Orn(N3)-Gly-NH2 (3a)
817.30205; found: 817.29744.
Chemical coupling; yield: 277 mg (80%). Enzymatic coupling;
yield: 325 mg (93%); Rf 0.39 (EtOAc).
IR (film): 3343, 2483, 2241, 2072, 1662 cm–1.
1H NMR (400 MHz, CDCl3/MeOD): d = 7.36–7.30 (m, 5 H), 5.13
(d, 12.3 Hz, 1 H), 5.08 (d, J = 12.3 Hz, 1 H), 4.13–4.10 (m, 1 H),
3.93 (d, J = 17.1 Hz, 1 H), 3.81 (d, J = 17.1 Hz, 1 H), 3.34–3.31 (m,
2 H), 1.94–1.62 (m, 4 H).
Cbz-L-Orn{4-(2,3,4,6-tetra-O-acetyl-b-D-glucosyl)[1,2,3]tri-
azol-1-yl}-Gly-NH2 (8)
Yield: 11 mg (62%); Rf 0.11 (EtOAc).
IR (KBr): 3351, 2953, 1754, 1675, 1525 cm–1.
1H NMR (400 MHz, CDCl3/MeOD): d = 7.71 (s, 1 H), 7.33–7.28
(m, 5 H), 5.33 (t, J = 9.4 Hz, 1 H), 5.20 (t, J = 9.6 Hz, 1 H), 5.15 (t,
J = 9.6 Hz, 1 H), 5.09 (d, J = 12.2 Hz, 1 H), 5.05 (d, J = 12.3 Hz, 1
H), 4.72 (d, J = 10.0 Hz, 1 H), 4.44–4.29 (m, 1 H), 4.26 (dd,
J = 12.4, 4.8 Hz, 1 H), 4.17–4.08 (m, 1 H), 4.11 (dd, J = 12.5, 1.9
Hz, 1 H), 3.94–3.75 (m, 2 H), 3.88 (ddd, J = 9.6, 4.7, 2.0 Hz, 1 H),
2.05 (s, 3 H), 2.04 (s, 3 H), 1.99 (s, 3 H), 1.98–1.90 (m, 2 H), 1.85
(s, 3 H), 1.82–1.71 (m, 1 H), 1.63–1.51 (m, 1 H).
13C NMR (75 MHz, CDCl3/MeOD): d = 172.3, 171.9, 170.8, 170.2,
169.9, 169.7, 156.7, 144.2, 135.8, 128.2, 128.0, 127.7, 122.7, 75.9,
73.6, 72.8, 71.4, 68.2, 66.9, 66.7, 62.0, 54.1, 42.0, 30.5, 28.5, 26.0,
20.3, 20.2, 20.1.
HRMS (ESI): m/z calcd for C15H20N6O4 + Na (M + Na): 371.14437;
found: 371.14351.
Cbz-L-propagylglycine-Gly-NH2 (3b)
Chemical coupling; yield: 181 mg (60%). Enzymatic coupling;
yield: 244 mg (80%); Rf 0.39 (EtOAc).
IR (film): 3291, 3066, 3036, 2941, 2409, 1658, 1532 cm–1.
1H NMR (400 MHz, CDCl3/MeOD): d = 7.34–7.27 (m, 5 H), 5.12
(d, J = 12.3 Hz, 1 H), 5.08 (d, J = 12.3 Hz, 1 H), 4.28 (t, J = 6.5 Hz,
1 H), 3.91 (d, J = 17.1 Hz, 1 H), 3.81 (d, J = 17.1 Hz, 1 H), 2.71
(ddd, J = 2.6, 6.2, 17.0 Hz, 1 H), 2.65 (ddd, J = 2.6, 6.8, 17.0 Hz, 1
H), 2.19 (t, J = 2.6 Hz, 1 H).
HRMS (ESI): m/z calcd for C31H40N6O13 + Na (M + Na):
727.25510; found: 727.25131.
HRMS (ESI): m/z calcd for C15H17N3O4 + Na (M + Na): 326.11168;
found: 326.11042.
Cbz-L-Orn{4-(b-D-glucosyl)[1,2,3]triazol-1-yl}-L-Phe-NH2 (9)
To a solution of glucodipeptide 6 (12 mg, 15 mmol) in MeOH (2
mL) was added K2CO3 (0.29 mg, 1.5 mmol). The mixture was stirred
overnight at r.t. It was neutralized with Amberlite IR-120 (5 mg)
and stirring was continued for another 10 min. Product 9 was isolat-
ed after filtration in pure form as a white solid (8.9 mg, 95%).
1H NMR (CDCl3/MeOD, 400 MHz): d = 7.86 (s, 1 H), 7.35–7.30
(m, 5 H), 7.26–7.16 (m, 5 H), 5.10 (d, J = 12.7 Hz, 1 H), 5.06 (d,
J = 12.0 Hz, 1 H), 4.62 (dd, J = 8.6, 5.8 Hz, 1 H), 4.41 (d, J = 8.6
Hz, 2 H), 4.33 (t, J = 6.8 Hz, 2 H), 4.06 (dd, J = 7.9, 5.8 Hz, 1 H),
3.88 (d, J = 11.3 Hz, 1 H), 3.72 (dd, J = 12.1, 4.7 Hz, 1 H), 3.60–
3.43 (m, 4 H), 3.18 (dd, J = 14.0, 5.8 Hz, 1 H), 2.92 (dd, J = 14.0,
8.8 Hz, 1 H), 1.86–1.75 (m, 2 H), 1.68–1.49 (m, 2 H).
Cbz-L-Orn{4-(2,3,4,6-tetra-O-benzyl-b-D-glucosyl)[1,2,3]tri-
azol-1-yl}-Gly-NH2 (3c)
Chemical coupling; yield: 361 mg (82%). Enzymatic coupling (us-
ing the standard procedure, but with 0.5 mmol of compound 1c);
yield: 43 mg (10%). Click-reaction; yield: 22 mg (96%); Rf 0.17
(2:1 EtOAc–heptane).
IR (film): 3343, 2945, 2824, 2483, 2232, 2072 cm–1.
1H NMR (400 MHz, CDCl3/MeOD): d = 7.73 (s, 1 H), 7.33–6.95
(m, 25 H), 5.10 (d, J = 12.3 Hz, 1 H), 5.09 (d, J = 12.3 Hz, 1 H),
4.91 (s, 2 H), 4.85 (d, J = 10.9 Hz, 1 H), 4.61–4.50 (m, 4 H), 4.47
(d, J = 12.0 Hz, 1 H), 4.34 (d, J = 7.0 Hz, 2 H), 4.23 (d, J = 11.0 Hz,
1 H), 4.14 (dd, J = 5.4, 8.4 Hz, 1 H), 3.91–3.70 (m, 7 H), 3.63–3.60
(m, 1 H), 2.02–1.60 (m, 4 H).
13C NMR (CDCl3/MeOD, 75 MHz): d = 173.7, 171.7, 156.4, 136.1,
135.6, 128.4, 127.6, 127.5, 127.3, 127.0, 125.9, 122.4, 79.9, 76.5,
73.5, 72.9, 69.4, 66.1, 61.0, 53.8, 53.3, 48.9, 36.7, 27.9, 25.2.
Synthesis 2006, No. 18, 3146–3152 © Thieme Stuttgart · New York

3152
S. Groothuys et al.
SPECIAL TOPIC
HRMS (ESI): m/z calcd for C30H38N6O9 + Na (M + Na): 649.25980;
found: 649.26012.
References
(1) Huisgen, R.; Szeimies, G.; Mobius, L. Chem. Ber. 1967,
100, 2494.
(2) (a) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless,
K. B. Angew. Chem. Int. Ed. 2002, 41, 2596. (b) Tornøe, C.
W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67,
3057.
Cbz-L-Ala{1-(2,3,4,6-tetra-O-acetyl-b-D-glucosyl)[1,2,3]triazol-
4-yl}-Gly-NH2 (11)
Yield: 18 mg (99%); Rf 0.52 (EtOAc).
IR (KBr): 3292, 2948, 1753, 1687, 1535 cm–1.
1H NMR (400 MHz, CDCl3/MeOD): d = 7.93 (s, 1 H), 7.42–7.25
(m, 5 H), 5.99–5.90 (m, 1 H), 5.53 (s, 1 H), 5.50–5.45 (m, 2 H),
5.34–5.23 (m, 1 H), 5.11 (d, J = 12.4 Hz, 1 H), 5.08 (d, J = 12.4 Hz,
1 H), 4.49 (t, J = 6.2, 6.2 Hz, 1 H), 4.32 (dd, J = 12.6, 4.8 Hz, 1 H),
4.18 (dd, J = 12.6, 2.1 Hz, 1 H), 4.16–4.10 (m, 1 H), 3.92 (d,
J = 17.1 Hz, 1 H), 3.84 (d, J = 17.0 Hz, 1 H), 3.29–3.18 (m, 2 H),
2.09 (s, 1 H), 2.08 (s, 1 H), 2.04 (s, 1 H), 1.83 (s, 1 H).
13C NMR (75 MHz, CDCl3/MeOD): d = 172.1, 171.5, 170.5, 169.7,
169.3, 168.9, 156.3, 135.6, 127.9, 127.6, 127.4, 121.6, 85.0, 74.2,
72.1, 70.2, 67.3, 66.6, 61.1, 54.1, 41.8, 27.1, 19.7, 19.6, 19.1.
(3) For reviews, see: (a) Kolb, H. C.; Sharpless, K. B. Drug
Discov. Today 2003, 8, 1128. (b) Bock, V. D.; Hiemstra, H.;
van Maarseveen, J. H. Eur. J. Org. Chem. 2006, 51.
(4) (a) Dirks, A. J.; van Berkel, S. S.; Hatzakis, N. S.; Opsteen,
J. A.; van Delft, F. L.; Cornelissen, J. J. L. M.; Rowan, A. E.;
van Hest, J. C. M.; Rutjes, F. P. J. T.; Nolte, R. J. M. Chem.
Commun. 2005, 4172. (b) Link, A. J.; Vink, M. K. S.;
Tirrell, D. A. J. Am. Chem. Soc. 2004, 126, 10598.
(c) Rijkers, D. T. S.; van Esse, G. W.; Merkx, R.; Brouwer,
A. J.; Jacobs, H. J. F.; Pieters, R. J.; Liskamp, R. M. J. Chem.
Commun. 2005, 4581.
(5) (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) Kuijpers, B.
H. M.; Dijkmans, G. C. T.; Groothuys, S.; Quaedflieg, P. J.
L. M.; Blaauw, R. H.; van Delft, F. L.; Rutjes, F. P. J. T.
Synlett 2005, 3059.
HRMS (ESI): m/z calcd for C29H36N6O13 + Na (M + Na):
699.22380; found: 699.22129.
Cbz-L-Ala{1-(2,3,4,6-tetra-O-acetyl-b-D-glucosyl)[1,2,3]triazol-
4-yl}-L-Phe-NH2 (12)
Yield: 18 mg (92%); Rf 0.60 (EtOAc).
IR (KBr): 3351, 2942, 1748, 1693, 1666, 1524 cm–1.
(6) (a) Joosten, J. A. F.; Tholen, N. T. H.; El Maate, F. A.;
Brouwer, A. J.; van Esse, G. W.; Rijkers, D. T. S.; Liskamp,
R. M. J.; Pieters, R. J. Eur. J. Org. Chem. 2005, 3182.
(b) Chittaboina, S.; Xie, F.; Wang, Q. Tetrahedron Lett.
2005, 46, 2331. (c) Dörner, S.; Westermann, B. Chem.
Commun. 2005, 2852.
1H NMR (400 MHz, CDCl3/MeOD): d = 7.74 (s, 1 H), 7.41–7.16
(m, 10 H), 5.96–5.87 (m, 1 H), 5.53 (s, 1 H), 5.49–5.41 (m, 2 H),
5.06 (s, 2 H), 4.63 (dd, J = 8.1, 5.9 Hz, 1 H), 4.42 (t, J = 6.4, 6.4 Hz,
1 H), 4.31 (dd, J = 12.6, 4.9 Hz, 1 H), 4.16 (dd, J = 12.6, 2.1 Hz, 1
H), 4.11 (ddd, J = 10.2, 4.8, 2.1 Hz, 1 H), 3.20–3.15 (m, 2 H), 3.05
(dd, J = 15.16, 6.86 Hz, 1 H), 2.96 (dd, J = 13.90, 8.27 Hz, 1 H),
2.09 (s, 3 H), 2.06 (s, 3 H), 2.04 (s, 3 H), 1.82 (s, 3 H).
13C NMR (75 MHz, CDCl3/MeOD): d = 173.6, 170.7, 170.5, 169.8,
169.4, 168.9, 156.1, 143.1, 136.2, 135.6, 128.6, 128.5, 128.0, 127.7,
127.4, 126.3, 121.4, 85.0, 74.3, 72.2, 70.1, 67.3, 66.6, 61.2, 53.9,
53.6, 36.8, 27.2, 19.8, 19.7, 19.2.
(7) Horne, W. S.; Stout, C. D.; Ghadiri, M. R. J. Am. Chem. Soc.
2003, 125, 9372.
(8) Dondoni, A.; Giovannini, P. P.; Massi, A. Org. Lett. 2004, 6,
2929.
(9) See for instance: Sewald, N.; Jakubke, H.-D. Peptides:
Chemistry and Biology; Wiley-VCH: Weinheim, 2002, 247–
253.
(10) For a review, see: Kumar, D.; Bhalla, T. C. Appl. Microbiol.
Biotechnol. 2005, 68, 726.
(11) Chen, S.-T.; Chen, S.-Y.; Wang, K.-T. J. Org. Chem. 1992,
57, 6960.
HRMS (ESI): m/z calcd for C36H42N6O13 + Na (M + Na):
789.27075; found: 789.26751.
(12) Chan, T. R.; Hilgraf, R.; Sharpless, K. B.; Fokin, V. V. Org.
Lett. 2004, 6, 2853.
(13) (a) Xu, J. W.; Egger, A.; Bernet, B.; Vasella, A. Helv. Chim.
Acta 1996, 79, 2004. (b) Dondoni, A.; Mariotti, G.; Marra,
A. J. Org. Chem. 2002, 67, 4475. (c) Shiozaki, M.; Arai,
M.; Macindoe, W. M.; Mochizuki, T.; Kurakata, S.; Maeda,
H.; Nishijima, M. Chem. Lett. 1996, 735.
Acknowledgment
SenterNovem (Ministry of Economic Affairs, The Netherlands) is
gratefully acknowledged for providing financial support. We thank
J. Andrien (DSM) for analytical support.
(14) Nyffeler, P. T.; Liang, C. H.; Koeller, K. M.; Wong, C.-H. J.
Am. Chem. Soc. 2002, 124, 10773.
(15) Wolf, L. B.; Sonke, T.; Tjen, K. C. M. F.; Kaptein, B.;
Broxterman, Q. B.; Schoemaker, H. E.; Rutjes, F. P. J. T.
Adv. Synth. Catal. 2001, 343, 662.
Synthesis 2006, No. 18, 3146–3152 © Thieme Stuttgart · New York