Synthesis of cyclic peptide analogues of the 310 helical Pro138-Gly144 segment of human aquaporin-4 by olefin metathesis

Article abstract of DOI:10.1039/b823559g

Four cyclic pentapeptides and two cyclic heptapeptides modelled on the 3₁₀ helical Pro138-Gly144 segment of the water channel aquaporin-4 (AQP4) postulated to mediate adhesive interactions between AQP4 tetramers were synthesised by olefin metat

Full text of DOI:10.1039/b823559g

View Article Online / Journal Homepage / Table of Contents for this issue
PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis of cyclic peptide analogues of the 310 helical Pro138-Gly144
segment of human aquaporin-4 by olefin metathesis†
Øyvind Jacobsen,*a Jo Klaveness,a Ole Petter Ottersen,b Mahmood Reza Amiry-Moghaddamb and Pa˚l Rongveda
Received 5th January 2009, Accepted 5th February 2009
First published as an Advance Article on the web 17th March 2009
DOI: 10.1039/b823559g
Four cyclic pentapeptides and two cyclic heptapeptides modelled on the 310 helical Pro138-Gly144
segment of the water channel aquaporin-4 (AQP4) postulated to mediate adhesive interactions between
AQP4 tetramers were synthesised by olefin metathesis. Three related acyclic pentapeptides
Boc-Ser(All)-Xaa1-Val-Ser(All)-Gly-OMe (Xaa1 = Val, Aib; Boc = tert-butoxycarbonyl; All = allyl)
and Boc-Ser(Bn)-Val-Val-Gly-Gly-OMe (Bn = benzyl) and two acyclic heptapeptides
Boc-Pro-Pro-Ser(All)-Val-Val-Ser(All)-Gly-OMe and Boc-Pro-Pro-Ser(Bn)-Val-Val-Gly-Gly-OMe
were also prepared. NMR, CD and IR data provided evidence that the peptides can access a 310 helical
structure in apolar solvents and pointed to a significant stabilising effect of the olefinic bridge on
helicity in an aqueous environment. Thus we could demonstrate the viability of using ring closing olefin
metathesis to stabilise short protein segments in the helical conformation that they adopt in their native
protein environment. Our approach provides access to a set of peptides with potential binding affinity
for AQP4.
Thus, despite the range of important physiological and patho-
Introduction
physiological roles played by aquaporins,1 we still lack specific
Aquaporins are water selective membrane channel proteins which
and reversible inhibitors of any member of this class of channel
allow rapid water transport across cell membranes at rates that can
proteins.
approach the diffusion limit.1,2 So far 13 mammalian isoforms have
Searching for a way to approach the problem of developing
been identified.3 The isoform aquaporin-4 (AQP4) is the predom-
AQP4 selective inhibitors we turned to the significant amount
inant water channel in the brain and is concentrated in astrocytic
of structural information that has accumulated in recent years
end-feet.4,5 AQP4 plays an important role in the regulation of
from electron diffraction (ED) and X-ray diffraction studies of
aquaporins and aquaglyceroporins.23–26 Recently, the first structure
water homeostasis in the brain and in the pathophysiology of
brain edema.6 It has been demonstrated that mice lacking AQP4 or
AQP4 anchoring proteins show less edema and suffer less damage
to the brain in an ischemic stroke model than wild-type mice,
validating AQP4 as a possible drug target for treatment of cerebral
edema.7–9
Few inhibitors of aquaporin function are known and all
are toxic, irreversible and/or unspecific.10 This is true for the
inorganic transition metal compounds, e.g. silver nitrate and
mercurials, that are established aquaporin inhibitors.11–13 In its
correct orientation in the membrane AQP4 is not even inhib-
ited by mercurials, hence its original name “mercury insen-
sitive water channel”.14 A few reports have appeared on the
inhibitory effect of small organic molecules like p-chloromercuri
benzenesulfonate,15 phloretin,16,17 carbonic anhydrase inhibitors
(of which acetazolamide is one example)18,19 and quaternary am-
monium salts.20 However, these results have been challenged.21,22
27
˚
of AQP4 was determined by ED to 3.2 A resolution. The
structure indicated that AQP4, like its isoform AQP0, may play
a role in cell-cell adhesion.27–29 Expressing AQP4 in L-cells, cells
with no surface adhesive molecules, causes the cells to aggregate,27
although this claim has recently been challenged.30 The primary
interaction between AQP4 molecules belonging to adjacent cells
is mediated by loop C, part of which takes up a 310 helical
conformation.27
We have initiated a program to explore the potential of using
synthetic peptides with similar primary and secondary structures
to loop C as molecular scaffolds for side chains that could
extend into the vestibule of the aquaporin and potentially block
it. In the perivascular AQP4 pool (i.e. the AQP4 pool that is
thought to constitute the main influx pathway for water during
the development of brain edema) the targeted binding site is not
engaged in binding to contiguous AQP4 tetramers and thus should
be freely available for ligand binding.31
aSchool of Pharmacy, University of Oslo, Sem Sælands vei 3, P.O. Box 1068
Blindern, 0316 Oslo, Norway. E-mail: oyvind.jacobsen@farmasi.uio.no;
Fax: +47 22854402; Tel: +47 92818344
Inspection of the reported structure revealed that Pro139 and
Val142 likely play important roles in mediating the interaction
between two AQP4 tetramers.27,29 Due to the mode of interaction
bCentre for Molecular Biology and Neuroscience, University of Oslo,
P.O. Box 1105 Blindern, 0317 Oslo, Norway. E-mail: o.p.ottersen@
medisin.uio.no; Fax: +47 22851299; Tel: +47 22851270/+47 22851180
(van der Waals/hydrophobic interactions and possibly non-
32
=
classical C–H–O C H-bonds ) it is likely of crucial importance
† Electronic supplementary information (ESI) available: Synthetic pro-
cedures for compounds fully characterised in the literature, partial
ROESY spectra of 29 and CD spectra recorded in MeOH. See DOI:
10.1039/b823559g
that the conformations of the synthetic peptides very closely repro-
duce the conformation of the segment Pro139-Val142. Hence, it
was deemed of great interest to explore methods that might afford
This journal is
The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 1599–1611 | 1599
©

View Article Online
some stabilisation of the 310 helical conformation of the segment
outside of its natural protein environment.
L-serine it seemed sensible to synthesise the fragments Boc-
Ser(Bn)-Xaa1-Val-OMe and Boc-Ser(All)-Xaa1-Val-OMe rather
than the fragments Boc-Pro-Pro-Ser(Bn)-OH and Boc-Pro-Pro-
Ser(All)-OH. This left us with a choice between synthesising the
N-terminal pentapeptidic fragments or the C-terminal pentapep-
tidic fragments indicated in Fig. 1. Because we initially did not
expect to be able to do the RCM already at the pentapeptide stage
both strategies seemed equally worthy of an attempt and it was
decided to synthesise the N-terminal fragments first. It turned out
that this strategy had to be revised in view of the problems incurred
by saponification of these fragments (Scheme 2, see Results and
Discussion) and the C-terminal pentapeptides were synthesised
instead.
Ring closing olefin metathesis (RCM) has been successfully
applied to conformationally restrain peptides in a range of
conformations.33–35 Peptides rich in a-aminoisobutyric acid (Aib)
residues are especially prone to form 310 helices.36,37 Recent work on
Aib rich peptides have demonstrated the feasibility of stabilising
a short peptide in a 310 helical conformation by construction of a
hydrocarbon bridge connecting the side chains of residues i and
i + 3, the key synthetic step being a ruthenium catalysed ring
closing olefin metathesis reaction using Grubbs’ catalysts.38,39 The
presence of a serine residue in loop C, i.e. Ser140, which could
easily be allylated, and a Gly residue at the i + 3 position which
did not seem to be involved in any key interactions made this an
especially attractive strategy in the current project. However, to the
best of our knowledge, this method had not been applied previ-
ously to short peptides containing only proteinogenic residues,
which have a less pronounced tendency than disubstituted
a-amino acids to adopt the required dihedral angles for a 310 helix,
or to 310 helical protein segments. One of our main objectives
is to demonstrate the applicability of this methodology to such
cases, thus providing access to a set of conformationally restrained
peptides with potential affinity for AQP4.
Several studies have demonstrated that diproline motifs often
precede helices in protein structures and can serve as folding
nuclei for short (310) helical peptides.40,41 The presence of a Pro
residue N-terminal to Pro139 in the relevant segment of AQP4
therefore strongly suggested including an additional Pro residue
in the synthetic peptides despite this not being involved in the
binding interaction. C-terminally to the Pro139-Val142 segment
we chose to include two residues, one whose function would be
to facilitate the construction of a bridge from the Ser residue,
and a Gly representing the Gly144 residue. This choice struck a
balance between including a sufficient number of residues to allow
formation of a stable helix, keeping the molecular weight low and
paying attention to the Lipinski Rule of Five,42 and havinga flexible
arm (Gly) for later derivatisation. As a result four heptapeptides
emerged as target compounds (Fig. 1).
Results and discussion
Solution phase synthesis of pentapeptides
The dipeptide fragment Boc-Pro-Pro-OMe 1 common to all the
target heptapeptides was synthesised in 82% yield by carbodiimide
coupling using EDC/HOBt.‡ Deprotection of the C-terminus by
saponification with lithium hydroxide in THF/H2O (2:1) afforded
the dipeptide Boc-Pro-Pro-OH 2 in 88% yield. The key building
block Boc-O-allyl L-serine 3 was synthesised by analogy to a
literature synthesis of Boc-O-benzyl L-serine by deprotonation
with NaH (2.20 eq.) and allylation employing a small excess
(1.10 eq.) of allyl bromide.43 Yields, which varied between 60–83%,
were generally comparable to literature values.38,39
The dipeptides Boc-Val-Val-OMe 4 and Boc-Aib-Val-OMe 5
were synthesised in excellent yields (87% and 95% respectively)
using EDC (1.10 eq.)/HOBt (1.00 eq.). Deprotection afforded
the trifluoroacetates 6 and 7 in 97% and 82% yield respectively
(Scheme 1).
Synthetic strategy
Scheme 1 Reagents and conditions: 6: (i) EDC, HOBt, DIPEA, DMF,
rt, 21 h, 87%; (ii) TFA, CH2Cl2, rt, 2 h, 97%; 7: (i) EDC, HOBt, DIPEA,
DMF, 47 h, 95%; (ii) TFA, CH2Cl2, rt, 2 h, 82%.
Because gram quantities of the various peptides were needed
for further studies and derivatisation a solution phase protocol
was adopted. The sequence similarity of the target heptapeptides
suggested adopting a fragment condensation strategy based on the
following retrosynthetic disconnections (Fig. 1).
The tripeptide 8 was synthesised by carbodiimide coupling
in 95% yield (Scheme 4) and the N-terminus deprotected with
neat formic acid. The resulting formate was not isolated, but
converted to the free amine 9. Carbodiimide coupling with 2
afforded pentapeptide 10 in 93% yield (Scheme 2). Disappoint-
ingly, saponification of 10 with NaOH in MeOH/H2O (2:1) to
form the free acid 11, in analogy to a literature procedure44 for
hydrolysis of 1, resulted in extensive epimerisation, as evidenced
by the appearance of too many amide NH signals in the 1H-NMR
spectrum of the isolated product. This forced a revision of our
Fig. 1 Retrosynthetic disconnections. Cyclic heptapeptides: Xaa1 = Val,
Aib; Acyclic heptapeptides: 31: Xaa2 = Ser(All), Xaa3 = Ser(All); 33:
Xaa2 = Ser(Bn), Xaa3 = Gly.
‡ HOBt: 1-hydroxybenzotriazole; EDC: N-(3-dimethylaminopropyl)-
N¢-ethylcarbodiimide; PyBOP: (benzotriazol-1-yloxy)tripyrrolidinophos-
phonium hexafluorophosphate; HATU: N,N,N¢,N¢-tetramethyl-O-(7-
azabenzotriazol-1-yl)uronium hexafluorophosphate
As the most convenient starting material for incorporation of
the O-allyl L-serine residue appeared to be N-tert-butoxycarbonyl
1600 | Org. Biomol. Chem., 2009, 7, 1599–1611
This journal is
The Royal Society of Chemistry 2009
©

View Article Online
large-scale solution phase preparation. Fortunately, it turned out
to be possible to obtain stereoisomerically pure (97–98%) material
by recrystallisation from absolute EtOH. The pentapeptides 23
and 24 were obtained analogously to 22 in 73% and 78% yield
respectively and with less epimerisation (Scheme 4).
Scheme 2 Reagents and conditions: (i) a. HCOOH, rt, 9 h; b. K2CO3,
H2O, 82%; (ii) 2, EDC, HOBt, DIPEA, DMF, 0 ◦C→rt, overnight, 93%;
(iii) NaOH, MeOH/H2O (2:1), rt, 8 h.
strategy and a decision was taken to synthesise the C-terminal
pentapeptides (Fig. 1) instead.
Tripeptides 12 and 13 were synthesised analogously to 8 by
condensation of 3 and 7 and N-tert-butoxycarbonyl O-benzyl
L-serine 14 and 6 in 78% (sum of diastereomers) and 95% yield
respectively (Scheme 4). The tripeptide 12 obtained was unfor-
tunately quite heavily contaminated with its diastereomer and
required recrystallisation twice from EtOAc/hexane (4:1) to afford
a product stereoisomerically pure enough to be used in subsequent
steps. To suppress epimerisation PyBOP‡ is a more preferred
reagent for coupling to Aib residues.45 However, due to the lower
cost of EDC, the relatively large amounts of coupling reagent
required and the simplicity of the purification procedure EDC was
deemed acceptable. Initially, epimerisation during deprotection
of the C-terminus of tripeptides 8, 12 and 13 was problematic.
Saponification of 8 using NaOH in MeOH/H2O (2:1) at room
temperature resulted in extensive (>30%) epimerisation. However,
lowering the temperature to 0 ◦C, using a very slight excess
of LiOH (1.10 eq.) in THF/H2O and taking care to neutralise
the excess LiOH with solid NaHCO3 before work-up afforded
the tripeptide 15 in acceptable yield (71%) and with only 2–3%
epimerisation (Scheme 4). Likewise, tripeptide 16 was obtained in
88% yield under similar conditions and with comparable extent
of epimerisation (Scheme 4). Tripeptide 17 on the other hand was
isolated in anomalously low yield (42%) and had to be purified
by dissolving the crude product in EtOAc and precipitating it by
addition of hexane (Scheme 4). The C-terminal dipeptides 18 and
19 were synthesised by the carbodiimide method in 84% and 70%
yield respectively and their N-termini deprotected with 50% TFA
in CH2Cl2 to afford the trifluoroacetates 20 and 21 (Scheme 3) as
viscous oils.
Scheme 4 Reagents and conditions: 22: (i) 6, EDC, HOBt, DIPEA, DMF,
0
◦C→rt, 22 h, 94%; (ii) LiOH, THF/H2O (2:1), 0 ◦C, 3 h, 71%; (iii) 20,
EDC, HOBt, DIPEA, CH2Cl2, 0 ◦C→rt, 16 h, 89%; 23: (i) 7, EDC, HOBt,
DIPEA, DMF, 0 ◦C→rt, 22 h, 78% (sum of diastereomers); (ii) LiOH,
THF/H2O (2:1), 0 ◦C, 3 h, 88%; (iii) 20, EDC, HOBt, DIPEA, CH2Cl2,
0
◦C→rt, 27 h, 73%; 24: (i) 6, EDC, HOBt, DIPEA, DMF, 0 ◦C→rt,
overnight, 95%; (ii) LiOH, THF/H2O (2:1), 0 ◦C, 3 h, 42%; (iii) 21, EDC,
HOBt, DIPEA, CH2Cl2, 0 ◦C→rt, 24 h, 78%.
Pentapeptide 22 was found to have special properties. In contrast
to 23 and 24 it precipitated from the reaction mixture and was
surprisingly poorly soluble in both CH2Cl2 and EtOAc. Aqueous
work-up consistently resulted in a troublesome emulsion. Because
of the poor solubility in the organic phase the suspension of 22
˚
had to be dried with 3 A molecular sieves before removal of the
solvent. Interestingly, direct injection of a solution of 22 in 0.5%
(v/v) aqueous DMSO into a mass spectrometer demonstrated that
dimers and, to a lesser extent, trimers were present even in the gas
phase at 365 ◦C, suggesting a quite strong interaction between the
monomers. All pentapeptides were synthesised on a multigram
scale to allow for the possibility of constructing a small library
based on the target heptapeptides as scaffolds. As an example, a
total of 17 g of the pentapeptide 22 was synthesised.
Ring closing olefin metathesis
At high dilution (~4 mM) 22 dissolved in CH2Cl2 and was
cyclised to 25 in 41% yield using Grubbs’ 2nd generation catalyst47
(20 mol%) (Scheme 6). The catalyst was added in two equally large
portions, with the second portion being added after 2–3 hours. The
E/Z selectivity was estimated to 14:1 from the 1H-NMR spectrum,
which compares nicely with previous work on Aib rich peptides
where a 20:1 E/Z selectivity was observed.38 The successful
cyclisation suggests that 22 can access a helical conformation in
solution. However, initially, purification of 25 from unreacted 22
by flash chromatography posed a serious problem. To minimise
problems with purification it was therefore essential that the RCM
was run under conditions which maximised the yield of cyclic
peptide. The pentapeptide 23 was similarly cyclised to yield the
cyclic product 26 (Scheme 6). However, the yield was higher (52%)
and lower catalyst loadings (15 mol%; 10 mol% + 5 mol% after
1–2 hours) were required.
Scheme 3 Reagents and conditions: 20: (i) EDC, HOBt, DIPEA, DMF,
rt, 24 h, 84%; (ii) TFA, CH2Cl2, rt, 2 h, 98%; 21: (i) EDC, HOBt, DIPEA,
DMF, rt, 15 h, 70%; (ii) TFA, CH2Cl2, rt, 2 h, quant.
Condensation of tripeptide 15 with the dipeptide 20 in CH2Cl2
afforded the pentapeptide 22 in 89% yield (Scheme 4, sum
of diastereomers). The carbodiimide coupling was somewhat
hampered by 10–20% epimerisation. Other coupling reagents, e.g.
HATU‡, might have suppressed epimerisation more efficiently,46
but the much lower cost of EDC made this more attractive in a
This journal is
The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 1599–1611 | 1601
©

View Article Online
Synthesis of heptapeptides
All pentapeptidic trifluoroacetates were efficiently purified from
excess TFA by washing with Et2O and were obtained as white
powders.
The cyclic pentapeptides 25 and 26 were deprotected to give the
trifluoroacetates 27 and 28 in 95% and 71% yield, respectively. Sub-
sequent coupling with 2 using the EDC/HOBt method afforded
the heptapeptides 29 and 30 in 78% and 82% yield, respectively
(Scheme 6). Also the acyclic analogue 31 was synthesised in order
to allow a study of possible conformational changes induced
on cyclisation, but, most importantly, for its general utility as a
possible AQP4 ligand in itself. Condensation of 2 and 32 provided
this heptapeptide in 73% yield (Scheme 5). The trifluoroacetate
32 was obtained in 96% yield from 22 by treatment with 50%
TFA in CH2Cl2. Finally, the heptapeptide 33, corresponding to
the native sequence of the relevant 310 helical segment of AQP4,
was synthesised analogously from trifluoroacetate 34 and 2 by way
of carbodiimide coupling (Scheme 5).
Structural studies
CD measurements
Circular dichroism (CD) spectra are commonly used to deter-
mine the presence of different secondary structure elements in
peptides and proteins. Far-UV CD spectra of several of the
neutral penta- and heptapeptides were recorded in the structuring
solvent 2,2,2-trifluoroethanol (TFE) (Figs. 2 and 3), MeOH† and
H2O/MeOH (39:1) (Fig. 4). In TFE all the spectra, with one
exception, displayed a strong negative minimum at 202–203 nm
corresponding to the p–p* transition and a much weaker negative
minimum at 218–222 nm corresponding to the n-p* transition. In
addition, the ratio R = H218–222/H202–203 was found to be approxi-
mately 0.3 for all pentapeptides but 26 (0.65) and approximately
Scheme 5 Reagents and conditions: 33: (i) TFA, CH2Cl2, rt, 1 h 30 min,
92%; (ii) 2, EDC, HOBt, CH2Cl2, 0 ◦C→rt, 23 h, 78%; 31: (i) TFA, CH2Cl2,
rt, 1 h 30 min, 96%; (ii) 2, EDC; HOBt, DIPEA, CH2Cl2, rt, 21 h, 73%.
The trifluoroacetate 34 was obtained from its N-protected
precursor 24 in 92% yield. The protecting groups were not
removed from the final products to avoid potential problems with
epimerisation on saponification of the C-terminus as seen for
the shorter peptides and because some evidence points towards
the Boc group having an important influence on the ability of
small peptides to adopt structured conformations in solution.48
Fig. 2 CD spectra of some of the synthesised pentapeptides in TFE.
Scheme 6 Reagents and conditions: 29: (i) Grubbs’ 2nd generation catalyst (20 mol%), CH2Cl2, rt, 7 h 30 min, 42%; (ii) TFA, CH2Cl2, rt, 2 h, 95%;
(iii) 2, EDC, HOBt, DIPEA, CH2Cl2, 0 ◦C→rt, 22 h, 78%; 30: (i) Grubbs’ 2nd generation catalyst (15 mol%), CH2Cl2, rt, 6 h, 52%; (ii) TFA, CH2Cl2, rt,
1 h, 71%; (iii) 6, EDC, HOBt, DIPEA, CH2Cl2, 0 ◦C→rt, 22 h, 82%.
1602 | Org. Biomol. Chem., 2009, 7, 1599–1611
This journal is
The Royal Society of Chemistry 2009
©

View Article Online
of roughly similar intensities, with the latter band indicating the
presence of H-bonding. Because only 2 out of 5 NH groups can
take part in intramolecular H-bonding in a pentapeptide with
310 helical structure the observed IR bands also supports the
possibility that 25 can adopt a helical structure in CDCl3.
NMR studies
For the dipeptide and tripeptide products only 1D spectra were
recorded, whereas for all the penta- and heptapeptides 2D COSY
and 2D TOCSY spectra were recorded in addition to aid residue
specific assignment of the proton resonances. For the heptapep-
tides 29 and 30 sequence specific information was obtained
from 2D ROESY spectra. In general, the 1H-NMR spectra
of the peptides containing a diproline moiety were complex.
Cis/trans isomerism around the carbamate bond and between two
proline residues is a well-known phenomenon.41,50,51 This flexibility
together with the presence of 14 unique proton types only in the
proline residues, of which 12 have similar chemical shifts, and the
presence of both (E)- and (Z)-isomers for the cyclic products all
contributed to the observed complexity of the spectra. However,
there were variations in the spectral complexity between the pro-
line containing products. For example, the 1H-NMR spectrum of
heptapeptide 30 had a simpler appearance than that of heptapep-
tide 29, especially evident in the NH region. This could be taken as
an indication of a higher degree of structural order in the former.
The 2D ROESY spectrum of heptapeptide 30 in CD2Cl2 clearly
demonstrated the presence of all possible NH(i)→NH(i + 1)
ROEs, which is indicative of a helical peptide (Fig. 5). These
ROEs are not specific for a 310-helix, but are also seen in
a-helical peptides.41,52,53 However, all possible CaH(i)→NH(i + 2)
ROEs, which are diagnostic for 310-helices, were also observed,
with the exception of a possible CaH(Val)→NH(Gly) ROE, which
was hard to distinguish due to overlap (Fig. 6).41,52,53 Finally,
two of three possible CaH(i)→NH(i + 3) correlations, typical of
mixed a-/310-helical conformations, were observed. Overlap with
a more intense peak again precluded a possible CaH(Ser(All)1)→
NH(Ser(All)2) ROE from being distinguishable. Evidence for a
310-helical conformation in CD2Cl2 could also be found in the
2D ROESY spectrum of 29. This included three short-range
NH(i)→NH(i + 1), four medium-range CaH(i)→NH(i + 2) and
two long-range CaH(i)→NH(i + 3) cross-peaks respectively.† Pos-
sible NH(Ser(All)2)→NH(Gly), CaH(Val2)→NH(Gly) and the
two CaH(Ser(All)1)→ NH(Ser(All)2) and CaH(Val1)→NH(Gly)
cross-peaks were not discernible due to overlap. In summary, the
2D ROESY data support the notion that both 29 and 30 can
access a 310-helical conformation in CD2Cl2. That neither peptide
can be perfectly helical is, however, seen by the presence of some
additional CaH(i)→NH(i + 1) ROEs. Finally, the intensity of the
CaH(Pro1)→NH(Ser(All)1) ROE is smaller for 29 than in the case
of 30. This may be taken in support of the hypothesis that the
proline residues of heptapeptide 29 are conformationally more
disordered than in 30.
Fig. 3 CD spectra of the synthesised heptapeptides in TFE.
Fig. 4 CD spectra of the heptapeptides 29 and 33 in H2O/MeOH (39:1).
0.4–0.5 for all heptapeptides. Similar values have been reported to
be characteristic of short 310-helical peptides.39
The spectrum of 10 on the other hand displayed a minimum at
197.5 nm and a very weak negative maximum at 215–217 nm,
which clearly demonstrated a much larger contribution from
random coil conformations (negative minimum at 195 nm and
weakly positive maximum at 212 nm). Similar spectra were
obtained in MeOH, with one notable exception for the acyclic
heptapeptide 33, whose strongest minimum was significantly
broadened. Further, the R value changed from approximately 0.4
in TFE to approximately 0.25 in MeOH. These results indicated
that the contribution from helical conformations might be con-
siderably smaller for the acyclic peptide 33 in a less structuring
solvent. This was corroborated by our findings in a predominantly
aqueous environment consisting of water/methanol (39:1) (Fig. 4).
Whereas the CD spectrum of 29 still resembled that in TFE and
was similar to the reported signature spectrum of a 310 helix in
water49 the changes were much more dramatic for 33 and thus
provided a compelling indication that the olefinic bridge may
stabilise the helical structure of 29 in aqueous solution.
IR measurements
The 310 helix is defined by intramolecular i → i + 3 H-bonds.
Peptides adopting a helical conformation in solution should
therefore show an absorption band in their IR spectra in the
range 3300–3400 cm-1, indicative of H-bonded amide NHs. The IR
spectrum of the cyclic pentapeptide 25 in CDCl3 shows two distinct
NH stretching bands at 3426 cm-1 and 3345 cm-1, respectively,
Conclusions
In summary the pentapeptides 10, 22–26 and heptapeptides
29–31 and 33, which are of potential significance for the develop-
ment of AQP4 inhibitors, have been synthesised by standard
This journal is
The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 1599–1611 | 1603
©

View Article Online
Fig. 5 Partial ROESY spectrum of 30 displaying NH(i)→NH(i + 1) ROEs.
Fig. 6 Partial ROESY spectrum of 30 displaying CaH(i)→NH(i + 2) and CaH(i)→NH(i + 3) cross-peaks (black and red circles, respectively).
solution phase techniques and ring-closing metathesis. We have
thus demonstrated the feasibility of scaling up and extending
the reported methodology on Aib rich peptides38,39 to peptides
containing only proteinogenic amino acids (i.e. with the obvious
exception of the O-allyl L-seryl residue, which is intrinsically
required in order to be able to employ the methodology) and
to protein segments. Despite quite serious initial problems with
epimerisation, in particular of tripeptides 8, 12 and 13 during
saponification of the methyl ester functionality, these challenges
have been addressed and the target penta- and heptapeptides have
been obtained in near stereoisomerically pure form on multigram
and gram scale, respectively. It has been demonstrated by CD, IR
and 2D ROESY experiments that there is a significant contribution
from a 310 helix in the ensemble of conformations available to the
peptides in apolar, structuring solvents like TFE and CD2Cl2,
supporting the secondary structure assigned to this segment in
the low-resolution ED structure of AQP4.27 Most importantly,
however, the CD spectra of 29 and 33 in H2O point to the olefinic
bridge having a stabilising effect on the helical conformation
of the protein segment in a realistic environment for an AQP4
inhibitor. Potential applications of the methodology unrelated to
AQP4 includes stabilisation of the 310 helical conformation of
a peptide corresponding to the N-terminal residues of the PB1
subunit of influenza virus RNA polymerase, an element recently
identified as responsible for an essential subunit interaction and a
potential lead structure for development of new influenza drugs.54
1604 | Org. Biomol. Chem., 2009, 7, 1599–1611
This journal is
The Royal Society of Chemistry 2009
©

View Article Online
Future work will focus on further structural elucidation of the
heptapeptides and establishing the affinity of the peptides for
AQP4 in a radioligand binding assay.
3.75 (1H, br d, J 5, CaH(Val1)), 3.64 (3H, s, OCH3), 2.16–2.01
(2H, m, CH(CH3)2), 0.96–0.90 (12H, m, CH(CH3)2); dC (75 MHz;
d6-DMSO) 171.4, 168.4, 158.4 (q, JCF 31), 117.1 (q, JCF 297), 57.7,
56.9, 51.7, 29.9, 29.6, 18.8, 18.1, 18.1, 17.4; m/z (ESI) 231.1710
(M+; C11H23N2O3 requires 231.1708).
Experimental
a,a-Dimethylglycyl L-valine methyl ester trifluoroacetate (7).
A
General
50% (v/v) solution of TFA in CH2Cl2 (350 mL) was added to
N-tert-butoxycarbonyl a,a-dimethylglycyl L-valine methyl ester
5 (33.50 g, 0.1059 mol) at room temperature and the reaction
mixture stirred for 2 hours. The solvent and bulk of excess TFA
were removed under reduced pressure. The residue was washed
with Et2O (3 ¥ 200 mL) and dried under reduced pressure
affording a clear oil. Dichloromethane (3 ¥ 200 mL) was added
and evaporated under reduced pressure affording a sticky white
solid (28.51 g, 82%); dH (300 MHz; d6-DMSO) 8.42 (1H, d, J 8,
NH), 8.26 (3H, br s, NH3+), 4.17 (1H, dd, J 8 and 8, CaH(Val)),
3.64 (3H, s, OCH3), 2.16–2.05 (1H, m, CH(CH3)2), 1.52 (3H, s,
CH3(Aib)), 1.50 (3H, s, CH3(Aib)), 0.91 (3H, d, J 6, CH3(Val)),
0.88 (3H, d, J 6, CH3(Val)); dC (75 MHz; d6-DMSO) 172.0, 171.6,
158.1 (q, JCF 32), 116.9 (q, JCF 297), 58.3, 56.5, 51.7, 29.5, 23.2,
23.1, 19.0, 18.6; m/z (ESI) 217.1556 (M+; C10H21N2O3 requires
217.1552).
Chemicals were purchased from Sigma-Aldrich Co. and used
as received unless otherwise stated. All solvents were of HPLC
quality and all reagents were more than 98% pure. Flash chro-
matography was carried out using Silica Gel 60 (particle size:
0.04–0.063 mm/230–400 mesh) from Aldrich Co. NMR spectra
were recorded in CDCl3, d6-DMSO or CD2Cl2 on a Bruker Avance
DPX200 or a Bruker Avance DPX300 instrument at 200 MHz
and 300 MHz respectively. All 2D spectra were recorded in phase
sensitive mode using the TPPI (time proportional phase incre-
mentation) method. The spectra were calibrated against internal
tetramethylsilane, residual CHCl3 (dH = 7.26 ppm, dC = 77.0 ppm),
CHDCl2 (dH = 5.32 ppm, dC = 54.00 ppm) or CHD2SOCD3
(dH = 2.50 ppm, dC = 39.43 ppm). For all compounds, with
the exceptions of 29 and 30, only residue specific assignments
were made. Three-letter amino acid abbreviations are given in
parenthesis where the assignment of signals otherwise could be
ambiguous. The residue O-allyl L-serine has been abbreviated
Ser(All) and the residue O-benzyl L-serine as Ser(Bn). Iden-
tical residues, e.g. two valines, are numbered starting from the
N-terminus. For the cyclic penta- and heptapeptides the chemical
shifts given are for the major stereoisomer. Coupling constant (J)
values are rounded off to the nearest whole number and given
in Hz. Levels of epimerisation were estimated from the ratios
of the NH integrals in the 1H-NMR spectra. High-resolution
mass spectrometric analyses were carried out on a Micromass
Q-Tof-2 instrument with electrospray ionisation. CD spectra were
measured on a JASCO J-810 spectropolarimeter between 185 and
260 nm using a 0.1 cm pathlength quartz cell and recording one
data point every 0.5 nm. All spectra were baseline corrected,
averaged over 5 scans, converted to a uniform scale of mean molar
ellipticity per residue (in 103 ¥ deg cm2 dmol-1) and plotted using
Microsoft Excel. The temperature was maintained at 20 ◦C and the
concentrations of all peptides were in the range of 200–300 mM. IR
spectra were recorded on a Perkin Elmer Spectrum BX FTIR
instrument.
N-tert-Butoxycarbonyl O-allyl L-seryl L-valyl L-valine methyl
ester (8). N-tert-Butoxycarbonyl O-allyl L-serine 3 (11.31 g,
46.10 mmol) was dissolved in DMF (40 mL). A solution of L-valyl
L-valine methyl ester trifluoroacetate 6 (15.87 g, 46.09 mmol) and
N,N-diisopropylethylamine (5.96 g, 46.1 mmol) in DMF (60 mL)
was added in one portion and the solution cooled to 0 ◦C (ice
bath). HOBt hydrate (7.06 g, 46.1 mmol) was added. Finally,
EDC hydrochloride (9.72 g, 50.7 mmol) was added together with
30 mL DMF. The reaction mixture was stirred for 22 hours before
the solvent was evaporated. The residue was taken up in EtOAc
(250 mL) and washed with 2 M aqueous H2SO4 (3 ¥ 100 mL),
7.5% (w/w) aqueous K2CO3 (3 ¥ 100 mL) and saturated brine
(100 mL). The solution was dried with anhydrous MgSO4 and the
solvent evaporated affording an off-white solid (19.82 g, 94%); dH
(300 MHz; d6-DMSO) 8.18 (1H, d, J 8, NH(Valx)), 7.62 (1H, d,
J 9, NH(Valy)), 7.03 (1H, d, J 8, NH(Ser(All))), 5.83 (1H, ddt, J
=
=
5, 10 and 17, CH CH2), 5.23 (1H, ddt, J 1, 3 and 17, CH CHH),
=
5.11 (1H, ddt, J 1, 3 and 10, CH CHH), 4.34 (1H, dd, J 7 and
9, CaH(Valy)), 4.22–4.17 (1H, m, CaH(Ser(All))), 4.13 (1H, dd,
a
=
J 6 and 8, C H(Valx)), 3.92 (2H, dt, J 1 and 5, CH2CH CH2),
3.61 (3H, s, OCH3), 3.53–3.49 (2H, m, CH2), 2.11–1.86 (2H, m,
CH(CH3)2), 1.38 (9H, s, (CH3)3), 0.90–0.79 (12H, m, CH3); dC
(75 MHz; d6-DMSO) 171.6, 171.0, 169.5, 155.1, 134.9, 116.3, 78.2,
70.9, 69.5, 57.4, 56.8, 54.5, 51.4, 31.1, 29.5, 28.0, 18.9, 18.8, 18.1,
17.7; m/z (ESI) 480.2666 ([M + Na]+; C22H39N3O7Na requires
480.2685).
Synthetic procedures†
L-Valyl L-valine methyl ester trifluoroacetate (6). N-tert-
Butoxycarbonyl L-valyl L-valine methyl ester
30.48 mmol) was treated with a 50% (v/v) TFA solution in
CH2Cl2 for 2 hours at room temperature. The solvent and bulk
of excess TFA were evaporated affording a clear, crystalline solid
4 (10.07 g,
O-Allyl L-seryl L-valyl L-valine methyl ester (9). N-tert-
Butoxycarbonyl O-allyl L-seryl L-valyl L-valine methyl ester 8
(6.80 g, 14.9 mmol) was stirred in formic acid (70 mL) at room
temperature for 9 hours and the formic acid evaporated. The
residue was diluted with water (110 mL) and the aqueous solution
washed with Et2O (3 ¥ 30 mL). The pH was adjusted to pH 9 by
addition of solid K2CO3 and the resulting solution extracted with
EtOAc (3 ¥ 55 mL). The combined organic extracts were washed
with saturated brine (30 mL) and dried with anhydrous MgSO4.
1
which contained TFA as judged by H-NMR. Small portions of
purified CHCl3 (i.e. without EtOH as stabiliser) were added and
evaporated under reduced pressure four times resulting in an off-
white solid. The solid was washed with Et2O (3 ¥ 50 mL) and dried
under reduced pressure affording a white solid (10.20 g, 97%). The
compound has been mentioned in the literature, but spectral data
was not provided55; dH (300 MHz; d6-DMSO) 8.62 (1H, d, J 7,
NH), 8.17 (3H, br s, NH3+), 4.19 (1H, dd, J 7 and 6, CaH(Val2)),
This journal is
The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 1599–1611 | 1605
©

View Article Online
The solvent was evaporated affording a white solid (4.35 g, 82%);
4.18–4.12 (2H, m, CaH(Ser(All))/CaH(Val)), 3.97–3.94 (2H, m,
=
dH (200 MHz; d6-DMSO) 8.26 (1H, d, J 8, NH(Valx)), 8.03 (1H,
d, J 9, NH(Valy)), 5.85 (1H, ddt, J 5, 10 and 17, CH CH2)),
CH2CH CH2), 3.62 (3H, s, OCH3), 3.57–3.46 (2H, m, CH2),
=
2.08–1.93 (1H, m, CH(CH3)2), 1.38 (9H, s, (CH3)3), 1.37 (3H, s,
CH3(Aib)), 1.35 (3H, s, CH3(Aib)), 0.83 (3H, d, J 7, CH3(Val)),
0.82 (3H, d, J 7, CH3(Val)); dC (75 MHz; d6-DMSO) 173.8,
171.7, 169.5, 155.2, 134.8, 116.3, 78.2, 70.9, 69.4, 57.4, 56.1, 54.3,
51.5, 29.9, 28.0, 25.6, 23.8, 18.8, 18.1; m/z 466.2514 ([M + Na]+;
C21H37N3O7Na requires 466.2529).
=
5.24 (1H, ddt, J 1, 3 and 17, CH CHH), 5.12 (1H, ddt, J 1,
a
=
3 and 10, CH CHH), 4.36 (1H, dd, J 6 and 9, C H(Valy)),
4.12 (1H, dd, J 6 and 8, CaH(Valx)), 3.93 (2H, dt, J 1 and 5,
=
CH2CH CH2), 3.61 (3H, s, OCH3), 3.49 (2H, d, J 5, CH2), 3.43–
3.39 (1H, m, CaH(Ser(All))), 2.38 (2H, br s, NH2), 2.12–1.86 (2H,
m, CH(CH3)2), 0.91–0.78 (12H, m, CH3).
N-tert-Butoxycarbonyl O-benzyl L-seryl L-valyl L-valine methyl
ester (13). L-Valyl L-valine methyl ester trifluoroacetate 6
(11.62 g, 33.75 mmol) was dissolved in DMF (60 mL). N-tert-
Butoxycarbonyl O-benzyl L-serine 14 (9.97 g, 33.8 mmol) was
added together with 10 mL DMF. The solution was cooled to 0 ◦C
(ice bath) before N,N-diisopropylethylamine (4.36 g, 33.7 mmol),
HOBt hydrate (5.17 g, 33.8 mmol) and EDC hydrochloride (7.12 g,
37.1 mmol) were added together with an additional 30 mL
DMF. The reaction mixture was stirred overnight and the solvent
evaporated. The residue was taken up in 200 mL EtOAc and
washed with 2 M aqueous H2SO4 (3 ¥ 80 mL), 7.5% (w/w) aqueous
K2CO3 (3 ¥ 80 mL) and saturated brine (80 mL). The solution was
dried with anhydrous MgSO4 and the solvent evaporated affording
a white solid (16.22 g, 95%); dH (300 MHz; d6-DMSO) 8.20 (1H,
d, J 8, NH(Valx)), 7.65 (1H, d, J 9, NH(Valy)), 7.35–7.23 (5H, m,
Ph), 7.08 (1H, d, J 8, NH(Ser(Bn)), 4.46 (2H, s, CH2Ph), 4.35 (1H,
dd, J 7 and 9, CaH(Valy)), 4.25 (1H, m, CaH(Ser(Bn))), 4.12 (1H,
dd, J 6 and 8, CaH(Valx)), 3.63–3.54 (2H, m, CH2), 3.60 (3H, s,
OCH3), 2.07–1.89 (2H, m, CH(CH3)2), 1.38 (9H, s, (CH3)3), 0.88–
0.80 (12H, m, CH3); dC (75 MHz; d6-DMSO) 171.6, 171.0, 169.4,
155.1, 138.0, 128.0, 127.3, 127.3, 78.2, 71.9, 69.8, 57.4, 56.8, 54.5,
51.4, 31.1, 29.5, 28.0, 18.9, 18.7, 18.2, 17.7; m/z (ESI) 530.2824
([M + Na]+; C26H41N3O7Na requires 530.2842).
N-tert-Butoxycarbonyl L-prolyl L-prolyl O-allyl L-seryl L-valyl
L-valine methyl ester (10). N-tert-Butoxycarbonyl L-prolyl
L-proline 2 (3.80 g, 12.2 mmol) and O-allyl L-seryl L-valyl L-valine
methyl ester 9 (4.35 g, 12.2 mmol) were dissolved in DMF (25 mL)
and the solution cooled to 0 ◦C. HOBt hydrate (1.87 g, 12.2 mmol)
was added under stirring. EDC hydrochloride (2.57 g, 13.4 mmol)
was added to the reaction mixture in small portions. The reaction
mixture was stirred overnight and the solvent evaporated. The
residue was taken up in EtOAc (50 mL) and washed with 2 M
HCl (3 ¥ 25 mL), 7.5% (w/w) K2CO3 (3 ¥ 25 mL) and saturated
brine (20 mL). The solution was dried with anhydrous MgSO4
and the solvent evaporated affording the title compound as a
white solid (7.34 g, 93%); dH (300 MHz; d6-DMSO) 8.10 (1H,
d, J 8, NH(Valx)), 7.98 (1H, d, J 8, NH(Ser(All))), 7.60 (1H, d,
J 9, NH(Valy), rotamer 1), 7.59 (1H, d, J 9, NH(Valy), rotamer
=
2), 5.83 (1H, ddt, J 5, 11 and 17, CH CH2), 5.24 (1H, ddt, J
=
=
1, 3 and 17, CH CHH), 5.14–5.09 (1H, m, CH CHH), 4.46–
4.37 (3H, m, CaH(Pro1)/CaH(Pro2)/CaH(Ser(All))), 4.32 (1H,
dd, J 7 and 9, CaH(Valy)), 4.15–4.10 (1H, m, CaH(Valx)), 3.93
=
(2H, m, J 1 and 5, CH2CH CH2), 3.67–3.50 and 3.36–3.24 (6H,
m, CdH2(Pro1)/CdH2(Pro2)/CH2(Ser(All))), 3.61 (3H, s, OCH3),
2.26–1.71 (10H, m, CH2(Pro)/CH(CH3)2), 1.37 (9H, s, (CH3)3,
rotamer 1), 1.30 (9H, s, (CH3)3, rotamer 2), 0.89–0.80 (12H, m,
CH3); dC (75 MHz; d6-DMSO) 171.6, 171.5, 171.3, 171.1, 170.9,
170.4, 169.1, 169.1, 153.3, 152.9, 134.8, 116.5, 78.3, 78.1, 71.1, 69.3,
59.1, 57.4, 57.2, 56.9, 52.9, 51.4, 46.5, 46.4, 46.3, 46.3, 30.9, 30.6,
29.6, 29.5, 29.4, 28.7, 28.5, 28.1, 27.9, 24.4, 24.3, 23.6, 23.0; m/z
(ESI) 674.3737 ([M + Na]+; C32H53N5O9Na requires 674.3741).
N-tert-Butoxycarbonyl O-allyl L-seryl L-valyl L-valine (15).
N-tert-Butoxycarbonyl O-allyl L-seryl L-valyl L-valine methyl
ester 8 (10.21 g, 22.31 mmol)◦was dissolved in THF (170 mL).
The solution was cooled to 0 C (ice bath). LiOH monohydrate
(1.03 g, 24.6 mmol) was dissolved in de-ionized water (85 mL)
and the solution cooled to 0 ◦C. The solution was then added
dropwise to the solution of 8 over 20 min. The reaction mixture
N-tert-Butoxycarbonyl O-allyl L-seryl a,a-dimethylglycyl L-
valine methyl ester (12). a,a-Dimethylglycyl L-valine methyl
ester trifluoroacetate 7 (19.20 g, 58.13 mmol) was dissolved
in DMF (100 mL). N-tert-Butoxycarbonyl O-allyl L-serine 3
(14.25 g, 58.12 mmol) and then N,N-diisopropylethylamine
(7.51 g, 58.1 mmol) were added and the solution cooled to
◦
was stirred for an additional 2 h 40 min at 0 C. Solid NaHCO3
(3.75 g, 44.6 mmol) was added and the mixture stirred for 5 min
before the THF was evaporated. The solution was diluted with
water (170 mL), washed with Et2O (2 ¥ 170 mL), acidified to pH 2
by addition of 2 M aqueous H2SO4 and extracted with EtOAc
(3 ¥ 250 mL). The combined organic fractions were dried with
anhydrous MgSO4 and the solvent evaporated affording the title
compound as a white solid (7.00 g, 71%); dH (300 MHz; d6-DMSO)
12.53 (1H, br s, COOH), 8.00 (1H, d, J 8, NH (Valx)), 7.62 (1H, d,
J 9, NH(Valy)), 7.02 (1H, d, J 8, NH(Ser(All))), 5.84 (1H, ddt, J
0
◦C (ice bath). HOBt hydrate (8.91 g, 58.2 mmol) and then
EDC hydrochloride (12.26 g, 63.95 mmol) were added together
with an additional 70 mL DMF. The reaction mixture was
stirred for 22 hours before the solvent was evaporated. The
residue was taken up in EtOAc (350 mL) and washed with 2 M
aqueous H2SO4 (3 ¥ 130 mL), 7.5% (w/w) aqueous K2CO3 (3 ¥
130 mL) and saturated brine (130 mL). The solution was dried
with anhydrous MgSO4 and the solvent evaporated affording an
=
=
5, 10 and 17, CH CH2), 5.23 (1H, ddt, J 1, 3 and 17, CH CHH)),
=
5.12 (1H, ddt, J 1, 3 and 10, CH CHH), 4.34 (1H, dd, J 7 and
1
off-white solid. H-NMR indicated 10–15% epimerization. The
9, CaH(Valy)), 4.21–4.15 (1H, m, CaH(Ser(All))), 4.10 (1H, dd,
a
=
residue was recrystallized twice from ethyl acetate/hexane (4:1) to
give the title compound as a stereopure off-white solid (20.15 g,
78% (sum of diastereomers)/13.49 g, 52%); dH (300 MHz; d6-
DMSO) 8.01 (1H, s, NH(Aib)), 7.29 (1H, d, J 8, NH(Val)),
J 6 and 8, C H(Valx)), 3.92 (2H, dt, J 1 and 5, CH2CH CH2),
3.59–3.45 (2H, m, CH2), 2.06–1.91 (2H, m, CH(CH3)2), 1.38
(9H, s, (CH3)3), 0.90–0.80 (12H, m, CH3); dC (75 MHz; d6-DMSO)
172.6, 170.9, 169.5, 155.1, 134.9, 116.3, 78.2, 71.0, 69.5, 57.2, 56.9,
54.6, 31.1, 29.6, 28.1, 19.0, 19.0, 17.9, 17.7; m/z (ESI) 466.2518
([M + Na]+; C21H37N3O7Na requires 466.2529).
=
6.89 (1H, d, J 7, NH(Ser(All))), 5.92–5.79 (1H, m, CH CH2),
=
=
5.29–5.22 (1H, m, CH CHH), 5.15–5.11 (1H, m, CH CHH),
1606 | Org. Biomol. Chem., 2009, 7, 1599–1611
This journal is
The Royal Society of Chemistry 2009
©

View Article Online
N-tert-Butoxycarbonyl O-allyl L-seryl a,a-dimethylglycyl
L-valine (16). N-tert-Butoxycarbonyl O-allyl L-seryl a,a-
dimethylglycyl L-valine methyl ester 12 (8.56 g, 19.3 mmol) was
dissolved in THF (150 mL). The solution was cooled to 0 ◦C (ice
bath). LiOH monohydrate (0.891 g, 21.2 mmol) was dissolved in
de-ionized water (75 mL) and the solution cooled to 0 ◦C. The
solution was added dropwise to the solution of 12 over 20 min.
The reaction mixture was stirred for an additional 2 h 40 min
before solid NaHCO3 (3.24 g, 38.6 mmol) was added. The mixture
was stirred for 5 min and the bulk of THF evaporated. The
solution was diluted with water (150 mL), washed with Et2O (2 ¥
150 mL), acidified to pH 2 by addition of 2 M aqueous H2SO4
and extracted with EtOAc (3 ¥ 300 mL). The combined organic
fractions were dried with anhydrous MgSO4 and the solvent
evaporated affording the title compound as a white solid (7.30 g,
88%); dH (300 MHz; d6-DMSO) 12.62 (1H, br s, COOH), 8.01
(1H, s, NH(Aib)), 7.12 (1H, d, J 9, NH(Val)), 6.85 (1H, d, J 8,
diisopropylethylamine (3.66 g, 28.3 mmol) added. The suspension
was then added in one portion to the solution of 3 together with
an additional 5 mL DMF. HOBt hydrate (4.33 g, 28.3 mmol) was
added. Finally, EDC hydrochloride (5.97 g, 31.1 mmol) was added
together with an additional 10 mL DMF. The reaction mixture
◦
was stirred at 0 C for 1 hour and then at room temperature for
23 hours before the solvent was evaporated. The residue was taken
up in EtOAc (150 mL) and washed with 1 M aqueous H2SO4 (3 ¥
100 mL), 7.5% (w/w) aqueous K2CO3 (3 ¥ 100 mL) and finally
saturated brine (100 mL). The solution was dried with anhydrous
MgSO4 and the solvent evaporated affording a clear and slightly
greenish/yellowish liquid (7.49 g, 84%); dH (200 MHz; d6-DMSO)
8.31 (1H, t, J 6, NH(Gly)), 6.85 (1H, d, J 8, NH(Ser(All))), 5.86
=
=
(1H, ddt, J 5, 11 and 17, CH CH2), 5.29–5.11 (2H, m, CH CH2),
4.22 (1H, ddd, J 8 and 12, CaH(Ser(All))), 3.94 (2H, dt, J 1 and
a
=
5, CH2CH CH2), 3.86 (1H, d, J 6, C HH(Gly)), 3.83 (1H, d, J 6,
CaHH(Gly)), 3.62 (3H, s, OCH3), 3.56–3.44 (2H, m, CH2), 1.38
(9H, s, (CH3)3); dC (50 MHz; d6-DMSO) 170.3, 170.0, 155.1, 134.9,
116.3, 78.2, 70.9, 69.6, 54.1, 51.6, 40.5, 28.1; m/z (ESI) 339.1541
([M + Na]+; C14H24N2O6Na requires 339.1532).
=
NH(Ser(All))), 5.92–5.79 (1H, m, CH CH2), 5.29–5.21 (1H, m,
=
=
CH CHH), 5.15–5.10 (1H, m, CH CHH), 4.19–4.10 (2H, m,
a
a
=
C H(Ser(All))/C H(Val)), 3.94 (2H, dt, J 1 and 5, CH2CH CH2),
3.57–3.45 (2H, m, CH2), 2.08–1.97 (1H, m, CH(CH3)2), 1.38
(12H, s, CH3(Aib)/(CH3)3), 1.35 (3H, s, CH3(Aib)), 0.84 (3H,
d, J 7, CH3(Val)), 0.81 (3H, d, J 7, CH3(Val)); dC (75 MHz;
d6-DMSO): 173.6, 172.7, 169.5, 155.1, 134.9, 116.3, 78.2, 70.9,
69.5, 57.0, 56.1, 54.4, 30.1, 28.1, 25.7, 23.8, 19.0, 17.8; m/z (ESI)
452.2357 ([M + Na]+; C20H35N3O7Na requires 452.2372).
O-Allyl L-seryl glycine methyl ester trifluoroacetate (20). N-
tert-Butoxycarbonyl O-allyl L-seryl glycine methyl ester 18 (5.86 g,
18.3 mmol) was dissolved in CH2Cl2 (30 mL) and TFA (30 mL)
added at room temperature. The reaction mixture was stirred for
2 hours before the solvent and bulk of excess TFA were evaporated
affording a brownish liquid (7.50 g). The residue was washed with
Et2O (2 ¥ 25 mL), redissolved in CH2Cl2 (25 mL), the solvents
evaporated and the residue dried under reduced pressure affording
a brownish, viscous liquid (6.01 g, 98%); dH (300 MHz; d6-DMSO)
9.02 (1H, t, J 6, NH), 8.33 (3H, br s, NH3+), 5.87 (1H, ddt, J 5, 10
N-tert-Butoxycarbonyl O-benzyl L-seryl L-valyl L-valine (17).
N-tert-Butoxycarbonyl O-benzyl L-seryl L-valyl L-valine methyl
ester 13 (14.60 g, 28.76 mmol) was dissolved in THF (220 mL)
and the solution cooled to 0 ◦C (ice bath). LiOH monohydrate
(1.33 g, 31.7 mmol) was dissolved in de-ionized water (110 mL).
The solution was cooled to 0 ◦C (ice bath) and added dropwise to
the solution of 13 over 25 min. The reaction mixture was stirred for
an additional 2 h 35 min before solid NaHCO3 (4.83 g, 57.5 mmol)
was added and the bulk of THF evaporated. The solution was
diluted with water (220 mL) and washed with Et2O (2 ¥ 220 mL).
The solution was then acidified to pH 2 by addition of 2 M
aqueous H2SO4 and extracted with EtOAc (3 ¥ 350 mL). The
combined organic fractions were dried with anhydrous MgSO4
and the solvent evaporated affording a slightly yellowish liquid.
The residue was redissolved in EtOAc and precipitated by addition
of hexane. The solvent was decanted off and the residue dried
affording the title compound as an off-white solid (5.95 g, 42%);
dH (200 MHz; d6-DMSO) 12.52 (1H, br s, COOH), 8.03 (1H, d, J
8, NH(Valx)), 7.65 (1H, d, J 9, NH(Valy)), 7.36–7.23 (5H, m, Ph),
7.10 (1H, d, J 8, NH(Ser(Bn)), 4.46 (2H, s, CH2Ph), 4.36 (1H, dd,
J 7 and 9, CaH(Valy)), 4.28–4.22 (1H, m, CaH(Ser(Bn))), 4.10 (1H,
dd, J 6 and 8, CaH(Valx)), 3.64–3.54 (2H, m, CH2), 2.08–1.91 (2H,
m, CH(CH3)2), 1.38 (9H, s, (CH3)2), 0.88–0.80 (12H, m, CH3);
dC (75 MHz; d6-DMSO) 172.6, 170.9, 169.4, 155.1, 138.1, 128.0,
127.4, 127.3, 78.3, 71.9, 69.8, 57.2, 56.8, 54.6, 31.1, 29.5, 28.1, 19.0,
18.9, 18.0, 17.7; m/z (ESI) 516.2663 ([M + Na]+; C25H39N3O7Na
requires 516.2685).
=
=
and 17, CH CH2), 5.29 (1H, ddt, J 1, 2 and 17, CH CHH), 5.21–
a
=
5.14 (1H, m, CH CHH), 4.10 (1H, m, C H(Ser(All))), 4.00 (2H,
ddd, J 1, 1 and 5, CH2CH CH2), 3.95 (2H, d, J 6, C H2(Gly)),
a
=
3.78–3.67 (2H, m, CH2), 3.64 (3H, s, OCH3); dC (75 MHz; d6-
DMSO) 169.7, 167.0, 158.4 (q, JCF 32), 134.4, 116.7 (q, JCF
296), 71.4, 68.0, 54.9, 52.3, 51.8, 40.7; m/z (ESI) 217.1194 (M+;
C9H17N2O4 requires 217.1188).
N-tert-Butoxycarbonyl O-allyl L-seryl L-valyl L-valyl O-allyl
L-seryl glycine methyl ester (22). O-Allyl L-seryl methyl ester
trifluoroacetate 20 (7.03 g, 21.3 mmol), N-tert-butoxycarbonyl
O-allyl L-seryl L-valyl L-valine 15 (9.45 g, 21.3 mmol) and
N,N-diisopropylethylamine (2.75 g, 21.3 mmol) were dissolved
◦
in CH2Cl2 (100 mL) and the solution cooled to 0 C (ice bath).
HOBt hydrate (3.26 g, 21.3 mmol) and then EDC hydrochloride
(4.49 g, 23.4 mmol) were added in small portions. The reaction
mixture was stirred for 1 h 30 min at 0 ◦C after which the ice
bath was removed and the mixture stirred for 14 h 30 min. The
volume was then increased by addition of CH2Cl2 (200 mL)
and the mixture stirred for another 7 hours before the volume
was increased to 400 mL. The suspension was washed with 2 M
aqueous H2SO4 (150 + 300 + 450 mL), 7.5% (w/w) aqueous
K2CO3 (150 + 300 + 450 mL), saturated brine (400 mL) and
water (400 + 600 mL). The suspension was then diluted with
˚
N-tert-Butoxycarbonyl O-allyl L-seryl glycine methyl ester
(18). Purified N-tert-butoxycarbonyl O-allyl L-serine 3 (6.94 g,
28.3 mmol) was dissolved in DMF (30 mL) and the solution
cooled to 0 ◦C (ice bath). Glycine methyl ester hydrochloride
(3.55 g, 28.3 mmol) was suspended in DMF (10 mL) and N,N-
CH2Cl2 (1.5 L) and dried with 3 A molecular sieves resulting
in a slightly turbid solution. The solvent was evaporated
affording a slightly yellowish solid, which was recrystallised
from EtOH affording the title compound as a white solid
(12.12 g, 89%); dH (300 MHz; d6-DMSO) 8.36 (1H, t, J 6,
This journal is
The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 1599–1611 | 1607
©

View Article Online
NH(Gly)), 7.98 (1H, d, J 8, NH(Ser(All)2)), 7.90 (1H, d, J
9, NH(Valx)), 7.68 (1H, d, J 9, NH(Valy)), 7.02 (1H, d, J 8,
d6-DMSO) 8.25–8.20 (2H, m, NH(Gly1)/NH(Gly2)), 7.93 (1H, d,
J 8, NH(Valx)), 7.70 (1H, d, J 9, NH(Valy)), 7.35–7.25 (5H, m,
Ph), 7.06 (1H, d, J 8, NH(Ser(Bn))), 4.47 (2H, s, CH2Ph), 4.32–
4.23 (2H, m, CaH(Ser(Bn))/CaH(Val)), 4.11 (1H, dd, J 8 and 8,
CaH(Val)), 3.89 (1H, dd, J 6 and 17, CaHH(Gly)), 3.82 (1H, dd,
J 6 and 17, CaHH(Gly)), 3.75 (2H, m, CaH2(Gly)), 3.62 (3H, s,
OCH3), 3.60–3.53 (2H, m, CH2), 2.01–1.88 (2H, m, CH(CH3)2),
1.38 (9H, s, (CH3)3), 0.85–0.78 (12H, m, CH3); dC (75 MHz; d6-
DMSO) 171.1, 170.9, 170.1, 169.6, 169.1, 155.2, 138.1, 128.0,
127.4, 127.3, 78.2, 71.9, 69.8, 58.1, 57.2, 54.5, 51.6, 41.6, 40.5,
30.8, 30.1, 28.1, 19.1, 18.2, 17.8; m/z (ESI) 644.3268 ([M + Na]+;
C30H47N5O9Na requires 644.3271).
=
NH(Ser(All)1)), 5.90–5.77 (2H, m, CH CH2), 5.27–5.10 (4H,
a
=
m, CH CH2), 4.55–4.49 (1H, m, C H(Ser(All)2)), 4.30–4.15
(3H, m, CaH(Valx)/CaH(Valy)/CaH(Ser(All)1)), 3.95–3.91 (4H,
a
=
m, CH2CH CH2), 3.86–3.84 (2H, m, C H2(Gly)), 3.61 (3H, s,
OCH3), 3.56–3.46 (4H, m, CH2), 2.02–1.89 (2H, m, CH(CH3)2),
1.38 (9H, s, (CH3)3), 0.84–0.78 (12H, m, CH3); dC (75 MHz;
d6-DMSO) 170.6, 169.9, 169.7, 169.5, 155.1, 134.9, 134.8, 116.5,
116.3, 79.8, 79.7, 78.2, 71.0, 69.5, 69.4, 57.4, 57.2, 57.2, 54.6, 54.5,
54.5, 52.3, 51.6, 40.5, 30.8, 30.3, 28.0, 19.0, 17.9, 17.8; m/z (ESI)
664.3526 ([M + Na]+; C30H51N5O10Na requires 664.3533).
N-tert-Butoxycarbonyl O-allyl L-seryl a,a-dimethylglycyl L-
valyl O-allyl L-seryl glycine methyl ester (23). O-Allyl L-seryl
glycine methyl ester trifluoroacetate 20 (4.42 g, 13.4 mmol)
was dissolved in CH2Cl2 (40 mL) and N-tert-butoxycarbonyl
O-allyl L-seryl a,a-dimethylglycyl L-valine 16 (5.75 g, 13.4 mmol)
added. N,N-diisopropylethylamine (1.73 g, 13.4 mmol) and HOBt
hydrate (2.05 g, 13.4 mmol) were added and the solution cooled
to 0 ◦C. EDC hydrochloride (2.82 g, 14.7 mmol) was added in
small portions together with 10 mL CH2Cl2. The reaction mixture
was stirred for 1 h 30 min at 0 ◦C after which the ice bath
was removed and the mixture stirred for an additional 26 hours.
The solution was diluted by addition of CH2Cl2 (60 mL) and
washed with 2 M aqueous H2SO4 (3 ¥ 90 mL), 7.5% (w/w)
aqueous K2CO3 (3 ¥ 90 mL) and saturated brine (90 mL). The
volume was increased by addition of CH2Cl2, the solution dried
with anhydrous MgSO4 and the solvent evaporated affording a
slightly orange solid (6.14 g, 73%); dH (300 MHz; d6-DMSO)
8.25 (1H, t, J 6, NH(Gly)), 8.14 (1H, s, NH(Aib)), 7.88 (1H,
d, J 8, NH(Ser(All))), 7.14 (1H, d, J 8, NH(Val)), 6.84 (1H,
Methyl 2-((3S,6S,9S,12S,E)-12-(tert-butoxycarbonylamino)-6,
9-diisopropyl-5,8,11-trioxo-1,14-dioxa-4,7,10-triazacyclooctadec-
16-ene-carboxamido)-acetate (25). N-tert-Butoxycarbonyl O-allyl
L-seryl L-valyl L-valyl O-allyl L-seryl glycine methyl ester 22
(0.642 g, 1.00 mmol) was dissolved in dry, degassed CH2Cl2
(225 mL). Grubbs’ 2nd generation catalyst (0.085 g, 0.10 mmol)
dissolved in dry, degassed CH2Cl2 (25 mL) was added by syringe
and the reaction mixture stirred under argon atmosphere for 2 h
45 min. A second portion of Grubbs’ 2nd generation catalyst
(0.085 g, 0.10 mmol) dissolved in CH2Cl2 (20 mL) was then
added and the reaction mixture stirred for an additional 4 h
45 min. Ethyl vinyl ether (1.5 mL) was added to quench the reac-
tion/remaining catalyst. After stirring for 25 min the solvent was
evaporated affording a dark residue. The residue was redissolved
in a minimal amount of CH2Cl2 and purified by flash column
chromatography (eluent: CH2Cl2/acetone (2:1)) affording the title
compound as an off-white solid (0.256 g, 42%); dH (300 MHz;
CDCl3) 7.23–7.20 (2H, m, NH(Valx)/NH(Gly)), 7.11 (1H, d,
J 8, NH(Ser(All)2)), 6.75 (1H, d, J 7, NH(Valy)), 5.92–5.80 (2H,
=
=
d, J 7, NH (Ser(All)1)), 5.92–5.74 (2H, m, CH CH2), 5.28–5.11
m, CH CH), 5.35 (1H, d, J 6, NH(Ser(All)1)), 4.71–4.65 (1H,
a
(4H, m, CH CH2), 4.50 (1H, m, C H(Ser(All)2)), 4.17–4.09 (2H,
m, CaH(Ser(All)2)), 4.37 (1H, dd, J 6 and 12, CaH(Ser(All)1)),
=
a
a
a
a
=
=
m, C H(Ser(All)1)/C H(Val)), 3.95 (4H, m, CH2CH CH2), 3.85
(2H, d, J 6, CaH2(Gly)), 3.61 (3H, s, OCH3), 3.58–3.46 (4H, m,
CH2), 2.06–1.95 (1H, m, CH(CH3)2), 1.38 (9H, s, (CH3)3), 1.35
(3H, s, CH3(Aib)), 1.34 (3H, s, CH3(Aib)), 0.83 (3H, d, J 7,
CH3(Val)), 0.78 (3H, d, J 7, CH3(Val)); dC (75 MHz; d6-DMSO)
173.9, 170.7, 169.9, 169.8, 169.7, 155.3, 134.8, 116.4, 116.3, 78.4,
71.0, 69.4, 58.2, 56.2, 54.8, 54.5, 52.5, 51.6, 40.6, 30.0, 28.0,
24.9, 24.7, 19.0, 17.8; m/z 650.3366 ([M + Na]+; C29H49N5O10Na
requires 650.3377).
4.18–4.11 (3H, m, CHHCH CH/C H(Valx)/C H(Valy)), 4.08
(1H, d, J 6, CaHH(Gly)), 4.02–3.91 (5H, m, CHH(Ser(All)2)/
a
=
=
CH2CH CH/CHHCH CH/C HH(Gly)), 3.77–3.69 (2H, m,
CH2(Ser(All)1)), 3.73 (3H, s, OCH3), 3.63 (1H, dd, J 5 and 9,
CHH(Ser(All)2)), 2.35–2.21 (2H, m, CH(CH3)2), 1.46 (9H, s,
(CH3)3), 1.02–0.90 (12H, m, CH3); dC (75 MHz; CDCl3) 172.0,
171.5, 171.3, 170.0, 156.3, 130.7, 128.7, 80.8, 77.2, 71.2, 70.0,
68.9, 67.7, 60.5, 60.4, 54.3, 53.3, 52.2, 41.3, 29.9, 29.3, 28.2, 19.5,
19.5, 18.0, 17.3; m/z (ESI) 636.3209 ([M + Na]+; C28H47N5O10Na
requires 636.3220).
N-tert-Butoxycarbonyl O-benzyl L-seryl L-valyl L-valyl glycyl
glycine methyl ester (24). N-tert-Butoxycarbonyl O-benzyl
L-seryl L-valyl L-valine 17 (2.20 g, 4.46 mmol) and glycyl
glycine methyl ester trifluoroacetate 21 (1.16 g, 4.44 mmol) were
dissolved in CH2Cl2 (15 mL). N,N-Diisopropylethylamine (0.57 g,
4.4 mmol) and HOBt hydrate (0.68 g, 4.4 mmol) were added
and the solution cooled to 0 ◦C. EDC hydrochloride (0.94 g,
4.9 mmol) was added slowly together with 5 mL CH2Cl2. The
reaction mixture was stirred for 1 h 30 min at 0 ◦C after which the
ice bath was removed. The mixture was stirred for an additional
23 hours before the volume was increased by addition of CH2Cl2
(20 mL). The solution was washed with 2 M aqueous H2SO4 (3 ¥
30 mL), 7.5% (w/w) aqueous K2CO3 (3 ¥ 30 mL) and saturated
brine (30 mL). The volume of the solution was increased, the
solution dried with anhydrous MgSO4 and the solvent evapo-
rated affording an off-white solid (2.14 g, 78%); dH (300 MHz;
Methyl 2-((3S,6S,12S,E)-12-(tert-butoxycarbonylamino)-6-iso-
propyl-9,9-dimethyl-5,8,11-trioxo-1,14-dioxa-4,7,10-triazacycloo-
ctadec-16-enecarboxamido)acetate (26). N-tert-Butoxycarbonyl
O-allyl L-seryl a,a-dimethylglycyl L-valyl O-allyl L-seryl glycine
methyl ester 23 (0.571 g, 0.910 mmol) was dissolved in dry CH2Cl2
(200 mL) and the solution degassed. Grubbs’ 2nd generation
catalyst (0.077 g, 0.091 mmol) was dissolved in dry, degassed
CH2Cl2 (25 mL) and added to the solution of 23 by syringe.
The reaction mixture was stirred under argon atmosphere for
3 hours. A new portion of Grubbs’ 2nd generation catalyst
(0.039 g, 0.046 mmol) dissolved in dry, degassed CH2Cl2 (10 mL)
was then added. After stirring for 3 hours ethyl vinyl ether
(1.0 mL) was added to quench the reaction/remaining catalyst.
The reaction mixture was stirred for an additional 30 min
before the solvent was evaporated. The residue was purified
1608 | Org. Biomol. Chem., 2009, 7, 1599–1611
This journal is
The Royal Society of Chemistry 2009
©

View Article Online
by flash column chromatography (eluent: CH2Cl2/acetone (2:1))
affording a glassy/transparent solid (0.286 g, 52%); dH (300 MHz;
CDCl3) 7.56–7.40 (2H, m, NH(Ser(All)2)/NH(Gly)), 7.13 (1H, s,
NH(Aib)), 6.93 (1H, d, J 5, NH(Val)), 5.81–5.65 (2H, m,
52.5, 51.6, 40.5, 30.8, 26.7, 22.7, 18.8, 18.1; m/z (ESI) 500.2713
(M+; C22H38N5O8 requires 500.2720).
(S)-tert-Butyl 2-((S)-2-((3S,6S,9S,12S,E)-6,9-diisopropyl-3-(2-
methoxy-2-oxoethylcarbamoyl)-5,8,11-trioxo-1,14-dioxa-4,7,10-
triazacyclooctadec-16-en-12-ylcarbamoyl)pyrrolidine-1-carbonyl)
pyrrolidine-1-carboxylate (29). Trifluoroacetate 27 (0.314 g,
=
CH CH), 5.49 (1H, d, J 5, NH(Ser(All)1)), 4.79–4.73 (1H,
m, CaH(Ser(All)2)), 4.25–4.19 (1H, m, CaH(Ser1)), 4.11–4.00
a
a
=
(4H, m, C H(Val)/C HH(Gly)/CH2CH CH), 3.89–3.74 (6H,
0.500 mmol), N-tert-butoxycarbonyl L-prolyl L-proline
2
a
=
m, CH2CH CH/C HH(Gly)/CH2(Ser(All)2))/CHH(Ser1)), 3.66
(0.157 g, 0.503 mmol) and HOBt hydrate (0.077 g, 0.50 mmol)
were dissolved in CH2Cl2 (5 mL). N,N-Diisopropylethylamine
(0.064 g, 0.50 mmol) dissolved in CH2Cl2 (1 mL) was added
and the mixture stirred for 5 min. EDC hydrochloride (0.106 g,
0.553 mmol) was added in portions together with CH2Cl2 (4 mL).
The reaction mixture was stirred for 1 hour at room temperature
before CH2Cl2 (5 mL) was added. Stirring was continued for
21 hours after which the volume was increased to 50 mL by
addition of CH2Cl2. The solution was washed with 1 M aqueous
H2SO4 (3 ¥ 20 mL), 7.5% (w/w) K2CO3 solution (3 ¥ 20 mL)
and saturated brine (20 mL). The solution was dried with
anhydrous MgSO4 and the solvent evaporated affording the title
compound as an off-white solid (0.316 g, 78%); dH (300 MHz;
CD2Cl2) 7.93 (1H, d, J 7, NH(Ser(All)1)), 7.77 (1H, d, J 5,
NH(Val1)), 7.36–7.31 (2H, m, NH(Ser(All)2)/NH(Gly)), 6.80
(3H, s, OCH3), 3.59–3.49 (1H, m, CHH(Ser1)), 2.42–2.25 (1H,
m, CH(CH3)2), 1.47–1.41 (15H, m, CH3(Aib)/(CH3)3), 1.00–0.92
(6H, m, CH3(Val)); dC (75 MHz; CDCl3) 175.6, 171.2, 171.0, 170.1,
169.9, 156.0, 131.0, 127.7, 80.7, 70.5, 69.5, 69.2, 67.0, 60.6, 57.3,
55.0, 53.8, 51.9, 41.1, 29.0, 28.1, 26.3, 23.7, 19.3, 17.4; m/z (ESI)
622.3054 ([M + Na]+; C27H45N5O10Na requires 622.3064).
(3S,6S,9S,12S,E)-6,9-Diisopropyl-3-(2-methoxy-2-oxoethylcar-
bamoyl)-5,8,11-trioxo-1,14-dioxa-4,7,10-triazacyclooctadec-16-
en12-aminium 2,2,2-trifluoroacetate (27). Cyclic pentapeptide 25
(0.403 g, 0.657 mmol) was dissolved in a 50% (v/v) solution of TFA
in CH2Cl2 (10 mL) at room temperature. The reaction mixture was
stirred for 2 hours before the solvent and excess TFA were evapo-
rated affording an off-white solid. Dichloromethane (10 mL) was
added and evaporated and the residue washed with Et2O (2 ¥ 5 mL)
to give the title compound as a slightly off-white solid (0.392 g,
95%); dH (300 MHz; d6-DMSO) 8.76 (1H, d, J 9, NH(Valx)), 8.58
(1H, t, J 6, NH(Gly)), 8.37–8.22 (4H, m, NH(Ser(All)2)/NH3+),
=
(1H, d, J 6, NH(Val2)), 5.93–5.73 (2H, m, CH CH), 4.73–4.66
(1H, m, CaH(Ser(All)1)), 4.64–4.58 (1H, m, CaH(Ser(All)2)), 4.40
(1H, dd, J 8 and 8, CaH(Pro2), 4.33–4.27 (1H, m, CaH(Pro1),
4.20–3.83 (11H, m, CaH(Val1)/CaH(Val2)/CaH2(Gly)/CH2(Ser
=
7.31 (1H, d, J 8, NH(Valy)), 5.80–5.67 (2H, m, CH CH), 4.84–
=
(All)1)/CHH(Ser(All)2)/CH2CH CH), 3.71–3.58 (3H, m,
4.77 (1H, m, CaH(Ser(All)2)), 4.32 (1H, dd, J 6 and 8, CaH(Valy)),
CHH(Ser(All)2)/CdH2(Pro)), 3.69 (3H, s, OCH3), 3.46–3.29 (2H,
m, CdH2(Pro)), 2.45–2.19 (4H, m, CH2(Pro)), 2.11–1.77 (6H, m,
CH(CH3)2/CH2(Pro)), 1.47 (9H, s, (CH3)3), 1.07–0.96 (12H, m,
CH3); dC (75 MHz; CD2Cl2) 173.9, 173.4, 172.5, 172.3, 172.0,
170.9, 170.5, 155.5, 132.8, 130.8, 81.6, 71.2, 70.7, 69.7, 67.3, 63.5,
63.1, 63.0, 61.2, 55.6, 54.6, 52.4, 47.7, 47.4, 41.8, 29.6, 29.5, 29.4,
29.4, 28.6, 26.7, 25.2, 19.8, 19.3, 19.0, 17.8; m/z (ESI) 830.4269
([M + Na]+; C38H61N7O12Na requires 830.4275).
a
a
=
4.13–3.93 (6H, m, CH2CH CH2/C H(Valx)/C H(Ser(All)1)),
3.90–3.87 (2H, m, CaH2(Gly)), 3.70 (1H, dd, J 4 and 10,
CHH(Ser(All)1)), 3.62 (3H, s, OCH3), 3.55–3.42 (3H, m,
CH2(Ser(All)2)/CHH(Ser(All)1)), 2.10–1.89 (2H, m, CH(CH3)2),
0.91–0.83 (12H, m, CH3); dC (75 MHz; d6-DMSO) 169.9, 169.8,
169.6, 169.5, 166.3, 130.6, 128.6, 70.9, 68.9, 68.7, 67.5, 59.8, 56.9,
52.6, 51.6, 50.8, 40.5, 31.4, 30.3, 19.2, 18.7, 18.3, 18.2; m/z (ESI)
514.2868 (M+; C23H40N5O8 requires 514.2876).
(S)-tert-Butyl 2-((S)-2-((3S,6S,9S,12S,E)-6-isopropyl-3-(2-met-
hoxy-2-oxoethylcarbamoyl)-9,9-dimethyl-5,8,11-trioxo-1,14-dio-
xa-4,7,10-triazacyclooctadec-16-en-12-ylcarbamoyl) pyrrolidine-
1-carbonyl)pyrrolidine-1-carboxylate (30). Trifluoroacetate 28
(0.238 g, 0.388 mmol), N-tert-butoxycarbonyl L-prolyl L-proline
2 (0.122 g, 0.391 mmol) and HOBt hydrate (0.059 g, 0.39 mmol)
were dissolved in CH2Cl2 (5 mL). N,N-Diisopropylethylamine
(0.050 g, 0.39 mmol) dissolved in CH2Cl2 (1 mL) was added
and the mixture cooled to 0 ◦C (ice bath). EDC hydrochloride
(0.082 g, 0.43 mmol) was added in portions together with more
CH2Cl2 (4 mL). The reaction mixture was stirred for 1 hour
at 0 ◦C, after which the ice bath was removed and stirring
continued for 24 h at room temperature. The volume was increased
to 50 mL by addition of CH2Cl2 and the solution washed
with 1 M aqueous H2SO4 (3 ¥ 20 mL), 7.5% (w/w) K2CO3
solution (3 ¥ 20 mL) and saturated brine (20 mL). The solution
was dried with anhydrous MgSO4 and the solvent evaporated
affording the title compound as a white solid (0.251 g, 82%);
dH (300 MHz; CD2Cl2) 7.95 (1H, s, NH(Aib)), 7.87 (1H, d, J
7, NH(Ser(All)1)), 7.59 (1H, d, J 9, NH(Ser(All)2)), 7.46 (1H, t,
J 6, NH(Gly)), 6.94–6.80 (1H, d, J 6, NH(Val)), 5.89–5.68 (2H,
(3S,6S,9S,12S,E)-6-Isopropyl-3-(2-methoxy-2-oxoethylcarbam-
oyl)-9,9-dimethyl-5,8,11-trioxo-1,14-dioxa-4,7,10-triazacycloocta-
dec-16-en-12-aminium 2,2,2-trifluoroacetate (28). Cyclic penta-
peptide 26 (0.426 g, 0.710 mmol) was dissolved in a 50% (v/v)
solution of TFA in CH2Cl2 (10 mL). The reaction mixture was
stirred for 1 hour at room temperature before the solvent and
bulk of excess TFA were evaporated. Et2O (10 mL) was added
to the liquid residue, which resulted in precipitation of a white
solid. After decanting off the Et2O the residue was washed with
additional portions of Et2O (2 ¥ 10 mL) and dried affording a
white solid (0.309 g, 71%); dH (300 MHz; d6-DMSO) 8.80 (1H, s,
NH(Aib)), 8.49 (1H, t, J 6, NH(Gly)), 8.28 (3H, br s, NH3+),
8.13 (1H, d, J 8, NH(Ser(All)2)), 7.11 (1H, d, J 7, NH(Val)),
a
=
5.84–5.72 (2H, m, CH CH), 4.68–4.61 (1H, m, C H(Ser(All)2)),
a
=
=
4.24–4.03 (4H, m, C H(Val)/CH2CH CH/CHHCH CH), 3.96–
3.91 (2H, m, C H(Ser(All)1)/CHHCH CH), 3.88 (2H, dd, J 6
a
=
and 6, CaH2(Gly)), 3.73 (1H, dd, J 4 and 10, CHH(Ser(All)1)),
3.62 (3H, s, OCH3), 3.54–3.39 (3H, m, CH2(Ser(All)2)/
CHH(Ser(All)1)), 2.04–1.93 (1H, m, CH(CH3)2), 1.43 (3H, s,
CH3(Aib), 1.38 (3H, s, CH3(Aib)), 0.83 (3H, s, CH3(Val)), 0.81
(3H, s, CH3(Val)); dC (75 MHz; d6-DMSO) 172.8, 170.1, 169.8,
169.7, 165.8, 131.0, 129.9, 70.4, 68.9, 67.8, 66.4, 57.4, 56.5, 53.6,
a
a
=
m, CH CH), 4.67–4.57 (2H, m, C H(Ser(All)2)/C H(Ser(All)1)),
4.41 (1H, m, CaH(Pro2)), 4.35–4.29 (1H, m, CaH(Pro1)),
This journal is
The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 1599–1611 | 1609
©

View Article Online
4.12–3.82 (10H, m, CaH(Val)/CaH2(Gly)/CH2(Ser(All)1)/CHH
5.77 (2H, m, CH CH2), 5.30–5.10 (4H, m, CH CH2), 4.56–4.50
=
=
(Ser(All)2)/CH2CH CH), 3.70–3.58 (3H, m, CHH(Ser(All)2)/
(1H, m, CaH(Ser(All)2)), 4.32 (1H, dd, J 7 and 9, CaH(Valx)), 4.24
=
CdH2(Pro)), 3.67 (3H, s, OCH3), 3.50–3.29 (2H, m,
CdH2(Pro)), 2.45–2.26 (3H, m, CH2(Pro)), 2.11–1.76 (6H, m,
CH(CH3)2/CH2(Pro)), 1.48–1.35 (15H, m, CH3(Aib)/(CH3)3),
1.05–0.97 (6H, m, CH3(Val)); dC (75 MHz; CD2Cl2) 177.0, 173.7,
172.4, 172.0, 171.3, 170.9, 170.4, 155.6, 133.1, 130.4, 81.6, 71.1,
70.0, 69.5, 66.9, 63.5, 63.2, 61.6, 58.0, 54.9, 54.5, 52.4, 47.8, 47.4,
41.7, 29.8, 29.4, 29.3, 28.6, 27.3, 26.7, 25.3, 23.6, 19.6, 17.7; m/z
(ESI) 816.4103 ([M + Na]+; C37H59N7O12Na requires 816.4119).
(1H, dd, J 7 and 8, CaH(Valy)), 4.10 (1H, br s, CaH(Ser(All)1)),
3.98–3.93 (4H, m, CH2CH CH2), 3.86–3.84 (2H, m, C H2(Gly)),
a
=
3.72–3.52 (4H, m, CH2(Ser(All)1)/CH2(Ser(All)2)), 3.61 (3H, s,
OCH3), 2.08–1.89 (2H, m, CH(CH3)2), 0.88–0.80 (12H, m, CH3);
dC (75 MHz; d6-DMSO) 170.6, 170.4, 169.9, 169.8, 166.2, 158.3
(q, JCF 37) 134.8, 134.4, 117.1, 116.9 (q, JCF 293), 116.5, 71.4, 71.0,
69.5, 68.3, 57.9, 57.6, 52.4, 52.2, 51.6, 40.6, 30.6, 30.4, 19.1, 18.1,
17.9; m/z (ESI) 542.3181 (M+; C25H44N5O8 requires 542.3189).
N-tert-Butoxycarbonyl L-prolyl L-prolyl O-allyl L-seryl L-valyl
L-valyl O-allyl L-seryl glycine methyl ester (31). O-Allyl L-
seryl L-valyl L-valyl O-allyl L-seryl glycine methyl ester triflu-
oroacetate 32 (0.524 g, 0.799 mmol), N-tert-butoxycarbonyl L-
prolyl L-proline 2 (0.250 g, 0.800 mmol) and HOBt hydrate
(0.122 g, 0.797 mmol) were dissolved in CH2Cl2 (5 mL). N,N-
Diisopropylethylamine (0.103 g, 0.797 mmol) dissolved in CH2Cl2
(5 mL) was added. At this stage the reaction mixture turned
into a gel. EDC hydrochloride (0.169 g, 0.882 mmol) was added
in portions together with an additional 5 mL of CH2Cl2 and
the reaction mixture vigorously stirred for 21 hours at room
temperature. The volume was subsequently increased to 50 mL
by addition of CH2Cl2. The solution was washed with 2 M
aqueous H2SO4 (3 ¥ 20 mL), 7.5% (w/w) K2CO3 solution (3 ¥
20 mL) and saturated brine (30 mL). The solution was dried with
anhydrous MgSO4 and the solvent evaporated affording the title
compound as an off-white solid (0.489 g, 73%); dH (300 MHz;
CDCl3) 7.50–7.37 (3H, m, NH(Valx)/NH(Gly)/NH(Ser(All)x)),
7.12 and 6.85 (1H, d, J 6, NH(Ser(All)y)), 7.02 (1H, d, J 5,
N-tert-Butoxycarbonyl L-prolyl L-prolyl O-benzyl L-seryl L-
valyl L-valyl glycyl glycine methyl ester (33). O-Benzyl L-seryl
L-valyl L-valyl glycyl glycine methyl ester trifluoroacetate 34
(0.364 g, 0.573 mmol), N-tert-butoxycarbonyl L-prolyl L-proline
2 (0.179 g, 0.573 mmol) and HOBt hydrate (0.088 g, 0.58 mmol)
were dissolved in CH2Cl2 (4 mL). N,N-Diisopropylethylamine
(0.074 g, 0.57 mmol) dissolved in CH2Cl2 (1 mL) was added and
the mixture cooled to 0 ◦C (ice bath). EDC hydrochloride (0.121 g,
0.631 mmol) was added in portions together with more CH2Cl2
(1 mL). The ice bath was removed after 1 hour and the reaction
mixture stirred for 22 hours at room temperature. The volume
was increased to 40 mL by addition of CH2Cl2 and the solution
washed with 2 M aqueous H2SO4 (3 ¥ 20 mL), 7.5% (w/w) K2CO3
solution (3 ¥ 20 mL) and saturated brine (20 mL). The solution was
dried with anhydrous MgSO4 and the solvent evaporated affording
the title compound as a slightly yellowish transparent/glassy
solid (0.364 g, 78%); dH (300 MHz; CDCl3) 7.56–7.51 (1H, m,
NH(Valx)), 7.48–7.37 (2H, m, NH(Glyx)/NH(Glyy)), 7.32–7.18
(6H, m, Ph/NH(Ser(Bn)), rotamer 1), 6.90 (1H, d, J 8, NH(Valy)),
6.82 (1H, d, NH(Ser(Bn)), rotamer 2), 4.52-4.27 (6H, m,
CaH(Valy)/CaH(Ser(Bn))/CaH(Prox)/CaH(Proy)/CH2Ph), 4.21-
4.11 (1H, m, CaH(Valx)), 4.07-3.85 (4H, m, CaH2(Gly)), 4.06–
3.75 (3H, m, CH2(Ser(Bn))/CdHH(Prox)), 3.73–3.58 (1H, m,
CdHH(Proy)), 3.64 and 3.62 (3H, s, OCH3), 3.52–3.23 (2H, m,
CdHH(Prox)/CdHH(Proy)), 2.49–2.36 (1H, m, CH(CH3)2), 2.34–
1.91 (6H, m, CH(CH3)2/CH2(Pro)), 1.87–1.49 (3H, m, CH2(Pro)),
1.40 and 1.33 (9H, s, (CH3)3), 1.03–0.98 (6H, m, CH3(Valx)),
0.93–0.89 (6H, m, CH3(Valy)); dC (75 MHz; CDCl3) 174.2, 173.2,
172.8, 172.5, 172.0, 172.0, 171.9, 171.8, 171.8, 171.8, 154.4, 153.1,
146.0, 137.2, 136.8, 128.4, 128.3, 128.1, 127.9, 127.8, 127.6, 80.3,
73.6, 73.1, 68.4, 62.2, 61.8, 61.6, 59.7, 58.7, 57.6, 55.7, 55.2, 53.4,
51.9, 51.8, 47.4, 47.3, 46.7, 46.4, 43.1, 40.9, 30.4, 29.4, 29.2, 29.1,
28.9, 28.3, 28.2, 25.6, 25.3, 24.4, 23.6, 19.5, 19.1, 19.0, 18.6, 18.3,
17.5; m/z (ESI) 838.4308 ([M + Na]+; C40H61N7O11Na requires
838.4326).
=
NH(Valy)), 5.89–5.71 (2H, m, CH CH2), 5.24–5.07 (4H, m,
a
=
CH CH2), 4.83–4.76 (1H, m, C H(Ser(All)x)), 4.48–4.38 (3H,
m, CaH(Ser(All)y)/CaH(Prox)/CaH(Proy)), 4.31–4.27 (1H, m,
CaH(Valy)), 4.23–4.19 (1H, m, CaH(Valx)), 4.09 (1H, dd, J 6 and
a
a
=
18, C HH(Gly)), 3.99–3.92 (5H, m, C HH(Gly)/CH2CH CH2),
3.87–3.37 (8H, m, CH2(Ser(All)x)/CH2(Ser(All)y)/CdH2(Prox)/
CdH2(Proy)), 3.68 (3H, s, OCH3), 2.34–1.81 (10H, m, CH2(Prox)/
CH2(Proy)/CH(CH3)2), 1.44 and 1.36 (9H, s, (CH3)3), 1.02–0.85
(12H, m, CH3); dC (75 MHz; CDCl3) 172.8, 172.6, 171.9, 171.9,
171.8, 171.8, 171.7, 171.3, 171.1, 170.5, 170.4, 170.1, 170.1, 154.5,
153.3, 134.7, 134.6, 133.8, 133.6, 118.0, 117.6, 116.7, 116.6, 80.2,
79.7, 72.3, 72.0, 71.7, 69.5, 68.4, 62.1, 61.6, 61.0, 60.9, 60.1,
59.2, 57.7, 57.7, 55.1, 54.6, 53.2, 53.0, 52.0, 51.9, 47.3, 46.7, 46.5,
41.3, 30.6, 29.9, 29.8, 29.4, 28.9, 28.9, 28.4, 28.3, 25.5, 25.3, 24.5,
23.8, 19.4, 19.0, 18.9, 18.2, 18.1, 18.0, 17.9; m/z (ESI) 858.4575
([M + Na]+; C40H65N7O12Na requires 858.4588).
O-Allyl L-seryl L-valyl L-valyl O-allyl L-seryl glycine methyl
ester trifluoroacetate (32). N-tert-Butoxycarbonyl O-allyl L-seryl
L-valyl L-valyl O-allyl L-seryl glycine methyl ester 22 (0.734 g,
1.14 mmol) was dissolved in a 50% (v/v) solution of TFA in
CH2Cl2 (16 mL). The reaction mixture was stirred for 1 h 30 min
before the solvent and bulk of excess TFA were evaporated
affording an oil. Addition of Et2O (25 mL) resulted in the
precipitation of a white solid. The mixture was centrifuged and the
Et2O decanted off. The residue was washed with an additional 2 ¥
25 mL Et2O and dried under reduced pressure overnight affording
a fine white powder (0.719 g, 96%); dH (300 MHz; d6-DMSO) 8.48
(1H, d, J 9, NH(Valx)), 8.41 (1H, t, J 6, NH(Gly)), 8.24 (3H,
br s, NH3+), 8.01–7.98 (2H, m, NH(Valy)/NH(Ser(All)2)), 5.90–
O-Benzyl L-seryl L-valyl L-valyl glycyl glycine methyl ester
trifluoroacetate (34). N-tert-Butoxycarbonyl O-benzyl L-seryl
L-valyl L-valyl glycyl glycine methyl ester 24 (0.525 g, 0.844 mmol)
was dissolved in a 50% (v/v) solution of TFA in CH2Cl2
(12 mL) at room temperature. The reaction mixture was stirred
for 1 h 30 min before the solvent and bulk of excess TFA
were evaporated. Et2O (10 mL) was added to the liquid residue,
which resulted in precipitation of a white solid. The Et2O was
decanted off and the residue washed with additional portions of
Et2O (2 ¥ 10 mL) and dried under reduced pressure overnight
affording the title compound as a white solid (0.494 g, 92%); dH
(300 MHz; d6-DMSO) 8.47 (1H, d, J 9, NH(Valx)), 8.29–8.20
(5H, m, NH3+/NH(Glyx)/NH(Glyy)), 8.00 (1H, d, J 8, NH(Valy))
1610 | Org. Biomol. Chem., 2009, 7, 1599–1611
This journal is
The Royal Society of Chemistry 2009
©

View Article Online
7.38–7.27 (5H, m, Ph), 4.51 (2H, s, CH2Ph), 4.34 (1H, dd, J 7
and 9, CaH(Valx)), 4.17–4.12 (2H, m, CaH(Valy)/CaH(Ser(Bn))),
3.88 (1H, dd, J 6 and 17, CaHH(Glyx)), 3.81 (1H, dd, J 6 and
17, CaHH(Glyx)), 3.76–3.64 (4H, m, CH2(Ser(Bn))/CaH2(Glyy)),
3.62 (3H, s, OCH3), 2.04–1.89 (2H, m, CH(CH3)2), 0.89–0.81
(12H, m, CH3); dC (75 MHz; d6-DMSO) 171.1, 170.6, 170.1, 169.2,
166.2, 158.3 (q, JCF 32) 137.5, 128.2, 127.6, 127.5, 116.9 (q, JCF
291), 72.5, 68.6, 58.1, 57.9, 52.2, 51.6, 41.6, 40.5, 30.6, 30.2, 19.1,
19.1, 18.2, 18.0; m/z (ESI) 522.2920 (M+; C25H40N5O7 requires
522.2927).
25 K. Murata, K. Mitsuoka, H. Kaoru, W. Teruhisa, T. Walz, P. Agre,
J. B. Heymann, A. Engel and Y. Fujiyoshi, Nature (London), 2000,
407(6804), 599–605.
26 A. D. Schenk, P. J. L. Werten, S. Scheuring, B. L. de Groot, L. Bert,
S. A. Mueller, H. Stahlberg, A. Philippsen and A. Engel, J. Mol. Biol.,
2005, 350(2), 278–289.
27 Y. Hiroaki, K. Tani, A. Kamegawa, N. Gyobu, K. Nishikawa, H.
Suzuki, T. Walz, S. Sasaki, K. Mitsuoka, K. Kimura, A. Mizoguchi
and Y. Fujiyoshi, J. Mol. Biol., 2006, 355(4), 628–639.
28 T. Gonen, Y. Cheng, J. Kistler and T. Walz, J. Mol. Biol, 2004, 342(4),
1337–1345.
29 A. Engel, Y. Fujiyoshi, T. Gonen and T. Walz, Curr. Opin. Struct. Biol.,
2008, 18(2), 229–235.
30 H. Zhang and A. S. Verkman, J. Mol. Biol., 2008, 382(5), 1136–
1143.
Notes and references
31 M. Amiry-Moghaddam and O. P. Ottersen, Nat. Rev. Neurosci., 2003,
1 P. Agre, L. S. King, M. Yasui, W. B. Guggino, O. P. Ottersen, Y.
Fujiyoshi, A. Engel and S. Nielsen, J. Physiol., 2002, 542, 3–16.
2 G. M. Preston, T. P. Carroll, W. B. Guggino and P. Agre, Science, 1992,
256(5055), 385–387.
3 A. Frigeri, G. P. Nicchia and M. Svelto, Curr. Pharm. Des., 2007, 13(23),
2421–2427.
4 J. S. Jung, R. V. Bhat, G. M. Gregory, W. B. Guggino, J. M. Baraban
and P. Agre, Proc. Natl. Acad. Sci. U. S. A., 1994, 91(26), 13052–13056.
5 S. Nielsen, E. A. Nagelhus, M. Amiry-Moghaddam, C. Bourque, P.
Agre and O. P. Ottersen, J. Neurosci., 1997, 17(1), 171–180.
6 Z. Zador, O. Bloch, X. Yao and G. T. Manley, Prog. Brain Res., 2007,
161, 185–194.
7 G. T. Manley, M. Fujimura, T. Ma, N. Noshita, F. Fliz, A. W. Bollen,
P. Chan and A. S. Verkman, Nat. Med. (N. Y.), 2000, 6(2), 159–
163.
4(12), 991–1001.
32 H. Guo, R. F. Beahm and H. Guo, J. Phys. Chem. B, 2004, 108(46),
18065–18072.
33 J. Lawrence, M. Jourdan, Y. Vallee and V. Blandin, Org. Biomol. Chem.,
2008, 6(24), 4575–4581.
34 H. E. Blackwell and R. H. Grubbs, Angew. Chem., Int. Ed., 1998,
37(23), 3281–3284.
35 A. Brik, Adv. Synth. Catal., 2008, 350(11 + 12), 1661–1675.
36 C. Toniolo, M. Crisma, F. Formaggio and C. Peggion, Biopolymers,
2001, 60(6), 396–419.
37 G. R. Marshall, E. E. Hodgkin, D. A. Langs, G. D. Smith, J. Zabrocki
and M. T. Leplawy, Proc. Natl. Acad. Sci. U. S. A., 1990, 87(1), 487–491.
38 A. K. Boal, I. Guryanov, A. Moretto, M. Crisma, E. L. Lanni, C.
Toniolo, R. H. Grubbs and D. J. O’Leary, J. Am. Chem. Soc., 2007,
129(22), 6986–6987.
8 Z. Vajda, M. Pedersen, E.-M. Fuchtbauer, K. Wertz, H. Stodkilde-
Jorgensen, E. Sulyok, T. Doczi, J. D. Neely, P. Agre, J. Frokiaer and
S. Nielsen, Proc. Natl. Acad. Sci. U. S. A., 2002, 99(20), 13131–
13136.
9 M. Amiry-Moghaddam, T. Otsuka, P. D. Hurn, R. J. Traystman, F. M.
Haug, S. C. Stanley, M. E. Adams, J. D. Neely, P. Agre, O. P. Ottersen
and A. Bhardwaj, Proc. Natl. Acad. Sci. U. S. A., 2003, 100(4), 2106–
2111.
39 H. E. Blackwell, J. D. Sadowsky, R. J. Howard, J. N. Sampson, J. A.
Chao, W. E. Steinmetz, D. J. O’Leary and R. H. Grubbs, J. Org. Chem.,
2001, 66(16), 5291–5302.
40 Y. V. Venkatachalapathi and P. Balaram, Nature (London), 1979,
281(5726), 83–84.
41 R. Rai, S. Aravinda, K. Kanagarajadurai, S. Raghothama, N. Shamala
and P. Balaram, J. Am. Chem. Soc., 2006, 128(24), 7916–7928.
42 A. Lipinski, F. Lombardo, B. W. Dominy and P. J. Feeney, Adv. Drug
Delivery Rev., 1997, 23(1–3), 3–25.
43 H. Sugano and M. Miyoshi, J. Org. Chem., 1976, 41(13), 2352–
2353.
10 N. A. Castle, Drug Discovery Today, 2005, 10(7), 485–493.
11 G. M. Preston, J. S. Jung, W. B. Guggino and P. Agre, J. Biol. Chem.,
1993, 268(1), 17–20.
12 C. M. Niemietz and S. D. Tyerman, FEBS Lett., 2002, 531(3), 443–447.
13 M. Zelenina, A. A. Bondar, S. Zelenin and A. Aperia, J. Biol. Chem.,
2003, 278(32), 30037–30043.
14 H. Hasegawa, T. Ma, W. Skach, M. A. Matthay and A. S. Verkman,
J. Biol. Chem., 1994, 269(8), 5497–5500.
15 K. Kuang, J. F. Haller, G. Shi, F. Kang, M. Cheung, P. Iserovich and J.
Fischbarg, Protein Sci., 2001, 10(8), 1627–1634.
16 H. Tsukaguchi, S. Weremowicz, C. C. Morton and M. A. Hediger,
Am. J. Physiol., 1999, 277(5), F685–F696.
17 H. T. Fan, S. Morishima, H. Kida and Y. Okada, Br. J. Pharmacol.,
2001, 133(7), 1096–1106.
18 J. Gao, X. Wang, Y. Chang, J. Zhang, Q. Song, H. Yu and X. Li, Anal.
Biochem., 2006, 350(2), 165–170.
19 V. J. Huber, M. Tsujita, M. Yamazaki, K. Sakimura and T. Nakada,
Bioorg. Med. Chem. Lett., 2007, 17(5), 1270–1273.
20 F. J. M. Detmers, B. L. De Groot, E. M. Mueller, A. Hinton, I. B. M.
Konings, M. Sze, S. L. Flitsch, H. Grubmueller and P. M. T. Deen,
J. Biol. Chem., 2006, 281(20), 14207–14214.
44 H. N. Gopi, R. S. Roy, S. R. Raghothama, I. L. Karle and P. Balaram,
Helv. Chim. Acta, 2002, 85(10), 3313–3330.
45 E. Frerot, J. Coste, A. Pantaloni, M. N. Dufour and P. Jouin,
Tetrahedron, 1991, 47(2), 259–270.
46 Y. Nishiyama, S. Ishizuka and K. Kurita, Tetrahedron Lett., 2001,
42(50), 8789–8791.
47 M. Scholl, S. Ding, C. W. Lee and R. H. Grubbs, Org. Lett., 1999, 1(6),
953–956.
48 S. Ganesh and R. Jayakumar, J. Pept. Res., 2002, 59(6), 249–256.
49 C. Toniolo, F. Formaggio, S. Tognon, Q. B. Broxterman, B. Kaptein,
R. Huang, V. Setnicka, T. A. Keiderling, I. H. McColl, L. Hecht and
L. D. Barron, Biopolymers, 2004, 75(1), 32–45.
50 E. Benedetti, C. Pedone, C. Toniolo, G. Nemethy, M. S. Pottle and
H. A. Scheraga, Int. J. Pept. Protein Res., 1980, 16(2), 156–172.
51 C. M. Deber, F. A. Bovey, J. P. Carver and E. R. Blout, J. Am. Chem.
Soc., 1970, 92(21), 6191–6198.
52 D. H. Williams, I. Fleming, Spectroscopic Methods in Organic Chem-
istry, 5th Edition, McGraw-Hill, 1995, pp. 140.
21 B. Yang, J. K. Kim and A. S. Verkman, FEBS Lett., 2006, 580(28–29),
6679–6684.
22 R. Sogaard and T. Zeuthen, Pfluegers Arch., 2008, 456(2), 285–292.
23 T. Gonen, P. Sliz, J. Kistler, Y. Cheng and T. Walz, Nature (London),
2004, 429(6988), 193–197.
53 K. Wu¨thrich, NMR of Proteins and Nucleic Acids, John Wiley & Sons,
New York, 1986.
54 E. Obayashi, H. Yoshida, F. Kawai, N. Shibayama, A. Kawaguchi,
K. Nagata, J. R. H. Tame and S.-Y. Park, Nature, 2008, 454(7208),
1127–1131.
24 D. Fu, A. Libson, L. J. W. Miercke, C. Weitzman, P. Nollert, J. Krucinski
and R. M. Stroud, Science, 2000, 290(5491), 481–486.
55 E. Mann, A. Chana, F. Sanchez-Sancho, C. Puerta, A. Garcia-Merino
and B. Herradon, Adv. Synth. Catal., 2002, 344(8), 855–867.
This journal is
The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 1599–1611 | 1611
©