Synthesis of amino-acid-, carbohydrate-, coumarin-, and biotin-labelled oligo[(R)-3-hydroxybutanoic acids] (OHB)

Article abstract of DOI:10.1002/(SICI)1522-2675(19981216)81:12<2414::AID-HLCA2414>3.0.CO;2-C

The benzyl esters 1- 3 of oligo[(R)-3-hydroxybutanoic acids] (OHB) containing 2, 16, or 32 HB units were coupled at the hydroxy terminus with arginine (by esterification with carbodiimide), with glucose (by acetalization with glucosyl trichloroacetimidate), and with 7- (dimethylamino)coumarin-4-acetic acid and biotin (by amide formation through a glycine linker) to give, after deprotection(s), the corresponding 'labelled' OHB acids 7-9, 12, 13, 25, 26, 33, and 34 (Schemes 1, 4, and 5). The respective novel 16- and 32mer derivatives exhibit distinct water solubility (Table) or may be detected (in minute amounts) by fluorescence spectroscopy, properties required for biochemical investigations.

Full text of DOI:10.1002/(SICI)1522-2675(19981216)81:12<2414::AID-HLCA2414>3.0.CO;2-C

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Helvetica Chimica Acta ± Vol. 81 (1998)
Synthesis of Amino-Acid-, Carbohydrate-, Coumarin-, and Biotin-Labelled
Oligo[(R)-3-Hydroxybutanoic Acids] (OHB)
1
by Monica G. Fritz ) and Dieter Seebach*
Laboratorium für Organische Chemie der Eidgenössischen Technischen Hochschule, ETH-Zentrum,
Universitätstrasse 16, CH-8092 Zürich
The benzyl esters 1 ± 3 of oligo[(R)-3-hydroxybutanoic acids] (OHB) containing 2, 16, or 32 HB units were
coupled at the hydroxy terminus with arginine (by esterification with carbodiimide), with glucose (by
acetalization with glucosyl trichloroacetimidate), and with 7-(dimethylamino)coumarin-4-acetic acid and biotin
(by amide formation through a glycine linker) to give, after deprotection(s), the corresponding ꢀlabelledꢁ OHB
acids 7 ± 9, 12, 13, 25, 26, 33, and 34 (Schemes 1, 4, and 5). The respective novel 16- and 32mer derivatives exhibit
distinct water solubility (Table) or may be detected (in minute amounts) by fluorescence spectroscopy,
properties required for biochemical investigations.
Introduction. ± Poly[(R)-3-hydroxybutanoic acid] (PHB) and related poly(3-
hydroxyalkanoates) (PHAs) are an ubiquitous class of biopolymers [1][2] occurring as
high-molecular-weight storage material (sPHB) in microorganisms [3] as well as in a
low-molecular-weight form (cPHB). cPHB has been detected in eucaryotic and
procaryotic cells, in human aorta tissue, and in blood plasma and has been further
characterized by 1H-NMR spectroscopy [4][5]. Apart from the well-established
ionophoric activity, the nature and function of cPHB as an integral cell constituent is
still unknown. Reusch and coworkers have shown that cPHB (ca. 150 units) and CaPPi
(calcium polyphosphate, ca. 75 units) form a complex which may function as an ion
channel in genetically competent Escherichia coli [5]. The ionophoric ability of this
non-proteinaceous calcium-selective channel was unambiguously demonstrated in
recent patch-clamp experiments, by comparison of the complex extracted from E. coli
membrane with a complex that was constituted from a synthetic oligo[(R)-3-
hydroxybutanoate] (OHB)2), containing 128 HB units, and inorganic polyphosphate
[6]. Furthermore, the cation-transport activity of OHBs themselves has been amply
demonstrated: they are able to transport cations across CH2Cl2 layers (ꢀbulk
membranesꢁ) in U-tubes [7], and they generate high-conductivity nonselective ion
channels as shown in patch-clamp experiments in lipid bilayers [8]. The ion-transport
activity of the 16mer and of longer-chain OHBs has been attributed to the formation of
hydrophobic aggregates in the lipid bilayer [8]; and these aggregates may have
structures related to the lamellar crystallites of ca. 50-Š thickness, characteristic of
OHB and PHB, in which the chains are arranged as 21-helix strings (pitch: 6 Š)
consisting of 16 HB units [9].
1
)
)
Part of the projected Ph.D. Thesis of M.G.F., ETH-Zürich, 1998.
The term oligomer refers to monodisperse compounds. Material with a molecular-weight distribution is
designated as polymer.
2

Helvetica Chimica Acta ± Vol. 81 (1998)
2415
One drawback for the use of OHBs in transport studies turned out to be their
peculiar solubility properties and the difficulty of their (quantitative) detection. Short-
chain OHBs ( ꢀ 8 units) are soluble in common organic solvents as well as in H2O3). In
contrast, long-chain OHBs are neither soluble in H2O nor in common polar or apolar
media, with the exception of polyhalogenated solvents (cf. CH2Cl2, CHCl3,
ClCH2CH2Cl, CF3CH2OH). Long-chain OHBs tend to form insoluble aggregates4)
in H2O. The fact that spontaneous incorporation of OHBs into lipid bilayers (planar
bilayers, vesicles, or cell membranes) does not occur might be a consequence of their
poor water solubility and lack of amphiphilic character, i.e., that they have low bio-
availability. The detection of sPHB and cPHB in biological systems is usually achieved
by an antibody test [5] or by a destructive analysis (heating in conc. H2SO4 solution to
generate crotonic acid, subsequently determined by HPLC) [11][12]5). Thus, a non-
destructive method to localize and to quantify OHB material in minute amounts had to
be developed.
In the present paper, we describe the synthesis of OHB derivatives with enhanced
water solubility, achieved by covalent coupling with an amino-acid or a carbohydrate
moiety. Furthermore, we present the synthesis of fluorescence-labelled OHBs as well as
of biotin-OHB conjugates which can be easily detected by an immunoassay with
fluorescence-labelled avidin [13]. The new compounds thus available were intended to
be used for ion-transport studies with liposomes [14].
Syntheses and Conclusion. ± Preamble. Monodisperse OHBs 1 ± 3 were synthesized
by a segment-coupling strategy previously developed by us [15] (Scheme 1). The
subsequent derivatization at the hydroxy and not at the carboxy terminus of the OHBs
was chosen for two reasons: i) previous patch-clamp experiments had indicated the
necessicity of a free carboxylate function for successful incorporation and stability of
OHBs in planar lipid membranes [8], and ii) OHBs tend to fragment via b-elimination
under basic conditions [15] (following a mechanism in which an OHB-species activated
at the carboxy terminus turned out to be a key intermediate). Thus, derivatization
involving the carboxy functionality would have been limited to reaction conditions used
in the segment coupling synthesis (via an acid chloride), whereas coupling at the
hydroxy end should allow the use of more diversified and more drastic reaction
conditions. Optimizations of derivatization conditions were carried out with the OHB
dimer 1 as a model system, while compounds derived from HB 16mer 2 and 32mer 3
were the actual target molecules.
Water-Soluble OHB Derivatives. In Scheme 1, the synthesis of arginine- and
glucose-coupled OHBs is outlined. For introducing arginine, we used the commercially
available tri-Z-protected l-arginine derivative6), which was coupled with OHB esters
1 ± 3, using DCC/DMAP (dicyclohexylcarbodiimide/4-(dimethylamino)pyridine) in
3
)
)
)
The water solubility of the cyclic triolide of HB was determined to be ca. 9 g/l [10].
The formation of defined supramolecular aggregates, as, e.g., micelles, has not been observed.
An analysis providing information about the amount of (R)- or (S)-3-hydroxybutanoate and -pentanoate
in biological samples has been developed in our laboratory and will be described elsewhere.
Z ˆ (Benzyloxy)carbonyl, Fmoc ˆ (9H-fluoren-9-ylmethoxy)carbonyl, Pmc ˆ (2,2,5,7,8-pentamethylchro-
man-6-yl)sulfonyl, Boc ˆ (tert-butoxy)carbonyl.
4
5
6
)

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Helvetica Chimica Acta ± Vol. 81 (1998)
CH2Cl2, to give the conjugates 4 (62%), 5 (64%), and 6 (36%). By using the N-Fmoc-
Nw-Pmc-protected arginine under otherwise similar conditions, the formation of
several by-products was observed. Coupling with the N-Z-Nw-NO2-protected arginine
derivative yielded the desired product in good yield (not shown), however, the
subsequent deprotection did not work in our case. Hydrogenolytic debenzylation of
compounds 4 ± 6 with H2 and Pd/C produced the fully deprotected compounds 7 ± 9 in
excellent yields.
Scheme 1. Synthesis of Water-Solubilized HB Derivatives
a) Tri-Z-l-arginine, DCC, DMAP, CH2Cl2, 08. b) H2, Pd/C, CF3CH2OH, r.t. c) 2,3,4,6-Tetra-O-benzyl-d-
glucosyl trichloroacetimidate, BF3 ´ Et2O, CH2Cl2, 08.
DCC ˆ dicyclohexylcarbodiimide, DMAP ˆ 4-(dimethylamino)pyridine
Coupling of an OHB with a carbohydrate moiety was achieved by following the
glycosylation procedure introduced by Schmidt [16]. Reaction of the hydroxy esters 1
and 2 with tetra-O-benzyl-d-glucosyl trichloroacetimidate, readily available in one step
from tetra-O-benzyl-d-glucose, gave the fully benzyl-protected conjugates 10 (93%)
and 11 (82%), respectively (Scheme 1). We were not able to separate the anomers by
flash chromatography, but for the intended studies, the configuration has no
significance. Hydrogenolytic cleavage of the benzyl ester and ethers in 10 and 11 with
H2 and Pd/C gave the desired products 12 (88%) and 13 (77%), respectively.
The water solubility of the OHB conjugates 8, 9, and 13 was compared with those of
their precursors 2 and 3 (Table). Thus, the solubility of the 16mer was enhanced from

Helvetica Chimica Acta ± Vol. 81 (1998)
2417
essentially zero (see 2) to 1.43 g/l by arginine and to 0.94 g/l by glucose derivatization
(see 8 and 13, resp.) Arginine modification of the 32mer 3 resulted in a water solubility
of 0.46 g/l (see 9). These values point to a reasonably amphiphilic character of the
OHB conjugates, Indeed, they are no longer soluble in pure CH2Cl2 as are their
precursors, and CH2Cl2/MeOH mixtures are required to dissolve them.
Table. Water Solubility of OHB Derivatives
n
Water solubilitya) [g/l]
2
3
8
9
13
16
32
16
32
16
ꢀ 0.1
ꢀ 0.1
1.43 (Æ 2%)
0.46 (Æ 8%)
0.94 (Æ 9%)
a) The values were determined as follows: A suspension of OHB material (in excess) in dist. H2O was stirred
for 1 d at r.t., then a part of the suspension was centrifugated, and an aliquot of the supernatant was taken.
9
The solvent was carefully evaporated (10 bar), and the amount of solid residue was analyzed by weight.
Fluorescence-Labelled OHB Derivatives. Coupling of the hydroxy ester 1 with
7-(dimethylamino)coumarin-4-acetic acid (ˆ 7-(dimethylamino)-2-oxo-2H-1-benzo-
pyran-4-acetic acid) was tried under various reaction conditions7) (Scheme 2). The
best result was obtained by applying the Mukaiyama method (Entry 5) [17] which
yielded compound 14 in 30% yield.
The unexpectedly low yields of 14 obtained under various reaction conditions might
be a consequence of the solvent mixtures employed (DMF/CH2Cl2; MeCN/CH2Cl2);
esterification rates often tend to be influenced by solvent effects. Coupling of OHB 2
under similar conditions (Scheme 2, Entry 5) gave the fluorescence-labelled compound
15 (21%) (Scheme 3).
Hydrogenolysis of 15 was carried out in DMF with Pd/C as the catalyst and
cyclohexa-1,4-diene as the hydrogen source [18] and gave acid 16 in 50% yield.
Attempts to debenzylate with H2 and Pd/C led to the destruction of the coumarin
moiety. Coupling of OHB 1 and 2 with dansyl chloride (ˆ 5-(dimethylamino)-
naphthalene-1-sulfonyl chloride) under basic conditions8) gave the fluorescent
compounds 17 (15%) and 18 (12%), attempted debenzylation of which, however,
failed completely9).
Because of the problems encountered with some of the coupling reactions (see
above), we decided to apply an alternative strategy: esterification of the OHBs 1 ± 3
with a glycine spacer and subsequent functionalization of the spacer amino group10
)
7
)
Attempts to prepare the acid chloride of 7-(dimethylamino)coumarin-4-acetic acid with oxalyl or thionyl
chloride failed.
8
9
)
)
The use of various bases, solvents, and reaction temperatures did not lead to better results.
Hydrogenolysis with H2 and Pd/C gave OHBs with free carboxy and hydroxy function, resulting from
benzyl-ester and S O bond cleavage. Catalytic transfer hydrogenolysis (Pd/C, cyclohexa-1,4-diene, DMF)
gave OHBs with a,b-unsaturated carbonyl functionalities, resulting from a b-elimination of dansyl sulfate.
Because of the higher nucleophilicity of the amino group and the enhanced stability of the newly formed
amide bond, the coupling reaction was expected to work better, what it actually did.
10
)

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Helvetica Chimica Acta ± Vol. 81 (1998)
Scheme 2. Coupling of the HB Dimer 1 with 7-(Dimethylamino)coumarin-4-acetic Acid
Entry
Reaction conditions
DMAP (0.1 equiv.), DCC
DMAP, (0.5 equiv.), DCC
di(2-pyridyl) disulfide, PPh3, CuBr2
BOP-Clb), Et3N
2-chloro-1-methylpyridinium iodide, Et3N
2-chloro-1-methylpyridinium iodide, DMAP (cat.)
PPh3, CCl4, pyridine
Yield [%]a) of 14
1
2
3
4
5
6
7
24
6
6
6
30
±
2
a) Based on material isolated from SiO2 chromatography. b) BOP-Cl ˆ bis(2-oxo-
oxazolidin-3-yl)phosphinic chloride.
Scheme 3. Synthesis of Fluorescent OHB Derivatives
a) 7-(Dimethylamino)coumarin-4-acetic acid, 2-chloro-1-methylpyridinium iodide, Et3N, CH2Cl2, MeCN, r.t.
b) Cyclohexa-1,4-diene, Pd/C, DMF, r.t. c) 5-(Dimethylamino)naphthalene-1-sulfonyl chloride, DMAP, 508,
ClCH2CH2Cl.
with the fluorescent building block (Scheme 4). For the introduction of the glycine
moiety, we coupled commercial N-Boc-glycine6) with OHBs 1 ± 3 using DCC/DMAP
in CH2Cl2, to give the conjugates 19 ± 21 in excellent yields of 89, 86, and 76%,
respectively. Removal of the Boc group (CF3COOH/CH2Cl2) and subsequent standard
peptide coupling of the trifluoroacetates derived from 19 ± 21, using EDC/HOBt (N-[3-

Helvetica Chimica Acta ± Vol. 81 (1998)
2419
(dimethylamino)propyl]-N'-ethylcarbodiimide
hydrochloride/1-hydroxy-1H-benzo-
triazole) [19] in CHCl3/DMF, with 7-(dimethylamino)coumarin-4-acetic acid gave
conjugates 22 (80%), 23 (96%), and 24 (70%), respectively. Catalytic transfer
hydrogenolysis (Pd/C, cyclohexa-1,4-diene, DMF) of 23 yielded 25 (50%), whereas
cleavage of the benzyl ester of 24 was performed with H2 and Pd/BaSO4 to afford the
free acid 26 in 55% yield.
Scheme 4. Synthesis of HB Derivatives Linked to a Coumarin via a Glycine Spacer
a) DCC, DMAP, CH2Cl2, 08. b) 1. CF3COOH, CH2Cl2, 08, 2. Et3N, CHCl3, 08; 3. 7-(dimethylamino)coumarin-4-acetic
acid, EDC, HOBt, DMF, CHCl3, 08 ! r.t. c) Cyclohexa-1,4-diene, Pd/C, DMF, r.t. or H2, Pd/BaSO4, CF3CH2OH, r.t.
EDC ˆ N-[3-(dimethylamino)propyl]-N'-ethylcarbodiimide hydrochloride
Analysis of the UV/VIS spectra of the coumarin-labelled compounds 25 and 26
revealed a coumarin-specific absorption with a maximum at 376 nm and a molar
1
extinction coefficient e of 15837 m 1 cm (Æ 2.5%) in CF3CH2OH11). The high molar
extinction coefficient and the coumarin absorption in a region, where most lipids do not
absorb, make these compounds well-suited for detection by UV/VIS or fluorescence
spectrometry in minute amounts in lipid membranes.
Biotin-Linked OHBs. For the reaction of an OHB hydroxy group with the acid
functionality of (‡)-biotin (ˆ(3aS,4S,6aR)-hexahydro-2-oxo-1H-thieno[3,4-d]imida-
zole-4-pentanoic acid), we intended to activate the acid by conversion to an acid
chloride, a process which succeeded neither with oxalyl chloride nor with thionyl
chloride. On the other hand, the (‡)-biotin ester with N-hydroxysuccinimide [20]
did not react with OHB 1 under various conditions tested. We managed to couple
(‡)-biotin with the OHBs 1 and 2, using DCC/DMAP in CH2Cl2/DMF, to give
the derivatives 27 and 28 in poor yields (Scheme 5). Hydrogenolytic cleavage over
11
)
For comparison: the 32mer HB with free carboxy and hydroxy function has a UV absorption maximum at
1
214 nm and a molar extinction coefficient e of 3326 m 1 cm (Æ 2.5%) in CF3CH2OH. The 376-nm
coumarin absorption does not give rise to a Cotton effect of compound 26.

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Helvetica Chimica Acta ± Vol. 81 (1998)
Pd/C12) of the benzyl esters in 27 and 28 gave the ꢀbiotinylatedꢁ acids 29 and 30, both in
ca. 82% yield. In view of the low coupling yields, we decided to use the strategy applied
for the introduction of coumarin: reaction of the glycinated OHB derivatives 19 and 20
with (‡)-biotin, using EDC/HOBt in CHCl3/DMF, gave the ꢀbiotinylatedꢁ compounds
31 (85%) and 32 (78%) (Scheme 5). Hydrogenolytic debenzylation of compounds 31
and 32 with H2 and Pd/C yielded the acids 33 (82%) and 34 (91%), respectively.
Scheme 5. Synthesis of ꢀBiotinylatedꢁ HB Derivatives
a) (‡)-Biotin, DCC, DMAP, DMF, CH2Cl2, 08. b) H2, Pd/C, CF3CH2OH, r.t. c) 1. CF3COOH, CH2Cl2, 08;
2. Et3N, CHCl3, 08; 3. (‡)-biotin, EDC, HOBt, DMF, CHCl3, 08 ! r.t.
In conclusion, the experiments described herein provide, for the first time, pure
(R)-3-hydroxybutanoate oligomers (OHBs) derivatized in such a way that they
become somewhat H2O-soluble, at least to the extent required for biochemical
experiments (0.5 ± 1.5 g/l). Furthermore, we have synthesized OHB derivatives which
can be detected in tiny amounts by direct (the ꢀcoumarinylatedꢁ ones) or by indirect
(the ꢀbiotinylatedꢁ ones) fluorescence measurements. This will enable us ± and
hopefully also others ± to investigate in more detail the intriguing and widely unknown
12
)
It is known that sulfur-containing compounds may poison the Pd/C catalyst, but in our work, this was not
the case [21].

Helvetica Chimica Acta ± Vol. 81 (1998)
2421
role which the simple biomacromolecules consisting of less than 200 3-hydroxybuta-
noate residues (cPHB) play in biology.
We thank Zeneca Bio Products, Billingham, GB, for supplying P(3-HB) and the Fonds der Basler
Chemischen Industrie for a scholarship granted to M.G.F.
Experimental Part
1. Abbreviations. BOP-Cl (bis(2-oxooxazolidin-3-yl)phosphinic chloride), DCC (dicyclohexylcarbodi-
imide), DMAP (4-(dimethylamino)pyridine), EDC (N-[3-(dimethylamino)propyl]-N'-ethylcarbodiimide hy-
drochloride), HOBt (1-hydroxy-1H-benzotriazole), h.v. (high vacuum, 10 4 ± 10 6 mbar).
2. General. All solvents were either puriss p.a. quality or distilled over appropriate drying reagents. CH2Cl2
and DMF were stored over 4-Š molecular sieves. All other reagents were used as purchased from Fluka or Senn.
Compounds 1 ± 3 were synthesized as described in [15]. For h.v. < 10 4 mbar, a turbomolecular pump Balzers
TSH065 was employed. TLC: Merck silica gel 60 F254 anal. plates; detection either with UV or by dipping into a
soln. of I2 (30 g), KI (2 g), EtOH (200 ml), H2O (200 ml) and drying in the air. Flash chromatography (FC):
Merck silica gel 60 (40 ± 63 mm). M.p.: Büchi 510; uncorrected. Optical rotations: 10-cm, 1-ml cell, at r.t.;
Perkin-Elmer-241 polarimeter. UV/VIS Spectra: Varian-Cary-1E-UV-VIS spectrophotometer; lmax in nm (e).
1
IR Spectra: Perkin-Elmer-1600-FT spectrophotometer. H-NMR: Bruker-AMX-II-500 (500 MHz), AMX-400
(400 MHz), ARX-300 (300 MHz), or Varian-Gem-200 (200 MHz) spectrometer. 13C-NMR: Bruker-AMX-500
(125 MHz), AMX-400 (100 MHz), ARX-300 (75 MHz), or Varian-Gem-200 (50 MHz) spectrometer; in
CDCl3, unless specified otherwise. MS: VG TRIBRID for EI; VG ZAB2-SEQ for liquid secondary ionization
(fast-atom bombardment) (LSI (FAB)) with 3-nitrobenzyl alcohol as matrix; Bruker-Reflex-MALDI TOF
spectrometer for matrix-assisted laser desorption time-of-flight (MALDI-TOF). Elemental analyses were
conducted by the Microanalytical Laboratory of the Laboratorium für Organische Chemie, ETH-Zürich.
3. General Procedure for the Coupling Using EDC (GP I). According to [19], a stirred soln. of the
trifluoroacetate salt (1 equiv.) in CHCl3 at 08 (ice-bath) under Ar was treated with Et3N (5 equiv.). This mixture
was added to a stirred soln. of the acid (1 equiv.) in DMF at 08 (ice-bath) under Ar. HOBt (1.2 equiv.) and EDC
(1.2 equiv.) were added successively. The mixture was allowed to warm to r.t., and stirring was continued for
16 h. The mixture was evaporated, the residue dissolved in CH2Cl2, the soln. washed with 1n HCl, aq. sat.
NaHCO3, and sat. NaCl soln., dried (MgSO4), and evaporated, and the residue purified by FC.
4. General Procedure for the Coupling Using DCC (GP II). According to [22], a 08-cold soln. of DCC
(1.1 equiv.) and DMAP (0.1 equiv.) in CH2Cl2 was added under Ar to a stirred soln. of the alcohol (1 equiv.) and
the acid (1 equiv.) in CH2Cl2 at 108 (ice/salt-bath). The mixture was allowed to warm to r.t., and stirring was
continued for 24 h. The mixture was filtered through Celite and washed with 1n HCl, aq. sat. NaHCO3, and NaCl
soln., the org. phase dried (MgSO4) and evaporated, and the residue purified by FC.
5. General Procedure for the Cleavage of Benzyl Ester and Benzyl Ether (GP III). GP IIIa: According to
[23], the benzyl-ester or -ether-protected oligomer was dissolved in CF3CH2OH at r.t., then 10% Pd/C was
added, and hydrogenation was carried out under H2 and vigorous stirring. The reaction went to completion
within 5 to 20 h. The mixture was filtered through Celite and evaporated. The residue was purified by FC.
GP IIIb: According to [23], the benzyl-ester or -ether-protected oligomer was dissolved in CF3CH2OH at
r.t., then 10% Pd/BaSO4 was added, and hydrogenation was carried out under H2 and vigorous stirring for 8 h.
The mixture was filtered through Celite and evaporated. The residue was purified by FC.
GP IIIc: According to [18], a soln. of the benzyl-ester-protected oligomer and cyclohexa-1,4-diene
(20 equiv.) in DMF was treated with 10% Pd/C under Ar. After 12 ± 24 h stirring, the mixture was filtered
through Celite and evaporated. The residue was purified by FC.
6. General Procedure for the Boc-Deprotection of Amino Acids (GP IV). According to [24], a stirred soln.
of the amino-acid derivative in CH2Cl2 was treated at 08 (ice-bath) under Ar with an equal volume of CF3CO2H.
The mixture was allowed to warm to r.t., and stirring was continued for 1.5 h. The mixture was evaporated and
the residue dried under h.v. The salts with CF3CO2H were used without further purification or characterization.
7. Arginine-HB Conjugates 4 ± 9. Benzyl (3R)-3-{{(3'R)-3'-{{(2S)-2-{[(Benzyloxy)carbonyl]amino}-5-{2,3-
bis[(benzyloxy)carbonyl]guanidino}pentanoyl}oxy}butanoyl}oxy}butanoate (4). Compound
1
(236 mg,
0.85 mmol) was transformed according to GP II with commercially available Z-Arg(Z2)-OH (490 mg,
0.85 mmol) in CH2Cl2 (20 ml). FC (CH2Cl2/Et2O 11:1) yielded 4 (440 mg, 62%). Colorless microcrystalline
solid. M.p. 87 ± 898. Rf 0.6 (CH2Cl2/Et2O 11:1). [a]D ˆ‡ 2.50 (c ˆ 0.6, CHCl3). IR (CHCl3): 3396w, 3008w,

2422
Helvetica Chimica Acta ± Vol. 81 (1998)
2976w, 1722s, 1611w, 1511w, 1455w, 1381w, 1101w, 1048w, 930w. 1H-NMR (400 MHz): 9.45 (br., NH); 9.26 (br.,
NH); 7.41 ± 7.29 (m, 20 arom. H); 5.63 (d, J ˆ 8.26, NH); 5.31 ± 5.26 (m, 2 CH); 5.12 (s, PhCH2O); 5.11
(s, PhCH2O); 5.08 (s, PhCH2O); 5.07 (s, PhCH2O); 4.40 ± 4.25 (m, CH); 4.01 ± 3.97 (m, CH2); 2.67, 2.52 (AB of
ABX, JAB ˆ 15.81, JAX ˆ 7.95, JBX ˆ 5.15); 2.51, 2.31 (AB of ABX, JAB ˆ 15.72, JAX ˆ 8.99, JBX ˆ 5.71); 1.84 ± 1.71
(m, CH2); 1.70 ± 1.62 (m, CH2); 1.27 (d, J ˆ 6.33, Me); 1.18 (d, J ˆ 6.26, Me). 13C-NMR (100 MHz): 171.23;
170.01; 169.07; 163.85; 160.50; 155.81; 136.91; 135.64; 135.65; 128.80; 128.55; 128.47; 128.37; 128.34; 128.31;
128.09; 128.04; 127.93; 127.79; 68.91; 68.51; 67.67; 66.96; 66.82; 66.51; 53.74; 44.19; 40.61; 40.51; 29.20; 24.58;
‡
‡
‡
19.75; 19.50. LSI-MS: 841.3 (1, [M ‡ 2 H] ), 840.3 (52, [M ‡ H] ), 839.3 (100, M ), 705.3(15), 687.3(24),
154.0(27), 137.0(19), 136.0(25), 107.0(16). Anal. calc. for C45H50N4O12: C 64.43, H 6.01, N 6.68; found:
C 64.26, H 5.86, N 6.79.
a-{(2S)-2-{[(Benzyloxy)carbonyl]amino}-5-{2,3-bis[(benzyloxy)carbonyl]guanidino}pentanoyl}-w-(benzyl-
oxy)hexadecakis[(R)-oxy(1-methyl-3-oxopropane-1,3-diyl)] (5). Compound 2 (1.49 g, 1.00 mmol) was trans-
formed according to GP II with commercially available Z-l-Arg(Z2)-OH (0.58 g, 1.00 mmol) in CH2Cl2 (50 ml).
FC (CH2Cl2/Et2O 3 :1) yielded 5 (1.3 g, 64%). White solid. M.p. 105 ± 1068. Rf 0.5 (CH2Cl2/Et2O 3 :1). [a]D
ˆ
‡ 1.62 (c ˆ 0.9, CHCl3). IR (CHCl3): 3387w, 3033w, 2985w, 1738s, 1611w, 1512w, 1457w, 1382w, 1306m,
1257m, 1178m, 1101m, 1059m, 979w. 1H-NMR (400 MHz): 9.43 (br., NH); 9.25 (br., NH); 7.38 ± 7.29
(m, 20 arom. H); 5.63 (d, J ˆ 8.39, NH); 5.32 ± 5.20 (m, 16 CH); 5.12 (s, 2 PhCH2O); 5.10 (s, PhCH2O); 5.06
(s, PhCH2O); 4.33 ± 4.31 (m, CH); 3.99 ± 3.97 (m, CH2); 2.71 ± 2.37 (m, 16 CH2); 1.81 ± 1.70 (m, CH2); 1.70 ± 1.64
(m, CH2); 1.28 ± 1.18 (m, 16 CH3). 13C-NMR (100 MHz): 171.20; 169.87; 169.21; 169.11; 169.02; 160.48; 155.80;
136.91; 136.39; 135.67; 128.78; 128.56; 128.46; 128.37; 128.31; 128.09; 128.05; 127.92; 127.77; 68.90; 68.50; 67.66;
67.57; 66.93; 66.54; 40.76; 40.66; 40.63; 40.60; 24.55; 19.78; 19.73; 19.70; 19.66; 19.53. LSI-MS: 2066.8 (1, [M ‡
‡
‡
‡
Na] ), 2065.6 (3, [M ‡ Na H] ), 2044.9 (100, [M ‡ H] ), 1910.6(37), 1892.7(11), 155.1(17), 154.1(12),
137.0(11), 136.0(11). Anal. calc. for C101H134N4O40: C 59.34, H 6.61, N 2.74; found: C 59.49, H 6.51, N 2.80.
a-{(2S)-2-{[(Benzyloxy)carbonyl]amino}-5-{2,3-bis[(benzyloxy)carbonyl]guanidino}pentanoyl}-w-(benzyl-
oxy)dotriacontakis[(R)-oxy(1-methyl-3-oxopropane-1,3-diyl)] (6). Compound 3 (872 mg, 0.31 mmol) was
transformed according to GP II with commercially available Z-l-Arg(Z2)-OH (192 mg, 0.37 mmol) in CH2Cl2
(50 ml). FC (CH2Cl2/Et2O 2 :1) yielded 6 (369 mg, 36%). White solid. M.p. 118 ± 1208. Rf 0.7 (CH2Cl2/Et2O
2 :1). [a]D ˆ‡ 1.32 (c ˆ 0.8, CHCl3). IR (CHCl3): 2988w, 2936w, 1738s, 1506w, 1457w, 1382w, 1306m, 1261m,
1178m, 1133w, 1100m, 1059m, 980w. 1H-NMR (500 MHz): 9.43 (br., NH); 9.24 (br., NH); 7.38 ± 7.28
(m, 20 arom. H); 5.63 (d, J ˆ 8.31, NH); 5.32 ± 5.21 (m, 32 CH); 5.12 (s, 2 PhCH2O); 5.10 (s, PhCH2O); 5.06
(s, PhCH2O); 4.34 ± 4.32 (m, CH); 3.98 ± 3.97 (m, CH2); 2.71 ± 2.38 (m, 32 CH2); 1.81 ± 1.72 (m, CH2); 1.68 ± 1.64
(m, CH2); 1.29 ± 1.18 (m, 32 Me). 13C-NMR (125 MHz): 171.25; 169.91; 169.25; 169.15; 169.07; 163.87; 160.52;
155.85; 136.95; 136.44; 135.72; 134.70; 128.83; 128.61; 128.50; 128.41; 128.35; 128.13; 128.09; 127.96; 127.82;
68.94; 68.54; 67.78; 67.70; 67.63; 67.46; 67.24; 66.98; 66.84; 66.49; 53.79; 44.22; 40.81; 40.71; 40.68; 40.64; 33.97;
‡
29.26; 25.63; 24.95; 24.60; 19.78; 19.74; 19.70; 19.57. MALDI-MS: 3445.7 ([M ‡ Na] ). Anal. calc. for
C
165H230N4O72: C 57.92, H 6.78, N 1.64; found: C 57.63, H 7.03, N 1.58.
(3R)-3-{{(3'R)-3'-{[(2S)-2-Amino-5-guanidinopentanoyl]oxy}butanoyl}oxy}butanoic Acid (7). Com-
pound 4 (83 mg, 99 mmol) was hydrogenated according to GP IIIa in CF3CH2OH (30 ml): 7 (34 mg, 98%).
Colorless, glassy solid. M.p. 84 ± 868. IR (NaCl film): 3675w, 3377w, 3029w, 2960w, 1737s, 1602m, 1519m, 1453m,
1384m, 1260m, 1102w, 1055w, 1012w. 1H-NMR (400 MHz, CDCl3/CD3OD): 7.35 ± 7.31 (m, NH); 5.30 ± 5.13
(m, 2CH); 4.17 ± 4.06 (m, CH); 3.62 ± 3.50 (m, CH2); 2.70 ± 2.23 (m, 2 CH2); 1.95 ± 1.87 (m, CH2); 1.87 ± 1.73
(m, CH2); 1.28 (d, J ˆ 6.10, Me); 1.21 (d, J ˆ 6.19, Me). 13C-NMR (100 MHz, CDCl3/CD3OD): 180.25; 178.35;
172.79; 158.42; 129.09; 70.65; 66.13; 66.29; 55.19; 46.40; 45.14; 44.99; 41.61; 29.26; 25.36; 23.11; 22.87; 20.29. ESI-
‡
MS: 347.3 (14, [M ‡ H] ), 298.3(100).
a-[(2S)-2-Amino-5-guanidinopentanoyl]-w-hydroxyhexadecakis[(R)-oxy(1-methyl-3-oxopropane-1,3-diyl)]
(8). Compound 5 (400 mg, 196 mmol) was hydrogenated according to GP IIIa in CF3CH2OH (20 ml): 8 (289 mg,
98%). White solid. M.p. 151 ± 1538. IR (KBr): 3677w, 3379w, 3029w, 2959w, 1737s, 1601w, 1519w, 1454w, 1384m,
1260m, 1104w, 1056w, 1012w. 1H-NMR (400 MHz, CDCl3/CD3OD): 5.41 ± 5.36 (m, NH); 5.30 ± 5.22 (m, 16 CH);
3.94 ± 3.87 (m, CH); 3.29 ± 3.25 (m, CH2); 2.72 ± 2.46 (m, 16 CH2); 2.04 ± 1.97 (m, CH2); 1.81 ± 1.76 (m, CH2); 1.36
(d, J ˆ 6.35, Me); 1.30 (d, J ˆ 6.47, Me); 1.28 (d, J ˆ 7.99, 4 Me); 1.28 (d, J ˆ 6.31, 10 Me). 13C-NMR (100 MHz,
CDCl3/CD3OD): 172.79; 169.76; 169.58; 169.44; 169.41; 169.40; 169.33; 69.82; 67.94; 67.84; 67.79; 67.71; 67.61;
‡
40.74; 40.64; 40.51; 40.40; 40.31; 27.47; 24.40; 19.66; 19.59. LSI-MS: 1580.2 (20); 1553.2 (68, [M ‡ 2 H] ), 1552.4
‡
‡
(100, [M ‡ H] ); 1551.7 (57, M ). Anal. calc. for C70H110NO34 ´ CF3CH2OH: C 50.69, H 6.63, N 3.20; found:
C 50.31, H 6.75, N 3.49.
a-[(2S)-2-Amino-5-guanidinopentanoyl]-w-hydroxydotriacontakis[(R)-oxy(1-methyl-3-oxopropane-1,3-
diyl)] (9). Compound 6 (200 mg, 58 mmol) was hydrogenated according to GP IIIa in CF3CH2OH (10 ml): 9

Helvetica Chimica Acta ± Vol. 81 (1998)
2423
(145 mg, 85%). M.p. 141 ± 1428. [a]D ˆ‡ 0.92 (c ˆ 0.8, CHCl3). IR (CHCl3): 2988w, 2936w, 1737s, 1459w,
1383m, 1306m, 1134w, 1101w, 1059w, 980w. 1H-NMR (500 MHz): 5.41 ± 5.38 (m, NH); 5.30 ± 5.22 (m, 32 CH);
3.48 ± 3.42 (m, CH); 3.18 ± 3.14 (m, CH2); 2.63 ± 2.45 (m, 32 CH2); 1.98 ± 1.81 (m, CH2); 1.74 ± 1.63 (m, CH2);
1.31 ± 1.21 (m, 32 Me). 13C-NMR (125 MHz): 176.50; 174.69; 169.71; 169.41; 169.34; 169.30; 169.27; 169.18;
160.27; 158.02; 128.36; 69.97; 68.03; 67.96; 67.87; 67.64; 67.25; 53.93; 49.20; 44.11; 41.08; 41.02; 40.80; 40.50;
‡
‡
33.95; 30.02; 25.63; 25.42; 24.95; 20.09; 19.78. MALDI-MS: 2953.9 ([M ‡ Na] ), 2930.1 ([M ‡ H] ).
8. Glycosylated HB Oligomers 10 ± 13. Benzyl (3R)-3-{{(3'R)-3'-[(2,3,4,6-Tetra-O-benzyl-a/b-d-glucopyr-
anosyl)oxy]butanoyl}oxy}butanoate (10). A soln. of 2,3,4,6-tetra-O-benzyl-a-d-glucosyl trichloroacetimidate
(520 mg, 0.76 mmol), prepared according to [16], and 1 (420 mg, 1.52 mmol) in CH2Cl2 (50 ml) at 208 under
Ar was treated slowly with BF3 ´ Et2O (0.17 ml, 0.76 mmol). After 2.5 h stirring, the mixture was washed with aq.
sat. NaHCO3 and NaCl soln. The org. phase was dried (MgSO4) and evaporated. FC (Et2O/pentane 4 : 3)
yielded 10 (567 mg, 93%), b-d/a-d 2.5 : 1. Colorless oil. Rf 0.6 (Et2O/pentane 4 : 3). [a]D ˆ‡ 3.67 (c ˆ 1.4,
CHCl3). IR (CHCl3): 3684w, 3621m, 3468w, 3008s, 2976s, 1734m, 1519m, 1477w, 1422m, 1391w, 1047m, 929m.
1H-NMR (400 MHz): 7.36 ± 7.22 (m, 25 arom. H); 5.32 ± 5.24 (m, CH); 5.09 (s, PhCH2O); 4.97 (d, J ˆ 3.73,
H
C(1)(a)); 4.95 (d, J ˆ 10.91, 1 H, PhCH2O); 4.91 (d, J ˆ 10.93, 1 H, PhCH2O); 4.82 (d, J ˆ 10.71, 1 H,
PhCH2O); 4.80 (d, J ˆ 10.54, 1 H, PhCH2O); 4.77 (d, J ˆ 10.84, 2 H, PhCH2O); 4.68 (d, J ˆ 10.98, 2 H,
PhCH2O); 4.62 (d, J ˆ 12.79, 1 H, PhCH2); 4.60 (d, J ˆ 12.15, 1 H, PhCH2); 4.55 (d, J ˆ 11.39, 1 H, PhCH2); 4.52
(d, J ˆ 10.83, 1 H, PhCH2O); 4.52 (d, J ˆ 12.27, 1 H, PhCH2O); 4.46 (d, J ˆ 7.75, H C(1)(b)); 4.46 (d, J ˆ 10.24,
1 H, PhCH2O); 4.46 (d, J ˆ 12.62, 1 H, PhCH2); 4.29 ± 4.21 (m, H C(b)); 4.16 ± 4.11 (m, H C(a)); 3.94 (dd
(ꢀtꢁ), J ˆ 9.32, H C(3)(a)); 3.86 ± 3.81 (m, H C(5)(a)); 3.73 (dd, J ˆ 3.69, 10.48, H C(6)(b)); 3.70 ± 3.63
(m, H C(3)(b), H C(4)(a), H C(6)(b), 2 H C(6)(a)); 3.60 (dd, J ˆ 8.67, 11.33, H C(4)(b)); 3.52 (dd, J ˆ
3.69, 9.71, H C(2)(a)); 3.43 ± 3.40 (m, H C(5)(b)); 3.39 (dd (ꢀtꢁ), J ˆ 8.38, H C(2)(b)); 2.81 ± 2.39 (m, 2
CH2); 1.28 (d, J ˆ 6.29, Me); 1.25 (d, J ˆ 6.17, Me). 13C-NMR (100 MHz): 170.10; 170.00; 169.94; 138.65; 138.46;
138.23; 138.18; 128.57; 128.55; 128.40; 128.34; 128.14; 128.05; 127.93; 127.87; 127.84; 127.75; 127.69; 127.65;
127.54; 127.50; 101.78; 84.73; 82.17; 81.92; 79.84; 76.68; 75.66; 75.60; 74.96; 74.84; 74.81; 73.44; 73.40; 71.87;
‡
68.89; 67.40; 66.41; 42.85; 41.73; 40.69; 40.62; 21.37; 19.80. LSI-MS: 826.3 (13, [M ‡ Na ‡ H] ), 825.3 (25, [M ‡
‡
‡
‡
‡
Na] ), 803.5 (15, [M ‡ H] ), 802.3 (37, M ), 801.2 (72, [M H] ), 182.1(19), 181.1(100), 107.0(17),
105.0(17). Anal. calc. for C49H54O10: C 73.30, H 6.78; found: C 73.26, H 6.87.
a-(2,3,4,6-Tetra-O-benzyl-a/b-d-glucopyranosyl)-w-(benzyloxy)hexadecakis[(R)-oxy(1-methyl-3-oxopro-
pane-1,3-diyl)] (11). A soln. of 2,3,4,6-tetra-O-benzyl-a-d-glucosyl trichloroacetimidate (0.44 g, 0.64 mmol)
and 2 (1.43 g, 0.97 mmol) in CH2Cl2 (80 ml) at 208 under Ar was treated slowly with BF3 ´ Et2O (0.14 ml,
0.65 mmol). After 2.5 h stirring, the mixture was washed with aq. sat. NaHCO3 and NaCl soln. The org. phase
was dried (MgSO4) and evaporated. FC (CH2Cl2/Et2O 6 : 1) yielded 11 (1.06 g, 82%), b-d/a-d 2.5 : 1. White
solid. M.p. 89 ± 908. Rf 0.3 (CH2Cl2/Et2O 6 : 1). [a]D ˆ‡ 2.01 (c ˆ 1.0, CHCl3). IR (CHCl3): 3520w, 2985w, 2937w,
1737s, 1456w, 1382m, 1306m, 1264m, 1178m, 1100m, 1060m, 979w. 1H-NMR (400 MHz): 7.36 ± 7.25
(m, 25 arom. H); 5.31 ± 5.22 (m, 15 CH); 5.12 (s, PhCH2O); 4.97 (d, J ˆ 3.28, H C(1)(a)); 4.96 (d, J ˆ 11.71,
1 H, PhCH2O); 4.92 (d, J ˆ 10.92, 1 H, PhCH2O); 4.90 (d, J ˆ 10.97, 2 H, PhCH2O); 4.82 (d, J ˆ 10.71, 1 H,
PhCH2O); 4.80 (d, J ˆ 10.83, 1 H, PhCH2O); 4.77 (d, J ˆ 10.97, 2 H, PhCH2O); 4.70 (d, J ˆ 12.08, 1 H,
PhCH2O); 4.68 (d, J ˆ 10.94, 1 H, PhCH2O); 4.61 (d, J ˆ 12.20, 1 H, PhCH2O); 4.61 (d, J ˆ 12.24, 1 H,
PhCH2O); 4.53 (d, J ˆ 10.85, 1 H, PhCH2O); 4.53 (d, J ˆ 12.09, 1 H, PhCH2O); 4.48 (d, J ˆ 7.81, H C(1)(b));
4.46 (d, J ˆ 12.81, 1 H, PhCH2O); 4.30 ± 4.26 (m, H C(b)); 4.17 ± 4.13 (m, H C(a)); 3.94 (dd (ꢀtꢁ), J ˆ 9.36,
H
H
H
H
C(3)(a)); 3.90 ± 3.82 (m, H C(5)(a)); 3.74 (dd, J ˆ 2.88, 9.68, H C(6)(b)); 3.72 (dd, J ˆ 2.13, 10.76,
C(6)(a)); 3.69 (dd, J ˆ 5.81, 10.11, H C(6)(b)); 3.67 ± 3.57 (m, H C(3)(b), H C(4)(a), H C(4)(b),
C(6)(a)); 3.53 (dd, J ˆ 3.65, 9.70, H C(2)(a)); 3.44 ± 3.41 (m, H C(5)(b)); 3.40 (dd (ꢀtꢁ), J ˆ 8.41,
C(2)(b)); 2.71 ± 2.38 (m, 16 CH2); 1.29 ± 1.24 (m, 9 Me); 1.27 (d, J ˆ 6.24, 6 Me); 1.24 (d, J ˆ 6.31, Me).
13C-NMR (100 MHz); 170.07; 169.91; 169.15; 128.60; 128.45; 128.37; 128.36; 128.17; 128.08; 127.96; 127.90;
127.73; 127.72; 127.66; 127.59; 101.83; 84.76; 82.18; 74.98; 74.86; 74.82; 73.42; 71.90; 67.70; 67.62; 67.57; 67.37;
‡
‡
66.49; 42.86; 40.87; 40.81; 40.68; 19.82; 19.78; 19.74. LSI-MS: 2140.8 (26, [M ‡ Cs] ), 2046.6 (4, [M ‡ K] ),
‡
‡
2030.5 (69, [M ‡ Na] ), 2007.0 (14, M ), 1576.1(30), 1485.7(39), 182.1(21), 181.1(85), 173.1(21), 156.1(20),
155.0(100), 154.0(56), 138.0(23), 137.0(46), 136.0(48). Anal. calc. for C105H138O38: C 62.80, H 6.93; found:
C 62.81, H 6.92.
(3R)-3-{{(3'R)-3'-[(a/b-d-Glucopyranosyl)oxy]butanoyl}oxy}butanoic Acid (12). Compound 10 (160 mg,
0.20 mmol) was hydrogenated according to GP IIIa in CF3CH2OH (40 ml). FC (CH2Cl2/MeOH/H2O
6 :2.5 :0.25) yielded 12 (62 mg, 88%), b-d/a-d 2.5 :1. Colorless oil. Rf 0.2 (CH2Cl2/MeOH/H2O 6 :2.5 :0.25).
IR (KBr): 3503w, 2979w, 2933w, 1738s, 1448w, 1383m, 1305m, 1178m, 1136m, 1100m, 1067s, 977w. 1H-NMR
(400 MHz, CD3OD): 5.28 ± 5.22 (m, CH); 4.94 (d, J ˆ 3.85, H C(1)(a)); 4.34 (d, J ˆ 7.75, H C(1)(b)); 4.29 ±

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Helvetica Chimica Acta ± Vol. 81 (1998)
4.23 (m, H C(b)); 4.20 ± 4.13 (m, H C(a)); 3.84 (dd, J ˆ 2.00, 11.88, H C(6)(b)); 3.79 (dd, J ˆ 2.09, 13.66,
H
H
C(6)(a)); 3.76 ± 3.62 (m, H C(3)(a), H C(5)(a), H C(6)(a), H C(6)(b)); 3.58 (dd (ꢀtꢁ), J ˆ 9.34,
C(3)(b)); 3.48 ± 3.24 (m, H C(4)(a), H C(4)(b), H C(5)(b)); 3.11 (dd (ꢀtꢁ), J ˆ 8.42, H C(2)(b)); 2.70,
2.50 (AB of ABX, JAB ˆ 15.88, JAX ˆ 6.58, JBX ˆ 5.76); 2.60, 2.44 (AB of ABX, JAB ˆ 15.40, JAX ˆ 7.89, JBX
ˆ
6.68); 1.29 (d, J ˆ 6.29, Me); 1.24 (d, J ˆ 6.21, Me). 13C-NMR (100 MHz, CD3OD): 172.65; 172.63; 102.40;
100.36; 78.05; 77.87; 75.16; 75.02; 74.06; 73.71; 72.68; 71.77; 71.70; 69.52; 62.85; 62.69; 43.81; 42.65; 41.89; 21.82;
‡
‡
‡
20.08; 19.97. LSI-MS: 1057.3 (13, [3M ‡ H] ), 493.1(21), 398.1 (22, [M ‡ 2Na] ), 397.1 (84, [M ‡ 2Na H] ),
‡
375.1 (36, [M ‡ Na] ), 329.1(21), 217.0(23), 199.9(26), 177.0(26), 176.0(100), 155.1(30), 154.1(41),
149.0(41), 137.0(35), 136.0(39), 131.0(26), 105.0(32). Anal. calc. for C14H24O10 ´ H2O: C 45.41, H 7.08; found:
C 45.64, H 6.90.
a-[(a/b-d-Glucopyranosyl)oxy]-w-hydroxyhexadecakis[(R)-oxy(1-methyl-3-oxopropane-1,3-diyl)] (13).
Compound 11 (400 mg, 198 mmol) was hydrogenated according to GP IIIa in CF3CH2OH (40 ml). FC
(CH2Cl2/MeOH/H2O 6 :2.5 :0.25) yielded 13 (238 mg, 77%), b-d/a-d 2.5 :1. White solid. M.p. 128 ± 1298. Rf 0.7
(CH2Cl2/MeOH/H2O 6 :2.5 :0.25). [a]D ˆ‡ 23.8 (c ˆ 0.7, CHCl3). IR (CHCl3): 3502w, 2982w, 2933w, 1737s,
1450w, 1382m, 1303m, 1178m, 1134m, 1101m, 1057s, 979w. 1H-NMR (400 MHz, CDCl3/CD3OD): 5.30 ± 5.24
(m, 15 CH); 4.97 (d, J ˆ 4.03, H C(1)(a)); 4.36 (d, J ˆ 7.75, H C(1)(b)); 4.30 ± 4.26 (m, H C(b)); 4.20 ± 4.13
(m, H C(a)); 3.86 (dd, J ˆ 2.87, 11.98,
H
C(6)(b)); 3.80 (dd, J ˆ 3.17, 11.88,
H C(6)(a)); 3.78 ± 3.67
(m, H C(3)(a), H C(5)(a), H C(6)(a), H C(6)(b)); 3.63 (dd (ꢀtꢁ), J ˆ 9.35, H C(3)(b)); 3.43 ± 3.27
(m, H C(4)(a), H C(4)(b), H C(5)(b)); 3.21 (dd (ꢀtꢁ), J ˆ 9.02, H C(2)(b)); 2.70 ± 2.43 (m, 16 CH2); 1.31
(d, J ˆ 6.43, 2 Me); 1.29 (d, J ˆ 6.33, 13 Me); 1.27 (d, J ˆ 6.27, Me). 13C-NMR (100 MHz, CDCl3/CD3OD):
‡
169.99; 169.97; 169.93; 68.20; 68.16; 68.12; 41.13; 41.03; 19.88. LSI-MS: 1580.1 (79, [M ‡ Na] ), 1417.7(11),
1396.6(56), 1395.5(75), 1285.7(32), 1283.6(56), 1282.5(22), 1281.5(21), 1193.6(22), 1191.6(40), 1190.5(20),
1189.5(20), 155.1(100), 154.0(43), 137.0(33), 136.0(34). Anal. calc. for C70H108O38 ´ H2O: C 53.36, H 7.03;
found: C 53.35, H 7.25.
9. Fluorescent HB Oligomers 14 ± 18. Benzyl (3R)-3-{{(3'R)-3'-{{[7-(Dimethylamino)-2-oxo-2H-1-benzo-
pyran-4-yl]acetyl}oxy}butanoyl}oxy}butanoate (14). To a suspension of 2-chloro-1-methylpyridinium iodide
(290 mg, 0.97 mmol) in CH2Cl2 (5 ml) under Ar, a soln. of 7-(dimethylamino)-2-oxo-2H-1-benzopyran-4-acetic
acid (200 mg, 0.81 mmol) and 1 (229 mg, 0.81 mmol) in CH2Cl2/MeCN 1:1 (6 ml) was added. After addition of
Et3N (270 ml, 1.93 mmol), stirring was continued for 12 h. The mixture was washed with 1n HCl, aq. sat.
NaHCO3, and NaCl soln. The org. phase was dried (MgSO4) and evaporated. FC (Et2O/pentane 3 :1) yielded 14
(122 mg, 30%). Green oil. Rf 0.4 (Et2O/pentane 3 :1). [a]D ˆ‡ 0.50 (c ˆ 1.0, CHCl3). IR (CHCl3): 1736s, 1618m,
1532w, 1403m, 1277w, 1177w, 1059w. 1H-NMR (300 MHz): 7.38 (d, J(5,6) ˆ 8.95, H C(5)); 7.37 ± 7.32
(m, 5arom. H); 6.61 (dd, J(5,6) ˆ 8.95, J(6,8) ˆ 2.56,
H
C(6)); 6.55 (d, J(6,8) ˆ 2.56,
H C(8)); 6.05
(s, H C(3)); 5.33 ± 5.22 (m, 2 CH); 5.08 (s, PhCH2O); 3.65 (s, CH2 C(4)); 3.05 (s, Me2N); 2.66 ± 2.37
(m, 2 CH2); 1.28 (d, J ˆ 6.36, Me); 1.24 (d, J ˆ 6.39, Me). 13C-NMR (75 MHz): 169.87; 169.15; 169.01; 168.29;
161.62; 155.98; 148.96; 135.71; 128.58; 128.33; 128.21; 125.36; 110.67; 109.01; 108.38; 98.35; 68.63; 67.77; 66.46;
‡
‡
40.88; 40.68; 40.64; 40.30; 19.79; 19.70. LSI-MS: 1021.2 (2, [M ‡ H]2 ), 1020.2 (7, [2M ‡ H] ), 1019.2 (12,
‡
‡
‡
‡
‡
[M]2 ), 1018.1 (6, [2M H] ), 511.1 (33, [M ‡ 2 H] ), 510.1 (100, [M ‡ H] ), 509.1 (88, M ), 508.1 (19, [M
‡
H] ), 203.1(10).
a-{[7-(Dimethylamino)-2-oxo-2H-1-benzopyran-4-yl]acetyl}-w-(benzyloxy)hexadecakis[(R)-oxy(1-methyl-
3-oxopropane-1,3-diyl)] (15). As described for 14, with 2-chloro-1-methylpyridinium iodide (97 mg,
0.32 mmol), CH2Cl2 (4 ml), 7-(dimethylamino)-2-oxo-2H-1-benzopyran-4-acetic acid (67 mg, 0.27 mmol), 2
(200 mg, 0.135 mmol), CH2Cl2/MeCN 1:1 (6 ml), and Et3N (61 ml, 0.44 mmol; stirring for 18 h). FC (CH2Cl2/
Et2O 3 :1) yielded 15 (49 mg, 21%). Yellow solid. M.p. 128 ± 1298. Rf 0.6 (CH2Cl2/Et2O 3 :1). [a]D
ˆ
0.55 (c ˆ
1.1, CHCl3). IR (CHCl3): 3011w, 1738s, 1605w, 1384w, 1305w, 1261w, 1178m, 1100w, 1056m. 1H-NMR (400 MHz):
7.39 (d, J(5,6) ˆ 8.94, H C(5)); 7.36 ± 7.32 (m, 5 arom. H); 6.62 (dd, J(5,6) ˆ 8.94, J(6,8) ˆ 2.56, H C(6)); 6.52
(d, J(6,8) ˆ 2.56, H C(8)); 6.05 (s, H C(3)); 5.33 ± 5.20 (m, 16 CH); 5.12 (s, PhCH2O); 3.66 (s, CH2 C(4));
3.06 (s, Me2N); 2.71 ± 2.37 (m, 16 CH2); 1.29 ± 1.25 (m, 4 Me); 1.29 (d, J ˆ 6.31, 2 Me); 1.27 (d, J ˆ 6.31, 8 Me);
1.24 (d, J ˆ 6.35, 2 Me). 13C-NMR (100 MHz): 169.88; 169.12; 169.06; 168.29; 161.65; 155.95; 152.96; 148.31;
135.70; 128.58; 128.32; 125.36; 110.77; 109.00; 108.48; 98.34; 68.55; 67.67; 67.60; 66.46; 40.78; 40.68; 40.66; 40.60;
‡
‡
‡
38.17; 19.79; 19.75; 19.70. LSI-MS: 1715.5 (32, [M ‡ H] ), 1714.6 (100, M ), 1713.3 (23, [M H] ), 155.0(12),
154.0(11). Anal. calc. for C84H115NO36: C 58.84, H 6.76, N 0.82; found: C 59.01, H 6.71, N 0.82.
a-{[7-(Dimethylamino)-2-oxo-2H-1-benzopyran-4-yl]acetyl}-w-hydroxyhexadecakis[(R)-oxy(1-methyl-3-
oxopropane-1,3-diyl)] (16). Compound 15 (30 mg, 18 mmol) was hydrogenated according to GP IIIc in 6 ml
N,N-dimethylacetamide. FC (CH2Cl2/MeOH 10 :1) yielded 16 (14 mg, 50%). Yellow solid. M.p. 115 ± 1168.
Rf 0.7 (CH2Cl2/MeOH 10 :1). [a]D
ˆ
1.15 (c ˆ 0.9, CHCl3). IR (CHCl3): 2986w, 1738s, 1602w, 1306w, 1265s,

Helvetica Chimica Acta ± Vol. 81 (1998)
2425
1178m, 1059w, 977w. 1H-NMR (400 MHz): 7.45 (d, J(5,6) ˆ 8.96, H C(5)); 6.63 (dd, J(5,6) ˆ 8.96, J(6,8) ˆ
2.56, H C(6)); 6.51 (d, J(6,8) ˆ 2.55, H C(8)); 6.08 (s, H C(3)); 5.32 ± 5.18 (m, 16 CH); 3.65 (s, CH2 C(4));
3.05 (s, Me2N); 2.66 ± 2.36 (m, 16 CH2); 1.27 ± 1.23 (m, 16 Me). 13C-NMR (100 MHz): 170.24; 162.57; 156.03;
148.38; 135.53; 128.41; 128.08; 125.61; 110.36; 110.09; 108.65; 98.38; 68.61; 67.67; 66.56; 41.65; 40.87; 40.54;
‡
‡
40.17; 40.04; 19.79; 19.63. LSI-MS: 1655.5(13), 1647.7 (12, [M ‡ Na] ), 1625.6 (100, [M ‡ H] ), 1624.7 (16,
‡
M ), 1417.6(22).
Benzyl
(3R)-3-{{(3'R)-3'-{{[5-(Dimethylamino)naphthalen-1-yl]sulfonyl}oxy}butanoyl}oxy}butanoate
(17). To a soln. of dansyl chloride (60 mg, 0.22 mmol) and 1 (41 mg, 0.15 mmol) in CH2Cl2 (15 ml) at 508
under Ar, a soln. of DMAP (15 mg, 0.15 mmol) in CH2Cl2 (5 ml) was added. After stirring was continued for 4 d,
the soln. was washed with 1n HCl, aq. sat. NaHCO3, and NaCl soln. The org. phase was dried (MgSO4) and
evaporated. FC (pentane/Et2O 1:1) yielded 17 (11 mg, 15%). Yellow, oily solid. M.p. 133 ± 1348. Rf 0.5
(pentane/Et2O 1:1). IR (CHCl3): 3038w, 1737s, 1573w, 1456w, 1358w, 1306w, 1175w, 1048w, 925w. 1H-NMR
(400 MHz): 8.58 (d, J(2,3) ˆ 8.54, H C(2)); 8.27 (d, J(3,4) ˆ 7.31, H C(4)); 8.22 (d, J(7,8) ˆ 9.61, H C(8));
7.58 ± 7.51 (m, H C(3), H C(7)); 7.37-7.31 (m, 5 arom. H); 7.17 (d, J(6,7) ˆ 7.58, H C(6)); 5.14 ± 5.09
(m, CH); 5.09 (s, PhCH2O); 4.94 ± 4.89 (m, CH); 2.88 (s, Me2N); 2.57, 2.50 (AB of ABX, JAB ˆ 15.69, JAX ˆ 7.31,
JBX ˆ 5.86); 2.64, 2.42 (AB of ABX, JAB ˆ 15.71, JAX ˆ 7.47, JBX ˆ 5.72); 1.21 (d, J ˆ 6.41, Me); 1.20 (d, J ˆ 6.39,
Me). 13C-NMR (100 MHz): 169.82; 168.33; 151.77; 135.64; 132.35; 131.45; 129.88; 129.82; 128.59; 128.54;
128.37; 128.34; 123.04; 119.50; 115.44; 67.95; 66.48; 45.39; 41.55; 40.46; 20.60; 19.64. LSI-MS: 1028.0 (8, [2M ‡
‡
‡
‡
‡
‡
‡
H] ), 1027.0 (15, [M]2 ), 1026.0 (5, [2M H] ), 514.9 (41, [M ‡ 2 H] ), 513.9 (91, [M ‡ H] ), 512.9 (100, M ),
‡
511.9 (22, [M H] ), 251.9(25), 250.9(66), 249.9(17), 170.0(25), 168.0(16), 154.0(26), 137.0(17), 136.0(22).
Anal. calc. for C27H31NO7S: C 63.14, H 6.08, N 2.72; found: C 63.16, H 6.25, N 2.76.
a-{[5-(Dimethylamino)naphthalen-1-yl]sulfonyl}-w-(benzyloxy)hexadecakis[(R)-oxy(1-methyl-3-oxopro-
pane-1,3-diyl)] (18). As described for 17, with dansyl chloride (188 mg, 0.7 mmol), 2 (520 mg, 0.35 mmol),
CH2Cl2 (60 ml), DMAP (44 mg, 0.37 mmol) in CH2Cl2 (15 ml), stirring for 2 d. FC (CH2Cl2/Et2O 3 :1) yielded
18 (67 mg, 12%). Yellow solid. M.p. 152 ± 1548. Rf 0.8 (CH2Cl2/Et2O 3 :1). [a]D
ˆ
1.07 (c ˆ 0.8, CHCl3). IR
(CHCl3): 3038w, 2985w, 1737s, 1580w, 1450w, 1382w, 1358w, 1306m, 1176m, 1129w, 1047w, 938w. 1H-NMR
(400 MHz): 8.57 (d, J(2,3) ˆ 8.53, H C(2)); 8.30 (d, J(3,4) ˆ 7.28, H C(4)); 8.23 (d, J(7,8) ˆ 9.61, H C(8));
7.59 ± 7.51 (m, H C(3), H C(7)); 7.36 ± 7.32 (m, 5 arom. H); 7.16 (d, J(6,7) ˆ 7.54, H C(6)); 5.31 ± 5.15
(m, 15 CH); 5.08 (s, PhCH2O); 4.95 ± 4.90 (m, CH); 2.87 (s, Me2N); 2.73 ± 2.36 (m, 16 CH2); 1.28 ± 1.20
(m, 16 Me). 13C-NMR (100 MHz): 172.00; 169.20; 169.12; 169.06; 168.28; 161.63; 155.95; 153.01; 148.41;
135.72; 132.45; 131.44; 129.89; 129.87; 128.60; 128.53; 128.39; 128.33; 122.98; 120.08; 115.43; 68.45; 67.65; 67.62;
‡
66.48; 45.41; 41.53; 40.76; 40.68; 40.65; 40.62; 19.79; 19.74; 19.64. LSI-MS: 1719.9 (25, [M ‡ H] ), 1718.8 (100,
‡
M ), 155.0(18).
10. Glycine-OHB Conjugates 19 ± 21. Benzyl (3R)-3-{{(3'R)-3'-{{2-{[(tert-Butoxy)carbonyl]amino}acetyl}-
oxy}butanoyl}oxy}butanoate (19). Compound 1 (700 mg, 2.50 mmol) was transformed according to GP II with
commercially available N-[(tert-butoxy)carbonyl]glycine (500 mg, 2.86 mmol) in CH2Cl2 (120 ml). FC (CH2Cl2/
Et2O 4 :1) yielded 19 (970 mg, 89%). Colorless oil. Rf 0.8 (CH2Cl2/Et2O 4 :1). [a]D ˆ‡ 0.81 (c ˆ 1.1, CHCl3). IR
1
(CHCl3): 2985w, 1737s, 1508w, 1456w, 1368w, 1262w, 1107m, 1102w, 1058m, 974w. H-NMR (400 MHz): 7.38 ±
7.31 (m, 5 arom. H); 5.34 ± 5.26 (m, 2 CH); 5.12 (s, PhCH2O); 5.06 (br., NH); 3.86 ± 3.84 (m, CH2NH); 2.68,
2.56 (AB of ABX, JAB ˆ 15.63, JAX ˆ 7.80, JBX ˆ 5.18); 2.55, 2.42 (AB of ABX, JAB ˆ 15.57, JAX ˆ 7.94, JBX
ˆ
5.60); 1.45 (s, t-Bu); 1.28 (d, J ˆ 6.34, Me); 1.27 (d, J ˆ 6.32, Me). 13C-NMR (100 MHz): 169.92; 169.52; 160.11;
155.64; 135.67; 128.57; 128.34; 128.33; 79.88; 68.34; 67.73; 66.51; 42.51; 40.77; 40.65; 28.30; 19.81; 19.77. LSI-MS:
‡
‡
‡
876.2 (11, [2M ‡ H] ), 875.2 (22, [M]2 ), 438.1 (16, [M ‡ H] ), 382.0(23), 339.1(29), 338.1(100), 154.0(11),
137.0(16). Anal. calc. for C22H31N8: C 60.40, H 7.14, N 3.20; found: C 60.24, H 7.19, N 3.47.
a-{2-{[(tert-Butoxy)carbonyl]amino}acetyl}-w-(benzyloxy)hexadecakis[(R)-oxy(1-methyl-3-oxopropane-
1,3-diyl)] (20). Compound 2 (2.0 g, 1.35 mmol) was transformed according to GP II with commercially available
N-[(tert-butoxy)carbonyl]glycine (350 mg, 2.0 mmol) in CH2Cl2 (90 ml). FC (CH2Cl2/Et2O 5 :1) yielded 20
(1.82 g, 82%). White solid. M.p. 128 ± 1298. Rf 0.5 (CH2Cl2/Et2O 5 :1). [a]D
ˆ
0.31 (c ˆ 1.0, CHCl3). IR
(CHCl3): 2985w, 1737s, 1504w, 1458w, 1382w, 1306m, 1266w, 1178m, 1136w, 1102w, 1060m, 978w. 1H-NMR
(400 MHz): 7.37 ± 7.31 (m, 5 arom. H); 5.35 ± 5.30 (br., NH); 5.29 ± 5.20 (m, 16 CH); 5.12 (s, PhCH2O); 3.88 ±
3.85 (m, CH2NH); 2.71 ± 2.37 (m, 16 CH2); 1.45 (s, t-Bu); 1.31 (d, J ˆ 6.33, 2 Me); 1.28 (d, J ˆ 5.70, 2 Me); 1.28
(d, J ˆ 6.31, 8 Me); 1.27 (d, J ˆ 6.13, 2 Me); 1.25 (d, J ˆ 6.34, 2 Me). 13C-NMR (100 MHz): 169.20; 169.15;
128.61; 128.35; 68.38; 67.70; 67.62; 66.49; 40.82M 40.69; 28.34; 19.84; 19.82; 19.76; 19.74. LSI-MS: 1664.4 (3,
‡
‡
‡
[M ‡ Na] ), 1642.3 (1, [M ‡ H] ), 1542.4 (100, [M Boc] ), 155.0(27). Anal. calc. for C78H115NO36: C 57.03,
H 7.06, N 0.85; found: C 56.99, H 7.29, N 1.04.

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Helvetica Chimica Acta ± Vol. 81 (1998)
a-{2-{[(tert-Butoxy)carbonyl]amino}acetyl}-w-(benzyloxy)dotriacontakis[(R)-oxy(1-methyl-3-oxopro-
pane-1,3-diyl)] (21). Compound 3 (840 mg, 0.29 mmol) was transformed according to GP II with commercially
available N-[(tert-butoxy)carbonyl]glycine (77 mg, 0.44 mmol) in CH2Cl2 (60 ml). FC (CH2Cl2/Et2O 2 :1)
yielded 21 (670 mg, 76%). White solid. M.p. 125 ± 1268. Rf 0.2 (CH2Cl2/Et2O 2 :1). [a]D ˆ‡ 1.45 (c ˆ 1.1,
CHCl3). IR (CHCl3): 2987w, 1739s, 1458w, 1383w, 1306m, 1262w, 1178m, 1135w, 1058m, 978w. 1H-NMR
(500 MHz): 7.37 ± 7.32 (m, 5 arom. H); 5.45 ± 5.42 (br., NH); 5.28 ± 5.20 (m, 32 CH); 5.12 (s, PhCH2O); 3.89 ±
3.85 (m, CH2NH); 2.71 ± 2.38 (m, 32 CH2); 1.45 (s, t-Bu); 1.31 (d, J ˆ 6.34, Me); 1.28 (d, J ˆ 6.32, 2 Me); 1.27
(d, J ˆ 6.32, 25 Me); 1.27 (d, J ˆ 6.03, 2 Me); 1.24 (d, J ˆ 6.31, 2 Me). 13C-NMR (125 MHz): 169.91; 169.20;
169.17; 169.15; 155.69; 135.72; 128.61; 128.35; 68.38; 67.78; 67.70; 67.63; 67.46; 66.49; 40.81; 40.68; 28.33; 19.91;
‡
19.84; 19.82; 19.78; 19.74; 19.61; 18.04. MALDI-MS: 3043.1 ([M ‡ Na] ). Anal. calc. for C124H211NO68: C 56.47,
H 7.04, N 0.46; found: C 56.22, H 7.21, N 0.51.
11. Fluorescent Glycine-OHB Conjugates 22 ± 26. Benzyl (3R)-3-{{(3'R)-3'-{{2-{{2-[7-(Dimethylamino)-2-
oxo-2H-1-benzopyran-4-yl]acetyl}amino}acetyl}oxy}butanoyl}oxy}butanoate (22). Boc deprotection of 19
(250 mg, 0.58 mmol) in CH2Cl2 (5 ml) according to GP IV. The obtained crude trifluoroacetate was treated
with 7-(dimethylamino)-2-oxo-2H-1-benzopyran-4-acetic acid (143 mg, 0.58 mmol) according to GP I. FC
(CH2Cl2/Et2O 2 :1) yielded 22 (262 mg, 80%). Green oil. Rf 0.3 (CH2Cl2/Et2O 2 :1). [a]D
ˆ
14.0 (c ˆ 0.9,
CHCl3). IR (CHCl3): 3011w, 2988w, 1738s, 1603w, 1384w, 1306m, 1265s, 1177m, 1135w, 1101w, 1059w, 976w.
1H-NMR (400 MHz): 7.47 (d, J(5,6) ˆ 8.98, H C(5)); 7.37 ± 7.32 (m, 5 arom. H); 6.61 (dd, J(5,6) ˆ 8.98,
J(6,8) ˆ 2.56, H C(6)); 6.49 (d, J(6,8) ˆ 2.56, H C(8)); 6.35 (dd, J ˆ 5.43, 1.17, NH); 6.08 (s, H C(3)); 5.33 ±
5.20 (m, 2 CH); 5.07 (s, PhCH2O); 3.97 (dd, J ˆ 5.43, 1.17, CH2NH); 3.66 (s, CH2CONH); 3.04 (s, Me2N);
2.64 ± 2.37 (m, 2 CH2); 1.27 (d, J ˆ 6.37, Me); 1.24 (d, J ˆ 6.36, Me). 13C-NMR (100 MHz): 170.10; 169.18;
168.55; 168.03; 161.59; 156.03; 149.15; 135.57; 128.57; 128.33; 128.26; 125.63; 110.58; 109.16; 108.31; 98.26; 68.63;
‡
67.78; 66.54; 41.62; 40.71; 40.65; 40.23; 40.06; 19.84; 19.72. LSI-MS: 1134.2 (3, [M ‡ H]2 ), 1133.2 (6, [2M ‡
‡
‡
‡
‡
‡
‡
H] ), 1332.1 (3, [M]2 ), 568.1 (34, [M ‡ 2 H] ), 567.1 (100, [M ‡ H] ), 566.1 (82, M ), 565.1 (25, [M H] ),
154.0(21), 136.0(27). Anal. calc. for C30H34N2O9: C 63.59, H 6.05, N 4.94; found: C 63.59, H 6.30, N 4.92.
a-{2-{{2-[7-(Dimethylamino)-2-oxo-2H-1-benzopyran-4-yl]acetyl}amino}acetyl}-w-(benzyloxy)hexadecakis-
[(R)-oxy(1-methyl-3-oxopropane-1,3-diyl)] (23). Boc deprotection of 20 (600 mg, 0.36 mmol) in CH2Cl2 (5 ml)
according to GP IV. The obtained crude trifluoroacetate was reacted according to GP I with 7-(dimethylamino)-
2-oxo-2H-1-benzopyran-4-acetic acid (99 mg, 0.4 mmol). FC (CH2Cl2/Et2O/MeOH 8 :2 :0.2) yielded 23
(557 mg, 86%). Yellow solid. M.p. 124 ± 1258. Rf 0.5 (CH2Cl2/Et2O/MeOH 8 :2 :0.2). [a]D
ˆ
2.5 (c ˆ 0.8,
CHCl3). IR (CHCl3): 2985w, 1737s, 1508w, 1456w, 1368w, 1262w, 1107m, 1102w, 1058m, 974w. 1H-NMR
(400 MHz): 7.44 (d, J(5,6) ˆ 8.97, H C(5)); 7.38 ± 7.30 (m, 5 arom. H); 6.60 (dd, J(5,6) ˆ 8.97, J(6,8) ˆ 2.57,
H
C(6)); 6.50 (d, J(6,8) ˆ 2.56, H C(8)); 6.34 (dd, J ˆ 5.43, 1.16, NH); 6.08 (s, H C(3)); 5.33 ± 5.20
(m, 16 CH); 5.07 (s, PhCH2O); 3.98 (dd, J ˆ 5.43, 1.16, CH2NH); 3.64 (s, CH2CONH); 3.05 (s, Me2N); 2.64 ±
2.37 (m, 16 CH2); 1.27 ± 1.22 (m, 16 Me). 13C-NMR (100 MHz): 170.11; 169.18; 168.57; 168.20; 161.62; 156.03;
149.14; 135.59; 128.55; 128.32; 128.27; 125.62; 110.58; 109.18; 109.03; 99.26; 68.62; 67.72; 66.43; 41.62; 40.71;
40.60; 40.33; 40.07; 19.84; 19.73. LSI-MS: 1772.6 (28, [M ‡ H] ), 1771.5 (100, M ), 1770.5 (22, [M H] ).
‡
‡
‡
Anal. calc. for C86H118N2O37: C 58.03, H 6.71, N 1.58; found: C 58.08, H 6.61, N 1.64.
a-{2-{{2-[7-(Dimethylamino)-2-oxo-2H-1-benzopyran-4-yl]acetyl}amino}acetyl}-w-(benzyloxy)dotriacon-
takis[(R)-oxy(1-methyl-3-oxopropane-1,3-diyl)] (24). Boc deprotection of 21 (80 mg, 26 mmol) in CH2Cl2
(2 ml) according to GP IV. The obtained crude trifluoroacetate was reacted according to GP I with 7-
(dimethylamino)-2-oxo-2H-1-benzopyran-4-acetic acid (13 mg, 50 mmol). FC (CH2Cl2/Et2O/MeOH 10 :5 :0.1)
yielded 24 (57 mg, 70%). Yellow solid. M.p. 125 ± 1268. Rf 0.6 (CH2Cl2/Et2O/MeOH 10 :5 :0.1). [a]D
ˆ
0.71
(c ˆ 0.7, CHCl3). IR (CHCl3): 3032w, 1738s, 1602w, 1383w, 1305w, 1178m, 1101w, 1059m, 974w. 1H-NMR
(500 MHz): 7.50 (d, J(5,6) ˆ 9.00, H C(5)); 7.46 ± 7.36 (m, 5 arom. H); 6.63 (dd, J(5,6) ˆ 9.00, J(6,8) ˆ 2.54,
H
C(6)); 6.52 (d, J(6,8) ˆ 2.54, H C(8)); 6.50 ± 6.48 (m, NH); 6.10 (s, H C(3)); 5.31 ± 5.20 (m, 32 CH); 5.07
(s, PhCH2O); 3.98 (dd, J ˆ 5.49, CH2NH); 3.69 (s, CH2CONH); 3.06 (s, Me2N); 2.71 ± 2.38 (m, 32 CH2); 1.29 ±
1.24 (m, 32 Me). 13C-NMR (100 MHz): 169.91; 169.43; 169.28; 169.24; 169.15; 168.62; 168.15; 161.60; 156.08;
153.13; 149.29; 135.72; 128.61; 128.35; 125.75; 110.64; 109.18; 108.45; 98.31; 68.67; 67.71; 67.67; 67.63; 66.48;
41.66; 40.93; 40.81; 40.68; 40.21; 40.13; 19.89; 19.78; 19.74. MALDI-MS: 3170.1 ([M ‡ Na] ). Anal. calc. for
‡
C
150H214N2O69 ´ 12 H2O: C 53.49, H 7.07, N 0.83; found: C 53.50, H 7.05, N 0.78.
a-{2-{{2-[7-(Dimethylamino)-2-oxo-2H-1-benzopyran-4-yl]acetyl}amino}acetyl}-w-hydroxyhexadecakis-
[(R)-oxy(1-methyl-3-oxopropane-1,3-diyl)] (25). Compound 23 (50 mg, 28 mmol) was hydrogenated according
to GP IIIc in DMF (4 ml). FC (CH2Cl2/MeOH 20 :1) yielded 25 (24 mg, 50%). Yellow solid. M.p. 115 ± 1168.
Rf 0.1 (CH2Cl2/MeOH 20 :1). [a]D
ˆ
1.13 (c ˆ 1.2, CHCl3). UV/VIS (CF3CH2OH): 215(1600), 378(15000).
IR (CHCl3): 3011w, 2987w, 1738s, 1603w, 1383w, 1306m, 1265s, 1177m, 1136w, 1101w, 1059w, 976w. 1H-NMR

Helvetica Chimica Acta ± Vol. 81 (1998)
2427
(400 MHz): 7.47 (d, J(5,6) ˆ 8.97, H C(5)); 6.63 (dd, J(5,6) ˆ 8.99, J(6,8) ˆ 2.55, H C(6)); 6.50 (d, J(6,8) ˆ
2.56, H C(8)); 6.38 (dd, J ˆ 5.43, 1.17, NH); 6.10 (s, H C(3)); 5.33 ± 5.18 (m, 16 CH); 3.95 (dd, J ˆ 5.41, 1.18,
CH2NH); 3.64 (s, CH2CONH); 3.04 (s, Me2N); 2.64 ± 2.37 (m, 16 CH2); 1.27 ± 1.22 (m, 16 Me). 13C-NMR
(100 MHz): 170.14; 162.59; 156.02; 148.35; 135.55; 128.48; 128.36; 128.16; 125.63; 110.38; 110.16; 108.61; 98.36;
‡
68.63; 67.68; 66.56; 41.67; 40.89; 40.55; 40.20; 40.03; 19.82; 19.62. LSI-MS: 1647.7 (12, [M ‡ Na] ), 1625.6 (100,
‡
‡
[M ‡ H] ), 1624.7 (16, M ), 1417.6(22), 155.1(77), 154.1(29), 137.1(26), 136.0(26).
a-{2-{{2-[7-(Dimethylamino)-2-oxo-2H-1-benzopyran-4-yl]acetyl}amino}acetyl}-w-hydroxydotriacontakis-
[(R)-oxy(1-methyl-3-oxopropane-1,3-diyl)] (26). Compound 24 (40 mg, 13 mmol) was hydrogenated according
to GP IIIb in CF3CH2OH (5 ml). FC (CH2Cl2/Et2O/MeOH 10 :3 :0.2) yielded 26 (22 mg, 55%). Yellow solid.
M.p. 123 ± 1248. Rf 0.3 (CH2Cl2/Et2O/MeOH 10 :3 :0.2). [a]D
ˆ
2.34 (c ˆ 0.9, CHCl3). UV/VIS (CF3CH2OH):
214 (3300), 376 (15000). IR (CHCl3): 3012w, 2987w, 1739s, 1600w, 1384w, 1306m, 1264s, 1178m, 1135w, 1102w,
1061w, 976w. 1H-NMR (500 MHz): 7.46 (d, J(5,6) ˆ 8.98, H C(5)); 6.62 (dd, J(5,6) ˆ 8.99, J(6,8) ˆ 2.54,
H
C(6)); 6.50 (d, J(6,8) ˆ 2.55, H C(8)); 6.37 (dd, J ˆ 5.44, 1.20, NH); 6.09 (s, H C(3)); 5.34 ± 5.20
(m, 32 CH); 3.95 (dd, J ˆ 5.42, 1.20, CH2NH); 3.64 (s, CH2CONH); 3.03 (s, Me2N); 2.65 ± 2.36 (m, 32 CH2);
1.27 ± 1.21 (m, 32 Me). 13C-NMR (125 MHz): 170.23; 162.62; 156.03; 148.48; 135.65; 128.49; 128.37; 128.16;
125.64; 110.41; 110.11; 108.59; 98.37; 68.64; 67.74; 67.69; 66.66; 41.68; 40.92; 40.45; 40.19; 40.02; 19.83; 19.60.
MALDI-MS: 3087.5 ([M ‡ Na] ).
‡
12. Biotin-OHB Conjugates 27 ± 30. Benzyl (3R)-3-{{(3'R)-3'-{{5-[(3aS,4S,6aR)-Hexahydro-2-oxo-1H-
thieno[3,4-d]imidazol-4-yl]pentanoyl}oxy}butanoyl}oxy}butanoate (27). Compound 1 (560 mg, 2 mmol) was
transformed according to GP II with commercial (‡)-biotin (537 mg, 2.2 mmol) in CH2Cl2/DMF 3 :5 (80 ml).
FC (AcOEt/MeOH 20 :1) yielded 27 (197 mg, 19%). Colorless oil. Rf 0.4 (AcOEt/MeOH 20 :1). [a]D ˆ‡ 15.92
(c ˆ 0.7, CHCl3). IR (CHCl3): 3468w, 3032w, 2934w, 1731s, 1456m, 1383w, 1304m, 1266m, 1177m, 1137w, 1101w,
1060w, , 976w. 1H-NMR (400 MHz): 7.36 ± 7.31 (m, 5 arom. H); 5.68 ± 5.64 (br., 2 NH); 5.32 ± 5.20 (m, 2 CH);
5.12 (s, PhCH2O); 4.50 ± 4.46 (m, H C(3a)); 4.31 ± 4.28 (m, H C(6a)); 3.16 ± 3.11 (m, H C(4)); 2.89
(dd, J(6,6') ˆ 12.79, J(6,6a) ˆ 5.18, H C(6)); 2.72 (d, J(6,6') ˆ 12.78, H' C(6)); 2.68, 2.56 (AB of ABX,
JAB ˆ 15.64, JAX ˆ 7.63, JBX ˆ 5.43); 2.53, 2.41 (AB of ABX, JAB ˆ 15.48, JAX ˆ 7.61, JBX ˆ 5.49); 2.28 (t, J ˆ 7.64,
CH2CO); 1.72 ± 1.62 (m, 2 CH2); 1.47 ± 1.40 (m, CH2); 1.29 (d, J ˆ 6.33, Me); 1.25 (d, J ˆ 6.33, Me). 13C-NMR
(100 MHz): 172.76; 170.00; 169.40; 163.39; 135.73; 128.61; 128.33; 67.72; 67.12; 66.50; 61.92; 60.10; 55.37; 40.93;
‡
‡
40.72; 40.55; 34.04; 28.28; 28.23; 24.78; 19.87; 19.84. LSI-MS: 1015.5 (4, [M ‡ H]2 ), 1014.4 (11, [2M ‡ H] ),
‡
‡
‡
‡
1013.4 (21, [M]2 ), 529.1 (7, [M ‡ Na] ), 508.1 (44, [M ‡ 2 H] ), 507.1 (100, [M ‡ H] ), 431.2(12). Anal. calc.
for C25H34N2O7S: C 59.27, H 6.76, N 5.53; found: C 59.30, H 6.61, N 5.63.
a-{5-[(3aS,4S,6aR)-Hexahydro-2-oxo-1H-thieno[3,4-d]imidazol-4-yl]pentanoyl}-w-(benzyloxy)hexadecakis-
[(R)-oxy(1-methyl-3-oxopropane-1,3-diyl)] (28). Compound 2 (700 mg, 0.47 mmol) was transformed according
to GP II with commercial (‡)-biotin (122 mg, 0.5 mmol) in CH2Cl2/DMF 1:2 (60 ml). FC (AcOEt/MeOH
20 :1) yielded 28 (90 mg, 11%). White solid. M.p. 129 ± 1308. Rf 0.6 (AcOEt/MeOH 20 :1). [a]D ˆ‡ 17.05 (c ˆ
0.7, CHCl3). IR (CHCl3): 3011m, 2935w, 1737s, 1602w, 1522w, 1449w, 1426w, 1383w, 1305m, 1178m, 1135w,
1100w, 1058m, 979w, 928w. 1H-NMR (400 MHz): 7.36 ± 7.34 (m, 5 arom. H); 5.28 ± 5.20 (m, 16 CH); 5.12
(s, PhCH2O); 4.98 ± 4.96 (br., NH); 4.66 ± 4.65 (br., NH); 4.54 ± 4.50 (m, H C(3a)); 4.35 ± 4.32 (m, H C(6a));
3.19 ± 3.15 (m, H C(4)); 2.93 (dd, J(6,6') ˆ 12.80, J(6,6a) ˆ 5.04, H C(6)); 2.73 (d, J(6,6') ˆ 12.79, H' C(6));
2.68 ± 2.41 (m, 16 CH2); 2.29 (t, J ˆ 7.33, CH2CO); 1.71 ± 1.64 (m, 2 CH2); 1.48 ± 1.42 (m, CH2); 1.28 (d, J ˆ 5.72,
2 Me); 1.27 (d, J ˆ 6.30, 8 Me); 1.27 (d, J ˆ 6.32, 4 Me); 1.24 (d, J ˆ 6.33, 2 Me). 13C-NMR (100 MHz): 172.62;
169.34; 169.13; 128.58; 128.32; 67.67; 67.60; 67.15; 66.46; 61.79; 60.02; 55.15; 40.88; 40.77; 40.64; 40.52; 33.94;
‡
‡
28.19; 24.69; 19.89; 19.75; 19.70. LSI-MS: 1818.4(23), 1713.8 (14, [M ‡ H] ), 1712.2 (100, M ), 155.1(18),
154.1(17), 137.0(13), 136.0(14). Anal. calc. for C81H118N2O35S: C 56.83, H 6.95, N 1.64, S 1.87; found: C 57.09,
H 6.78, N 1.79, S 2.07.
(3R)-3-{{(3'R)-3'-{{5-[(3aS,4S,6aR)-Hexahydro-2-oxo-1H-thieno[3,4-d]imidazol-4-yl]pentanoyl}oxy}bu-
tanoyl}oxy}butanoic Acid (29). Compound 27 (30 mg, 55 mmol) was hydrogenated according to GP IIIa in
CF3CH2OH (5 ml). FC (CH2Cl2/MeOH 2 :1) yielded 29 (24 mg, 81%). Colorless oil. Rf 0.7 (CH2Cl2/MeOH 2 :1).
1
IR (KBr): 3276w, 2932w, 1735s, 1702s, 1448w, 1386w, 1302w, 1265m, 1178w, 1061w, 970w. H-NMR (300 MHz,
CD3OD): 5.30± 5.22 (m, 2 CH); 4.53± 4.50 (m, H C(3a)); 4.34± 4.31 (m, H C(6a)); 3.19± 3.14 (m, H C(4));
2.95 (dd, J(6,6') ˆ 12.82, J(6,6a) ˆ 5.03, H C(6)); 2.74 (d, J(6,6') ˆ 12.81, H' C(6)); 2.61, 2.58 (AB of ABX,
JAB ˆ 15.63, JAX ˆ 7.81, JBX ˆ 5.51); 2.57, 2.47 (AB of ABX, JAB ˆ 15.41, JAX ˆ 7.79, JBX ˆ 5.40); 2.28 (t, J ˆ 7.35,
CH2CO); 1.74 ± 1.64 (m, 2 CH2); 1.45 ± 1.39 (m, CH2); 1.28 (d, J ˆ 5.98, Me); 1.25 (d, J ˆ 6.34, Me). 13C-NMR
(75 MHz, CD3OD): 171.24; 170.54; 169.82; 169.81; 163.30; 61.64; 56.94; 42.26; 42.12; 41.81; 41.12; 36.38; 29.61;
‡
‡
‡
29.42; 26.66; 20.09; 20. LSI-MS: 455.2 (7, [M ‡ K] ), 439.7 (13, [M ‡ Na] ), 416.7 (100, [M ‡ H] ).

2428
Helvetica Chimica Acta ± Vol. 81 (1998)
a-{5-[(3aS,4S,6aR)-Hexahydro-2-oxo-1H-thieno[3,4-d]imidazol-4-yl]pentanoyl}-w-hydroxyhexadecakis-
[(R)-oxy(1-methyl-3-oxopropane-1,3-diyl)] (30). Compound 28 (35 mg, 20 mmol) was hydrogenated according
to GP IIIa in CF3CH2OH (5 ml). FC (CH2Cl2/MeOH 5 :1) yielded 30 (27 mg, 82%). White solid. M.p. 138 ±
1398. Rf 0.7 (CH2Cl2/MeOH 5 :1). [a]D
ˆ
2.67 (c ˆ 0.9, MeOH). IR (KBr): 3277w, 2934w, 1737s, 1698s, 1452w,
1382w, 1302w, 1264m, 1176w, 1060w, 970w. 1H-NMR (300 MHz, CD3OD): 5.28 ± 5.19 (m, 16 CH); 4.55 ± 4.50
(m, H C(3a)); 4.35 ± 4.31 (m, H C(6a)); 3.19 ± 3.15 (m, H C(4)); 2.94 (dd, J(6,6') ˆ 12.81, J(6,6a) ˆ 5.02,
H
C(6)); 2.76 (d, J(6,6') ˆ 12.79, H' C(6)); 2.65 ± 2.41 (m, 16 CH2); 2.28 (t, J ˆ 7.36, CH2CO); 1.73 ± 1.64
(m, 2 CH2); 1.47 ± 1.40 (m, CH2); 1.28 (d, J ˆ 5.92, 2 Me); 1.27 (d, J ˆ 6.31, 8 Me); 1.27 (d, J ˆ 6.32, 4 Me); 1.23
(d, J ˆ 6.33, 2 Me). 13C-NMR (75 MHz, CD3OD): 169.25; 169.20; 169.16; 169.09; 68.60; 67.69; 67.66; 67.63;
67.60; 66.43; 61.68; 60.12; 55.29; 41.19; 40.76; 40.71; 40.65; 40.43; 35.52; 28.10; 28.01; 25.45; 19.81; 19.74; 19.05.
‡
‡
‡
LSI-MS: 1728.4(78), 1660.5 (2, [M ‡ K] ), 1644.2 (11, [M ‡ Na] ), 1622.4 (100, [M ‡ H] ), 1592.3(34).
13. ꢀBiotinylatedꢁ Glycine-OHB Conjugates 31 ± 34. Benzyl (3R)-3-{{(3'R)-3'-{{2-{{5-[(3aS,4S,6aR)-Hexa-
hydro-2-oxo-1H-thieno[3,4-d]imidazol-4-yl]pentanoyl}amino}acetyl}oxy}butanoyl}oxy}butanoate (31). Boc de-
protection of 19 (400 mg, 0.92 mmol) in CH2Cl2 (2 ml) according to GP IV. The obtained crude trifluoroacetate
was treated with (‡)-biotin (225 mg, 0.92 mmol) according to GP I. FC (AcOEt/MeOH 7:1) yielded 31
(440 mg, 85%). White, gelatinous solid. M.p. 94 ± 958. Rf 0.4 (AcOEt/MeOH 7:1). [a]D ˆ‡ 3.56 (c ˆ 0.9,
1
CHCl3). IR (CHCl3): 3278w, 2936w, 1738s, 1700s, 1457w, 1382w, 1304w, 1265m, 1177w, 1057w, 974w. H-NMR
(400 MHz): 7.37 ± 7.32 (m, 5 arom. H); 6.91 ± 6.90 (br., NH); 6.44 ± 6.43 (br., NH); 5.38 ± 5.36 (br., NH); 5.32 ±
5.24 (m, 2 CH); 5.12 (s, PhCH2O); 4.51 ± 4.48 (m, H C(3a)); 4.32 ± 4.29 (m, H C(6a)); 4.06 (dd, J ˆ 18.16,
5.87, 1 H, CH2NH); 3.91 (dd, J ˆ 18.16, 5.18, 1 H, CH2NH); 3.15 ± 3.11 (m, H C(4)); 2.90 (dd, J(6,6') ˆ 12.80,
J(6,6a) ˆ 4.97, H C(6)); 2.72 (d, J(6,6') ˆ 12.80, H' C(6)); 2.66, 2.57 (AB of ABX, JAB ˆ 15.69, JAX ˆ 7.79,
JBX ˆ 5.29); 2.55, 2.43 (AB of ABX, JAB ˆ 15.69, JAX ˆ 7.60, JBX ˆ 5.49); 2.30 ± 2.26 (m, CH2CO); 1.77 ± 1.65
(m, 2 CH2); 1.47 ± 1.41 (m, CH2); 1.29 (d, J ˆ 6.34, Me); 1.28 (d, J ˆ 6.35, Me). 13C-NMR (100 MHz): 173.52;
170.05; 169.97; 169.20; 163.89; 135.66; 128.60; 128.36; 128.31; 68.54; 67.79; 66.52; 61.68; 60.18; 55.45; 41.20;
‡
‡
40.70; 40.66; 40.47; 35.54; 28.07; 25.47; 19.82; 19.78. LSI-MS: 1128.4 (15, [M ‡ H]2 ), 1127.4 (25, [2M ‡ H] ),
‡
‡
‡
‡
587.1 (14, [M ‡ Na ‡ H] ), 586.1 (41, [M ‡ Na] ), 565.1 (40, [M ‡ 2 H] ), 564.1 (100, [M ‡ H] ), 227.0(14).
Anal. calc. for C27H37N3O8S: C 57.53, H 6.62, N 7.45, S 5.69; found: C 57.40, H 6.47, N 7.53, S 5.66.
a-{2-{{5-[(3aS, 4S,6aR)-Hexahydro-2-oxo-1H-thieno[3,4-d]imidazol-4-yl]pentanoyl}amino}acetyl}-w-(benzyl-
oxy)hexadecakis[(R)-oxy(1-methyl-3-oxopropane-1,3-diyl)] (32). Boc deprotection of 20 (600 mg, 0.37 mmol)
in CH2Cl2 (5 ml) according to GP IV. The obtained crude trifluoroacetate was treated with (‡)-biotin (111 mg,
0.40 mmol) according to GP I. FC (AcOEt/MeOH 8 :1) yielded 32 (505 mg, 78%). White solid. M.p. 133 ± 1348.
Rf 0.3 (AcOEt/MeOH 8 :1). [a]D ˆ‡ 1.04 (c ˆ 1.0, CHCl3). IR (CHCl3): 1737s, 1458w, 1382w, 1306w, 1267w,
1178w, 1135w, 1101w, 1060w, 978w. 1H-NMR (400 MHz): 7.37 ± 7.32 (m, 5 arom. H); 6.77 ± 6.75 (br., NH);
6.03 ± 6.01 (br., NH); 5.36 ± 5.32 (br., NH); 5.30 ± 5.20 (m, 16 CH); 5.12 (s, PhCH2O); 4.54 ± 4.50
(m, H C(3a)); 4.36 ± 4.32 (m, H C(6a)); 4.06 (dd, J ˆ 18.15, 5.83, 1 H, CH2NH); 3.94 (dd, J ˆ 18.5, 5.19,
1 H, CH2NH); 3.18 ± 3.13 (m, H C(4)); 2.93 (dd, J(6,6') ˆ 12.81, J(6,6a) ˆ 5.03, H C(6)); 2.74 (d, J(6,6') ˆ
12.79, H C(6)); 2.71 ± 2.41 (m, 16 CH2); 2.31 ± 2.27 (m, CH2CO); 1.76 ± 1.64 (m, 2 CH2); 1.51 ± 1.43 (m, CH2);
1.29 (d, J ˆ 6.33, Me); 1.28 (d, J ˆ 6.09, 3 Me); 1.27 (d, J ˆ 6.31, 7 Me); 1.27 (d, J ˆ 5.85, 3 Me); 1.24 (d, J ˆ 6.32,
2 Me). 13C-NMR (100 MHz): 169.23; 169.20; 169.18; 169.12; 128.57; 128.32; 68.56; 67.70; 67.67; 67.66; 67.60;
66.45; 61.68; 60.14; 55.28; 41.23; 40.78; 40.70; 40.65; 40.46; 35.50; 28.12; 28.01; 25.41; 19.80; 19.75; 19.06. LSI-
‡
‡
‡
MS: 1791.0 (3, [M ‡ Na] ), 1769.2 (100, [M ‡ H] ), 1768.9 (92, M ), 155.0(35), 154.0(16), 137.0(13),
136.0(15). Anal. calc. for C83H121N3O36S: C 56.36, H 6.89, N 2.38, S 1.81; found: C 56.32, H 6.90, N 2.46, S 1.90.
(3R)-3-{{(3'R)-3'-{{2-{{5-[(3aS,4S,6aR)-Hexahydro-2-oxo-1H-thieno[3,4-d]imidazol-4-yl]pentanoyl}ami-
no}acetyl}oxy}butanoyl}oxy}butanoic Acid (33). Compound 31 (100 mg, 0.18 mmol) was hydrogenated
according to GP IIIa in CF3CH2OH (10 ml). FC (CH2Cl2/MeOH 2 :1) yielded 33 (144 mg, 82%). M.p. 141 ±
1448. Rf 0.6 (CH2Cl2/MeOH 2 :1). IR (KBr): 3279w, 2934w, 1738s, 1697s, 1455w, 1383w, 1302w, 1266m, 1177w,
1060w, 971w. 1H-NMR (400 MHz, CD3OD): 5.31 ± 5.23 (m, 2 CH); 4.51 ± 4.48 (m, H C(3a)); 4.34 ± 4.31
(m, H C(6a)); 3.90 (s, CH2NH); 3.24 ± 3.19 (m, H C(4)); 2.93 (dd, J(6,6') ˆ 12.74, J(6,6a) ˆ 4.97, H C(6));
2.70 (d, J(6,6') ˆ 12.35, H' C(6)); 2.61, 2.57 (AB of ABX, JAB ˆ 15.65, JAX ˆ 7.78, JBX ˆ 5.53); 2.58, 2.49 (AB of
ABX, JAB ˆ 15.33, JAX ˆ 7.81, JBX ˆ 5.41); 2.30 ± 2.26 (m, CH2CO); 1.77 ± 1.58 (m, 2 CH2); 1.52 ± 1.45 (m, CH2);
1.29 (d, J ˆ 6.40, Me); 1.27 (d, J ˆ 6.38, Me). 13C-NMR (100 MHz, CD3OD): 176.57; 171.25; 170.74; 169.83;
169.81; 163.28; 61.65; 56.93; 42.27; 42.14; 41.79; 41.04; 36.40; 29.57; 29.41; 26.68; 20.08; 20.00. LSI-MS:
‡
‡
‡
‡
992.4 (3, [M ‡ Na]2 ), 991.4 (4, [2M ‡ 2Na H] ), 970.4 (2, [2M ‡ Na] ), 947.3 (2, [2M ‡ H] ), 497.0 (25,
‡
‡
‡
‡
‡
[M ‡ Na ‡ H] ), 496.0 (100, [M ‡ Na] ), 495.0 (7, [M ‡ Na H] ), 475.1 (18, [M ‡ H] ), 474.0 (60, M ),
‡
473.1 (5, [M H] ).

Helvetica Chimica Acta ± Vol. 81 (1998)
2429
a-{2-{{5-[(3aS,4S,6aR)-Hexahydro-2-oxo-1H-thieno[3,4-d]imidazol-4-yl]pentanoyl}amino}acetyl}-w-hy-
droxyhexadecakis[(R)-oxy(1-methyl-3-oxopropane-1,3-diyl)] (34). Compound 32 (300 mg, 0.17 mmol) was
hydrogenated according to GP IIIa in CF3CH2OH (30 ml). FC (CH2Cl2/MeOH 8 :1) yielded 34 (260 mg, 91%).
White solid. M.p. 140 ± 1428. Rf 0.7 (CH2Cl2/MeOH 8 :1). IR (KBr): 3280w, 2931w, 1738s, 1698s, 1454w, 1383w,
1301w, 1267m, 1177w, 1060w, 970w. 1H-NMR (400 MHz, CD3OD/CDCl3): 5.30 ± 5.20 (m, 16 CH); 4.55 ± 4.50
(m, H C(3a)); 4.35 ± 4.32 (m, H C(6a)); 3.97 (s, CH2NH); 3.18 ± 3.11 (m, H C(4)); 2.91 (dd, J(6,6') ˆ 12.79,
J(6,6a) ˆ 5.02, H C(6)); 2.73 (d, J(6,6') ˆ 12.79, H' C(6)); 2.72 ± 2.40 (m, 16 CH2); 2.31 ± 2.26 (m, CH2CO);
1.76 ± 1.64 (m, 2 CH2); 1.51 ± 1.46 (m, CH2); 1.29 ± 1.23 (m, 16 Me). 13C-NMR (100 MHz): 172.62; 128.58;
128.32; 68.56; 67.71; 67.64; 67.60; 66.44; 62.38; 60.09; 55.21; 41.24; 40.78; 40.68; 40.65; 40.46; 35.40; 28.12; 28.01;
‡
‡
‡
26.41; 19.82; 19.76; 19.07. LSI-MS: 1718.0 (16, [M ‡ K] ), 1702.3 (41, [M ‡ Na ‡ H] ), 1701.0 (46, [M ‡ Na] ),
‡
1679.4 (100, [M ‡ H] ), 1650.1(13), 1649.1(12), 132.9(14). Anal. calc. for C76H115N3O36S: C 54.40, H 6.85,
N 2.51, S 1.91; found: C 54.28, H 6.87, N 2.62, S 1.98.
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Received August 14, 1998