New staves for old barrels: Regioisomeric (12,2 2,33,42,53,62,7 3,82)-p-octiphenyl rods with an NMR tag

Article abstract of DOI:10.1039/b512276g

The usefulness of rigid-rod p-octiphenyls with the novel 1 ²,2²,3³,4²,5³,6 ²,7³,8²-motif as staves in rigid-rod β-barrel pores is evaluated. Comparison with the known

Full text of DOI:10.1039/b512276g

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PAPER
www.rsc.org/njc | New Journal of Chemistry
New staves for old barrels: regioisomeric (12,22,33,42,53,62,73,82)-
p-octiphenyl rods with an NMR tagw
´
Dawn Ronan, Damien Jeannerat, Andre Pinto, Naomi Sakai and Stefan Matile*
Received (in St Louis, MO, USA) 30th August 2005, Accepted 7th September 2005
First published as an Advance Article on the web 28th September 2005
DOI: 10.1039/b512276g
The usefulness of rigid-rod p-octiphenyls with the novel 12,22,33,42,53,62,73,82-motif as staves in
rigid-rod b-barrel pores is evaluated. Comparison with the known characteristics of isomeric
pores with the common 13,23,32,43,52,63,72,83-sequence indicates that the self-assembly and the
supramolecular chirality of rigid-rod b-barrels are independent of the substitution pattern of the
p-octiphenyl stave. NMR tags added at the rod termini are shown to facilitate product
characterization.
1
Introduction
struction of synthetic ion channels and pores. For selected
recent reviews of and publications in this field, please see ref.
10–18 and 19–32, respectively. The objective of this report is to
explore whether or not the self-assembly of rigid-rod b-barrel
pores like 1 is limited to the 13,23,32,43,52,63,72,83-substitution
pattern of conventional p-octiphenyl staves like 7.1,2 To
Conventional rigid-rod b-barrels—the only artificial b-barrels
known today—are made by self-assembly of rigid-rod p-
octiphenyl staves with peptide strands attached to the posi-
tions 13, 23, 32, 43, 52, 63, 72 and 83 (Fig. 1 and 2).1,2 The non-
planarity of the rigid-rod staves is thought to be important to
preorganize cylindrical self-assembly into supramolecular oli-
gomers and to prevent linear self-assembly into supramolecu-
lar polymers. The orthogonal orientation of neighboring
amino-acid residues in a b-sheet conformation can be used
to functionalize both the outer and inner surfaces of rigid-rod
b-barrels in a straightforward, versatile and rational manner
(Fig. 1). For the construction of synthetic multifunctional
pores 1–6, outer surface design using amino acids 1, 3 and 5
has been of use to maximize the subtle interplay between pore
stability and bilayer affinity, to maximize water solubility (4),3
and for ligand gating (5).4 Histidines (H), arginines (R), lysines
(K), and aspartates (D) have been positioned at the inner pore
surface to interact with ions and molecules that move through
the pore from one side of a bilayer membrane to the other.1–5
Because of their multifunctionality, synthetic pores 1–6 turned
out to be of use as supramolecular hosts,1–8 sensors6–8 and
catalysts.4,9
do so, isomeric p-octiphenyl staves
8 with a novel
12,22,33,42,53,62,73,82-motif were synthesized and character-
ized (Scheme 1). Nearly identical characteristics found for
the nearly isomeric multifunctional pores 1 and 9 suggested
that the self-assembly of rigid-rod b-barrels is independent of
the relative arene substitution pattern in p-octiphenyl staves.
Pores 1 and 9 are not exactly structural isomers because
terminal methyl groups were added to the new stave. These
methyls were introduced as NMR tags to facilitate the char-
acterization of synthetic intermediates and products and to
provide access to new structural insights.
2
Results and discussion
2.1 Synthesis of isomeric rigid p-octiphenyl rods
The isomeric, NMR-labelled p-octiphenyl rod 8 was prepared
in eight steps from commercial biphenyls (Scheme 1). The
selected route was of interest because the synthesis of the
terminally activated p-sexiphenyl 10 in only three steps had
been developed previously as an invaluable shortcut to multi-
ply functionalized p-oligophenyl rods.33 Namely, nucleophilic
aromatic Sandmeyer substitution of the biphenyl bis(diazo-
nium) salt 11 with KI gave biphenyl diiodide 12, which was
readily converted into the biphenyl boronate esters 13. A high
purity of biphenyl 13 was critical for the success of the
following Suzuki coupling with excess diiodide 12. Because
of its poor solubility in the reaction mixture, p-sexiphenyl 10
could be accumulated, isolated and purified in up to 10%
yield. Not impressive as such, this yield is much higher than
the yield expected for the stepwise synthesis of p-sexiphenyl 10
by repeated couplings, not to mention so far unresolved
problems in such a multistep exercise, such as direct terminal
iodination of p-sexiphenyls.
These results identified p-octiphenyl b-barrels as an attrac-
tive supramolecular barrel-stave motif for the adaptable con-
Department of Organic Chemistry, University of Geneva, Geneva,
Switzerland. E-mail: stefan.matile@chiorg.unige.ch;
Fax: þ41(0)22 379 3215; Tel: þ41(0)22 379 6523
w Abbreviations. Arg, R: L-arginine; BOC: tert-butoxycarbonyl; CD:
circular dichroism; DMF: N,N-dimethylformamide; EC50: effective
cM (monomer concentration to reach 50% pore activity); EYPC-
LUVs*ANTS/DPX: egg yolk phosphatidylcholine (EYPC) large
unilamellar vesicles (LUVs) loaded with 8-amino-1,3,6-naphthalene-
trisulfonate (ANTS) and p-xylenebis(pyridinium) bromide (DPX);
Gla, G: –OCH2CO– (H-Gla-OH: glycolic acid); HATU: N-(dimethyl-
amino-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene)-N-methylmetha-
naminium hexafluorophosphate N-oxide; His, H: L-histidine; IC50:
inhibitory cB (blocker concentration to reduce to 50% pore activity);
ICR: internal charge repulsion; Leu, L: L-leucine; NOESY: nuclear
Overhauser effect spectroscopy; Pmc: 2,2,5,7,8,-pentamethylchromane-
6-sulfonyl; TEA: triethylamine; TFA: trifluoroacetic acid; Trt: trityl.
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The terminal NMR tags were prepared from nitroanisol 14
following a recent report by Coleman et al.34 Reduction gave
amine 15, which was subjected to Sandmeyer treatment via the
diazonium salt to afford iodoanisol 16.34,35 Transformation
into the pinacol boronate ester 17 prepared for the Suzuki
coupling with p-sexiphenyl 10. The methoxys along the scaf-
fold of the resulting p-octiphenyl 18 were then cleaved with
BBr3, and the obtained octaphenol 19 was reacted with tert-
butyl bromoacetate. Deprotection of p-octiphenyl 20 liberated
the carboxylic acids along the rigid-rod scaffold of 21 for
coupling with the N-terminus of the previously reported
pentapeptide 22.36 Side-chain deprotection gave target rod 8
as a TFA salt.
Fig. 1 Axial view of six previously made and characterized multi-
functional rigid-rod b-barrel pores with b-sheets shown as solid (back-
bone) and dotted lines (hydrogen bonds) and amino-acid residues at
the outer (1, 3, 5) and inner (2, 4) barrel surfaces dark on white and
white on dark, respectively (single-letter abbreviations). For side views
of rigid-rod b-barrel pores, see Fig. 2.
The target molecule 8 was purified by semi-preparative RP-
HPLC. HPLC chromatograms and mass spectra of isomers 7
and 8 and their protected precursors showed qualitatively
Fig. 2 Self-assembly of conventional and isomeric p-octiphenyl-peptide conjugates 7 and 8 into conventional and isomeric multifunctional rigid-
rod b-barrel pores 1 and 9, respectively. Amino-acid residues are shown in neutral form. Under conditions relevant for function, arginine (R)
residues are expected to be fully protonated and complexed with exchangeable counteranions, mainly inorganic phosphates,37 whereas histidine (H)
residues are partially protonated below pH 6 only. Arrows represent b-sheets (N-C). For axial views of rigid-rod b-barrel pores, see Fig. 1, 6 and 8.
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Scheme 1 (a) KI, 70%;33 (b) 4,4,5,5-tetramethyl-[1,3,2]-dioxaborolane, PdCl2(dppf), 70%;33 (c) Pd(PPh3)4, Na2CO3, 10%;33 (d) Pd/C, H2,
quant.;34 (e) 1. NaNO2, H2SO4, 2. KI, 71%;34,35 (f) 4,4,5,5-tetramethyl-[1,3,2]-dioxaborolane, PdCl2(dppf), TEA, CH3CN, 85 1C, 2.5 h, not
isolated; (g) Pd(PPh3)4, K2CO3, DMSO–H2O 4 : 1, microwave, 150 1C, 15 min, 60%; (h) BBr3, CH2Cl2, ꢁ78 1C to rt, 14 h; (i) Cs2CO3, DMF,
80 1C, 1 h, then tert-butyl bromoacetate, 60 1C, 3 h, 49% from 18; (j) TFA, rt, 3 h; (k) 22,36 HATU, TEA, DMF, rt, 2.5 h, 16% from 20; (l) TFA,
rt, 2 h, quant.
identical behavior. The key characteristic in the mass spectra,
that is the spontaneous counterion exchange from the ex-
pected TFA to the detected phosphates, was reproduced.37
This unexpected observation was the key to understand the
similarly unexpected low conductance and cation selectivity of
pore 1 above pH 5 and marked the beginning of a general
investigation of the contribution of counteranions to the
functions of oligoarginines such as the cell-penetrating pep-
tides in lipid bilayer membranes and live cells.38,39
of the chiral lateral pentapeptides results in the loss of the
center of symmetry in p-octiphenyl 8.
2.2 Activity of b-barrel pores with isomeric rigid p-octiphenyl
staves
Synthetic multifunctional pores formed by rigid-rod b-barrels
1 with conventional 13,23,32,43,52,63,72,83-staves have been
characterized in detail in both spherical36,40 and planar bilayer
membranes.37 The activity of the isomeric rigid-rod b-barrels 9
with the novel 12,22,33,42,53,62,73,82-staves was determined in
large unilamellar vesicles (LUVs) composed of egg yolk
phosphatidylcholine (EYPC) and loaded with the fluorescent
probe 8-amino-1,3,6-naphthalenetrisulfonate (ANTS) and
the quencher p-xylenebis(pyridinium) bromide (DPX), i.e.,
The NMR tags produced the expected resonances in silent
regions of the NMR spectrum of target rod 8 (Fig. 3). Namely,
the electron-donating 16 and 86 methyls shifted the 15 and 85
hydrogens in the ortho and the 13 and 83 hydrogens in the para
positions upfield to 6.5 and 6.3 ppm, respectively. Comparison
of the integrals of these isolated p-octiphenyl signals with those
of peptide resonances provided facile and clearcut experimen-
tal confirmation of MS results that rod 8 is defect-free (i.e., the
presence of eight pentapeptides per rod rather than seven plus
a defect such as a methyl ester).
EYPC-LUVs*ANTS/DPX.36,40
After
addition
of
12,22,33,42,53,62,73,82-rod 8, release of either fluorophore or
quencher from EYPC-LUVs*ANTS/DPX was readily de-
tectable as an increase of ANTS emission (Fig. 4A). For pores
with an interior large enough to allow for efflux of organics
like ANTS and DPX, the ANTS/DPX assay is, in our hands,
the assay of choice to determine cM and pH profiles because of
its minimal dependence on pH and anion/cation selectivity.
According to the ANTS/DPX assay, the activity of pores 9
increased with decreasing pH (Fig. 4B). The pH profiles of the
isomeric pores 9 and 140 were nearly identical. pH-gated pore
opening has been interpreted previously with the ICR model,
i.e., maximal pore activity at intermediate internal charge
repulsion (ICR).41,42 Contributions from internal arginines
to ICR are expected to be weak because of charge neutraliza-
tion by counteranion scavenging. The observed pore opening
below pH 6 is therefore thought to originate chiefly from the
stabilization of the internal space of pores 9 and 1 by inter-
mediate ICR between increasingly protonated histidine resi-
dues below their intrinsic pKa ¼ 6.0.
In all common NMR solvents, these signals were, however,
as broad as all other signals. Below findings demonstrated that
symmetry losses with chiral lateral substitution contributed to
this signal broadening besides the known polymorphous self-
assembly at the comparably high millimolar concentrations
needed in NMR spectroscopy. Namely, denaturation with
TFA resulted in the expected line-sharpening. However, ap-
parent triplets rather than doublets were observed for the 15/85
and the 13/83 hydrogens under these extreme, denaturing
conditions (Fig. 3). Moreover, the 16/86 methyls appeared as
an apparent doublet at 1.8 ppm. 2D NMR NOESY revealed
that the found signal fine structure originated from two
separate spin systems with the expected doublet multiplicities
from 3J couplings. This chemical non-identity of the two
terminal arenes A and H demonstrated that the introduction
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Fig. 4 pH profile of isomeric rigid-rod b-barrel pore 9 (B) with
original traces (A) showing fractional change in ANTS emission I
(lex 360 nm, lem 520 nm) as a function of time during addition of rod 8
(final monomer concentration cM ¼ 60 nM; r15 nM tetrameric pores)
to EYPC-LUVs*ANTS/DPX in buffer (B130 mM EYPC, 100 mM
KCl, 10 mM MES) at, with decreasing activity, pH 4.2, 4.5, 5.1, 5.5,
5.8, 6.2, 6.8 and 7.5, calibrated by final lysis (excess triton X-100).
relevant for function. The apparent difference to n o 1
behavior of isomeric pore 1 (n ¼ 0.6)3,40 may suggest that
the
fibrillogenesis43
of
rigid-rod
b-barrels
with
13,23,32,43,52,63,72,83-staves is hindered in isomeric rigid-rod
b-barrels with 12,22,33,42,53,62,73,82-staves. The apparent dif-
ference in the cM profiles of pores 1 and 9 is, however, with all
likelihood, of minor relevance because the differentiation
between n E 1 and n o 1 behavior is often a question of
subtle experimental conditions (e.g., solvent and concentration
of stock solutions). These reservations do not apply to the, in
our experience, unambiguous differentiation of endergonic
and exergonic self-assembly using n 4 1 and n r 1 behavior.
The effective monomer concentration EC50, i.e., the con-
centration where 50% pore activity is observed under given
conditions, was EC50 ¼ 90 nM. For a tetrameric pore 9, this
Fig. 3 Part of the 2D NOESY NMR spectrum of target molecule 8 in
denaturing CF3COOD–CD3OD 3 : 1 (A and H indicate the non-
identical terminal arenes 1 and 8, s ¼ solvent).
Around optimal pH, the dependence of the activity of pores
9 in EYPC-LUVs*ANTS/DPX on the concentration cM of
monomeric rods 8 was nearly linear below the apparent half
effective concentration EC50 under these conditions (Fig. 5A).
A fit to the Hill equation for self-assembly >eqn (1)]:
log Y ¼ n ꢂ log cM ꢁ n ꢂ log EC50
(1)
gave a Hill coefficient n E 1 (Y ¼ fractional pore activity)3. As
discussed elsewhere,3 the interpretation of results from Hill
analysis in the context of the self-assembly of functional
supramolecules can be qualitatively classified into three cate-
gories:
n 4 1 is observed with endergonic self-assembly of unstable
functional supramolecules formed by n monomers,
n E 1 is observed with exergonic self-assembly of stable
functional supramolecules (n is not indicative of the number of
monomers per supramolecule),
n o 1 is observed with exergonic self-assembly of metastable
functional supramolecules that transform into inactive higher
assemblies at high concentration (e.g., precipitate; n is not
indicative of the number of monomers per supramolecule).
The found Hill coefficient n E 1, therefore, suggested that
the self-assembly of pores 9 is exergonic under conditions
Fig. 5 Dependence of the fractional activity I of pore 9 on the
concentration cM of monomer 8 with linear curve fit below the effective
concentration EC50 (cM profile, A, pH 4.0) and the concentration cB
of poly(L-glutamic acid) blocker 24 [cB profile, B, cM ¼ 60 nM (r15
nM tetrameric pores), pH 4.5] with curve fit to Hill eqn (1); method
and other conditions as in Fig. 3.
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Fig. 6 Blockage of isomeric 12,22,33,42,53,62,73,82-p-octiphenyl pores 9 by molecular recognition of a-helical poly(L-glutamic acid) 24 is expected
to occur by formation of the supramolecular inclusion complex 25.
was calculated to be a remarkable EC50 ¼ 23 nM. This is the
highest apparent activity observed for a rigid-rod b-barrel
pore. Assessment of the EC50 of the isomeric pore 1 was not
meaningful because of the onset of saturation below EC50
with n r 1 behavior.40
complex with pore 1 as improved a-helix recognition with
12,22,33,42,53,62,73,82-staves was questionable. Additional cau-
tion was indicated in comparisons of apparent IC50s near
stoichiometric binding because they could, depending on
experimental conditions, appear higher than they are in rea-
lity.47
2.3 Multifunctionality of b-barrel pores with isomeric rigid
p-octiphenyl staves
2.4 Chirality of b-barrel pores with isomeric rigid p-octiphenyl
staves
Molecular recognition by synthetic multifunctional pores 1–6
is attractive because host–guest chemistry within pores coin-
cides with changes in pore activity and can therefore be
controlled and detected by several attractive methods that
are not available in isotropic media.1,2 The recognition of a-
helical guests turned out to be particularly attractive because
the dimensions of the internal nanospaces of rigid-rod b-barrel
pore hosts like 1 match those of, e.g., a-helical poly(L-glutamic
acid) 24 (Fig. 6).1–4
Denaturation experiments revealed that most monomeric p-
oligophenyl staves are nearly CD silent.48,49 This finding was
in agreement with free atropisomerism around the arene–arene
single bonds in monomeric rods 7 and 8. The circular dichro-
ism (CD) Cotton effects for the exciton-coupled low-energy
La-transitions in the p-oligophenyl stave report, therefore,
selectively on the supramolecular chirality of the self-as-
sembled rigid-rod b-barrel. This supramolecular chirality is
obviously induced by the stereogenic centers in the enantio-
pure pentapeptides. The CD spectrum of the regioisomeric,
NMR-labelled 12,22,33,42,53,62,73,82-rod 8 in bilayer mem-
branes was nearly superimposable with that of the original
13,23,32,43,52,63,72,83-rod 7 (Fig. 7). This similarity demon-
strated that the supramolecular chirality of classical rigid-rod
a-Helix recognition was, therefore, selected as an example of
scientific relevance44 to corroborate the functional similarity
of the isomeric multifunctional pores 1 and 9. To do so, the
activity of synthetic multifunctional pore 9 in spherical EYPC-
LUVs*ANTS/DPX was assessed in the presence of increas-
ing concentrations of helix 24 at pH 4.5. As with isomer 1,15
the activity of pores 9 decreased with increasing concentra-
tions of helix 24 (Fig. 5B). Fit of the obtained dose–response
curve to the Hill equation for blockage [eqn (2)]:
log Y ¼ n ꢂ log cB ꢁ n ꢂ log IC50
(2)
gave an inhibitory concentration IC50 ¼ 12 ꢃ 2 nM and a Hill
coefficient n ¼ 0.8 ꢃ 0.1. These results for blockage of pores 9
by a-helix recognition were similar to those obtained with
isomeric pores 136 and, therefore, suggestive of the formation
of inclusion complex 25 (Fig. 6) with slightly negative coop-
erativity. The recent crystal structure of the translocator
domain of the bacterial autotransporter NaIP from Neisseria
meningitidis suggests that similar functional suprastructural
architecture exists in nature.45 Recent findings demonstrated
that replacement not only of the biological b-barrel pore with
synthetic multifunctional rigid-rod pores but also of that of
the a-helix blocker with shape-persistent, supramolecular
rigid-rod a-helix mimics is possible.46
Fig. 7 Circular dichroism (CD) spectra of p-octiphenyls 7 (5 mM,
dotted) and isomer 8 (5 mM, solid) in lipid bilayer membranes [EYPC-
LUVs (250 mM EYPC), 100 mM KCl, 10 mM MES, pH 4.5].
The meaningfulness of an interpretation of the 10-times
lower IC50 obtained for complex 25 compared to the isomeric
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conformational change. The b-sheet expansion achieved by
such an anti - gauche isomerization in M-9 implied hoop–
stave matching (rather than glycolate conformation) as the
dominant driving force for the enantioselective self-assembly
of M-barrels 1 and 9 (Fig. 8A). With classical barrels, the
same anti - gauche isomerization is hindered by increasing
hoop–stave mismatch with the b-sheet compression from the
favored M-1 to the unfavored P-1 (Fig. 8B). Whatever the
final suprastructural explanation will be, the bottom line at
this stage is that the identical supramolecular chirality of
regioisomeric barrels 1 and 9 implies conformational differ-
ences in the respective peptide hoops (e.g., glycolate spacer)
that occur for experimentally unverified reasons (e.g., hoop–
stave matching). The implied dependence of absolute barrel
chirality on hoop conformation is in agreement with the
well-documented sensitivity of supramolecular p-oligophenyl
chirality to chemical modification, chemical stimulation, and
heat.50
3
Conclusions
For reasons discussed elsewhere,11,42,51,52 there is increasing
awareness in the field that the most reliable insights on active
suprastructures of synthetic and biological ion channels and
pores may come from studies on function. According to this
notion, we report that the self-assembly of rigid-rod b-barrels
is roughly independent of the substitution pattern along the
p-octiphenyl scaffold. Specifically, synthetic multifunc-
tional pores formed by rigid-rod b-barrels with classical
13,23,32,43,52,63,72,83-staves (i.e., 7) and regioisomeric
12,22,33,42,53,62,73,82-staves (i.e., 8) have similar pH profiles,
similar cM profiles and exhibit similar dose–response curves
for blockage by a-helical poly(L-glutamic acid). NMR tags
added to the new stave 8 are found to facilitate the character-
ization of synthetic products and demonstrate the loss of
central p-oligophenyl symmetry upon symmetric attachment
of chiral substituents along the rigid-rod scaffold. Circular
dichroism spectroscopy indicates that regioisomeric staves do
not invert the absolute supramolecular chirality of the result-
ing barrel.
Fig. 8 Identical supramolecular barrel chirality with regioisomeric
staves is proposed to originate from hoop–stave matching with
expanded b-sheets (A) in M-supramolecules with glycolate spacers
(red) in either anti (M-1) or gauche conformation (M-9, see Fig. 6).
b-barrel 1 and that of isomeric rigid-rod b-barrel 9 are
identical (Fig. 2, 6 and 8).
This finding is not trivial. In rigid-rod b-barrels, the first and
last amino-acid residues 1 and 5 of all pentapeptides are
oriented to the outer barrel surface to avoid steric interference
in the p-octiphenyl turns. With the given absolute pentapep-
tide chirality and glycolate spacers having C–O bonds in an
anti conformation, this peripheral crowding enantioselectively
freezes classical atropisomerizing p-octiphenyl staves in a zig-
zag M,P,M,P,M,P,M-conformation. The resulting residual
M-‘‘helicity’’ of classical barrels like M-1 has been verified in
molecular mechanics simulations (Fig. 8).3,4 In the isomeric
12,22,33,42,53,62,73,82-stave 8, the preserved pentapeptide chir-
ality and an anti conformation of the glycolate spacers should
induce the P,M,P,M,P,M,P-conformation of a barrel P-9 with
a mirror-imaged CD spectrum compared to M-1. The found
identical CD spectra implied the stereoselective self-assembly
of barrel M-9 instead. This inversion of supramolecular
chirality implied a conformational change in the lateral pep-
tide strands. With inflexible b-sheets, rotation of the glycolate
spacers from an anti conformation in P-9 to a gauche in
M-9 remained as the only reasonable possibility for such a
4
Experimental section
4.1 General
As in ref. 4. Reagents for synthesis, poly(glutamic acid)
(M 13 600 g molꢁ1), buffers and salts were from Sigma, Fluka,
Aldrich or Acros, amino acid derivatives from Novabiochem,
EYPC from Avanti, ANTS and DPX from Molecular Probes.
Solvents were dried by use of a solvent purification system
from Solv-tek. Microwave reactions were carried out using a
Biotage Initiatort microwave synthesizer. Column chromato-
graphy was carried out on silica gel 60 (Fluka, 40–63 mm).
Sephadex LH-20 was from Amersham Biosciences. Analytical
(TLC) and preparative thin layer chromatography (PTLC)
were performed using silica gel 60 (Fluka, 0.2 mm) and silica
gel GF (Analtech, 1000 mm), respectively. Purity of the
products was confirmed by HPLC by using either Jasco HPLC
system (PU-980, UV-970, FP-920) or Agilent 1100 Series. ESI-
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MS was performed on either an Applied Biosystems API 150
Ex or a Finnigan MAT SSQ 7000 instrument. APCI-MS was
performed using the latter instrument. 1H and 13C NMR
spectra were recorded (as indicated) on either a DRX Bruker
300 MHz, 400 MHz or 500 MHz spectrometer and are
reported as chemical shifts (d) in ppm relative to TMS (d ¼
0). Spin multiplicities are reported as singlet (s), doublet (d) or
triplet (t), with coupling constants (J) given in Hz. The 500
MHz NOESY spectrum of 8 was acquired using a square
window of 9.4 ppm. In the first dimension, 256 increments
were acquired resulting in a resolution of 18.35 Hz per point.
In the direct dimension, the number of time data points was 4k
resulting in a digital resolution of 1.15 Hz per point. After
processing using 512 ꢂ 8k points and no window function,
cross peaks and diagonal signals had the same signs indicating
an NOE in the slow motion regime. The mixing time was
500 ms and the recovery period 1.83 s. UV-Vis measurements
were preformed on a Varian Cary 1 Bio spectrophotometer,
CD measurements on a Jasco J-715 spectropolarimeter and
fluorescence measurements were preformed on a Fluoro-
Max-3, Jobin Yvon-Spex, equipped with an injector port, a
stirrer and a temperature controller (25 1C). pH, cM and cB
profiles of pore 9 were obtained as reported previously for
pore 1.35,40,46
6H), 3.77 (s, 6H), 2.12 (s, 3H); 13C NMR (100 MHz, CDCl3): d
157.5 (s, 8ꢂ), 142.4 (s, 2ꢂ), 142.3 (s, 2ꢂ), 142.0 (s, 2ꢂ), 138.8
(s, 2ꢂ), 132.1 (d, 2ꢂ), 132.0 (d, 2ꢂ), 131.9 (d, 2ꢂ), 128.3 (d,
2ꢂ), 127.3 (s, 2ꢂ), 126.8 (s, 2ꢂ), 126.6 (s, 2ꢂ), 125.9 (s, 2ꢂ),
122.5 (d, 2ꢂ), 119.7 (d, 4ꢂ), 119.6 (d, 2ꢂ), 110.6 (d, 2ꢂ), 110.5
(d, 2ꢂ), 110.4 (d, 2ꢂ), 108.7 (d, 2ꢂ), 56.2 (q, 2ꢂ), 56.1 (q, 4ꢂ),
56.0 (q, 2ꢂ), 20.3 (q, 2ꢂ); MS (APCI, CH2Cl2): m/z (%) 880
(100, [M þ H]1).
16,86-Dimethyl-12,22,33,42,53,62,73,82-octakis(Gla-OtBu)-p-
octiphenyl (20). To a solution of octaanisole 18 (24 mg,
27 mmol) in CH2Cl2 (5 ml), was added boron tribromide
(0.7 ml, 1 M solution in CH2Cl2, 0.7 mmol) at ꢁ78 1C. The
solution was allowed to warm to room temperature over 14 h,
quenched with MeOH (1 ml) and then concentrated in vacuo
to give crude octaphenol 19. Crude 19 was dissolved in DMF
(5 ml), treated with Cs2CO3 (211 mg, 0.65 mmol) at 80 1C for
1 h, and then with tert-butyl bromoacetate (96 ml, 0.65 mmol)
at 60 1C for 3 h. The resulting suspension was diluted with
CH2Cl2 (15 ml), washed with brine (20 ml ꢂ 3), dried over
Na2SO4, and concentrated in vacuo. Flash column chromato-
graphy (CH2Cl2–MeOH 100 : 1) followed by PTLC (CH2Cl2–
toluene–MeOH 7 : 3 : 0.1) gave pure 20 as a colorless solid
(22 mg, 49%). TLC (CH2Cl2–toluene–MeOH 7 : 3 : 0.1): Rf
1
4
0.69; H NMR (400 MHz, CDCl3): d 7.54 (dd, J ¼ 3.0 Hz,
3J ¼ 7.8 Hz, 4H), 7.33–7.29 (m, 8H), 7.22 (t, 3J ¼ 8.0 Hz, 2H),
4.2 Synthesis
3
7.13 (s, 4H), 7.10 (s, 2H), 6.96 (d, J ¼ 7.6 Hz, 2H), 6.72 (d,
2-Iodo-3-methylanisole (16). This compound was prepared
in 2 steps from 14 following previously reported proce-
dures.34,35
3J ¼ 8.3 Hz, 2H), 4.59 (s, 8H), 4.50 (s, 4H), 4.45 (s, 4H), 1.49
(s, 18H), 1.48 (s, 18H), 1.47 (s, 18H), 1.45 (s, 18H); 13C NMR
(100 MHz, CDCl3): d 168.6 (s, 2ꢂ), 168.5 (s, 2ꢂ), 168.3 (s, 4ꢂ),
156.1 (s, 2ꢂ), 156.0 (s, 2ꢂ), 155.9 (s, 4ꢂ), 141.9 (s, 2ꢂ), 141.8
(s, 2ꢂ), 141.5 (s, 2ꢂ), 139.4 (s, 2ꢂ), 132.6 (d, 2ꢂ), 132.5 (d,
2ꢂ), 132.4 (d, 2ꢂ), 128.2 (d, 2ꢂ), 127.4 (s, 2ꢂ), 126.8 (s, 2ꢂ),
126.6 (s, 2ꢂ), 126.2 (s, 2ꢂ), 123.4 (d, 2ꢂ), 120.5 (d, 2ꢂ), 120.4
(d, 2ꢂ), 120.3 (d, 2ꢂ), 111.7 (d, 4ꢂ), 111.6 (d, 2ꢂ), 109.8 (d,
2ꢂ), 82.2 (s, 4ꢂ), 82.1 (s, 2ꢂ), 82.0 (s, 2ꢂ), 66.8 (t, 8ꢂ), 28.3 (q,
4ꢂ), 28.2 (q, 2ꢂ), 28.2 (q, 2ꢂ), 20.5 (q, 2ꢂ); MS (ESI, CH2Cl2
–MeOH 9 : 1): m/z (%) 1698 (100, [M þ H3O]1), 858 (40, [M þ
H þ H3O]21).
14,64-Diiodo-13,23,32,43,52,63-hexamethoxy-p-sexiphenyl (10).
This compound was prepared in 3 steps from 11 following
previously reported procedures.33
16,86-Dimethyl-12,22,33,42,53,62,73,82-octamethoxy-p-octiphe-
nyl (18). To a solution of 16 (89 mg, 0.36 mmol) in degassed
anhydrous acetonitrile (4 ml), were added PdCl2(dppf) (10
mol%), TEA (300 ml, 2.16 mmol) and 4,4,5,5-tetramethyl-
[1,3,2]-dioxaborolane (160 ml, 1.08 mmol) successively and the
solution set to stir at 85 1C for 2.5 h. The reaction was cooled
to room temperature and activated charcoal was added to the
reaction mixture. The mixture was sonicated, filtered and the
filtrate concentrated in vacuo to yield crude 2-(4,4,5,5-tetra-
methyl-[1,3,2]-dioxaborolane)-3-methylanisole 17. To a 20 ml
glass microwave vessel was added 10 (40 mg, 45 mmol) in
DMF–H2O (4 : 1) (10 ml). Following the addition of
Pd(PPh3)4 (10 mol%), K2CO3 (37 mg, 0.27 mmol) and crude
17 obtained above (assumed 0.36 mmol), the solution was
degassed and put under N2. The reaction was microwaved at
150 1C while stirring during 15 min, cooled to room tempera-
ture, the product extracted into CH2Cl2 (15 ml), washed with
brine (15 ml ꢂ 3) and concentrated in vacuo. Purification of the
product first by column chromatography (CH2Cl2) and then
by preparative thin layer chromatography (CH2Cl2–petroleum
ether 10 : 1) gave 18 (24 mg, 60%) as a colorless solid. TLC
(CH2Cl2–petroleum ether 10 : 1): Rf 0.44; 1H NMR (400 MHz,
16,86-Dimethyl-12,22,33,42,53,62,73,82-octakis(Gla-OH)-p-oc-
tiphenyl (21). To 20 (11 mg, 6.5 mmol) was added TFA (1 ml)
and the solution was allowed to stir for 1 h 30 min, after which
toluene (1 ml) was added and the solution concentrated in
vacuo. The product was washed with hexane (1 ml ꢂ 3),
following which TFA (1 ml) was again added and the solution
allowed to stir for an additional 1 h 30 min. Toluene (1 ml) was
added, the solution concentrated in vacuo, washed with hexane
(1 ml ꢂ 3) and dried to give 21 (8 mg, quant.) as a colorless
1
3
solid. H NMR (300 MHz, CD3OD): d 7.46 (d, J ¼ 7.6 Hz,
4H), 7.36 (d, 3J ¼ 8.0 Hz, 6H), 7.29–7.18 (m, 10H), 6.95
3
3
(d, J ¼ 7.5 Hz, 2H), 6.80 (d, J ¼ 8.3 Hz, 2H), 4.74 (s, 8H,
CH2), 4.64 (s, 4H), 4.56 (s, 4H), 2.14 (s, 6H); MS (ESI, CH2Cl2
–MeOH 50 : 1): m/z (%) 1254 (61, [M þ Na]1), 1249 (100,
[M þ H3O]1), 1232 (24, [M þ H]1).
4
3
CDCl3): d 7.43 (dd, J ¼ 2.3 Hz, J ¼ 7.6 Hz, 4H), 7.34–7.26
Leu-Arg(Pmc)-Leu-His(Trt)-Leu-NH2 (22). This compound
was prepared in 8 steps following previously reported proce-
dures.36
(m, 14H), 7.20 (d, 3J ¼ 7.6 Hz, 2H), 6.94 (d, 3J ¼ 7.6 Hz, 2H),
3
6.87 (d, J ¼ 8.1 Hz, 2H), 3.93 (s, 6H), 3.92 (s, 6H), 3.85 (s,
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174 | New J. Chem., 2006, 30, 168–176

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16,86-Dimethyl-12,22,33,42,53,62,73,82-octakis[Gla-Leu-Arg
(Pmc)-Leu-His(Trt)-Leu-NH2]-p-octiphenyl (23). TEA (43 ml,
0.31 mmol) was added to a DMF (1 ml) solution containing 21
(8 mg, 6.5 mmol), 22 (84 mg, 73 mmol) and HATU (28 mg, 73
mmol). The reaction was allowed to stir at room temperature
for 2 h 30 min, before being concentrated in vacuo. Flash
column chromatography (CH2Cl2–MeOH 4 : 1), followed by
size exclusion chromatography (Sephadex LH-20, MeOH) and
then PTLC (CHCl3–MeOH 9 : 1) gave 23 as a colorless solid
(11 mg, 16%). HPLC (YMC-Pack silica, 250 ꢂ 10 mm,
CH2Cl2–MeOH 90 : 10, 2 ml minꢁ1, tR ¼ 6.55 min); 1H
NMR (400 MHz, CDCl3–CD3OD 4 : 1): d 7.57–6.91 (several
m, 148H), 6.81–6.66 (several m, 10H), 5.07–4.31 (several m,
56H), 3.25–2.77 (several m, 32H), 2.6–2.35 (m, 64H), 2.10–1.98
(several m, 30H), 1.83–1.08 (several m, 168H), 0.93–0.49 (m,
144H); MS (ESI, [CH3CN–H2O–AcOH 74 : 24 : 2]–[MeOH]
9 : 1): m/z (%) 2589 (100, [M þ 4H]41), 2529 (85, [M þ
4H ꢁ Trt]41), 2468 (15, [M þ 4H ꢁ 2Trt]41), 2072 (10,
[M þ 5H]51).
4 V. Gorteau, F. Perret, G. Bollot, J. Mareda, A. N. Lazar, A. W.
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7 N. Sorde, G. Das and S. Matile, Proc. Natl. Acad. Sci. USA, 2003,
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127, 9316–9317.
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and S. Matile, J. Am. Chem. Soc., 2003, 125,
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16,86-Dimethyl-12,22,33,42,53,62,73,82-octakis(Gla-Leu-Arg-
Leu-His-Leu-NH2)-p-octiphenyl (8). Compound 23 (11 mg, 1
mmol) was dissolved in TFA (1 ml). After stirring for 1 h at
room temperature toluene (1 ml) was added and the reaction
mixture concentrated in vacuo. The product was washed with
hexane (1 ml ꢂ 3), following which TFA (1 ml) was again
added and the solution allowed to stir for 1 h. Toluene (1 ml)
was added and the product concentrated in vacuo. The product
was dissolved in methanol and impurities were removed by
extracting with hexane (1 ml ꢂ 3) to give 8 as a colorless solid
(7 mg, quant.). RP HPLC (YMC-Pack ODS-A, 250 ꢂ 10 mm,
[MeOH þ 1% TFA]–[H2O þ 1% TFA] 85 : 15, 2 ml minꢁ1, tR
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1
¼ 5.93 min); H NMR (500 MHz, CF3COOD–CD3OD 3 : 1):
d 7.14–6.56 (several m, 36H), 6.48 (d, 3J ¼ 8.2, 1H), 6.46 (d, 3J
¼ 8.0, 1H), 6.32 (d, 3J ¼ 7.9, 1H), 6.30 (d, 3J ¼ 7.4, 1H), 4.50–
3.65 (several m, 56H), 2.73–2.52 (several m, 32H), 1.57 (s, 3H),
1.54 (s, 3H), 1.33–0.55 (several m, 104H), 0.42–0.01 (m, 144H);
MS (ESI, [CH3CN–H2O–AcOH 74 : 24 : 2]–[MeOH] 9 : 1): m/z
(%) 1297 (15, [M þ 2H3PO4 þ 5H]51), 1277 (9, [M þ 1H3PO4
þ 5H]51), 1258 (2, [M þ 5H]51), 1066 (15, [M þ 1H3PO4 þ
6H]61), 1049 (3, [M þ 6H]61), 899 (100, [M þ 7H]71), 913 (37,
[M þ 1H3PO4 þ 8H]81), 908 (40, [M þ 1AcOH þ 8H]81), 787
(15, [M þ 8H]81).
Acknowledgements
32 C. Pe
´
rez, C. G. Espı
´
nola, C. Foces-Foces, P. Nu
´ nez-Coello, H.
We thank M. Juillard for assistance in organic synthesis, P.
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33 N. Sakai, D. Gerard and S. Matile, J. Am. Chem. Soc., 2001, 123,
´
Perrottet and the group of F. Gulac¸ ar for MS measurements,
¨
2517–2524.
and the Swiss NSF for financial support (including the Na-
tional Research Program ‘‘Supramolecular Functional Mate-
rials’’ 4047–057496).
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