Solid-phase synthesis and biological evaluation of Joro spider toxin-4 from Nephila clavata

Article abstract of DOI:10.1021/np100746w

Polyamine toxins from orb weaver spiders are attractive pharmacological tools particularly for studies of ionotropic glutamate (iGlu) receptors in the brain. These polyamine toxins are biosynthesized in a combinatorial manner, providing a plethora of related, but structurally complex toxins to be exploited in biological studies. Here, we have used solid-phase synthetic methodology for the efficient synthesis of Joro spider toxin-4 (JSTX-4) (1) from Nephila clavata, providing sufficient amounts of the toxin for biological evaluation at iGlu receptor subtypes using electrophysiology. Biological evaluation revealed that JSTX-4 inhibits iGlu receptors only in high μM concentrations, thereby being substantially less potent than structurally related polyamine toxins.

Full text of DOI:10.1021/np100746w

NOTE
pubs.acs.org/jnp
Solid-Phase Synthesis and Biological Evaluation of Joro Spider Toxin-4
from Nephila clavata
Anne F. Barslund, Mette H. Poulsen, Tinna B. Bach, Simon Lucas, Anders S. Kristensen, and
Kristian Strømgaard*
Department of Medicinal Chemistry, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark
S Supporting Information
b
ABSTRACT: Polyamine toxins from orb weaver spiders are attractive pharmacological tools
particularly for studies of ionotropic glutamate (iGlu) receptors in the brain. These polyamine
toxins are biosynthesized in a combinatorial manner, providing a plethora of related, but
structurally complex toxins to be exploited in biological studies. Here, we have used solid-phase
synthetic methodology for the efficient synthesis of Joro spider toxin-4 (JSTX-4) (1) from
Nephila clavata, providing sufficient amounts of the toxin for biological evaluation at iGlu
receptor subtypes using electrophysiology. Biological evaluation revealed that JSTX-4 inhibits
iGlu receptors only in high μM concentrations, thereby being substantially less potent than
structurally related polyamine toxins.
he ionotropic glutamate (iGlu) receptors are ligand-gated
ion channels that mediate the majority of excitatory synaptic
T
transmission in the vertebrate brain and are crucial for normal brain
function. Dysfunctional iGlu receptor signaling is involved in a range
of neurological and psychiatric diseases, and iGlu receptors are
considered important drug targets for the treatment of brain
diseases.1 In particular, inhibition of iGlu receptors is a promising
therapeutic strategy in neurodegenerative diseases such as stroke
and Alzheimer's disease.1 Very few drugs targeting iGlu receptors
have been approved, among them memantine, an open-channel
blocker of the N-methyl-D-aspartate (NMDA) subtype of iGlu
receptors, used in the treatment of Alzheimer's disease.2,3
Polyamine toxins are a group of small-molecule natural
products found in spiders and wasps that like memantine are
open-channel blockers of iGlu receptors.4-6 Polyamine toxins
have found valuable use as neuropharmacological tools based on
their high affinity and selectivity for iGlu receptors,4,5 in parti-
cular because of their ability to discriminate Ca2þ permeable
from impermeable R-amino-3-hydroxy-5-methyl-4-isoxazole-
propionic acid (AMPA) and kainate receptor subtypes.7 To date
most structure-activity relationship (SAR) studies of polyamine
toxins have used philanthotoxin-433 (PhTX-433, Figure 1), a
wasp toxin identified by Nakanishi and co-workers,8 as a template
for synthetic analogues. In contrast, the structurally more com-
plex polyamine toxins from spiders have generally been used in
their native forms only. Polyamine toxins from spiders are
biosynthesized in a combinatorial manner, providing a cocktail
of structurally related compounds, the majority of which remain
to be explored biologically.9,10 The spider polyamine toxins have
the general structure as depicted for the prototypical polyamine
toxin argiotoxin-636 (ArgTX-636, Figure 1), composed of an
aromatic headgroup, an optional amino acid linkage, a polyamine
backbone, and an optional amino acid tail.10
Figure 1. General structure of polyamine toxins found in wasps and
spiders, illustrated with PhTX-433 and ArgTX-636 where the four
variable regions are indicated. JSTX-3 and JSTX-4 (1) are structurally
related toxins to ArgTX-636.
We recently developed a synthetic methodology for the prepara-
tion of ArgTX-636 and analogues using solid-phase synthesis.11
However, the overall yield for the synthesis of ArgTX-636 was only
around 2%, thus not rendering the method applicable for synthesis
Special Issue: Special Issue in Honor of Koji Nakanishi
Received: October 16, 2010
Published: December 28, 2010
r
Copyright
2010 American Chemical Society and
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dx.doi.org/10.1021/np100746w J. Nat. Prod. 2011, 74, 483–486
American Society of Pharmacognosy
|

Journal of Natural Products
NOTE
Scheme 1. Solid-Phase Synthesis of JSTX-4 (1)a
a Reagents and conditions: (a) (i) DFPE resin, DMF/acetic acid (9:1), (ii) NaBH(OAc)3; (b) Fmoc-L-Asn(Trt)-OH, HATU, DIPEA, CH2Cl2/DMF
(9:1); (c) 20% piperidine in DMF; (d) 2-(2,4-bis(benzyloxy)phenyl)acetic acid, HATU, DIPEA, CH2Cl2/DMF (9:1); (e) TBAF, THF, 55 °C; (f)
Fmoc-L-Orn(Boc)-OH, HATU, DIPEA, CH2Cl2/DMF (9:1); (g) 20% piperidine in DMF; (h) Boc-L-Arg(Pbf)-OH, HATU, DIPEA, CH2Cl2/DMF
(9:1); (i) TFA/CH2Cl2/TIPS/H2O (75:20:2.5:2.5); (j) Pd(OH)2/C, H2, HOAc.
of larger amounts of toxins required for advanced biological studies
or for generation of libraries of polyamine toxins in general. Thus, we
wanted to apply the synthetic methodology to a related toxin, which
could be prepared in a significantly higher overall yield and subse-
quently be used in chemical and biological studies. Initially, we
explored the synthesis of JSTX-3 (Figure 1), which is also a pro-
totypical polyamine toxin with potent inhibitory activity at
iGlu receptors.12,13 However the synthesis of JSTX-3 was even
more challenging than for ArgTX-636, in particular introduction of
the 3-aminopropanoic acid moiety (data not shown). Instead,
we took a different approach and became inspired by the wealth
of polyamine toxins isolated from the Joro spider Nephila clavata10
and in particular JSTX-4 (1, Figure 1),14 which can be considered a
hybrid between JSTX-3 and ArgTX-636. Moreover, JSTX-4 had
been neither synthesized nor biologically characterized before, and
we envisioned that the synthesis of JSTX-4 could be efficiently
achieved using our previously developed protocol.11
The synthetic strategy for obtaining JSTX-4 was related to that
of ArgTX-636: Starting from a backbone amide linker (BAL) resin,
2-(trimethylsilyl)ethyl 5-aminopentylcarbamate was attached by
reductive amination, and the aromatic headgroup was generated
by subsequent couplings of protected asparagine and 2-(2,4-bis-
(benzyloxy)phenyl)acetic acid15 to furnish 2 (Scheme 1). Next, the
N-Teoc protection group was selectively cleaved by treatment with
tetrabutylammonium fluoride (TBAF), and appropriately protected
ornithine was coupled to the free, primary amino group to give 3.
This was followed by Fmoc removal and coupling of protected
arginine to the primary amine, giving fully protected, resin-bound
JSTX-4 (4, Scheme 1). The target compound was obtained by
treating resin 4 with trifluoroacetic acid (TFA), triisopropylsilane
(TIPS), and water and subsequent deprotection of the aromatic O-
benzyl groups with Pd(OH)2/C and H2 in solution. Gratifyingly the
10-step synthetic sequence and purification by preparative HPLC
provided JSTX-4 (1) in >98% purity as determined by LC-MS and
an overall yield of 39%, equaling >90% yield per step.
electrophysiology on Xenopus oocytes expressing the NMDA
receptor subtype GluN1/2A and the AMPA receptor subtype
GluA1. We tested the ability of increasing concentrations of JSTX-
4 to inhibit agonist-evoked receptor currents at a membrane
potential of -60 mV, which is close to the typical neuronal resting
potential. Generally, we observed that high μM concentrations
of JSTX-4 were needed to produce a weak inhibitory effect
(Figure 2). The inhibitory effect of JSTX-4 at the two iGlu
receptor subtypes was generally similar, and a 100 μM concentr-
ation of JSTX-4 produced approximately 25% inhibition at both
receptor subtypes; however IC50 values could not be determined
for the concentration range used. The low inhibitory potency of
JSTX-4 at the GluN1/2A and GluA1 subtypes was in contrast to
ArgTX-636, which has IC50 values of 74 and 77 nM at these
subtypes, respectively, when tested under similar conditions;11
thus clearly demonstrating a dramatic difference in the biological
activity of ArgTX-636 and JSTX-4. The primary structural differ-
ences are the overall length of the molecule and the number of
secondary amino groups; ArgTX-636 is a longer molecule and may
therefore bind deeper in the ion channel region of iGlu receptors,
as required for biological activity.11 Moreover, in a previous study
we demonstrated the importance of the secondary amino groups
in ArgTX-636 for selectivity among AMPA and NMDA recep-
tors, which could largely be explained by changes in their three-
dimensional structure due to different internal hydrogen-bonding
patterns.11 We suggest that the surprisingly weak inhibitory activity
of JSTX-4 at iGlu receptors is caused by the absence of secondary
amino groups combined with a shorter distance between the
aromatic headgroup and the terminal guanidinium groups.
In conclusion, we have developed a highly efficient solid-phase
synthesis of JSTX-4, where the 10-step sequence provides the
compound in a yield of 39%, hence being substantially more
efficient than the previously reported synthesis for ArgTX-636.
Biological evaluation of JSTX-4 revealed that the compound
inhibits only iGlu receptors in high μM concentrations, thus
being substantially less potent than structurally related polya-
mine toxins such as ArgTX-636.
The potency of JSTX-4 to block NMDA- and AMPA-type
iGlu receptors was evaluated using two-electrode voltage-clamp
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Journal of Natural Products
NOTE
1
UV detection at 214 and 254 nm. H and 13C NMR spectra were
recorded on a Bruker Avance AV-300 spectrometer. Solid-phase synthe-
sis was carried out using Bohdan MiniBlock (Mettler-Toledo). Unless
otherwise stated, reactions were carried out at room temperature, all
washings were performed with 3 ꢀ 4 mL of solvent, and resins were
dried in vacuo after washings.
2-(Trimethylsilyl)ethyl 5-aminopentylcarbamate:. yield 77%;
1H NMR (300 MHz, CDCl3) δ 4.64 (bs, 1H), 4.16 (t, J = 8.3 Hz, 2H), 3.19
(q, J = 6.3 Hz, 2H), 2.71 (t, J = 6.6 Hz, 2H), 1.33-1.60 (m, 6H), 1.28 (bs,
2H), 1.00 (t, J = 8.3 Hz, 2H), 0.06 (s, 9H); EIMS m/z 247.1 [M þ H]þ;
purity (ELSD), 91%.
(S)-N1-(5-((S)-5-Amino-2-((S)-2-amino-5-guanidinopentana-
mido)pentanamido)pentyl)-2-(2-(2,4-dihydroxyphenyl)aceta-
mido)succinamide (JSTX-4, 1). DFPE resin (0.15 mmol) was swelled
in DMF (4 mL) for 1 h. The resin was drained and treated with a solution
of 2-(trimethylsilyl)ethyl-5-aminopentylcarbamate (1.50 mmol) in dry
DMF/acetic acid (9:1, 4 mL), and after 2 min NaBH(OAc)3 (1.50 mmol)
was added. The mixture was agitated overnight, the solvents were drained,
and the resin was washed successively with DMF, DIPEA (10% in DMF),
DMF, CH2Cl2, CH3OH, and CH2Cl2. A solution of Fmoc-L-Asn(Trt)-OH
(0.75 mmol) and N-[(dimethylamino)-1H-1,2,3-triazol[4,5-b]pyridin-1-
ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide
(HATU, 0.75 mmol) in dry CH2Cl2/DMF (9:1, 4 mL) was added to the
resin followed by addition of N,N-diisopropylethylamine (DIPEA, 1.50
mmol). The mixture was agitated overnight, and the resin was subsequently
washed with DMF, CH2Cl2, CH3OH, and CH2Cl2. The resulting resin was
treated with 20% piperidine in dry DMF (v/v, 4 mL) for 2 and 20 min and
washed with DMF in between. The resin was washed with DMF, CH2Cl2,
CH3OH, and CH2Cl2, drained, and treated with a solution of 2-(2,4-
bis(benzyloxy)phenyl)acetic acid (0.75 mmol) and HATU (0.75 mmol) in
dry CH2Cl2/DMF (9:1, 4 mL), followed by treatment with DIPEA (1.50
mmol). The mixture was agitated for 2 h and washed with DMF, CH2Cl2,
CH3OH, and CH2Cl2 and subsequently suspended in THF (4 mL) for 30
min. Then it was heated to 55 °C before a solution of TBAF (1 M in THF,
0.60 mmol) was added slowly, and the mixture was agitated at 55 °C for
30 min. The resin was drained, washed with DMF, CH2Cl2, CH3OH, and
CH2Cl2, and treated with a solution of Fmoc-L-Orn(Boc)-OH (0.75
mmol) and HATU (0.75 mmol) in dry CH2Cl2/DMF (9:1, 4 mL),
followed by DIPEA (1.50 mmol). The mixture was agitated for 2 h, washed
with DMF, CH2Cl2, CH3OH, and CH2Cl2, then treated with 20%
piperidine in dry DMF (v/v, 4 mL) for 2 and 20 min, and washed with
DMF in between. The resin was then washed with DMF, CH2Cl2,
CH3OH, and CH2Cl2, treated with a solution of Boc-L-Arg(Pbf)-OH
(0.75 mmol) and HATU (0.75 mmol) in dry CH2Cl2/DMF (9:1, 4 mL),
followed by DIPEA (1.50 mmol), agitated for 2 h, and washed with DMF,
CH2Cl2, CH3OH, and CH2Cl2. Finally, the resin was treated with a mixture
of TFA/CH2Cl2/TIPS/H2O (75:20:2.5:2.5 v/v, 4 mL) for 2 h, drained,
and washed with CH3OH (2 mL) and CH2Cl2 (2 mL). The combined
solvents were evaporated to give a sticky yellow solid (2,4-Di-O-Bn-JSTX-
4), which was purified by preparative HPLC. The purified 2,4-Di-O-Bn-
JSTX-4 was dissolved in glacial acetic acid (4 mL) and transferred to a screw
cap vial, and Pd(OH)2/C (10% w/w) was added. Hydrogen was bubbled
through the solution for 1 min, and the mixture was stirred under an
atmosphere of hydrogen until the reaction was completed. The reaction
mixture was filtered and washed with CH3OH, and water (10 mL) was
added. The combined washings were removed by freeze-drying to give a
colorless solid, which was purified by preparative HPLC to give the product
as a colorless solid (56.9 mg, 39%): 1H NMR (300 MHz, CD3OD) δ 6.89
(d, J = 8.3 Hz, 1H), 6.31 (d, J = 2.2 Hz, 1H), 6.23 (dd, J = 8.0, 2.2, Hz, 1H),
4.64 (t, J = 6.3 Hz, 1H), 4.33 (t, J = 6.3 Hz, 1H), 3.95 (t, J = 6.1 Hz, 1H),
3.46 (d, J = 14.9 Hz, 1H), 3.38 (d, J = 15.1 Hz, 1H), 3.01-3.21 (m, 6H),
2.85-2.98 (m, 2H), 2.66 (d, J = 6.3 Hz, 2H), 1.83-1.96 (m, 2H),
1.51-1.83 (m, 6H), 1.33-1.51 (m, 4H), 1.15-1.33 (m, 2H); 13C NMR
(75 MHz, CD3OD) δ 175.2, 175.0, 173.2, 173.1, 169.9, 159.0, 158.6, 157.3,
Figure 2. Biological evaluation of JSTX-4. (A) Representative current
trace obtained from electrophysiological recording of the membrane
current from a Xenopus oocyte expressing GluA1 receptors held at a
membrane potential of -60 mV. The agonist-evoked response (Glu;
100 μM) is observed as a downward deflection of the membrane current.
Co-application of increasing concentrations of JSTX-4 is indicated by
gray bars. (B) Summary of the effect of various concentrations of JSTX at
GluN1/2A (black) and GluA1 (gray) receptors. Data represent mean
((SEM) from experiments at 9 to 12 oocytes normalized to the agonist-
evoked current response in the absence of JSTX-4.
’ EXPERIMENTAL SECTION
General Experimental Procedures. Unless otherwise stated,
starting materials were obtained from commercial suppliers and were
used without further purification. The BAL-resin used was 2-(3,5-
dimethoxy-4-formylphenoxy)ethyl (DFPE) polystyrene with a loading
of 0.5-1.0 mmol/g and was purchased from Novabiochem. 2-(2,4-
Bis(benzyloxy)phenyl)acetic acid was prepared according to a published
procedure,15 and 2-(trimethylsilyl)ethyl 5-aminopentylcarbamate was
prepared following procedures for related aliphatic diamines.16 Tetra-
hydrofuran (THF) was distilled under N2 from sodium/benzophenone
immediately before use. N,N-Dimethylformamide (DMF) and dichlor-
omethane were dried using AldraAmine trapping packets.
LC-MS analysis was performed on an Agilent 6410 Triple Quadru-
pole mass spectrometer with electron spray coupled to an Agilent 1200
HPLC system (ESI-LC/MS) with autosampler and diode-array detector
using a linear gradient of the binary solvent system of water/acetonitrile/
formic acid (A: 95/5/0.1 and B: 5/95/0.086) with a flow rate of 1 mL/min.
During ESI-LC/MS analysis evaporative light scattering (ELS) traces
were obtained with a Sedere Sedex 85 light scattering detector, which
were used for estimation of the purity of the final products. Preparative
HPLC was performed on a Agilent 1100 system using a C18 reversed-
phase column (Zorbax 300 SB-C18, 21.2 ꢀ 250 mm) with a linear
gradient of the binary solvent system of water/acetonitrile/TFA (A: 95/
5/0.086 and B: 5/95/0.086) with a flow rate of 20 mL/min and
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Journal of Natural Products
NOTE
132.8, 114.0, 108.1, 103.9, 54.5, 53.9, 52.0, 42.0, 40.3, 40.4, 38.8, 37.6, 30.2,
29.9, 29.8, 29.8, 25.2, 24.9; EIMS m/z 637.3 [M þ H]þ; purity (ELSD),
98.6%; HRMS m/z 637.3766 (calcd for C28H49N10O7, 637.3786).
Biology. In Vitro cRNA Transcription. The cDNA encoding rat
GluA1i or GluN1/2A subunits was inserted into the vector pGEM-HE
or pCI-IRES-BLAS, respectively, for preparation of high-expression
cRNA transcripts. Plasmids were grown in Top10 E. coli bacteria
(Invitrogen, Carlsbad, CA) and isolated by using column purification
(Qiagen, La Jolla, CA). The cRNA was synthesized from the above
cDNAs by in vitro transcription using the mMESSAGE mMACHINE
T7 mRNA-capping kit (Ambion, Austin, TX) according to the protocol
supplied by the manufacturer.
Oocyte Electrophysiology. Mature female Xenopus laevis
(Nasco, Modesto, CA) were anaesthetized using 0.1% ethyl 3-amino-
benzoate, and their ovaries were surgically removed. The ovarian tissue
was dissected and treated with Collagenase IV (Worthington, Lake-
wood, NJ) in Ca2þ-free Barth's medium (2 mg/mL) for 1-2 h at room
temperature. On the second day, oocytes were injected with 25 nL of
cRNA (1 ng/nL GluA1i or 0.05 ng/nL GluN1 and GluN2A) and
incubated at 17 °C in Barth's medium (in mM: 88 NaCl, 1 KCI, 0.33
Ca(NO3)2, 0.41 CaC12, 0.82 MgSO4, 2.4 NaHCO3, 10 HEPES; pH 7.4)
with gentamicin (0.10 mg/mL). Oocytes were used for recordings 3-6
days postinjection and were voltage-clamped with the use of a two-
electrode voltage clamp (OC-725C, Warner Instruments, Hamden, CT)
with both microelectrodes filled with 3 M KCl. Recordings were made
while the oocytes were continuously superfused with frog Ringer's
solution (in mM: 115 NaCl, 2 KCI, 1.8 BaC12, 5 HEPES; pH 7.6).
The test compounds were dissolved in frog Ringer's solution and added
by bath application. Recordings were performed at room temperature at
holding potentials of -60 mV. Inhibition of agonist-evoked currents was
measured by recording the maximal current induced by a saturating
concentration of agonist (300 μM glutamate for GluA1i, 300 μM
glutamate and 100 μM glycine for GluN1/2A) and then applying
increasing concentrations of test compound in the presence of the
’ DEDICATION
Dedicated to Dr. Koji Nakanishi of Columbia University for his
pioneering work on bioactive natural products.
’ REFERENCES
(1) Traynelis, S. F.; Wollmuth, L. P.; McBain, C. J.; Menniti, F. S.;
Vance, K. M.; Ogden, K. K.; Hansen, K. B.; Yuan, H.; Myers, S. J.;
Dingledine, R. Pharmacol. Rev. 2010, 62, 405–496.
(2) Lipton, S. A. Nat. Rev. Neurosci. 2007, 8, 803–808.
(3) Cosman, K. M.; Boyle, L. L.; Porsteinsson, A. P. Expert Opin.
Pharmacother. 2007, 8, 203–214.
(4) Strømgaard, K.; Mellor, I. R. Med. Res. Rev. 2004, 24, 589–620.
(5) Strømgaard, K.; Jensen, L. S.; Vogensen, S. B. Toxicon 2005, 45,
249–254.
(6) Fleming, J. J.; England, P. M. Nat. Chem. Biol. 2010, 6, 89–97.
(7) Liu, S. J.; Zukin, R. S. Trends Neurosci. 2007, 30, 126–134.
(8) Eldefrawi, A. T.; Eldefrawi, M. E.; Konno, K.; Mansour, N. A.;
Nakanishi, K.; Oltz, E.; Usherwood, P. N. R. Proc. Natl. Acad. Sci. U. S. A.
1988, 85, 4910–4913.
(9) Itagaki, Y.; Nakajima, T. J. Toxicol.-Tox. Rev. 2000, 19, 23–52.
(10) Palma, M. S. Toxin Rev. 2005, 24, 209–234.
(11) Nelson, J. K.; Frølund, S. U.; Tikhonov, D. B.; Kristensen, A. S.;
Strømgaard, K. Angew. Chem., Int. Ed. 2009, 48, 3087–3091.
(12) Blaschke, M.; Keller, B. U.; Rivosecchi, R.; Hollmann, M.;
Heinemann, S.; Konnerth, A. Proc. Natl. Acad. Sci. U. S. A. 1993, 90,
6528–6532.
(13) Iino, M.; Koike, M.; Isa, T.; Ozawa, S. J. Physiol. (London) 1996,
496, 431–437.
(14) Toki, T.; Yasuhara, T.; Aramaki, Y.; Hashimoto, Y.; Shudo, K.;
Kawai, N.; Nakajima, T. Jpn. J. Sanit. Zool. 1990, 41, 9–14.
(15) Chiba, T.; Akizawa, T.; Matsukawa, M.; Nishi, M.; Kawai, N.;
Yoshioka, M. Chem. Pharm. Bull. 1996, 44, 972–979.
(16) Olsen, C. A.; Witt, M.; Jaroszewski, J. W.; Franzyk, H. J. Org.
Chem. 2004, 69, 6149–6152.
appropriate agonist. For ArgTX-636, the concentration-inhibition data
50-log[antagonist])nH
were fitted to the equation I = Imax/[1 þ 10(log IC
],
where I is the response observed at a given antagonist concentration and
Imax is the response in the absence of antagonist. The parameters Imax
(maximal current observed at infinite agonist concentration), nH (Hill
coefficient), and IC50 (concentration of antagonist producing 50% of
Imax) were determined by an iterative least-squares fitting routine.
’ ASSOCIATED CONTENT
1
Supporting Information. Copies of H and 13C NMR
S
b
spectra for JSTX-4 (1). This can be accessed free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: þ45 3533 6114. Fax: þ45 3533 6041. E-mail: krst@
farma.ku.dk.
’ ACKNOWLEDGMENT
We thank the Alfred Benzon Foundation for a postdoctoral
scholarship (to S.L.) and the Aase and Ejnar Danielsens Founda-
tion, the Brødrene Hartmanns Foundation, the Torben & Alice
Frimodts Fond, and the Direktør Ib Henriksens Foundation
for instrument grants (to A.S.K.). Dr. D. Tikhonov, Sechenov
Institute of Evolutionary Physiology and Biochemistry, Russian
Academy of Science, St. Petersburg, Russia, is thanked for input
to the manuscript.
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