Synthesis of a stable-isotope-labeled biotinylated pentasaccharide conjugate (EP217609), a dual-effect anticoagulant drug

Article abstract of DOI:10.1002/jlcr.1900

EP217609 is a neutralizable dual-effect anticoagulant under clinical investigation in cardiopulmonary bypass during cardiac surgery. Stable-isotope-labeled EP217609 was synthesized as an internal standard for mass spectrometry in support of bioassays. EP2

Full text of DOI:10.1002/jlcr.1900

Research Article
Received 11 January 2011,
Revised 28 March 2011,
Accepted 20 April 2011
Published online 11 August 2011 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1900
Synthesis of a stable‐isotope‐labeled
biotinylated pentasaccharide conjugate
(EP217609), a dual‐effect anticoagulant drug
a
a
b
*
Sébastien Richard, Ahmed El Hadri, Marcel Schreuder Goedheijt,
Michel Heisen,b Jan Vader,b Peter Wiegerinck,b and Maurice Petitoua
EP217609 is a neutralizable dual‐effect anticoagulant under clinical investigation in cardiopulmonary bypass during cardiac
surgery. Stable‐isotope‐labeled EP217609 was synthesized as an internal standard for mass spectrometry in support of
bioassays. EP217609 was obtained in six steps starting from three building blocks in an overall yield of 42%, with a chemical
purity of >99%. Thus, coupling between the N‐protected labeled biotin‐lysine 4, prepared in three steps from [13C,15N]‐L‐
lysine 2, and the pentasaccharide‐spacer‐amine 6 was first performed. Removal of the Cbz protective group to 8 followed by
coupling of the activated peptidomimetic building‐block 10 gave the immediate precursor of EP217609, which could be
obtained after catalytic hydrogenolysis of 1,2,4‐oxadiazol‐5(2H)‐one into amidine.
Keywords: antithrombotic; anticoagulant; isotope labeling; biotin; labeled lysine; pentasaccharide; heparin mimetic
Other considerations are less relevant, such as improving the
signal‐to‐noise ratio as the same signal‐to‐noise ratio can be
Compounds labeled with stable isotopes are commonly used as
obtained by the addition of more SIL compounds to the biological
Introduction
internal standards for mass spectroscopy (MS) studies of
biological fluids during development of new drugs, wherein
sample. Among the stable isotopes used, C‐13 and N‐15 are
preferred by bioanalysts because the labeled molecules seldom, if
ever, fractionate from their unlabeled isotopomers under typical
chromatographic conditions, in contrast to H‐2‐labeled com-
using isotopically labeled compound allows a more accurate
determination of the concentration of the drug.1–11 The stable
isotope labels used in these studies are typically C‐13, N‐15, and
H‐2. The oxygen isotope O‐18 is seldom used due to the
propensity of oxygen atoms to exchange with water; however,
pounds, which can have different retention times in LC if the label
is not properly located.13 This effect can be minimized by
avoiding placing the deuterium label within two bonds of
nitrogen.13 The H‐2 label must also be placed in a location that
in a few elegant studies, O‐18‐labeled compounds have been
utilized for mechanistic elucidation.12 The stable‐isotope‐labeled
is nonexchangeable (e.g. not alpha to a carbonyl).
(SIL) compound is generally used as an internal standard to
quantitate the parent compound in biological matrixes. A known
amount of the SIL drug is added to the biological sample, and
after processing to prepare the sample for liquid chromatography‐
MS (LC‐MS), the ratio of SIL to unlabeled drug is determined. As by
adding the SIL compound before processing, the loss of unlabeled
compound due to these manipulations is accounted for, this ratio
allows the calculation of the amount of unlabeled drug present in
the biological sample.
A number of precautions have to be taken when designing an
SIL version of a drug candidate. These particularly deal with the
location of the label in the structure and the difference in atomic
mass. Concerning the location of the label in the structure, as the
molecule will be studied in LC‐MS/MS, it is necessary to have the
isotopic label in the daughter fragment. The minimum mass
increase required for pentasaccharide analogs to achieve appro-
priate resolution is 6, while 8 or more is preferred. The isotopes
incorporated are not limited to one or two elements. A key
consideration and requirement of the labeling synthesis is that
the isotopically labeled material must not contain (or only very
low levels in the range of<0.1%) any unlabeled material.
Although the starting materials for preparing SIL compounds
are expensive, a wide range is commercially available. The
synthesis of SIL compounds is very similar to unlabeled synthesis
with a few notable exceptions. The synthesis often needs to be
altered from the previously developed routes to allow for late‐
stage incorporation of isotope labeling into the target molecule.
This allows for a higher chemical yield because of fewer reaction
steps and generates less label material waste.
In this article, we report the synthesis and characterization of
(13C,15N)‐labeled EP217609, a dual‐action anticoagulant, which
contains a pentasaccharide analog, fondaparinux, covalently
bound to a peptidomimetic thrombin inhibitor and to a biotin
aEndotis Pharma, Biocitech, 102 Avenue Gaston Roussel, 93230 Romainville,
France
bMSD, Department of Process Chemistry, Molenstraat 110, 5340 BH Oss, the
Netherlands
*Correspondence to: Dr. Ahmed El Hadri, Chemistry Department, Endotis Pharma,
102 Avenue Gaston Roussel, 93230 Romainville, France.
E‐mail: ahmed.elhadri@endotis.com
J. Label Compd. Radiopharm 2011, 54 637–644
Copyright © 2011 John Wiley & Sons, Ltd.

S. Richard et al.
entity.14,15 This new anticoagulant drug is currently under clinical EP217609. All reactions were carried out under an atmosphere of
trial in cardiopulmonary bypass during cardiac surgery, a clinical nitrogen unless stated otherwise. All compounds were homo-
setting requiring a potent, rapidly neutralizable anticoagulant. The geneous by thin layer chromatography (TLC) and had spectral
fast neutralization of the anticoagulant activity of EP217609 is properties consistent with their assigned structures. Purifications
carried out by injection of avidin, a protein displaying high affinity in organic solvents were performed by flash column chromato-
for biotin (Kd ~1015 M−1).
graphy on a Merck cartridge (GX0171511110LK [554–3646]; EVF
D17, Si60 15–40 µm; 10 g) using the Isolera™ flash purification
system (Biotage). Gel‐permeation chromatography with aqueous
eluents was performed using Sephadex G‐25 (GE Healthcare).
Compound purity was checked by TLC on silica gel 60 F254
(E. Merck) with detection by charring with sulfuric acid. The chem-
ical purity of all labeled compounds was determined by HPLC
and ESI‐TOF or LC‐MS. 1H‐ and 19F‐NMR spectra were recorded on
a Brucker 400‐MHz instrument; chemical shifts were expressed
relative to internal tetramethylsilane (TMS; spectra recorded in
organic solvents) or trimethylsilyl propionate (TSP; spectra
recorded in D2O) unless stated otherwise. MS analyses were
performed on a QSTAR® instrument 40 MCA Scans (MDS‐Sciex and
Applied Biosystems Inc.). Before analysis in D2O, samples were
passed through a Chelex (Bio‐Rad) ion exchange column and
lyophilized three times from D2O. All prepared SIL compounds
contained no unlabeled material by ESI‐TOF MS analysis.
Results and discussion
On the basis of the molecular weight of EP217609 and its MS
spectrum, it was concluded that the minimum atomic molecular
mass increase required was at least 6 in order to avoid any
interference with the unlabeled material. Considering the
structure, it is particularly attractive to use a commercially
available (13C,15N) lysine that allows adding 8 amu (atomic mass
units) at the very end of the building block assembly process.
EP217609 was originally synthesized in about 60 steps by De
Kort and Van Boeckel.15 In this route, the final coupling step was
performed between the pentasaccharide building block 6 and a
building block containing the peptidomimetics and the biotin
entity (Scheme 1). As a small amount of undesired racemisation
product was formed at the lysine alpha‐carbon during the late
step of this coupling reaction, a new approach to the synthesis of
isotopically labeled EP217609 has been explored (Schemes 2–4),
in which the final coupling involves the activation of a carboxylate
function in alpha‐position to a methylenic carbon.
HPLC analysis
HPLC was performed on a Waters 600 Controller in combination
In the first step, [13C,15N]‐L‐lysine 2 was treated with p‐ with a Waters 2996 dual absorbance detector, set at 200 to
anisaldehyde to form a Schiff base. Addition of benzyloxycarbonyl 400 nm. An Altima 55–45 C18 (250 × 4.6 mm, 5 µm) column was
chloride in the presence of sodium hydroxide followed by direct used, and chromatograms were processed using MassLynx Soft.
hydrolysis afforded Cbz‐Lys‐OH 3 in a 78% yield (Scheme 2). The following HPLC conditions were used: mobile phase A,
Coupling between the activated D‐(+)‐biotin 1 and 3 was carried 25 mM ammonium acetate; mobile phase B, 25 mM ammonium
out in dimethylformamide (DMF) to afford the desired 4 in a acetate in MeOH. Elution was carried out at 0.90 ml/min.
practically quantitative yield.
The building block 4 was coupled to the oligosaccharide‐
spacer 615 (Scheme 3) using a standard peptide coupling method
(EDCI/HOBt) to give compound 7 in 88% yield. After hydro-
genolysis, compound 8 was isolated quantitatively.
Eluent
% Ammonium acetate (A) 100
Time (min)
45 50 55 60 75
0
The peptidomimetic building‐block 916 was activated as
pentafluorophenolyl ester and coupled with 8 to give com-
pound 11 in 67% yield (Scheme 4). Deprotection of the amidine
moiety of 11 by hydrogenolysis in the presence of Pd/C afforded
EP217609 in quantitative yield.
45 40
5
55 55
% Ammonium acetate in
MeOH (B)
0
55 60 95 45 45
In summary, 13C‐ and 15N‐labeled EP217609 was synthesized
in six steps starting from 13C‐ and 15N‐labeled L‐lysine. The
overall yield of the labeling steps was 42% with a chemical
purity higher than 99% (Figure 1) as determined by HPLC. This
new approach for the synthesis of EP217609 allowed access to a
pure labeled compound without racemisation of peptide
reaction step and high isotopic enrichment.
The ESI‐MS spectrum of labeled EP217609 (Figure 2) showed
peaks corresponding to four and three multiple charged
ions identical to that observed for the unlabeled compound
with an increase of 2 and 2.6amu, respectively, for molecular ions
[M− 4H]4− and [M − 3H]3−. Interestingly, no overlapping on peaks
multiplicity of molecular ion between labeled and unlabeled
compounds was observed.
Chemistry
Nα‐Benzyloxycarbonyl‐L‐(13C6,15N2)lysine (Cbz‐Lys‐OH, 3)
To an aqueous LiOH solution (2N, 0.58 mL) at room temperature,
isotope‐labeled L‐lysine monohydrochloride (2 ; 0.2 g, 1.05
mmoL) was added, and the mixture was cooled below 4 °C. p‐
Anisaldehyde (0.16 g, 1.15 mmoL) was added in three portions of
about 50 mg each. The reaction mixture was stirred for 3 h,
maintaining the temperature below 4 °C; then the thick paste
was kept overnight below 5 °C. After filtration and washing with
cold acetonitrile (0.3 mL), the wet cake of Nε‐anisilidine‐L‐
lysine Lys[N═CH‐(p‐C6H4OMe)]‐OH was used in the next step
without further purification. It was suspended at 0 °C in a 1N
aqueous NaOH/ethanol mixture [1:1 (v/v), 1.9 mL], cooled to
−20 °C, and benzyloxycarbonyl chloride (0.64 g, 3.75 mmoL) and
a cold 1N aqueous NaOH/ethanol solution [2:2.5 (v/v), 2 mL]
were then added in portions while the temperature of the
Experimental
General
Labeled L‐lysine was purchased from Sigma‐Aldrich. The inter- reaction mixture was maintained at −20 °C. The reaction mixture
mediates 5 and 9 were obtained from MSD, the manufacturer of was stirred for about 2 h, allowing the temperature to rise to
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Copyright © 2011 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2011, 54 637–644

S. Richard et al.
Scheme 1. Original synthesis of unlabeled EP217609.15
about −5 °C. The pH of the reaction mixture was then adjusted white suspension. Nα‐Benzyloxycarbonyl‐L‐lysine 3 (0.2 g, 0.69
to 2 by adding concentrated HCl (0.23 mL) to hydrolyze the mmoL) and diisopropyl‐ethylamine (0.73 mL, 4.16 mmoL, 6
intermediate Schiff base. Ethanol was then evaporated at around equivalents) were added, and the reaction mixture was stirred
50 °C, and the aqueous phase was then washed with dichlor- overnight at room temperature. After 20 h, TLC analysis
omethane (4 × 5 ml) and concentrated. The pH was then (dichloromethane/methanol 9:1) of the yellow solution indi-
adjusted to 6.5–7 using aqueous 1N NaOH, and the solution cated a complete reaction. Water (3 mL) was added, and the
was concentrated under vacuum. Purification on silica gel reaction mixture was stirred for 4 h at room temperature (slight
column using dichloromethane/methanol (9:1) as eluent af- rise in temperature after addition water). The white precipitate
forded 3 (0.21 g, 78%). 1H NMR (400 MHz, D2O) δ 1.13–1.88 was filtered, washed twice with water (1 mL) and twice with
(m, 6 H), 2.82 (m, 2 H), 3.97 (m, 1 H,), 4.98 (s, 2 H), 4.99 (d, 1 H, ether (2 mL), and dried under vacuum at 40 °C to afford 4
J = 24 Hz, NH‐CO‐O), 7.29 (m, 5 H).
(0.32 g, 95%). LC‐MS (ESI): m/z 515.3, purity 99%; 1H NMR
(400 MHz, DMSO‐d6) δ 1.13–1.61 (m, 12H), 2.04 (t, 2 H, J = 8.0 Hz),
2.57 (d, 1 H, J = 12.0 Hz), 2.82 (m, 2 H), 3.08 (m, 1 H), 4.10 (m, 3 H),
Nα‐Benzyloxycarbonyl‐[D‐(+)‐biotinyl]‐L‐(13C6,15N2)lysine (4)
In a 50‐mL round‐bottomed flask, compound 1 (0.23 g, 0.66 4.30 (m, 1 H), 5.03 (s, 2 H), 6.35 (bs, 1 H), 6.42 (bs, 1 H), 7.34 (m,
mmoL) was suspended in 6 mL of anhydrous DMF to give a 5 H), 7.63 (bs, 1 H), 7.86 (bs, 1 H).
J. Label Compd. Radiopharm 2011, 54 637–644
Copyright © 2011 John Wiley & Sons, Ltd.
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S. Richard et al.
Scheme 2. (a) p‐Anisaldehyde, 2N LiOH, 4 °C, overnight, not isolated; (b) 1N NaOH/EtOH, Cbz‐Cl, −20 °C to −5 °C, conc. HCl, 78% over a and b; (c) DIEA, DMF, 95%.
Methyl O‐2,3‐di‐O‐methyl‐4‐O‐(12‐aza‐3,6,9‐trioxa‐dodecyl)‐6‐O‐
sulfo‐alpha‐D‐glucopyranosyl‐(L‐>4)‐O‐2,3‐di‐O‐methyl‐beta‐D‐
glucopyranuronosyl‐(L‐>4)‐O‐2,3,6‐tri‐O‐sulfo‐alpha‐D‐
glucopyranosyl‐(L‐>4)‐O‐2,3‐di‐O‐methyl‐alpha‐L‐
idopyranuronosyl‐(L‐>4)‐3‐O‐methyl‐2,6‐di‐O‐sulfo‐alpha‐D‐
glucopyranoside octakis sodium salt (6)
3.67–3.86 (m, 4 H), 3.92–4.01 (m, 2 H), 4.07–4.28 (m, 6 H), 4.38–4.52
(m, 2 H), 4.90 (s, 2 H), 4.91–5.04 (m, 2 H), 5.22 (d, 1 H, J = 4.0Hz), 5.30
(d, 1 H, J = 4.0Hz), 7.28 (m, 5 H).
Methyl O‐2,3‐di‐O‐methyl‐4‐O‐(12‐[{Nε‐(D‐(+)‐biotinyl)}‐L‐
(13C6,15N2)lysyl]‐aza‐3,6,9‐trioxa‐dodecyl)‐6‐O‐sulfo‐alpha‐D‐
glucopyranosyl‐(L‐>4)‐O‐2,3‐di‐O‐methyl‐beta‐D‐
To a solution of compound 5 (0.15 g, 0.077 mmoL) in water glucopyranuronosyl‐(L‐>4)‐O‐2,3,6‐tri‐O‐sulfo‐alpha‐D‐
(10 mL) was added 10% Pd/C (0.07 g), and the mixture was glucopyranosyl‐(L‐>4)‐O‐2,3‐di‐O‐methyl‐alpha‐L‐
stirred at room temperature under a continuous stream of idopyranuronosyl‐(L‐>4)‐3‐O‐methyl‐2,6‐di‐O‐sulfo‐alpha‐D‐gluco
hydrogen. After 4 h, the catalyst was removed by filtration and pyranoside octakis sodium salt (8)
the filtrate was concentrated. Lyophilisation gave pure 6
To a solution of 7 (0.088 g, 0.038 mmoL) in water (8 mL) was
(0.138 g, 100%), which was used for the next step without
added 10% Pd/C (0.04 g), and the mixture was stirred at room
further purification.
temperature under a continuous stream of hydrogen. After 4 h,
the catalyst was removed by filtration, and the filtrate was
Methyl O‐2,3‐di‐O‐methyl‐4‐O‐(12‐[Nα‐benzyloxycarbonyl‐{Nε‐(D‐
concentrated and lyophilized to give 8 (0.082 g, 96%). ESI‐MS: m/
(+)‐biotinyl)}‐L‐(13C6,15N2)lysyl]‐aza‐3,6,9‐trioxa‐dodecyl)‐6‐O‐sulfo‐
z calcd for C62H104N5O52S7, 660.7955; found, 660.8155 [M − 3H]3
;
J = 7.2 Hz), 2.62 (d, 1 H, J = 15.6 Hz), 2.85 (m, 2 H), 3.07–3.66 (m,
58 H), 3.72–3.87 (m, 4 H), 3.94–4.02 (m, 2 H), 4.07–4.19 (m, 4 H),
4.26 (m, 2 H), 4.45–4.52 (m, 2 H), 4.90 (d, 1 H, J = 4.0 Hz), 5.13 (s,
1 H), 5.23 (d, 1 H, J = 4.0 Hz), 5.31 (d, 1 H, J = 4.0 Hz).
alpha‐D‐glucopyranosyl‐(L‐>4)‐O‐2,3‐di‐O‐methyl‐beta‐D‐
−
1H NMR (400 MHz, D2O) δ 1.02–1.68 (m, 12 H), 2.10 (t, 2 H,
glucopyranuronosyl‐(L‐>4)‐O‐2,3,6‐tri‐O‐sulfo‐alpha‐D‐
glucopyranosyl‐(L‐>4)‐O‐2,3‐di‐O‐methyl‐alpha‐L‐
idopyranuronosyl‐(L‐>4)‐3‐O‐methyl‐2,6‐di‐O‐sulfo‐alpha‐D‐
glucopyranoside octakis sodium salt (7)
To a suspension of compound 4 (0.039 g, 0.075 mmoL) in
anhydrous DMF (1 mL) in a 5‐mL round‐bottomed flask under
nitrogen, 1‐hydroxybenzotriazole (0.011 g, 0.079 mmoL) and 1‐
4‐[4‐[[(1R)‐1‐[[4‐(1,2,4‐Oxadiazol‐5(2H)‐one)‐phenyl]methyl]‐2‐
oxo‐2‐(1‐piperidinyl) ethyl]amino]‐3‐[[(4‐methoxy‐2,3,6‐
trimethylphenyl)sulfonyl]amino]‐1,4(S)‐dioxobutyl] amino]‐
butanoic acid pentafluorophenyl ester (10)
(3‐dimethylaminopropyl)‐3‐ethylcarbodiimide
hydrochloride
(0.015 g, 0.079 mmoL) were added, and the mixture was stirred
for 1 h at room temperature. A solution of compound 6 (0.09 g, To a solution of 9 (0.15 g, 0.206 mmoL) in anhydrous DMF (1.5mL)
0.05 mmoL) in anhydrous DMF (3 mL) was added dropwise, and in a 5‐mL round‐bottomed flask under nitrogen, 2,3,4,5,6‐
the mixture was stirred for 2 h at room temperature. TLC pentafluorophenol (0.042 g, 0.226 mmoL) and 1‐(3‐dimethylami-
(spraying with 10% H2SO4 in ethanol) and HPLC analyses nopropyl)‐3‐ethylcarbodiimide hydrochloride (0.051 g, 0.268
showed complete reaction. The mixture was poured into ethyl mmoL) were added, and the mixture was stirred for 16h at room
acetate (2 mL), stirred for 15 min at room temperature, and temperature. TLC and HPLC analyses showed complete reaction.
filtered. The cake was washed twice with 2 mL of ethyl acetate Water (2 mL) was added to the mixture, and 10 was extracted
dissolved in water and desalted by passing through Sephadex with CH2Cl2 (3 × 15 mL). The organic layer was concentrated and
G‐25 to afford 7 (0.101 g, 88%) in 93% purity; ESI‐MS: m/z calcd purified on a silica gel column to afford 10 (0.152 g, 87%). 1H NMR
for C70H109N5O54S7, 528.8558; found, 528.8385 [M − 4H]4−; calcd (400MHz, CDCl3) δ 1.60–1.69 (m, 6 H), 1.86 (m, 2 H), 1.99 (t, 2 H,
for C70H110N5O54S7, 705.4745; found, 705.4567 [M − 3H]3− 1H J = 8.0Hz), 2.15 (s, 3 H, Me), 2.57 (s, 3 H, Me), 2.67 (s, 3 H, Me), 2.76
;
NMR (400 MHz, D2O) δ 1.01–1.58 (m, 12 H), 2.08 (t, 2 H, J = 7.0 Hz), (t, 2 H, J = 8.0Hz), 2.82 (m, 1 H), 3.06 (dd, 1 H, J = 20.0 and 4.0 Hz),
2.58 (d, 1 H, J = 12.0 Hz), 2.80 (m, 2 H), 3.07–3.64 (m, 58 H), 3.42 (dd, 2 H, J = 16.0 and 8.0Hz), 3.61 (m, 4 H), 3.88 (s, 3 H, OMe),
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J. Label Compd. Radiopharm 2011, 54 637–644

S. Richard et al.
Scheme 3. (a) H2, 10% Pd/C, H2O, room temperature, 4 h, 100%; (b) EDCI/HOBt, DMF, room temperature, 2 h, 88%; (c) H2, 10% Pd/C, H2O, room temperature, 4 h (96%).
4.12 (m, 1 H), 5.20 (m, 1 H), 5.34 (s, 2 H), 6.50 (bs, 1 H), 6.61 (s, 1 H), at 40 °C for 16 h. HPLC and TLC analyses showed complete
7.12 (bs, 1 H), 7.30 (d, 2 H, J= 12.0 Hz), 7.55 (d, 2 H, J = 12.0 Hz). 19F reaction. The mixture was poured into ethyl acetate (10 mL) and
NMR (CDCl3, 400 MHz) δ −162.4 (dt, 2 F, J = 28.57 and 3.2 Hz), stirred for 10 min at room temperature. After filtration and
−158.1 (t, 1 F, J = 28.57 Hz), −152.6 (dd, 2 F, J = 28.57 and 3.2 Hz).
washing with ethyl acetate (2 mL), the solid was dissolved in
water and desalted by passage through Sephadex G‐25 to afford
11 (0.067 g, 67%) as a white solid. HPLC purity 92%; ESI‐MS: m/z
Methyl O‐2,3‐di‐O‐methyl‐4‐O‐«<12‐N‐«Nε‐(D‐(+)‐biotinyl)‐N‐<)]‐
{4‐[[4‐[[(1R)‐L‐[(4‐[1,2,4‐oxadiazol‐5(2H)‐one]‐phenyl)methyl]‐2‐
oxo‐2‐(L‐piperidinyl)ethyl]amino]‐3‐[[(4‐methoxy‐2,3,6‐
trimethylphenyl)sulfonyl]amino]‐l,4‐(S)‐dioxo‐butyl]amino]‐
butanoyl}‐L‐(13C6,15N2)lysyl»‐12‐aza‐3,6,9‐trioxa‐dodecyl>»‐6‐O‐
sulfo‐alpha‐D‐glucopyranosyl‐(L‐>4)‐O‐2,3‐di‐O‐methyl‐beta‐D‐
glucopyranuronosyl‐(L‐>4)‐O‐2,3,6‐tri‐O‐sulfo‐alpha‐D‐
glucopyranosyl‐(L‐>4)‐O‐2,3‐di‐O‐methyl‐alpha‐L‐
calcd for C96H145N11O61S8, 672.9150; found, 673.1746 [M − 4H]4−
;
1H NMR (400 MHz, D2O) δ 1.01–1.73 (m, 20 H), 2.04 (s, 3 H), 2.08
(t, 2 H, J = 8.0 Hz), 2.19 (m, 2 H), 2.25 (m, 2 H), 2.41 (s, 3 H), 2.51 (s,
3 H), 2.64 (m, 2 H), 2.70–2.99 (m, 5 H), 3.10–3.68 (m, 57 H), 3.75–
3.83 (m, 8 H), 3.91 (m, 2 H), 4.02–4.10 (m, 3 H), 4.17–4.34 (m, 7 H),
4.46 (m, 2 H), 4.58 (m, 1 H), 4.70 (m, 1 H), 4,81 (m, 1 H), 4.98 (m,
1 H), 5.30 (m, 1 H), 5.37 (d, 1 H, J = 4.0 Hz), 6.75 (s, 1 H), 7.19 (d, 2 H,
J = 8.0 Hz), 7.65 (d, 2 H, J = 8.0 Hz).
idopyranuronosyl‐(L‐>4)‐3‐O‐methyl‐2,6‐di‐O‐sulfo‐alpha‐D‐
glucopyranoside octakis sodium salt (11)
Methyl O‐2,3‐di‐O‐methyl‐4‐O‐«<12‐N‐«Nε‐(D‐(+)‐biotinyl)‐N‐<)]‐
To a solution of 10 (0.051 g, 0.057 mmoL) in 4 mL of anhydrous {4‐[[4‐[[(1R)‐L‐[[4‐ (aminoiminomethyl)phenyl]methyl]‐2‐oxo‐2‐
DMF, in a 5‐mL round‐bottomed flask, compound 8 (0.076 g, (L‐piperidinyl)ethyl]amino]‐3‐[[(4‐methoxy‐ 2,3,6‐trimethylphenyl)
0.035 mmoL) and diisopropyl‐ethylamine (0.01 mL, 0.056 mmoL, sulfonyl]amino]‐l,4‐(S)‐dioxo‐butyl]amino]‐butanoyl}‐L‐(13C6,15N2)
1.6 equivalent) were added, and the reaction mixture was stirred lysyl»‐12‐aza‐3,6,9‐trioxa‐dodecyl>»‐6‐O‐sulfo‐alpha‐D‐
J. Label Compd. Radiopharm 2011, 54 637–644
Copyright © 2011 John Wiley & Sons, Ltd.
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S. Richard et al.
Scheme 4. (a) Pentafluorophenol, EDCI, DMF, room temperature, 16 h, 87%; (b) DMF, 40 °C, 16 h, 67%; (c) H2, 1% Pd/C, H2O, room temperature, 4 d (quantitative).
glucopyranosyl‐(L‐>4)‐O‐2,3‐di‐O‐methyl‐beta‐D‐
glucopyranuronosyl‐(L‐>4)‐O‐2,3,6‐tri‐O‐sulfo‐alpha‐D‐
glucopyranosyl‐(L‐>4)‐O‐2,3‐di‐O‐methyl‐alpha‐L‐
idopyranuronosyl‐(L‐>4)‐3‐O‐methyl‐2,6‐di‐O‐sulfo‐alpha‐D‐
glucopyranoside octakis sodium salt (EP217609).
To a solution of 11 (0.017 g, 0.006 mmoL) in water (2mL) was added
1% Pd/C (0.01g), and the mixture was stirred at room temperature
under a continuous stream of hydrogen for 4 days. HPLC analysis
showed complete reaction. The catalyst was removed by filtration,
and the filtrate was desalted by passage through a G‐25 Sephadex
column. The combined fractions of interest were pooled and
concentrated. Lyophilisation afforded pure EP217609 as a white
solid in quantitative yield. HPLC purity>99%; ESI‐MS: m/z calcd for
C95H147N11O59S8, 662.4214; found, 662.6471 [M − 4H]4−; calcd for
1
C95H148N11O59S8, 883.5619; found, 883.8674 [M − 3H]3−; H NMR
(400MHz, D2O) δ 1.03–1.83 (m, 19 H), 1.90 (m, 2), 2.01 (s, 3 H),
2.02–2.08 (m, 4 H), 2.20–2.32 (m, 2 H), 2.40 (s, 3 H), 2.51 (s, 3 H), 2.65
(d, 1 H, J = 17.1Hz), 2.76 (m, 1 H), 2.80–2.90 (m, 2 H), 2.92–3.07
(m, 2 H), 3.07–3.70 (m, 52 H), 3.70–3.88 (m, 8 H), 3.92 (m, 2 H),
Figure 1. HPLC profile of synthetic stable‐isotope‐labeled EP217609.
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S. Richard et al.
Figure 2. ESI‐MS spectra of unlabeled (A) and labeled EP217609 (B) with an example of expanded region of molecular ion m/z [M − 4H]4− showing peaks from 660.4432
to 662.1850 for unlabeled compound (A) and peaks from 662.3958 to 664.1537 for labeled compound (B). DBA, dibutylamine.
J. Label Compd. Radiopharm 2011, 54 637–644
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S. Richard et al.
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Acknowledgements
The authors thank Djamel Abderrahmani for mass spectrometry
and Francisco Alvarez for NMR spectroscopy.
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J. Label Compd. Radiopharm 2011, 54 637–644