Synthesis of Deuterated Aminocaproyl Linkers

Article abstract of DOI:10.1071/CH01179

Gramicidin A (gA) analogues with a biotin attached to the C-terminus by an aminocaproyl linker are used as ion channels in the AMBRI biosensor and are referred to as biotinylated gA. In order to examine the conformation and dynamics of the aminocaproyl linker by ²H solid-state nuclear magnetic resonance, the synthesis of deuterated aminocaproic acid is required. We report on the synthesis of (D₁₀)-6-aminocaproic acid from commercially available perdeuterated cyclohexanol and its covalent attachment to the C-terminal group of gA to form gAX_DXB.

Full text of DOI:10.1071/CH01179

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Full Papers
Aust. J. Chem. 2001, 54, 747–750
Synthesis of Deuterated Aminocaproyl Linkers
Aphrodite Anastasiadis,A Frances SeparovicA,B and Jonathan WhiteA
A School of Chemistry, University of Melbourne, Vic. 3010, Australia.
B Author to whom correspondence should be addressed (e-mail: fs@unimelb.edu.au).
Gramicidin A (gA) analogues with a biotin attached to the C-terminus by an aminocaproyl linker are used as ion
channels in the AMBRI biosensor and are referred to as biotinylated gA. In order to examine the conformation and
2
dynamics of the aminocaproyl linker by H solid-state nuclear magnetic resonance, the synthesis of deuterated
aminocaproic acid is required. We report on the synthesis of (D10)-6-aminocaproic acid from commercially
available perdeuterated cyclohexanol and its covalent attachment to the C-terminal group of gA to form gAXDXB.
Manuscript received: 15 November 2001.
Final version: 11 January 2002.
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Introduction
group, which is covalently attached to the C-terminus of gA
by an aminocaproyl linker (X). These modified gA
molecules or biotinylated gramicidins (Fig. 2) function best
in the AMBRI biosensor with five aminocaproyl groups
Biotinylated gramicidins are an important component of the
AMBRI biosensor[1] created by Ambri Pty Ltd, Sydney,
N.S.W., Australia(Fig. 1). The AMBRI biosensor has a vast
range of potential applications, from medical diagnosis to
the development of pharmaceutical products and
environmental monitoring systems. The biosensor consists
of a receptor attached to gramicidin A (gA, a peptide ion
channel) embedded in a lipid membrane. The gA ion channel
functions as a dimer, while the receptor has the ability to bind
to a ligand. When binding takes place, dimer formation is
disrupted and, therefore, ions do not readily flow across the
lipid membrane. The receptor is linked to gA by a biotin
(gA5XB), but not with shorter linkers (gA2XB).[2]
A
knowledge of the conformation and dynamics of these linker
groups in lipid bilayers is essential in order to determine the
optimum linker groups for biotinylated gA used in the
AMBRI biosensor. A means of obtaining this information
would utilize deuterium (2H) nuclear magnetic resonance
(NMR) of specifically deuterated linkers. This paper reports
on the synthesis of deuterated aminocaproyl linkers and their
attachment to gA.
Vogt and coworkers[3] have reported the synthesis and
structural studies of gA analogues using specifically
deuterated and perdeuterated acyl gramicidins. The chain
order and dynamics of these derivatives were investigated in
h
i
i
2
aligned lipid bilayers using solid-state H NMR.[4] Using
similar methods, we propose to investigate the conformation
and dynamics of the aminocaproyl groups, in oriented
bilayers, as a function of linker length. We report the
g
e
f
d
b
c
a
Fig. 1. Diagrammatic representation of the AMBRI biosensor,[1]
consisting of a gold electrode (a) with an attached lipid bilayer (b).
Embedded in the lipid bilayer are two gA analogues, one fixed (c)
and
a mobile gA (d) with biotin (e) attached through the
aminocaproyl linker (f). Biotin binds strongly to streptavidin (g).
When a molecule of a desired analyte (h) binds to the biotinylated
receptor (i), the ion channel is disrupted and interrupts ion current
flow to the gold electrode below.
Fig. 2. Biotinylated gA, consisiting of a gramicidin A molecule
with an attached biotin moiety linked by n aminocaproic acid groups
(n = number of aminocaproyl groups).
© CSIRO 2001
10.1071/CH01179
0004-9425/01/120747

748
A. Anastasiadis, F. Separovic and J. White
synthesis of deuterated aminocaproic acid necessary for 2H
NMR studies, and its covalent attachment to the C-terminal
group of gA to form gAXDXB (Fig. 3).
Fig. 3. Deuterated biotinylated gA derivative gAXDXB (8).
Results and Discussion
Deuterated aminocaproic acid was prepared from
commercially available perdeuterated cyclohexanol in four
steps as summarized in Scheme 1. All the intermediates were
characterized by 2H and 13C NMR spectroscopy and
electrospray (EI) and matrix-assisted laser-desorption
ionization (MALDI) mass spectrometry (MS). The
perdeuterated ketone (1) was synthesized by the oxidation of
(D12)cyclohexanol using pyridinium chlorochromate (PCC)
absorbed on alumina.[5] The product showed a molecular ion
at 108 consistent with the presence of 10 deuterons in the
formula. In order to optimize the conditions for the synthesis
of (D10)cyclohexanone, protonated cyclohexanone was first
synthesized following the literature procedure.[5] When the
same conditions were used for the preparation of
(D10)cyclohexanone, the reaction was incomplete. Longer
reaction times and more vigorous conditions (the reaction
mixture was heated to reflux overnight rather than left
stirring at room temperature for 2 h) were required to oxidize
(D12)cyclohexanol. This apparent difference in reactivities is
presumably a result of the deuterium isotope effect.[6] The
ketone (1) was then converted into the oxime derivative (2)
by modifying a previously recorded method.[7] Oxime (2)
was heated in deuterated sulfuric acid (D2SO4),[8,9] affording
the Beckmann product, deuterated ⑀-caprolactam* (3).
D2SO4, rather than H2SO4, was used to avoid proton
incorporation into the product. Hydrolysis of (3)[10] in 30%
DCl/D2O gave (D10)-6-aminocaproic acid† as its
deuterochloride salt (4). The 2H and 13C NMR spectra of all
Scheme 1. Synthesis of deuterated aminocaproyl linker.
gAXDBOC (6), as shown in Scheme 2. The conjugate was
purified by Sephadex gel-permeation chromatography[12]
1
2
followed by silica gel chromatography. H and H NMR
spectra confirmed covalent attachment of the deuterated
linker to gA. Deprotection of gAXDBOC (6) using tri-
fluoroacetic acid gave (N-6-amino(D10)caproyl)gramicidin,
gAXD (7). Biotinamidocaproate-N-hydroxysuccinimide was
coupled onto this deprotected derivative (7) to form [6-
(aminocaproyl)-N-biotinyl(D10)(aminocaproyl)]gramicidin,
gAXDXB (8), which was purified by Sephadex gel-
permeation chromatography followed by silica gel
chromatography. The final product (8) was characterized by
1H, 2H NMR and MALDI MS which showed the presence of
an ion at 2346, which corresponded to the molecular weight
of gAXDXB. 1H NMR as well as thin-layer chromatography
(TLC) data for the final product were almost identical to
native gA, confirming that gAXDXB had similar structural
characteristics. Two-dimensional NMR spectroscopy has
already shown that gA2XB has the same backbone
conformation and function as native gA.[13] 1H NMR
confirmed that deuteration of the linker in the final product
was greater than 98%. The conformation and dynamics of
the linker reconstituted into aligned phospholipid bilayers
will be studied by solid-state NMR techniques[14] and
reported elsewhere.
1
of these deuterated derivatives were compared with the H
and 13C NMR of the equivalent protonated compounds and
they were in good agreement.
The primary amino group in (4) was Boc protected
with [2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile
(BOC-ON) to form 6-[N-(tert-butoxycarbonylamino)](D10)-
caproic acid, XDBOC (5).[11] This was then coupled to the
ethanolamine moiety at the C-terminus of gA to form
[6-[N-(tert-butoxycarbonylamino)](D10)caproyl]gramicidin,
* ⑀-Caprolactam refers to 2-oxohexamethyleneimine.
† Caproic acid refers to hexanoic acid.

Synthesis of Deuterated Aminocaproyl Linkers
749
(St Louis, MO, U.S.A.). Sephadex LH20 was purchased from
Amershaw Pharmacia Biotech AB (Uppsula, Sweden).
Pyridinium Chlorochromate (PCC) on Alumina
To a suspension of chromium trioxide (6 g, 39.5 mmol), in 6N
deuterated hydrochloric acid (11 mL), at 40°C, was added pyridine
(4.75 g, 60.1 mmol) within 10 min. The mixture was cooled to 10°C
then reheated to 40°C and aluminium oxide (50 g, 490.4 mmol) was
added. A yellow solid was obtained and dried under vacuum.
(D10)Cyclohexanone (1)
To a solution of (D12)cyclohexanol (2.5 g, 22 mmol) in hexane (56 mL),
fresh PCC on alumina was added (56 g). The mixture was heated to
reflux overnight. The solid was filtered off and washed with diethyl
ether (5 × 50 mL). The diethyl ether fractions were combined and
evaporated under vacuum to yield (D10)cyclohexanone (1) (1.53 g, 14
mmol, 64%) as a yellow oil. Mass spectrum m/z 108 [MH+]. 2H NMR
(CDCl3) δ 2.24, m, 4D; 1.77, m, 4D; 1.61, m, 2D. 13C NMR (CDCl3) δ
211.7, CO; 40.1 CD2CO; 23.5, 22.0, CD2.
(D10)Cyclohexanone Oxime (2)
(D10)Cyclohexanone (1) (1.53 g, 14 mmol) was dissolved in
(D4)methanol (34 mL), followed by the addition of hydroxylamine
hydrochloride (0.48 g, 6.9 mmol) and pyridine (0.48 g, 6.1 mmol). The
mixture was stirred for 5 h under N2. D2O (43 mL) was added and the
product was extracted with ether (3 × 43 mL) and the extracts were
washed with saturated copper sulfate solution to remove pyridine. The
combined ether solutions were dried with magnesium sulfate, filtered
and dried under high vacuum to yield (D10)cyclohexanone oxime (2)
(0.94 g, 7.6 mmol, 55%) as white crystals. Mass spectrum m/z 124
[MH+]. 2H NMR (CDCl3) δ 1.86, m, 2D; 1.64, m, 4D; 0.97, m, 4D. 13
NMR (CDCl3) δ 166.3, CO; 30.7, CD2CO; 23.7, 22.0, CD2.
C
(D10)-⑀-Caprolactam (3)
To (D10)cyclohexanone oxime (1) (0.34 g, 2.76 mmol) was added 85%
D2SO4 in D2O (1 mL). The mixture was heated until bubbles first
appeared and then allowed to cool down to 0°C. The mixture was made
slightly alkaline with the addition of 24% potassium hydroxide
solution. Potassium sulfate precipitated out and was filtered off and
washed with deuterated chloroform. The solution containing (3) was
then extracted with chloroform (5 × 10 mL). The combined chloroform
solutions were washed with deuterated water and then evaporated under
vacuum to yield (D10)-⑀-caprolactam (3) (0.18 g, 1.46 mmol, 53%) as
a yellow oil. Mass spectrum m/z 124 [MH+]. 2H NMR (CDCl3) δ 3.20,
m, 2D; 2.40, m, 2D; 1.60, m, 6D. 13C NMR (CDCl3) δ 180.1, CO; 43.1,
CD2NH; 36.0, CD2CO; 29.4, 28.3, 22.0, CD2.
Scheme 2. Synthesis of gAXDXB.
(D10)-6-Aminocaproic Acid·DCl (4)
Experimental
To (D10)-⑀-caprolactam (3) (0.142 g, 1.15 mmol) was added 30%
deuterated hydrochloric acid. The mixture was heated to reflux for 2 h.
The solution was then evaporated under vacuum to yield (D10)-6-
aminocaproic acid·DCl (4) (0.11 g, 0.78 mmol, 68%) as white crystals.
Mass spectrum m/z 141 [MH+]. 2H NMR (D2O) δ 3.08, m, 2D; 2.32, m,
2D; 1.60, m, 2D; 1.46, m, 2D; 1.34, m, 2D. 13C NMR (D2O) δ 174.2,
CO; 34.8, CD2NH; 20.9, 19.4, 18.2, CD2.
Instrumentation
2H and 13C NMR experiments were carried out using a Varian Unity
400 MHz (Palo Alto, CA, U.S.A.) spectrometer. Spectra were acquired
in chloroform (CDCl3), unless otherwise specified. The 1H NMR
spectrum of each deuterium derivative showed no evidence of proton
exchange. Therefore, 98+ atom % D was assumed. EI MS was obtained
on a micromass Quattro II instrument recorded at 30 V (solvent system
used: acetonitrile/water, 50 : 50). MALDI MS experiments were
recorded on a PerSeptive Voyager D.E. spectrometer, Perkin Elmer
(MA, U.S.A.).
6-[N-(tert-Butoxycarbonylamino)](D10)caproic Acid (5)
(D10)-6-Aminocaproic acid (4) (70 mg, 0.39 mmol) was dissolved in
50% aqueous dioxan (1.6 mL) and triethylamine (82 µL). The mixture
was stirred at room temperature and BOC-ON (107 mg, 0.43 mmol)
was added. The mixture was then stirred for 1 h at 45°C. H2O (5 mL)
was added and the mixture was washed with ethyl acetate (3 × 10 mL).
The aqueous layer was acidified with HCl (3 M). The product was then
extracted with ethyl acetate (3 × 10 mL), the solution dried with sodium
sulfate and then evaporated under vacuum to yield deuterated 6-[N-
(tert-butoxycarbonylamino]caproic acid (5) (45 mg, 0.19 mmol, 48%)
Reagents
(D12)Cyclohexanol (98+ atom % D) was purchased from Aldrich
(St Louis, MO, U.S.A.) and used without further purification.
Deuterated solvents were used throughout the synthesis to prevent
deuterium–hydrogen exchange. All were purchased from Aldrich.
BOC-ON, 1,3-dicyclohexylcabodiimide (DCC), 4-dimethylamino-
pyridine (DMAP) and alumina were also purchased from Aldrich
(St Louis, MO, U.S.A.). Gramicidin D (gD) was obtained from Sigma
2
as a yellow oil. Mass spectrum m/z 241 [MH+]. H NMR (CDCl3) δ
3.08, m, 2D; 2.32, m, 2D; 1.60, m, 2D; 1.46, m, 2D; 1.34, m, 2D. 13
C

750
A. Anastasiadis, F. Separovic and J. White
NMR δ 174.2, CO2H; 150.05, CO; 85.87, C(CH3)3; 34.8, CCO; 27.7,
column eluted with dichloromethane/methanol/water (800 : 100 : 8) to
afford a major fraction of [6-(aminocaproyl)-N-biotinyl(D10)(amino-
caproyl)]gramicidin (gAXDXB) (8) (6.3 mg, 2.69 × 10–3 mmol, 39%),
RF 0.35. MALDI MS 2346. 1H NMR (CD3OD) δ 0.3–1.8, m, 80H; 2.05,
m, 4H; 2.05–2.20, m, 8H; 2.78, d, 1H; 2.90, dd, 1H; 3.0–3.7, m, 19H;
3.88, dd, 2H; 3.90, s, 2H; 3.9–4.8, m, 16H; 6.8–7.6, m, 20H; 8.15, s, 1H.
(CH3)3; 20.9, 19.4, 18.2, CD2.
[6-[N-(tert-Butoxycarbonylamino)](D10)caproyl]gramicidin (6)
gA was isolated from the natural mixture gD using a flash silica column
eluted with dichloromethane/methanol/water (800 : 60 : 4), RF 0.6
(modified procedure).[12,15] TLC plates were developed in
dichloromethane/methanol/water/acetic acid (400 : 60 : 4 : 4) and
visualized with the tryptophan reagent, N,N-dimethylamino-
benzaldehyde.[16] To the purified gA (22 mg, 0.02 mmol) was coupled
6-[N-(tert-butoxycarbonylamino](D10)-6-caproic acid (19.6 mg,
0.07 mmol) and DMAP (3 mg, 0.02 mmol). Dry dichloromethane
(5 mL) and DCC (13 mg, 0.06 mmol) were added.[15] The solvent was
evaporated, then the crude product was dissolved in methanol and
passed down a Sephadex LH20 column. The eluent was dried and then
Acknowledgments
We gratefully acknowledge Dr Chris Burns and Doreen Bali,
Ambri Pty Ltd, for their helpful discussions.
References
[1] B. A. Cornell, V. L. B. Braacch-Maksvytis, L. G. King, P. D. J.
Osman, B. Raguse, L. Wieczorek, R. Pace, Nature 1997, 387,
580.
further purified on
dichloromethane/methanol/water (800 : 60 : 6) to afford
a
flash silica column and eluted with
major
a
fraction of [6-[N-(tert-butoxycarbonylamino)](D10)caproyl]gramicidin
(6) (15 mg, 7.07 × 10–3 mmol, 36%), RF 0.44. 1H NMR (CD3OD) δ 0.3–
1.8, m, 72H (note: a sharp singlet at 1.5 from BOC methyl 1H was
difficult to integrate due to overlap of aliphatic proton resonances from
gA); 2.05–2.40, m, 9H; 3.0–3.7, m, 16H; 3.8–4.8, m, 14H; 6.8–7.6, m,
20H; 8.15, s, 1H.
[2] T. I. Rokitskaya, Y. N. Antonenko, E. A. Kotova, A. J.
Anastasiadis, F. Separovic, Biochemistry 2000, 39, 13053.
[3] T. C. B. Vogt, J. A. Killian, R. A. Demel, B. De Kruijff, Biochim.
Biophys. Acta 1991, 1069, 157.
[4] T. C. B. Vogt, J. A. Killian, B. De Kruijff, Biochemistry 1994, 33,
2063.
[5] Y. Cheng, W. Liu, S. Chen, Synthesis 1980, 3, 223.
[6] J. March, Advanced Organic Chemistry 1968, pp. 213–216
(McGraw Hill: New York).
(N-6-Amino(D10)caproyl)gramicidin (gAXD) (7)
[6-[N-(tert-Butoxycarbonylamino)](D10)caproyl]gramicidin (6) was
deprotected using trifluoroacetic acid (3 mL) and the solution was
evaporated to dryness and dried under high vacuum to yield
(N-6-amino(D10)caproyl)gramicidin (gAXD) (7). 1H NMR (CD3OD) δ
0.3–1.8, m, 72H; 2.05–2.40, m, 9H; 3.0–3.7, m, 16H; 3.8–4.8, m, 14H;
6.8–7.6, m, 20H; 8.15, s, 1H.
[7] L. Semon, Org. Synth. 1941, 1, 318.
[8] J. C. Eck, C. S. Marvel, Org. Synth. 1943, 2, 76.
[9] J. C. Eck, C. S. Marvel, Org. Synth. 1943, 2, 371.
[10] J. C. Eck, Org. Synth. 1960, 4, 39.
[11] I. Masumi, H. Daijiro, K. Takashi, Tetrahedron Lett. 1975, 49,
4393.
[12] W. R Veatch, E. Fossel, E. R. Blout, Biochemistry 1974, 13, 5249.
[13] F. Separovic, S. Barker, M. Delahunty, R. Smith, Biochim.
Biophys. Acta 1999, 1416, 48.
[14] F. Separovic, T. Gehrmann, T. Milne, B. A. Cornell, S. Y. Lin, R.
Smith, Biophys. J. 1994, 67, 1495.
[15] D. Bali, L. King, S. Kim, Bioconjugate Chem. unpublished
results.
[16] J. P. Greenstein, M. Winitz, Chemistry of the Amino Acids 1961,
Vol. 3, p. 2323 (John Wiley: New York).
[6-(Aminocaproyl)-N-Biotinyl(D10)(aminocaproyl)]gramicidin
(gAXDXB) (8)
To gAXD (7) (15 mg, 7.07 × 10–3 mmol) was added dichloromethane/
methanol (2 : 1) (2 mL) and biotinamidocaproate-N-hydroxysucci-
nimide (8.4 mg, 0.02 mmol). The reaction mixture was stirred overnight
at room temperature. The solvent was evaporated, and then the crude
product was dissolved in methanol and passed down a Sephadex LH20
column. The eluent was dried and then further purified on a flash silica