Synthesis of heparin-like antithrombotics having perphosphorylated thrombin binding domains

Article abstract of DOI:10.1016/S0968-0896(99)00139-X

The synthesis of three heparin analogues (i.e. compounds VI-VIII) having perphosphorylated thrombin binding domains (TBDs) is reported. These compounds were tested in vitro for their antithrombin III (ATIII)-mediated anti-Xa and antithrombin activities. Conjugates VI and VIII show a remarkable increase in antithrombin activity compared to the structurally related conjugates with persulfated TBDs (i.e. compounds IV and V), whereas compound VII displays a diminished activity.

Full text of DOI:10.1016/S0968-0896(99)00139-X

Bioorganic & Medicinal Chemistry 7 (1999) 1881±1890
Synthesis of Heparin-Like Antithrombotics Having
Perphosphorylated Thrombin Binding Domains
Rogier C. Buijsman, a Jan E. M. Basten, b Cornelia M. Dreef-Tromp, b
Gijsbert A. van der Marel, a Constant A. A. van Boeckel b and Jacques H. van Boom a,*
aLeiden Institute of Chemistry, Gorlaeus Laboratories, PO Box 9502, 2300 RA, Leiden, The Netherlands
bLead Discovery Unit, N.V. Organon, PO Box 20, 5340 BH, Oss, The Netherlands
Received 17 November 1998; accepted 15 March 1999
AbstractÐThe synthesis of three heparin analogues (i.e. compounds VI±VIII) having perphosphorylated thrombin binding
domains (TBDs) is reported. These compounds were tested in vitro for their antithrombin III (ATIII)-mediated anti-Xa and
antithrombin activities. Conjugates VI and VIII show a remarkable increase in antithrombin activity compared to the structurally
related conjugates with persulfated TBDs (i.e. compounds IV and V), whereas compound VII displays a diminished activity. # 1999
Elsevier Science Ltd. All rights reserved.
Introduction
designated as the ATIII binding domain (ABD), after
which thrombin interacts with a remote thrombin bind-
ing domain (TBD) of heparin. Compared to the highly
speci®c binding of the ABD of heparin with ATIII the
interaction between thrombin and the TBD of heparin
is less speci®c in nature.12 The latter phenomenon was
nicely illustrated13,14 by the fact that synthetic glyco-
conjugates containing persulfated cellobiose and mal-
totriose as TBD (i.e. compounds IV and V, Fig. 1) show
a signi®cant antithrombin activity (Table 1). These
heparin analogues could be obtained by attachment of a
TBD to the non-reducing end of an ABD via a spacer
spanning a length of about 50 atoms (Fig. 2).11,12
For more than half a century the sulfated glycosami-
noglycan heparin has been used for the prophylaxis and
treatment of thrombotic disorders.1 Heparin enhances
the inhibitory potency of the plasma protein ATIII
towards factor Xa and thrombin, two essential serine
proteases in blood coagulation.2
It is well recognized that ATIII undergoes a conforma-
tional change3±5 upon binding with a unique penta-
saccharide (PS) domain of heparin6,7 (synthetic
counterpart: compound I, Fig. 1). This conformational
transition is sucient to promote the inhibition of factor
Xa, while the activity of thrombin remains una€ected.
Studies towards the structure±activity relationships
(SAR)8,9 of the PS domain not only led to more potent
analogues, but also to easily accessible O-sulfated/
O-methylated PS analogues (e.g. compound II, Fig. 1).
In a recent paper15 it was reported that a T18-oligo-
deoxynucleotide (T18-ODN) could also function as
TBD (PS-ODN conjugate III, Figs 1 and 2). The latter
result was an incentive to explore in which way phos-
phorylated TBDs in¯uence the pharmacological prop-
erties of the heparin analogues compared to their
sulfated counterparts. In order to evaluate this e€ect
conjugates containing perphosphorylated cellobiosyl
and maltotriosyl moieties (i.e. conjugate VI and VIII,
respectively, Figs 1 and 2) were prepared. In addition, it
was anticipated that introduction of lipophilic groups in
the TBD may facilitate interaction with thrombin as it
was found that in case of the ABD O-alkylated analo-
gues interact more strongly with ATIII than their nat-
ural counterparts.8 To this end, a glycoconjugate was
synthesized bearing stable isopropyl phosphodiesters on
its TBD (i.e. compound VII).
In order to enhance thrombin inhibition, heparin frag-
ments should comprise in addition to the PS domain at
least 10 to 12 consecutive saccharide units to allow for-
mation of a ternary complex with ATIII and thrombin
as depicted in Figure 2.10,11 During this formation
ATIII ®rst strongly binds to the PS domain of heparin,
Key words: Antithrombotics; enzyme inhibitor; heparin; oligo-
saccharides; thrombin.
* Corresponding author. Tel.: +31-715-274-274; fax: +31-715-274-
307; e-mail: j.boom@chem.leidenuniv.nl
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00139-X

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R. C. Buijsman et al. / Bioorg. Med. Chem. 7 (1999) 1881±1890
Figure 2. Schematic representation of the ternary complex of throm-
bin and ATII with heparin, the sulfated glycoconjugates, a PS-ODN
conjugate and the proposed phosphorylated glycoconjugates.
Reduction of the azido group at this stage prevented a
Staudinger reaction18 during phosphitylation of the
TBDs later on in the synthesis (Schemes 2 and 3). Thus,
treatment of compound 5 with PPh3 in pyridine and
subsequently with ammonium hydroxide (25%)19 gave
amine 6, which was protected with a benzyloxycarbonyl
(Z) group using Z-OSu in H2O/DMF under the in¯u-
ence of N,N-diisopropylethylamine (DiPEA) to yield
spacer 7 (50% overall yield from 3).
Figure 1.
Assembly of the TBDs 16a, 16b and 24
Results and Discussion
Trichloroacetimidate donor 10 (Scheme 2) was prepared
from commercially available cellobiose octaacetate (8)
by anomeric deacetylation20 and subsequent reaction of
9 with trichloroacetonitrile in the presence of cesium
carbonate.21 Trimethylsilyl tri¯uoromethanesulfonate
(TMSOTf) assisted condensation of cellobiose donor 10
with spacer 7 gave the b-linked glycoside 11 exclusively.
Unmasking the hydroxyl functions in 11 by Zemplen
deacetylation yielded partially deprotected 12. It was
anticipated that the introduction of the seven phosphate
monoesters in target TBD 16a could be accomplished
using the powerful phosphoramidite method. Indeed,
1H-tetrazole assisted phosphitylation of 12 with amidite
1322 and in situ oxidation of the intermediate phosphite-
The assembly of the target glycoconjugates VI±VIII
comprises four stages: (i) synthesis of the N-benzyloxy-
carbonyl-1-amino-1-deoxyhexaethyleneglycol spacer 7
(Scheme 1); (ii) assembly of the TBDs 16a, 16b and 24
(Schemes 2 and 3); (iii) functionalization of the TBDs
with a thiophilic iodoacetyl linker (Scheme 4); and (iv)
one-pot deprotection of known ABD 28 and conjuga-
tion of its corresponding thiol with the functionalized
TBDs (Scheme 4).
Synthesis of spacer 7
The requisite TBDs were equipped with the same
1-amino-1-deoxyhexaethyleneglycol spacer used earlier14
in the construction of glycoconjugates IV and V. Since
the reported16 four-step protocol for this type of func-
tionalized spacers requires expensive hexaethyleneglycol
as starting material, an alternative route was devised
using the cheap starting materials 1 and 3 (Scheme 1).
Tetraethyleneglycol (1) was treated with p-tosyl chloride
and a catalytic amount of 4-dimethylaminopyridine
(DMAP) in pyridine to a€ord tetraethyleneglycol di-p-
tosylate (2). Known17 2-(2-tetrahydropyran-2-yloxy-
ethanoxy)ethanol (3) was subsequently alkylated with
an excess of 2 in the presence of sodium hydride in
DMF. Reaction of the intermediate tosyl derivative 4
with lithium azide gave, after removal of the tetra-
hydropyranyl (THP) group, the azido alcohol 5.
Table 1. In vitro ATIII-mediated anti-Xa and antithrombin activites
of glycoconjugates VIÐVIII and earlier reported conjugates III±V
Compound
TBD moietya
Anti-Xa
activity
Antithrombin
activity
1
1
(U mg
)
(U mg
)
III
IV
V
VI
VII
VIII
T
18-Oligonucleotide
Cellobiose-(7-S)
173
740
490
500
690
1000
5
10
64
22
5
Maltotriose-(10-S)
Cellobiose-(7-P)
Cellobiose-(7-IP)
Moltotriose (10-P)
167
a
The number and nature of charges in the TBD is in parentheses
(S=sulfate, P=phosphate, IP=isopropyl phosphate).

R. C. Buijsman et al. / Bioorg. Med. Chem. 7 (1999) 1881±1890
1883
Scheme 1. (i) Tos-Cl, pyridine, DMAP, 16 h, 71%; (ii) THPO(CH2CH2O)2H (3), NaH, DMF, 50ꢀC, 2 h; then (iii) (a) LiN3, 80ꢀC, 16h; (b) AcOH, THF,
H2O (4:2:1, v/v/v), 4 h, 59% (three steps); (iv) PPh3, pyridine, 2 h; then 25% NH4OH, 2 h; (v) Z-OSu, DiPEA, DMF/H2O, 30 min, 85% (two steps).
Scheme 2. (i) NH2NH2, HOAc, DMF, 1 h; (ii) CCl3CN, Cs2CO3, CH2Cl2, 2 h, 87% (two steps); (iii) 7, TMSOTf, CH2Cl2, 20ꢀC, 10 min, 75%; (iv)
KOtBu, MeOH, 1 h, 88%; (v) (a) 13 (10 equiv), 1H-tetrazole, (14 equiv), CH3CN, 1 h, then tert-BuOOH, 0ꢀC, 45 min, 92%, (b) 14 (10 equiv), 1H-
tetrazole, (14 equiv), CH3CN, 1 h, then tert-BuOOH, 0ꢀC, 45 min, 58%; (vi) H2, Pd/C, tert-BuOH, H2O, 3 h, 98% (16a), 97% (16b).
triesters with tert-butylhydroperoxide proceeded
smoothly to give after work up and silica gel column
chromatography fully protected 15a. Removal of the
benzyl and the benzyloxycarbonyl protective groups by
hydrogenolysis gave TBD 16a, which was isolated as its
sodium salt (52% overall yield from 8). The presence of
the seven phosphate monoesters in 16a was ®rmly
of the resulting 22 with 13 gave after oxidation the fully
protected decakisphosphate 23. Hydrogenolysis of 23
gave the requisite TBD 24 (28% overall yield from 17).
Functionalization with thiophilic linker 25
The excellent results obtained with the iodoacetyls as
thiophilic groups in the synthesis of the earlier repor-
ted14,15 conjugates urged us to exploit sulfo-SIAB2 (25)
in the derivatization of the TBDs 16a, 16b and 24
(Scheme 4). Thus, treatment of the TBDs with 25 in a
bu€ered medium gave, after size exclusion chromato-
graphy, the iodoacetyl derivatized TBDs 26a, 26b and
27.
1
established by two-dimensional (2-D) H-31P correlated
NMR spectroscopy (Fig. 3).
Introduction of the seven isopropyl phosphodiesters in
target TBD 16b proceeded in a similar manner as
described for TBD 16a. Phosphitylation of 12 with
phosphoramidite 14 (prepared according to refs 23 and
24) under the in¯uence of 1H-tetrazole and subsequent
tert-butyl hydroperoxide-mediated oxidation of the
intermediate phosphite-triesters gave 15b. Hydro-
genolysis of all protecting groups in 15b furnished
homogeneous 16b (Na+-salt, 32% overall yield from 8).
One-pot deprotection of ABD 28 and conjugation with
functionalized TBDs
The ®nal stage in the assembly of the glycoconjugates
VI±VIII entails coupling of the ABD and TBD units. In
a one-pot procedure known ABD 2814 was deprotected
using a bu€ered solution of 0.05 M NH2OH and con-
jugated to the derivatized TBD units by forming a
stable thioether bond (Scheme 4). Traces of symmetrical
disul®de formed during the reaction were reduced with
dithiothreitol and removed from the crude mixture by
size exclusion chromatography to yield the requisite
conjugates VI±VIII in excellent purity and good yield
(51±80%). The analytical data of the conjugates (NMR
TBD 24 (Scheme 3) was prepared following the same
sequence of reactions as described for the preparation of
the TBD 16a. Commercially available maltotriose (17)
was peracetylated with acetic anhydride and a catalytic
amount of DMAP in pyridine. Anomeric deacetylation
of the resulting peracetate 18 and subsequent conversion
into the corresponding trichloroacetimidate donor 20
was followed by glycosylation with spacer 7 to give tri-
saccharide 21. Deacetylation of 21 and phosphitylation

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R. C. Buijsman et al. / Bioorg. Med. Chem. 7 (1999) 1881±1890
Scheme 3. (i) Ac2O, pyridine, DMAP, 5 h, 91%; (ii) NH2NH2, HOAc, DMF, 1 h, 92%; (ii) CCl3CN, Cs2CO3, CH2Cl2, 2 h, 71%; (iv) 7, TMSOTf,
CH2Cl2, 20ꢀC, 30 min, 56%; (v) KOtBu, MeOH, 1 h, 95%; (vi) 13 (15 equiv), 1H-tetrazole (20 equiv), dioxane:CH3CN (1:1, v/v) 2 h, then tert-
BuOOH, 0ꢀC, 45 min, 92%; (vii) H2, Pd/C, tert-BuOH, H2O, 3 h, 96%.
Scheme 4. (i) DMF, H2O, 1 h; (ii) 0.1 M aqueous Na2HPO4 (pH 7.0), 0.05 M NH2OH, 2 h (26a!VI, 68%), (26b!VII, 80%), (27!VIII, 51%).

R. C. Buijsman et al. / Bioorg. Med. Chem. 7 (1999) 1881±1890
1885
Figure 3. 1H-31P-Correlated NMR spectrum of compound 16a.
spectroscopy, mass spectrometry) were in complete
accordance with the proposed structures.
ATIII,8 but resulted in an opposite e€ect in the anity
between TBD and thrombin. Apart from this, it should
be emphasized that the thrombin inhibitory activity is
mainly governed by the charge density of the TBD, a
phenomenon also observed in other studies towards the
SAR of the TBD.14,15,27 Furthermore, the glycoconju-
gates VI and VIII are pointed out as promising antith-
rombotics.
The glycoconjugates VI±VIII were tested25,26 in vitro
for ATIII-mediated anti-Xa and antithrombin activity
and the results were compared with the conjugates IV
and V (Table 1). Glycoconjugates VI and VIII having
phosphate monoesters on their TBDs display strong
antithrombin activities, which clearly exceed those of
their counterparts with persulfated TBDs. The higher
charge densities at the TBD most likely account for the
increase in antithrombin activity. Conjugate VII, carry-
ing phosphodiesters on its TBD, shows a surprisingly
low antithrombin activity. Apparently, the bulky iso-
propyl groups on the TBD hamper an appropriate
interaction with thrombin.
Experimental
Materials and methods
1H and 13C NMR spectra were recorded with a Jeol
JNM-FX-200 (200 and 50.1 MHz, respectively). 2-D
1
1
1
(1H-1H COSY, H-1H TOCSY, H-13C COSY, H-31P
COSY) NMR spectra were recorded at 300 MHz on a
Bruker DPX-300 or at 600 MHz on a Bruker DMX-
1
600. H NMR chemical shifts in organic solvents are
Conclusion
reported in ppm (d relative to tetramethylsilane and 31P
chemical shifts are given relative to 85% H3PO4 as an
external standard. For proton spectra in aqueous solu-
tions (D2O) the residual HDO peak was set at 4.80 ppm.
Optical rotations were determined at 20ꢀC by means of
a Propol polarimeter. Mass spectra were recorded on a
Finnigan MAT TSQ-70 equipped with an electrospray
interface.
From the results presented in this paper it can be con-
cluded that the TBD of heparin shows completely dif-
ferent structure±activity relationships than the ABD of
heparin. The anity of the TBD for thrombin is
increased with the introduction of phosphate mono-
esters, whereas replacement of only one sulfate group
for a phosphate monoester in the ABD already
destroyed the anity of the ABD for ATIII.8 The
introduction of lipophilic groups, although at di€erent
positions, showed an increase in anity of the ABD for
Dichloromethane, pyridine, diisopropylamine and die-
thyl ether were re¯uxed with CaH2 for 3 h, distilled and

1886
R. C. Buijsman et al. / Bioorg. Med. Chem. 7 (1999) 1881±1890
stored over molecular sieves (4 A). N,N-Dimethylfor-
mamide was stirred with CaH2 for 16 h and then dis-
tilled under reduced pressure and stored over molecular
sieves (4 A). Acetonitrile (Rathburn, HPLC-grade), iso-
propanol (Baker, p.a.), benzylalcohol (Merck, p.a.),
tetrahydrofuran (THF, Biosolvent, HPLC-grade) were
stored over molecular sieves (4 A).
Tos, J=8.2 Hz), 4.13 (t, 4H, CH2OTos, J=4.6 Hz), 3.65
(t, 4H, CH2CH2OTos, J=4.6 Hz), 3.53 (bs, 8H, CH2O
TEG), 2.45 (s, 6H, CH3 Tos); 13C {1H} NMR (CDCl3) d
144.5 (Carom Tos), 132.5 (Carom Tos), 129.5 (CHarom
Tos), 127.3 (CHarom Tos), 69.7±68.0 (CH2O TEG), 21.0
(CH3 Tos).
1-Azido-1-deoxyhexaethylene glycol (5). Compound 3
(1.51 g, 8.0 mmol) and compound 2 (12 g, 23.8 mmol)
were dissolved in DMF (100 mL) and heated to 50ꢀC.
Sodium hydride (0.48 g, 12 mmol, 60 wt% suspension)
was added and the reaction mixture was stirred for 2 h.
After TLC analysis indicated a conversion of 2 into 4,
lithium azide (3.92 g, 80 mmol) was added and the reac-
tion temperature was elevated to 80ꢀC. After stirring for
16 h, the excess sodium hydride was quenched with
methanol (4 mL). The reaction mixture was con-
centrated and the residue was dissolved in diethyl ether
(300 mL) and washed twice with aq NaHCO3 (1 M,
2Â150 mL), dried with MgSO4, and concentrated in
vacuo. The crude 1-azido-6-tetrahydropyranyl-1-deoxy-
hexaethyleneglycol thus obtained was treated with
AcOH:THF:H2O (160 mL, 4:2:1, v/v/v). After stirring
for 4 h, TLC analysis revealed the reaction to be com-
plete and the reaction mixture was neutralized with 1 N
NaOH, concentrated to a smaller volume and extracted
three times with diethyl ether (75 mL). The combined
organic layers were dried (MgSO4) and concentrated
under reduced pressure. The residue was chromato-
graphed on silica gel (100:0 to 90:10 EtOAc:MeOH) to
Trimethylsilyl tri¯uoromethanesulfonate, dithiothreitol
(Aldrich), sodium hydride (60% dispersion in mineral
oil), hydrazine monohydrate, trichloroacetonitrile,
cesium carbonate, hydroxylamine hydrochloride, tri-
phenylphosphine (Acros), p-toluenesulfonyl chloride,
phosphor trichloride, potassium tert-butylate, tert-
butylhydroperoxide (Merck), acetic anhydride (Baker,
p.a.), N-(benzoyloxycarbonyloxy)-succinimide (Fluka)
and sulfo-SIAB2 (Pierce) were used as received.
Column chromatography was performed on Merck
Kieselgel 60 (230±400 Mesh). Gel permeation chroma-
tography was accomplished on Sephadex LH20 (Phar-
macia), Superdex 30 prep. grade (Pharmacia), Sephadex
G25 and G50 (Pharmacia). TLC analysis was per-
formed on DC Fertigfolien (Schleicher and Schull,
F1500, LS254). Compounds were visualized by UV
absorption (254 nm), exposure to sublimated iodine
crystals or charring with 20% H2SO4 in MeOH or 3%
ninhydrin in ethanol.
High performance liquid chromatography (HPLC)
(anion exchange) was conducted on a Waters 600E
(system controller) single pump gradient system using a
MonoQ prepacked HR5/5 column (Pharmacia) (anion
exchange). A Waters model 484 variable wavelength
UV detector was used for detection at 214 nm. An IBZ
Messtechnik Chiralyser was used for optical rotation
detection. Gradient elution in anion exchange was
performed at 20ꢀC by building up a gradient starting
with bu€er A 20% CH3CN in H2O and applying bu€er
B 2.0 M NaCl in 20% CH3CN in H2O at a ¯ow rate of
1 mL/min.
1
give pure 5 (1.34 g, 59% yield). H NMR (CDCl3) d
3.75±3.59 (m, 22H, CH2O HEG), 3.39 (t, 2H, CH2N3,
J=5.4 Hz); 13C {1H} NMR (CDCl3) d 72.4±69.7 (CH2O
HEG), 61.1 (CH2CH2N3), 50.3 (CH2N3).
N-Benzyloxycarbonyl-1-amino-1-deoxyhexaethylene glycol
(7). Compound 5 (1.34 g, 4.36 mmol) and triphenyl-
phosphine (1.83 g, 7.00 mmol) were dissolved in pyridine
(5 mL). After stirring for 2 h, TLC indicated a complete
conversion of the azide to the corresponding phosphi-
nimine. Concentrated NH4OH (25%, 10 mL) was then
added and the solution was allowed to stand for 2 h,
after which the reaction mixture was concentrated
under reduced pressure. The crude 6 was dissolved in
DMF (10 mL) and H2O (5 mL) and treated with N-
(benzoyloxycarbonyloxy)-succinimide (Z-OSu) (1.19 g,
4.80 mmol) and N,N-diisopropylethylamine (1.09 mL,
6.28 mmol). After 30 min, TLC analysis showed the
complete disappearance of 6. The reaction mixture was
extracted three times with ether (25 mL), dried
(MgSO4), and concentrated in vacuo. The oily residue
was puri®ed by silica gel column chromatography
(100:0 to 90:10 CH2Cl2:MeOH) to give 7 (1.53 g, 85%
yield). 1H NMR (CDCl3) d 7.34±7.28 (bs, 5H, Harom Z),
5.29 (bs, 1H, NH), 5.10 (s, 2H, CH2 Z), 3.72±3.53 (m,
22H, CH2O HEG), 3.39 (t, 2H, CH2NH); 13C{1H}
NMR (CDCl3) d 155.9 (CˆO Z), 136.1 (Carom Z),
127.7±127.3 (CHarom Z), 71.8±69.2 (CH2O HEG), 65.7
(CH2 Z), 60.7 (CH2CH2NH), 40.1 (CH2NH).
Reactions were run at ambient temperature unless
otherwise stated. Prior to reactions that require anhy-
drous conditions, traces of water were removed by
coevaporation with pyridine, toluene, dioxane or 1,2-
dichloroethane.
Tetraethyleneglycol-di-p-tosylate (2). To a stirred solu-
tion of tetraethylene glycol (1) (6.8 g, 35 mmol) in pyri-
dine (175 mL) were added p-toluenesulfonyl chloride
(20.0 g, 105 mmol) and a catalytic amount of DMAP
(0.42 g, 3.5 mmol). After 16 h, TLC analysis indicated a
complete conversion of 1 into 2. The reaction mixture
was concentrated and the residue was dissolved in
EtOAc (400 mL) and washed three times with water
(100 mL), where after the water layers were extracted
twice with EtOAc (2Â100 mL). The organic layers were
combined, dried with MgSO4 and concentrated in
vacuo. The crude product was chromatographed on
silica gel (50:50 to 30:70 light petroleum:EtOAc) to
Benzyl isopropyl N,N-diisopropylphosphoramidite (14).
To a solution of phosphor trichloride (61 mL, 0.70 mol)
in CH3CN (24 mL) was added dropwise a solution of
1
a€ord pure 2 (12 g, 71% yield). H NMR (CDCl3) d
7.76 (d, 4H, Harom Tos, J=8.2 Hz), 7.30 (d, 4H, Harom

R. C. Buijsman et al. / Bioorg. Med. Chem. 7 (1999) 1881±1890
1887
benzylalcohol (10 mL, 0.10 mol) in 40 mL of the same
solvent. After complete addition (5 min), the solution
was left at ambient temperature for 10 min. After
this period the reaction mixture was concentrated to
remove unreacted PCl3. 31P NMR showed one signal (d
177.7 ppm). Crude benzyl phosphorodichloridite was
dissolved in Et2O (75 mL) and cooled to 10ꢀC. To this
solution diisopropylamine (85 mL, 0.6 mol) was added
dropwise and the reaction mixture was stirred over-
night. 31P NMR showed the presence of one resonance
(d 123.1 ppm). The reaction mixture was diluted with
diethylether (100 mL) and washed with aq NaHCO3 (1
M, 2Â50 mL), dried with MgSO4, and concentrated
under reduced pressure to a€ord crude benzyl bis-(N,N-
diisopropyl) phosphoramidite. To a solution of iso-
propanol (2.7 mL, 35 mmol) and sym-collidine hydro-
chloride (0.32 g, 2 mmol) in CH2Cl2 (25 mL) was added
the crude bisamidite (7.5 g, 25 mmol). After 8 h, TEA
(2.5 mL) was added and the mixture was concentrated.
Puri®cation of the residue by column chromatography
(light petroleum:TEA 19:1, v/v) yielded pure 14 (2.1 g,
32% yield (3 steps)). 31P NMR (CDCl3) d 145.75; 13C
{1H} NMR (CDCl3) d 128.1±126.8 (CHarom Bn), 66.8,
66.5 (OCH(CH3)2), 65.2, 64.9 (CH2 Bn), 43.0, 42.7
(NCH(CH3)2), 24.7±24.4 (NCH(CH3)2, OCH(CH3)2).
sieves 4 A (500 mg). The solution was cooled to 20ꢀC
and a solution of trimethylsilyl tri¯uoromethane-
sulfonate (26 mL) in CH2Cl2 (1 mL) was added dropwise
to the reaction mixture. After 10 min, TLC analysis (5%
MeOH in CH2Cl2) showed the presence of one product.
Solid NaHCO3 (0.5 g) was added to the reaction mix-
ture, which was stirred for 10 min and then ®ltrated.
The ®ltrate was diluted with CH2Cl2 (50 mL), subse-
quently washed with aq NaHCO3 (1 M, 2Â25 mL),
dried (MgSO4), and concentrated in vacuo. The residue
was chromatographed on silica gel (CH2Cl2:MeOH,
100:0 to 95:5, v/v) yielding pure 11 (1.03 g, 75% yield).
1H NMR (CDCl3) d 7.44±7.41 (m, 5H, Harom Z), 5.15
(s, 2H, CH2 Z), 5.36±5.11 (m, 3H, H3, H30, H40), 5.01±
4.91 (m, 2H, H2, H20) 4.59, 4.56 d 2 (d, 2H, H1, H10,
0
0
0
J1,2=J1 ,2 =7.5 Hz), 4.61±4.16 (m, 2H, H6a, H6b, H6 a,
H60b), 3.78±3.60 (m, 25H, H4, H5, H50, CH2O HEG),
3.46 (t, 2H, CH2NH, J=5.2 Hz), 2.18±2.04 (7Âs, 21H,
CH3 Ac); 13C{1H} NMR (CDCl3) d 170.4±168.9
(7ÂCˆO Ac), 156.5 (CˆO Z), 136.8 (Carom Z), 128.4±
127.8 (CHarom Z), 100.7, 100.5 (C1, C10), 76.5, 72.9,
72.6, 72.5, 71.8, 71.6, 67.8 (C2, C20, C3, C30, C4, C40,
C5, C50), 70.5±68.9 (CH2O HEG), 66.4 (CH2 Z), 62.0,
61.6 (C6, C60), 40.9 (CH2NH), 20.8±20.2 (7ÂCH3 Ac);
ES±MS: [M+H]+ 1034.6, [M+Na]+ 1056.7, [M+K]+
1072.6.
4-O-(2,3,4,6-Tetra-O-acetyl-ꢀ-D-glucopyranosyl)-2,3,6-
tri-O-acetyl-ꢁ/ꢀ-D-glucopyranosyl trichloroacetimidate
(10). Compound 8 (1.0 g, 1.5 mmol) was treated with a
0.1 M solution of hydrazine acetate in DMF (16.8 mL,
1.68 mmol) for 1 h. TLC analysis (EtOAc:light petro-
leum, 2:1, v/v) showed the reaction to be complete.
After concentration in vacuo, the reaction mixture was
diluted with EtOAc (50 mL), washed with NaHCO3 (1
M, 3Â25 mL), dried (MgSO4), and concentrated under
reduced pressure. Column chromatography (EtOAc:
light petroleum, 2:1 to 1:1, v/v) of the residual oil gave
pure 9. Compound 9 was dissolved in CH2Cl2 (8 mL)
and trichloroacetonitrile (0.76 mL) together with a cat-
alytic amount of Cs2CO3 (99 mg, 0.30 mmol) were
added. After 2 h, TLC analysis of the reaction mixture
indicated a complete conversion of 9 into 10. The reac-
tion mixture was ®ltrated and the ®ltrate was con-
centrated under reduced pressure. Silica gel column
chromatography (EtOAc:light petroleum:TEA, 50:49:1
to 75:24:1, v/v) furnished homogeneous 10 (1.04 g, 87%
yield (two steps)). 1H NMR (CDCl3) d 8.68 (s, 1H,
NHimidate), 6.48 (d, 1H, aH1, J1,2=3.6 Hz), 5.57±4.89
N-Benzyloxycarbonyl-1-amino-1-deoxyhexaethylene glycol
4-O-cellobioside (12). Compound 11 (0.40 g, 0.39 mmol)
was treated with a solution of potassium tert-butylate
(27 mg, 10 mg per mmol Ac) in MeOH (15 mL). After
1 h, TLC analysis (EtOAc:pyridine:AcOH:water,
5:7:4:1.6, v/v/v/v) indicated a complete conversion of 11
into 12. The reaction was neutralized with Dowex 50
WX4-H+ resin. The resin was removed by ®ltration and
the ®ltrate was concentrated under reduced pressure to
a€ord 12 (0.25 g, 88% yield), which was used without
further puri®cation. 1H NMR (CD3OD) d 7.36±7.29 (m,
5H, Harom Z), 4.40 (d, 1H, H1, J1,2=7.9 Hz), 4.31 (d,
1H, H10, J1 ,2 =7.9 Hz), 3.89 (dd, 1H, H6a, J5,6a
=
=
0
0
2.5 Hz, J6a,6b=12.0 Hz), 3.86 (dd, 1H, H60a, J5 ,6 a
0
0
3.2 Hz, J6 a,6 b=11.7 Hz), 3.70±3.65 (m, 2H, H6b, H60b),
0
0
3.63±3.59 (m, 22H, CH2O HEG), 3.56 (dd, 1H, H40,
0
J3 ,4 =J4 ,5 =9.0 Hz), 3.53 (t, 2H, CH2CH2NH, J=
0
0
0
5.8 Hz), 3.51 (dd, 1H, H30, J2 ,3 =8.9 Hz), 3.40 (ddd,
0
0
1H, H50, J5 ,6 b=6.6 Hz), 3.36 (dd, 1H, H3, J2,3
J3,4=8.8 Hz), 3.33 (ddd, 1H, H5, J4,5=7.7 Hz, J5,6b
=
=
0
0
5.7 Hz), 3.31±3.28 (m, 3H, CH2NH, H4), 3.26 (dd, 1H,
H20), 3.22 (dd, 1H, H2); 13C{1H} NMR (CD3OD) d
155.9 (CˆO Z), 136.1 (Carom Z), 129.4±128.8 (CHarom
Z), 104.5, 104.1 (C1, C10), 80.6, 78.0, 77.7, 76.4, 76.2,
74.8, 74.7 (C2, C20, C3, C30, C4, C40, C5, C50), 71.4±69.6
(CH2O HEG), 67.3 (CH2 Z), 62.3±61.8 (C6, C60,
CH2CH2NH), 41.7 (CH2NH); ES±MS: [M+H]+ 740,
[M+NH4]+ 757, [M+Na]+ 763.
(m, 5H, H2, H20, H3, H30, H40), 4.55 (d, 1H, bH010,
0
0
0
J1 ,2 =7.9 Hz), 4.57±3.66 (m, 6H, H5, H5 , H6a, H6 a,
H6b, H60b), 3.85 (dd, 1H, H4, J3,4=J4,5=9.3 Hz), 2.15±
2.01 (7Âs, 21H, CH3 Ac); 13C{1H} NMR (CDCl3) d
169.9±168.5 (7ÂCˆO Ac), 160.2 (CˆNH), 100.3 (bC10),
92.3 (aC1), 90.2 (CCl3), 75.6, 72.5, 71.4, 71.2, 70.5, 69.4,
68.8, 67.3 (C2, C20, C3, C30, C4, C40, C5, C50), 61.1, 61.0
(C6, C60), 20.3±20.1 (7ÂCH3 Ac).
N-Benzyloxycarbonyl-1-amino-1-deoxyhexaethylene glycol
4-O-cellobiosyl-heptakis(dibenzyl phosphate) (15a). A
solution of 1H-tetrazole (54 mg, 0.77 mmol) in CH3CN
(1 mL) was added to a mixture of 12 (45 mg, 56 mmol)
and 13 (185 mg, 0.59 mmol) in CH3CN (2 mL). After
stirring for 1 h at 20ꢀC, the reaction mixture was cooled
with an ice bath and tert-butylhydroperoxide (0.5 mL)
N-Benzyloxycarbonyl-1-amino-1-deoxyhexaethylene glycol
4-O-(2,3,4,6-tetra-O-acetyl-ꢀ-D-glucopyranosyl)-2,3,6-
tri-O-acetyl-ꢀ-D-glucopyranoside (11). A solution of
donor 10 (1.04 g, 1.33 mmol) and acceptor 7 (0.55 g,
1.33 mmol) in CH2Cl2 (3 mL) was stirred for 1 h under a
¯ow of argon in the presence of activated molecular

1888
R. C. Buijsman et al. / Bioorg. Med. Chem. 7 (1999) 1881±1890
was added. Stirring was continued for 45 min, after
which TLC analysis showed the presence of one main
product. Puri®cation by silica gel column chromato-
graphy (100:0 to 95:5, CH2Cl2:MeOH, v/v) furnished
pure 15a (130 mg, 92% yield). 31P NMR (CDCl3) d
0.09, 0.57, 0.71, 1.19, 1.45, 1.60, 2.15.
0.06; 1H NMR (D2O, 600 MHz, HH-COSY) d 3.58,
3.57, 3.51, 3.50, 3.48, 3.47 (7Âs, 21H, CH3O), 4.40±4.00
(H6a/H6b sugar); ring D: 5.41 (d, 1H, H1, J1,2=3.6 Hz),
3.24 (dd, 1H, H2, J2,3=9.3 Hz), 3.49 (m, 1H, H3), 3.35
(t, 1H, H4, J3,4=J4,5=9,6 Hz), 3.84 (m, 1H, H5); ring E:
4.62 (d, 1H, H1, J1,2=7.5 Hz), 3.21 (dd, 1H, H2,
J2,3=8.5 Hz), 3.48 (m, 1H, H3), 3.86 (m, 1H, H4), 3.69
(m, 1H, H5); ring F: 5.37 (d, 1H, H1, J1,2=3.5 Hz), 4.27
1-Amino-1-deoxyhexaethylene glycol 4-O-cellobiosyl-
heptakisphosphate (16a). Compound 15a (130 mg,
51 mmol) was dissolved in tert-butanol:H2O (6:1, v/v,
20 mL) containing a few drops of acetic acid. The solu-
tion was stirred under a continuous stream of hydrogen
in the presence of 10% Pd/C (100 mg). After 3 h the Pd/
C catalyst was removed by ®ltration and the ®ltrate was
concentrated in vacuo. Dowex 50 WX4-Na+ ion-
exchange then furnished 16a (64.5 mg, 98% yield). 31P
NMR (D2O, 600 MHz, 31P-1H COSY) d 2.58 (30-O-p),
1.42 (60-O-p), 0.85 (6-O-p), 0.38 (3-O-p), 0.22 (40-O-p),
(dd, 1H, H2, J2,3=9.3 Hz), 4.49 (t, 1H, H3, J3,4
=
9.3 Hz), 3.94 (t, 1H, H4, J4,5=9.3 Hz), 4.17 (m, 1H, H5);
ring G: 5.04 (d, 1H, H1, J1,2=3.0 Hz), 3.48 (m, 1H, H2),
3.71 (m, 1H, H3), 4.15 (m, 1H, H4), 4.73 (m, 1H, H5);
ring H: 5.11 (d, 1H, H1, J1,2=3.5 Hz), 4.32 (dd, 1H, H2,
J2,3=9.3 Hz), 4.62 (t, 1H, H3, J3,4=9.3 Hz), 3.94 (t, 1H,
H4, J4,5=9.3 Hz), 4.05 (m, 1H, H5); cellobiose: (redu-
cing end) 4.59 (d, 1H, H1, J1,2=7.5 Hz), 3.97 (m, 1H,
3
H2), 4.36 (ddd, 1H, H3, J2,3=J3,4=10.7, JHP
=
17.4 Hz), 3.92 (m, 1H, H4), 3.78 (m, 1H, H5); (non-
reducing end) 3.99 (m, 1H, H20), 4.27 (m, 1H, H30), 4.20
(m, 1H, H40); spacer: 7.78, 7.59 (2Âd, 4H, Harom SIAB),
4.01 (s, 2H, OCH2C(O)), 3.69±3.55 (m, 54H, CH2O
HEG/TEG), 2.84 (t, 2H, CH2S, J=5.0 Hz); ES±MS:
[M+H]+ 3265.3.
0
1
0.18 (2-O-p), 0.09 (2 -O-p); H NMR (D2O, 600 MHz,
0
HH-COSY) d 4.76 (d, 1H, H10, J1 ,2 =8.0 Hz), 4.61 (d,
1H, H1, J1,2=7.8 Hz), 4.32 (ddd, 1H, H3, J2,3=
0
J3,4=9.0 Hz, JHP=18.6 Hz), 4.27 (ddd, 1H, H30,
3
3
0
J2 ,3 =J3 ,4 =9.0 Hz, JHP=18.4 Hz), 4.26 (ddd, 1H,
0
0
0
3
H6a, J5,6a=3.9 Hz, J6a,6b=11.5 Hz, JHP=14.2 Hz),
3
4.19 (ddd, 1H, H40, J3 ,4 =J4 ,5 =9.7 Hz, JHP
=
N-Benzyloxycarbonyl-1-amino-1-deoxyhexaethylene glycol
4-O-cellobiosyl-heptakis(benzyl isopropyl phosphate)
(15b). Prepared as described for 15a, using 12 and 14
as starting materials. Puri®cation by silica gel column
chromatography (0±7% MeOH in CH2Cl2) gave 15b
(133 mg, 58% yield). 31P NMR (CDCl3) d 1.07, 1.19,
1.40, 1.51, 1.90, 1.91, 1.92.
0
0
0
0
19.1 Hz), 4.15 (ddd, 1H, H6b), 4.14 (ddd, 1H, H60a),
4.04 (ddd, 1H, H60b), 4.01 (m, 1H, H12a HEG), 3.97
(ddd, 1H, H20, JHP=10.7 Hz), 3.94 (ddd, 1H, H2,
3
3JHP=17.4 Hz), 3.85 (m, 1H, H12b HEG), 3.79 (m, 1H,
H5), 3.75±3.65 (m, 21H, H50, CH2O HEG), 3.18 (t, 2H,
CH2NH, J=5.0 Hz); FAB±MS: [M 2H+Na] 1186,
[M 3H+2Na] 1208, [M 4H+3Na] 1230, [M 5H
+4Na] 1252, [M 6H+5Na] 1274.
1-Amino-1-deoxyhexaethylene glycol 4-O-cellobiosyl-
heptakis(isopropyl phosphate) (16b). Prepared as descri-
bed for 16a, using 15b as starting material (94 mg, 97%
yield). 31P NMR (D2O, 600 MHz, 31P-1H COSY) d 0.05
(60-O-p/60-O-p), 0.83 (40-O-p/20-O-p), 1.13 (2-O-p),
Condensation of 28 with 16a. To a solution of 16a
(12 mg, 8.3 mmol) in 0.1 M Na2HPO4 bu€er (2.5 mL, pH
7.5) was added sulfo-SIAB2 (25) (31 mg, 62 mmol).
After stirring for 3 h in the dark at 20ꢀC, the reaction
mixture was applied onto a Superdex 30 column eluted
with 10% CH3CN. The appropriate fractions were
pooled and concentrated under reduced pressure at low
temperature (25ꢀC). The derivatized cellobiose-heptakis-
phosphate 26a and pentasaccharide 28 (11.5 mg,
5.5 mmol) were dissolved in a 0.05 M hydroxylamine
solution in 0.1 M Na2HPO4 bu€er (1.6 mL, pH 7.0),
which was degassed by passing through helium and by
soni®cation. The reaction was stirred in an argon
atmosphere. HPLC analysis (MonoQ anion exchange)
indicated the reaction to be complete after 2 h. The
reaction mixture was applied onto a Sephadex G-50
column eluted with 0.05 M NaCl in 10% CH3CN in
water. The appropriate fractions were pooled and con-
centrated to a small volume. To remove symmetrical
disul®de from conjugate VI, the residue was treated
with dithiothreitol (5 mg) dissolved in 0.1 M Na2HPO4
bu€er (1 mL, pH 8.3). Size exclusion chromatography
on a Sephadex G-50 column was followed by a gel ®l-
tration on a Superdex 30 column eluted with 10%
CH3CN in water. Concentration of the appropriate
fractions and subsequent Dowex 50 WX-Na+ ion-
exchange gave after lyophilization conjugate homo-
geneous VI (13.8 mg, 68% yield). [a]d +27.4 (c 0.5,
H2O). 31P NMR (D2O) 2.95, 2.08, 0.80, 0.77, 0.76, 0.39,
1.30 (3-O-p),
1.38 (30-O-p); 1H NMR (D2O,
600 MHz, HH-COSY) d 4.94 (d, 1H, H10, J1 ,2
5.7 Hz), 4.83 (d, 1H, H1, J1,2=6.7 Hz), 4.54±4.04 (m,
0
0
=
9H, H3, H30, CH(CH3)2), 4.36 (ddd, 1H, H40, J3 ,4
0
0
=
J4 ,5 =3.5 Hz, JHP=9.9 Hz), 4.23±4.19 (m, 3H, H2,
3
0
0
H20, H5), 4.15 (ddd, 1H, H6a, J5,6a=7.7 Hz, J6a,6b
=
10 Hz, 3JHP=11 Hz), 4.12±4.10 (m, 2H, H60a, H50), 4.07
(ddd, 1H, H4), 4.06±4.02 (m, 2H, H12a HEG, H60b),
3.87±3.77 (m, 20H, OCH2 HEG), 3.21 (t, CH2NH,
J=4.9 Hz), 1.32±1.27 (m, 42H, CH(CH3)2); ES±MS:
[M+H]+ 1461, [M+Na]+ 1483, [M+2Na H]+ 1505,
[M+3Na 2H]+ 1527.
Condensation of 28 with 16b. Prepared as described for
conjugate VI, using 16b and 28 as starting materials.
Puri®cation of the crude conjugate VII proceeded in a
similar fashion as described for conjugate VI (18 mg,
84% yield). [a]d +22.9ꢀ (c 0.5, H2O). H NMR (D2O,
1
600 MHz, HH-COSY) d 3.60, 3.59, 3.58, 3.52, 3.51,
3.49, 3.43 (7Âs, 21H, CH3O), 4.28±4.09 (H6a/H6b
sugar); ring D: 5.47 (d, 1H, H1, J1,2=3.5 Hz), 3.31 (dd,
1H, H2, J2,3=8.7 Hz), 3.57 (m, 1H, H3), 3.40 (t, 1H,
H4, J3,4=J4,5=7.8 Hz), 3.88 (m, 1H, H5); ring E: 4.68
(d, 1H, H1, J1,2=7.5 Hz), 3.25 (dd, 1H, H2, J2,3=
8.5 Hz), 3.56 (m, 1H, H3), 3.82 (m, 1H, H4), 3.73 (m,
1H, H5); ring F: 5.42 (d, 1H, H1, J1,2=3.6 Hz), 4.32 (dd,

R. C. Buijsman et al. / Bioorg. Med. Chem. 7 (1999) 1881±1890
1889
1H, H2, J2,3=8.5 Hz), 4.56 (m, 1H, H3), 4.00 (t, 1H,
H4, J3,4=J4,5=8.4 Hz), 4.21 (m, 1H, H5); ring G: 5.17
(bs, 1H, H1), 3.56 (m, 1H, H2), 3.79 (m, 1H, H3), 4.19
(m, 1H, H4); ring H: 5.17 (d, 1H, H1, J1,2=3.2 Hz), 4.32
(dd, 1H, H2, J2,3=8.7 Hz), 4.67 (m, 1H, H3), 3.98 (t,
1H, H4, J3,4=J4,5=8.7 Hz), 4.10 (m, 1H, H5); cello-
biose: (reducing end) 4.85 (d, 1H, H1, J1,2=6.0 Hz), 4.23
(m, 1H, H2), 4.52 (m, 1H, H3), 4.09 (m, 1H, H4); (non-
J=5.2 Hz), 2.30±1.83 (m, 30H, CH3 Ac); 13C{1H}
NMR (CDCl3) d 170.4±169.2 (10ÂCˆO Ac), 154.3
(CˆO Z), 136.8 (Carom Z), 128.5±127.9 (CHarom Z),
100.2 (C1), 95.7, 95.6 (C10, C100), 75.2, 73.9, 72.7, 72.1,
71.9, 71.6, 69.3, 68.9, 68.5, 68.0 (C2, C20, C200, C3, C30,
C300, C4, C40, C400, C5, C50, C500), 70.4±68.9 (CH2O
HEG), 66.3 (CH2 Z), 63.4, 63.1, 62.4, 61.4 (C6, C60,
C600, CH2CH2NH), 40.8 (CH2NH), 20.8±20.5 (10ÂCH3
Ac); ES±MS: [M+H]+ 1322.8, [M+Na]+ 1344.8.
reducing end) 4.95 (d, 1H, H10, J1 ,2 =7.1 Hz), 4.22 (m,
1H, H20), 4.53 (m, 1H, H30), 4.36 (ddd, 1H, H40); 4.28±
4.09 (H5, cellobiose), 4.54±4.04 (m, 7H, CH(CH3)2),
1.38±1.31 (m, 42H, CH(CH3)2); spacer: 7.80, 7.62 (2Âd,
4H, Harom SIAB), 4.09 (s, 2H, OCH2C(O)), 3.75±3.62
(m, 54H, CH2O HEG/TEG), 2.91 (t, 2H, CH2S,
J=5.2 Hz); ES±MS: [M+H]+ 3561.2.
0
0
N-Benzyloxycarbonyl-1-amino-1-deoxyhexaethylene glycol
4-O-(4-O-(ꢁ-D-glucopyranosyl)-ꢁ-D-glucopyranosyl)-ꢀ-
D-glucopyranoside (22). Prepared as described for 12,
1
using 21 as starting material (177 mg, 95% yield). H
NMR (CD3OD) d 7.35 (bs, 5H, Harom Z), 5.15 (d, 1H,
00
H10, J1 ,2 =2.9 Hz), 5.14 (d, 1H, H1 , J1 ,2 =3.8 Hz),
5.07 (s, 2H, CH2 Z), 4.33 (d, 1H, H1, J1,2=7.8 Hz), 3.98
(m, 1H, H12a HEG), 3.87 (m, 1H, H6a), 3.83 (m, 1H,
H60a), 3.81 (m, 1H, H600a), 3.77 (m, 1H, H6b), 3.76 (m,
1H, H60b), 3.73 (m, 1H, H600b), 3.71 (m, 1H, H12b
HEG), 3.68±3.62 (m, 4H, H3, H30, H300, H500), 3.62±3.58
(m, 18H, CH2O HEG), 3.55±3.48 (m, 6H, H20, H4, H40,
0
0
00 00
4-O-(4-O-(2,3,4,6-Tetra-O-acetyl-ꢁ-D-glucopyranosyl)-
2,3,6-tri-O-acetyl-ꢁ-D-glucopyranosyl)-2,3,6-tri-O-acetyl-
ꢁ/ꢀ-D-glucopyranosyl trichloroacetimidate (20). To a
stirred solution of maltotriose (17) (2.0 g, 4.0 mmol) in
pyridine (100 mL) was added acetic anhydride (6.2 mL,
65 mmol) and a catalytic amount of DMAP (0.79 g,
6.5 mmol). After 5 h the reaction mixture was poured
into aq NaHCO3 (1 M, 250 mL) and extracted three
times with EtOAc (200 mL). The combined organic lay-
ers were dried on MgSO4 and concentrated in vacuo.
The product was puri®ed by column chromatography
(light petroleum:EtOAc, 1:1 to 0:1, v/v) giving 18 as a
white foam (3.5 g, 91% yield). Anomeric deacetylation
of 18 (3.0 g, 3.1 mmol) was achieved in a similar fashion
as described for the synthesis compound 9. Puri®cation
by silica gel column chromatography (light petroleum:
EtOAc, 3:2 to 1:0, v/v) gave 19 (2.7 g, 92% yield).
Trichloroacetimidate 20 was subsequently prepared as
described for compound 10, using 19 as starting
material. Puri®cation of the crude 20 by column chro-
matography (light petroleum:EtOAc:TEA, 50:49:1 to
0:99:1, v/v/v) yielded pure 20 as white foam (1.9 g, 71%
H50, CH2CH2NH), 3.45 (dd, 1H, H200, J2 ,3 =9.5 Hz),
00 00
3.38 (ddd, 1H, H5, J4,5=6.7 Hz, J5,6a=4.7 Hz, J5,6b
=
3.0 Hz), 3.31±3.27 (m, 3H, CH2NH, H400), 3.25 (dd, 1H,
H2, J2,3=9.3 Hz); 13C{1H} NMR (CD3OD) d 154.4
(CˆO Z), 136.8 (Carom Z), 129.8±128.8 (CHarom Z),
104.2 (C1), 102.7, 102.6 (C10, C100), 81.3, 77.6, 76.5, 75.0,
74.9, 74.8, 74.7, 74.6, 74.1, 73.7, 73.3 (C2, C20, C200, C3,
C30, C300, C4, C40, C400, C5, C50, C500), 71.4±70.9 (CH2O
HEG), 67.3 (CH2 Z), 62.7±62.1 (C6, C60, C600,
CH2CH2NH), 41.7 (CH2NH); ES±MS: [M H] 900.
N-Benzyloxycarbonyl-1-amino-1-deoxyhexaethylene glycol
4-O-maltotriosyl-decakis(dibenzyl phosphate) (23). Pre-
pared as described for 15a, using 22 as starting material,
which was dissolved in CH3CN:dioxane (2:1, v/v)
(216 mg, 92% yield). 31P NMR: d 0.16, 0.08, 0.76,
0.90, 0.92, 1.21, 1.25, 1.37, 1.82.
1
yield). H NMR (CDCl3) d 8.67 (s, 1H, NHimidaat), 6.48
(d, 1H, aH1, J1,2=3.7 Hz), 5.64±5.07 (m, 6H, H2, H20,
H200, H3, H30, H300, H400), 5.02, 4.85, 4.75 (3Âdd, 3H,
H6a, H60a, H600a), 4.50 (m, 3H, H10, H100, H6b), 4.32±
3.90 (m, 7H, H60b, H600b, H5, H50, H500, H4, H40), 2.30±
1.80 (m, 30H, CH3 Ac); 13C{1H} NMR (CDCl3) d
169.9±168.7 (10ÂCˆO Ac), 159.9 (CˆNH), 95.4, 95.1
(C10, C100), 92.1 (C1), 90.1 (CCl3), 73.0, 72.3, 71.1, 70.1,
69.9, 69.5, 68.7, 68.5, 67.9, 67.4 (C2, C20, C200, C3, C30,
C300, C4, C40, C400, C5, C50, C500), 62.0, 61.0, 59.6 (C6,
C60, C600), 20.2±19.7 (10ÂCH3 Ac).
1-Amino-1-deoxyhexaethylene glycol 4-O-maltotriosyl-
decakisphosphate (24). Prepared as described for 16a,
using 23 as starting material (131 mg, 96% yield). 31P
NMR (D2O, 600 MHz, 31P-1H COSY) d 1.85 (300-O-p0),
1.68 (20-O-p), 1.01 (200-O-p), 1.60, 0.60, 0.50 (600-O-p/60-
O-p/6-O-p), 0.68 (2-O-p), 0.21 (400-O-p), 0.52 (30-O-p),
1
0.69 (3-O-p); H NMR (D2O, 600 MHz, HH-COSY)
0
5.78 (d, 1H, H100, J1 ,2 =3.4 Hz), 5.60 (d, 1H, H1 ,
00 00
0
0
0
0
0
0
0
J1 ,2 =3.8 Hz), 4.68 (ddd, 1H, H3 , J2 ,3 =J3 ,4 =7.3 Hz,
3JHP=16.3 Hz), 4.65 (d, 1H, H1, J1,2=7.2 Hz), 4.48
3
N-Benzyloxycarbonyl-1-amino-1-deoxyhexaethylene glycol
4-O-(4-O-(2,3,4,6-tetra-O-acetyl-ꢁ-D-glucopyranosyl)-
2,3,6-tri-O-acetyl-ꢁ-D-glucopyranosyl)-2,3,6-tri-O-acetyl-
ꢀ-D-glucopyranoside (21). Prepared as described for 11,
using 20 and 7 as starting materials. Puri®cation by
silica gel column chromatography (0±4% MeOH in
(ddd, 1H, H3, J2,3=J3,4=8.3 Hz, JHP=16.8 Hz), 4.43
(ddd, 1H, H300, J2 ,3 =J3 ,4 =7.3 Hz, JHP=16.8 Hz),
4.26 (m, 1H, H20), 4.24 (m, 1H, H400), 4.21±4.10 (m, 6H,
H6a, H60a, H600a, H6b, H60b, H600b), 4.19 (m, 1H, H200),
3
00 00
00 00
4.07 (dd, 1H, H40, J4 ,5 =9.3 Hz), 4.03 (ddd, 1H, H2,
3JHP=17.9 Hz), 3.99 (m, 1H, H4), 3.88±3.81 (m, 3H,
H5, H50, H500), 3.75 (t, 2H, CH2CH2NH, J=5.6 Hz),
3.70±3.66 (m, 20H, CH2O HEG), 3.19 (t, 2H, CH2NH);
ES±MS: [M+H]+ 1568, [M+Na]+ 1590, [M+2Na
H]+ 1612, [M+3Na 2H]+ 1634.
0
0
1
EtOAc) gave 21 (0.57 g, 56% yield). H NMR (CDCl3)
d 7.35±7.29 (m, 6H, Harom Z, NH), 5.66±4.96 (m, 7H,
H2, H20, H200, H3, H30, H300, H400), 5.11 (s, 2H, CH2 Z),
4.92±4.66 (m, 3H, H6a, H60a, H600a), 4.62 (d, 1H,
J1,2=8.5 Hz), 4.55±4.38 (m, 3H, H10, H100, H6b), 4.35±
3.84 (m, 7H, H4, H40, H5, H50, H500, H60b, H600b), 3.78±
3.48 (m, 22H, CH2O HEG), 3.40 (t, 2H, CH2NH,
Condensation of 28 with 24. Prepared as described for
conjugate VI, using 24 and 28 as starting materials.

1890
R. C. Buijsman et al. / Bioorg. Med. Chem. 7 (1999) 1881±1890
Puri®cation of the crude conjugate VIII proceeded in a
similar fashion as described for conjugate VI (10.4 mg,
References
ꢀ
1
1. Lane, D. A.; Lindahl, U., Eds. Heparin: Chemical and
Biological Properties, Clinical Applications; Edward Arnold:
London, 1989.
2. Rosenberg, R. D.; Damus, P. S. J. Biol. Chem. 1973, 248,
6490.
3. Olson, S. T.; Bjork, I.; She€er, R.; Craig, P. A.; Shore, J.
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4. van Boeckel, C. A. A.; Grootenhuis, P. D. J.; Visser, A.
Nature Struc. Biol. 1994, 1, 423.
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N.; Carrell, R. W. Proc. Natl. Acad. Sci. USA 1997, 94, 14683.
6. Tunberg, L.; Backstrom, G.; Lindahl, U. Carbohydrate Res.
1982, 100, 393.
7. Choay, J.; Petitou, M.; Lormeau, J.-C.; Sinay, P.; Casu, B.;
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8. van Boeckel, C. A. A.; Petitou, M. Angew. Chem., Int. Ed.
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9. Petitou, M.; van Boeckel, C. A. A. Prog. Chem. Org. Nat.
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10. Lane, D. A.; Denton, J.; Flynn, A. M.; Thunberg, L.;
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51% yield). [a]d +42.8 (c 0.5, H2O). H NMR (D2O,
600 MHz, HH-COSY) d 3.68, 3.67, 3.66, 3.60, 3.59,
3.57, 3.51 (7Âs, 21H, CH3O), 4.47±4.20 (H6a/H6b
sugar); ring D: 5.51 (d, 1H, H1, J1,2=3.4 Hz), 3.34 (dd,
1H, H2, J2,3=8.1 Hz), 3.67 (m, 1H, H3), 3.45 (t, 1H,
H4, J3,4=J4,5=8.5 Hz), 3.92 (m, 1H, H5); ring E: 4.70
(d, 1H, H1, J1,2=7.5 Hz), 3.30 (t, 1H, H2, J2,3=7.5 Hz),
3.67 (m, 1H, H3), 3.94 (m, 1H, H4), 3.78 (m, 1H, H5);
ring F: 5.45 (d, 1H, H1, J1,2=3.5 Hz), 4.36 (dd, 1H, H2,
J2,3=8.4 Hz), 4.58 (t, 1H, H3, J3,4=8.4 Hz), 4.02 (t, 1H,
H4, J4,5=8.4 Hz), 4.26 (m, 1H, H5); ring G: 5.12 (bs,
1H, H1), 3.65 (m, 1H, H2), 3.83 (m, 1H, H3), 4.23 (m,
1H, H4); ring H: 5.20 (d, 1H, H1, J1,2=3.4 Hz), 4.40
(dd, 1H, H2, J2,3=8.5 Hz), 4.71 (t, 1H, H3, J3,4
=
8.5 Hz), 4.00 (t, 1H, H4, J4,5=8.5 Hz), 4.16 (m, 1H, H5);
maltotriose: (reducing end) 4.05 (m, 1H, H2), 4.54 (m,
1H, H3), 3.95 (m, 1H, H4); 5.58 (bs, 1H, H10), 4.26 (m,
1H, H20), 4.10 (m, 1H, H40); (non-reducing end) 5.72
(bs, 1H, H100), 4.22 (m, 1H, H200), 4.52 (m, 1H, H300),
4.24 (m, 1H, H400); 3.91±3.84 (H5, maltotriose); spacer:
7.85, 7.67 (2Âd, 4H, Harom SIAB), 4.10 (s, 2H,
OCH2C(O)), 3.76±3.70 (m, 54H, CH2O HEG/TEG),
2.93 (t, 2H, CH2S, J=5.0 Hz). ES±MS: [M+H]+
3668.4.
12. Olson, S. T.; Bjork, I. J. Biol. Chem. 1991, 266, 6353.
13. Grootenhuis, P. D.; Westerduin, P.; Meulenman, D.; Peti-
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14. Westerduin, P.; Basten, J.; Broekhoven, M.; de Kimpe, V.;
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15. Buijsman, R. C.; Kuijpers, W. H. A.; Basten, J. E. M.;
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A.; van Boom, J. H. Chem. Eur. J. 1996, 2, 1572.
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22. Bannwarth, W.; Trzeciak, A. Helv. Chim. Acta 1987, 71,
175.
Measurements of the ATIII-mediated antithrombin and
anti-Xa activities. The inhibitory potency of heparin
and its analogues was measured amidolytically accord-
ing to a modi®ed procedure described by Teien and
Lie.25,26 In this procedure thrombin is incubated for a
short time with a bu€ered solution of human ATIII and
the heparin analogue (VI±VIII). After incubation the
residual thrombin activity is determined by addition of
the chromogenic substrate S-2238 (H-d-Phe-Pip-Arg-
pNA). After 2 and 7 min, respectively, the release of p-
nitroaniline is measured spectrophotometrically at
405 nm and the di€erence in optical density (ÁOD) is
calculated. The antithrombin activity of the compound
was read from a calibration curve of standard heparin,
1
which has an activity of 160 U mg according to the
Fourth International Standard of Heparin Calibration.
23. Claessen, C. A. A.; Segers, R. P. A. M.; Tesser, G. I. Recl.
Trav. Chim. Pays-Bas 1985, 104, 119.
24. Nielsen, J.; Dahl, O. Nucl. Acids Res. 1987, 15, 3626.
25. Teien, A. N.; Lie, M. Thromb. Res. 1977, 10, 399.
26. Lie-Larsen, M.; Bildgaard, U.; Teien, A. N.; Gjesdal, K.
Thromb. Res. 1978, 13, 285.
Similar experiments were carried out to measure the
anti-Xa activity of heparin and its analogues. This
requires the S-2222 chromogenic substrate (Bz-Ile-Glu-
Gly-Arg-pNA) to determine the residual Xa activity. In
this case the anti-Xa activity was compared with the
`natural' pentasaccharide (I), which has an activity of
1
27. Basten, J. E. M.; Dreef-Tromp, C. M.; de Wijs, B.; van
Boeckel, C. A. A. Bioorg. Med. Chem. Lett. 1998, 8, 1201.
700 U mg
.