Ring-opening Cross-metathesis (ROCM) as a novel tool for the ligation of peptides

Article abstract of DOI:10.1002/chem.200601183

The development of ring-opening cross-metathesis (ROCM) as a novel tool for the site-specific ligation of peptide units is reported. The resulting structural units at the site of ligation resulting from ROCM resemble proline as well as other known ss-turn

Full text of DOI:10.1002/chem.200601183

DOI: 10.1002/chem.200601183
Ring-Opening Cross-Metathesis (ROCM) as a Novel Tool
for the Ligation of Peptides
Simon Michaelis and Siegfried Blechert*[a]
Abstract: The development of ring-
opening cross-metathesis (ROCM) as a
novel tool for the site-specific ligation
of peptide units is reported. The result-
ing structural units at the site of liga-
tion resulting from ROCM resemble
proline as well as other known b-turn
stabilising structural units. ROCM
under mild reaction conditions be-
tween a variety of peptides bearing a
cyclic olefin with amino acids or pep-
tides results in high yields. The peptidic
cross-partners for metathesis are equip-
ped with double bonds via the N and
the C terminus and the side chain, re-
spectively, to allow the synthesis of
linear as well as non-linear and
branched peptides. The ligation in this
manner succeeds with low catalyst
loadings, with no need for any excess
of one reaction partner and with a high
compatibility with a wide range of
functional groups. Furthermore, the
stereochemical outcome of the ROCM
can easily be controlled by using a
Hoveyda-type chiral catalyst. Fluores-
cence labelling of peptides is possible
in the same manner when using a
cyclic olefin equipped with a fluores-
cence marker.
Keywords: bioorganic chemistry ·
diastereoselectivity · ligation techni-
ques · metathesis · peptidomimetics
Introduction
acids. Ideally, ligation should be carried out stereoselectively
under mild conditions with low catalyst loadings in high
yields. As both substrates (for example, two peptide building
blocks) are expensive biomolecules, there should be no need
for any excess of one reaction partner. Furthermore, the li-
gation technique should allow the synthesis of a linear as
well as a non-linear and a branched peptide.
Numerous techniques have been developed to synthesise
large peptide fragments that have led to the elucidation of
gene function and structure-function relationships, making
possible the discovery of novel biology and new therapeutic
agents. The segmental approach that ligates building blocks
in peptide synthesis without using any coupling reagents is a
superior strategy to the stepwise approach.[1] As a result it
has become more common during the last 15 years.
An alternative strategy in the synthesis of peptide seg-
ments involves appending two peptide building blocks to
the amine and carboxyl group of a secondary structural unit
via the C and N termini of these fragments, by using known
coupling reagents. The use of these non-peptidic structural
units results in a stabilisation of b-turn regions and bioactive
conformations, and therefore many peptide analogues con-
taining such structures exhibit higher biological activity.[2]
Both ligation techniques require chemoselectivity, avoid-
ing reaction with the manifold functional groups of amino
ꢀ
Olefin metathesis is one of the most versatile tools for C
C-bond formation and meets all the above demands.[3] We
were therefore interested in applying this classic organome-
tallic reaction to the biochemical field of ligation and deri-
vatization of peptides. We wished to exploit the advantages
of the atom-economical ring-opening cross-metathesis
(ROCM) for the segmental ligation of peptide building
blocks. The resulting structural unit from ROCM should in
principal be able to mimic the role of b-turn stabilising sys-
tems, such as proline, leading to a ligation approach towards
b-turn mimetics.
Results and Discussion
Recently, two interesting ligation techniques were reported.
One takes advantage of the Huisgen azide cycloaddition,[4]
the other of the Diels–Alder reaction.[5] Both techniques
offer an advantageous new opportunity for the ligation of
proteins and peptides. They proceed under mild conditions
[a] Dr. S. Michaelis, Prof. S. Blechert
Institut für Chemie, Technische Universität Berlin
Strasse des 17. Juni 135, 10623 Berlin (Germany)
Fax : (+49)30-314-29745
E-mail: blechert@chem.tu-berlin.de
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Chem. Eur. J. 2007, 13, 2358 – 2368

FULL PAPER
with high selectivity and are compatible with most function-
al groups found in proteins.
Herein, we present the development of a new concept for
the efficient ligation of peptides by ROCM. Cyclic amines,
such as 1, equipped with a peptide segment, undergo
ROCM with different metathesis cross-partners 2, 3 or 4,
leading to ligation of the peptide segments (Scheme 1). Con-
ceptually, the ligation can be achieved in a linear (5), non-
linear (6) or branched (7) manner by using cross-partners of
types A–C. Instead of expendable separations, a homogene-
ous and stereochemically well-defined product should be
achieved by using chiral Hoveyda-type metathesis catalysts
and perhydrogenation of the resulting double bonds in 5, 6
and 7. Cross-partners 2 and 3 can be easily prepared by
amide bond formation with the peptide N terminus in 2 and
esterification with the peptide C terminus in 3, respectively.
Cross-partner 4 is a peptide containing allyl-glycine as one
amino acid. Amide bond formation of amine 11, accessible
by sequential Diels–Alder reaction of cyclobutadiene 8 and
maleimide 9 and reduction, leads to the cyclo-olefin 1
(Scheme 2).
Scheme 2. Synthesis of cyclo-olefin 1. a) Diethyl ether, 238C, 2 h, 96 %;
b) LiAlH4 (4.1 equiv), THF, 48 h, reflux, 84%; c) general procedure: 0.83
to 1.0 equiv amino acid or peptide (Fmoc-protected), 1.0 to 2.0 equiv
TBTU, dichloromethane/DMF, 08C/0.5 h, 238C/1 h.
Scheme 3. [Ru]-catalyzed ROCMs of 12 and 13. [a] Isolated as a mixture
of E and Z isomers as well as a diastereoisomeric mixture.
To optimise the reaction conditions, we first coupled the
cyclic amine 11 with Fmoc-protected glycine (Fmoc=9-fluo-
renylmethoxycarbonyl) and used the glycine-ester 13a as
the coupling partner for metathesis (Scheme 3). As shown
in Table 1, a solution of a 1:1 mixture of 12a and 13a in
either dichloromethane or 1,2-dichlorethane was stirred at
different temperatures by using 1 mol% of the metathesis
catalysts [Ru-1] to [Ru-4]. The conversion was monitored by
HPLC-ESI. Ahistogram of the relative product distribution
of 14a, versus olefin 12a and peptide 13a is shown in
Figure 1. After chromatography, the product 14 was isolated
as a mixture of E and Z isomers as well as a diastereoiso-
meric mixture.
We found the first-genera-
tion Hoveyda catalyst [Ru-3]
to be superior to the others, as
the more active second-gener-
ation catalysts [Ru-2] and [Ru-
4] led to significant polymeri-
sation as well as rapid con-
sumption of the starting mate-
rial 12a (Figure 1).
To demonstrate the compati-
bility of the ROCM method
with various functional groups,
we investigated the ligation of
peptides 12a–k with 13a–c
(Table 2). In accordance with
our optimized reaction condi-
tions, the cross-partners were
stirred
in
dichlormethane
(0.1m) for 17 h at 408C by
using generally 1–5 mol% of
the metathesis catalyst [Ru-3].
Selected examples are given in
Table 2.
As expected for the afore-
mentioned example (entry 1)
and its homologues (entries 2
and 3), we were able to isolate
the coupled product 14 in high
Scheme 1. General concept of peptide ligation by means of ROCM.
Chem. Eur. J. 2007, 13, 2358 – 2368
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
2359

S. Blechert and S. Michaelis
Table 1. ROCMs of 12a (R1 =Fmoc-Gly-) and 13a (R2 =Fmoc-Gly-).
yields following sequential purification by silica gel and Se-
phadex chromatography. The UV chromatographs of the
crude product 14b (entry 2) as well as for the product after
each purification step are shown in Figure 2 as an example.
The presence of two peaks Aand B in the product (reten-
tion time 6.5–8.0 min) represents the E/Z isomers of product
14b. This was supported by perhydrogenation of pure 14b,
the product of which displayed a single peak by HPLC-ESI
Entry
Catalyst (1 mol%)
[Ru-1][a]
[Ru-2]
[Ru-2]
[Ru-3]
[Ru-3]
[Ru-3]
[Ru-3]
[Ru-4]
[Ru-4]
T [8C]
Solvent
c [mol Lꢀ1
]
1
2
3
4
5
6
7
8
9
C
40
40
60
40
40
60
60
40
60
dichloromethane
dichloromethane
DCE
dichloromethane
dichloromethane
DCE
DCE
dichloromethane
DCE
0.01
0.01
0.01
0.1
0.01
0.1
0.01
0.01
0.01
[a] 2 mol% of [Ru-1] was used.
Figure 2. UV chromatograph of 14b. a) crude product, b) after column
chromatography, c) after Sephadex chromatography.
Figure 1. Results for the [Ru]-catalyzed ROCMs of 12a and 13a.
Table 2. Results for the peptide ligation by means of ROCM.
Entry
12
R1
13[a]
R2
Catalyst (mol%)
[Ru-3] (2)
[Ru-3] (2)
[Ru-3] (2)
[Ru-2] (1)
[Ru-4] (1)
[Ru-3] (1)
[Ru-5] (1)
[Ru-3] (1)
[Ru-3] (1)
[Ru-3] (5)
[Ru-3] (1)
[Ru-3] (5)
[Ru-4] (5)
[Ru-3] (2)
[Ru-3] (2)
[Ru-3] (2)
Product
14/cdp[b]
Yield [%]
E/Z[c]
1
2
3
12a
12b
12c
12d
12d
12d
12d
12e
12 f
12g
12h
12i
Fmoc-Gly-
Fmoc-Phe-
Fmoc-Val-
Fmoc-Gly-Gly-Gly-
Fmoc-Gly-Gly-Gly-
Fmoc-Gly-Gly-Gly-
Fmoc-Gly-Gly-Gly-
Fmoc-Phe-Gly-Gly-
Fmoc-Asn-Gly-Gly-
13a
13a
13a
13a
13a
13a
13a
13a
13a
13a
13a
13a
13a
13b
13c
13b
Fmoc-Gly-
Fmoc-Gly-
Fmoc-Gly-
Fmoc-Gly-
Fmoc-Gly-
Fmoc-Gly-
Fmoc-Gly-
Fmoc-Gly-
Fmoc-Gly-
Fmoc-Gly-
Fmoc-Gly-
Fmoc-Gly-
Fmoc-Gly-
G
14a
14b
14c
14d
14d
14d
14d
14e
14 f
14g
14h
14i
80:20
80
74
82
35
51
75
80
83
37
80
83
<5
30
82
66
74
78:22
63:37
65:35
G
85:10[d]
83:11[d]
ACHTREUNG
4[e]
5[e]
6
ACHTREUNG
AHCTREUNG
E
93:7
48:52
7[f]
8
ACHTREUNG
U
91:9
64:36
67:33
64:36
64:36
–
9
G
77:23
90:10
>95:<5
–
10
11
12
13[g]
14
15
16
Fmoc-Lys
Fmoc-Cys
N
ACHTREUNG
A
ACHTREUNG
Fmoc-Cys-Gly-Gly-
Fmoc-Cys-Gly-Gly-
Fmoc-Phe-Gly-Gly-
Fmoc-Leu-Ala-Pro-
Fmoc-Leu-Ala-Pro-
ACHTREUNG
12i
H
14i
14k
14l
12e
12k
12k
Fmoc-Leu-Ala-Pro-
Fmoc-Cys(Trt)-Gly-Gly-
Fmoc-Leu-Ala-Pro-
U
U
ACHTREUNG
C
14m
[a] Cross-partner. [b] Determined by HPLC-ESI, cdp: crossed-dimer product. [c] Determined by 1H NMR spectroscopy. [d] 5–6% double-crossed prod-
uct (dcp). [e] 0.01m, 1 h. [f] 6 h. [g] 2 equiv cross-partner, 1,2-dichlorethane, 708C, 0.012m, 1 h.
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Chem. Eur. J. 2007, 13, 2358 – 2368

Ligation of Peptides
FULL PAPER
analysis. This fulfilled our desire for access to a stereochemi-
cally well-defined product by simple perhydrogenation.
Peaks C1 and C2 (retention time 10–12 min) correspond to
the double crossed product (dcp), and peaks at 13–16 min
(D1 and D2) to the crossed dimer product (cdp), as judged
by their mass spectral analysis. The appearance of a should-
er in product peak B led us to attempt HPLC methods to
revolve these peaks. By using more polar eluents, four peaks
were displayed, representing the diasteroisomers in addition
to the E/Z isomers. We did not try to separate the diastereo-
meric product mixture but instead investigated the influence
of chiral metathesis catalysts.
underwent ROCM with [Ru-3] giving the coupling products
14k–m in 66–82% yields.
Next, we tried to expand the new methodology for liga-
tion with type C cross-partners (Schemes 1 and 4). N- and
C-protected allylglycine 15 was, therefore, used as a type C
cross-partner model and we investigated its ROCM with
cyclic olefins 12 (Table 3).
To probe the reaction further, peptides with a Gly-Gly
spacer were synthesized and used as ROCM substrates (en-
tries 4–14). Glycine is one of the most common amino acids
present in native b-turn regions because of the increased
flexibility that accommodates the conformation of the pep-
tide. Additionally, we observed that lower catalyst loadings
(1 instead of 2 mol%) could be employed for substrates
bearing a Gly-Gly spacer (comparing entries 1/6 and 2/8).
We attribute this to the decreased interactions between the
potentially coordinating Fmoc
Scheme 4. [Ru]-catalyzed ROCMs of 12 and type C cross-partner 15.
[a] Isolated as a mixture of E and Z isomers as well as a diastereoisomer-
ic mixture.
groups and the reaction centre
Table 3. Results for the [Ru]-catalyzed ROCMs with type C cross-partner 15.
during ROCM. In these Gly-
Gly substrates, [Ru-3] dis-
played again the best reactivi-
ty, as demonstrated by compar-
ison of entries 4 to 6. However,
[Ru-5],[6] a Blechert-group cat-
alyst gave a higher yield still
(80%) of ligated peptide 14d,
in a dramatically reduced reac-
tion time (entry 7). Astarting
material containing asparagine
(12 f, entry 9) resulted in the
Entry
12
R1
15
Catalyst (mol%)
16
c [molLꢀ1
]
12/16/cdp[b]
AHCTREUNG
1
2
3
4
5
6
7
12a
12a
12a
12d
12d
12d
12d
Fmoc-Gly-
Fmoc-Gly-
Fmoc-Gly-
Fmoc-Gly-Gly-Gly-
Fmoc-Gly-Gly-Gly-
Fmoc-Gly-Gly-Gly-
Fmoc-Gly-Gly-Gly-
1
2
1
1
1
2
1
N
16a
16a
16a
16b
16b
16b
16b
0.1
0.02
0.1
>98:<2:0
>99:<1:0
5:56:39
0.1
84:11:5
0.1
1:72:27
0.02
0.1
1:59:40
2:60:38
[a] Cross-partner. [b] Determined by HPLC-ESI, cdp: crossed-dimer product.
formation of large amounts of dimers and polymeric side
products as observed as an insoluble material in the reaction
flask. Nevertheless, 37% of coupling product 14 f was isolat-
ed. To investigate the compatibility of nucleophelic func-
tional groups with ROCM, we examined the coupling reac-
tion with peptidic cross-partners bearing either protected or
unprotected SH groups. Whereas the trityl-protected pep-
tide 12h (entry 11) reacted as expected, the unprotected cys-
teine derivative 12i (entry 12) was not converted when
using [Ru-3]. Areasonable explanation is the deactivation
or decomposition of the catalyst by the strongly nucleophilic
SH group, inhibiting ROCM. By using the more active and
functional-group compatible catalyst of the second genera-
tion [Ru-4] at optimized conditions (5 mol% catalyst, 708C,
0.012m, 1 h) gave more than 50% of dimers, <10% starting
material and 30% of the coupling product 14i, which is an
acceptable result for olefin metathesis with these substrates.
Higher yields might result in the case of bigger peptides if
strongly nucleophilic functional groups are internally located
in peptides.
In general, we found that the type C cross-partner gave a
branched peptidelike product 16 (Table 3) as a mixture of E
and Z isomers as well as a diastereoisomeric mixture. As
the optimised reaction conditions for type B cross-partners
did not lead to any significant conversion to peptides 16 (en-
tries 1 and 4), we increased the amount of catalyst [Ru-3]
(entry 5) which gave large amounts of dimers as detected by
HPLC-ESI. Further investigation showed that the second-
generation catalyst [Ru-4] gave the best results (entries 6
and 7) when using two equivalents of cross-partner 15. By
using these conditions, we were able to isolate 33% of the
ring-opened cross-product 16b after purification on silica
gel and Sephadex. The good results of the peptide ligation
with type B cross-partners as well as the observations with
different spacers in the manner of fluorescence labelling
(see below), indicates that higher yields of coupled products
are obtainable when decreasing the steric hindrance of the
substrates or by increasing the length of the side chain to
the double bond.
Additionally, we investigated the ability of asymmetric
metathesis catalysts to influence the stereochemical outcome
of ROCM as it is important for stereoselective synthesis (of
We next prepared hexapeptides with a wide variety of
amino acids (entries 14–16). The tripeptides 12e,k and 13b,c
Chem. Eur. J. 2007, 13, 2358 – 2368
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2361

S. Blechert and S. Michaelis
pharmaceutical compounds). To investigate this, we analysed
the stereochemical reaction outcomes of our peptide ligation
reactions by HPLC-ESI when
the crude mixture, the product was purified by preparative
TLC.
The fluorescence-labelled product 18b was isolated in
67% yield (Table 5, entry 2) and identified by NMR spec-
troscopy and MS. We found the conversion of the metathe-
sis reaction was dependant on the spacer as the conversion
using the Hoveyda-type chiral
catalyst [Ru-6]*.[7] This nonphos-
phine Ru carbene bears a biden-
tate styrene ether ligand and is
currently one of the best asym-
metric catalysts which enables
reactions to be carried out under
air and with undistilled solvents.
Table 5. Results for the fluorescence labelling by means of ROCM.
Entry
13[a]
18
n
Conv. [%][b]
Yield [%]
1
2
3
13d[c]
13e[c]
13 f[c]
18a
18b
18c
2
4
9
60
80
60
43
67
49
In
preliminary
reactions
(Scheme 3, Table 4, entries 1
and 3), we found that the conversion of 12 and 13 needed
elevated reaction temperatures of 608C, probably as a result
of various steric and electronic factors of [Ru-6]* in contrast
[a] Cross partner. [b] Determined by 1H NMR spectroscopic analysis.
[c] R2 =Trt-Cys
(Mmt)-Ser-Val-.
AHCTREUNG
Table 4. Results for the asymmetric [Ru]*-catalyst ROCMs.
as well as the isolated yields of
the reactions with shorter
(entry 1) or longer spacers
(entry 3) decreased dramatical-
ly. Nevertheless, these results
show that peptides can be
easily labelled by ROCM with
the olefin 17 (Scheme 5).
Entry 12
R1
13[a] R2
14
Catalyst/
mol%
13/
de
E/
14[b]
[%][b]
Z[b]
1
2
3
4
5
12b Fmoc-l-Phe-
12l Fmoc-d-Phe-
12c Fmoc-l-Val-
12m Fmoc-d-Val-
12k Fmoc-Leu-Ala-
Pro-
13a Fmoc-Gly-
13a Fmoc-Gly-
13a Fmoc-Gly-
13a Fmoc-Gly-
13b Fmoc-Leu-Ala-
Pro-
14b
14n
14c
14o
14m
3
3
2
2
2
90:10
45:55
38:62
54:46
85:15
74
70
62
63
61
–
87:13
84:16
84:16
87:13
[a] Cross-partner. [b] Determined by HPLC-ESI.
to the achiral catalysts. Furthermore, the selectivity of [Ru-
6]*-catalyzed ROCMs is not dependent on the solvent.
While ROCM in toluene gave lower conversion and diaste-
reoisomeric excess, ROCM carried out in 1,2-dichlorethane
and THF gave better comparative results. For this, we ana-
lysed the catalytic activity of [Ru-6]* on a range of reactions
in 1,2-dichlorethane.
As shown in Table 4, olefin metathesis of 12 and 13 led to
products with excellent diastereoisomeric excesses (de) com-
pared to the examples reported by the group of Hovey-
da.[7–9] For example, the cyclic olefin 12b bearing N-Fmoc-
phenylalanine reacted with cross-partner 13a in 55% yield
and 70% de to give 14b when using 3 mol% of [Ru-6]*. In
contrast to Hoveydaꢁs work, we used one instead of five
equivalents of the cross-partner and lower amounts of cata-
lyst, which might be the reason for incomplete conversions.
As the ROCM stereochemical outcome using d or l starting
materials (entries 1–2 and 3–4) lie within a close range, the
influence of the substrate was considered to be negligible.
Finally, we wished to expand the concept to fluorescence
labelling of peptides as it is an important application in pep-
tide chemistry. The fluorescence-labelled cyclic olefin 17 is
easily accessible from amine 11 and commercially available
dansyl chloride. Sulfonamide 17 and one equivalent of the
cross-partner 13 were treated with 1 mol% of the second-
generation Grubbs catalyst [Ru-2] and stirred at 408C. After
30 min, the reaction was quenched with ethyl vinylsilane,
Scheme 5. [Ru]-catalyzed ROCMs of fluorescence-labelled 17 and 13.
[a] Isolated as a mixture of E and Z isomers as well as a diastereoisomer-
ic mixture.
Conclusion
ꢀ
Our results demonstrate the power of ROCM in C C-bond
formation as a means of ligating smaller peptide units. By
this method, the ligation can be achieved in a linear as well
as in a non-linear and a branched sense, in good yields and
under mild conditions, with low catalyst loadings and with
no need for any excess of one reaction partner. We were
able to demonstrate a high compatibility with a wide range
of functionalised amino acids. The first generation Hoveyda
catalyst [Ru-3] as well as its variants were superior to the
more active catalysts of the second generation, which led to
higher amounts of polymeric byproducts. The stereochemi-
cal outcome can be controlled by using the chiral catalyst
[Ru-6]*. Relative to the published results of Hoveyda,[7,9] we
1
and after monitoring the conversion by H NMR analysis of
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Chem. Eur. J. 2007, 13, 2358 – 2368

Ligation of Peptides
FULL PAPER
acid (solvents mentioned in examples). The reaction mixture was stirred
for 0.5 h at 08C and additionally for 1 h at 238C. It was diluted with di-
chloromethane and washed with H2O (2” with the same volume). The
combined organic layers were dried over Na2SO4. The drying agent was
removed by filtration and the solvents were removed under vacuum. The
crude product was purified by column chromatography and, if noted, ad-
ditionally by Sephadex chromatography.
were able to obtain the ROCM products with excellent dia-
stereomeric excesses, which should lead to a homogenous
and stereochemically well-defined product after perhydroge-
nation.
Currently, we are investigating the peptide ligation in
aqueous media, as peptides with a higher number of amino
acids have a limited solubility in organic solvents. Further-
more, we are interested in the ability of the secondary struc-
tural unit at the site of ligation to stabilize or initiate b-
turns.
Cyclic olefins
Imine (10): Freshly distilled cyclopentadiene (2.8 mL, 33.3 mmol,
1.01 equiv) was added to a solution of maleimide (3.2 g, 33 mmol) in di-
ethyl ether (80 mL). The reaction mixture was stirred for 2 h at 238C.
After this time, the solid product was separated by filtration and dried
under air. Recrystallisation from diethyl ether/methanol 2:1 gave product
10 (5.2 g, 31.7 mmol, 96%) as white crystals. M.p. 170–1728C; 1H NMR
(400 MHz, CDCl3): d=1.53 (d, J=9.0 Hz, 1H), 1.76 (d, J=9.0 Hz, 1H),
3.24–3.44 (m, 4H), 6.21 (2H, AB-syst.), 7.62 ppm (s, 1H); 13C NMR
(100.6 MHz, CDCl3): d=45.0 (2CH), 47.4 (2CH), 52.4 (2CH2), 134.6
(2CH), 178.5 ppm (2Cq); IR (ATR): n˜ =3158, 3063, 2991, 2962, 2954,
1753, 1718, 1699, 1354, 1167, 736 cmꢀ1; MS (EI): m/z (%): 163 (13), 98
(34), 91 (53), 66 (100); HRMS: calcd for C9H9O2N: 163.0633; found:
163.0631 [M+].
Experimental Section
General methods and materials: Unless otherwise noted, all reactions
were performed in dried glassware under air. All solvents were dried and
distilled prior to use. Commercially available reagents were used as re-
ceived without further purification. Column chromatography was carried
out by using 60 mm silica gel from Merck. Sephadex LH-20 was supplied
from Pharma Biotech. Analytical reversed-phase HPLC-(MS) was car-
ried out on a Varian ProStar system (autosampler model 410; UV-visible-
detector model 320; solvent delivery module model 210; fraction collec-
tor model 701) by using a Waters X-Terra (RP-18, 4.6*100 mm) column
and on a Hewlett–Packard system series 1100 on a Waters Symmetry,
RP-18, 3.9*150 mm column in combination with Bruker-Daltonics es-
Amine (11): Under a nitrogen atmosphere in a thoroughly dried appara-
tus containing a three-necked flask, a dropping funnel and a condenser, a
solution of 10 (1.63 g, 10 mmol) in THF (35 mL) was added dropwise to
a suspension of LiAlH4 (1.56 g, 41 mmol, 4.1 equiv) in THF (35 mL)
through the dropping funnel. The reaction mixture was stirred for 48 h
under reflux. After cooling down, diethyl ether (150 mL) was added and
the mixture was hydrolysed by the successive addition of ethyl acetate,
methanol and water until a white solid precipitated. The solid was re-
moved by filtration, and solvents were removed under vacuum. Kugel-
rohrdestillation (3”10ꢀ2 mbar, 458C) of the pale-yellow crude product
quire 2000 (ESI), detection at 210 and 254 nm and a linear gradient of
ꢀ1
solvent B in solvent Aat 0.5 mLmin
flow rate. HPLC-grade solvents
were delivered by Acros and Fisher Scientific. Calculated masses were
obtained by using the program molecular weight calculator by Matthew
gave amine 11 (1.14 g, 8.4 mmol, 84%) as
a white, waxlike solid.
1
Monroe Version 6.35. H and 13C NMR spectra were recorded on an AM
1H NMR (500 MHz, CDCl3): d=1.52 (d, J=9.0 Hz, 1H), 1.65 (d, J=
8.8 Hz 1H), 2.85 (dd, J=12.0, 2.6 Hz, 2H), 2.96 (d, J=1.0 Hz, 2H), 3.03–
3.09 (m, 2H), 3.16–3.22 (m, 2H), 6.43 ppm (s, 2H); 13C NMR
(100.6 MHz, CDCl3): d=46.4 (2CH), 48.0 (2CH), 49.6 (2CH2), 53.1
(2CH2), 135.9 ppm (2CH); IR (ATR): n=3221, 3056, 2960, 2937, 2866,
1713, 1623, 1570, 1350, 735 cmꢀ1; MS (EI): m/z (%): 135 (12), 94 (18), 68
(100), 66 (13); HRMS: calcd for C9H13N: 135.1048; found: 135.1054 [M+].
400 (400 MHz) and DRX 500 spectrometer (500 MHz) from Bruker.
Chemical shifts are reported in ppm relative to CHCl3 (d=7.27 ppm) for
1H NMR and the central resonance of CDCl3 (d=77.0 ppm) for
13C NMR spectra. IR spectra were obtained on a Perkin–Elmer 881 and
on a Nicolet Magna 750 Series FTIR. HRMS were obtained on a Finni-
gan MAT 95 SQ mass spectrometer. Optical rotations were measured on
a polarimeter as solutions in a 10 cm unit cell at 589 nm. Trt=triphenyl-
methyl; Mmt=monomethoxytrityl.
Fmoc-Gly-cyclo-olefin (12a): According to the general TBTU method A,
amine 11 (100 mg, 0.74 mmol) in dichloromethane (2 mL) and TBTU
(285 mg, 0.89 mmol, 1.2 equiv) were added to a solution of Fmoc-protect-
ed glycine (220 mg, 0.74 mmol) in DMF (3.5 mL) at 08C. The reaction
mixture was stirred for 0.5 h at 08C and additionally for 1 h at 238C.
After aqueous workup and column chromatography (hexanes/ethyl ace-
tate 1:1), 12a (142 mg, 0.34 mmol, 46%) was obtained as a white crystal-
line solid. M.p. 70–728C; 1H NMR (400 MHz, CDCl3): d=1.45 (d, J=
8.5 Hz, 1H), 1.58 (d, J=8.5 Hz, 1H), 2.90–3.04 (m, 5H), 3.28–3.38 (m,
3H), 3.82 (d, J=3.2 Hz, 2H), 4.23 (t, J=7.3 Hz, 1H), 4.35 (d, J=7.3 Hz,
2H), 5.81 (s, 1H), 6.19 (m, 2H), 7.31 (dt, J=7.5, 1.0 Hz, 2H), 7.39 (t, J=
7.5 Hz, 2H), 7.60 (d, J=7.5 Hz, 2H), 7.74 ppm (d, J=7.5 Hz, 2H);
13C NMR (100.6 MHz, CDCl3): d=43.5 (CH2), 43.8 (CH), 45.8 (CH),
46.7 (CH), 47.1 (CH), 47.9 (CH2), 48.2 (CH2), 51.9 (CH2), 67.1 (CH2),
119.9 (CH), 125.2 (CH), 127.1 (CH), 127.7 (CH), 134.7 (CH), 136.0
(CH), 141.3 (Cq), 143.9 (Cq), 156.2 (Cq), 165.4 ppm (Cq); IR (ATR): n˜ =
3267, 3062, 3037, 3028, 2966, 2944, 2870, 1713, 1627, 1532, 1451, 1246,
1032, 741 cmꢀ1; LRMS (70 eV, EI): m/z (%): 414 (41), 367 (44), 368
(100), 313 (21), 284 (42), 281 (23), 178 ppm (>100); HRMS: calcd for
C26H26O3N2: 414.1963; found: 414.1952 [M+].
General procedure for peptide synthesis
DCC method: The coupling partner (5-hexene-1-ol or C-protected amino
acid; 1.0–1.2 equiv), 1,3-dicyclohexylcarbodiimide (DCC, 1 to 2 equiv)
and 4-dimethylaminopyridine (DMAP, 0.17 equiv) were added to a solu-
tion of the Fmoc-protected amino acid (unless otherwise noted: 0.2m in
dichloromethane) at 08C. The reaction mixture was stirred for 2 h at 08C
and additionally for 12 h at 238C. The solid was removed by filtration
and the solution was transferred to a separatory funnel. It was quenched
by addition of water (same volume as the organic layer). After separa-
tion, the aqueous layer was extracted with dichloromethane twice. The
combined organic layers were dried over Na2SO4. The drying agent was
removed by filtration and solvents were removed under vacuum. The
crude product was purified by column chromatography and, if noted, ad-
ditionally by Sephadex chromatography.
TBTU method A: The coupling partner (11 or C-protected amino acid;
1 equiv) and O-benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium tetra-
fluoroborate (TBTU, 1 to 2 equiv) at 08C were added to a solution of the
Fmoc-protected amino acid (solvents mentioned in examples). The reac-
tion mixture was stirred for 0.5 h at 08C and additionally for 1 h at 238C.
It was diluted with dichloromethane and washed with H2O (2” with the
same volume). The organic layer was dried over Na2SO4. The drying
agent was removed by filtration and the solvents were removed under
vacuum. The crude product was purified by column chromatography and,
if noted, additionally by Sephadex chromatography.
Fmoc-Phe-cyclo-olefin (12b): According to the general DCC method,
amine 11 (270 mg, 2 mmol), DCC (825.3 mg, 4 mmol, 2 equiv) and
DMAP (41.5 mg, 0.34 mmol, 0.17 equiv) were added successively to a so-
lution of Fmoc-protected phenylalanine (775 mg, 2 mmol) in dichlorome-
thane (4 mL) at 08C. The reaction mixture was stirred for 2 h at 08C and
additionally for 12 h at 238C. After filtration, aqueous workup and
column chromatography (hexanes/ethyl acetate 3:1!1:1) 12b (477 mg,
0.94 mmol, 47%) was obtained as a white crystalline solid. M.p. 77–
TBTU method B: The TFAsalt of the C-protected peptide (1 equiv),
TBTU (1.0 to 2.0 equiv) and N,N-diisopropylethylamine (DIPEA,
2.2 equiv) at 08C were added to a solution of the Fmoc-protected amino
1
798C; [a]2D0 =+6.88 (c=0.99 in CH2Cl2); H NMR (500 MHz, CDCl3): d=
Chem. Eur. J. 2007, 13, 2358 – 2368
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
2363

S. Blechert and S. Michaelis
1.33 (d, J=8.6 Hz, 0.5H), 1.39 (d, J=8.6 Hz, 0.5H), 1.46–1.53 (m, 1H),
2.57 (t, J=10.0 Hz, 0.5H), 2.65 (dd, J=10.6, 3.6 Hz, 1H), 2.70–2.77 (m,
0.5H), 2.79 (s, 0.5H), 2.82 (s, 0.5H), 2.87 (s, 2H), 2.90 (d, J=6.0 Hz,
0.5H), 2.92–2.97 (m, 1H), 2.98–3.05 (m, 1H), 3.07 (dd, J=11.0, 2.0 Hz,
0.5H), 3.14 (dd, J=13.0, 9.4 Hz, 0.5H), 3.28 (dd, J=12.6, 3.0 Hz, 0.5H),
3.34 (dd, J=12.6, 9.2 Hz, 0.5H), 3.54 (t, J=10.0 Hz, 0.5H), 4.15- 4.22 (m,
1H), 4.24–4.40 (m, 2H), 4.55 (dd, J=7.4, 6.6 Hz, 0.5H), 4.59 (dd, J=8.0,
6.0 Hz, 0.5H), 5.64 (dd, J=5.0, 2.6 Hz, 0.5H), 6.02 (dd, J=5.0, 2.0 Hz,
0.5H), 6.07–6.10 (m, 0.5H), 6.11–6.15 (m, 0.5H), 7.17 (t, J=7.6 Hz, 2H),
7.22–7.33 (m, 5H), 7.37–7.42 (m, 2H), 7.53–7.60 (m, 2H), 7.73–7.80 ppm
(m, 2H); 13C NMR (100.6 MHz, CDCl3): d=39.2 (CH2), 40.0 (CH2), 43.5
(CH), 44.0 (CH), 45.6 (CH), 46.0 (CH), 46.4 (CH), 46.4 (CH), 46.5 (CH),
46.6 (CH), 47.2 (CH2), 47.9 (CH2), 48.5 (CH2), 48.8 (CH2), 49.2 (CH2),
51.8 (CH2), 52.0 (CH2), 53.6 (CH), 53.8 (CH), 67.1 (CH2), 120.0 (CH),
125.2 (CH), 125.3 (CH), 127.0 (CH), 127.1 (CH), 127.8 (CH), 128.5
(CH), 128.6 (CH), 129.5 (CH), 129.7 (CH), 135.2 (CH), 135.4 (CH),
135.5 (CH), 135.8 (CH), 136.3 (Cq), 136.4 (Cq), 141.4 (Cq), 144.0 (Cq),
155.7 (Cq), 155.8 (Cq), 168.7 (Cq), 169.1 ppm (Cq); IR (ATR): n˜ =3260,
3062, 2964, 2870, 1715, 1628, 1533, 1451, 1247, 742 cmꢀ1; MS (EI): m/z
(%): 504 (2), 191 (14), 179 (96), 178 (100), 120 (29), 69 (36); HRMS:
calcd for C33H32O3N2: 504.2413; found: 504.2417 [M+].
(CH), 45.6 (CH), 47.0 (CH), 47.1 (CH), 48.8 (CH2), 48.9 (CH2), 51.5
(CH2), 115.4 (Cq), 118.3 (Cq), 135.7 (CH), 136.0 (CH), 163.3 (Cq), 163.6
(Cq), 167.6 (Cq), 167.8 ppm (Cq); IR (ATR): n˜ =3284, 3090, 3062, 2966,
2936, 2873, 2654, 1677, 1638, 1541, 1464, 1203, 721 cmꢀ1; ESIMS: m/z:
calcd for C13H20O2N3: 250.16; found: 249.9 [M++HꢀC2O2F3], 271.9 [M+
+Na], 192.9 [M++HꢀGly], 136.0 [M++Hꢀ2Gly].
Fmoc-Gly-Gly-Gly-cyclo-olefin (12d): According to the general TBTU
method B, TFAsalt 19 (150 mg, 0.41 mmol) in DMF (1 mL), TBTU
(145.9 mg, 0.45 mmol, 1.1 equiv) and DIPEA(158 mL, 0.91 mmol,
2.2 equiv) were added to a solution of Fmoc-protected glycine (122.7 mg,
0.41 mmol) in DMF (2.5 mL) at 08C. The reaction mixture was stirred
for 0.5 h at 08C and additionally for 1 h at 238C. After aqueous workup
and column chromatography (dichloromethane/MeOH 50:1!25:1), 12d
(179.3 mg, 0.34 mmol, 82%) was obtained as a white crystalline solid.
M.p. 103–1058C; 1H NMR (400 MHz, CDCl3): d=1.39 (d, J=8.3 Hz,
1H), 1.53 (d, J=8.3 Hz, 1H), 2.75–3.0 (m, 5H), 3.10–3.23 (m, 3H), 3.77
(s, 2H), 3.90 (d, J=4.8 Hz, 2H), 3.97 (d, J=5.2 Hz, 2H), 4.18 (t, J=
7.0 Hz, 1H), 4.36 (d, J=7.0 Hz, 2H), 5.96 (s, 1H), 6.10 (s, 1H), 7.28 (d,
J=7.5 Hz, 2H), 7.37 (t, J=7.5 Hz, 2H), 7.58 (d, J=7.5 Hz, 2H),
7.73 ppm (d, J=7.5 Hz, 2H); 13C NMR (100.6 MHz, CDCl3): d=42.0
(CH2), 42.7 (CH2), 43.7 (CH), 44.36 (CH2), 45.8 (CH), 46.6 (CH), 47.0
(CH), 48.0 (CH2), 48.3 (CH2), 51.9 (CH2), 53.4 (CH2), 67.3 (CH2), 119.9
(CH), 125.1 (CH), 127.1 (CH), 127.7 (CH), 134.8 (CH), 136.0 (CH),
141.2 (Cq), 143.8 (Cq), 156.9 (Cq), 165.5 (Cq), 169.0 (Cq), 170.0 ppm (Cq);
IR (ATR): n˜ =3301, 3065, 2968, 2942, 2872, 1674, 1642, 1531, 1451, 1203,
741 cmꢀ1; ESIMS: m/z: calcd for C30H33O5N4: 529.24; found: 529.0 [M+
+H], 551.0 [M++Na], 307.0 [M++HꢀFmoc], 192.9 [M++HꢀFmoc-
GlyꢀGly], 135.9 [M++HꢀFmocGlyꢀ2Gly].
Fmoc-Phe-Gly-Gly-cyclo-olefin (12e): According to the general TBTU
method B, TFAsalt 19 (36.3 mg, 0.1 mmol) in DMF (0.25 mL), TBTU
(32.1 mg, 0.1 mmol, 1 equiv) and DIPEA(38.3 mL, 0.22 mmol, 2.2 equiv)
were added to a solution of Fmoc-protected phenylalanine (38.7 mg,
0.1 mmol) in DMF (0.4 mL) at 08C. The reaction mixture was stirred for
0.5 h at 08C and additionally for 1 h at 238C. After aqueous workup and
column chromatography (dichloromethane/MeOH 40:1), 12e (48.3 mg,
0.078 mmol, 78%) was obtained as a white crystalline solid. [a]2D0 =10.58
(c=2.3 in CH2Cl2); 1H NMR (400 MHz, CDCl3): d=1.43 (d, J=8.6 Hz,
1H), 1.55 (d, J=8.6 Hz, 1H), 2.84–3.36 (m, 10H), 3.74–3.91 (m, 3H),
3.92–4.05 (m, 1H), 4.17 (t, J=6.9 Hz, 1H), 4.28–4.53 (m, 3H), 5.44 (s,
1H), 6.15 (m, 2H), 6.59 (s, 1H), 6.87 (s, 1H), 7.12–7.31 (m, 7H), 7.39 (t,
J=7.4 Hz, 2H), 7.52 (t, J=7.4 Hz, 2H), 7.75 ppm (d, J=7.5 Hz, 2H);
13C NMR (100.6 MHz, CDCl3): d=38.5 (CH2), 42.1 (CH2), 42.8 (CH2),
43.8 (CH), 45.8 (CH), 46.6 (CH), 47.1 (CH), 47.9 (CH2), 48.3 (CH2), 51.9
(CH2), 56.1 (CH), 67.1 (CH2), 119.9 (CH), 125.1 (CH), 127.0 (CH), 127.1
(CH), 127.7 (CH), 128.7 (CH), 129.3 (CH), 134.8 (CH), 136.0 (CH),
136.5 (Cq), 141.3 (Cq), 143.8 (Cq), 143.8 (Cq), 156.0 (Cq), 165.0 (Cq), 168.1
(Cq), 171.3 ppm (Cq); IR (ATR): n˜ =3297, 3063, 3028, 2965, 2942, 2870,
1635, 1529, 1451, 1248, 741 cmꢀ1; ESIMS: m/z: calcd for C37H39O5N4:
619.28; found: 619.0 [M++H], 641.0 [M++Na], 393.0 [M++HꢀFmoc],
249.9 [M++HꢀFmocPhe], 192.9 [M++HꢀFmocPheꢀGly].
Fmoc-Val-cyclo-olefin (12c): According to the general TBTU method A,
amine 11 (100 mg, 0.74 mmol) in dichloromethane (2 mL) and TBTU
(285 mg, 0.89 mmol, 1.2 equiv) were added to a solution of Fmoc-protect-
ed valine (251 mg, 0.74 mmol) in DMF (3.5 mL) at 08C. The reaction
mixture was stirred for 0.5 h at 08C and additionally for 1 h at 238C.
After aqueous workup and column chromatography (hexanes/ethyl ace-
tate 2:1!3:2), 12c (178 mg, 0.39 mmol, 53%) was obtained as a white
crystalline solid. [a]D20 =+1.28 (c=0.49 in CH2Cl2); 1H NMR (400 MHz,
CDCl3): d=0.89–0.96 (m, 6H), 1.36–1.58 (m 2H), 1.90–2.00 (dq, J=6.7,
3.3 Hz, 1H), 2.91–2.96 (m, 4H), 3.14–3.38 (m, 2H), 3.39–3.43 (m, 2H),
4.16–4.22 (m, 2H), 4.30–4.39 (m, 2H), 5.52 (m, 1H), 6.17 (m, 2H), 7.31
(m, 2H), 7.40 (m, 2H), 7.59 (m, 2H), 7.77 ppm (dd, J=7.5, 2.5 Hz, 2H);
13C NMR (100.6 MHz, CDCl3): d=17.6 (CH3), 17.6 (CH3), 18.5 (CH3),
18.6 (CH3), 31.1 (CH), 31.3 (CH), 43.5 (CH), 43.8 (CH), 45.7 (CH), 45.8
(CH), 46.6 (CH), 46.6 (CH), 46.7 (CH), 47.1 (CH), 47.9 (CH), 48.1
(CH2), 48.7 (CH2), 49.7 (CH2), 49.8 (CH2), 51.7 (CH2), 52.0 (CH2), 57.4
(CH), 57.5 (CH), 67.0 (CH2), 76.7 (CH2), 119.9 (CH), 125.2 (CH), 127.0
(CH), 127.7 (CH), 135.1 (CH), 135.4 (CH), 136.0 (CH), 141.3 (Cq), 144.0
(Cq), 156.2 (Cq), 165.4 ppm (Cq); IR (ATR): n˜ =3260, 3062, 3038, 2964,
2938, 2871, 1714, 1628, 1450, 1238, 1031, 740 cmꢀ1; LRMS (70 eV, EI):
m/z (%): 456 (3), 390 (3), 294 (4), 260 (4), 179 (91), 178 (100), 165 (34);
HRMS: calcd for C29H32O3N2: 456.2413; found: 456.2420 [M+].
TFA·Gly-Gly-cyclo-olefin (19): Aqueous NaOH (10 mL, 1n) and
(Boc)2O (Boc=tert-butoxycarbonyl, 2.4 g, 11 mmol, 1.1 equiv) were
added to a solution of Gly-Gly (1.32 g, 10 mmol) in dioxane and water
(2:1, 30 mL) at 08C. The reaction mixture was stirred for 1 h at 238C.
The dioxane was removed under reduced pressure. The aqueous layer
was diluted with ethyl acetate (20 mL) and the pH was adjusted to pH 2–
3 by using KHSO4. The layers were separated and the aqueous layer was
extracted with ethyl acetate (5”20 mL). The combined organic layers
were washed with water (50 mL) and dried over Na2SO4. Removing the
drying agent by filtration and the solvent under reduced pressure deliv-
Fmoc-Asn-Gly-Gly-cyclo-olefin (12 f): According to the general TBTU
method B, TFAsalt 19 (36.3 mg, 0.1 mmol) in DMF (0.25 mL), TBTU
(32.1 mg, 0.1 mmol, 1 equiv) and DIPEA(38.3 mL, 0.22 mmol, 2.2 equiv)
ered
a crude product (1.5 g, 6.5 mmol, 65%). Amine 11 (1.14 g,
were added to
a solution of Fmoc-protected asparagine (35.4 mg,
8.45 mmol, 1.3 equiv) in dichloromethane (26 mL), DCC (1.61 g,
7.8 mmol, 1.2 equiv) in dichloromethane (2 mL) and DMAP (140 mg,
1.1 mmol, 0.17 equiv) were added to a solution of the crude product in
THF (26 mL) at 08C, according to the general DCC method. The reac-
tion mixture was stirred for 2 h at 08C and additionally for 12 h at 238C.
0.1 mmol) in dioxane/DMF 3:1 (1 mL) at 08C. The reaction mixture was
stirred for 0.5 h at 08C and additionally for 1 h at 238C. After aqueous
workup and column chromatography (dichloromethane/MeOH 15:1),
12 f (17.8 mg, 0.03 mmol, 30%) was obtained as a white crystalline solid.
1
[a]2D0 =+9.38 (c=0.9 in CH2Cl2); H NMR (400 MHz, CDCl3): d=1.40 (d,
Removing the drying agent and solvent delivered
a crude product
J=8.3 Hz, 1H), 1.53 (d, J=8.3 Hz, 1H), 2.65 (dd, J=15.2, 5.5 Hz, 1H),
2.75–3.0 (m, 5H), 3.09–3.32 (m, 3H), 3.79 (d, J=3.2 Hz, 2H), 3.98 (d, J=
4.8 Hz, 2H), 4.18 (t, J=7.0 Hz, 1H), 4.36 (d, J=7.0 Hz, 2H), 4.62 (q, J=
5.3 Hz, 1H), 6.11 (s, 2H), 6.15 (s, 1H), 6.60 (s, 1H), 6.67 (s, 1H), 7.29 (d,
J=7.5 Hz, 2H), 7.37 (t, J=7.5 Hz, 2H), 7.41 (s, 1H), 7.58 (d, J=7.5 Hz,
2H), 7.73 ppm (d, J=7.5 Hz, 2H); 13C NMR (100.6 MHz, CDCl3): d=
37.4 (CH2), 42.0 (CH2), 43.1 (CH2), 43.8 (CH), 45.8 (CH), 46.6 (CH), 46.6
(CH), 47.1 (CH), 48.0 (CH2), 48.3 (CH2), 51.9 (CH2), 53.4 (CH2), 67.3
(CH2), 119.9 (CH), 125.2 (CH), 127.1 (CH), 127.7 (CH), 134.9 (CH),
(1.82 g, 5.2 mmol, 80%) which was dissolved in dichloromethane
(15 mL). To the solution was added TFA(21 mL) at 0 8C and the reaction
mixture was stirred for 1 h at 238C. After removal of the solvent and
column chromatography (dichloromethane/MeOH 10:1), 19 (0.98 g,
2.7 mmol, 52%) was obtained as a white crystalline solid. 1H NMR
(400 MHz, D2O): d=1.44 (q, J=9.0 Hz, 2H), 3.00 (m, 3H), 3.03 (m,
1H), 3.18 (dd, J=11.6, 2.5 Hz, 1H), 3.24 (d, J=4.8 Hz, 2H), 3.41 (dd, J=
11.6, 9.2 Hz, 1H), 3.86 (s, 2H), 3.94 (d, J=7.6 Hz, 2H), 6.23 ppm (m,
2H); 13C NMR (100.6 MHz, CDCl3): d=40.8 (CH2), 42.1 (CH2), 43.6
2364
www.chemeurj.org
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2007, 13, 2358 – 2368

Ligation of Peptides
FULL PAPER
136.0 (CH), 141.2 (Cq), 143.7 (Cq), 143.8 (Cq), 156.3 (Cq), 165.7 (Cq),
169.0 (Cq), 171.7 (Cq), 173.5 ppm (Cq); IR (ATR): n˜ =3306, 3063, 2966,
2944, 2872, 1665, 1639, 1531, 1451, 1256, 741 cmꢀ1; ESIMS: m/z: calcd for
C32H36O6N5: 586.26; found: 586.0 [M++H], 608.0 [M++Na].
Fmoc-Leu-Ala-Pro-cyclo-olefin (12k): Leu-Ala-Pro-OH
(500 mg,
1.67 mmol) was dissolved in aq Na2CO3 and diluted with dioxane
(2.5 mL). Under rapid stirring Fmoc-Cl (432 mg, 1.67 mmol) was added
in portions at 08C. The reaction mixture was stirred for 4 h at 08C and
additionally for 8 h at 238C. The mixture was diluted with water
(100 mL) and extracted with tert-butyl methyl ether (MTB, 3”17 mL).
The combined organic layers were cooled to 08C and the pH was adjust-
ed to pH 3–4 by using a concentrated solution of HCl. Asolid product
precipitated and was separated by filtration. After removal of the solvent
under reduced pressure, a product (623 mg, 1.2 mmol, 72%) was ob-
tained as a white solid. To a solution of this crude product (51.8 mg,
0.38 mmol) in dichloromethane (7 mL) were added amine 11 (200 mg,
0.38 mmol), DCC (95 mg, 0.46 mmol, 1.2 equiv) and DMAP (8 mg,
0.07 mmol, 0.17 equiv) at 08C, according to the general DCC method.
The reaction mixture was stirred for 2 h at 08C and additionally for 12 h
at 238C. Removing the drying agent and solvent delivered a crude prod-
uct (1.82 g, 5.2 mmol, 80%) which was dissolved in dichloromethane
(15 mL). To the solution was added TFA(21 mL) at 0 8C and the reaction
mixture was stirred for 1 h at 238C. After removal of the solvent and
column chromatography (dichloromethane/MeOH 80:1!20:1), 12k
(152 mg, 0.24 mmol, 62%) was obtained as a white solid. [a]2D0 =43.58
(c=0.78 in CH2Cl2); 1H NMR (400 MHz, CDCl3): d=0.92 (d, J=7.0 Hz,
6H), 1.20–2.2 (m, 9H), 2.75–3.4 (m, 8H), 3.65–3.71 (m, 2H), 4.21 (m,
2H), 4.38 (m, 2H), 4.68 (m, 1H), 5.18 (m, 1H), 6.13 (m, 2H), 6.83 (m,
1H), 7.30 (tq, J=7.5, 1.1 Hz, 2H), 7.39 (m, 2H), 7.59 (m, 2H), 7.75 ppm
(d, J=7.5 Hz, 2H); 13C NMR (100.6 MHz, CDCl3): d=18.0 (CH3), 18.1
(CH3), 21.8 (CH3), 23.1 (CH3), 24.7 (CH2), 24.9 (CH2), 28.0 (Cq), 28.2
(Cq), 42.1 (CH2), 43.7 (CH), 43.9 (CH), 46.0 (CH), 46.3 (CH), 46.3 (CH),
46.6 (CH), 46.6 (CH), 47.0 (CH), 47.1 (CH2), 47.2 (CH), 48.3 (CH2), 48.3
(CH2), 48.6 (CH2), 48.8 (CH2), 51.8 (CH2), 51.9 (CH2), 53.4 (CH), 57.8
(CH), 57.9 (CH), 67.0 (CH2), 119.9 (CH), 125.1 (CH), 125.2 (CH), 127.1
(CH), 127.7 (CH), 134.8 (CH), 135.2 (CH), 135.9 (CH), 136.2 (CH),
141.3 (Cq), 143.7 (Cq), 144.0 (Cq), 156.0 (Cq), 168.8 (Cq), 168.9 (Cq), 170.3
(Cq), 171.3 ppm (Cq); IR (ATR): n˜ =3281, 3063, 2962, 2871, 1719, 1633,
1537, 1450, 1245, 741 cmꢀ1; ESIMS: m/z: calcd for C38H46O5N4Na: 661.34;
found: 661.0 [M++Na].
Fmoc-Lys(Boc)-Gly-Gly-cyclo-olefin (12g): According to the general
A
TBTU method B, TFAsalt 19 (36.3 mg, 0.1 mmol) in DMF (0.2 mL),
TBTU (32.1 mg, 0.1 mmol, 1 equiv) and DIPEA(38.3 mL, 0.22 mmol,
2.2 equiv) were added to a solution of Fmoc-protected (Boc)-lysine
(46.9 mg, 0.1 mmol) in DMF (0.3 mL) at 08C. The reaction mixture was
stirred for 0.5 h at 08C and additionally for 1 h at 238C. After aqueous
workup and column chromatography (dichloromethane/MeOH 50:1!
25:1), 12e (52.8 mg, 0.075 mmol, 75%) was obtained as a white crystal-
line solid. M.p. 94–968C; [a]2D0 =~8.88 (c=0.89 in CH2Cl2); 1H NMR
(400 MHz, CDCl3): d=1.32–1.52 (m, 14H), 1.55 (d, J=8.5 Hz, 1H),
1.62–1.91 (m, 2H), 2.82–3.01 (m, 5H), 3.02–3.28 (m, 2H), 3.29–3.34 (m,
2H), 3.80 (d, J=3.0 Hz, 2H), 3.97 (m, 2H), 4.19 (m, 2H), 4.38 (m, 2H),
4.88 (s, 1H), 5.67 (s, 1H), 6.13 (m, 2H), 6.98 (s, 1H), 7.05 (s, 1H), 7.39 (t,
J=7.5 Hz, 2H), 7.58 (t, J=7.5 Hz, 2H), 7.64 (m, 2H), 7.75 ppm (d, J=
7.5 Hz, 2H); 13C NMR (100.6 MHz, CDCl3): d=22.5 (CH2), 28.5 (CH3),
29.6 (CH2), 31.9 (CH2), 39.9 (CH2), 42.0 (CH2), 42.7 (CH2), 43.8 (CH),
45.8 (CH), 46.6 (CH), 47.1 (CH), 47.9 (CH2), 48.3 (CH2), 51.9 (CH2), 53.4
(Cq), 55.0 (CH), 67.1 (CH2), 79.1 (Cq), 119.9 (CH), 125.1 (CH), 127.1
(CH), 127.7 (CH), 134.8 (CH), 136.0 (CH), 141.3 (Cq), 143.8 (Cq), 143.8
(Cq), 156.2 (Cq), 165.1 (Cq), 168.4 (Cq), 172.2 ppm (Cq); IR (ATR): n˜ =
3306, 3064, 2969, 2938, 2869, 1702, 1640, 1526, 1451, 1249, 741 cmꢀ1
;
ESIMS: m/z: calcd for C39H49O7N5Na: 722.36; found: 722.0 [M++Na],
600.0.
Fmoc-Cys(Trt)-Gly-Gly-cyclo-olefin (12h): According to the general
A
TBTU method B, TFAsalt 19 (36.3 mg, 0.1 mmol) in DMF (0.25 mL),
TBTU (32.1 mg, 0.1 mmol, 1 equiv) and DIPEA(38.3 mL, 0.22 mmol,
2.2 equiv) were added to a solution of Fmoc-protected (Trt)-cystein
(58.6 mg, 0.1 mmol) in dichloromethane (0.4 mL) at 08C. The reaction
mixture was stirred for 0.5 h at 08C and additionally for 1 h at 238C.
After aqueous workup and column chromatography (dichloromethane/
MeOH 40:1), 12e (54.9 mg, 0.067 mmol, 67%) was obtained as a white
crystalline solid. M.p. 125–1278C; [a]2D0 =+1.58 (c=0.90 in CH2Cl2);
1H NMR (400 MHz, CDCl3): d=1.42 (d, J=8.5 Hz, 1H), 1.54 (m, 1H),
2.68 (m, 2H), 2.82–2.99 (m, 5H), 3.15–3.30 (m, 3H), 3.74–3.85 (m, 3H),
3.86–3.92 (m, 2H), 4.19 (t, J=6.7 Hz, 1H), 4.38 (d, J=6.7 Hz, 2H), 5.18
(t, J=6.6 Hz, 1H), 6.13 (m, 2H), 6.62 (s, 1H), 6.93 (s, 1H), 7.12–7.21 (m,
3H), 7.21–7.32 (m, 8H), 7.34–7.43 (m, 8H), 7.57 (t, J=7.0 Hz, 2H),
7.74 ppm (t, J=7.0 Hz, 2H); 13C NMR (100.6 MHz, CDCl3): d=33.8
(CH2), 38.6 (CH), 42.0 (CH2), 42.7 (CH2), 43.8 (CH), 45.8 (CH), 46.6
(CH), 47.1 (CH), 47.8 (CH2), 48.2 (CH2), 51.9 (CH2), 53.4 (CH2), 53.4
(CH), 67.1 (Cq), 67.3 (CH2), 119.9 (CH), 125.1 (CH), 125.2 (CH), 126.9
(CH), 127.1 (CH), 127.7 (CH), 128.1 (CH), 129.6 (CH), 134.8 (CH),
136.0 (CH), 141.2 (Cq), 143.7 (Cq), 143.8 (Cq), 144.3 (Cq), 156.0 (Cq),
165.0 (Cq), 168.0 (Cq), 170.3 ppm (Cq); IR (ATR): n˜ =3291, 3058, 3032,
2966, 2942, 2870, 1714, 1632, 1521, 1445, 1246, 741 cmꢀ1; ESIMS: m/z:
calcd for C50H48O5N4SNa: 839.34; found: 638.9 [M++Na].
Dansyl-cyclo-olefin (17): Asolution of NaHCO (58.8 mg, 0.70 mmol,
3
2 equiv) and 11 (47.3 mg, 0.35 mmol) in water (2.7 mL) were added to a
solution of dansylchloride (94.4 mg, 0.35 mmol) in acetonitrile (7 mL).
The reaction mixture was stirred for 1.5 h at 238C. The solvents were re-
moved under reduced pressure. After chromatography on silica gel (di-
chloromethane/MeOH 50:1), 17 (75.2 mg, 0.2 mmol, 58%) was obtained
1
as a light-green solid. H NMR (500 MHz, CDCl3): d=1.34 (d, J=8.4 Hz,
1H), 1.47 (dt, J=8.4 Hz, J=1.6 Hz, 1H), 2.80 (m, 2H), 2.81–2.95 (m,
13H), 3.27–3.41 (m, 2H), 5.87 (AB-syst., 2H), 7.18 (dd, J=7.2 Hz,
1.0 Hz, 1H), 7.43–7.57 (m, 2H), 8.16 (dd, J=7.2 Hz, 1.0 Hz, 1H), 8.46 (d,
J=9.0 Hz, 1H), 8.54 ppm (dd, J=9.0 Hz, 1.0 Hz, 1H); 13C NMR
(100.6 MHz, CDCl3): d=45.5 (CH3), 45.7 (CH3), 46.2 (CH), 49.8 (CH2),
52.2 (CH2), 115.2 (CH), 120.4 (CH), 123.2 (CH), 127.7 (CH), 130.0 (CH),
130.2 (Cq), 130.5 (CH), 130.7 (Cq), 133.8 (Cq), 135.5 (CH), 151.5 ppm
(Cq); IR (ATR): n˜ =3058, 2962, 2940, 2942, 2867, 2832, 2788, 1588, 1573,
1519, 1477, 1454, 1334, 1320, 1162, 1145, 793 cmꢀ1; MS (EI): m/z (%): 368
(16), 171 (100), 170 (24), 154 (6), 69 (7); HRMS: calcd for C21H24N2O2S:
368.1559; found: 368.1561 [M+].
Fmoc-Cys-Gly-Gly-cyclo-olefin (12i): TFA(21 mL, 0.274 mmol, 5 equiv)
was added to a solution of 12 h (45.3 mg, 0.055 mmol) and triethylsilane
(44 mL, 0.274 mmol, 5 equiv) in dichloromethane (0.2 mL) at 08C. The re-
action mixture was stirred for 2 h at 238C. After chromatography on
silica gel (dichloromethane/MeOH 25:1) and Sephadex, 12i (28.9 mg,
0.051 mmol, 92%) was obtained as a white crystalline solid. [a]2D0 =5.28
(c=1.45 in CH2Cl2); 1H NMR (400 MHz, CDCl3): d=1.40 (d, J=8.5 Hz,
1H), 1.54 (m, 1H), 1.54 (m, 1H), 2.75–3.10 (m, 7H), 3.15–3.35 (m, 3H),
3.82 (d, J=2.6 Hz, 2H), 4.02 (s, 2H), 4.20 (t, J=6.9 Hz, 1H), 4.35–4.55
(m, 3H), 6.10 (m, 2H), 7.19 (s, 1H), 7.29 (m, 2H), 7.39 (t, J=7.5 Hz,
2H), 7.58 (m, 2H), 7.74 ppm (d, J=7.5 Hz, 2H); 13C NMR (100.6 MHz,
CDCl3): d=27.2 (CH2), 42.0 (CH2), 43.0 (CH2), 43.8 (CH), 45.9 (CH),
46.7 (CH), 47.2 (CH), 48.0 (CH2), 48.4 (CH2), 52.0 (CH2), 53.4 (CH2),
56.3 (CH), 67.3 (CH2), 120.0 (CH), 125.2 (CH), 127.2 (CH), 127.8 (CH),
134.9 (CH), 136.1 (CH), 141.4 (Cq), 143.8 (Cq), 143.8 (Cq), 165.2 (Cq),
168.4 (Cq), 170.3 ppm (Cq); IR (ATR): n˜ =3298, 3064, 2965, 2935, 2871,
Cross-partners for ROCM
Fmoc-Gly-hexenol (13a): According to the general DCC method, 5-
hexen-1-ol (117.8 mL, 0.93 mmol; 1.11 equiv), DCC (208 mg, 1 mmol,
1.2 equiv) and DMAP (10.3 mg, 0.08 mmol, 0.1 equiv) were added succes-
sively to a solution of Fmoc-protected glycine (250 mg, 0.84 mmol) in
THF (3 mL) at 08C. The reaction mixture was stirred for 2 h at 08C and
additionally for 12 h at 238C. After filtration, aqueous workup and
column chromatography (hexanes/ethyl acetate 5:1), 13a (255 mg,
0.69 mmol, 80%) was obtained as
a
white waxlike solid. 1H NMR
(400 MHz, CDCl3): d=1.46 (m, 2H), 1.67 (m, 2H), 2.08 (tq, J=7.0,
1.3 Hz, 2H), 4.00 (d, J=5.5 Hz, 2H), 4.18 (t, J=6.7 Hz, 2H), 4.24 (t, J=
7.0 Hz, 1H), 4.40 (d, J=7.0 Hz, 2H), 4.93–5.06 (m, 2H), 5.28 (t, J=
4.5 Hz, 1H), 5.78 (m, 1H), 7.32 (dt, J=7.5, 1.2 Hz, 2H), 7.41 (t, J=
7.5 Hz, 2H), 7.60 (d, J=7.5 Hz, 2H), 7.76 ppm (d, J=7.5 Hz, 2H);
1717, 1636, 1529, 1451, 1248, 742 cmꢀ1
C31H34O5N4SNa: 597.22; found: 597.0 [M++Na].
; ESIMS: m/z: calcd for
Chem. Eur. J. 2007, 13, 2358 – 2368
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
2365

S. Blechert and S. Michaelis
13C NMR (100.6 MHz, CDCl3): d=25.0 (CH2), 27.9 (CH2), 33.1 (CH2),
42.8 (CH2), 47.1 (CH), 65.5 (CH2), 67.2 (CH2), 115.0 (CH2), 120.0 (CH),
125.1 (CH), 127.1 (CH), 127.7 (CH), 138.1 (CH), 141.3 (Cq), 143.8 (Cq),
156.2 (Cq), 170.1 ppm (Cq); IR (ATR): n˜ =3417, 3339, 3066, 3041, 3018,
2937, 2858, 1749, 1727, 1532, 1451, 1195, 1052, 741 cmꢀ1; LRMS (70 eV,
EI): m/z (%): 379 (100), 252 (20), 250 (5), 178 (>100); HRMS: calcd for
C23H25O4N: 379.1784; found: 379.1789 [M+].
169.5 (Cq), 170.4 ppm (Cq); IR (ATR): n˜ =3306, 3062, 3032, 3019, 2973,
2935, 2859, 1701, 1648, 1529, 1446, 1245, 1207, 741 cmꢀ1; ESI-MS: m/z:
calcd for C47H47O6N3SNa: 804.32; found: 803.9 [M++Na].
AHCTREUNG
a
8.69 mmol) in methanol (80 mL) at 08C. The reaction mixture was stirred
for 12 h at 238C. After removal of the volatile compounds under reduced
pressure, the crude product was dissolved in acetonitrile (33 mL). DMAP
(0.2 g, 1.74 mmol, 0.2 equiv), (Boc)2O (2.01 g, 9.56 mmol, 1.1 equiv) and
NEt3 (1.5 mL) were added to this solution. The reaction mixture was stir-
red for 2 h at 238C and was then washed sequentially with NaHCO3 solu-
tion, water and NaCl solution. After removal of the solvent under
vacuum and silica gel chromatography (hexanes/MTB 6:1), 15 (0.62 g,
2.6 mmol, 30%) was obtained as a white solid. 1H NMR (400 MHz,
CDCl3): d=1.43 (s, 9H), 2.50 (m, 2H), 3.79 (s, 3H), 4.37 (q, J=7.4 Hz,
1H), 5.02 (d, J=7.4 Hz, 1H), 5.16 (m, 2H), 5.67 ppm (m, 1H); 13C NMR
(100.6 MHz, CDCl3): d=28.3 (CH3), 28.5 (CH3), 36.8 (CH2), 52.2 (CH3),
52.9 (CH), 79.9 (Cq), 119.1 (CH2), 132.3 (CH), 155.2 (Cq), 172.6 ppm
(Cq); IR (ATR): n˜ =3439, 3359, 3080, 3005, 2979, 2955, 2933, 2848, 1747,
1716, 1504, 1438, 1367, 1163 cmꢀ1; LRMS (70 eV, EI): m/z (%): 189 (80),
148 (74), 146 (32), 81 (47), 69 (100), 57 (71); HRMS: calcd for
C8H15O4N: 189.1001; found: 189.1001 [M+ꢀC3H4].
Fmoc-Leu-Ala-Pro-hexenol (13b): According to the general DCC
method, 5-hexen-1-ol (50.8 mL, 0.38 mmol; 1.11 equiv), DCC (158.2 mg,
0.76 mmol, 2 equiv) and DMAP (7.9 mg, 0.07 mmol, 0.17 equiv) were
added successively to
a solution of Fmoc-Leu-Ala-Pro-OH (200 mg,
0.38 mmol) in dichloromethane (2 mL) at 08C. The reaction mixture was
stirred for 2 h at 08C and additionally for 12 h at 238C. After filtration,
aqueous workup and column chromatography (hexanes/ethyl acetate
2:1), 13b (117 mg, 0.19 mmol, 50%) was obtained as a white waxlike
solid. [a]2D0 =~58.18 (c=1.1 in CH2Cl2); 1H NMR (400 MHz, CDCl3): d=
0.92 (d, J=5.6 Hz, 6H), 1.20–1.70 (m, 10H), 1.92–2.12 (m, 5H), 2.18–2.29
(m, 1H), 3.58–3.71 (m, 2H), 4.03–4.17 (m, 2H), 4.18–4.26 (m, 2H), 4.33–
4.42 (m, 2H), 4.51 (m, 1H), 4.70 (m, 1H), 4.91–5.03 (m, 2H), 5.21 (d, J=
7.0 Hz, 1H), 5.77 (m, 1H), 6.81 (d, J=6.3 Hz, 1H), 7.33 (t, J=7.5 Hz,
2H), 7.39 (t, J=7.5 Hz, 2H), 7.59 (m, 2H), 7.76 ppm (d, J=7.5 Hz, 2H);
13C NMR (100.6 MHz, CDCl3): d=18.0 (CH3), 21.8 (CH3), 23.1 (CH3),
24.7 (CH), 24.9 (CH2), 25.1 (CH2), 28.0 (CH2), 29.0 (CH2), 33.2 (CH2),
42.1 (CH2), 46.8 (CH2), 46.9 (CH), 53.5 (CH), 58.9 (CH), 65.2 (CH2), 67.0
(CH2), 114.9 (CH2), 119.9 (CH), 120.0 (CH), 125.1 (CH), 127.1 (CH),
127.7 (CH), 138.2 (CH), 141.3 (Cq), 143.7 (Cq), 143.9 (Cq), 156.0 (Cq),
170.7 (Cq), 171.4 (Cq), 171.8 ppm (Cq); IR (ATR) n˜ =3282, 3068, 2955,
2935, 2870, 1744, 1719, 1638, 1541, 1451, 1241, 741 cmꢀ1; ESIMS: m/z:
calcd for C35H45O6N3Na: 626.32; found: 626.0 [M++Na].
ROCMs
General procedure for ROCM: The cyclic olefin, the cross-partner for
metathesis (1 equiv) and the Ru catalyst (1 to 5 mol%) were placed in a
screw-top HPLC vial (1.5 mL) and dissolved in dichloromethane (or 1,2-
dichlorethane, DCE) to get a 0.1m solution. The reaction mixture was
stirred for the mentioned time at 408C (708C, respectively). The conver-
sion was monitored by HPLC. The solvents were removed under vacuum
and the crude product was purified by column chromatography and, if
noted, additionally by Sephadex chromatography.
Fmoc-Cys(Trt)-Gly-Gly-hexenol (13c): Aqueous NaOH (10 mL, 1n) and
A
(Boc)2O (2.4 g, 11 mmol, 1.1 equiv) were added to a solution of GlyGly
(1.32 g, 10 mmol) in dioxane and water (2:1, 30 mL) at 08C. The reaction
mixture was stirred for 1 h at 238C. The dioxane was removed under re-
duced pressure. The aqueous layer was diluted with ethyl acetate
(20 mL) and the pH was adjusted to pH 2–3 by using KHSO4. The layers
were separated and the aqueous layer was extracted with ethyl acetate
(5”20 mL). The combined organic layers were washed with water
(50 mL) and dried over Na2SO4. Removing the drying agent by filtration
and the solvent under reduced pressure delivered a crude product (1.5 g,
6.5 mmol, 65%). 5-Hexen-1-ol (1 mL, 8.45 mmol, 1.3 equiv), DCC
(1.61 g, 7.8 mmol, 1.2 equiv) in dichloromethane (10 mL) and DMAP
(140 mg, 1.1 mmol, 0.17 equiv) were added to a solution of the crude
product in THF (26 mL) at 08C, according to the general DCC method.
The reaction mixture was stirred for 2 h at 08C and additionally for 12 h
at 238C. Removal of the drying agent and solvent delivered a crude prod-
uct (1.92 g, 6.1 mmol, 72%). Aportion of the crude product (650 mg,
2.07 mmol) was dissolved in dichloromethane (6 mL). To this solution
was added TFA(8 mL) at 0 8C and the reaction mixture was stirred for
15 min at 08C and additionally for 45 min at 238C. After removing of the
volatile compounds and column chromatography (dichloromethane/
MeOH 25:1!10:1), 20 (0.66 g, 2 mmol, 97%) was obtained as a yellow
oil. Aportion of this oil (200 mg, 0.61 mmol) was dissolved in dichloro-
Metathesis product (14a): According to the general procedure for
ROCM, 12a (10.36 mg, 0.025 mmol), 13a (9.49 mg, 0.025 mmol) and [Ru-
3] (0.3 mg, 0.5 mmol,
2 mol%) were dissolved in dichloromethane
(0.25 mL) and stirred for 17 h at 408C. The solvent was removed under
reduced pressure. After chromatography on silica gel (dichloromethane/
MeOH 50:1!25:1) and Sephadex, 14a (15.88 mg, 0.02 mmol, 80%) was
obtained as a white crystalline solid. M.p. 65–678C; IR (ATR): n˜ =3293,
3065, 3039, 2953, 2934, 2872, 1721, 1639, 1534, 1451, 1244, 742 cmꢀ1
;
ESIMS: m/z: calcd for C49H52O7N3: 794.37; found: 794.2 [M++H], 816.2
[M++Na], 572.2 [M++HꢀFmoc], 515.2 [M++HꢀFmocGly], 293.2 [M+
+HꢀFmocGlyꢀFmoc], 350.2 [M++Hꢀ2Fmoc].
Metathesis product (14b): According to the general procedure for
ROCM, 12b (12.62 mg, 0.025 mmol), 13a (9.49 mg, 0.025 mmol) and
[Ru-3] (0.3 mg, 0.5 mmol, 2 mol%) were dissolved in dichloromethane
(0.25 mL) and stirred for 17 h at 408C. The solvent was removed under
reduced pressure. After chromatography on silica gel (dichloromethane/
MeOH 50:1!25:1) and Sephadex, 14b (16.3 mg, 0.019 mmol, 74%) was
obtained as a white crystalline solid. [a]2D0 =12.08 (c=0.82 in CH2Cl2); IR
(ATR): n˜ =3315, 3065, 3039, 2927, 2855, 1717, 1631, 1527, 1451, 1248,
741 cmꢀ1; ESIMS: m/z: calcd for C56H58O7N3: 884.42; found: 884.3 [M+
+H], 906.3 [M++Na], 662.3 [M++HꢀFmoc], 515.2 [M++HꢀFmocPhe],
293.2 [M++HꢀFmocPheꢀFmoc], 440.2 [M++Hꢀ2Fmoc].
Metathesis product (14c): According to the general procedure for
ROCM, 12c (11.42 mg, 0.025 mmol), 13a (9.49 mg, 0.025 mmol) and [Ru-
3] (0.3 mg, 0.5 mmol, 2 mol%) were dissolved in dichloromethane
(0.25 mL) and stirred for 17 h at 408C. The solvent was removed under
reduced pressure. After chromatography on silica gel (dichloromethane/
MeOH 50:1!25:1) and Sephadex, 14c (17.2 mg, 0.021 mmol, 82%) was
obtained as a white crystalline solid. [a]2D0 =~4.28 (c=0.86 in CH2Cl2); IR
(ATR): n˜ =3321, 3066, 3040, 2958, 2935, 2872, 1719, 1630, 1527, 1450,
1196, 741 cmꢀ1; ESIMS: m/z: calcd for C52H58O7N3: 836.42; found: 836.3
[M++H], 858.3 [M++Na], 614.3 [M++H-Fmoc], 515.2 [M++H-Fmoc-
Val], 293.2 [M++HꢀFmocPheꢀFmoc], 640.3 [M++NaꢀFmoc].
methane and DMF (2:1, 3 mL). Fmoc-Cys(Trt) (356.7 mg, 0.61 mmol),
A
TBTU (234.7 mg, 0.73 mmol, 1.2 equiv) and DIPEA(234 mL, 1.34 mmol,
2.2 equiv) were added to this solution at 08C. The reaction mixture was
stirred for 0.5 h at 08C and additionally for 1 h at 238C. After filtration,
aqueous workup and column chromatography (hexanes/ethyl acetate
1:1), 13c (248 mg, 0.31 mmol, 50%) was obtained as a white crystalline
solid. [a]2D0 =+2.38 (c=1.0 in CH2Cl2); 1H NMR (400 MHz, CDCl3): d=
1.42 (m, 2H), 1.60 (m, 2H), 2.06 (tq, J=6.4, 1.3 Hz, 2H), 2.64–2.73 (m,
2H), 3.65 (q, J=6.4 Hz, 1H), 3.80–3.99 (m, 4H), 4.07 (t, J=6.7 Hz, 2H),
4.18 (t, J= Hz, 1H), 4.41 (m, 2H), 4.91–5.03 (m, 2H), 5.77 (m, 1H), 6.36
(t, J=5.8 Hz, 1H), 6.82 (m, 1H), 7.20 (tt, J=7.2, 1.3 Hz, 3H), 7.23–7.32
(m, 8H), 7.39 (m, 8H), 7.57 (m, 2H), 7.75 ppm (m, 2H); 13C NMR
(100.6 MHz, CDCl3): d=25.0 (CH2), 27.9 (CH2), 33.2 (CH2), 33.4 (CH2),
38.6 (CH), 40.9 (CH2), 43.0 (CH2), 47.1 (CH), 53.4 (Cq), 54.3 (CH), 65.4
(CH2), 67.0 (Cq), 67.5 (CH2), 115.0 (CH2), 120.0 (CH), 124.9 (CH), 125.0
(CH), 127.1 (CH), 127.8 (CH), 127.8 (CH), 128.1 (CH), 129.5 (CH),
138.2 (CH), 141.3 (Cq), 143.6 (Cq), 144.2 (Cq), 156.2 (Cq), 168.6 (Cq),
Metathesis product (14d): According to the general procedure for
ROCM, 12d (13.2 mg, 0.025 mmol), 13a (9.49 mg, 0.025 mmol) and [Ru-
3] (0.15 mg, 0.25 mmol, 1 mol%) were dissolved in dichloromethane
(0.25 mL) and stirred for 17 h at 408C. The solvent was removed under
2366
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ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2007, 13, 2358 – 2368

Ligation of Peptides
FULL PAPER
reduced pressure. After chromatography on silica gel (dichloromethane/
MeOH 50:1!25:1) and Sephadex, 14d (17.0 mg, 0.019 mmol, 75%) was
obtained as a white crystalline solid. M.p. 95–978C; [a]2D0 =~0.18 (c=0.75
in CH2Cl2); IR (ATR): n˜ =3313, 3066, 3041, 3017, 2937, 2891, 1724, 1642,
1529, 1451, 1250, 741 cmꢀ1; ESIMS: m/z: calcd for C53H58O9N5: 908.42;
found: 908.2 [M++H], 930.2 [M++Na], 686.2 [M++HꢀFmoc], 572.2
[M++HꢀFmocGlyꢀGly], 515.2 [M++HꢀFmocGlyꢀ2Gly], 293.2 [M+
+HꢀFmocGlyꢀ2GlyꢀFmoc], 708.2 [M++NaꢀFmoc], 684.2 [M+
+Naꢀ2Fmoc].
754.2 [M++NaꢀFmoc], 515.2 [M++HꢀFmocCysꢀ2Gly], 293.2 [M+
+HꢀFmocCysꢀ2GlyꢀFmoc].
Metathesis product (14k): According to the general procedure for
ROCM, 12e (9.3 mg, 0.015 mmol), 13b (9.1 mg, 0.015 mmol) and [Ru-3]
(0.18 mg, 0.3 mmol, 2 mol%) were dissolved in dichloromethane
(0.15 mL) and stirred for 17 h at 408C. The solvent was removed under
reduced pressure. After chromatography on silica gel (dichloromethane/
MeOH 50:1!25:1) and Sephadex, 14k (15.1 mg, 0.012 mmol, 82%) was
obtained as a white crystalline solid. M.p. 91–938C; [a]2D0 =~31.28 (c=
0.76 in CH2Cl2); IR (ATR): n˜ =3293, 3066, 2953, 2932, 2871, 1720, 1639,
1534, 1451, 1246), 741 cmꢀ1; ESIMS: m/z: calcd for C72H83O11N7Na:
1244.61; found: 1244.3 [M++Na], 1022.3 [M++NaꢀFmoc], 816.2 [M+
Metathesis product (14e): According to the general procedure for
ROCM, 12e (15.5 mg, 0.025 mmol), 13a (9.49 mg, 0.025 mmol) and [Ru-
3] (0.15 mg, 0.25 mmol, 1 mol%) were dissolved in dichloromethane
(0.25 mL) and stirred for 17 h at 408C. The solvent was removed under
reduced pressure. After chromatography on silica gel (dichloromethane/
MeOH 50:1!25:1) and Sephadex, 14e (20.7 mg, 0.021 mmol, 83%) was
obtained as a white crystalline solid. M.p. 758C; [a]2D0 =~6.38 (c=1.0 in
CH2Cl2); IR (ATR): n˜ =3303, 3065, 3039, 3000, 2941, 2893, 1723, 1640,
1528, 1451, 1250, 741 cmꢀ1; ESIMS: m/z: calcd for C60H64O9N5: 998.47;
found: 998.2 [M++H], 1020.2 [M++Na], 572.2 [M++HꢀFmocPheꢀGly],
+HꢀFmocLeuAla],
390.2
[M++HꢀFmocLeuAlaꢀFmocPheꢀGly],
333.1 [M++HꢀFmocLeuAlaꢀFmocPheꢀ2Gly].
Metathesis product (14l): According to the general procedure for
ROCM, 12k (9.6 mg, 0.015 mmol), 13c (11.7 mg, 0.015 mmol) and [Ru-3]
(0.18 mg, 0.3 mmol, 2 mol%) were dissolved in dichloromethane
(0.15 mL) and stirred for 17 h at 408C. The solvent was removed under
reduced pressure. After chromatography on silica gel (dichloromethane/
MeOH 50:1!25:1) and Sephadex, 14l (14 mg, 0.01 mmol, 66%) was ob-
tained as a white crystalline solid. M.p. 105–1068C; [a]2D0 =~14.08 (c=0.7
in CH2Cl2); IR (ATR): n˜ =3294, 3064, 3018, 2953, 2935, 2870, 1720, 1653,
1637, 1532, 1449, 1246, 741 cmꢀ1; ESIMS: m/z: calcd for C85H93O11N7SNa:
1442.66; found: 1442.3 [M++Na], 1199.2 [M++NaꢀTrt], 1199.2 [M+
+HꢀFmocLeuAla], 977.2 [M++NaꢀTrtꢀFmoc], 772.2 [M++HꢀFmoc-
LeuAlaꢀTrt].
Metathesis product (14m): According to the general procedure for
ROCM, 12k (9.6 mg, 0.015 mmol), 13b (9.1 mg, 0.015 mmol) and [Ru-3]
(0.18 mg, 0.3 mmol, 2 mol%) were dissolved in dichloromethane
(0.15 mL) and stirred for 17 h at 408C. The solvent was removed under
reduced pressure. After chromatography on silica gel (dichloromethane/
MeOH 50:1!25:1) and Sephadex, 14m (13.9 mg, 0.011 mmol, 74%) was
obtained as a white crystalline solid. M.p. 110–1128C; [a]2D0 =~45.28 (c=
0.7 in CH2Cl2); IR (ATR): n˜ =3287, 3066, 2954, 2871, 1721, 1639, 1537,
1451, 1243, 741 cmꢀ1; ESIMS: m/z: calcd for C73H91O11N7Na: 1264.67;
found: 1264.3 [M++Na], 1042.3 [M++NaꢀFmoc], 836.3 [M++H-Fmo-
cLeuAla], 739.2 [M++H-FmocLeuAlaPro], 430.2 [M++H-FmocLeuAla-
FmocLeuAla], 333.1 [M++H-FmocLeuAlaPro-FmocLeuAla].
515.2
[M++HꢀFmocPheꢀ2Gly],
293.2
[M++HꢀFmoc-
Pheꢀ2GlyꢀFmoc], 798.2 [M++NaꢀFmoc].
Metathesis product (14 f): According to the general procedure for
ROCM, 12 f (14.6 mg, 0.025 mmol), 13a (9.49 mg, 0.025 mmol) and [Ru-
3] (0.15 mg, 0.25 mmol, 1 mol%) were dissolved in dichloromethane
(0.25 mL) and stirred for 17 h at 408C. The solvent was removed under
reduced pressure. After chromatography on silica gel (dichloromethane/
MeOH 50:1!25:1) and Sephadex, 14 f (8.9 mg, 0.009 mmol, 37%) was
obtained as a white crystalline solid. M.p. 87–898C); [a]2D0 =2.98 (c=0.45
in CH2Cl2); IR (ATR): n˜ =3321, 3065, 3041, 3017, 2933, 2855, 1720,
1669, 1643, 1531, 1451, 1252, 741 cmꢀ1
; ESIMS: m/z: calcd for
C55H61O10N6: 965.44; found: 965.2 [M++H], 987.2 [M++Na], 629.3
[M++HꢀFmocAsn], 572.2 [M++HꢀFmocAsnꢀGly], 515.2 [M++
HꢀFmocPheꢀ2Gly], 765.2 [M++NaꢀFmoc].
Metathesis product (14g): According to the general procedure for
ROCM, 12g (17.5 mg, 0.025 mmol), 13a (9.49 mg, 0.025 mmol) and [Ru-
3] (0.75 mg, 1.25 mmol, 5 mol%) were dissolved in dichloromethane
(0.25 mL) and stirred for 17 h at 408C. The solvent was removed under
reduced pressure. After chromatography on silica gel (dichloromethane/
MeOH 50:1!25:1) and Sephadex, 14g (21.6 mg, 0.02 mmol, 80%) was
obtained as a white crystalline solid. M.p. 63–658C; [a]2D0 =~4.78 (c=1.1
in CH2Cl2); IR (ATR): n˜ =3316, 3066, 3040, 2972, 2937, 2864, 1710, 1642,
1524, 1451, 1270, 741 cmꢀ1; ESIMS: m/z: calcd for C62H76O11N6: 1080.55;
found: 1079.2 [M++H], 1101.2 [M++Na], 979.3 [M++HꢀBoc], 757.2
[M++HꢀBocꢀFmoc], 572.2 [M++HꢀBocꢀFmocLysꢀGly], 515.2
[M++HꢀBocꢀFmocLysꢀ2Gly], 293.2 [M++HꢀBocꢀFmocLysꢀGlyꢀ
2Fmoc], 779.2 [M++NaꢀBocꢀFmoc].
Metathesis product (16b): According to the general procedure for
ROCM, 12d (26.4 mg, 0.05 mmol), 15 (22.9 mg, 0.1 mmol, 2 equiv) and
[Ru-4] (0.03 mg, 0.5 mmol, 1 mol%) were dissolved in dichloromethane
(0.15 mL) and stirred for 17 h at 408C. The solvent was removed under
reduced pressure. After chromatography on silica gel (dichloromethane/
MeOH 50:1!25:1) and Sephadex, 16b (12.5 mg, 0.017 mmol, 33%) was
obtained as a white crystalline solid. IR (ATR): n˜ =3309, 3069, 2974,
2950, 2884, 1709, 1642, 1525, 1451, 1249, 742 cmꢀ1; ESIMS: m/z: calcd for
C41H51O9N5Na: 780.36; found: 780.1 [M++Na], 724.1 [M++NaꢀtBu],
680.1 [M++NaꢀBoc], 658.2 [M++HꢀBoc], 458.1 [M++Naꢀ
Metathesis product (14h): According to the general procedure for
ROCM, 12h (20.4 mg, 0.025 mmol), 13a (9.49 mg, 0.025 mmol) and [Ru-
3] (0.15 mg, 0.25 mmol, 1 mol%) were dissolved in dichloromethane
(0.25 mL) and stirred for 17 h at 408C. The solvent was removed under
reduced pressure. After chromatography on silica gel (dichloromethane/
MeOH 50:1!25:1) and Sephadex, 14h (24.7 mg, 0.021 mmol, 83%) was
obtained as a white crystalline solid. M.p. 84–868C; [a]2D0 =~0.28 (c=1.2
in CH2Cl2); IR (ATR): n˜ =3301, 3063, 3018, 2942, 2895, 1723, 1638, 1523,
1450, 1249, 742 cmꢀ1; ESIMS: m/z: calcd for C73H73O9N5SNa: 1218.50;
BocꢀFmoc],
436.2
[M++HꢀBocꢀFmoc],
322.1
[M+
+HꢀBocꢀFmocGlyꢀGly], 265.1 [M++HꢀBocꢀFmocGlyꢀ2Gly].
Metathesis product (18a): In a thoroughly dried flask, 13d (19.3 mg,
22 mmol) and a solution of [Ru-2] (0.19 mg, 0.22 mmol, 1 mol%) in di-
chloromethane (0.1 mL) were added to a solution of 17 (8.1 mg, 22 mmol)
in dichloromethane (0.4 mL) and stirred for 0.5 h at 408C under a nitro-
gen atmosphere. The solvent was removed under reduced pressure. After
preparative TLC (dichloromethane/MeOH 40:1), 18a (11.6 mg, 9.5 mmol,
43%) was obtained as a white crystalline solid. 1H NMR (500 MHz,
CDCl3): d=0.75–0.95 (m, 6H), 1.27–1.50 (m, 1H), 1.50–1.70 (m, 3H),
1.90–2.10 (m, 2H), 2.27–2.40 (m, 1H), 2.50–2.65 (m, 1H), 2.65–2.7 (m,
3H), 2.85–2.90 (m, 7H), 2.95–3.00 (m, 1H), 3.00–3.25 (m, 4H), 3.35–3.45
(m, 1H), 3.70 (s, 3H), 3.75–3.95 (m, 2H), 4.05–4.15 (m, 2H), 4.25–4.35
(m, 1H), 4.85–5.05 (m, 2H), 5.10–5.25 (m, 1H), 5.25–5.35 (m, 1H), 5.60–
5.85 (m, 1H), 6.55–6.70 (m, 2H), 6.90–6.95 (m, 1H), 7.10–7.40 (m, 28H),
7.50–7.60 (m, 2H), 8.20–8.25 (m, 1H), 8.40–8.60 ppm (m, 2H); 13C NMR
(100.6 MHz, CDCl3): d=14.2 (CH1), 18.1 (CH3), 19.2 (CH), 25.9 (CH2),
28.6 (CH2), 29.8 (CH2), 30.6 (CH), 32.1 (CH2), 33.0 (CH2), 35.0 (CH2),
36.9 (CH2), 45.5 (CH3), 49.4 (CH2), 49.5 (CH2), 54.1 (CH), 55.2 (CH3),
55.3 (CH), 56.4 (CH), 62.4 (CH2), 65.3 (CH2), 66.4 (Cq), 71.3 (Cq), 113.3
found: 1218.2 [M++Na], 975.1 [M++NaꢀTrt], 753.1
[M+
+NaꢀTrtꢀFmoc].
Metathesis product (14i): To a solution of 12i (50 mg, 0.087 mmol) in
DCE (7.25 mL) was added 13a (66 mg, 0.147 mmol, 2 equiv) and [Ru-4]
(2.7 mg, 4.35 mmol, 5 mol%) in a thoroughly dried flask, and the resulting
mixture was stirred for 1 h at 708C under a nitrogen atmosphere. After
this time, the solvent was removed under reduced pressure. After chro-
matography on silica gel (dichloromethane/MeOH 100:1!25:1) and Se-
phadex, 14i (24.9 mg, 0.026 mmol, 30%) was obtained as a white crystal-
line solid. M.p. 96–998C; [a]D20 =~3.98 (c=0.9 in CH2Cl2); IR (ATR): n˜ =
3308, 3067, 3041, 2940, 2893, 1721, 1640, 1527, 1451, 1248, 741 cmꢀ1
ESIMS: m/z: calcd for C54H59O9N5SNa: 976.39; found: 976.2 [M++Na],
;
Chem. Eur. J. 2007, 13, 2358 – 2368
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
2367

S. Blechert and S. Michaelis
(CH), 115.0 (CH2), 115.2 (CH), 115.6 (CH2), 115.7 (CH2), 120.2 (CH),
120.2 (CH), 123.2 (CH), 126.7 (CH), 126.8 (CH), 127.2 (CH), 127.3
(CH), 127.9 (CH), 127.9 (CH), 128.0 (CH), 128.0 (CH), 128.0 (CH),
129.0 (CH), 129.3 (CH), 129.4 (CH), 129.5 (CH), 130.6 (CH), 131.8
(CH), 144.9 (Cq), 145.8 (Cq), 146.9 (Cq), 147.2 (Cq), 158.2 (Cq), 170.5 (Cq),
171.7 (Cq), 174.0 ppm (Cq); IR (ATR): n˜ =3493, 3343, 3083, 3057, 3031,
2958, 2931, 2871, 2849, 2791, 1738, 1653, 1607, 1588, 1575, 1508, 1251,
1182, 1161, 1144, 1033, 744, 706 cmꢀ1; FABMS: m/z (%)=[norm 1245]:
1245 (100) [M]+, 970 (55), [norm 243]: 273 (90), 243 (100), 147 (23), 73
(46).
1H), 8.40–8.60 ppm (m, 2H); 13C NMR (100.6 MHz, CDCl3): d=18.1
(CH3), 19.2 (CH), 25.9 (CH2), 28.6 (CH2), 29.2 (CH2), 29.5 (CH2), 29.8
(CH), 30.6 (CH), 32.1 (CH2), 33.3 (CH2), 35.0 (CH2), 36.9 (CH2), 39.4
(CH), 44.7 (CH2), 45.5 (CH3), 45.8 (CH), 46.1 (CH), 46.3 (CH), 46.7
(CH), 49.4 (CH2), 49.5 (CH2), 54.1 (CH), 55.2 (CH3), 56.3 (CH), 56.3
(CH), 57.8 (CH), 62.4 (CH2), 65.3 (CH2), 66.4 (Cq), 71.3 (Cq), 113.3
(CH), 115.0 (CH2), 115.2 (CH), 115.6 (CH2), 115.7 (CH2), 120.2 (CH),
120.2 (CH), 123.2 (CH), 126.6 (CH), 126.8 (CH), 127.2 (CH), 127.3
(CH), 127.8 (CH), 127.9 (CH), 128.0 (CH), 128.1 (CH), 129.0 (CH),
129.3 (CH), 129.4 (CH), 129.5 (CH), 130.6 (CH), 131.0 (CH), 131.0
(CH), 144.9 (Cq), 145.6 (Cq), 146.9 (Cq), 158.2 (Cq), 170.5 (Cq), 171.7 (Cq),
174.0 ppm (Cq); IR (ATR): n˜ =3493, 33.43, 3083, 3057, 3031, 2958, 2931,
2871, 2849, 2791, 1738, 1653, 1607, 1588, 1575, 1508, 1251, 1182, 1161,
1144, 1033, 744, 706 cmꢀ1; FABMS: m/z (%)=[norm 1344]: 1344 [M]+
(100), 1069 (60), 997 (23), [norm 243]: 273 (82), 243 (100), 55 (20).
Metathesis product (18b): In a thoroughly dried flask, 13e (19.9 mg,
22 mmol) and a solution of [Ru-2] (0.19 mg, 0.22 mmol, 1 mol%) in di-
chloromethane (0.1 mL) were added to
a solution of 17 (8.1 mg,
22 mmmol) in dichloromethane (0.4 mL) and stirred for 0.5 h at 408C
under nitrogen atmosphere. The solvent was removed under reduced
pressure. After preparative TLC (dichloromethane/MeOH 40:1), 18b
(18.8 mg, 14.7 mmol, 67%) was obtained as a white crystalline solid.
1H NMR (500 MHz, CDCl3): d=0.75–0.95 (m, 6H), 1.27–1.5 (m, 3H),
1.50–1.70 (m, 5H), 1.85–1.90 (m, 1H), 1.90–2.10 (m, 2H), 2.27–2.40 (m,
1H), 2.50–2.65 (m, 1H), 2.65–2.7 (m, 3H), 2.85–2.90 (m, 7H), 2.95–3.00
(m, 1H), 3.00–3.25 (m, 4H), 3.35–3.45 (m, 1H), 3.70 (s, 3H), 3.75–3.95
(m, 2H), 4.05–4.15 (m, 2H), 4.25–4.35 (m, 1H), 4.85–5.05 (m, 2H), 5.10–
5.25 (m, 1H), 5.25–5.35 (m, 1H), 5.60–5.85 (m, 1H), 6.55–6.70 (m, 2H),
6.90–6.95 (m, 1H), 7.10–7.4 (m, 28H), 7.50–7.60 (m, 2H), 8.20–8.25 (m,
1H), 8.40–8.60 ppm (m, 2H); 13C NMR (100.6 MHz, CDCl3): d=18.1
(CH3), 19.2 (CH), 25.2 (CH2), 25.7 (CH2), 25.9 (CH2), 27.3 (CH2), 28.0
(CH2), 28.1 (CH2), 28.2 (CH2), 29.8 (CH2), 30.6 (CH), 32.1 (CH2), 33.3
(CH2), 35.0 (CH2), 36.5 (CH2), 36.9 (CH2), 39.4 (CH), 44.7 (CH), 45.5
(CH3), 45.8 (CH), 46.0 (CH), 46.1 (CH), 46.3 (CH), 46.5 (CH), 46.7
(CH), 49.4 (CH2), 49.5 (CH2), 54.1 (CH), 55.2 (CH3), 56.3 (CH), 57.8
(CH), 62.4 (CH2), 65.3 (CH2), 66.4 (Cq), 71.3 (Cq), 113.3 (CH), 115.0
(CH2), 115.2 (CH), 115.6 (CH2), 115.7 (CH2), 120.2 (CH), 120.2 (CH),
123.2 (CH), 126.7 (CH), 126.8 (CH), 127.9 (CH), 127.9 (CH), 128.0
(CH), 128.1 (CH), 128.1 (CH), 129.0 (CH), 129.1 (CH), 129.4 (CH),
129.5 (CH), 130.0 (CH), 130.3 (CH), 130.5 (CH), 130.6 (CH), 130.8
(CH), 130.8 (CH), 130.9 (CH), 131.0 (CH), 133.0 (Cq), 136.6 (Cq), 138.0
(CH), 138.1 (CH), 138.3 (CH), 144.7 (Cq), 144.9 (Cq), 145.6 (Cq), 151.7
(Cq), 158.2 (Cq), 170.5 (Cq), 171.7 (Cq), 174.0 ppm (Cq); IR (ATR): n˜ =
3493, 33.43, 3083, 3057, 3031, 2958, 2931, 2871, 2849, 2791, 1738, 1653,
Acknowledgements
Donation of [Ru-6]* from Professor Hoveyda is gratefully acknowledged.
The authors also thank Professor Waldmann, Dr. Anja Watzke and Dr.
Lucas Brunsveld for the donation of peptides 13d–f and for their support
in peptide synthesis.
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X. Jiaxi, K. D. Eom, Biopolymers 2001, 60, 194; P. E. Dawson,
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BioconjugateTechniques, AcademicPress, SanDiego 1996; J. W. Bode,
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4564; Angew. Chem. Int. Ed. 2005, 44, 4490; R. H. Grubbs, Tetrahe-
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1607, 1588, 1575, 1508, 1251, 1182, 1161, 1144, 1033, 744, 706 cmꢀ1
;
FABMS: m/z (%)=[norm 1273]: 1273 [M]+ (100), 998 (40), 952 (10), 905
(18), [norm 243]: 273 (90), 243 (100), 154 (10).
Metathesis product (18c): In a thoroughly dried flask, 13 f (21.4 mg,
22 mmol) and a solution of [Ru-2] (0.19 mg, 0.22 mmol, 1 mol%) in di-
chloromethane (0.1 mL) were added to
a solution of 17 (8.1 mg,
22 mmmol) in dichloromethane (0.4 mL) and stirred for 0.5 h at 408C
under nitrogen atmosphere. The solvent was removed under reduced
pressure. After preparative TLC (dichloromethane/MeOH 40:1), 18c
(15.3 mg, 10.8 mmol, 49%) was obtained as a white crystalline solid.
1H NMR (500 MHz, CDCl3): d=0.75–0.95 (m, 6H), 1.27–1.50 (m, 12H),
1.50–1.70 (m, 7H), 1.85–1.90 (m, 1H), 1.90–2.10 (m, 2H), 2.27–2.40 (m,
1H), 2.50–2.65 (m, 1H), 2.65–2.70 (m, 3H), 2.85–2.90 (m, 7H), 2.95–3.00
(m, 1H), 3.00–3.25 (m, 4H), 3.35–3.45 (m, 1H), 3.70 (s, 3H), 3.75–3.95
(m, 2H), 4.05–4.15 (m, 2H), 4.25–4.35 (m, 1H), 4.85–5.05 (m, 2H), 5.10–
5.25 (m, 1H), 5.25–5.35 (m, 1H), 5.60–5.85 (m, 1H), 6.55–6.70 (m, 2H),
6.90–6.95 (m, 1H), 7.10–7.40 (m, 28H), 7.50–7.60 (m, 2H), 8.20–8.25 (m,
Received: August 14, 2006
Published online: December 7, 2006
2368
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ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2007, 13, 2358 – 2368