Ring-closing metathesis for the synthesis of side chain knotted pentapeptides inspired by vancomycin

Article abstract of DOI:10.1039/b408820d

A versatile method for the synthesis of bicyclic side chain knotted peptides inspired by vancomycin is described. The synthetic approach is based on the incorporation of a central amino acid derivative 3 having two allylic groups - introduced by a Stille

Full text of DOI:10.1039/b408820d

View Article Online / Journal Homepage / Table of Contents for this issue
A R T I C L E
Ring-closing metathesis for the synthesis of side chain knotted
pentapeptides inspired by vancomycin
Hefziba T. ten Brink, Dirk T. S. Rijkers, Johan Kemmink, Hans W. Hilbers and
Rob M. J. Liskamp*
Department of Medicinal Chemistry, Utrecht Institute for Pharmaceutical Sciences,
Faculty of Pharmaceutical Sciences, Utrecht University, P.O. Box 80082, 3508 TB Utrecht,
The Netherlands. E-mail: R.M.J.Liskamp@pharm.uu.nl; Fax: +31 30 2536655;
Tel: +31 30 2537396
Received 11th June 2004, Accepted 3rd August 2004
First published as an Advance Article on the web 24th August 2004
A versatile method for the synthesis of bicyclic side chain knotted peptides inspired by vancomycin is described.
The synthetic approach is based on the incorporation of a central amino acid derivative 3 having two allylic
groups—introduced by a Stille coupling—into pentapeptide 8 containing two allylated serine residues. Treatment of
this bis-ring-closing metathesis precursor with 2nd generation Grubbs catalyst results in the formation of a bicyclic
pentapeptide with the correct side chain to side chain connectivity pattern as observed in vancomycin: i − 2 → i, i →
i + 2. Modelling studies using MacroModel hint at a cavity-like structure of the bicyclic pentapeptide which may
bind suitable ligands.
In the previously reported applications concerning ring-
Introduction
closing metathesis one might consider the alkene resulting from
the metathesis reaction as a stable alternative of the disulfide
bridge.6 Here we wish to report the results of ring-closing
metathesis towards the construction of a “multiple side chain
knotted” system as is present in vancomycin. One of the aims
is to develop vancomycin mimics, possibly possessing cavity or
shell-like structures, which—because of their pre-organisation—
may tightly bind other molecules. In the case of vancomycin
mimics this may be D-Ala-D-Ala or D-Ala-D-lactate7,8 but the
variations of the building blocks used in the synthesis of our
molecular constructs including combinatorial approaches might
also allow the development of molecules capable of binding
other biologically relevant targets.
Nature has found many intriguing and elegant ways to reduce
the flexibility of peptides in order to control shape. Reduction
of the flexibility by disulfide bridges is the most well-known way
to control the three dimensional structure.1 One of the most
sophisticated ways is probably “multiple side chain knotting”,
in which a number of side chains are tied together in such a
way that conformational mobility is minimised. Outstanding
examples in this respect are the glycopeptide antibiotics of
which vancomycin is an important representative2,3 (Fig. 1).
So far only “monocyclic”—representing the right or the left
macrocycle—mimics of vancomycin have been prepared.9–11
Here we present a convenient approach which, for the first time,
allows construction of the “bicyclic multiple side chain knotted”
framework, featuring a Stille coupling12 followed by tandem ring
closing metathesis13,14 (Scheme 1).
Results and discussion
An important design consideration in the synthesis of our target
bicyclic pentapeptide 9 was the right choice of the central amino
acid residue in the ring-closing metathesis precursor. This residue
should possess two allylic groups, one for the macrocycle to be
localised on the right and one for the macrocycle on the left hand
side of the bicyclic target molecule. For this purpose, 4-hydroxy-
phenylglycine turned out to be a suitable starting compound
(Scheme 1). After proper protection, the resulting amino acid
derivative was easily iodinated on both the 3- and 5-positions
by treatment with N-iodosuccinimide in acetone15 to give 1. In
order to obtain good yields in the C–C-coupling reaction in
which both iodine atoms were substituted, the hydroxyl function
had to be converted into an electron withdrawing substituent
by conversion into acetate 2 using acetic anhydride. Now 2
was subjected to a Stille coupling12 with allyltributylstannane
and Pd(PPh3)2Cl2 in DMF at 100 °C to give 3 in 96% yield.
Unfortunately, 3 was obtained as a racemate possibly due
to the high reaction temperature. However, when the Stille
coupling was carried out at a lower reaction temperature,
racemization was not prevented but also resulted in a complex
reaction mixture of mono- and dialkylated products, which was
Fig. 1 Target bicyclic peptide as inspired by the vancomycin CDE
ring system.
We are interested in applying selective C–C-coupling
reactions in order to pre-organise peptides and ultimately
to achieve control over their conformation. So far we have
employed ring-closing metathesis (RCM) for this purpose4 and
other approaches for selective C–C-bond forming reactions to
constrain the structure of peptides such as Sonogashira and
Heck reactions are currently being explored in our laboratory.5
2 6 5 8
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 6 5 8 – 2 6 6 3
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4

View Article Online
Scheme 1 Synthesis of bicylic pentapeptide 9 via a tandem ring-closing metathesis.
difficult to purify. Apparently, the temperature was not the only
factor causing racemization and perhaps other factors include
decomposition of DMF and basicity of the organometallic
reagent or the racemization-proneness of the phenylglycine-
derived ester. Nevertheless, bisallyl derivative 3 was treated with
TFA to remove the Boc group followed by coupling of Boc-D-
Ala-OH in the presence of BOP/DIPEA to afford dipeptide 4
in 94% yield. TFA treatment was repeated and the obtained
TFA-salt was subjected to BOP-coupling with Boc-Ser(All)-
OH16 to yield tripeptide 5 (84%). In the first approach towards
the synthesis of bicyclic pentapeptide 9, tripeptide 5 was
treated with 2nd generation Grubbs catalyst17 in DCM in
order to construct 10a containing the macrocycle on the right.
Unfortunately, treatment of cyclization precursor 5 resulted
not only in the desired macrocycle 10a but also the isomerized
product 10b was isolated as a major side product in a total yield
of 69%.18 This isomerization reaction19 could be suppressed by
the addition of hydride scavengers,4b albeit not completely and
it was difficult to separate the isomerized product 10b from the
desired product 10a. Therefore, this route was abandoned and it
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 6 5 8 – 2 6 6 3
2 6 5 9

View Article Online
was decided to complete the synthesis of the pentapeptide first
and then attempt tandem ring-closing metathesis.
Thus, tripeptide 5 was saponified followed by BOP-coupling
with dipeptide 7 to give pentapeptide 8 in 52% yield (two steps).
Treatment of ring-closing metathesis precursor 8 with 2nd
generationGrubbscatalystinTCEwascarriedoutinthepresence
of a small amount DMF because of the poor solubility of 8. In
contrast to most RCM reactions a stoichiometric amount of
catalyst was required since the used DMF deactivated part of the
catalyst by coordination to ruthenium.20 The cyclization reaction
was monitored by mass spectrometry, since the differences in
Rf-values of 8, the monocyclic intermediates and the desired
bicyclic product 9 were too small. Monitoring by HPLC was
too slow because the reaction turned out to be complete within
10 min. The cyclization proceeded via a monocyclic intermediate
with a molecular mass of 771 Da. During the course of the
reaction, this peak disappeared completely and a new peak at
m/z = 743 Th appeared, corresponding to the mass of bicyclic
product 9. After workup this bicyclic peptide 9 was isolated in
67% yield. In principle, a mass value of 771 Da could also be due
to formation of a monocyclic intermediate in which one allyl
functionality was isomerized. However, this intermediate was
unlikely, since this would probably not be converted to bicyclic
pentapeptide 9.
Fig. 2 Lowest energy conformation of bicyclic peptide 9.
Unfortunately, further structural characterisation by 1H and
13C NMR (COSY, TOCSY, HMBC, HSQC) was rather difficult
since the product is probably composed of eight possible stereo-
isomers: E/Z for each of the double bonds and R/S for the
central phenylglycine derivative. However, the mass data were
very unambiguous since HR-MS and MALDI-TOF analyses
confirmed the molecular mass to be (M + H)+ 743.39 Da, thus
any cyclic dimers could be rigorously excluded. Moreover, a
strong long-range coupling between the ~O–CH2–CH and
the CH–CH2-arom could be observed in a TOCSY spectrum
which is characteristic for the correct side chain to side chain
connectivity pattern. Thus, the NMR and mass data were
unambiguous and other possibilities for ring-closing metathesis
could be excluded.
In order to get some qualitative insight into the 3D-structure
of bicyclic peptide 9, the eight possible stereoisomers were
subjected to a Monte Carlo conformational search procedure
in combination with molecular mechanics calculations using
MacroModel21 to find the global minimum energy conforma-
tion of each stereoisomer. Modelling experiments suggest that
the bicyclic peptide 9 possessing the Z,S,Z configuration was
the isomer with the lowest energy. Assuming for the time being
that this stereoisomer was formed in excess, its 3D-structure is
depicted in Fig. 2, showing the cavity-like appearance, which
might host suitable ligands.
1
Peak assignments are based on H NMR COSY spectra. 13C
NMR spectra were recorded on a Varian G-300 (75.5 MHz)
or a Varian INOVA-500 (125 MHz) spectrometer and chemi-
cal shifts are given in ppm relative to CDCl3 (77.0 ppm). The
13C NMR spectra were recorded using the attached proton test
(ATP) sequence (G-300) or using HMBC and HSQC sequences
(I-500). Electrospray ionization mass spectrometry (EI-MS)
was measured on a Shimadzu LCMS-QP8000 single quadruple
bench-top mass spectrometer operating in a positive ionization
mode. Analytical HPLC runs were performed on Shimadzu au-
tomated HPLC system equipped with a UV/VIS detector oper-
ating at 220/254 nm and an evaporative light scattering detector
(Polymer Laboratories ELS 1000) on an Alltech Adsorbosphere
C8 column (particle size 5 lm, pore size 90 Å, 250 × 4.6 mm)
at a flow rate of 1 mL min−1 using a linear gradient from 100%
buffer A (0.1% TFA in water) to 100% buffer B (0.085% TFA
in acetonitrile/water 95:5 v/v) in 40 min. High resolution mass
spectra (HR-MS) were measured on a Micromass Q-TOF hy-
brid mass spectrometer, with pentaphenylalanine as reference.
MALDI-TOF analysis was performed on a Kratos Axima
CFR apparatus, with bradykinin(1–7) as external reference
and a-cyano-4-hydroxycinnamic acid as matrix. Optical rota-
tions were measured at 20 °C using a Jasco P-1010 polarimeter.
Elemental analyses were performed by Kolbe Microanalytisches
Labor (Mülheim an der Ruhr, Germany). Rf values were deter-
mined by thin layer chromatography (TLC) on Merck pre-
coated silica gel 60 F254 glass plates. Spots were visualized by UV
quenching, ninhydrin or Cl2/TDM.22 Column chromatography
was performed on ICN Silica 60 Å (70–230 mesh).
In conclusion, a bicyclic macrocyclic ring-system inspired
by the multiple side chain knotted structure of vancomycin
was constructed by tandem ring-closing metathesis, thereby
providing access to many peptidic systems where more control of
the three dimensional structure is required or at least beneficial
for e.g. binding properties. Furthermore, the synthetic route is
amenable to combinatorial approaches since a variety of other
amino acids can be introduced, variation of the stereochemistry
of these building blocks is possible and a solid phase procedure
is within reach.
S-Na-(tert-Butyloxycarbonyl)-4-hydroxy-3,5-diiodo-
phenylglycine methyl ester (1). SOCl2 (6 mL) was carefully
added to MeOH (60 mL) at 0 °C and to this mixture S-4-
hydroxy-phenylglycine (5 g, 29.9 mmol) was added portionwise.
After refluxing for 4 h, the reaction was complete according to
TLC and the solvent was evaporated in vacuo. Then, the crude
product was dissolved in H2O/dioxane (100 mL, 1:1 v/v), Boc2O
(6.52 g, 29.9 mmol) was added and TEA was added portionwise
to keep the pH at 8.5. After stirring for 5 h the reaction was
complete according to TLC and the solvents were removed
at reduced pressure. The residue was dissolved in EtOAc
and subsequently washed with 1 N KHSO4, 1 M NaHCO3,
brine and dried (Na2SO4). Ethyl acetate was removed under
reduced pressure and S-Na-(tert-butyloxycarbonyl)-4-hydroxy-
phenylglycine methyl ester was obtained as a white solid in 88%
yield (7.39 g) after crystallization from hexane/EtOAc. Rf(2:1
Material and methods
General
Chemicals were obtained from commercial sources and used
without further purification, unless stated otherwise. DIPEA
was distilled consecutively from ninhydrin and KOH. Other
dry solvents were obtained as peptide grade solvents from
1
Biosolve and were stored on molecular sieves (4 Å). H NMR
spectra were recorded on a Varian G-300 (300 MHz) or a Var-
ian INOVA-500 (500 MHz) spectrometer and chemical shifts
are given in ppm (d) relative to TMS or DMSO-d6 (2.39 ppm).
2 6 6 0
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 6 5 8 – 2 6 6 3

View Article Online
reached. The coupling was complete after 2 h and the solvent
was removed under reduced pressure. The residue was dissolved
in EtOAc and the solution was washed with 1 N KHSO4, 1 M
NaHCO3, brine and dried (Na2SO4). Ethyl acetate was removed
under reduced pressure and dipeptide 4 was obtained as an oil
(620 mg, 94%) which slowly solidified (after purification by
column chromatography (1:2 EtOAc/hexane → 1:1 EtOAc/
hexane)). Rf(1:1 EtOAc/hexane): 0.89; HPLC showed that the
product was 100% pure (based on two diastereomers) as judged
by ELSD; dH (300 MHz, CDCl3) 1.35–1.37 (3H, t, bCH3-Ala),
1.42 and 1.45 (9H, double s, Boc), 2.30 (3H, s, CH3), 3.24 and
3.38 (4H, 2 × d, 3-CH2/5-CH2), 3.71 and 3.73 (3H, double s,
OMe), 4.24 (1H, broad s, aCH-Ala), 5.04–5.12 (5H, m, CH2-
allyl/NH-urethane), 5.50 and 5.78 (1H, d, aCH), 5.78–5.92 (1H,
m, CH-allyl), 7.09 and 7.10 (3H, 2 × s, 2-H/6-H/NH-amide); dC
(75.5 MHz, CDCl3) 18.1, 20.6, 28.2, 34.9, 49.8, 52.8, 55.8, 80.2,
116.7, 127.2, 133.2, 133.8, 135.3, 136.9, 147.6, 168.8, 170.9, 171.3;
ES-MS calcd for C25H34N2O7: 474; found: m/z [M + H]+ 475,
[M + Na]+ 497, [(M − C4H8) + H]+ 419, [(M − C5H8O2) + H]+
375; HR-MS [M + H]+ found: m/z 475.2437, calcd 475.2444;
Anal. calcd for C25H34N2O7: C, 63.27; H, 7.22; N, 5.90; found: C,
63.19; H, 7.24; N, 5.79%.
EtOAc/hexane): 0.73; dH 1.44 (9H, s, Boc), 3.70 (3H, s, OMe),
5.22 (1H, d, aCH), 5.65 (1H, d, NH), 6.73 (2H, d, arom H), 7.01
(1H, broad s, OH), 7.15 (2H, d, arom H); dC 28.3, 52.7, 57.0,
80.6, 115.8, 127.9, 128.4, 155.1, 156.4, 172.0. This Boc-protected
ester (1.5 g, 5.3 mmol) was dissolved in acetone (60 mL). The
mixture was cooled to −79 °C and the the mixture was protected
from light by aluminium foil. To this mixture a solution of N-
iodosuccinimide (2.64 g, 11.74 mmol) in acetone (30 mL) was
added dropwise over 5 h at −79 °C.15 After stirring overnight
the reaction was complete according to TLC and the solvent
was evaporated in vacuo. The residue was dissolved in EtOAc,
washed with a saturated solution of Na2S2O3, H2O, brine
and dried (Na2SO4). The solvent was removed under reduced
pressure and the diiodo compound 1 was obtained as a yellowish
solid (2.24 g, 80%) after purification by column chromatography
(1:4 acetone/hexane). Rf(1:2 EtOAc/hexane): 0.76; [a]D +100 (c
1, CHCl3); dH 1.43 (9H, s, Boc), 3.74 (3H, s, OMe), 5.18 (1H, d,
aCH), 5.67 (1H, d, NH), 5.95 (1H, broad s, OH), 7.66 (2H, s, 2-
H, and 6-H); dC 28.2, 53.0, 55.5, 80.5, 82.5, 131.9, 137.8, 153.7,
154.5, 170.8.
S-Na-(tert-Butyloxycarbonyl)-4-acetoxy-3,5-diiodo-phenyl-
glycine methyl ester (2). Diiodo compound 1 (4.3 g, 8 mmol) was
dissolved in acetone (100 mL) in the presence of K2CO3 (1.67 g,
12.1 mmol) and acetic anhydride (1.06 mL, 11.2 mmol) was
added dropwise and the obtained reaction mixture was stirred
for 2 h. The solvent was evaporated in vacuo and the residue was
dissolved in EtOAc and the organic layer was washed with 1 N
KHSO4, 1 M NaHCO3, brine and dried (Na2SO4). The solvent
was removed under reduced pressure and acetate 2 was obtained
as a white solid (1.63 g, 97%) after purification by column
chromatography (1:2 EtOAc/hexane). Rf(1:4 acetone/hexane):
0.63; [a]D +59 (c 1, CHCl3); dH 1.44 (9H, s, Boc), 2.43 (3H, s,
CH3), 3.76 (3H, s, OMe), 5.24 (1H, d, aCH), 5.67 (1H, d, NH),
7.78 (2H, s, 2-H/6-H); dC 21.3, 28.2, 53.2, 55.6, 80.7, 90.7, 138.2,
138.3, 151.6, 154.5, 167.3, 170.3; ES-MS calcd for C16H19I2NO6:
575; found: m/z [M + Na]+ 598; Anal. calcd for C16H19I2NO6: C,
33.41; H, 3.33; N, 2.44; found: C, 33.35; H, 3.47; N, 2.45%.
Na-(tert-Butyloxycarbonyl)-seryl(allyl)-D-alanyl-RS-4-
acetoxy-3,5-bisallyl-phenylglycine methyl ester (5). Dipeptide 4
(590 mg, 1.24 mmol) was dissolved in DCM (25 mL) and TFA
(5 mL) was added. After 2 h the deprotection was complete as
judged by TLC and the reaction mixture was evaporated to
dryness and subsequently coevaporated with DCM (twice) to
remove any residual TFA. The crude product was dissolved in
DCM (50 mL) and to this solution were added BOP (606 mg,
1.37 mmol), Boc-Ser(All)-OH (335 mg, 1.37 mmol) followed by
DIPEA until pH 8.5 was reached. The coupling was complete
after 2 h and the solvent was evaporated in vacuo. The residue
was dissolved in EtOAc and the solution was washed with 1 N
KHSO4, 1 M NaHCO3, brine and dried (Na2SO4). Subsequently,
ethyl acetate was removed under reduced pressure. Tripeptide 5
was obtained as a colorless oil (626 mg, 84%) which crystallized
after purification by column chromatography (1:2 EtOAc/
hexane → 1:1 EtOAc/hexane). Rf(1:1 EtOAc/hexane): 0.37;
HPLC showed that the product was 100% pure (based on two
diastereomers) as judged by ELSD; dH (300 MHz, CDCl3)
1.36–1.41 (3H, t, bCH3-Ala), 1.44 (9H, s, Boc), 2.29 (3H, s,
CH3), 3.23 (4H, d, 3-CH2/5-CH2), 3.47–3.56 (1H, m, bCH2-
Ser (1H)), 3.70 and 3.72 (3H, double s, OMe), 3.77–3.91 (3H,
m, ~O-CH2-allyl/bCH2-Ser (1H)), 4.27 (1H, broad s, aCH-Ser),
4.50–4.53 (1H, m, aCH-Ala), 5.05–5.24 (6H, m, CH2-allyl), 5.41
(1H, broad s, NH-urethane), 5.45–5.49 (1H, m, aCH), 5.74–5.91
(3H, m, CH-allyl), 6.92 (1H, d, NH-amide (Ala)), 7.08 (2H, s,
2-H/6-H), 7.08 and 7.24 (1H, 2 × d, NH-amide); dC (75.5 MHz,
CDCl3) 17.9, 18.1, 28.1, 34.8, 48.6, 52.7, 54.2, 55.7, 55.9, 69.4,
72.0, 80.2, 116.6, 117.5, 127.1, 127.2, 133.1, 133.6, 133.7, 135.3,
147.5, 147.6, 155.3, 168.8, 170.2, 170.3, 170.8, 171.2; ES-MS
calcd for C31H43N3O9: 601; found: m/z [M + H]+ 602, [M + Na]+
624, [(M − C4H8) + H]+ 546, [(M − C5H8O2) + H]+ 502; HR-MS
[M + H]+ found: m/z 602.3065, calcd 602.3077; Anal. calcd for
C31H43N3O9: C, 61.88; H, 7.20; N, 6.98; found: C, 61.98; H, 7.34;
N, 6.78%.
RS-Na-(tert-Butyloxycarbonyl)-4-acetoxy-3,5-bisallyl-
phenylglycine methyl ester (3). In a flame-dried nitrogen filled
flask, acetate 2 (723 mg, 1.26 mmol) was dissolved in DMF
(14 mL). After addition of allyltributylstannane (1.53 mL,
5 mmol) the mixture was heated to 100 °C under a weak nitrogen
flow. Then Pd(PPh3)2Cl2 (88 mg, 0.13 mmol) was added. After
2 h the reaction was complete according to TLC and the solvent
was evaporated in vacuo. The residue was dissolved in EtOAc,
and the solution was washed with dilute ammonia, brine
and dried (Na2SO4). After removal of EtOAc under reduced
pressure, bisallyl compound 3 was obtained as a yellowish oil
(487 mg, 96%) after purification by column chromatography
(hexane → 1:4 acetone/hexane). Rf(1:4 acetone/hexane): 0.43;
HPLC showed that the product was 99% pure (based on both
enantiomers) by ELSD; dH (300 MHz, CDCl3) 1.43 (9H, s, Boc),
2.29 (3H, s, CH3), 3.24 (4H, d, 3-CH2/5-CH2), 3.71 (3H, s, OMe),
5.04–5.11 (4H, m, CH2-allyl), 5.26 (1H, d J = 6 Hz, aCH), 5.49
(1H, d J = 6 Hz, NH), 5.78–5.90 (2H, m, CH-allyl), 7.10 (2H,
s, 2-H/6-H); dC (75.5 MHz, CDCl3) 20.6, 28.2, 35.8, 52.7, 57.0,
80.2, 116.6, 127.1, 133.1, 134.4, 135.4, 147.5, 154.7, 168.9, 171.5;
ES-MS calcd for C22H29NO6: 403; found: m/z [M + Na]+ 426;
HR-MS [M + NH4]+ found: m/z 421.2343, calcd 421.2339.
Monocyclic tripeptide (10a and 10b). Tripeptide 5 (100 mg,
0.166 mmol) was dissolved in TCE (66 mL) to obtain a final
concentration of 2.5 mM. The solution was purged with
nitrogen gas (25 min) and heated until reflux followed by the
addition Ru catalyst (2nd generation Grubbs; 14 mg, 17 lmol).
After 10 min ring-closing was complete according to TLC and
EI-MS. Subsequently, the solvent was removed under reduced
pressure and the residue was purified by column chromato-
graphy (2:1 EtOAc/hexane). The product was obtained as a
mixture of 10a and 10b (1:1) as a colorless oil (66 mg, 69%).
Rf(1:1 EtOAc/hexane): 0.11; dH (500 MHz, CDCl3), for clarity
the two different products are assigned together, only the allyl
Na-(tert-Butyloxycarbonyl)-D-alanyl-RS-4-acetoxy-3,5-
bisallyl-phenylglycine methyl ester (4). TFA (5 mL) was added
to bisallyl compound 3 (560 mg, 1.39 mmol) dissolved in DCM
(25 mL). After 2 h deprotection was complete as judged by
TLC and the reaction mixture was evaporated to dryness and
subsequently coevaporated with DCM (twice) to remove any
residual TFA. The crude product was dissolved in DCM (50 mL)
and to this solution BOP (633 mg, 1.43 mmol), Boc-D-Ala-OH
(270 mg, 1.43 mmol) and DIPEA were added until pH 8.5 was
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 6 5 8 – 2 6 6 3
2 6 6 1

View Article Online
function of the aromate in 10a and 10b are indicated separately,
1.35–1.49 (12H, m, Boc/bCH3-Ala), 1.89 and 1.96 (1.5 H, double
s, CH3-CH = (10b)), 2.28–2.41 (3H, m, CH3), 3.10–3.26 (3H, m,
3-CH2/5-CH2), 3.52–3.59 (1H, m, bCH2-Ser (1H)), 3.68–3.81
(4H, m, OMe/~O-CH2-allyl (1H)), 4.05 (2H, m, ~O-CH2-allyl
(1H)/bCH2-Ser (1H)), 4.65 (1H, broad s, aCH-Ala), 5.08–5.11
(1H, m, CH2-allyl (10a)), 5.30–5.80 (4H, m, -CHCH-/NH-
urethane/aCH), 5.85 (0.5 H, m, CH-allyl (10a)), 6.29 (1H,
m, 2 × CH-allyl (10b)), 6.75 (1H, broad s, NH-amide (Ala)),
6.97–7.41 (3H, m, 2-H/6-H/NH-amide); ES-MS calcd for
C29H39N3O9: 573, found: m/z [M + H]+ 574, [M + Na]+ 596,
[(M − C4H8) + H]+ 518, [(M − C5H8O2) + H]+ 474; HR-MS
[M + H]+ found: m/z 574.2724, calcd 574.2764.
to TLC and the solvents were evaporated in vacuo. The residue
was dissolved in H2O and the aqueous solution was acidified
with 1 N KHSO4 and extracted with EtOAc. The collected
organic layers were dried (Na2SO4) and ethyl acetate was
removed under reduced pressure. The saponified tripeptide was
obtained as a white solid (158 mg, 86%). dH (300 MHz, CDCl3)
1.34–1.36 (3H, m, bCH3-Ala), 1.41 and 1.42 (9H, double s,
Boc), 3.53–3.69 (1H, m, bCH2-Ser (1H)), 3.34 (4H, d, 3-CH2/
6-CH2), 3.85–3.89 (3H, m, ~O-CH2-allyl/bCH2-Ser (1H)),
4.34 (1H, broad s, aCH-Ser), 4.74 (1H, broad s, aCH-Ala),
5.09–5.20 (6H, m, CH2-allyl, 3 × 2H)), 5.40–6.01 (5H, m, CH-
allyl (3 × 1H)/NH-urethane/aCH), 7.01 (2H, d, 2-H/6-H), 7.47
(1H, d, NH-amide (Ala)), 7.68 (1H, broad s, OH), 7.78 (1H, d,
NHMe); dC (75.5 MHz, CDCl3) 18.12, 28.1, 35.1, 48.6, 54.2,
56.0, 55.1, 69.3, 72.0, 80.4, 116.5, 117.4, 126.2, 127.2, 127.8,
133.7, 133.8, 136.1, 152.7, 152.8, 155.5, 170.7, 171.6, 173.1.
Next, dipeptide 6 (135 mg, 0.36 mmol) was dissolved in DCM
(2 mL) and TFA (2 mL). After 1 h Boc-removal was complete
as jugded by TLC and the reaction mixture was evaporated
in vacuo and coevaporated with DCM (twice) to remove any
residual TFA. Without further purification the saponified
peptide and TFA salt 7 were dissolved in DCM/DMF (15:2
v/v) followed by the addition of BOP (136 mg, 0.31 mmol) and
DIPEA (176 lL, 1.12 mmol). The coupling was complete in
3 h. After removal of the solvents, the product was triturated
with EtOAc until pentapeptide 8 was obtained as a white solid
(151 mg, 61%). dH (300 MHz, DMSO-d6) 0.65–0.86 (6H, m,
d/dCH3-Leu), 1.16–1.22 (3H, t, bCH3-Ala), 1.35 (12H, s, Boc/
cCH-Leu/bCH2-Leu), 2.53 (3H, broad s, NHCH3), 3.27–3.29
(4H, m, 3-CH2/5-CH2), 3.48 (4H, m, bCH2-Ser (2 × 2H)), 3.89
(4H, m, ~O-CH2-allyl (2 × 2H)), 4.16 (2H, broad s, aCH-Ser),
4.96–4.29 (2H, m, aCH-Ala and aCH-Leu), 4.96–5.23 (8H, m,
CH2 (allyl, 4 × 2H)), 5.37–5.34 (1H, m, aCH), 5.75–5.93 (4H,
m, CH-allyl (4 × 1H)), 6.76 (1H, d, NH-urethane), 6.90–6.93
(2H, m, 2-H/6-H), 7.94–8.42 (4H, m, NH-amide (4 × H));
ES-MS calcd for C41H62N6O10: 798; found: m/z [M + H]+ 799,
[M + Na]+ 821, [(M − C4H8) + H]+ 743, [(M − C5H8O2) + H]+
699; HR-MS [M + H]+ found: m/z 799.4583, calcd 799.4605;
Anal. calcd for C41H62N6O10: C, 61.63; H, 7.82; N, 10.52; found:
C, 61.72; H, 7.75; N, 10.40%.
Na-(tert-Butyloxycarbonyl)-D-leucyl-serine(allyl)
methyl
amide (6). Boc-Ser(All)-OH (1.02 g, 4.65 mmol) was dissolved
in DCM (50 mL). To this solution BOP (2.06 g, 4.65 mmol)
and DIPEA (813 lL, 5.12 mmol) were added, followed by the
addition of methylamine (0.8 M in THF, 577 lL, 4.65 mmol).
After 2 h of stirring at room temperature the amide formation
was complete according to TLC and the solvent was evaporated
in vacuo. The residue was dissolved in EtOAc and this solution
was washed with 1 N KHSO4, 1 M NaHCO3, brine and dried
(Na2SO4). Ethyl acetate was removed under reduced pressure
and after purification by column chromatography (DCM →
95:5 DCM/MeOH) Boc-Ser(allyl)-NHMe was obtained as
a white solid (0.90 g, 75%). Rf(2:1 EtOAc/hexane): 0.43; dH
(300 MHz, CDCl3) 1.45 (9H, s, Boc), 2.83 (3H, d, NHCH3),
3.51–3.57 (1H, m, bCH2-Ser (1H)), 3.81 and 3.86 (1H, dd,
bCH2-Ser (1H)), 4.01 (2H,d, ~O-CH2-allyl), 4.26 (1H, bs, aCH-
Ser), 5.16–5.29 (2H, m, CH2-allyl), 5.51 (1H, d, NH-urethane),
5.82–5.91 (1H, m, CH-allyl), 6.67 (1H, broad s, NHMe); dC
(CDCl3, 75.5 MHz) 26.2, 28.2, 53.8, 69.6, 72.0, 80.0, 117.3,
133.9, 155.4, 170.8.
Boc-Ser(All)-NHMe (254 mg, 0.98 mmol) was dissolved in
DCM (3 mL) and 6 N HCl in diethyl ether (10 mL) was added.
After 10 min Boc-removal was complete as judged by TLC and
the reaction mixture was evaporated to dryness and subse-
quently coevaporated with DCM (twice) to remove any residual
acid. The crude product was dissolved in DMF (15 mL) and
BOP (563 mg, 1.27 mmol), Boc-D-Leu-OH (250 mg, 1.08 mmol)
and DIPEA (312 lL, 1.96 mmol) were added. The coupling
reaction was completed after 3 h and the solvent was removed
under reduced pressure. The residue was dissolved in EtOAc and
the solution was washed with 1 N KHSO4, 1 M NaHCO3, brine
and dried (Na2SO4). Ethyl acetate was removed under reduced
pressure and after purification by column chromatography
(EtOAc/hexane 1:4 v/v → EtOAc/hexane 1:1 v/v) dipeptide
6 was obtained as a colorless oil (345 mg, 98%). Rf(95:20:3
CHCl3/MeOH/AcOH): 0.85; Rf(60:45:20 CHCl3/MeOH/
25%NH4OH): 0.50; HPLC showed that the product was 100%
pure according to ELSD and UV; dH (CDCl3, 300 MHz) 0.93
and 1.44 (6H, dd, d/dCH3-Leu), 1.45 (9H, s, Boc), 1.53–1.68
(3H, m, cCH-Leu/bCH2-Leu), 2.76 (3H, d, NHCH3), 3.54–3.58
(1H, m, bCH2-Ser (1H)), 3.99–4.04 (4H, m, bCH2-Ser (1H)/~O-
CH2-allyl/aCH-Leu), 4.57–4.60 (1H, m, aCH-Ser), 5.17–5.28
(2H, m, CH2-allyl), 5.38 (1H, d, NH-urethane), 5.80–5.90
(1H, m, CH-allyl), 6.98 (1H, d, NH-amide), 7.24 (1H, broad s,
NHMe); dC (CDCl3, 75.5 MHz) 22.0, 22.6, 24.5, 26.2, 28.1, 40.3,
52.8, 53.9, 68.9, 72.0, 80.2, 117.3, 134.0, 156.2, 170.0, 172.7;
ES-MS calcd for C18H33N3O5: 371; found: m/z [M + H]+ 372,
[M + Na]+ 394, [(M − C4H8) + H]+ 316, [(M − C5H8O2) + H]+
272; HR-MS [M + H]+ found: m/z 372.2518, calcd 372.2498;
Anal calcd for C18H33N3O5: C, 58.20; H, 8.95; N, 11.31; found:
C, 58.33; H, 8.86; N, 11.28%.
Bicyclic pentapeptide (9). Pentapeptide 8 (20 mg, 0.025 mmol)
was dissolved in TCE (12 mL). The solution was purged with
nitrogen gas (25 min) and heated to 80 °C followed by the
addition of Ru catalyst (2nd generation Grubbs, 21 mg,
0.025 mmol) and DMF (0.5 mL). After 10 min the reaction
was complete according to TLC and ES-MS. Subsequently, the
solvent was removed under reduced pressure and the residue
was purified by column chromatography (DCM → 5:95
MeOH/DCM). The tandem ring-closed pentapeptide 9 was
obtained as a slightly brownish solid in 67% yield (13.4 mg).
HPLC showed that the product was 92% pure (based on eight
diastereomers) according to ELSD; dH (500 MHz, DMSO-d6)
0.83–0.87 (6H, m, d/dCH3-Leu), 1.39 (9H, s, Boc), 1.39–1.58
(6H, m, bCH3-Ala/cCH-Leu/bCH2-Leu), 2.27–2.49 (3H,
m, NHCH3), 3.17–4.62 (16H, m, 3-CH2/5-CH2/bCH2-Ser
(2 × 2H)/~O-CH2-allyl
(2 × 2H)/aCH-Ser/aCH-Ala/aCH-
Leu), 4.62–5.90 (5H, m, CHCH (2 × 2H)/aCH), 6.75–8.59
(8H, m, NH-urethane, 2-H/6-H/NH-amide (Ser)/NHMe/NH-
amide (Ala)/NH-amide (Leu)/NH-amide); ES-MS calcd for
C37H45N6O10: 742; found: m/z [M + H]+ 743, [M + Na]+ 765,
[(M − C4H8) + H]+ 687, [(M − C5H8O2) + H]+ 643; HR-MS
[M + H]+ m/z 743.3969 calcd 743.3979; MALDI-TOF found:
m/z (M + H)+ 743.39.
Acknowledgements
Na-(tert-Butyloxycarbonyl)-seryl(allyl)-D-alanyl-RS-4-
hydroxy-3,5-bisallyl-phenylglycyl-D-leucyl-serine(allyl) methyl
amide (8). Tripeptide 5 (200 mg, 0.33 mmol) was dissolved in
MeOH/H2O (30 mL 5:1 v/v) and LiOH (32 mg, 0.76 mmol)
was added. After 20 min saponification was complete according
These investigations were supported by the council for Chemi-
cal Sciences of The Netherlands—Organization for Scientific
Research (CW-NWO). We thank Cees Versluis for performing
the HR-MS experiments.
2 6 6 2
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 6 5 8 – 2 6 6 3

View Article Online
13 For general reviews concerning ring closing metathesis:
(a) R. H. Grubbs and S. Chang, Tetrahedron, 1998, 54, 4413;
(b) S. K. Armstrong, J. Chem. Soc., Perkin Trans. 1, 1998, 371;
(c) A. Fürstner, Angew. Chem., Int. Ed., 2000, 39, 3012.
References and notes
1 A. K. Galande, J. O. Trent and A. F. Spatola, Biopolymers, 2003, 71,
534.
2 D. H. Williams and B. Bardsley, Angew. Chem., Int. Ed., 1999, 39,
1172.
3 K. C. Nicolaou, H. J. Mitchell, N. F. Jain, N. Winssinger, R. Hughes
and T. Bando, Angew. Chem., Int. Ed., 1999, 38, 240.
4 (a) J. F. Reichwein, B. Wels, J. A. W. Kruijtzer, C. Versluis and
R. M. J. Liskamp, Angew. Chem., Int. Ed., 1999, 38, 3684;
(b) J. F. Reichwein and R. M. J. Liskamp, Eur. J. Org. Chem., 2000,
2335; (c) J. F. Reichwein, C. Versluis and R. M. J. Liskamp, J. Org.
Chem., 2000, 65, 6187.
5 (a) H. T. ten Brink, D. T. S. Rijkers and R. M. J. Liskamp,
manuscript in preparation; (b) for a preliminary communication see:
R. M. J. Liskamp, J. F. Reichwein, B. Wels, J. A. W. Kruijtzer and
C. Versluis, in Peptides: The Wave of the Future, eds. M. Lebl and
R. A. Houghten, Proc. 2nd Int. Pept. Symp/17th Am. Pept. Symp.,
Kluwer Academic Publishers, Dordrecht, The Netherlands, 2001,
p. 127.
14 For recent examples of tandem ring closing metathesis:
(a) F.-D. Boyer, I. Hanna and L. Ricard, Org. Lett., 2004, 6, 1817;
(b) R. Garcia-Fandiño,
E. M. Codesido,
E. Sobarzo-Sánchez,
L. Castedo and J. R. Granja, Org. Lett., 2004, 6, 193; (c) T. Honda,
H. Namiki, K. Kaneda and H. Mizutani, Org. Lett., 2004, 6, 87;
(d) R. A. J. Wybrow, N. G. Stevenson and J. P. A. Harrity, Synlett,
2004, 140; (e) M. Rosillo, L. Casarrubios, G. Domínguez and
J. Pérez-Castelles, Org. Biomol. Chem., 2003, 1, 1450; (f) A. E. Sutton,
B. A. Seigal, D. F. Finnegan and M. L. Snapper, J. Am. Chem. Soc.,
2002, 124, 13390; (g) F.-D. Boyer and I. Hanna, Tetrahedron Lett.,
2002, 43, 7469; (h) P. Børsting and P. Nielsen, Chem. Commun., 2002,
2140; (i) T.-L. Choi and R. H. Grubbs, Chem. Commun., 2001, 2648.
15 S. K. Nishiyama, H. Moon and S. Yamamura, Tetrahedron. Lett.,
1994, 35, 8397.
16 H. Sugano and M. Miyoshi, J. Org. Chem., 1976, 41, 2352.
17 M. Scholl, S. Ding, C. W. Lee and R. H. Grubbs, Org. Lett., 1999, 1,
953.
18 Compounds 7a and 7b could not be separated, however from the
1H-NMR spectrum, the ratio of the two products was calculated to
be 1:1.
6 (a) S. J. Miller and R. H. Grubbs, J. Am. Chem. Soc., 1995, 117,
5855; (b) S. J. Miller, H. E. Blackwell and R. H. Grubbs, J. Am.
Chem. Soc., 1996, 118, 9606; (c) J. L. Stymiest, B. F. Mitchell,
S. Wong and J. C. Vederas, Org. Lett., 2003, 5, 47.
7 T. D. H. Bugg, S. Dutka-Malen, M. Arthur, P. Courvalin and
C. T. Walsh, Biochemistry, 1991, 30, 2017.
8 I. A. D. Lessard and C. T. Walsh, Proc. Natl. Acad. Sci. USA, 1999,
96, 11028.
9 R. Xu, G. Greiveldinger, L. E. Marenus, A. Cooper and J. A.
Ellman, J. Am. Chem. Soc., 1999, 121, 4898.
10 R. J. Pieters, Tetrahedron Lett., 2000, 41, 7541.
11 C. J. Arnusch and R. J. Pieters, Eur. J. Org. Chem., 2003, 3131.
12 J. M. Beierle, E. B. Osimboni, C. Metallinos, Y. Zhao and T. R.
Kelly, J. Org. Chem., 2003, 68, 4970.
19 D. Bourgeois, A. Pancrazi, S. P. Nolan and J. Prunet, J. Organomet.
Chem., 2002, 643–644, 247.
20 M. S. Sanford and J. A. Love, in Handbook of Metathesis, ed. R. H.
Grubbs, WILEY-VCH Verlag GmbH & Co KgaA, Weinheim, 2003,
vol. 1, p. 120.
21 F. Mohamadi, N. C. J. Richards, W. C. Guida, R. Liskamp,
M. Lipton, C. Caufield, G. Chang, T. Hendrickson and W. C. Still,
J. Comput. Chem., 1990, 11, 440.
22 E. Von Arx, M. Faupel and M. J. Bruggen, J. Chromatogr., 1976,
120, 224.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 6 5 8 – 2 6 6 3
2 6 6 3