Cyclohexylether δ-Amino Acids: New Leads for Selectivity Filters in Ion Channels

Full text of DOI:10.1002/1521-3773(20010601)40:11<2076::AID-ANIE2076>3.0.CO;2-G

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[9] a) R. Irie, Y. Ito, T. Katsuki, Synlett 1991, 265; b) N. S. Finney, P. J.
Pospisil, S. Chang, M. Palucki, R. G. Konsler, K. B. Hansen, E. N.
Jacobsen, Angew. Chem. 1997, 109, 1798; Angew. Chem. Int. Ed. Engl.
1997, 36, 1720.
[10] It should be pointed out that the role of spin contamination in Kohn ±
Sham theory is not clear. For a more extensive discussion see: W.
Koch, M. C. Holthausen, A Chemistꢀs Guide to Density Functional
Theory, Wiley-VCH, Weinheim, 2000.
[11] These would also be similar to the ªbentº conformation proposed for
chromium ± salen complexes: K. M. Ryan, C. Bousquet, D. G. Gil-
heany, Tetrahedron Lett. 1999, 40, 3613.
[12] Further evidence for the electronic nature of this effect comes from
preliminary B3LYP/3-21G* calculations of the catalyst bearing an
axial OCl ligand. As in the case of trimethylamine N-oxide, the
ligation of hypochlorite leads to highly nonplanar conformations.
Similarly, inspection of the calculated structure of a Cl -ligated Mn ±
salen model catalyst depicted in ref. [7b] reveals a strong tendency for
a nonplanar conformation.
Scheme 1. From d,l-dipeptides to the stereoequivalent d-amino acid 1, a
new building block for cation channels.
‡
The selectivity filters of biological K and Ca2‡ channels are
lined with backbone atoms.[8a,b] In the ion channel active, but
weakly selective, b6.3-helical dimer of the d,l-peptide grami-
cidin A (gA) only backbone amides are exposed towards the
[13] K. Miura, T. Katsuki, Synlett 1999, 783.
[14] For examples of such calculations, see: a) J. N. Harvey, M. Aschi, H.
Schwarz, W. Koch, Theor. Chem. Acc. 1998, 99, 95; b) S. Mitchell, M.
Blitz, P. Siegbahn, M. Svensson, J. Chem. Phys. 1994, 100, 423;
compare also: c) D. Schröder, S. Shaik, H. Schwarz, Acc. Chem. Res.
2000, 33, 139.
Cyclohexylether d-Amino Acids: New Leads
for Selectivity Filters in Ion Channels**
Hans-Dieter Arndt, Andrea Knoll, and Ulrich Koert*
Biological ion channels are key molecules for cellular
regulation and communication. They couple (bio)molecular
events to electric signals.[1] This property of natural pore-
forming substances has been utilized in engineering biosen-
sors.[2] In order to use synthetic channel structures[3] as sensors
or implants in biological systems, they have to meet require-
ments in two areas: ion selectivity[3e, 4] and gating.[5] We report
here on novel oligomers made from d-amino acids that led to
interior.[8c] The incorporation of d-AA 1 with its ether oxygens
should offer additional binding sites for a cation in the
lumen.[9] Our target compounds 2 ± 4 were chosen by combin-
ing di-, tetra-, and hexameric d-AA segments with function-
ally important sequences from gA to yield structures with the
approximate total length of the gA dimer.[10, 11]
The synthesis of the compounds incorporating d-AA 1
starts from cyclohexene epoxide 5, which was transformed to
the azido alcohol according to the method of Jacobsen and co-
workers[12] (94% yield, 93% ee under optimized conditions,
Scheme 2). Alkylation with tert-butylbromoacetate using
phase-transfer catalysis[13] gave masked d-AA monomer 6
(95%; R ˆ tBu),[14] which was homodimerized to 9 via a mixed
‡
‡
H - and NH4 -selective ion channels.
On substituting the central amide bond of a dipeptide, a d-
amino acid is obtained (Scheme 1).[6] Besides offering struc-
tural diversity at four positions, a d-amino acid allows
incorporation of a heteroatom in the continuous backbone.[7]
If one chooses an oxygen and constricts the degrees of
conformational freedom with a cyclohexane ring, the ether
amino acid (AA) 1 results.
anhydride (R ˆ COCMe3; formed from
7
(step e in
Scheme 2)). Dimer 9 could be obtained isomerically pure by
crystallization (n-hexane/Et2O (7:1), Figure 2). After elonga-
tion with a succinate building block, the resulting diester 10
was connected at both termini with the a-peptide 11[11] to yield
the target compound 2. Compounds 3 and 4 were similarly
synthesized (see Supporting Information).[14]
[*] Prof. Dr. U. Koert, Dipl.-Chem. H.-D. Arndt, Dr. A. Knoll
Institut für Chemie der Humboldt-Universität zu Berlin
Hessische Strasse 1 ± 2, 10115 Berlin (Germany)
Fax : (‡49)30-2093-7266
E-mail: koert@chemie.hu-berlin.de
The compounds 2 ± 4 were then examined for their ion
channel forming activity.[15, 16] The compounds with tetra- and
hexameric d-peptide units, 3 and 4, did not form detectable
cation channels. But they induced short-lived proton channels
when applied in concentrations above 100 nm (Figure 1a, b).
Probably, a bottleneck conformation permits only protons to
pass.[17]
[**] Financial support by the Fonds der Chemischen Industrie (FCI), the
Volkswagen Foundation, the Pinguin Foundation, and Schering AG is
gratefully acknowledged. H.-D.A. thanks the FCI for a PhD fellow-
ship. We thank Dr. B. Ziemer (Humboldt-Universität zu Berlin) for
the X-ray structure analysis and Dr. P. Franke (Freie Universität
Berlin) for MALDI-TOF mass spectra.
Supporting information for this article is available on the WWW under
http://www.angewandte.com or from the author.
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Scheme 2. Synthesis of 2: a) 1. TMSN3 (1.1 equiv), 2 mol% [Cr(N3)(sa-
len)],[12] 20 mol% iPrOH, Et2O (2m), 08C, 36 h, 94%; 2. 0.1% TFA in
MeOH, 208C, 1 h, >99%; b) tBuOCOCH2Br, nBu4Br, 12m NaOH/
toluene, 208C, 24 h, 95%; c) TFA/CH2Cl2 (2:1), 208C, 1 h, >99%;
d) MeOH, cat. Pd/C (5%), H2 (1 bar), 2 h, >99%; e) 7 (R ˆ H) in DMF,
NEt3, PivCl, 08C, 30 min, then 8, NEt3, 08C !208C, 1 h, 77%; f) pNBnO-
Succ, EDC, HOBt, EtNiPr2, CH2Cl2/DMF (10:1), 08C !208C, 12 h, 90%;
g) 11, HATU, HOAt, EtNiPr2, CH2Cl2/DMF (3:1), 08C !208C, 6 h;
h) LiOH, THF/H2O 3:1, 08C, 1 h. DMF ˆ N,N-dimethylformamide,
EDC ˆ 3-(3-dimethyl-aminopropyl)-1-ethylcarbodiimide hydrochloride,
Figure 1. Representative current traces of compounds 2 ± 4 in planar soya-
bean lecithin lipid bilayers: a) 4, conductivity L ˆ 355/198 pS, dwell time
t ˆ 3.7 ms; b) 3, L ˆ 255/107 ps, t ˆ 8 ms (c(3, 4) ˆ 500 nm, U ˆ ‡100 mV;
HATU ˆ O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexa-
fluorophosphate, HOAt ˆ 7-aza-1-hydroxy-1H-benzotriazole, HOBt ˆ 1-
hydroxy-1H-benzotriazole monohydrate, H2Salen ˆ (R,R)-N,N'-bis(3,5-di-
tert-butylsalicylidene)cyclohexane-1,2-diamine, Piv ˆ pivaloyl (COCMe3),
pNBn ˆ 4-nitrobenzyl, Succ ˆ succinyl (COCH2CH2CO2H), TFA ˆ tri-
fluoroacetic acid.
‡
‡
‡
‡
in 1m HCl); c) 2, H ; d) 2, K ; e) 2, Cs ; f) 2, NH4 (c(2) ˆ 10 pm, U ˆ
‡
‡
‡
‡
‡
‡200 mV; in 1m MCl, M ˆ H , K , Cs , NH4 ); g) amplitude histogram
‡
for NH4 -channels of compound 2 at 120 mV (A ˆ amplitude, N ˆ number
of results). Experimental details can be found in the Supporting Informa-
tion.
In contrast, compound 2 formed well-defined channels with
‡
a remarkable selectivity among monovalent cations: Cs and
but the proton-channel dwell time is only 8 ms (Fig-
ure 1c).[5b, 19]
With the exception of Cs , two levels of conductance were
found (Table 1, Figure 1g). Asymmetric compounds such as
2 ± 4 may adopt at least two orientations within the mem-
brane, with respect to the membrane normal. A maximum of
two detectable conductance levels supports the assumptions
that a) unimolecular channels are present and b) the d-AAs
do indeed influence the ions pathway.
‡
‡
NH4 were conducted well, but K single-channel events were
‡
‡
‡
at the detection limit (Figure 1d ± f). For Na and Li , single-
channel events could no longer be resolved any more. The
analysis of the permeability ratios revealed a pronounced
‡
‡
‡
‡
‡
Eisenman-I selectivity:[1b] H ꢀ NH4 ꢀ Cs > Rb > K >
‡
‡
Na ꢁ Li (Table 1). The selectivity (Prel, Lrel) of 2 is three
to four times higher than that of gA. The absolute conduc-
tance is decreased by the incorporated d-AAs, which indicates
a narrowed pore or a reinforced binding.[18] The mean dwell
times of the cation channels of 2 are between 200 and 350 ms,
These results introduce the new d-AA 1 as a novel lead
‡
‡
structure in the synthesis of H - and NH4 -selective ion
Table 1. Permeability ratios P and conductivity figures L of 2 and gA.
‡
M
Prel(gA)[a]
Prel(2)[a]
L(gA)[b]
L1(2)[b]
L2(2)[b]
Lrel(gA)[c]
Lrel(2)[c]
‡
Li
0.051 Æ 0.001
0.105 Æ 0.003
0.248 Æ 0.004
0.401 Æ 0.012
0.507 Æ 0.009
1
0.035 Æ 0.002
0.036 Æ 0.002
0.078 Æ 0.008
0.122 Æ 0.004
0.273 Æ 0.025
1
2.93 Æ 0.07
±
±
±
±
0.07
0.34
0.60
1.00
1.01
1
±
±
‡
Na
14.80 Æ 0.08
26.0 Æ 0.2
43.1 Æ 0.3
43.6 Æ 0.2
43.3 Æ 0.2
561 Æ 17
‡
K
1.06 Æ 0.11
2.38 Æ 0.13
6.48 Æ 0.11
7.43 Æ 0.07
90 Æ 15
0.66 Æ 0.06
1.85 Æ 0.11
±
0.14 (0.12)
0.32 (0.34)
0.87
‡
Rb
Cs
‡
‡
NH4
5.49 Æ 0.09
1
‡
H
3.99 Æ 0.19
4.77 Æ 0.18
67.2 Æ 0.8
13.0
12.1 (12.2)
[a] Determined from the reversal potentials of 1m MCl solutions relative to a 1m NH4Cl solution.[1b] This is the mean of all conductivity levels because sum
‡
currents were measured (see Supporting Information). [b] Conductivity [pS], determined from the slope of the current-voltage curve at U ˆ 0 mV. For Cs
‡
only one level was found. [c] Conductivity relative to NH4 conductivity. The second conductivity level is given in parentheses.
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[6] b-Peptides: a) D. Seebach, J. L. Matthews, Chem. Commun. 1997, 21,
2015 ± 2022; b) S. H. Gellman, Acc. Chem. Res. 1998, 31, 173 ± 180; g-
peptides: c) T. Hintermann, K. Gademann, B. Jaun, D. Seebach, Helv.
Chim. Acta 1998, 81, 983 ± 1002; d) S. Hanessian, X. L. Luo, R.
Schaum, S. Michnick, J. Am. Chem. Soc. 1998, 120, 8569 ± 8570.
[7] As opposed to their b- and g-counterparts, d-peptides should
generally interact easily with the ªnaturalº a-peptide world. In
essence, the a-peptides form a subset of all possible d-peptides.
Examples for d-peptides can be found in: a) U. Koert, J. Prakt. Chem.
2000, 342, 325 ± 333; b) F. Machetti, A. Ferrali, G. Menchi, E. G.
Occhiato, A. Guarna, Org. Lett. 2000, 2, 3987 ± 3990, and references
therein.
channels. Synthetic compound 2 shows a more than threefold
‡
‡
increase in NH4 /K selectivity compared to gA. The X-ray
crystal structure analysis of dimer 9 (Figure 2) gives a hint of
the role of d-AA 1 in the channel:[20] The d-peptide 9 adopts
[8] a) D. Doyle, J. M. Cabral, R. A. Pfuetzner, A. Kuo, J. M. Gulbis, S. L.
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[9] Modifications of the sequence and terminal groups of gramicidin A
are well studied: a) P. Läuger, Angew. Chem. 1985, 97, 939 ± 959;
Angew. Chem. Int. Ed. Engl. 1985, 24, 905 ± 925; b) C. J. Stankovic,
S. L. Schreiber, Chemtracts: Org. Chem. 1991, 4, 1 ± 19; c) R. E.
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[10] THF-d-AAs give functional ion channels: A. Schrey, A. Vescovi, A.
Knoll, C. Rickert, U. Koert, Angew. Chem. 2000, 112, 928 ± 931;
Angew. Chem. Int. Ed. 2000, 39, 900 ± 902.
[11] Short minigramicidins from the terminal 11-mer of gA form ion
channels dependent on membrane thickness: H.-D. Arndt, A. Knoll,
U. Koert, ChemBioChem 2001, 2, 221 ± 223.
[12] S. E. Schaus, J. F. Larrow, E. N. Jacobsen, J. Org. Chem. 1997, 62,
4197 ± 4199.
Figure 2. X-ray crystal structure analysis of d-dipeptide 9 (ellipsoids with
50% probability). Selected distances [pm]: N4-O1 256.3(6), N4-O4
315.8(7). Please note the right-handed helical conformation (N1-O1-N4-
O3-O5).
the conformation of a right-handed helix in the solid state.
Thus, it should be ideally suited to propagate the right-handed
b6.3-helices of the d,l-peptide segments in their ion-conduct-
ing conformation.[8c, 11] The central bifurcated hydrogen bond
has to open to allow ions to pass vertically in the plane of
display. Such ªgatingº could account for the dwell times,
which are short compared with known examples.[9b, 11, 19]
[13] M. Pietraszkiewicz, J. Jurczak, Tetrahedron 1984, 40, 2967 ± 2970.
[14] All new compounds were characterized by NMR spectroscopy,
HPLC, elemental analysis, or HR-EI/HR-MALDI-TOF/ESI mass
spectrometry (see Supporting Information). For ion-channel analysis
all compounds were purified by repetitive semipreparative HPLC
(C8, CH3CN/iPrOH/H2O).
Received: December 18, 2000 [Z16287]
[15] P. Mueller, D. Rudin, Nature 1968, 217, 713 ± 719.
[16] We could not find evidence for anion conductivity. Variation of the
[1] a) E. Neher, B. Sakmann, Nature 1976, 260, 779 ± 802; b) B. Hille,
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[3] Reviews on artificial ion channels: a) Y. Kobuke in Advances in
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2
anion (Cl /SO4 ) did not affect the reversal potentials.
[17] Distinct proton-channel activity was only observed at high concen-
trations, which could indicate that aggregates of these compounds
form the active channels. This possibility is under active investigation.
[18] The bulk of the hydration shell of an ion is stripped off on entering the
gA channel.[9] A comparison of the changed selectivities with the
effective ionic radii[1b] reveals that particularly the smaller, harder
cations pass the novel channel 2 more slowly (effective ionic radii are
‡
‡
‡
given in parentheses): Li (60 pm) < Na (95 pm) < K (133 pm) <
‡
‡
‡
Rb (148 pm) ꢁ NH4 (150 pm) < Cs (169 pm).
[19] gA proton channels are long lived: S. Cukierman, E. P. Quigley, D. S.
Crumrine, Biophys. J. 1997, 73, 2489 ± 2502.
[20] Crystallographic data (excluding structure factors) for the structure
reported in this paper have been deposited with the Cambridge
Crystallographic Data Centre as supplementary publication no.
CCDC-154390. Copies of the data can be obtained free of charge on
application to CCDC, 12 Union Road, Cambridge CB21EZ, UK (fax:
(‡44)1223-336-033; e-mail: deposit@ccdc.cam.ac.uk).
[5] a) C. J. Stankovic, S. H. Heinemann, S. L. Schreiber, Biochim. Bio-
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