N-Monoalkylation of Tetra-O-benzyl-D-arabinonamide: Synthesis of Some Open-Chain Analogues of N-Acetylneuraminic Acid and Their Evaluation as Sialidase Inhibitors

Article abstract of DOI:10.1002/(sici)1522-2675(19981007)81:10<1896::aid-hlca1896>3.0.co;2-5

N-Arabinonoylglycine 2, its phospho analogue (arabinonoylamino)methylphosphonate 14, N-arabinonoyltaurine salt 18, and [2-(arabinonoylamino)ethylidene]bis[phosphonic acid] 22 have been synthesized from D-arabinose in seven (2 or 14), and eight steps (18 o

Full text of DOI:10.1002/(sici)1522-2675(19981007)81:10<1896::aid-hlca1896>3.0.co;2-5

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Helvetica Chimica Acta ± Vol. 81 (1998)
N-Monoalkylation of Tetra-O-benzyl-d-arabinonamide: Synthesis of Some
Open-Chain Analogues of N-Acetylneuraminic Acid and Their Evaluation as
Sialidase Inhibitors
by Thomas Storz1) and Andrea Vasella*
Laboratorium für Organische Chemie, ETH-Zentrum, Universitätstr. 16, CH-8092 Zürich
N-Arabinonoylglycine 2, its phospho analogue (arabinonoylamino)methylphosphonate 14, N-arabino-
noyltaurine salt 18, and [2-(arabinonoylamino)ethylidene]bis[phosphonic acid] 22 have been synthesized from
d-arabinose in seven (2 or 14), and eight steps (18 or 22a), respectively. With the exception of the salt 22b, none
of these compounds showed a significant inhibitory activity in vitro against the sialidases of Vibrio cholerae,
Salmonella typhimurium, or Influenza A (N9), or B (B/Lee/40) virus. Ammonolysis of the oxosulfonate 8
obtained by oxidation of the hydrogensulfite adduct 7 of 2,3,4,5-tetra-O-benzyl-aldehydo-d-arabinose (6)
yielded the primary amide 9 (64% from 6), which was alkylated with the triflates 10 or 11 of benzyl glycolate and
dibenzyl hydroxymethylphosphonate, respectively, to give the protected N-arabinonoylglycinate 12 and the
(arabinonoylamino)methylphosphonate 13 (45 and 90%, resp.). N-Alkylation of 9 with 2-bromoethyl triflate 15
followed by nucleophilic displacement with sodium sulfite yielded the protected taurine analogue 17 (21% from
9), whereas the protected tetraethyl bis[phosphonate] 20 was formed in 90% yield by 1,4-addition of 9 to
tetraethyl ethenylidenebis[phosphonate] 19. Debenzylation of 12 and 13, followed by purification by reversed-
phase HPLC gave the triethylammonium salt of N-(d-arabinonoyl)glycine (2) and triethylammonium (d-
arabinonoylamino)methylphosphonate (14b), respectively, whereas the deprotection of 17 afforded the N-(d-
arabinonoyl)taurine salt 18. Debenzylation of 20, followed by treatment with Me3SiBr and hydrolysis of the
resulting silyl ester gave the bis[phosphonic acid] 22a (3 steps, 88%).
1. Introduction. ± In the context of the reported inhibitor activity of the d-lactone 1
against several sialidases (B/Berkeley/3/69) [1], we became interested in the synthesis
of the hydrolysis product 2, derived from d-arabinonamide, and its analogues as
potential inhibitors of the neuraminidases of Vibrio cholerae and Salmonella
typhimurium. The activity of 1 is surprising, considering the absence of an acidic
function in 1, known to be important for the inhibitory activity [2] [3], and of the
pyranoside ring in its hydrolysis product 2, both structural elements characterizing the
archetypical inhibitor 3 (DANA) [4] [5]. Indeed, the Glaxo group has reported the
inactivity of 2 as inhibitor against Influenza sialidases [6]. The glycine derivative 2 was
prepared by treating dicyclohexylcarbodiimide (DCC)-activated arabinonic acid with
tert-butyl glycinate. Following a different strategy for the synthesis of 2, i.e.,
N-monoalkylation of the benzyl-protected arabinonamide 9 (cf. Scheme 2), we have
developed a convenient large-scale preparation of this amide. In the following, we
report the experimental details and the results of the inhibition of additional
sialidases.
1
)
New address: Novartis Pharma AG, Chemical & Analytical Development, Process R&D, P. O. Box, CH-
4002 Basel.

Helvetica Chimica Acta ± Vol. 81 (1998)
1897
2. Results and Discussion. ± Selective N-alkylations of carbohydrate-derived
primary amides have not been reported to date. Examples of intermolecular mono-N-
alkylations of primary amides are scarce, and most of them involve acidic amides, such
as b-heteroaryl-amide [7], a-(trifluoromethyl)-b-aryl-amide [8], and trifluoro- or
trichloroacetamides [9 ± 11]. This may be due to the formation of mixtures by mono-
and dialkylation [12] (O- and N-alkylation2)), by epimerization in a-position [13] [18],
and by b-elimination [18][19]. However, N-alkylation may be advantageous when the
electrophile is readily accessible.
While attempts to perbenzylate potassium arabinonate led to mixtures, the
benzylated arabinose dithioacetal 5 [20] was readily hydrolyzed to the aldehyde 6 3)
(85% from the dithioacetal 4 [22]; see Scheme 1). Considering that 2,3,4,5,6-penta-O-
benzylgluconoyl chloride cyclizes spontaneously to the corresponding 1,4-lactone [23],
we avoided the preparation of an acyl chloride. The heavy-metal-free oxidation of
aldehyde hydrogen sulfite adducts, described by Wuts and Bergh in 1986 [24] and
apparently fallen into oblivion, appeared ideally suited for our needs. The resulting acyl
sulfonates are mildly activated acyl derivatives that are readily transformed into esters
or amides [24]. Indeed, tetra-O-benzyl-d-arabinonamide 9 is readily accessible on a
preparative scale (> 30 g) by ammonolysis of the oxosulfonate 8 obtained by a Pfitzner-
Moffatt-type oxidation of the hydrogen sulfite adduct 7 (64% over three steps, no
purification of intermediates; Scheme 1).
The arabinonamide 9 was treated with BuLi at 788, and monoalkylated with the
triflate 10 [25] derived from benzyl glycolate to afford 45% of the glycine derivative 12.
The moderate yield reflects the instability of the triflate 10 under the basic reaction
conditions. Similarly, mono-N-alkylation by 11 [26] derived from dibenzyl hydroxy-
methylphosphonate yielded 90% of the phosphonate 13. No traces of a-epimerized,
dialkylated, or elimination products could be detected in either case. Both 12 and 13
were smoothly hydrogenolysed to the N-arabinonoylglycine 2 and the isosteric
(arabinonoylamino)methylphosphonate 14, respectively (Scheme 2).
Efforts to synthesize the b-sulfonate analogue 18 by alkylation of 9 with sodium 2-
bromoethanesulfonate failed under a range of conditions. The desired N-arabinonoyl-
taurine analogue 18a was, however, obtained by nucleophilic substitution with sodium
sulfite [27 ± 29] of the N-(2-bromoethyl)arabinonamide 16, followed by hydrogenolytic
2
)
Sometimes a-C-alkylated products are obtained as well; for reviews, see [13] [14]. The ratio of O- vs. N-
alkylated products is in many cases correctly predicted by Gompperꢀs rule [13] [15] [16]. For N-
monoadditions of primary amides to vinyl ethers, yielding a-alkoxy- or a-(aryloxy)-methyl amides, cf. [17].
The aldehyde 6 has been reported by Schreiber et al. [21] who prepared it by a de novo synthesis. However,
the reported specific rotation was ‡28, while we found ‡9.78 for a d-arabinose-derived sample (see
Exper. Part).
3
)

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Helvetica Chimica Acta ± Vol. 81 (1998)
Scheme 1
a) NaH, BnBr, Bn4NI (0.05 equiv.), DMF, 20 to 258; quant. b) HgCl2, CaCO3, MeCN/H2O, 258, 85%.
c) NaOH, SO2 (sat.), H2O/Et2O 3:5, 0 ± 258; quant. d) DMSO/Ac2O, 258, 24 h; quant. e) NH3 (sat.), 4 to 388;
64%.
Scheme 2
a) BuLi, 1.4 equiv. of TfOCH2COOBn (10), THF/DMPU 3:1, 85 to 208; 45%. b) BuLi, 1.4 equiv. of
TfOCH2PO3Bn2 (11), THF/DMPU 3:1, 85 to 108; 90%. c) H2 (10 bar), 20% Pd(OH)2/C, t-BuOH/H2O 4:1,
258; 91% of 2, or 82% of 14a.

Helvetica Chimica Acta ± Vol. 81 (1998)
1899
debenzylation of the intermediate 17 (Scheme 3). The bromide 16 was prepared by
monoalkylating 9 with excess 2-bromoethyl triflate4) (15) [30].
Finally, we synthesized the bis[phosphonate] 22 by mono-1,4-addition of 95) to
tetraethyl ethenylidenebis[phosphonate] 19 [36 ± 38]. Deprotection of the resulting 20,
first by hydrogenolysis to the tetrol 21 and then by transesterification with Me3SiBr and
hydrolysis of the silyl esters [35] [39 ± 42] gave the bis[phosphonic acid] 22a in 79%
overall yield from 9 (Scheme 3).
Scheme 3
a) t-BuLi, 3.1 equiv. of Br(CH2)2OTf (15), THF/DMPU 2:1, 78 to 258; 42% of 16 and 43% of 9. b) Na2SO3,
EtOH/H2O 3:1, 808; 50%. c) H2 (10 bar), 20% Pd(OH)2/C, t-BuOH/H2O 4:1, 258; 88%. d) t-BuOK, t-BuOH,
40 ± 508; 90%. e) H2 (10 bar), 20% Pd(OH)2/C, t-BuOH/H2O 4:1, 258; quant. f ) 1. Me3SiBr, CH2Cl2, 10 to
258; 2. MeOH/H2O 6:1, 258; 88%.
Compounds 2 ´ NEt3, 14b, 18b, and 22b were tested against the sialidases of Vibrio
cholerae, Salmonella typhimurium (LT2 strain), Influenza A (N2), and Influenza B (B/
Lee/70) virus, with DANA (3) [4] [5] as a reference inhibitor and using the Warren-
thiobarbituric acid assay [43 ± 45]6). Neither N-arabinonoylglycine 2 nor the isosteric
phosphonate 14b or the homologated sulfonate 18b showed significant inhibition
against any of the four sialidases tested. The homologated bis[phosphonate] 22b
showed a weak inhibition of the Influenza B enzyme (Table).
4
)
Treatment of 9 with BuLi and only 1.2 equiv. of 15 led to the corresponding N-vinyl-amide (data not
shown), isolated in low yields besides starting material.
For 1,4-additions to ethenylidenephosphonates, see [31 ± 35].
We thank Prof. Laver (Influenza Research Unit, Australian National University, Canberra, Australia) for
his help in establishing the assay and for generous gifts of Influenza A and B, and S. typhimurium sialidases.
5
6
)
)

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Helvetica Chimica Acta ± Vol. 81 (1998)
Table. Inhibition of Sialidases by 2 ´ NEt3, 14b, 18b, 22b, and 3. Enzyme activity in % of control at a uniform
inhibitor concentration of 2.5 ´ 10 3m.
Inhibitor
V. cholerae
S. typhimurium
Influenza A
Influenza B
2 ´ NEt3
14b
18b
22b
3
not tested
100
90
90
10
not tested
100
100
100
20
100
100
90
100
5
100
80
100
50
5
We thank the Swiss National Science Foundation and F. Hoffmann-La Roche AG, Basel, for financial
support.
Experimental Part
1. General. Reagents were obtained in the best available commercial quality. Solvents for reactions and
chromatography were distilled or dried according to standard procedures [46]. Molecular sieves were activated
by heating at 2008 for 48 h in high vacuum. Reactions involving alkyllithium were routinely conducted under Ar
in flame-dried glasware [47]. TLC: Merck silica gel 60 precoated (F254) aluminum plates; compound detection
by treating with ꢁMo-stainꢀ soln. (400 ml of 10% H2SO4, 20 g (NH4)6Mo7O24 ´ 6 H2O, and 0.4 g of Ce(SO4)2),
anisaldehyde stain (4 ml of anisaldehyde, 4 ml of conc. H2SO4, and 200 ml of AcOH) [48], or ninhydrin stain
[48] (sat. ninhydrin soln. in EtOH) and heating at ca. 2008. Flash chromatography (FC): Merck silica gel 60
(40 ± 63 mm). Anal. HPLC: Merck-Hitachi-system (HPLC Pump L-6200 A, D-2500 Chromato-integrator); 4 Â
250-mm columns; either refractometric (Merck differential refractometer RI-71 or LDC anal. refracto-monitor
IV) or UV detection (Merck UV/VIS detector L-4250; l 254 nm). Prep. HPLC: Dupont-8800 or Merck-Hitachi
system (HPLC pump L-6250); 20 Â 250-mm columns: refractometric (Merck differential refractometer RI-71)
or UV detection (Merck-Hitachi UV detector L-4000). HPLC Columns: RP-18 (anal.: Merck LiChroSpher 100,
5 mm; prep. Merck Hibar LiChroSorb-RP 18, 7 mm); RP-8 (anal.: Merck LiChroSpher 100, 5 mm; prep.: Merck
Hibar LiChroSorb-RP 8, 7 mm); CN (Macherey-Nagel Nucleosil-5-CN, 5 mm); NH2 (anal.: Merck LiChroSpher
100, 5 mm; prep.: Merck Hibar LiChroSorb-NH2, 7 mm); SAX (Knauer SpheriSorb-SAX, 5 mm), silica gel 60
(Knauer SpheriSorb-SW, 5 mm). M.p.: uncorrected; Büchi 510 apparatus; in open capillaries. Optical-rotation
values [a]2D5 1-dm cuvette. IR Spectra [cm 1]: Perkin-Elmer 297
: Perkin-Elmer 241 polarimeter, in a
spectrometer. NMR Spectra: Varian-XL-200 spectrometer (1H: 200 MHz, 13C: 50 MHz, 31P: 81 MHz); Varian
XL-300 spectrometer (1H: 300 MHz, 13C: 75 MHz, 31P: 121.5 MHz); Bruker-AMX-400 spectrometer (1H:
400 MHz, 13C: 100 MHz, 31P: 162 MHz); Bruker AMX-500 spectrometer (1H: 500 MHz, 13C: 125 MHz, 31P:
202.5 MHz), and Bruker AMX-600 spectrometer (1H: 600 MHz, 13C: 150 MHz, 31P: 243 MHz); in deuterated
solvents. d Values in ppm relative to TMS as internal standard or external reference (solvent signal); for 31P-
NMR external reference H3PO4, coupling constants J (first order) in Hz; in ambiguous cases, assignments were
secured by appropriately chosen 2D experiments, multiplicities by DEPT experiments; both 13C- and 31P-NMR
spectra broadband-decoupled with respect to the 1H nucleus. Mass spectra: Varian MAT-112S or Finnigan MAT-
90 (EI: 70 eV; CI: isobutane (C4H10) or NH3 as carrier gas, ionized with 100±150 eV), VG ZAB2-SEQ (FAB:
bombardment with 35-keV Cs-atoms (3-nitrobenzyl-alcohol, (NOBA) matrix), or Finnigan TSQ 700 triple stage
quadrupole spectrometer (ESI: Analytica ion source, Brandford, Inc.; N2 as drying gas at 1108, 3.0 ± 3.2 kV U at
the electrospray probe); in (m/z) (rel. %). Elemental analyses were carried out by the Mikroanalytisches
Laboratorium, Laboratorium für Organische Chemie, ETH Zürich.
2. Syntheses. 2,3,4,5-Tetra-O-benzyl-d-arabinose (6). Molecular sieves (3 Š; 10 g) were added to a soln. of
d-arabinose diethyl dithioacetal 4 [22] (27.5 g, 107.25 mmol) in DMF (550 ml). Under cooling (ice bath), NaH
(Aldrich, 95%; 14.44 g, 571.64 mmol) was added in portions under vigorous stirring. After the H2 evolution had
ceased, Bu4NI (2.033 g, 5.50 mmol) was added, followed by dropwise addition of BnBr (55.9 ml, 471.94 mmol) in
DMF (150 ml) within 100 min. Stirring was continued at 08 for 75 min. The mixture was kept at 258 for 12 h,
warmed to r.t., and quenched by cautiously adding MeOH (50 ml), while stirring. After stirring for 2 h, the
brown suspension was poured into a NH4Cl/ice 1:1 (ca. 300 g). After addition of sat. aq. NaCl soln. (150 ml), the
suspension was extracted with Et2O (4 Â 450 ml). The Et2O phases were washed with sat. aq. NaCl soln. (4 Â
150 ml) and H2O (2 Â 150 ml). The aq. phases were extracted with Et2O (150 ml) and discarded. After drying
(MgSO4), the org. phases were taken to dryness under reduced pressure. Further drying in high vacuum (20 h):
crude 5 [20] as yellow oil (59.5 g, 90%). A vigourously stirred soln. of this material in MeCN/H2O 9:1 (800 ml)

Helvetica Chimica Acta ± Vol. 81 (1998)
1901
was treated with CaCO3 (48.27 g, 482.25 mmol). HgCl2 (115.22 g, 424.39 mmol) was added in portions within
15 min. The resulting white suspension was stirred vigourously under Ar at r.t. for 22 h. After suction-filtration
through a sand/Celite/sand bed, the clear filtrate was concentrated i.v. (Tꢀ 308) to a turbid, yellowish soln. The
filter cake was washed with AcOEt (5 Â 300 ml) and the filtrate added to the concentrated suspension, which
was then washed consecutively with 1m aq. KI (5 Â 180 ml) ( ! org. phase turned lemon-yellow), 30% aq.
Na2S2O3 soln. (2 Â 200 ml), and sat. aq. NaCl soln. (200 ml). The aq. phases were extracted with AcOEt (2 Â
120 ml). The combined org. phases were dried (Na2SO4), evaporated, and the residue was further dried (24 h)
at under high vacuum at 08 to yield a yellow oil (163.4 g) that was purified by FC (hexane/AcOEt 6:1 !1:1): 6 as
a slightly yellow honey (41.78 g, 85% over two steps). An anal. sample was obtained by HPLC (hexane/AcOEt
10:1, flow: 16 ml/min, detectionat 254 nm, tR 11.2 min). Rf (hexane/AcOEt 6:1) 0.32. Rf (toluene/AcOEt 5:1)
0.80. [a]2D1
ˆ
9.7 (c ˆ 1.092, CHCl3) ([21] [a]D
ˆ
2.1 (c ˆ 1.0, CHCl3). IR (CHCl3): 3060/3020/3000w, 2940/
1
2910w, 2860w-m, 1725s, 1492m-s, 1450/1360/1330/1250m, 1095/1075vs, 1025s, 695s. H-NMR (500 MHz, C6D6):
3.64 (dd, 2J(5,5') ˆ 10.6, 3J(5,4) ˆ 4.5 H C(5)); 3.71 (dd, 2J(5',5) ˆ 10.6, 3J(5',4) ˆ 3.0, H' C(5)); 3.88 (not
fully resolved, ddd, H C(4)); 4.06 (dd, 3J(2,1) ˆ 1.7, J(2,3) ˆ 4.0, H C(2)); 4.21 (dd, 3J(3,2) ꢁ 3.6, J(3,4) ꢁ
3
3
6.9,
H C(3)); 4.20 ± 4.35 (m, 4H, PhCH2); 4.47 ± 4.58 (m, 4H, PhCH2); 7.02 ± 7.33 (m, 20 arom. H);
9.66 (d, 3J(1,2) ˆ 1.7, H C(1)). 13C-NMR (125 MHz, C6D6): 69.21 (t, C(5)); 72.21, 73.25, 73.44, 74.19 (4
t, PhCH2); 78.27 (d, C(4)); 79.42 (d, C(3)); 85.03 (d, C(2)); 127.73, 127.75, 127.98, 128.53, 128.55, 128.60,
128.61 (7d, arom. C); 138.20, 138.70, 138.94, 139.02 (4 s, Cipso(Ph)); 201.27 (s, C(1)). CI-MS (NH3): 530(7),
‡
‡
‡
529(35, [M‡ 2 ‡ NH3] ), 528(100, [M ‡1 ‡ NH3] ), 421([M ‡ 2 ‡ NH3 BnOH] ), 420 (63, [M‡ 1 ‡
‡
NH3 BnOH] ), 403(10).
2,3,4,5-Tetra-O-benzyl-d-arabinonamide (9). SO2 Gas was passed at 08 through a soln. of NaOH (20.0 g,
0.500 mol) in H2O (150 ml) until the pH dropped to 7.0±6.5. A soln. of 6 (32.544 g, 63.73 mmol) in Et2O (250 ml) was
added dropwise at 08 under vigorous stirring. The ice bath was removed after 1 h. Stirring was continued at r.t.
for 63 h (TLC monitoring). Then, the aq. phase was extracted with Et2O (3 Â 50 ml). The combined Et2O phases
were washed with H2O dest. (50 ml), dried (Na2SO4), and evaporated. The residual yellow oil was co-evaporated
with dry benzene (3 Â 100 ml) and dried under high vacuum for 24 h: 7 (36.378 g, 93%) as a solid, yellow foam.
The foam was taken up in dry CH2Cl2 (250 ml) and evaporated, and the residue dried (37±408, 0.05 Torr) for ca. 18 h.
A soln. of this material in abs. DMSO (250 ml) under Ar was stirred in an ice-bath. Ac2O (44 ml, 465.5 mmol)
was injected through a septum, and the resulting soln. was stirred at r.t. for 24 h. The resulting soln. of 8 was
cooled to 48 and diluted with dry MeCN (150 ml). Dry NH3 was bubbled through the turbid soln. for ca.
30 min (Caution: strongly exothermic reaction!). The temp. of the yellow soln. reached ‡388. Stirring and cooling
were continued until the temp. fell to 15 ± 188. The soln. was poured onto NH4Cl/ice 1:1 (ca. 500 g). The pH was
immediately adjusted to 6.5 ± 7.0 with dil. AcOH. Extraction with Et2O (5 Â 300 ml). The combined org. phases
were washed with sat. aq. NaCl soln. (400 ml) and H2O (200 ml), dried (MgSO4), and evaporated. The residue was
further dried under high vacuum for 24 h to give crude 9 (41.18 g, 123%), as a brown honey which was purified
by FC (hexane/AcOEt 3:1 !1:1.25): 9 (21.36 g, 64%) as slighly yellow, microcrystalline solid. An anal. sample
was obtained by prep. HPLC (hexane/AcOEt 2:1, flow: 16 ml/min, detection at 254 nm, tR 16.6 min). M.p. 618.
Rf (hexane/AcOEt 1:1) 0.51. Rf (toluene/AcOEt 3:1) 0.27. [a]2D1 ˆ ‡5.1 (c ˆ 1.144, CHCl3). IR (CHCl3): 3510/
3395w-m, 3060/3020/3000w, 2960/2920/2860w, 1730w, 1685vs, 1565/1495/1452m, 1390/1370/1330/1310/1250w-m,
1190m(sh), 1150 ± 1080s(br.), 1068vs, 1028s, 698s. 1H-NMR (400 MHz, C6D6): 3.69 (dd, 2J(5,5') ˆ 10.7,
3
3
3J(5,4) ˆ 4.4, H C(5)); 3.78 (dd, 2J(5',5)ˆ 10.7, J(5',4)ˆ 2.1, H' C(5)); 4.00 (ddd, 3J(4,3) ˆ 8.4, J(4,5) ˆ 4.5,
3J(4,5') ˆ 2.15, H C(4)); 4.16 (d, 2Jgem ˆ 11.5, 1 H, PhCH2); 4.27 ± 4.32 (m, 4 H, PhCH2); 4.43 (d, 3J(2,3) ˆ 2.4,
H
C(2)); 4.50 (dd, 3J(3,2) ˆ 2.4, 3J(3,4) ˆ 8.4, H C(3)); 4.62 (d, 2Jgem ˆ 11.1, 1 H, PhCH2); 4.66 (d, 2Jgem
ˆ
11.9, 1 H, PhCH2); 4.83 (d, 2Jgem ˆ 11.1, 1 H, PhCH2); 6.39 (br. d, 2Jgem ˆ 3.57; with D2O ! br. s at 6.40
(ca. 30% exchange, 1 H, CONH2); 6.72 (br. d, 2Jgem ˆ 3.52; with D2O ! br. s at 6.71 (ca. 30% exchanged,
1 H, CONH2); 7.03 ± 7.16 (m, 14 arom. H); 7.24 ± 7.32 (m, 6, arom. H). 13C-NMR (100 MHz, C6D6):
69.31 (t, C(5)); 71.93, 73.37, 73.72, 75.13 (4t, PhCH2); 78.27 (d, C(4)); 79.87 (d, C(3)); 80.69 (d, C(2)); 127.54,
127.61, 127.66, 127.70, 127.87, 127.95, 127.98, 128.35, 128.47, 128.52, 128.57, 128.64 (12d, arom. C); 138.11,
‡
138.99, 139.08, 139.34 (4s, Cipso(Ph)); 174.84 (s, C(1)). CI-MS (NH3): 528(7), 527(37, [M ‡ 2] ), 526(100,
‡
[M ‡ 1] ), 166(8), 148(19). Anal. calc. for C33H35NO5 (525.65): C 75.41, H 6.71, N 2.66; found: C 75.53, H 6.90,
N 2.65.
7
)
The concentration of the BuLi solns. did not have to be determined beforehand, since deprotonation of 9
was very conveniently followed: the colour of the soln. changed from yellow to orange after addition of
exactly 1.0 equiv. of BuLi, as during the titration of BuLi with 1,3-diphenylacetone tosylhydrazone [49].

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Helvetica Chimica Acta ± Vol. 81 (1998)
2,3,4,5-Tetra-O-benzyl-N-[2-(benzyloxy)-2-oxoethyl]-d-arabinonamide ( ˆ Benzyl N-(2,3,4-Tetra-O-ben-
zyl-d-arabinonoyl)glycinate; 12). A soln. of 9 (520 mg, 0.989 mmol) in abs. THF/DMPU 14:8 (22 ml) at
858 was titrated with BuLi (Fluka, ca. 1.35m, in hexane, ca. 1.0 ml), until the colour of the soln. changed from
yellow to orange7). A soln. of benzyl [(trifluoromethyl)sulfonyloxy]acetate (10; 415 mg, 1.391 mmol) in abs.
THF (2 ml) was added after 5 min in one portion. Stirring was continued until the soln. had reached 258 (ca.
3.5 h). After 60 h at 258, the mixture was poured on sat. aq. NH4Cl soln. (25 ml) and diluted with Et2O
(25 ml). The org. phase was separated and the aq. phase extracted with Et2O (4 Â 30 ml). The combined org.
phases were washed with sat. aq. NaCl soln. (15 ml) and H2O (15 ml), dried (MgSO4), and evaporated, and the
residue was purified by FC (toluene/AcOEt 10:1): 12 (302 mg, 45%). Slightly yellowish oil. Rf (toluene/AcOEt
5:1) 0.46. [a]2D1
ˆ
10.4 (c ˆ 1.140, CHCl3). IR (CHCl3): 3405m, 3050/3020/2990/2970/2930/2895/2860w, 1742s,
1670s, 1520m, 1495w-m, 1450m, 1385/1355w, 1255/1235w-m, 1185m-s, 1100s(br.), 1065s, 1025m, 695s. 1H-NMR
(500 MHz, C6D6): 3.29 (dd, 2Jgem ˆ 18.0, 3J(NH) ˆ 4.6, 1 H, NCH2COOBn); 3.71 (dd, 2J(5,5') ˆ 10.7, J(5,4) ˆ
3
4.5, H C(5)); 3.81 (dd, 2J(5',5) ˆ 10.7, 3J(5',4) ˆ 2.15, H' C(5)); 4.06 ± 4.13 (m, 3J(NH) ˆ 7.1, Jgem ˆ 18.0,
2
2 H, H C(4), NCH2COOBn); 4.25 (d, 2Jgem ˆ 11.3, 1 H, PhCH2); 4.34 (s, 2 H, CH2Ph); 4.38 (d, 2Jgem ˆ 11.9,
1 H, CH2Ph); 4.49 ± 4.54 (m, 3J(2,3) ꢁ 2.55, 3J(3,4) ꢁ 8.2, 3 H, PhCH2, H C(2), H C(3)); 4.63 ± 4.89 (m, 5 H,
PhCH2); 6.94 (br. dd, 3J(N,H) ˆ 7.1, 4.7; with D2O no exchange, CONHCH2); 7.02 ± 7.16 (m, 18, arom. H);
7.26 ± 7.34 (m, 7, arom. H). 13C-NMR (125 MHz, C6D6): 40.74 (t, CH2N); 66.81 (t, PhCH2 (COOBn)); 69.43 (t,
C(5)); 72.00, 73.39, 73.94, 75.11 (4t, PhCH2 (OBn)); 78.31 (d, C(4)); 80.12 (d, C(3)); 80.94 (d, C(2)); 127.56,
127.61, 127.65, 127.67, 128.09, 128.30, 128.54, 128.55, 128.58, 128.65, 128.69, 128.71 (12d, arom. C); 136.03,
138.14, 139.04, 139.30, 139.36 (5s, Cipso(Ph)); 169.53 (s, C(1)); 171.52 (s, COOBn). CI-MS (NH3): 676(18),
‡
‡
675(35, [M ‡ 2] ), 674(100, [M ‡ 1] ). Anal. calc. for C42H43NO7 (673.81): C 74.87, H 6.43, N 2.08; found:
C 74.77, H 6.40, N 1.97.
2,3,4,5-Tetra-O-benzyl-N-[bis(benzyloxy)phosphinyl]methyl-d-arabinonamide ( ˆ Dibenzyl [(2,3,4,5-Tet-
ra-O-benzyl-d-arabinonoyl)amino]methylphosphonate; 13). A soln. of 9 (588 mg, 1.186 mmol) in abs. THF/
DMPU 2:1 (22.5 ml) at 788 was titrated with BuLi (Fluka, 1.4m, in hexane) until the colour of the soln.
changed from yellow to orange (ca. 1.20 ml) and stirred at 788 for 10 min. A soln. of ([bis(benzyloxy)phos-
phinyl]methyl trifluoromethanesulfonate (11; 548 mg, 1.291 mmol) in abs. THF (2 ml) was injected through a
septum. Stirring was continued until the cooling-bath temp. reached 258. After 19 h at 258 (TLC: incomplete
conversion), the soln. was cooled to 788, and t-BuLi (0.26 ml, Aldrich, 1.33m in pentane) and additional 11
(160 mg, 0.377 mmol) were added. Stirring was continued until the cooling-bath temp. had reached 108.
Workup was the same as described for 12. FC (toluene/AcOEt 3:1) of the crude product afforded 13 (811 mg, 90%)
as a colourless, viscous oil. An anal. sample was obtained by prep. HPLC (hexane/AcOEt 1.7:1, flow: 16 ml/min,
detection at 254 nm; tR 34.6 min). Rf (toluene/AcOEt 1:1) 0.60. [a]D21
3405m, 3050/3020/2990/2940/2920/2890/2860w, 1675s, 1515m-s, 1495w-m, 1450m, 1390/1375/1360/1330/1305w-m,
1240s, 1140 ± 1065s (br.), 1025/1005/995s, 695s. 1H-NMR (500 MHz, C6D6): 3.15 (ddd, 2Jgem ˆ 15.7, 2J(H,P) ˆ
ˆ
10.5 (c ˆ 0.917, CHCl3). IR (CHCl3):
3
3
10.2, J(N,H)ˆ 4.6; with P decoupling ! dd, 2Jgem ˆ 15.8, J(N,H)ˆ 4.6, 1 H, NCH2PO3Bn2); 3.68 (dd, 2J(5,5') ˆ
10.7, 3J(5,4) ˆ 4.5,
H
C(5)); 3.78 (dd, 2J(5',5) ˆ 10.7, 3J(5',4) ˆ 2.2, H' C(5)); 4.01 (ddd, 3J(4,3) ˆ 7.5,
3J(4,5) ˆ 4.7, 3J(4,5') ˆ 2.5, H C(4)); 4.09 (ddd, 2Jgem ˆ 15.75, 2J(H,P) ˆ 13.0, 3J(N,H) ˆ 8.0, P decoupling
! dd, 2Jgem ˆ 15.75, 3J(N,H) ˆ 8.0, 1 H, NCH2PO3Bn2); 4.15 (d, 2Jgem ˆ 11.15, 1 H, PhCH2); 4.31 (s, 2 H,
PhCH2); 4.35 (d, 2Jgem ˆ 11.1, 1 H, PhCH2); 4.36 (d, 2Jgem ˆ 11.8, 1 H, PhCH2); 4.47 ± 4.49 (m, H C(2),
H
C(3)); 4.59 (d, 2Jgem ˆ 11.4, 1 H, PhCH2); 4.69 (d, 2Jgem ˆ 11.8, 1 H, PhCH2); 4.73 (d, 2Jgem ˆ 11.4, 1 H,
PhCH2); 4.82 ± 4.92 (m (2 AB, strong signal overlap); P decoupling ! 1 AB, 4 H, P(OCH2Ph)2); 6.96 (br.
t, 3J(N,H) ꢁ 5.5, additional 3J(H,P) not resolved, CONHCH2); 6.98 ± 7.29 (m, 30, arom. H). 13C-NMR
(125 MHz, C6D6): 35.04 (dt, 1J(H,P) ˆ 155.6, CH2N); 67.71, 67.76 (2dt, 2J(C,P) ˆ 2.0, 2.1, P(OCH2Ph)2);
69.37 (t, C(5)); 69.37, 73.39, 73.97, 75.09 (4t, PhCH2 (OBn)); 78.32 (d, C(4)); 80.08 (d, C(3)); 80.95 (d, C(2));
127.57, 127.62, 127.63, 127.68, 128.08, 128.11, 128.45, 128.48, 128.50, 128.54, 128.58, 128.73, 128.75, (13d, arom.
C); 136.81 (d, 3J(C,P) ˆ 5.9, Cipso(Ph)); 137.92, 139.01, 139.21, 139.37 (4s, Cipso(OBn)); 171.17 (d, 3J(C,P) ˆ 4.3,
C(1)). 31P-NMR (205.8 MHz, C6D6): 24.15 (s). CI-MS (NH3): 801(13, [M ‡ 2] ), 800(25, [M ‡ 1] ), 386(9),
‡
‡
‡
296(30), 295(11), 278(10), 277(7), 109(8), 108(100, [BnOH] ). Anal. calc. for C48H50NO8P (799.90): C 72.07,
H 6.30, N 1.75; found: C 71.90, H 6.20, N 1.87.
N-(d-Arabinonoyl)glycine (2). A soln. of 12 (358 mg, 0.531 mmol) in t-BuOH (7 ml) was added to Pd(OH)2/
C (20%, 206 mg) in the same solvent (5 ml) that had been stirred under H2 at 10 bar for 30 min. The suspension
was diluted with H2O (3 ml), stirred vigourously under H2 at 10 bar until UV-active compounds were no longer
detected by TLC (ca. 110 h), diluted with H2O (5 ml), treated with Celite (ca. 50 mg), and centrifuged. After
collection of the supernatant, the sediment was thoroughly mixed with H2O (10 ml) and centrifuged again. This
procedure was repeated twice. The combined supernatants were lyophilized. The residue was taken up in H2O

Helvetica Chimica Acta ± Vol. 81 (1998)
1903
(ca. 2 ml), filled in a syringe, pressed through a membrane filter (0.2 mm, Merck Anotop) and again lyophilized.
The free carboxylic acid 2 was obtained as colourless foam (108 mg, 91%). Prep. HPLC on a derivatized RP-
phase (NH2, 0.1m (Et3NH)HCO3 buffer, pH 7.1 adjusted with AcOH, 1% MeCN, refract. detection, flow 5 ml/
min; tR 26 min) yielded the triethylammonium salt 2 ´ Et3N (152 mg, 86%). White powder. [a]D21
ˆ
25.9 (c ˆ
1.125, H2O). IR (KBr): 3400vs(br.), 2970/2930/2870m-w, 2720/2675/2490m, 1742s, 1655s, 1600s(br.), 1540m-s,
1470m, 1430/1400m-s, 1330/1315/1285w-m, 1170w, 1130/1115/1090/1055/1025s, 910/885w. 1H-NMR (500 MHz,
D2O): 1.24 (t, 3J ˆ 7.3, 9 H, MeCH2N); 3.16 (q, 3J ˆ 7.3, 6 H, MeCH2N); 3.64 (dd, 2J(5,5') ˆ 11.8, J(5,4) ˆ 6.1,
3
H
C(5)); 3.69 ± 3.76 (m, 2Jgem ˆ 17.2, 2 H, H C(4), NCH2COO ); 3.80 ± 3.95 (m, 2Jgem ˆ 17.5, 3J(3,4) ˆ 8.9,
3J(2,3) not resolved, 3J(5',4) ˆ 2.5, 3 H, NCH2COO , H' C(5), H C(3)); 4.42 (br. s, 3J(2,3) < 1, H C(2)).
13C-NMR (125 MHz, D2O): 10.95 (q, MeCH2N); 45.73 (t, NCH2COO ); 49.36 (t, MeCH2N); 65.70 (t, C(5));
73.28 (d, C(4)); 73.64 (d, C(3)); 74.23 (d, C(2)); 177.88 (s, C(1)); 179.14 (s, COO ). FAB-MS (neg. mode): 445
(32.5, [2M ‡ H ]), 223 (13.8, [M ‡ H] ), 222 (100, M ), 183 (14, glycerol matrix), 132 (13.8), 91 (15.4, glycerol
matrix). Anal. calc. for C13H28N2O7 ´ 2 H2O (360.40): C 43.32, H 8.94, N 7.77; found: C 43.65, H 8.34, N 7.98.
(d-Arabinonoylamino)methylphosphonate (14). To a suspension of Pd(OH)2/C (20%, 390 mg) in t-BuOH/
H2O 4:1 (10 ml), 13 (615 mg, 0.7688 mmol) was added. The suspension was stirred vigourously under H2 (9 bar),
until TLC failed to detect UV-active products (ca. 70 h). Workup followed the procedure described for 2. A
soln. of the crude lyophilisate in H2O (2 ml) was passed through a cation-exchange resin column (Amberlyst 15,
‡
Na form, ca. 5 g). Product-containing fractions were collected and lyophilized. The residue was taken up in
H2O dest. (3 ml), filled in a syringe and pressed through a membrane filter (0.2 mm, Merck Anotop). The filtered
soln. was lyophilized. The resulting sodium salt 14a (191 mg, 82%) was purified by prep. HPLC on a quaternary
ion exchange resin column (SAX; 0.1m (Et3NH)HCO3 buffer, pH 6.5 adjusted with AcOH, detection at 200 nm,
flow 10 ml/min, tR 13.2 min) to afford the triethylammonium salt 14b (118 mg, 33%; purification not optimized)
as colourless foam. Anal. HPLC (SAX, 0.1m (Et3NH)HCO3 buffer, pH 6.5 adjusted with AcOH, detection at
200 nm, flow: 2 ml/min): tR 4.8 min. [a]D21
ˆ
23.1 (c ˆ 1.116, H2O). IR (KBr): 3400vs(br.), 2970/2935/2850/
2840w, 2680m, 2490w-m, 1645s(br.), 1540m-s, 1475/1435/1400m, 1360w, 1300w(br.), 1170/1140/1080/
1035s(br.), 975/915m(br.). 1H-NMR (400 MHz, D2O): 1.27 (t, 3J ˆ 7.3, 13 H, MeCH2N); 3.19 (q, 3J ˆ 7.3,
9 H, MeCH2N); 3.34 (dd, 2Jgem ˆ 15.0, 2J(H,P) ˆ 10.4, 1 H, NCH2PO3); 3.46 (dd, 2Jgem ˆ 15.0, 2J(H,P) ˆ 12.9,
1 H, NCH2PO3); 3.67 (dd, 2J(5,5') ˆ 11.7, 3J(5,4) ˆ 6.1,
H
C(5)); 3.76 (ddd, 3J(4,3) ˆ 9.0, 3J(4,5) ˆ 6.1,
3J(4,5') ˆ 2.8, H C(4)); 3.85 (dd, 2J(5',5) ˆ 11.7, 3J(5',4) ˆ 2.7, H' C(5)); 3.91 (dd, 3J(3,2) ˆ 1.6; 3J(3,4) ˆ
5
9.0, H C(3)); 4.45 (br. t, 3J(2,3) ꢁ J(H,P) ꢁ 1.3, H C(2)). 13C-NMR (100 MHz, D2O): 10.90 (q, MeCH2N);
40.35 (dt, 1J(C,P) ˆ 142.2, NCH2P); 49.34 (t, MeCH2N); 65.67 (t, C(5)); 73.29 (d, C(4)); 73.64 (d, C(3));
74.14 (d, C(2)); 177.60 (d, 3J(C,P) ˆ 6.05, C(1)). 31P-NMR (121.5 MHz, D2O): 14.99 (s). FAB-MS (neg. mode):
517 (27.2%, [2M ‡ H ]), 259 (15.0, [MH] ), 258 (100, M ), 242 (5.1, [phospholactone ‡ H] ), 241 (2.2,
[phospholactone] ), 240 (4.1), 183 (18.0, glycerol matrix), 167(14.7), 110(7.2), 91 (14.5, glycerol matrix).
Anal. calc. for C12H29N2O8P (mono(triethylammonium) salt)/C18H44N3O8P (bis(triethylammonium) salt) 1:1 ´
1.5 H2O (437.96): C 41.14, H 9.09, N 8.00; gef.: C 41.64, H 8.72, N 7.53.
2,3,4,5-Tetra-O-benzyl-N-(2-bromoethyl)-d-arabinonamide (16). A soln. of 9 (1400 mg, 2.663 mmol) in dry
THF/DMPU 2:1 (30 ml) at 788 was titrated with t-BuLi (Aldrich, in pentane, ca. 1.25m) under vigourous
stirring until the colour of the soln. changed from yellow to orange (ca. 1.8 ml) and stirred for 10 min at 788. A
precooled ( 208) soln. of 2-bromoethyl trifluoromethanesulfonate (15)8) (2118 mg, 8.24 mmol) in dry THF
(2.2 ml) was added dropwise. After the addition was complete, the colour of the soln. changed from orange to
lemon-yellow. Stirring was continued until the cooling-bath temp. had reached 258. The mixture was poured
into sat. aq. NH4Cl/sat. aq. NaCl soln. 1:1 (50 ml). The pH was adjusted to 7.0 with 1m phosphate buffer.
Extraction with Et2O (5 Â 50 ml) was followed by washing of the combined org. phases with sat. aq. NaCl soln.
(2 Â 20 ml) and H2O (10 ml). Drying (Na2SO4) and evaporation under reduced pressure gave a yellow oil which
was further dried under high vacuum (20 h) and purified by FC (toluene/AcOEt 3:1): 16 (710 mg, 42%) as
slightly yellowish oil, besides starting material 9 (600 mg, 43%). An anal. sample was obtained by prep. HPLC
(hexane/AcOEt 4:1, flow: 16 ml/min, detection at 254 nm, tR: 20.6 min). Colorless oil. Rf (hexane/AcOEt 3:1)
0.28. Rf (toluene/AcOEt 1:1) 0.79. [a]D21
ˆ
13.2 (c ˆ 0.535, CHCl3) 13.28. IR (CHCl3): 3405m, 3070/3050/
2990/2920/2860w, 1670s, 1515m-s, 1492/1450/1435w-m, 1360/1330w, 1300w-m, 1240m, 1190/1090/1065s, 1025/
1010m(sh), 695s. 1H-NMR (500 MHz, C6D6): 2.67 ± 2.70 (m, 1 H, CH2N); 2.87 ± 2.94 (m, CH2Br); 3.41 ±
3.48 (m, 1 H, CH2N); 3.70 (dd, 2J(5,5') ˆ 10.7, 3J(5,4) ˆ 4.45, H C(5)); 3.81 (dd, 2J(5',5) ˆ 10.7, 3J(5',4) ˆ
8
)
Prepared according to the procedure of Welsh and co-workers [30]: yield 61%; colourless liquid, purified
by distillation in vacuo. B.p. 33 ± 358/0.25 Torr ([30]: 508/0.5 Torr).

1904
Helvetica Chimica Acta ± Vol. 81 (1998)
2.15, H' C(5)); 4.05 (ddd, 3J(4,3) ˆ 8.3, 3J(4,5) ˆ 4.4, 3J(4,5') ˆ 2.2, H C(4)); 4.24 (d, 2Jgem ˆ 11.35, 1 H,
PhCH2); 4.31 ± 4.33 (m, 3 H, PhCH2); 4.37 (d, 2Jgem ˆ 11.8, 1 H, PhCH2); 4.46 (d, 3J(2,3) ˆ 2.5, H C(2));
4.50 (dd, 3J(3,2) ˆ 2.5, 3J(3,4) ˆ 8.3, H C(3)); 4.62 ± 4.69 (m (AB), 2 H, PhCH2); 4.72 (d, 2Jgem ˆ 11.8, 1 H,
PhCH2); 6.77 (br. t, 3J(H,N) ꢁ 5.7, CONH); 7.05 ± 7.17 (m, 12, arom. H); 7.25 ± 7.31 (m, 8, arom. H). 13C-NMR
(125 MHz, C6D6): 31.91 (t, CH2Br); 40.72 (t, CH2N); 69.32 (t, C(5)); 71.94, 73.42, 74.18, 75.06 (4t, PhCH2);
78.30 (d, C(4)); 80.10 (d, C(3)); 81.06 (d, C(2)); 127.62, 127.64, 127.67, 127.72, 127.88, 128.19, 128.50, 128.57,
128.60, 128.76 (10d, arom. C); 138.01, 138.98, 139.20, 139.32 (4s, Cipso(Ph)); 171.15 (s, C(1)). CI-MS (NH3): 634
‡
‡
‡
‡
‡
(0.83, [M ‡ 2] ), 632 (0.75, M ) (M (79Br)/[M ‡ 2] (81Br) ca. 1:1 ! 1 Br), 554 (8, [M ‡ 2 HBr] ), 553 (40,
‡
‡
‡
[M ‡ 1 HBr] ), 552 (100, [M H79Br] or [M ‡ 2 H81Br] ). Anal. calc. for C35H38BrNO5 (632.59): C 66.45,
H 6.05, Br 12.63, N 2.21; found: C 66.51, H 6.02, Br 12.33, N 2.39.
N-(2,3,4,5-Tetra-O-benzyl-d-arabinonoyl)taurine ( ˆ Sodium 2-[(2,3,4,5-Tetra-O-benzyl-d-arabinonoyl)-
amino]ethanesulfonate; 17) and N-(d-Arabinonoyl)taurine Salt ( ˆ Sodium or Triethylammonium (d-
Arobinonylamino)ethanesulfonate; 18). To a soln. of 16 (555 mg, 0.877 mmol) in EtOH (99%, 10 ml) was
added Na2SO3 (111 mg, 0.877 mmol) and H2O (3.6 ml). The white suspension was boiled under reflux at 808
under Ar for 17 h, cooled to r.t., diluted with MeOH (20 ml), and evaporated. The residue was purified by FC
(CH2Cl2/MeOH 20:1) to give 179) (290 mg, 50%) as slightly yellow oil, which was dissolved in t-BuOH (7 ml)
and added to a suspension of Pd(OH)2/C (20%, 300 mg) in t-BuOH (5 ml) that had been stirred under 10 bar H2
for 30 min. After dilution with H2O (3.0 ml), the suspension was stirred vigourously at r.t. under H2 (10 bar) for
17 h. Workup as described for 2 afforded the sodium salt 18a (113 mg, 88%) as colourless solid. Further
purification by prep. HPLC (NH2, isocratic 0.1m (Et3NH)HCO3 buffer, pH 6.8 ± 7.0 adjusted with AcOH; 1%
MeCN; detection at 200 nm, flow: 5 ml/min, tR 19.5 min) afforded the triethylammonium salt 18b (128 mg,
78%) as colourless, amorphous solid.
Data of 17: Rf (CH2Cl2/MeOH 20:1) 0.23. 13C-NMR (75 MHz, CD3OD): 42.64 (t, CH2N); 61.40 (t,
CH2SO3Na); 69.11 (t, C(5)); 78.86 (d, C(4)); 80.62 (d, C(3)); 80.95 (d, C(2)); 128.78, 129.19, 129.35, 129.46,
129.57 (5d, arom. C); 130.92, 138.62, 139.48, 139.93 (4s, Cipso(Ph)); 174.19 (s, C(1)).
Data of 18b: Anal. HPLC (NH2, 0.1m (Et3NH)HCO3 buffer; pH 7.2 adjusted with AcOH; 2% MeCN;
refract. detection, flow, 0.5 ml/min): tR 5.9 min. [a]D21
ˆ
29.6 (c ˆ 0.706, H2O). IR (KBr): 3480/3380vs(br.),
2970/2930/2880w, 2680/2570/2500/2420m-s, 1645vs, 1560s, 1445/1425m, 1370/1330w, 1290/1260/1230/1205m, 1110/
1090/1080/1055/1040s. 1H-NMR (500 MHz, D2O): 1.24 (t, 3J ˆ 7.3, 9 H, MeCH2N); 3.16 (q, 3J ˆ 7.35, 6 H,
MeCH2N); 3.34 ± 3.43 (m (AA'), CH2SO3); 3.62 ± 3.66 (m, CONHCH2, H C(5)); 3.72 (not fully resolved ddd,
3J(4,5) ˆ 6.2, 3J(4,5') ˆ 2.8, H C(4)); 3.82 (dd, 2J(5',5) ˆ 11.8, 3J(5',4) ˆ 2.8, H' C(5)); 3.86 (dd, 3J(3,2) ˆ 1.6;
3J(3,4) ˆ 9.1, H C(3)); 4.40 (d, 3J(2,3) ˆ 1.6, H C(2)). 13C-NMR (125 MHz, D2O): 10.96 (q, MeCH2N);
43.89 (t, CONCH2); 49.37 (t, MeCH2N); 62.73 (t, CH2SO3); 65.68 (t, C(5)); 73.33 (d, C(4)); 73.59 (d, C(3));
‡
74.18 (d, C(2)); 178.32 (s, C(1)). FAB-MS (neg. mode): 417 (9.9, [2(M SO2) ‡ H] ), 304(12.9), 209 (10.5),
208 (100, [M SO2] ), 206(9.7), 183 (26.8, glycerol matrix), 168(12.3), 165(35.7), 153(11.8), 118(11.4), 91
(17.5, glycerol matrix). Anal. calc. for C13H30N2O8S ´ 0.5 H2O (383.46): C 40.72, H 8.15, N 7.31; found: C 40.72,
H 8.12, N 7.48.
2,3,4,5-Tetra-O-benzyl-N-[2,2-bis(diethoxyphosphinyl)ethyl]-d-arabinonamide ( ˆ Tetraethyl {2-[(2,3,4,5-
Tetra-O-benzyl-d-arabinonoyl)amino]ethylidene}bis[phosphonate]; 20).
A soln. of t-BuOK in t-BuOH
(Aldrich, 1.0m, 0.47 ml, ca. 0.47 mmol) was added at 408 to a soln. of 9 (1232 mg, 2.343 mmol) in dry t-
BuOH (25 ml) under Ar. Stirring was continued for 5 min. A soln. of tetraethyl ethenylidenebis[phosphonate]
(19)10) (774 mg, 2.578 mmol) in t-BuOH (2.5 ml) was injected through a septum. After stirring at 408 for 43 h,
the temp. was increased to 508, and additional 19 (70 mg, 0.2343 mmol, 0.1 equiv.) was added. After 67 h, the
same amount of 19 and more t-BuOK soln. (0.235 ml, 0.234 mmol, 0.1 equiv.) were added. After a total reaction
time of 92 h, the soln. was cooled to r.t. and poured onto a mixture of sat. aq. NH4Cl soln. (25 ml), sat. aq. NaCl
soln. (15 ml), and Et2O (25 ml). The org. phase was separated. The aq. phase was diluted with sat. aq. NaCl soln.
(25 ml), and extracted with Et2O (4 Â 25 ml). The combined org. phases were washed with sat. aq. NaCl soln.
(10 ml), dried (MgSO4), and evaporated. The resulting amber oil was purified by FC (toluene/acetone 1:1): 20
9
)
The intermediate tetra-O-benzyl-protected sodium sulfonate 17 is readily soluble in common org. solvents
(THF, Et2O, AcOEt, CH2Cl2) and was conveniently purified by FC.
Synthesized from tetraethyl ethylenebis[phosphonate] according to the procedure of Charlton and
Alauddin [50], and Degenhardt and Burdsall [36] [38], and purified by fractional distillation in vacuo: B.p.
120 ± 1258/0.4 Torr; Rf (toluene/acetone 1:1) 0.22. 13C-NMR (C6D6): 147.9 (t, CH2 ˆ ). 31P-NMR (C6D6):
13.3 (s).
10
)

Helvetica Chimica Acta ± Vol. 81 (1998)
1905
(1.753 g, 90%) as slightly yellow oil. An anal. sample was obtained by prep HPLC (CHCl3/Et2O 10:1).
Colourless oil. Rf (toluene/acetone 1:1) 0.35. [a]D21
ˆ
11.98 (c ˆ 0.852, CHCl3). IR (CHCl3): 3400m, 3060/2985/
2920/2890/2850w, 1665s, 1515m-s, 1495w-m, 1450m, 1390/1375/1350w-m, 1250s, 1090/1060/1020/970s, 695s.
1H-NMR (500 MHz, C6D6): 1.00 ± 1.09 (m, 12 H, MeCH2OP); 2.61 (tt, 3J(CH,CH2) ˆ 5.9, 2J(CH,P) ˆ
2J(CH,P') ˆ 23.1 P decoupling ! t, 3J(CH,CH2) ˆ 5.9, CH(PO3Et2)2); 3.71 (dd, 2J(5,5') ˆ 10.7, 3J(5,4) ˆ 4.6,
H
C(5)); 3.80 (dd, 2J(5',5) ˆ 10.7, 3J(5',4) ˆ 2.1, H' C(5)); 3.92 ± 4.12 (m, P decoupling ! strongly changed
m, 10 H, H C(4), CH2N, MeCH2OP); 4.29 ± 4.32 (m, 3 H, PhCH2, CH2N); 4.39 (d, 2Jgem ˆ 12.1, 1 H, PhCH2);
4.57 (d, 3J(2,3) ˆ 2.5, H C(2)); 4.60 (dd, 3J(3,2) ˆ 2.5, 3J(3,4) ˆ 8.2,
H
C(3)); 4.66 (d, 2Jgem ˆ 11.4, 1 H,
PhCH2); 4.71 (d, 2Jgem ˆ 11.9, 1 H, PhCH2); 4.79 (d, 2Jgem ˆ 11.0, 1 H, PhCH2); 4.81 (d, 2Jgem ˆ 11.3, 1 H, PhCH2);
7.05 ± 7.49 (m, 20, arom. H); 8.09 (br. t, 3J(NH,CH2)ꢁ 5.8, additional 4J to P-atoms not resolved, P decoupling
! sharp dd, 3J(NH,CH2) ˆ 5.1, 6.7, NHCO). 13C-NMR (125 MHz, C6D6): 16.34, 16.36, 16.39, 16.42 (4q,
MeCH2OP); 38.67 (td, 1J(CH,P) ˆ 1J(CH,P') ˆ 131.3, CH2CH(PO3Et2)2); 39.90 (tt, 2J(CH2,P) ˆ 4.6, CH2N);
62.64 (dt, 2J(CH2,P) ˆ 6.4, MeCH2OP); 62.80 (dt, 2J(CH2,P) ˆ 7.4, MeCH2OP); 62.85 (dt, 2J(CH2,P) ˆ 6.7,
MeCH2OP); 62.98 (dt, 2J(CH2,P) ˆ 6.3, MeCH2OP); 69.57 (t, C(5)); 71.97, 73.36, 74.36, 75.04 (4t, PhCH2);
78.50 (d, C(4)); 80.27 (d, C(3)); 81.20 (d, C(2)); 127.49, 127.51, 127.57, 127.61, 127.92, 128.46, 128.53, 128.55,
128.71 (9d, arom. C); 138.43, 139.12, 139.39, 139.52 (4s, Cipso(Bn)); 171.08 (s, C(1)). 31P-NMR (205.8 MHz,
C6D6): 21.72 (d, 2J(P,P') ˆ 1.65); 21.81 (d, 2J(P',P) ˆ 1.44). CI-MS (NH3): 828(13), 827 (49, [M ‡ 2] ), 826
‡
‡
‡
‡
(100, [M ‡ 1] ), 526 (20, [M ‡ 1 CH2CH(PO3Et2)] ), 301 (13, [CH2CH(PO3Et2)] ). Anal. calc. for
C43H57NO11P2 (825.87): C 62.54, H 6.96, N 1.69, P 7.50; found: C 62.28, H 6.70, N 1.72, P 7.21.
N-(2,2-Diphosphonoethyl)-d-arabinonamide (ˆ [2-(d-Arabinonoylamino)ethylidene]bis[phosphonic acid];
22a). A soln. of 20 (2.259 g, 2.73 mmol) in t-BuOH (25 ml) was added to a suspension of Pd(OH)2/C (20%,
980 mg) in t-BuOH (15 ml) and prehydrogenated under 10 bar H2 for 30 min. After dilution with H2O (10 ml),
the suspension was stirred vigourously under 10 bar H2 for 6d. Celite (100 mg) was added, and the Pd catalyst
was removed by centrifugation. The supernatant was removed and the residue suspended in MeOH (60 ml) and
again centrifuged. This procedure was repeated twice. The combined supernatants were concentrated to a small
volume and pressure-filtered over a 0.2-mm membrane filter (Merck, Anotop). The filtrate was evaporated. The
residue was dissolved in 50 ml of H2O and lyophilized. The resulting yellow gum of 21 was dried under high
vacuum (96 h) over P4O10 and dissolved in dry CH2Cl2 (60 ml) under Ar, and the soln. was stirred at 158 for
15 min. Me3SiBr (8.83 ml, 68.25 mmol, 25 equiv.) was added dropwise through a septum within 15 min. The
cooling bath was removed after 1 h. After stirring at r.t. for 48 h, the yellow soln. was evaporated and kept under
high vacuum for 2 h, before the solid foamy residue was taken up in H2O/MeOH 1:6 (50 ml) and shaken
vigourously. After a few min, the white suspension turned into a clear yellow soln., which was suction-filtered
through a sand/Celite/sand bed, concentrated to a small volume, and lyophilized. The residue was taken up in
H2O (60 ml), pressure-filtered through a 0.2-mm membrane filter (Merck, Anotop) and lyophilized again to
afford the free acid 22a (1.786 g, 88%) as colourless foam. An anal. sample was obtained by prep. HPLC (CN,
0.1m (Et3NH)HCO3 buffer, 1% MeCN, pH 7.15 adjusted with AcOH, flow: 5 ml/min, refract. detection tR
13.2 min): 210 mg of 22a yielded 98 mg of the tetrakis(triethylammonium) salt 22b as colourless fluffy solid
(purification not optimized). M.p. 135 ± 1408 (dec.). [a]2D1 ˆ ‡6.6 (c ˆ 0.738, H2O). IR (KBr): 3400s(br.), 2970/
2930w-m, 2740/2675w, 1780m, 1630m, 1495/1435/1395m, 1170s(br.), 1035s. 1H-NMR (500 MHz, D2O):
1.25 (t, 3Jvic ˆ 7.3, 36 H, MeCH2N); 2.24 (br. t, 2J(CH,P) ˆ 2J(CH,P') ˆ 20.9, additional coupling to CH,N ca.
6; P decoupling ! br. d, 1 H, CH2CH(PO3H2)2); 3.17 (q, 3Jvic ˆ 7.3, 24 H, MeCH2N); 3.39 (td, 3Jvic ˆ 6.3;
3J(CH2,P) ˆ 3J(CH2,P') ˆ 14.9; P decoupling ! br. d, CH2N); 3.63 (dd, 2J(5,5') ˆ 11.7, 3J(5,4) ˆ 6.4, H C(5));
3.70 (m,
H
C(4)); 3.81 (dd, 3J(3,2) ˆ 3J(3,4) ˆ 8.9,
H
C(3)); 3.82 (dd, 2J(5',5) ˆ 11.7, 3J(5',4) ˆ 2.6,
H' C(5)); 4.20 (d, 3J(2,3) ˆ 1.7,
H
C(2)). 13C-NMR (125 MHz, D2O): 10.94 (q, MeCH2N); 39.73 (td,
1J(CH,P) ˆ 1J(CH,P') ˆ 115.1, CH2CH(PO3H2)2); 40.16 (t, CONHCH2); 49.38 (t, MeCH2N); 65.82 (t, C(5));
73.92 (d, C(4)); 74.24 (d, C(3)); 74.91 (d, C(2)); 183.69 (s, C(1)). FAB-MS (neg. mode, amide bond is cleaved
under FAB conditions, i.e., only A , the corresponding (2-aminoethylidene)bis[phosphonate] is observable):
818 (1.3, [A4H3] ), 613 (3.3, [A3H2] ), 408 (33.0, [A2H] ), 388(6.5), 296(16.7), 275 (6.5, glycerol matrix),
232(10), 204 (100, A ), 186(12.1), 183 (43.9, glycerol matrix), 153(5.4), 91 (25.4, glycerol matrix).
3. Thiobarbituric Acid Assay [43 ± 45]. According to the procedure of Schauer (ꢀMicro-Warrenꢀ) [51] in
phosphate buffer of pH 5.9 with a 15 min inhibitor preincubation period. S. typhimurium, Influenza A (N9), and
Influenza B (B/Lee/40) sialidase, and the fetuin (from fetal calf serum) used as enzyme substrate were kindly
provided by Prof. Laver (Influenza Research Unit, Australian National University, Canberra, Australia). Vibrio
cholerae sialidase was obtained in protease-free quality from Boehringer Mannheim. Enzyme dilutions with
suitable activity for the assay conditions were found from preliminary experiments without inhibitor. Values in
the Table reflect the relative enzyme activities as estimated from visual inspection of the relative colour intensity

1906
Helvetica Chimica Acta ± Vol. 81 (1998)
of the pink b-formylpyruvate/thiobarbituric-acid complex formed in the enzymatic reaction in inhibitor
experiments (15 min preincubation) as compared to reference experiments without inhibitors (ˆ 100%). For
better comparison, a reference inhibitor (DANA) was always tested in the same assay. In addition to blank
experiments to account for nonenzymatic hydrolysis, separate inhibitor blanks were run in parallel to check for
inhibitor-mediated substrate hydrolysis, which was not observed for any of the inhibitors tested. The activities
observed are the result of duplicate experiments.
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Received August 3, 1998