Targeting of phosphonoacetate to the liver: Synthesis of a phosphonoacetate-trilactoside conjugate

Article abstract of DOI:10.1560/8QKA-DFYF-FJ70-E1CX

As part of continuing studies on drug delivery to the human liver, a phosphonoacetate-trilactoside conjugate (12) has been synthesized in an overall yield of 23%. The shortness of the synthesis of this conjugate hinges upon a successful Michaelis-Arbuzov reaction of bromoacetamide 8 with tris-(trimethylsilyl) phosphite.

Full text of DOI:10.1560/8QKA-DFYF-FJ70-E1CX

Targeting of Phosphonoacetate to the Liver: Synthesis of a
Phosphonoacetate–Trilactoside Conjugate1
SHRIDHAR BHAT, ANDREW R. VAINO, AND WALTER A. SZAREK*
Department of Chemistry, Queen’s University, Kingston, Ontario K7L 3N6, Canada
(Received 30 July 2000)
Abstract. As part of continuing studies on drug delivery to the human liver, a
phosphonoacetate–trilactoside conjugate (12) has been synthesized in an overall
yield of 23%. The shortness of the synthesis of this conjugate hinges upon a
successful Michaelis–Arbuzov reaction of bromoacetamide 8 with tris-
(trimethylsilyl) phosphite.
INTRODUCTION
of binding of a host of molecules containing various
Phosphonoacetic acid is a broad-spectrum antiviral numbers of terminal D-galactopyranosyl residues has
agent2 that acts by inhibiting specifically viral-induced demonstrated that, as the number of these residues in-
DNA polymerase and by suppressing the replication of creased, the binding increased as well.11a,b Previous stud-
DNA viruses including herpes simplex virus,3 cytome- ies in this laboratory have elicited a consistent incre-
galovirus,4 varicella-zoster virus,5 Epstein–Barr virus,6 ment of hepatophilicity in adding one, two, or three
and vaccinia virus.7 Infection with human cytomega- lactose units to a glycerol backbone.11c–e Subsequently, a
lovirus has been associated with severe diseases, includ- tethering of cytotoxic nucleoside analogs to the
ing neonatal hemochromatosis,8a cytomegalovirus- trilactoside of the well-known buffer, tris(hydroxy-
hepatitis,8b especially in immuno-suppressed patients methyl)methylamine (TRIS), was realized.12 In the
such as recipients of liver transplants8c or patients posi- present work, TRIS was modified such that, after addi-
tive for HIV,8d,e and also in infants and children.8f
A
tion of three lactose units, the antiviral agent, phospho-
drawback with the highly charged phosphonoacetic acid noacetic acid, could be readily introduced.
is its difficult permeation through biomembranes, a fea-
ture that eventually escalates its effective dose to an
RESULTS AND DISCUSSION
undesirable amount.5 A method of resolving this issue
The synthesis of delivery vehicle 6 (Scheme 1) began
involves derivatizing phosphonoacetic acid while keep-
with readily available 6-aminohexanoic acid (1). Cou-
ing the phosphonic acid moiety unaltered.9 In the
pling of TRIS with the protected hexanoic acid 2 was
present work, we describe not only a derivatization of
performed using 2-ethoxy-1-ethoxycarbonyl-1,2-di-
phosphonoacetic acid, but also, more importantly, a
hydroquinoline (EEDQ).13 Glycosylation of the three
derivatization designed to target the acid directly to the
primary hydroxy groups of 3 with 2,3,6,2′,3′,4′,6′-
liver (see 12).
hepta-O-acetyl-α-lactosyl bromide (4), prepared by the
The mammalian hepatocytes express the asialo-
method of Kartha and Jennings,14 was achieved in 73%
glycoprotein receptor (ASGP-R), a unique integral
yield using the Helferich15 modification of the Königs–
membrane receptor exhibiting specificity for terminal,
Knorr reaction. The structure of this oligolactoside was
nonreducing β-D-galactopyranosyl or 2-acetamido-2-
elucidated using Fourier-transform, proton chemical-
deoxy-β-D-galactopyranosyl residues.10 This specific
shift correlation spectroscopy (COSY) and hetero-
binding to liver cells has been examined with a variety
nuclear correlation spectroscopy (HETCOR). The β-D
of oligogalactosides and oligolactosides. The evaluation
configuration for the newly formed glycosidic linkages
Dedicated to the memory of Wolf Prize laureate Professor
Raymond U. Lemieux in recognition of his inspiration and *Authortowhomcorrespondenceshouldbeaddressed. E-mail:
significant contributions to carbohydrate chemistry.
szarekw@chem.queensu.ca
Israel Journal of Chemistry
Vol. 40
2000 pp. 335–341

336
Scheme 1. Reagents: i, BnOCOCl, 4 N NaOH, 1,4-dioxane–H2O (1:3); HCl; ii, (HOCH2)3C-NH2, EEDQ, EtOH; iii, 4 (3.5
equiv), Hg(CN)2, 1:1 MeNO2–C6H6; iv, H2, 5% Pd/C (Degussa type), HCO2H, dil HCl, MeOH; v, XCH2COX, Et3N (2.1 equiv),
–20 °C, CH2Cl2.
was discerned from a characteristic coupling constant Chloroacetamide 9 could be easily transformed into the
(J1,2 = 7.9 Hz).16 Deprotection of 5 was readily accom- phosphonatoacetamide 10 in 78% yield by heating in
plished in quantitative yield by catalytic hydrogenolysis the presence of triethylphosphite.18 Surprisingly, the
and the product was isolated as the hydrochloride salt trilactosylated chloroacetamide 7 under the same reac-
(6) in order to avert the possibility of transacetylation of tion conditions failed to undergo the Michaelis–
the free amine. Since the direct coupling of the free Arbuzov reaction. Neither prolonged heating (24 h) nor
amine of compound 6 with commercially available di- elevated temperatures (to 130 °C) effected the desired
ethyl phosphonatoacetic acid could not be achieved in reaction; the chloroacetamide 7 was recovered un-
good yields using the common methods of coupling changed. However, the corresponding bromoacetamide
such as DCC or its variants17 or EEDQ,13 we were com- 8 readily formed the phosphonate 11 in 94% yield under
pelled to assemble the target phosphonoacetate–conju- identical reaction conditions. The recalcitrant chloro-
gate by way of a carbon–phosphorus bond-forming re- acetamide 7 also succumbed to the Michaelis–Arbuzov
action.
reaction when a catalytic amount of lithium iodide was
In order to validate the approach, the chloro- utilized. These startling results may stand as one of the
acetamide 9, an archetype of compound 7 lacking the finest examples of the remark made about the Michaelis–
trilactoside moiety of 7 (Scheme 2), was synthesized. Arbuzov reaction, “The usual reactivity scale of iodide–
Israel Journal of Chemistry
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2000

337
Scheme 2. Reagents: i, (EtO)3P, 100 °C, 5 h; ii, (EtO)3P, catalytic LiI, 100 °C, 6 h.
bromide–chloride applies to this reaction, as does the tensive glycosidic cleavage occurred). Consequently,
general principle of decreasing reactivity with the the trilactosylated bromoacetamide 8 (Scheme 3) was
growth of molecular size in any particular class”19 (mol heated in the presence of tris(trimethylsilyl) phosphite
wt of 9 is 235.65 and that of 7 is 2166.41).
under a rapid bubbling of argon (in order to expel
Although phosphonate 11 was synthesized in good bromotrimethylsilane), and the trimethylsilyl groups in
yield, removal of the ethyl groups on the phosphonate the resulting phosphonate were removed by the addition
proved elusive to either base-mediated (only the acetyl of pyridine–methanol; the reaction mixture was then
groups were removed) or acid-mediated hydrolysis (ex- treated with 2 N sodium hydroxide, and the phosphonic
Scheme 3. Reagents: i, P(OSiMe3)3, PhCH3, 120 °C; ii, pyridine, MeOH; iii, 2 N NaOH, MeOH–H2O; dil HCl.
Bhat, Vaino, and Szarek / Phosphonoacetate–Trilactoside Conjugate

338
(lit.23 82–83 °C); νmax (KBr): 3319.55 (br, OH, NH), 2938.31,
acid 12 was obtained by neutralization with dilute hydro-
chloric acid. Phosphonic acid 1220 was purified by size-
exclusion chormatography using a Bio-gel P2 column.
We have demonstrated the synthesis of a novel
phosphonoacetate derivative that is secured to a liver-
targeting vehicle; it is expected that derivative 12 would
be hydrolyzed in the liver releasing phosphonoacetic
acid. Thus, 12 could be a promising drug-conjugate to
fight cytomegalovirus infection in the liver. The results
of an evaluation of the binding of 12 and related com-
pounds to the ASGP-R and of antiviral studies will be
reported in due course.
2861.61 (CH), 1688.44 (NHCOO), 1621.13 (CONH),
1
1557.35, and 1526.23 cm–1; H NMR (300 MHz, CDCl3) δ:
1.27–1.42 (m, 2 H, CH2-4), 1.45–1.57 (m, 2 H, CH2-5), 1.62
(qt, 2 H, J3,2 = J3,4 = 7.2 Hz, CH2-3), 2.23 (t, 2 H, J2,3 = 7.2 Hz,
CH2-2), 3.17 (br d, 2 H, J6,NH = 4.2 Hz, CH2-6), 3.62 (br s, 6 H,
3 CH2OH), 4.95 (br s, 1 H, NHCOO), 5.08 (br s, 5 H, CH2Ph, 3
OH), 6.65 (s, 1 H, NHCO), 7.31 (br s, 5 H, Ph); 13C NMR (50
MHz, CDCl3) δ: 25.2 (CH2-3), 25.99 (CH2-4), 29.46 (CH2-5),
36.45 (CH2-2), 40.66 (CH2-6), 61.78 (3 CH2OH), 61.91 (Cquat),
66.54 (CH2Ph), 127.98, 128.1, 128.5 (Car-2,3,4,5,6), 136.5
(Car-1), 156.5 (NHCOO), 175.55 (NHCO); ESIMS (positive-
ion) m/z: 407.2 (M+K)+, 391.2 (M+Na)+, 369.2 (M+H)+, 351.2
(M–OH)+, 212.1, 206.1, 117.0. Anal. Calcd for C18H28N2O6: C,
58.68, H, 7.66, N, 7.60. Found: C, 58.62, H, 7.87, N, 7.86.
EXPERIMENTAL
N-[1,3-Bis-O-(2,3,6,2′,3′,4′,6′-hepta-O-acetyl-β-lactosyl)-2-
{(2,3,6,2′,3′,4′,6′-hepta-O-acetyl-β-lactosyl)methyl}prop-2-
yl]-6-[(benzyloxy)carbonylamino]hexanamide (5)
General
Melting points were determined using a Mel-Temp II ap-
paratus, and are uncorrected. NMR spectra were obtained on
Brüker AVANCE-500, 400, 300, and AM-200 spectrometers.
Proton and carbon chemical shifts are given relative to Me4Si
(δ = 0); phosphorus chemical shifts are relative to H3PO4.
Chemical-shift and coupling assignments were made with the
aid of 2-D experiments, namely COSY and HETCOR. The
multiplicities given are those observed within the resolving
power of the spectrometer, and are not necessarily true multi-
plicities. Infrared spectra were recorded using a BOMEM
MB-120 FTIR spectrophotometer and optical rotations with a
Perkin–Elmer model 241 automatic polarimeter. M-H-W
Laboratories, Phoenix, Arizona, performed elemental analy-
ses. Thin-layer chromatography (TLC) was performed using
glass-backed E. Merck silica gel 60 F-254 plates and column
chromatography on E. Merck silica gel 60. The developed
plates were sprayed with 1% ceric sulfate and 1.5% molybdic
acid in 10% aqueous sulfuric acid and heated at about 150 °C.
The lactosyl bromide 4 (26.6 g, 37.9 mmol), triol 3 (4 g,
10.86 mmol), and Hg(CN)2 (10.56 g, 41.81 mmol) were added
to a 1:1 mixture of dry benzene and nitromethane (100 mL)
and the reaction mixture was stirred at 40 °C under argon for
72 h. The mixture was diluted with toluene (50 mL), then
washed sequentially with a saturated aqueous solution of
NaHCO3 (3 × 30 mL) and water (50 mL), and dried (Na2SO4);
the solvent was removed under vacuum. Chromatography us-
ing 1:1 (v/v) toluene–EtOAc as eluent gave 5 as an amorphous
solid (17.63 g, 73%); Rf 0.043 (1:1 CHCl3–EtOAc, 2 drops of
isopropanol); mp 99 °C; [α]D – 4.9° (c 1.02, CHCl3); νmax
(KBr): 3396.83 (br, NH), 2944.33 (CH), 1752.7 (br, OAc),
1675.17 (CONH), 1532, 1372, 1215.8, 1045.24, 901.3, 753.9,
and 602.4 cm–1; 1H NMR (400 MHz, CDCl3) δ: 1.2–1.3 (m, 2
H, CH2-4), 1.41–1.54 (m, 4 H, CH2-3,5), 1.90–2.15 (multiple
s’s, 65 H, CH2-2, 21 OAc), 3.1–3.20 (m, 2 H, CH2-6), 3.53–
3.59 (m, 3 H, H-6′(3)), 3.61 (d, 3 H, J5,4 = 10.3 Hz, H-5,5,5),
3.73 (t, 3 H, J4,3 ≈ J4,5 = 9.4 Hz, H-4,4,4), 3.83 (t, 3 H, J6′,6′ ≈ J6′,5′
= 6.67 Hz, H-6′(3)), 4.0–4.11 (m, 12 H, TRIS CH2O, H-6(6)),
4.33 (d, 3 H, J1,2 = 7.91 Hz, H-1,1,1), 4.39 (d, 3 H, J5′,6′ = 10.9
Hz, H-5′,5′,5′), 4.44 (d, 3 H, J1′,2′ = 7.87 Hz, H-1′,1′,1′), 4.79 (t,
3 H, J2,3 ≈ J2,1 = 8.06 Hz, H-2,2,2), 4.91 (dd, 3 H, J3′,2′ = 10.4
and J3′,4′ = 3.35 Hz, H-3′,3′,3′), 5.02–5.08 (m, 5 H, H-2′(3),
CH2Ph), 5.11 (t, 3 H, J3,4 ≈ J3,2 = 9.37 Hz, H-3,3,3), 5.21 (br s,
1 H, HNCOO), 5.3 (d, 3 H, J4′,3′ = 3.3 Hz, H-4′,4′,4′), 5.72 (s, 1
H, HNCO), 7.28-7.32 (br s, 5 H, Ph); 13C NMR (100 MHz,
CDCl3) δ: 20.40 20.52, 20.60, 20.67, 20.75, 21.04 (OAc),
24.89 (CH2-3), 26.02 (CH2-4), 29.47 (CH2-5), 36.49 (CH2-2),
40.74 (CH2-6), 58.89, 60.65 (CH2O), 60.13 (Cquat), 61.81 (C-
5′), 66.35 (PhCH2O), 66.50 (C-4′), 68.32 (C-6), 68.96 (C-5),
69.12 (C-2′), 70.53 (C-6′), 70.85 (C-3′), 71.54 (C-2), 72.39
Materials
All solvents were purified by distillation under argon as
follows: benzene and toluene from blue sodium benzyl ketyl
solution; ethanol from Mg(OEt)2; nitromethane was dried over
calcium chloride and then fractionally distilled. Routine dry-
ing during processing of all compounds was accomplished
over Na2SO4 unless otherwise stated. 6-[(Benzyloxy)-
carbonylamino]hexanoic acid (2),21 2,3,6,2′,3′,4′,6′-hepta-
O-acetyl-α-lactosyl bromide (4), and ethyl 6-aminohex-
anoate22 were prepared according to procedures delineated in
the literature.
N-[1,3-Dihydroxy-2-(hydroxymethyl)prop-2-yl]-6-
[(benzyloxy)carbonylamino]hexanamide (3)
EEDQ (12.82 g, 51.83 mmol) was added to a solution of (C-6′), 72.55 (C-3), 76.03 (C-4), 100.63 (C-1), 100.91 (C-1′),
the protected hexanoic acid 2 (12.5 g, 47.12 mmol) in absolute 127.96, 128.40 (Car-2,3,4,5,6), 136.62 (Car-1), 156.32
ethanol (200 mL) and the reaction solution was stirred for 30 (HNCOO), 168.94, 169.46, 169.52, 169.94, 170.01, 170.21
min. Tris(hydroxymethyl)methylamine (6.28 g, 51.83 mmol) (OAc), 172.98 (HNCO); ESIMS (positive-ion) m/z: expected
was added and the mixture was heated at reflux temperature for (M+Na)+: 2247.7; found: 2247.5; also 2263.5 (M+K)+,
for 6 h. The solvent was evaporated and the residue was 2222.7 (M–1)+, 1956.8, 1669.1, 1609.1, 1235.0, 1140.8,
chromatographed using ethyl acetate as eluent to give 3 as a 1048.9, 1033.1. Anal. Calcd for C96H130N2O57: C, 51.84, H,
white solid (10.42 g, 60%); Rf 0.06 (EtOAc); mp 81–82 °C 5.89, N, 1.26. Found: C, 51.68, H, 5.91, N, 1.32.
Israel Journal of Chemistry
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339
N-[1,3-Bis-O-(2,3,6,2′,3′,4′,6′-hepta-O-acetyl-β-lactosyl)-2-
{(2,3,6,2′,3′,4′,6′-hepta-O-acetyl-β-lactosyl)methyl}prop-2-
yl]-6-aminohexanamide hydrochloride (6)
(OAc), 24.78 (CH2-3), 25.97 (CH2-4), 28.82 (CH2-5), 36.37
(CH2-2), 39.50 (CH2-6), 42.59 (CH2Cl), 58.89, 60.64 (CH2O),
60.25 (Cquat), 61.80 (C-5′), 66.50 (C-4′), 68.09 (C-6), 68.95 (C-
To a solution of a sample of the benzyl carbamate 5 ob- 5), 70.53 (C-6′), 70.54 (C-2′), 70.84 (C-3′), 71.53 (C-2), 72.37
tained above (5 g, 2.248 mmol) in methanol (100 mL) were (C-6′), 72.56 (C-3), 76.01 (C-4), 100.63 (C-1), 100.89 (C-1′),
added formic acid (4 mL, to make an approximately 4% 165.77 (HNCOCH2Cl), 168.91, 169.43, 169.49, 169.91,
solution) and hydrochloric acid (4 mL of 1 N HCl, 1.8 equiv) 169.98, 170.18 (OAc), 172.85 (HNCO); ESIMS (positive-ion)
and the mixture was degassed; the catalyst, 5% palladium-on- m/z: expected for (M+Na)+ and (M+H)+: 2188.7 and 2166.7,
carbon (Degussa type, 1.5 g, 0.3 equiv), was added. (Caution: respectively; found: 2188.1 and 2166.4; also 2089.8, 2051.9,
extreme fire hazard!) The reaction mixture was shaken under 1977.9, 1900.7, 1706.3, 1622.8, 1611.5, 1557.1, 1235.1,
a hydrogen atmosphere (55 psig) for 1.5 h; the mixture was 953.1, 617.2, 559.1, 331.0, 210.9. Anal. Calcd for
filtered through a pad of Celite and the residue and pad were C90H125ClN2O56: C, 49.89, H, 5.82, Cl, 1.64, N, 1.29. Found: C,
washed with EtOH. The combined filtrate and washings were 50.05, H, 5.68, Cl, 1.89, N, 1.37.
evaporated and the residue was coevaporated with EtOH sev-
N-[1,3-Bis-O-(2,3,6,2′,3′,4′,6′-hepta-O-acetyl-β-lactosyl)-2-
eral times to obtain 6 as a white solid (4.685 g, 98%); mp
120 °C; νmax (KBr): 3392.65 (br, NH, NH3+), 2943.99 (CH),
1757.32 (br, OAc), and 1672.98 (CONH) cm–1; 1H NMR (300
MHz, CDCl3) δ: 1.23–1.35 (m, 2 H, CH2-4), 1.5–1.62 (m, 4 H,
CH2-3,5), 1.93–2.25 (multiple s’s, 65 H, CH2-2, 21 OAc),
3.58–3.68 (m, 5 H, H-6′(3), CH2-5), 3.72–3.98 (m, 8 H, CH2-6,
H-4(3), H-6′(3)), 4.01–4.2 (m, 12 H, TRIS CH2O, H-6(6)),
4.35–4.58 (m, 9 H, H-1(3), H-5′(3), H-1′(3)), 4.78–4.88 (m, 3
H, H-2,2,2), 4.91–5.01 (m, 3 H, H-3′,3′,3′), 5.05–5.19 (m, 6 H,
H-2′(3), H-3(3)), 5.35 (br s, 3 H, H-4′,4′,4′), 6.05 (br s, 1 H,
HNCO), 8.08 (br s, 3 H, NH3+); ESIMS (positive-ion) m/z:
2090.9 (M–Cl)+, 1800.8, 1512.8, 1492.8, 1234.7, 1067.1,
940.8.
{(2,3,6,2′,3′,4′,6′-hepta-O-acetyl-β-lactosyl)methyl}prop-2-
yl]-6-[N-(bromoacetyl)amino]hexanamide (8)
Compound 8 was prepared from 6 in a manner analogous
to that for chloroacetamide 7 by using bromoacetyl bromide
instead of chloroacetyl chloride. The bromoacetamide 8 was
isolated, after purification by column chromatography using
EtOAc as eluent, as a white solid (2.246 g, 72%); Rf 0.4
(EtOAc); mp 118 °C; [α]D –11.25° (c 9.25, CHCl3); νmax
(KBr): 3385.53 (NH) 3023.26, 2942.23 (CH) 1749.98 (br,
OAc), 1673.94 (CONH), 1534.34, 1432.09, 1370.31, 1200,
1057.55, and 755.69 cm–1; 1H NMR (400 MHz, CDCl3) δ: 1.2–
1.3 (m, 2 H, CH2-4), 1.41–1.58 (m, 4 H, CH2-3,5), 1.87–2.10
(multiple s’s, 65 H, CH2-2, 21 OAc), 3.20 (qt, 2 H, J6,5 ≈ J6,NH
6.61 Hz, CH2-6), 3.5–3.54 (m, 3 H, H-6′(3)), 3.56 (d, 3 H, J5,4
=
=
N-[1,3-Bis-O-(2,3,6,2′,3′,4′,6′-hepta-O-acetyl-β-lactosyl)-2-
{(2,3,6,2′,3′,4′,6′-hepta-O-acetyl-β-lactosyl)methyl}prop-2-
yl]-6-[N-(chloroacetyl)amino]hexanamide (7)
10.38 Hz, H-5,5,5), 3.70 (t, 3 H, J4,3 ≈ J4,5 = 9.41 Hz, H-4,4,4),
3.79 (s, 2 H, CH2Br), 3.81 (t, 3 H, J6′,6′ ≈ J6′,5′ = 6.69 Hz, H-
6′(3)), 3.95–4.11 (m, 12 H, TRIS CH2O, H-6(6)), 4.30 (d, 3 H,
J1,2 = 7.9 Hz, H-1,1,1), 4.35 (d, 3 H, J5′,6′ = 11.02 Hz, H-
5′,5′,5′), 4.41 (d, 3 H, J1′,2′ = 7.87 Hz, H-1′,1′,1′), 4.75 (dd, 3 H,
A mixture of 6 (3 g, 1.41 mmol) and dry triethylamine (415
µL, 2.96 mmol) in dry CH2Cl2 (25 mL) was cooled to –20 °C
and freshly distilled chloroacetyl chloride (125 µL, 1.55
mmol) was introduced dropwise under an argon atmosphere.
The reaction mixture was stirred for 1.5 h, warmed to room
temperature, and diluted with CH2Cl2 (20 mL); the organic
layer was washed sequentially with dilute HCl (10 mL) and
water (2 × 20 mL), dried (Na2SO4), and evaporated to dryness.
The residue was chromatographed using EtOAc as eluent to
afford 7 as a white solid (2.256 g, 84%); Rf 0.42 (EtOAc); mp
107 °C; [α]D –14.3° (c 0.65, CHCl3); νmax (KBr): 3380.53 (NH)
2927.21 (CH) 1749.26 (br, OAc), 1670.0 (CONH), 1530.41,
J2,3 = 9.32 and J2,1 = 8.28 Hz, H-2,2,2), 4.87 (dd, 3 H, J3′,2′
=
10.4 and J3′,4′ = 3.37 Hz, H-3′,3′,3′), 5.0 (dd, 3 H, J2′,3′ = 10.3
and J2′,1′ = 7.94 Hz, H-2′,2′,2′), 5.07 (t, 3 H, J3,4 ≈ J3,2 = 9.35 Hz,
H-3,3,3), 5.25 (d, 3 H, J4′,3′ = 3.25 Hz, H-4′,4′,4′), 5.71 (s, 1 H,
HNCO), 6.67 (t, 1 H, JNH,CH -6, = 5.6 Hz, HNCOCH2Br); 13C
2
NMR (100 MHz, CDCl3) δ: 20.45, 20.54, 20.59, 20.68, 20.85,
21.38, 21.51 (OAc), 24.68 (CH2-3), 25.88 (CH2-4), 28.65
(CH2-5), 29.11 (CH2Br), 36.30 (CH2-2), 39.73 (CH2-6), 58.83,
60.59 (TRIS CH2O), 60.23 (Cquat), 61.75 (C-5′), 66.44 (C-4′),
68.22 (C-6), 68.88 (C-5), 70.46 (C-6′), 70.52 (C-2′), 70.77 (C-
3′), 71.46 (C-2), 72.29 (C-6′), 72.49 (C-3), 75.93 (C-4),
100.56 (C-1), 100.81 (C-1′), 165.37 (HNCOCH2Br), 168.85,
169.37, 169.44, 169.85, 169.92 (OAc), 172.82 (HNCO);
ESIMS (positive-ion) m/z: expected for (M+Na)+ and (M+H)+:
2233.6 and 2211.6, respectively; found: 2234.1 and 2212.1;
also 2215.1, 2214.0, 2213.0, 2211.2, 2210.1, 2186.3, 2031.5,
1725.4, 1592.5, 1127.7, 618.9, 559.0, 330.9, 210.9. Anal.
Calcd for C90H125BrN2O56: C, 48.89, H, 5.70, Br, 3.61, N, 1.27.
Found: C, 49.06, H, 5.75, Br, 3.81, N, 1.27.
1
1369.93, and 1223.37 cm–1; H NMR (400 MHz, CDCl3) δ:
1.22–1.31 (m, 2 H, CH2-4), 1.47–1.57 (m, 4 H, CH2-3,5),
1.87–2.10 (multiple s’s, 65 H, CH2-2, 21 OAc), 3.24 (qt, 2 H,
J6,5 ≈ J6,NH = 6.7 Hz, CH2-6), 3.52–3.60 (m, 3 H, H-6(3)), 3.58
(d, 3 H, J5,4 = 10.33 Hz, H-5,5,5), 3.71 (t, 3 H, J4,3 ≈ J4,5 = 9.43
Hz, H-4,4,4), 3.82 (t, 3 H, J6′,6′ ≈ J6′,5′ = 6.91 Hz, H-6′(3)), 3.98
(s, 2 H, CH2Cl), 4.0–4.12 (m, 12 H, TRIS CH2O, H-6(6)), 4.32
(d, 3 H, J1,2 = 7.93 Hz, H-1,1,1), 4.37 (d, 3 H, J5′,6′ = 10.6 Hz, H-
5′,5′,5′), 4.43 (d, 3 H, J1′,2′ = 7.9 Hz, H-1′,1′,1′), 4.77 (dd, 3 H,
J2,3 = 9.57 and J2,1 = 8.05 Hz, H-2,2,2), 4.89 (dd, 3 H, J3′,2′
=
10.4 and J3′,4′ = 3.41 Hz, H-3′,3′,3′), 5.03 (dd, 3 H, J2′,3′ = 10.35
and J2′,1′ = 7.9 Hz, H-2′,2′,2′), 5.09 (t, 3 H, J3,4 ≈ J3,2 = 9.37 Hz, Ethyl 6-(chloroacetamido)hexanoate (9)
H-3,3,3), 5.27 (d, 3 H, J4′,3′ = 3.34 Hz, H-4′,4′,4′), 5.72 (s, 1 H,
Hexanoate 9 was prepared from ethyl 6-aminohexanoate
HNCO), 6.74 (t, 1 H, JNH,CH2-6, = 5.6 Hz, NHCOCH2Cl); 13C by following the procedure described for the preparation of
NMR (100 MHz, CDCl3) δ: 20.39, 20.51, 20.60, 20.65, 20.75 chloroacetamide 7 from 6. Chromatography (1:4 EtOAc–hex-
Bhat, Vaino, and Szarek / Phosphonoacetate–Trilactoside Conjugate

340
anes) of the crude product obtained after processing of the 8.33 Hz, H-2,2,2), 4.90 (dd, 3 H, J3′, 2′ = 10.38 and J3′, 4′ = 3.28
reaction mixture afforded hexanoate 9 as as an oil (87%); Rf Hz, H-3′,3′,3′), 5.04 (dd, 3 H, J2′, 3′ = 10.13 and J2′, 1′ = 8.02 Hz,
1
0.24 (3:2 hexanes–EtOAc); H NMR (200 MHz, CDCl3) δ: H-2′,2′,2′), 5.097 (t, 3 H, J3, 4 ≈ J3, 2 = 9.35 Hz, H-3,3,3), 5.285
1.08 (t, 3 H, J = 6.4 Hz, OCH2CH3), 1.14–1.29 (m, 2 H, CH2- (d, 3 H, J4′, 3′ = 3.11 Hz, H-4′,4′,4′), 5.79 (s, 1 H, HNCO), 6.81
4), 1.32–1.56 (m, 4 H, CH2-5, CH2-3), 2.14 (t, 2 H, J = 7.77 (t, 1 H, JNH, CH2-6 = 5.8 Hz, HNCOCH2P); 13C NMR (100 MHz,
Hz, CH2-2), 3.13 (td, 2 H, JNH, 6 ≈ J5, 6 = 5.94 Hz, CH2-6), 3.87 CDCl3) δ: 16.0 and 16.07 (POCH2CH3), 20.25, 20.41, 20.53,
(s, 2 H, CH2Cl), 3.95 (q, 2 H, J = 6.4 Hz, 2 H, OCH2CH3), 6.81 20.62, 20.68, 20.77 (OAc), 24.7 (CH2-3), 26.20 (CH2-4), 28.98
(br s, 1 H, NH); 13C NMR (50 MHz, CDCl3) δ: 13.76 (CH2-5), 33.32 (CH2-2), 35.4 (d, JC, P = 128.0 Hz, CH2-P),
(OCH2CH3), 24.01 (CH2-3), 25.8 (CH2-4), 28.49 (CH2-5), 39.57 (CH2-6), 58.9 and 60.65 (TRIS CH2O), 60.91 (Cquat),
33.51 (CH2-2), 39.2 (CH2-6), 42.2 (CH2Cl) 59.80 (OCH2CH3), 61.85 (C-5′), 62.63 and 62.70 (POCH2CH3), 66.52 (C-4′),
165.86 (CONH), 173.1 (COO).
68.34 (C-6), 68.97 (C-5), 70.43 (C-6′), 70.53 (C-2′), 70.88 (C-
3′), 71.56 (C-2), 72.41 (C-6′), 72.57 (C-3), 76.06 (C-4),
100.66 (C-1), 100.92 (C-1′), 164.3 (HNCOCH2P), 168.95,
169.46, 169.53, 169.95, 170.02, 170.21, 170.23 (OAc), 173.1
(HNCO); 31P NMR (162 MHz, CDCl3) δ: 23.69; ESIMS (posi-
tive-ion) m/z: expected for (M+Na)+ and (M+H)+: 2290.7 and
2268.7, respectively; found: 2290.3 and 2268.8; also 2226.9,
2155.4, 2058.1, 1980.5, 1812.6, 1671.1, 1649.4, 1582.4,
1341.4, 1251.2, 1235.2. Anal. Calcd for C94H135N2O59P: C,
49.78, H, 5.99, N, 1.23, P, 1.36. Found: C, 49.66, H, 5.83, N,
1.18, P, 1.12.
Ethyl 6-[(diethyl phosphonato)acetamido]hexanoate (10)
A mixture of chloroacetamide 9 (2.212 g, 9.4 mmol) and
triethyl phosphite (10 mL) was heated at 100 °C under argon
for 5 h and the excess of (EtO)3P was removed under high
vacuum. The crude product was subjected to column chroma-
tography on silica gel using EtOAc as eluent, and the phos-
phonatoacetamide 10 was obtained as a viscous oil (2.452 g,
1
78%); Rf 0.5 (1:9 MeOH–CHCl3); H NMR (400 MHz,
CDCl3) δ: 1.075 (t, J = 7 Hz, 3 H, CO2CH2CH3), 1.15–1.23 (m,
8 H, CH2-4, 2 POCH2CH3), 1.31–1.40 (m, 2 H, CH2-5), 1.42–
1.5 (m, 2 H, CH2-3), 2.12 (t, 2 H, J = 7.45 Hz, CH2-2), 2.69 (d,
2 H, JH,P = 20.8 Hz, CH2P), 3.07 (td, 2 H, JNH, 6 ≈ J5, 6 = 6.1 Hz,
CH2-6), 3.75 (m, 6 H, CO2CH2CH3, 2 POCH2CH3), 6.98 (br t,
1 H, J = 6.1 Hz, NH); 13C NMR (100 MHz, CDCl3) δ: 13.41
(CO2CH2CH3), 15.46 and 15.58 (POCH2CH3), 23.75 (CH2-3),
25.53 (CH2-4), 28.21 (CH2-5), 33.27 (CH2-2), 34.33 (d, JC, P
131.25 Hz, CH2P), 38.74 (CH2-6), 59.27 (CO2CH2CH3), 61.65
and 61.78 (POCH2CH3), 163.45 (CONH), 172.5 (CO2Et); 31
N-[1,3-Bis-O-(2,3,6,2′,3′,4′,6′-hepta-O-acetyl-β-lactosyl)-2-
{(2,3,6,2′,3′,4′,6′-hepta-O-acetyl-β-lactosyl)methyl}prop-2-
yl]-6-[N-(phosphonoacetyl)amino]hexanamide (12)
Tris(trimethylsilyl) phosphite (380 µL, 1.13 mmol) was
added to a solution of 8 (1 g, 0.4523 mmol) in dry toluene (15
mL) at 60 °C, with argon bubbling through the solution. The
bubbling mixture was heated to 120 °C and held at that tem-
perature for 4 h, then cooled to room temperature and treated
with a solution of pyridine (0.5 mL) in methanol (2 mL). The
solvent was evaporated and the residue was dissolved in 1:1
(v/v) MeOH–H2O (10 mL); solid NaOH (0.75 g, 40 equiv)
was added and the reaction mixture was stirred overnight. The
mixture was acidified to ca. pH 3 by adding dilute HCl and
=
P
NMR (162 MHz, CDCl3) δ: 23.59; ESIMS (positive-ion) m/z:
expected for (M+Na)+ and (M+H)+: 360 and 338, respectively;
found: 360.2 and 338.3; also 283.1, 225.0, 166.9.
N-[1,3-Bis-O-(2,3,6,2′,3′,4′,6′-hepta-O-acetyl-β-lactosyl)-2-
{(2,3,6,2′,3′,4′,6′-hepta-O-acetyl-β-lactosyl)methyl}prop-2-
yl]-6-[N-{(diethyl phosphonato)acetyl}amino]hexanamide (11) then concentrated to dryness by lyophilization; the residue
Phosphonatoacetamide 11 was prepared from the bromo- was dissolved in a minimum amount of water and the solution
acetamide 8 by following the procedure described above for was subjected to size-exclusion chromatography using Bio-
the preparation of phosphonatoacetamide 10 from chloro- Gel P2 and eluting with distilled and degassed water. The
acetamide 9. Phosphonatoacetamide 11 was also made start- fractions containing 12 were pooled and lyophilized to afford
ing from the chloroacetamide 7 by adding a catalytic amount a fluffy white solid (601 mg, 76%); Rf 0.4 (reverse phase TLC,
of lithium iodide (10 mol%) to the reaction mixture. Column 2:3 MeOH–H2O; detection by spraying with anisaldehyde–
chromatography of the crude product on silica gel using 2:3 H2SO4 reagent followed by heating to ca. 150 °C); mp 188 °C
CH2Cl2–EtOAc as eluent afforded pure phosphonate 11 as a (dec); [α]D –16.4° (c 1.25, H2O); 1H NMR (500 MHz, D2O) δ:
white solid (94% from 8 and 90% from 7); Rf 0.04 (EtOAc); 1.21–1.29 (m, 2 H, CH2-4), 1.43 (qt, 2 H, J5,4 ≈ J5,6 = 7.5 Hz,
mp 102 °C; [α]D –13.56° (c 15.25, CHCl3); νmax (thin film): CH2-5), 1.46–1.54 (m, 2 H, CH2-3), 2.16 (br t, 2 H, J2,3 = 7.18
3395 (NH), 2983.85, 2938.55, 1752.26 (OAc), 1672.83 Hz, CH2-2), 2.60 (d, 2 H, JP,H = 20.32 Hz, CH2P), 3.08 (t, 2 H,
1
(CONH), 1538.38, 1433.93, 1370.48, and 1229.84 cm–1; H J6,5 = 6.9 Hz, CH2-6), 3.22–3.27 (m, 3 H, H-2,2,2), 3.42–3.46
NMR (500 MHz, CDCl3) δ: 1.18–1.25 (m, 2 H, CH2-4), 1.28 (m, 3 H, H-2′,2′,2′), 3.47–3.51 (m, 3 H, H-4′,4′,4′), 3.52–3.59
(overlapping t’s, 6 H, J = 7.0 Hz, 2 POCH2CH3), 1.41–1.54 (m, (m, 12 H, H-5(3), H-3′(3), unexchanged OHs, NH), 3.61–3.64
4 H, CH2-3,5), 1.85–2.15 (multiple s’s, 65 H, CH2-2, 21 OAc), (m, 3 H, H-5′,5′,5′), 3.65–3.75 (m, 12 H, TRIS CH2O, H-6(6)),
2.79 (d, 2 H, JH, P = 20.61 Hz, CH2P), 3.20 (qt, 2 H, J6, 5 ≈ J6, NH 3.79–3.85 (m, 6 H, H-3(3), H-4(3)), 3.85–3.91 (m, 3 H, H-
= 6.61 Hz, CH2-6), 3.50–3.56 (m, 3 H, H-6′(3)), 3.58 (d, 3 H, 6′(3)), 4.16 (d, 3 H, J6′,6′ = 10.47 Hz, 3 H, H-6′(3)), 4.345 (d, 3
J5, 4 = 10.4 Hz, H-5,5,5), 3.72 (t, 3 H, J4, 3 ≈ J4, 5 = 9.4 Hz, H- H, J1′,2′ = 7.97 Hz, H-1′,1′,1′), 4.384 (d, 3 H, J1,2 = 7.95 Hz, H-
4,4,4), 3.83 (t, 3 H, J6′, 6′ ≈ J6′, 5′ = 6.69 Hz, H-6′(3)), 3.99–4.16 1,1,1); 13C NMR (100 MHz, D2O) δ: 25.12 (CH2-3), 25.82
(m, 12 H, TRIS CH2O, H-6(6)), 4.33 (d, 3 H, J1,2 = 7.9 Hz, H- (CH2-4), 28.24 (CH2-5), 36.58 (CH2-2), 37.5 (d, JC,P = 120 Hz),
1,1,1), 4.38 (d, 3 H, J5′, 6′ = 11.33 Hz, H-5′,5′,5′), 4.44 (d, 3 H, 39.83 (CH2-6), 57.73, 59.98 (TRIS CH2O), 60.23 (Cquat),
J1′, 2′ = 7.84 Hz, H-1′,1′,1′), 4.78 (dd, 3 H, J2,3 = 9.23 and J2,1
=
60.39, 60.79, 61.02, 61.31 (C-6,6′), 68.86 (C-4), 71.25 (C-2′),
Israel Journal of Chemistry 40 2000

341
72.98 (C-2), 73.01 (C-3), 74.53 (C-3′), 75.07 (C-5′), 75.64 (C-
5), 78.75 (C-4′), 103.01 (C-1′), 103.25 (C-1), 170.5 (HNCO),
177.61 (HNCOCH2P); 31P NMR (121.5 MHz, D2O) δ: 15.64;
ESIMS (positive-ion) m/z: expected for (M)+: 1327.4; found:
1327.3; also 1329.2, 1328.1, 1326.2, 1325.2, 1164.9, 1079.5,
1003.1, 754.8, 565.7, 120.7. Anal. Calcd for C48H85N2O38P·1.5
H2O: C, 42.51, H, 6.54, N, 2.065, P, 2.28. Found: C, 42.22, H,
6.31, N, 2.41, P, 2.76.
Phair, J.P. Am. J. Epidemiol. 1996, 143, 943. (f)
Rosenberg, H.S.; Kohl, S.; Vogler, C. Monogr. Pathol.
1981, 133.
(9) Mao, J.C.-H.; Otis, E.R.; von Esch, A.M.; Herrin, T.R.;
Fairgrieve, J.S.; Shipkowitz, N.L.; Duff, R.G.
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Acknowledgments. The authors are grateful to the Natural
Sciences and Engineering Research Council of Canada for
financial support in the form of a grant to W.A.S. and to Mr.
Austin Chen for some preliminary studies.
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1
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Bhat, Vaino, and Szarek / Phosphonoacetate–Trilactoside Conjugate