Delayed fibril formation of amylin(20-29) by incorporation of alkene dipeptidosulfonamide isosteres obtained by solid phase olefin cross metathesis

Article abstract of DOI:10.1016/j.bmcl.2007.11.009

The synthesis of a new peptidomimetic structure, the alkene dipeptidosulfonamide isostere, is described. The synthesis is based on a cross metathesis reaction between two allylic building blocks, both in solution and on the solid phase. This method was also applicable to the solid phase synthesis of alkene dipeptide isosteres. Derivatives of amylin(20-29) containing the alkene dipeptidosulfonamide isostere as well as the alkene dipeptide isostere were successfully synthesized using the solid phase cross metathesis method. Investigation of relations between structure and fibril formation of these amylin(20-29) derivatives showed retardation of fibril formation and altered secondary structures, compared to native amylin(20-29).

Full text of DOI:10.1016/j.bmcl.2007.11.009

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 78–84
Delayed fibril formation of amylin(20–29) by incorporation
of alkene dipeptidosulfonamide isosteres obtained by solid
phase olefin cross metathesis
Arwin J. Brouwer, Ronald C. Elgersma, Monika Jagodzinska,
Dirk T. S. Rijkers and Rob M. J. Liskamp*
Department of Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University,
PO Box 80082, NL-3508 TB Utrecht, The Netherlands
Received 21 September 2007; revised 1 November 2007; accepted 2 November 2007
Available online 6 November 2007
Abstract—The synthesis of a new peptidomimetic structure, the alkene dipeptidosulfonamide isostere, is described. The synthesis is
based on a cross metathesis reaction between two allylic building blocks, both in solution and on the solid phase. This method was
also applicable to the solid phase synthesis of alkene dipeptide isosteres. Derivatives of amylin(20–29) containing the alkene dipept-
idosulfonamide isostere as well as the alkene dipeptide isostere were successfully synthesized using the solid phase cross metathesis
method. Investigation of relations between structure and fibril formation of these amylin(20–29) derivatives showed retardation of
fibril formation and altered secondary structures, compared to native amylin(20–29).
ꢀ 2007 Elsevier Ltd. All rights reserved.
The replacement of a backbone amide bond in peptides
is a strategy which has been widely used to study peptide
backbone interactions as well as for stabilization of pep-
tides toward enzymatic degradation. An amide bond
surrogate that mimics the geometry of the amide bond
(A, Fig. 1) is the (E)-alkene dipeptide isostere (B,
Fig. 1).1
O
R'
R'
O
R'
O
O
H
N
H
N
H
N
S
N
H
A
N
H
N
H
R
R
O
O
R
R
C
R'
O
H
N
H
N
H
N
H
N
S
O
This isostere has been mainly applied in the synthesis of
dipeptide mimics.1 Only a few papers describe the incor-
poration of the alkene dipeptide isostere in longer pep-
tides probably partly due to the difficulties involved in
the synthesis of the isosteres. Another amide bond sur-
rogate is the sulfonamide bond (C), which increases
the flexibility of the backbone and is resistant to enzy-
matic degradation.2c Although the sulfonamide-group
is a weaker hydrogen bond acceptor, it still can form
hydrogen bonds, mainly via the NH, which is a better
hydrogen bond donor than an amide NH. The sulfon-
amide peptidomimetic is conveniently accessible3 and
has been used for incorporation into peptides.2 A disad-
vantage of peptidosulfonamides (C, Fig. 1) might be the
B
D
Figure 1. General structures of a dipeptide (A), an alkene dipeptide
isostere (B), a peptide-peptidosulfonamide (C), and an alkene dipept-
idosulfonamide isostere (D).
presence of an additional carbon atom in each amino
sulfonic acid residue, which is needed for stability
reasons.4 As a result, there is no exact match of a pept-
idosulfonamide and its parent peptide. We envisioned
that by combining the alkene dipeptide isostere with
the sulfonamide a new useful peptidomimetic could be
designed and synthesized: the alkene dipeptidosulfona-
mide isostere (D, Fig. 1). This isostere has the same
backbone length as the parent (di)peptide and also con-
tains a sulfonamide moiety. For the synthesis of the al-
kene dipeptidosulfonamide isostere it was decided to
modify a known cross metathesis procedure for the
Keywords: Alkenedipeptidosulfonamide isosteres; Alkenedipeptide
isosteres; Peptidomimetics; Solid phase cross metathesis; Amyloid.
*
Corresponding author. E-mail: R.M.J.Liskamp@uu.nl
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.009

A. J. Brouwer et al. / Bioorg. Med. Chem. Lett. 18 (2008) 78–84
79
synthesis of alkenedipeptide isosteres.1j From the avail-
able methods described in the literature, this was the
most attractive one because of the relatively few reaction
steps, and since its direct applicability to the synthesis of
peptides containing a variety of sequences. Several other
methods for the synthesis of alkene dipeptide isosteres
described in the literature require many, sometimes te-
dious, reaction steps. In addition, these procedures
mostly have been optimized for the synthesis of a spe-
cific sequence, and optimization is needed for the syn-
thesis of other sequences.
derived allylamine 1b, to give isostere 4b in 39% yield.10
When proline-derived allylamine 1c was used the yield
of the cross metathesis product (4c) was even higher,
that is, 64%.10 This is in agreement with the literature
yields for the synthesis of alkene dipeptide isosteres via
cross metathesis.1j The E/Z ratios for the isosteres (4a–
c) were found to be higher than 10:1 (1H NMR analysis)
which is also comparable to ratios found in the literature
for alkene dipeptide isosteres.1j Although it was ex-
pected that the yields could be improved by optimizing
the molar ratio of the cross metathesis building blocks,
it was decided to translate the optimal procedure in
solution to application in solid phase synthesis and sub-
sequently to optimize the ratio of the cross metathesis
reaction partners.
Besides cross metathesis in solution, we were also inter-
ested in application of cross metathesis on the solid
phase, since peptides are usually synthesized on a solid
support. The advantage of this approach would be that
in principle any peptide sequence can be prepared using
easily accessible building blocks.
Although solid phase cross metathesis reactions have
been hardly described in the literature,11 it has some
advantages compared to cross metathesis in solution,
which are reminiscent to solid phase synthesis. The ole-
fin can be added in excess to the resin for driving the
reaction to completion, and its non-desired homodimer
can be easily removed by filtration. Another possible
advantage is less homodimerization of the resin-bound
olefin due to its attachment to the solid support.11a
However, there are reports that the majority of the re-
sin-bound olefin is able to come within reacting distance,
leading to homodimerization. Yields reported in the lit-
erature vary from low/moderate (11–25%),11a,b possibly
due to homodimerization, to reasonable/good (54–81%).
The latter yields were obtained using simple styrene
derivatives with similar reactivities.11c
For cross metathesis in solution, the N-terminal build-
ing block derived from glycine (1a) was prepared by
Boc-protection of allylamine. Side chain containing N-
terminal building blocks 1b and 1c (Scheme 1) were eas-
ily prepared from Boc-protected amino acids. Reduction
to the amino alcohols was followed by a Swern oxida-
tion to give the corresponding aldehydes.5–7
A Wittig olefination with methyl triphenylphosphonium
bromide afforded Boc-protected allylic amines derived
from phenylalanine (1b) and proline (1c).7 For the
‘S-’terminal building block, allylsulfonic acid was con-
verted into benzyl sulfonamide 2.8 The benzylsulfona-
mide is stable and is easily detected by UV.
For cross metathesis on the solid phase Fmoc-protected
allylamines were chosen to allow determination of the
coupling efficiency by measuring the dibenzofulvene-
piperidine adduct obtained after cleavage of the Fmoc-
group.12 Fmoc-protected allylamine 6a was prepared
by Fmoc-protection of allylamine,13 and Fmoc-pro-
tected allylamine 6b (Scheme 2) was prepared from
Boc-protected allylamine 1b by cleavage of the Boc-
group followed by introduction of the Fmoc-group
using Fmoc-chloride.
First, cross metathesis was carried out using equimolar
amounts of Boc-allylamine (1a) and sulfonamide 2
(Scheme 1) with Grubbs second generation catalyst 39
(10%) and dichloromethane as a solvent. This reaction
afforded mainly homodimerized Boc-allylamine and
only a small amount of product (4a). From the literature
it is known that the molar ratio of cross metathesis part-
ners is related to their differential reactivities.1j When
two equivalents of Boc-allylamine (1a) were used, isoste-
re 4a was isolated in 12% yield.10 We expected that less
Boc-allylamine homodimerization would occur using a
more sterically hindered allylamine. In line with this
assumption, sulfonamide 2 reacted with phenylalanine-
Resin 5 was used for optimization of the cross metathe-
sis on the solid phase, which was obtained after coupling
H
NHFmoc
N
+
S
H
N
O
O
R
5
6a-b
S
Bn
2
O
O
+
CH2Cl2, Δ
Boc
N
H
N
Mes N N Mes
R'
S
Boc
N
Cl
Cl
Mes N N Mes
O
O
3 or 8
R
Ru
R'
1a-c
Cl
Cl
Ph
4a-c
Ru
PCy3
R
O
8
3
H
N
NHFmoc
R
7a (R=H)
7b (R=Bn)
4
a
b
c
R, R'
H, H
Isostere Yield(%)
S
O
Gly-Glys
12
39
64
Boc
N
O
O
CH2Ph, H Phe-Glys
-(CH2)3- Pro-Glys
R'
OH
R
Scheme 1. Synthesis of alkene dipeptidosulfonamide isosteres 4a–c in
solution.
Scheme 2. Solid phase synthesis of alkene dipeptidosulfonamide
isosteres 7a and 7b.

80
A. J. Brouwer et al. / Bioorg. Med. Chem. Lett. 18 (2008) 78–84
Table 1. Reaction conditions of the optimization of the solid phase synthesis of alkene dipeptidosulfonamides
Entry
6 R (equiv)
(Ru) %
Lewis acid
Solvent
ꢂC
Time (h)
Loading (%)b
1
H
H
H
H
H
H
H
H
H
Bn
Bn
5
10
5
3, 50
3, 100
3, 50
8, 50
3, 20
3, 20
3, 20
3, 20
8, 20
8, 20
8, 50
DCM
DCM
40
40
16
16
16
16
0.5
0.5
0.5
16
16
16
16
17
11
0
2
3
DCM/MeOH
DCM/MeOH
DCM
40
4
5
40
11
16
32
21
19
35
13
26
5a
6a
7a
8
5
150a
150a
150a
80
5
Toluene
Toluene
Toluene
Toluene
Toluene
Toluene
5
Cy2BCl
Cy2BCl
Cy2BCl
Cy2BCl
Cy2BCl
5
9
5
80
80
10
11
5
5
80
a Experiment performed in a dedicated microwave reactor.
b By spectroscopic determination of the dibenzofulvene piperidine adduct.12
of freshly prepared allylsulfonyl chloride to a TentaGelꢁ
S NH2 resin. In the first cross metathesis attempt five
equivalents of Fmoc-allylamine (6a) and 50% Grubbs
catalyst (3) were used. After refluxing overnight in
dichloromethane the Fmoc-loading of resin 7a was only
17% of the total available (Table 1, entry 1).
Amylin(20–29) (18, Scheme 3) was chosen for this pur-
pose, since this is a peptide with a high tendency to form
(anti)parallel b-sheets, which leads to fibril formation.
Investigation of the relations between structure and fi-
bril formation is an important issue in our research.
Amylin(20–29) is the highly amyloidogenic region of
amylin, also known as human islet amyloid polypeptide,
a 37-mer peptide hormone which is involved in the path-
ogenesis of type II diabetes. So far we have incorporated
several peptidomimetic moieties in amylin(20–29) to
function as structure breaking entities.17 Incorporation
of the b-peptidosulfonamide moiety resulted in a com-
plete loss of fibril formation.17c Instead, supramolecular
folding morphologies were observed. Since the alkene
dipeptidosulfonamide isostere contains both a sulfon-
amide and an alkene isostere we were interested in the
structural effects upon incorporation in amylin(20–29).
In the literature there are only few examples of oligopep-
tides containing alkene dipeptide isosteres.1m–r To our
knowledge, the only example in which this isostere was
incorporated into a peptide that forms fibrils is the Alz-
heimer’s amyloid peptide (Ab (1–40)).1q This led to a
different secondary structure as compared to the native
Ab (1–40) and also spherical aggregates were found in-
stead of fibrils. Based on these results, we were also
interested in incorporation of the alkene dipeptide iso-
stere, which can be prepared similarly to the alkene dip-
eptidosulfonamide isostere by using an allylic carboxylic
acid instead of an allylic sulfonic acid. Based on the lit-
erature18 and our previous results,17a it was decided to
introduce the isosteres at position 27 and 28, occupied
by the amino acids leucine and serine. Previously it
was found that the serine at position 28 can be replaced
by a glycine without losing the aggregation properties of
the peptide.17a
Unexpectedly, doubling the amounts of allylamine 6a
and catalyst gave an even lower loading (11%, entry 2).
It was observed that the reaction mixture contained a
white precipitate, which was found to be the homodimer
of allylamine 6a. When this homodimer precipitates the
equilibrium of the reaction will be shifted toward dimer-
ization leading to less cross coupled product 7a.
When the reaction was performed in a methanol/dichlo-
romethane mixture (1/4, v/v) no precipitate was ob-
served but almost no cross coupled product was found
(entry 3) and most of the allylamine (6a) had not re-
acted, probably because of the instability of the catalyst
in methanol. The Hoveyda Grubbs catalyst (8),14 which
is more stable in polar solvents (Scheme 2), did not lead
to a significant increase of Fmoc-loading (11%, entry 4).
According to the literature a cross metathesis reaction in
solution in the presence of a Lewis acid can increase the
yields significantly.15 Indeed, when 10% Cy2BCl was
used for the synthesis of 7a, the loading increased to
19% (entry 8) with Grubbs catalyst 3, and with Hoveyda
Grubbs catalyst 8 to 35% (entry 9). Microwave assis-
tance of the cross metathesis reaction did not show sig-
nificant improvements leading to a loading of 16%
(entry 5, 30 min at 150 ꢂC in dichloromethane), 32% (en-
try 6, in toluene) or 21% (entry 7, in toluene with Lewis
acid Cy2BCl).16 Using the above optimized conditions
for the solid phase cross metathesis obtained with
Fmoc-allylamine 6a, it was assumed that the more steri-
cally hindered Fmoc-allylamine derived from phenylala-
nine, 6b, would give a higher efficiency. Unfortunately
the obtained loading was only 13% (entry 10), which in-
creased to 26% when the amount of Hoveyda Grubbs
catalyst was raised from 20% to 50% (entry 11). Despite
these modest results it was decided to apply this opti-
mized cross metathesis procedure (entry 11) to the incor-
poration of an alkene dipeptidosulfonamide into a
peptide.
First, resin bound H-Ser(tBu) (9) was reacted with
freshly prepared allylsulfonyl chloride (10) or with
BOP activated 3-butenoic acid (11), to yield both sulfon-
amide 12 and peptide 13 (Scheme 3).
The next step was the cross metathesis reaction with
Fmoc-leucine-derived allylamine 6c, using the above
optimized conditions (Table 1, entry 11). The
Fmoc-loadings12 found were 20% for alkene dipeptido-
sulfonamide isostere 14 and only 10% for alkene

A. J. Brouwer et al. / Bioorg. Med. Chem. Lett. 18 (2008) 78–84
81
OtBu
H
Rink
N
H2N
O
9
O
O
O
S
BOP,
DiPEA,
CH2Cl2
OH
Cl
NMM,
CH2Cl2
10
11
OtBu
OtBu
O
O
O O
S
H
H
Rink
Rink
N
N
N
N
H
H
O
O
12
13
6c, 8, Cy2BCl,
toluene, 80˚C
6c, 8, Cy2BCl,
toluene, 80˚C
FmocN
H
6c
OtBu
OtBu
H
O O
S
H
Rink
N
N
Rink
FmocN
H
FmocN
H
N
H
N
H
O
O
14
15
1. SPPS
2. TFA
TIS
1. SPPS
2. TFA, TIS, H2O
H2O
O
NH2
OH
O
O
O
O
H
O
O
O
O
H
N
H
N
H2N
N
N
S
NH2
N
N
H
N
H
N
16
H
H
H
O
H2N
O
O
O
O
O
HO
O
O
NH2
H
N
OH
NH2
O
O
O
O
O
O
H
N
H
N
H2N
N
H
N
H
N
H
N
H
N
H
17
O
H2N
O
O
O
HO
O
O
NH2
H
N
OH
O
O
H
N
H
N
H
H2N
N
NH2
N
H
N
H
N
H
N
H
N
H
18
O
H2N
O
O
HO
OH
O
Scheme 3. Solid phase synthesis of amylin(20–29) incorporated with an alkene dipeptidosulfonamide isostere (16) and with an alkene dipeptide
isostere (17). TIS, triisopropylsilane.
dipeptide isostere 15. However, the latter loading was an
unoptimized one. Then, the synthesis was continued
using the Fmoc/tBu solid phase peptide synthesis proto-
cols.19 Peptides 16 and 17 were cleaved from the resin
and deprotected using TFA in the presence of triisopro-
pylsilane (TIS) and H2O as scavengers.20 Purification
using solid phase extraction column chromatography
and HPLC afforded alkene dipeptidosulfonamide isoste-
re 16 and alkene dipeptide isostere 17 in 1% and 2%
yield, respectively.21 These yields are comparable to
the overall yields described in the literature for the syn-
thesis of peptides containing alkene dipeptide isosteres,
which were prepared using preformed alkene dipeptide
isostere building blocks. The overall yields of these
building blocks vary from 2% to 29%.1n,o,q One example
describes 11% overall yield for the synthesis of the
dipeptide mimic and 36% for the peptide synthesis,
leading to an overall yield of 4%.1n The syntheses of
other peptides using the alkene dipeptide isostere
building blocks are reported without yields.1o,q,r Our
overall yields for the peptide synthesis include the
preparation of the dipeptide isostere, which in fact is
part of the solid phase peptide synthesis.
Both peptides (16 and 17) were characterized by electro-
spray mass spectrometry.
To study the aggregation behavior, both peptides were
dissolved in 0.1% TFA/H2O to obtain a concentration
of 10 mg/mL and rapid gel formation was observed.
Gel formation of sulfonamide isostere 16 was clearly
slower than alkene dipeptide isostere 17 (60 vs
30 min), while both were significantly slower than native
amylin(20–29) (<10 min)17a (18). Retardation in fibril
formation can be explained by removal of both hydro-
gen bond donor and acceptor in the peptide backbone.
Moreover, in case of the sulfonamide containing isostere

82
A. J. Brouwer et al. / Bioorg. Med. Chem. Lett. 18 (2008) 78–84
3
18
2
1
0
195
205
215
225
235
245
255
-1
-2
-3
17
16
Wavelength (nm)
Figure 3. CD spectra of amylin(20–29) (18) and amylin(20–29)
incorporated with an alkene dipeptidosulfonamide isostere (16) and
with an alkene dipeptide isostere (17). Peptide concentrations: 1 mg/
mL in 0.1% TFA/H2O.
Figure 2. TEM images of amylin(20–29) (A, 18), amylin(20–29)
incorporated with an alkene dipeptide isostere (B, 17) and with an
alkene dipeptidosulfonamide isostere (C, 16).22 Scale bars represent
100 nm.
In conclusion, we have developed a method for the prep-
aration of a new peptidomimetic structure, the alkene
dipeptidosulfonamide isostere. This isostere was success-
fully prepared using alkene cross metathesis both in
solution and on the solid phase. The yields and conver-
sions mainly depend on the reactivity and steric factors
of the alkenes in the cross metathesis reaction in solu-
tion. The solid phase synthesis gives overall similar
yields as compared to literature yields for the synthesis
of peptides containing an alkene dipeptide isostere. An
important advantage of the solid phase cross metathesis
method described here is that no time-consuming multi-
step synthesis is required for the preparation of dipep-
tide peptidomimetic building blocks. The method was
found to be also applicable to the solid phase synthesis
of alkene dipeptide isosteres. Both isosteres were suc-
cessfully introduced in the peptide amylin(20–29). Incor-
poration resulted in retardation of fibril formation for
both peptides and altered secondary structures com-
pared to native amylin(20–29) as was confirmed by
FTIR and CD.
(16), the sulfonamide is a weaker hydrogen bond accep-
tor which is an additional inhibitory factor in the forma-
tion of a hydrogen bond network. The presence of
amyloid fibrils was confirmed by transmission electron
microscopy (TEM) (Fig. 2).
The morphology of both peptide isosteres was similar
compared to native amylin(20–29) (18). However, the
TEM images of peptide isosteres 16 and 17 showed sig-
nificantly less fibrils than the native amylin(20–29) (18).
This is in accordance with the delay found in the gel for-
mation of peptide isosteres 16 and 17. The Fourier
transform infrared spectra showed amide I absorptions
at 1640 cmꢀ1 for both peptides, which indicates either
an unordered structure (1640–1648 cmꢀ1) or a b-sheet
secondary structure (1625–1640 cmꢀ1).23
The absorptions clearly shifted to a higher frequency as
compared to native amylin(20–29) (1629 cmꢀ1),17a indi-
cating b-sheet secondary structures containing less or
weaker hydrogen bonds, which can be ascribed to the
presence of a double bond in the isosteres. Unexpect-
edly, circular dichroism spectroscopy (CD) showed a
random coil-like absorption (Fig. 3).
Acknowledgments
We thank Annemarie Dechesne for measuring some of
the mass spectra and Dr. George Posthuma (UMC, Utr-
echt) for assistance with the TEM measurements.
Since fibrils were formed according to the TEM images,
a random coil-like secondary structure was not very
likely. A combined interpretation of the CD spectra,
shifts in the infrared spectra, and TEM images shows
that a random coil is not likely as the secondary struc-
ture, concluding that a twisted b-sheet is the most prob-
able secondary structure.24
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48, 380.
obtained as white solids. Characterization data for 4b: Rf
(MeOH/CH2Cl2, 2/98, v/v): 0.54; 1H NMR (CDCl3):
d = 1.38 (s, 9H, C(CH3)3, 2.75 (dd, 1H, PhCHa,
Jvic = 7.4 Hz, Jgem = 13.5 Hz), 2.86 (dd, 1H, PhCHb,
Jvic = 6.9 Hz, Jgem = 13.5 Hz), 3.53 (d, 2H, CH2SO2),
4.20 (d, 2H, NCH2Ph), 4.29 (m, 1H, CHCH2Ph), 4.71
(d, 1H, NHBoc), 5.09 (m, 1H, NHBn), 5.48 (m, 2H,
CH@CH), 7.13–7.36 (m, 10H, 2 · Ph); 13C NMR
(CDCl3): d = 28.2 (C(CH3)3), 40.8 (CHCH2Ph), 47.2
(NCH2PH), 53.2 (CHCH2Ph), 55.6 (CH2SO2), 79.8
(C(CH3)3), 118.6 (NCHCH@), 126.7, 128.0, 128.1, 128.5,
128.7, 129.4, 136.9, 137.0 (Ar-C), 139.5 (=CHCH2SO2),
155.2 (C@O (Boc)); ESI-MS: calculated for C23H30N2O4S-
Na [M+Na]+: 453.18, found: 453.55.
11. (a) Testero, S. A.; Mata, E. G. Org. Lett. 2006, 8, 4783; (b)
Tang, Q.; Wareing, J. R. Tetrahedron Lett. 2001, 42, 1399;
(c) Chang, S.; Na, Y.; Shin, H. J.; Choi, E.; Jeong, L. S.
Tetrahedron Lett. 2002, 43, 7445; (d) Ghalit, N.; Kemm-
ink, J.; Hilbers, H. W.; Versluis, C.; Rijkers, D. T. S.;
Liskamp, R. M. L. Org. Biomol. Chem. 2007, 5, 924.
12. Meienhofer, J.; Waki, M.; Heimer, E. P.; Lambros, T. J.;
Makofske, R. C.; Chang, C.-D. Int. J. Pep. Protein Res.
1979, 13, 35.
13. Volkmer-Engert, R.; Hoffmann, B.; Schneider-Mergener,
J. Tetrahedron Lett. 1997, 38, 1029.
14. Barber, S. B.; Kingsbury, J. S.; Gray, B. L.; Hoveyda, A.
H. J. Am. Chem. Soc. 2000, 122, 8168.
15. Vedrenne, E.; Dupont, H.; Oualef, S.; Elka¨ım, L.;
Grimaud, L. Synlett 2005, 670.
16. For microwave-enhanced cross metathesis in solution, see
for example: Morris, T.; Sanman, D.; Caddick, S. Org.
Biomol. Chem. 2007, 5, 1025.
2. (a) De Bont, D. B. A.; Dijkstra, G. D. H.; den Hartog, J.
A. J.; Liskamp, R. M. J. Bioorg. Med. Chem. Lett. 1996, 6,
3035; (b) De Bont, D. B. A.; Moree, W. J.; Liskamp, R.
M. J. Bioorg. Med. Chem. 1996, 4, 667; (c) De Bont, D. B.
A.; Sliedregt-Bol, K. M.; Hofmeyer, L. J. F.; Liskamp, R.
M. J. Bioorg. Med. Chem. 1999, 7, 1043; (d) Liskamp, R.
M. J.; Kruijtzer, J. A. W. Mol. Divers. 2004, 8, 79; (e) Di-
segni, S.; Giordano, C.; Rahimipour, S.; Ben-aroya, N.;
Koch, Y.; Fridkin, M. J. Pept. Sci. 2005, 11, 45.
3. Brouwer, A. J.; Monnee, M. C. F.; Liskamp, R. M. J.
Synthesis 2000, 1579.
4. Paik, S.; White, E. H. Tetrahedron 1996, 52, 5303, and
references cited therein.
17. (a) Rijkers, D. T. S.; Ho¨ppener, J. W. M.; Posthuma, G.;
Lips, C. J. M.; Liskamp, R. M. J. Chem. Eur. J. 2002, 8,
4285; (b) Elgersma, R. C.; Meijneke, T.; Posthuma, G.;
Rijkers, D. T. S.; Liskamp, R. M. J. Chem. Eur. J. 2006,
12, 3714; (c) Elgersma, R. C.; Meijneke, T.; de Jong, R.;
Brouwer, A. J.; Posthuma, G.; Rijkers, D. T. S.; Liskamp,
R. M. J. Org. Biomol. Chem. 2006, 4, 3587; (d) Elgersma,
R. C.; Mulder, G. W.; Kruijtzer, J. A. W.; Posthuma, G.;
Rijkers, D. T. S.; Liskamp, R. M. J. Bioorg. Med. Chem.
Lett. 2007, 17, 1837.
18. Moriarty, D. F.; Raleigh, D. P. Biochemistry 1999, 38,
1811.
19. Fields, C. G.; Lloyd, D. H.; Macdonald, R. L.; Otteson,
K. M.; Noble, R. L. Pept. Res. 1991, 4, 95.
5. Rodriquez, M.; Llinares, M.; Doulut, S.; Heitz, J.;
Martinez, J. Tetrahedron Lett. 1991, 32, 923.
20. Peptide synthesis. Peptide isosteres 16 and 17 were
synthesized on a 0.26 mmol scale on Tentagel Fmoc-
Rink-Amide resin in order to obtain C-terminally ami-
dated peptides. Each synthetic cycle consisted of Na-Fmoc
removal by a 1 h treatment with 20% piperidine in NMP,
washings with NMP (2 · 2 min) and CH2Cl2 (5 · 2 min), a
45-min coupling step with preactivated Fmoc-amino acid
(1.04 mmol), and washings with NMP (2 · 2 min) and
CH2Cl2 (5 · 2 min). Na-Fmoc amino acids were activated
in situ with HBTU/HOBt (1.04 mmol, 0.21 M in NMP) in
the presence of DiPEA (2.08 mmol). 3-Butenoic acid was
coupled using normal amino acid coupling conditions for
3 h. Allylsulfonyl chloride (1.04 mmol), freshly prepared
from the corresponding sodium sulfonate using phosgene
(purification: silicagel plug, CH2Cl2; 85% yield; clear
colorless oil),3 was coupled using CH2Cl2 and DiPEA,
followed by normal washings. The cross metathesis
reaction was performed using allyl amine 6c (1.3 mmol),
Hoveyda Grubbs catalyst (8, 0.13 mmol), and Cy2BCl
(26 lmol) in toluene (20 mL, degassed (N2)). After stirring
overnight at 80 ꢂC, the resin was washed with methanol
(1 · 2 min), NMP (2 · 2 min), and CH2Cl2 (5 · 2 min).
The peptides were detached from the resin and
6. Monache, G. D.; Misiti, D.; Salvatore, P.; Zappia, G.
Tetrahedron: Asymmetry 2000, 11, 1137.
7. (a) Luly, J. R.; Dellaria, J. F.; Plattner, J. J.; Soderquist, J.
L.; Yi, N. J. Org. Chem. 1987, 52, 1487; (b) Briot, A.;
Bujard, M.; Gouverneur, V.; Mioskowski, C. Eur. J. Org.
Chem. 2002, 139.
8. (a) Hanson, P. R.; Probst, D. A.; Robinson, R. E.; Yau,
M. Tetrahedron Lett. 1999, 40, 4761; (b) Barton, W. R. S.;
Paquette, L. A. Can. J. Chem. 2004, 82, 113.
9. Scholl, M.; Ding, S.; Lee, C. W.; Grubbs, R. H. Org. Lett.
1999, 1, 953.
10. Synthesis of alkene dipeptidosulfonamide isosteres in solu-
tion. To a degassed (N2) solution of 1a–c (2.0 mmol) and 2
˚
(1.0 mmol) in CH2Cl2 (5 mL, dried on molsieves (4 A))
was added Grubbs catalyst 3 (42 mg, 50 lmol). The flask
was fitted with a reflux condenser and the mixture was
refluxed for 16 h while N2 gas bubbling through. Another
portion of catalyst 3 (42 mg, 50 lmol) was added and
stirring was continued for 24 h. The mixture was concen-
trated in vacuo and purified directly using silica gel
column chromatography (eluent: gradient EtOAc/hex-
anes, start: 1/6 ! 1/4 ! 1/2, v/v). Isosteres 4a–c were

84
A. J. Brouwer et al. / Bioorg. Med. Chem. Lett. 18 (2008) 78–84
deprotected by treatment with TFA/H2O/TIS (95/2.5/2.5,
Peptides 16 and 17 were characterized by mass spectrom-
etry. Yields: 16, 1% (2 mg); 17, 2% (5 mg). ESI-MS mass
found (calcd) [M+H]+: 16, 961.78 (961.51); 17, 997.40
(997.48). HPLC analysis tR: 16, 16.90 min; 17, 16.78 min.
Electrospray ionization mass spectrometry (ESI-MS) was
performed on a Shimadzu LC MS QP8000 single quad-
v/v/v) for 3h. The peptides were precipitated with MTBE/
hexane (1/1, v/v) at ꢀ20 ꢂC, dissolved in H2O, and
separated from brown ruthenium impurities by a SPE
column (C4, eluent = H2O) and finally lyophilized.
21. Purification and analysis. The peptides were purified by
dissolving the crude material in a minimal amount of
buffer A and loaded onto an Adsorbosphere XL C8 HPLC
rupole bench-top mass spectrometer operating in
positive ionization mode.
a
˚
column (90 A pore size, 10 lm particle size, 2.2 · 25 cm).
22. TEM sample preparation. Peptide isosteres were dis-
solved in 0.1% TFA/H2O to obtain a concentration of
10 mg/mL. These solutions were stored at 4 ꢂC during 3
weeks. The obtained gels were used for TEM
measurements.
The peptides were eluted with a flow rate of 10 mL/min
using a linear gradient of buffer B (50% in 40 min) from
100% buffer A (buffer A: 0.1% TFA in CH3CN/H2O, 5/95,
v/v; buffer B: 0.1% TFA in CH3CN/H2O, 95/5, v/v). The
purity was determined by analytical HPLC on an Adsorb-
23. Jackson, M.; Mantsch, H. M. Crit. Rev. Biochem. Mol.
Biol. 1995, 30, 95.
˚
osphere XL C8 column (90 A pore size, 5 lm particle size,
0.46 · 25 cm) at a flow rate of 1.0 mL/min using a linear
gradient of buffer B (100% in 20 min) from 100% buffer A.
24. Johnson, W. C., Jr. Ann. Rev. Biophys. Biophys. Chem.
1988, 17, 145, and references cited therein.