Synthesis of tools for raising antibodies against moenomycin epitopes and initial immunological studies

Tetrahedron 58 (2002) 7741–7760

Synthesis of tools for raising antibodies against moenomycin

epitopes and initial immunological studies

Andrij Buchynskyy,^aKatherina Stembera,^aDietmar Knoll,^aStefan Vogel,^aUwe Kempin,^a

a

Astrid Biallaß (nee Donnerstag), Lothar Hennig, Matthias Findeisen, Dietrich Muller

a

b

´

¨

and Peter Welzel^a

,

*

a

¨ ¨

Fakultat fur Chemie und Mineralogie, Institut fur Organisshe Chemie, Universitat Leipzig, Talstr. 35, D-04103 Leipzig, Germany

¨

Institut fur Analytische Chemie, Ruhr-Universitat Bochum, D-44780 Bochum, Germany

¨

b

¨

Received 26 April 2002; revised 10 June 2002; accepted 9 July 2002

Abstract—The moenomycins A and C₁as well as penta-, di- and monosaccharide analogues have been conjugated to BSA and biotin,

respectively. The moenomycin A–BSA conjugates have been used to raise polyclonal antibodies. It has been demonstrated that the antisera

1. Introduction

domain of the bifunctional high-molecular PBPs). Based on

structure–activity relationships,¹⁴it is assumed that they are

anchored to the cytoplasmic membrane via the lipid part¹⁵

and that the C–E–F trisaccharide part then blocks highly

selectively the binding site of the growing polysaccharide

strand at the enzyme.^4,16,17

In view of the problem of antibiotic resistance¹anti-

infectives with novel modes of action are desperately

needed. The transglycosylation reaction,²the second last

step in the biosynthesis of peptidoglycan (the main

structural polymer of the bacterial cell wall) occurs at the

outside of the cytoplasmic membrane and is catalyzed by

membrane proteins designated as bifunctional (class A)

high molecular mass penicillin-binding proteins (PBPs).³

The reaction came recently into focus as a promising new

target for antiinfectives for a number of reasons: (i) the

methods for isolating the enzyme(s) that are involved have

improved considerably,^4,5(ii) one of the substrates of the

transglycosylation step, the so-called lipid II, can now be

made in sufﬁcient amounts,⁶and (iii) new and efﬁcient in

vitro test systems have been developed which conveniently

allow to monitor the inhibition of the incorporation of lipid

II into uncross-linked peptidoglycan^6,7and binding of

inhibitors to the enzyme, respectively.⁸

The moenomycins are, thus, unique tools for investigating

both the transglycosylation step and the corresponding

enzyme(s) as well as for the development of new

transglycosylase inhibitors. For some time we have been

involved in exploiting these tools based on the chemistry

which permits to prepare compounds 2a,¹⁸2b,¹⁹and 9b.²⁰

Both the amino groups in 2b and 9b, and the thiol group in

2a are orthogonal in their reactivities to all other functional

groups present in 2b, 9b, and 2c and have been used for

immobilization reactions at chromatographic supports⁵and

surface plasmon resonance (SPR) sensor chips,⁸

respectively, and for the introduction of reporter groups

allowing (i) to isolate PBP 1b by afﬁnity chromatography,⁵

(ii) to study binding of moenomycin analogues to PBP 1b by

SPR,⁸(iii) to investigate the interaction of moenomycin

with artiﬁcial membranes making use of ﬂuorescence

methods,¹⁵(iv) to perform initial studies aimed at identify-

ing the moenomycin binding site at the enzyme by afﬁnity

labeling,²¹(v) to isolate aptamers that bind to moenomycin

in the nanomolar range,²²and (vi) to execute ﬂuorescence

correlation spectroscopy measurements.²³

The assembly of the peptidoglycan polysaccharide strands

from lipid II is blocked by certain glycopeptides,⁹

ramoplanin,¹⁰lantibiotics such as nisin.¹¹and the moeno-

mycin-type antibiotics.¹²Ramoplanin and nisin interfere

with lipid II. The mechanism of the glycopeptides that

inhibit the transglycosylation reaction is not clear.¹³The

moenomycins (see Scheme 1) are the only compounds

known to inhibit the enzyme itself²(i.e. the transglycosylase

The incentive of selecting moenomycin aptamers arose

from the idea that it would be useful to be able to detect

moenomycin and moenomycin analogues (for example

biosynthetic precursors²⁴or metabolites) with high selec-

tivity and sensitivity. Parallel to the selection of aptamers

Keywords: antibiotics; antibodies; carbohydrates; oxocarbon acid

derivatives.

*

Corresponding author. Tel.: þ-49-341-97-36551; fax: þ49-341-97-

36599; e-mail: welzel@organik.chemie.uni-leipzig.de

PII: S0040-4020(02)00838-4

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A. Buchynskyy et al. / Tetrahedron 58 (2002) 7741–7760

Scheme 1.

Scheme 2.

A. Buchynskyy et al. / Tetrahedron 58 (2002) 7741–7760

7743

Scheme 3.

and with the same intention we have prepared moeno-

mycin–bovine serum albumin (BSA) conjugates and have

used them to raise anti-moenomycin antibodies. This is the

subject of the present publication.

It seemed desirable to develop a conjugation procedure not

demanding a capping step, which means changing the

sequence of the two conjugation steps. Squaric acid diethyl

ester was used for this purpose as a bifunctional linker. It is

known that under neutral conditions only one of the ethoxy

groups can be replaced by primary or secondary amines

whereas the second one reacts only under basic conditions

to form the corresponding diamide.^26,27In a model

experiment BSA was treated with 133 equiv. of squaric

acid amide ester 6 (prepared from 5-amino-N-[2-ami-

noethyl)-2-nitrobenzamide and diethyl squarate) at pH 9.0

for 64 h at 208C. After removing inorganic salts and the

excess of 6 by ultraﬁltration the residue was analyzed by

MALDI TOF MS. From the molecular mass difference

(center of the conjugate molecular mass around m/z¼78,369

and the BSA molecular mass (internal standard) at

m/z¼68,932) it was concluded that an average of 30

residues of 6 were attached to BSA, a value well in accord

with the fact that out of the 59 lysine groups present in BSA

30–35 are available for conjugation reactions (Scheme 3).²⁸

2. Results and discussion

2.1. Coupling of moenomycin to BSA

In order to exploit the SH-labeled moenomycin derivative

2a, BSA was activated by reaction of the lysine amino

groups with the heterobifunctional reagent 3. SDS-PAGE

showed a new band and MALDI TOF indicated that about

30–32 lysine residues had been converted to 3-malein-

imidopropionamides. BSA derivative 4 was subsequently

treated with the thiol 2a (in fact a thiol mixture obtained

from the moenomycin complex in which moenomycin A

(1a) is the major component²⁵) for 1 d at 58C.

Puriﬁcation was achieved by ultraﬁltration. An aliquot was

analyzed by MALDI TOF MS. The molecular ion signal

was centered at m/z¼79,000 indicating that on an average

three moenomycin units were coupled to BSA. 2-Mercapto-

ethanol was then added to cap all unreacted maleimide

groups (1 d at 208C) (Scheme 2).

Compound 2b (obtained from pure moenomycin A) was

ﬁrst converted to squaric acid amide ester 2c at pH 7.3 and

this in turn coupled to BSA at pH 9.0 using an excess of

5 equiv. (a), 10 equiv. (b), and 20 equiv. (c) of 2c. After

removing inorganic salts and the excess of 2c by ultraﬁltra-

tion at 48C the conjugates 8 were analyzed by MALDI TOF

Figure 1. MALDI-TOF MS spectra of BSA and moenomycin–BSA conjugates 8 (experiments a–c, see text).

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A. Buchynskyy et al. / Tetrahedron 58 (2002) 7741–7760

Scheme 4.

MS with BSA as external standard (m/z¼66,418). The

molecular ion signal of the conjugates 8 were centered at

m/z¼73,749 (a), m/z¼77,126 (b) and m/z¼83,141 (c)

corresponding to average loading values of 3.9, 5.6 and

8.8, respectively (see Fig. 1). The ESI ICR MS with the

deconvoluted molecular ion peak of 8 (from experiment a)

centered at m/z¼74,000 (BSA at m/z¼66,400) conﬁrmed

the MALDI results (Scheme 4).

(1:9 (a), 1:18 (b), 1:43 (c)) to give 9d. According to the

MALDI TOF MS analysis on an average from 2.2 to 4.7

hapten units were attached to BSA (see Fig. 2).

2.3. Coupling of the moenomycin C₁and A₁₂type

disaccharide 18b to BSA

It has been speculated that the branching methyl group

present in unit F of moenomycin A (1a) and related

antibiotics is introduced at a late stage of the biosynthesis

and that compounds of the moenomycin C₁and A₁₂type^25,

2.2. Coupling of pentasaccharide 9c to BSA

It is known from structure–activity studies¹⁴as well as from

surface plasmon resonance measurements⁸that the anti-

biotic activity of the moenomycins is critically dependent

on the sugar units and that there is a speciﬁc binding

interaction of the sugar components with PBP 1b. It seemed,

thus, useful to prepare conjugates of BSA and haptens

consisting of moenomycin carbohydrate units that would

allow to raise antibodies against selected areas of the

moenomycin sugar chain (Scheme 5).

(see 1b and 1c) with a D-galacturonamide part F are the

29

biogenetic precursors.²⁴With this in mind we have prepared

a number of ^D-galacturonic acid-derived conjugates. In the

course of this study some linker chemistry was tested. The

oleﬁnic linkers 11a and 11b were obtained from 10a³⁰and

4-pentenoic acid and 6-heptenoic acid, respectively, after

activating the carboxylic acids with carbonyldiimidazole.

Compound 12 was obtained from the Boc derivative of

3-bromopropylammonium bromide and 4-penten-1-ol by

Williamson etheriﬁcation. Finally, 3-mercaptopropionic

acid (13a) was converted into hydroxysuccinimide

derivative 13b.³¹The latter compound on reaction with

10b³²furnished 13c which was deprotected to give 13d

(Scheme 6).

The known moenomycin pentasaccharide degradation

product 9b²⁰was coupled to diethyl squarate at pH 7.0 to

give ester amide 9c in 61% yield. High resolution mass

spectra were in accord with structure 9c. As usual a mixture

of obviously two conformers was observed by NMR giving

rise to two N-CH₃-3 signals at d¼37.72 and 37.91 ppm.²⁶

Subsequently, 9c was coupled to BSA at pH 9.0. Three

experiments were performed with different 9c–BSA ratios

Disaccharide 16a on ozonolysis in methanol provided

aldehyde 14 in 94% yield. 14 gave 15b after reductive

amination with 10a (!15a, 76%) and hydrogenolytic

Scheme 5.

A. Buchynskyy et al. / Tetrahedron 58 (2002) 7741–7760

7745

Scheme 6.

deprotection (61%). Allyl derivative 16a on attempted

metathesis reaction in CHCl₃solution using the ﬁrst

generation Grubbs catalyst³³failed to react with 11a. The

oleﬁn 11b with a longer distance between the double bond

and the amide group provided 17a in 21% yield as a mixture

of stereoisomers. The cross metathesis of 16a with 12 gave

the best results in this series (!17b, 37%). 17a and 17b on

hydrogenation of the double bond and concomitant removal

of the protecting groups led to 19a (84%) and 19b (93%),

respectively. Addition of 13d to the allyl group of the

disaccharide 16b by irradiation at 254 nm³⁴provided 18a in

71% yield. 18a on reaction with diethyl squarate at pH 7.2

furnished the ester amide 18b in 66% yield. The structure of

18b was in agreement with the NMR and high resolution

mass spectra. Again, the ¹³C NMR spectra showed two sets

of signals for some of the carbons indicating the existence of

two conformers of 18b in solution. 18b (15 equiv.) was

coupled to BSA at pH 9.0 to furnish 18c. After puriﬁcation

by ultraﬁltration the MALDI TOF MS analysis revealed that

an avarage of 12–13 units of 18b were loaded onto BSA

(Scheme 7).

2.4. Coupling of 21e, a monosaccharide analogue of

moenomycins A₁₂and C₁, to BSA

Reaction of the known galacturonic acid derived compound

20a³⁵with the acid chloride of allyl hydrogen succinate

yielded 20b (77% yield). Subsequently, the acetal protect-

ing group was removed quantitatively by treatment with

20% acetic acid at 508C. The NMR spectra of 21a had to be

taken in polar solvents such as methanol-d₄or pyridine-d₅,

since in CDCl₃solution only broad and unresolved signals

resulted. The carbamoyl group was then introduced using

the well-established two-step procedure:³⁵(i) reaction with

trichloroacetyl isocyanate (1.2 equiv., 268C) to give the

trichloroacetyl urethane, and (ii) reductive removal of the

trichloroacetyl group with Zn dust in methanol. 21b was

obtained in 69% yield alongside with the 4-isomer 21c (2%)

and the dicarbamoyl derivative 21d (10%). The structures

followed from the characteristic ¹H and ¹³C NMR chemical

shifts (see Section 3). Selective cleavage of the allyl ester of

21b was achieved according to Kunz and co-workers³⁶by

Pd(0)-mediated allyl transfer onto a soft nucleophile. Thus,

Figure 2. MALDI-TOF MS spectra of BSA and pentasaccaride–BSA conjugates 9d (experiments a–c, see text).

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A. Buchynskyy et al. / Tetrahedron 58 (2002) 7741–7760

Scheme 7.

21b furnished 21e on treatment with 10 mol% of freshly

prepared Pd[PPh₃]₄and morpholine in THF. After removal

of N-allyl morpholine and excess of morpholine with

Dowex 50 (H^þ) 21e was isolated in 89% yield (Scheme 8).

7.2) was added. The reaction mixture was left at 208C for

several days. The adduct 21g was puriﬁed either by careful

dialysis against water or by ultraﬁltration. SDS-PAGE (see

Fig. 3) clearly showed the conversion of BSA into a new

product. A more speciﬁc characterization was achieved by

MALDI TOF mass spectrometry. Fig. 4 shows the co-mass

spectra of the BSA conjugate 21g and BSA which was

added as an internal standard. Setting the BSA molecular

mass to 66,497 the (somewhat broader) adduct peak had its

intensity maximum at m/z¼72,541 demonstrating that an

average of 17 molecules of 21e had been coupled to one

BSA molecule (see Fig. 4).

Compound 21e was activated through reaction with

N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-diamino-

propyl)carbodiimide (EDC) in DMF and coupled in a model

experiment to glycine benzyl ester to give 21f in moderate

yield (not optimized). For the conjugation to BSA 21e was

activated in DMF solution as described above. Then BSA

(3£10²³equiv., dissolved in 50 mM phosphate buffer, pH

A. Buchynskyy et al. / Tetrahedron 58 (2002) 7741–7760

7747

Scheme 8.

2.5. Some biotin conjugates

A number of moenomycin-derived supports for afﬁnity

chromatography have already been reported.^5,22We wish to

add three more compounds which could be useful for the

afﬁnity puriﬁcation of anti-moenomycin antibodies. Moeno-

mycin A derivatives 2a and 2b have been coupled to biotin

previously.^18,19Following the reported protocol¹⁸moeno-

mycin C₁(1b) was converted into thiol derivative 22b via

22a. The thiol grouping in 22b was used to prepare the

moenomycin C₁–biotin adduct 22c by 1,4-additions to a

known biocetin-derived maleimide (cf. formula 22c). 22c

Figure 3. SDS PAGE of the conjugation experiment. Lanes 1, 2: reaction

product, lane 3: BSA.

Figure 4. MALDI TOF MS spectrum of conjugate 21g. BSA was added as an internal reference.

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A. Buchynskyy et al. / Tetrahedron 58 (2002) 7741–7760

Scheme 9.

exhibited somewhat reduced antibiotic properties when

compared with moenomycin A (Scheme 9).

mycin–KLH (obtained from 2a and maleimide-activated

KLH), moenomycin–BSA 8, and the complex of strepta-

vidin and biotinylated moenomycin derivative 16c,

respectively. Antiserum was added in serial dilutions. For

the ELISAs anti-rabbit antibodies conjugated with peroxi-

dase were employed. Each of these different ELISA

strategies showed increasing antibody concentrations

recognizing moenomycin during the immunization pro-

cedure (Fig. 5(A)). After 20 injections, the immunization

procedure was stopped (Fig. 5(B)). Alternatively, a Western

blot analysis was performed with 8 and anti-moenomycin

antiserum as primary antibody. After incubation with

alkaline phosphatase-conjugated anti-rabbit antibodies the

Western blot showed speciﬁc coloring but not in control

experiments with BSA conﬁrming the presence of anti-

bodies recognizing moenomycin (Fig. 6). Furthermore, 2b

was coupled to a SPR sensor chip as described previously.⁵

SPR experiments with antiserum as soluble analyte

conﬁrmed the speciﬁcity of the antiserum for moenomycin

The pentasaccharide-derived biotin derivative 23 was

obtained from 9a and 24a by reductive amination as

reported above. Analogously the disaccharide-derived

biotin adduct 25b was prepared from 16a by a sequence

of (i) ozonolysis, (ii) reductive amination with 24b, and (iii)

hydrogenolytic removal of the benzyl protecting groups

(Schemes 10 and 11).

2.6. Biochemical results

Rabbits were immunized by injection of moenomycin–

BSA conjugates 8 (from experiments a–c) to raise

polyclonal antisera against moenomycin. First screening

of the blood sera for anti-moenomycin antibodies was

performed in enzyme-linked immunosorbent assays

(ELISA). Microtiter plates were coated with moeno-

Scheme 10.

A. Buchynskyy et al. / Tetrahedron 58 (2002) 7741–7760

7749

Scheme 11.

Figure 5. ELISA. (A) Determination of the titer after 5, 10, 15 and 20 injections of 8. Microtiter plates were coated with 8. (B) Percentual increasing of the

immune response (dilution 1:1600) during the immunization procedure according to the ELISA.

Figure 7. SPR sensorgram of the injection of rabbit anti-moenomycin

Figure 6. Western blotting for the determination of anti-moenomycin

antibodies. Incubation of antiserum was performed in the presence of 1%

BSA for neutralization of anti-BSA antibodies. (1) 15 mg BSA. (2) 15 mg

moenomycin–BSA.

antibodies. The graph displays the chance of the response during and after

the injection of antiserum. Control experiments were done with preserum.

Running and binding buffer: HBS-EP, pH 7.4. Protein concentrations:

2.5 mg/mL, ﬂow 20 mL/min, contact time 45 s, 258C.

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A. Buchynskyy et al. / Tetrahedron 58 (2002) 7741–7760

not coupled to BSA (Fig. 7). However, afﬁnities have not

yet been determined. In all experiments anti-BSA antibodies

which were also produced during the immunization

procedure were neutralized with BSA.

the second is the mono-isotopic mass. IR: Genesis FTIR

(ATI Mattson).

3.2. Reaction of BSA with 3

To a stirred solution of BSA (SERVA, 10 mg) in phosphate

buffer (9 mL, pH 7.0, 50 mmol) a solution of 3-(2,5-dioxo-

2,5-dihydro-pyrrol-1-yl) propionic acid 2,5-dioxo-pyrroli-

din-1-yl ester (3) (10 mg, 0.038 mmol) in dimethylform-

amide (1 mL) was added. The reaction mixture was stirred

at 58C for 3 d. Removal of low molecular weight reaction

products was accomplished by dialysis against phosphate

buffer (Pierce Slide-A-Lyzere 10 K Dialysis Cassette). The

MALDI TOF MS (matrix: sinapic acid, internal calibration

with BSA, m/z¼66,892) displayed an average m/z of 71,441,

corresponding to an average loading value of 32.

3. Experimental

3.1. Methods and materials

For ﬂash chromatography (FC), see Ref. 37. The matrix for

the FAB mass spectra was 3-nitrobenzyl alcohol. For HPLC

the following instrumentation was used: Analytical HPLC:

Jasco PU-980 pump with Uniﬂows Degasys DG-1310

system, Sepsil column (C18, 5 mm, 250 mm£2.1 mm),

Sepsil precolumn (C18, 5 mm, 20 mm£2.1 mm), ﬂow rate

0.5 mL/min, sample volume 20 mL, eluent: a 63:37

mixture of buffer (KH₂PO₄(0.6 g), K₂HPO₄£3H₂O

(26.2 g), 1-heptanesulfonic acid, sodium salt monohydrate

(3.0 g), water, ﬁnal volume 1 L) and acetonitrile (adjusted to

pH 8 with phosphoric acid),³⁸detection with the Jasco

MD-910 diode array detector, data processing with the

DP-L910-V software. Preparative HPLC: Jasco PU-987

pump, Jasco 875-UV UV–Vis detector, Sepsil column

(C18, 10 mm, 250 mm£20 mm), 0.5 mL sample volume.

Either an Amicon gas-pressurized cell (model 8050) with an

Amicon membrane YM 3 (3000 Da cut-off) or reversed

phase chromatography (HP-20 resin, swollen in methanol

for 12 h, washed with acetone, water, 0.1 M NaOH, water,

0.1 M HCl, water, methanol, water) or gel ﬁltration (PD-10

prepacked columns, Sephadex G-25M, 9.1 mL) were used

for the removal of low molecular weight impurities and

inorganic salts. Microsep centrifugal concentrators (PALL

FILTRON, 30 kDa cut-off, Beckman Avanti 30 centrifuge,

ﬁxed-angle rotor at 258, 7500g, 48C) and Amicon gas-

pressurized cell (model 8050) with an Amicon membrane

YM 30 (30 kDa cut-off) were used for the puriﬁcation of the

BSA preparations. For ion exchange chromatography

Dowex W 50 and Lewatit S 100, respectively, were used.

NMR equipment: NMR: UNITY 400 (Varian), DRX 400

(Bruker), DRX 600 (Bruker), GEMINI 200 (Varian),

GEMINI 2000 (Varian). All spectra have been analyzed

as described in the individual procedures. For brevity,

signals of protecting groups and spacers are described only

once. Mass spectrometry: EI MS: MAT-731 (Varian), FAB

MS: VG Autospec (Fisons, matrix: 3-nitrobenzylalcohol),

ESI MS: FT-ICR-MS Apex II (Bruker Daltonics, water–

methanol). The MALDI TOF mass spectra were acquired

with the Reﬂexe (Bruker) and the Voyagere (PerSeptive

Biosystems, nitrogen laser at 337 nm, Delayed Extra-

ctione), matrices: sinapic acid, solvent: 30% acetonitrile

in ultrapure water (18 MV cm)þtriﬂuoroacetic acid (3%);

2,4,6-trihydroxyacetophenone (THAP) (10 mg/1 mL) dis-

solved in water–acetonitrile 1:1, the solutions of analytes

were applied to dried matrix (fast-evaporation method); 2,5-

dihydroxybenzoic acid (DHB) dissolved in ultrapure water

(ca 10 mg/1 mL), the sample of BSA–moenomycin con-

jugates in ultrapure water (1 mg/1 mL) was premixed with

the matrix solution in a 1:1 v/v ratio and applied onto the

plate (1 mL) (dried-droplet method). Following the mol-

ecular formula two masses are always communicated, the

ﬁrst was calculated using the International Atomic Masses,

3.3. Flavomycin–BSA conjugate 5

To a solution of the above activated BSA (4) in phosphate

buffer (5 mL, pH 7.0, 50 mM) a solution of 2a¹⁸(prepared

from Flavomycin^w, vide supra, 10 mg, 78 equiv.) in

phosphate buffer was added. The reaction mixture was

stirred for 1 d at 58C. An aliquot was dialyzed against water

(Pierce Slide-A-Lyzere 10K Dialysis Cassette). The

MALDI TOF MS (DHB matrix, external calibration with

BSA, m/z¼66,418) displayed a mean m/z of 77,015,

corresponding to an average protein loading of 3.0. Then

mercaptoethanol (0.1 mL) was added and stirring was

continued for 1 d. Removal of low molecular weight

reaction products was accomplished by dialysis against

phosphate buffer (Pierce Slide-A-Lyzere 10K Dialysis

Cassette).

3.3.1. 5-Amino-N-[2-(2-ethoxy-3,4-dioxo-1-cyclobuten-1-

ylamino)ethyl]-2-nitrobenzamide (6). A solution of 5-

amino-N-(2-aminoethyl)-2-nitro-benzamide

(15 mg,

0.067 mmol) and 3,4-diethoxy-3-cyclobutene-1,2-dione

(10 mg, 0.058 mmol) in ethanol (2.2 mL) was stirred at

ambient temperature. Progress of the reaction was con-

trolled by TLC (CHCl₃–methanol 10:2, R_f¼0.15). After

16 h all diethyl squarate was consumed. Solvent evapor-

ation and FC (CHCl₃–methanol 5:1) provided 20 mg (99%)

of pure 6. ¹H NMR (200 MHz, CD₃OD, homo decoupling):

d¼1.45 (t, J¼7.0 Hz, 3H, OCH₂CH₃), 3.50–3.85 (m, 4H,

CH₂-1^Ar, CH₂-2^Ar), 4.72 (m, 2H, OCH₂CH₃), 6.52 (d, J_6–4

¼

2.4 Hz, 1H, 6^Ar-H), 6.65 (dd, J_4–3¼9.2 Hz, J_4–6¼2.4 Hz,

1H, 4^Ar-H), 7.96 (d, J_3–4¼9.2 Hz, 1H, 3^Ar-H). ¹³C NMR

(50 MHz, CD₃OD): d¼16.0 (OCH₂C H₃), 41.1, 41.4

(C-1^DAE), 44.8 (C-2^DAE), 70.9 (OC H₂CH₃), 113.2, 114.2

(C-6^Ar, C-4^Ar), 128.9 (C-3^Ar), 135.1 (C-1^Ar), 137.6 (C-2^Ar),

156.5 (C-5^Ar), 171.7 (CONH^Ar), the squaric acid signals

could not be identiﬁed. C₁₅H₁₆N₄O₆(348.31, 348.10698),

FAB MS: m/z¼371.1 [MþNa]^þ, 349.1 [MþH]^þ.

3.4. Coupling of 6 to BSA

To a solution of BSA (5 mg, 0.075 mmol) in borax buffer

(0.8 mL, 0.01 M, pH 9.0) a suspension of 6 (3.5 mg,

0.01 mmol) in borax buffer (0.6 mL, 0.01 M, pH 9.0) with a

few drops of methanol and EtOH (to make the solution

homogeneous) was added and the mixture was stirred at

208C under argon. After 64 h the solution was ﬁltered and

A. Buchynskyy et al. / Tetrahedron 58 (2002) 7741–7760

7751

freed from inorganic salts and starting 6 by ultraﬁltration.

Freeze drying gave 4 mg of 7. MALDI TOF MS (THAP

matrix, internal standard BSA, m/z¼68,932): m/z¼78,369

[M]^þ, 39,688 [M]^2þindicating an average loading of BSA

with 30 residues of 6.

76.0, 76.2, 76.6, 76.7 (C-5^C, C-4^D, C-4^B, C-2^B, C-3^B, C-3^C,

C-5^F, C-6^E, C-5^B, C-3^E, C-2^F, C-4^F, C-5^E, C-2^D, C-3^F, C-5^D,

C-3^D), 80.1 (C-4^E), 83.3 (C-4^C), 94.6 (d, C-1^F), 101.4,

102.3, 102.8, 103.4 (C-1^C, C-1^E, C-1^B, C-1^D), 158.4

(OCONH^F₂), 172.9, 173.4, 174.4, 174.8 (CONH₂^B, CONH^F₂,

NHCOCH^E₃, NHCOCH^C₃), 176.6 (C-3^H). The signals of the

quaternary carbons of the squaric acid part were not found.

³¹P NMR (81 MHz, D₂O): d¼22.54. C₄₈H₇₅N₆O₃₅P

(1327.12, 1326.40), ESI MS: m/z¼1347.3748 (1347.3758)

[MþNa22H]², 1325.3941 (1325.3938) [M2H]²,

3.5. Preparation of BSA–moenomycin conjugates 8

Compound 2c (1.7 mg, 0.87 mmol (experiment a), 6.1 mg,

3.0 mmol (experiment b), 23.6 mg, 12 mmol (experiment

c)), and BSA (Serva, 10 mg, 0.15 mmol (experiment a),

20 mg, 0.3 mmol (experiment b), 40 mg, 0.6 mmol (experi-

ment c)) in borax buffer, pH 9.03 (prepared from 100 mL

0.025 M borax and 80 mL 0.1 M KHCO₃, 1 mL (experi-

ment a), 2 mL (experiment b), 3 mL (experiment c)) were

stirred under argon at 208C for 7 d. Inorganic salts were

removed by ultraﬁltration. After lyophilization products 8,

9.3 mg (experiment a), 23.3 mg (experiment b) and 60.0 mg

(experiment c) were obtained. MALDI TOF MS (DHB

matrix, BSA as external standard with m/z¼66,418) showed

662.1917 (662.1933) [M22H]²²

.

3.6. Coupling of 9c to BSA

To a solution of BSA (20 mg, 0.3 mmol) in borate buffer

(from 0.05 M sodium tetraborate (50 mL) and 0.2 M boric

acid (50 mL), pH 9.0, 2 mL) a solution of 9c (a) 3.6 mg

(2.7 mmol), (b) 7.2 mg (5.4 mmol), (c) 18 mg (13 mmol) in

borate buffer (pH 9.0, 2 mL) was added and the mixtures

were stirred at 208C for 48 h. The reaction mixture was then

directly passed through a Sephadex^wG-15 column (elution

with water). After solvent evaporation and lyophilization

the conjugates 9d (a) (21 mg), (b) (19.8 mg) and (c) (22 mg)

were obtained and analysed by MALDI TOF MS (DHB

matrix, BSA as external standard with m/z¼66,346): 9d (a)

m/z¼69,274 [M]^þ, 34,895 [M]^2þ, corresponding to an

average protein loading of 2.3; 9d (b) m/z¼69,167 [M]^þ,

34,455 [M]^2þ, corresponding to an average protein loading

for experiment a: m/z¼73,749 [M]^þ, 36,938 [M]^2þ

;

experiment b: m/z¼77,126 [M]^þ, 38,783 [M]^2þand

experiment c: m/z¼83,141 [M]^þ, 41,985 [M]^2þthat

correspond to an average protein loading of 3.9 (experiment

a), 5.6 (experiment b) and 8.8 (experiment c). ESI FT ICR

MS (BSA as external standard with deconvoluted peak at

m/z¼66,400): 8a (from experiment a) deconvoluted peak at

m/z¼74,000 that correspond to an average protein loading

of 4.0.

of 2.2 and 9d (c) m/z¼72,352 [M]^þ, 36,272 [M]^2þ

,

corresponding to an average protein loading of 4.7.

3.5.1. (R )-2-{2-[(2-Ethoxy-3,4-dioxo-1-cyclobuten-1-

yl)methylamino]ethoxy}-3-({b-^D-galactopyranuron-

amidosyl-(1!4)-2-acetamido-2,6-dideoxy-b-^D-glucopyr-

anosyl-(1!4)-[b-^D-glucopyranosyl-(1!6)]-2-acetamido-

2-deoxy-b-^D-glucopyranosyl-(1!2)-3-O-carbamoyl-4-

C-methyl-a-^D-glucopyranuronamidosyl-oxy}-hydroxy-

phosphoryloxy)-propionic acid (9c). To a solution of

amine 9b (100 mg, 83 mmol) in phosphate buffer (5 mL, pH

7.0, 1 M) 3,4-diethoxy-3-cyclobuten-1,2-dion (141 mg,

830 mmol) was added dropwise and the mixture was stirred

at 208C for 24 h (during that time 3,4-diethoxy-3-cyclo-

buten-1,2-dions was dissolved slowly). In regular time

intervals the pH was adjusted to 7.0 by adding saturated

NaHCO₃solution. The reaction mixture was then directly

passed through Sephadex^wLH-20 (elution with 1:4 water–

CH₃OH). All fractions containing product were combined

and the solvent was evaporated. The residue was adsorbed

onto Kieselguhr (500 mg) and placed on top of a FC

column. Elution with ethyl acetate–isopropanol–water

4:5:4, solvent evaporation, ﬁltration of the product-contain-

ing fractions through a Sephadex^wLH-20 column (elution

with 1:4 water–methanol), solvent evaporation, and

lyophilization provided 9c (67 mg, 61%). ¹H NMR

(400 MHz, H,H COSY, D₂O): d¼1.18 (s, CH₃-4^F), 1.34

(bs, a doublet in the 200 MHz spectrum, CH₃-6^C), 1.36–

1.40 (CH₃-6^K), 1.97, 2.04 (s, NHCOCH₃^E, s, NHCOCH₃^C),

3.24 (m, 2^D-H), 4.16 (s, 4^B-H, 5^B-H), 4.45 (d,

J_1D–2D¼7.3 Hz, 1^D-H), 4.96 (m, J_2F–3F¼9.8 Hz, 3^F-H),

5.73 (m, 1^F-H). ¹³C NMR (50 MHz, D₂O): d¼14.9

(CH₃-4^F), 15.3 (CH₃-6^K), 16.9 (CH₃-6^C), 22.6 (NHCOC H^E₃,

NHCOC H^C₃, C-5^K), 37.7–37.9 (CH₃-3^I, two steroisomers),

55.4–55.7 (C-2^E, C-2^C), 61.0 (C-6^D), 67.3 (C-1^I), 69.1,

69.9, 70.8, 71.3, 72.4, 72.7, 73.0, 73.3, 73.7, 74.5, 75.1,

3.6.1. Benzyl {2-[2-(2-pent-4-enoylamino-ethoxy)-

ethoxy]-ethyl}-carbamate (11a). A solution of 4-pentenoic

acid (306 mg, 3.06 mmol) and N,N⁰-carbonyldiimidazole

(545 mg, 3.36 mmol) in dry pyridine (20 mL) was stirred for

1 h at 208C. 10a (949 mg, 3.36 mmol) was added and the

mixture was stirred at 208C for 2 h. Solvent evaporation and

FC (CHCl₃–methanol 19:1) furnished 11a (902 mg, 81%,

based on 4-pentenoic acid) as a pale yellow oil. R_f¼0.27

(CHCl₃–methanol 19:1). IR (ﬁlm): 3315, 2927, 2870, 1714,

1

1651, 1543, 1257, 1134, 1115 cm²¹. H NMR (300 MHz,

CDCl₃): d¼2.15–2.44 (m, 4H, CH₂CONH, CH₂vCHCH₂),

3.31–3.63 (m, 12H, 2£CH₂NH, 4£CH₂O), 4.92–5.01 (m,

1H, CH HvCHCH₂), 5.05–5.12 (m, 3H, CH₂^Cbz

,

CHHvCHCH₂), 5.40 (bs, 1H, NH), 5.67–5.89 (m, 1H,

CH₂vCHCH₂), 6.10 (bs, 1H, NH), 7.26–7.36 (m, 5H,

Ar–Hs^Cbz). ¹³C NMR (75 MHz, CDCl₃, APT): d¼29.6 (þ,

CH₂vCHCH₂), 35.7 (þ, CH₂CONH), 39.1, 40.8 (þ,

2£CH₂NH), 66.7 (þ, CH^C₂^bz), 69.9–70.2 (þ, CH₂O-

signals), 115.4 (þ, C H₂vCHCH₂), 128.2–128.5 (2, Ar–

CH^Cbz), 136.5 (þ, Ar–Cq^Cbz), 137.1 (2, CH₂vC HCH₂),

156.5 (þ, CvO^Cbz), 172.4 (þ, CONH). C₁₉H₂₈N₂O₅

(364.44, 364.20), FAB MS: m/z¼365.2 [MþH]^þ.

3.6.2. Benzyl {2-[2-(2-hept-6-enoylamino-ethoxy)-

ethoxy]-ethyl}-carbamamate (11b). 11b was prepared as

described for 11a. R_f¼0.25 (petroleum ether–ethyl

1

acetate–methanol 10:10:3). H NMR (300 MHz, CDCl₃,

H,H COSY): d¼1.37–1.51 (m, 2H, CH₂vCHCH₂CH₂),

1.60–1.74 (m, 2H, CH₂CH₂CONH), 2.04–2.14 (m, 2H,

CH₂vCHCH₂), 2.14–2.25 (m, 2H, CH₂CONH), 3.39–3.52

(m, 4H, 2£CH₂NH), 3.53–3.71 (m, 8H, 4£CH₂O), 4.94–

5.08 (m, 2H, CH₂vCHCH₂), 5.14 (bs, 2H, CH^C₂^bz), 5.34 (bs,

7752

A. Buchynskyy et al. / Tetrahedron 58 (2002) 7741–7760

1H, NH), 5.75–5.90 (m, 1H, CH₂vCHCH₂), 5.99 (bs, 1H,

NH), 7.35–7.43 (m, 5H, Ar–Hs^Cbz). ¹³C NMR (75 MHz,

CDCl₃, H,C COSY, APT): d¼25.4 (þ, C H₂CH₂CONH),

28.7 (þ, CH₂vCHCH₂C H₂), 33.7 (þ, CH₂vCHC H₂),

36.7 (þ, C H₂CONH), 39.4, 41.1 (þ, 2£CH₂NH), 67.0

(þ, CH^C₂^bz), 70.3–70.5 (þ, CH₂O-signals), 114.9 (þ,

C H₂vCHCH₂), 128.4–128.8 (2, Ar–CH^Cbz), 136.7 (þ,

Ar–Cq^Cbz), 138.8 (2, CH₂vC HCH₂), 156.9 (CvO^Cbz),

173.5 (CONH). C₂₁H₃₂N₂O₅(392.50, 392.23), FAB MS:

m/z¼393.2 [MþH]^þ, 415.2 [MþNa]^þ.

3.25–3.33 (m, 2H, CH₂NHBoc), 3.41–3.49 (m, 2H, CH_2-

NHCO), 3.50–3.64 (2£CH₂O, OCH₂CH₂O), 5.05 (bs, 1H,

NH Boc), 6.25 (bs, 1H, NHCO). ¹³C NMR (75 MHz,

CDCl₃): d¼20.6 (CH₂SH), 28.5 (C(CH₃)₃), 39.4, 40.3,

40.5 (CH₂CO, CH₂NHCO, CH₂NHboc), 69.9, 70.2, 70.3,

70.4 (2£CH₂O, OCH₂C H₂O), 79.5 (C(CH₃)₃), 156.1

(COC(CH₃)₃), 170.9 (CONH). C₁₄H₂₈N₂O₅S (336.45,

336.17), ESI ICR MS: m/z¼337.1793 (calcd 337.1792)

[MþH]^þ, 359.1919 (calcd 359.1611) [MþNa]^þ, 671.3354

(calcd 671.3354) [disulﬁdeþH]^þ.

3.6.3. Benzyl (3-pent-4-enyloxy-propyl)-carbamamate

(12). The mixture of sodium hydride (60% in oil, 697 mg,

17.4 mmol) and 4-penten-1-ol (1.0 g, 11.6 mmol) in dry

DMF (5 mL) was stirred at 208C for 30 min. At 08C a

solution of tert-butyl 3-bromopropylcarbamate³⁹(4.14 g,

17.4 mmol) in dry DMF (10 mL) was added dropwise. The

mixture was stirred at 08C for 15 min and at 208C for 4 h.

Work-up and FC (cyclohexane–CH₂Cl₂–ethyl acetate

5:5:1) gave tert-butyl 3-pent-4-enyloxy-propylcarbamate

(17 g, 41%, based on 4-penten-1-ol) as a colorless oil. The

latter compound was deprotected by treatment with 20%

TFA in CH₂Cl₂, to give, after work-up 3-pent-4-enyloxy-

propylamine (309 mg, 95%). To a solution of this amine

(250 mg, 1.74 mmol) and triethylamine (266 mL,

1.92 mmol) in CH₂Cl₂(30 mL) at 2508C a solution of

benzyl chloroformate (271 mL, 1.92 mmol) in dichloro-

methane (50 mL) was added dropwise within 30 min. The

mixture was then stirred at 2508C for 30 min and at 208C

for 2 h. Work-up and FC (cyclohexane–CH₂Cl₂–ethyl

acetate 5:5:1) furnished 12 (412 mg, 85%) as a colorless oil.

R_f¼0.28 (cyclohexane–CH₂Cl₂–ethyl acetate 5:5:1). IR

(ﬁlm): 3332, 2937, 2864, 1718, 1703, 1529, 1255,

3.6.5. N-{2-[2-(2-Aminoethoxy)ethoxy]ethyl}mercapto-

acetamide (13d). Compound 13c (500 mg, 1.49 mmol)

was dissolved in a 20:80 mixture of triﬂuoroacetic acid and

dichloromethane (5 mL) and the mixture was stirred at 208C

for 4 h. Solvent evaporation and FC (CHCl₃–methanol–

water 10:10:2) furnished 490 mg of 13d (as a salt) as a

yellow oil. R_f¼0.33 (CHCl₃–methanol–water 10:10:2). IR

(ﬁlm): 3373, 1676, 1641, 1441, 1188, 1138 cm²¹. ¹H NMR

(300 MHz, D₂O, H,H COSY): d¼2.55 (t, J¼6.6 Hz, 2H,

CH₂CONH), 2.76 (t, 2H, CH₂SH), 3.19 (m, 2H, CH₂NH₂),

3.41 (t, J¼5.5 Hz, 2H, CH₂NHCO), 3.64 (t, 2H, CH₂O),

3.70 (s, 4H, OCH₂CH₂O), 3.74 (t, J¼5.1 Hz, 2H, CH₂O).

¹³C NMR (75 MHz, D₂O): d¼20.1 (CH₂SH), 39.0, 39.3,

39.5 (C H₂CONH, CH₂NH, CH₂NH₂), 66.6, 69.0, 69.6, 69.8

(4£CH₂O), 174.7 (CONH). C₉H₂₀N₂O₃S (236.33, 236.12),

FAB MS: m/z¼237.1 [MþH]^þ, 259.1 [MþNa]^þ.

3.6.6. Allyl-2-O-(2-acetamido-3,4,6-O-tribenzyl-2-deoxy-

b-^D-glucopyranosyl)-3-O-carbamoyl-a-D-galactopy-

ranosiduronamide (16a). The compound was prepared as

described for allyl-2-O-(2-acetamido-3,4,6-O-triacetyl-2-

deoxy-b-^D-glucopyranosyl)-3-O-carbamoyl-a-D-galacto-

pyranosiduronamide.³⁵R_f¼0.17 (CHCl₃–methanol 6:1). ¹H

NMR (400 MHz, pyridine-d₅, H,H COSY): d¼2.19 (s, 3H,

NHCOCH₃), 3.59–3.62 (m, 1H, 5^E-H), 3.76–3.96 (m, 4H,

2^E-H, 4^E-H, CH₂-6^E), 4.11 (dd, J¼13.0, 5.1 Hz, 1H,

CH₂vCHCHH ), 4.23 (dd, 1H, CH₂vCHCH H), 4.56–

5.08 (m, 10H, CH₂– Hs^benzyl, 2^F-H, 5^F-H, 3^E-H,

CHHvCHCH₂), 5.33 (d, J¼17.1 Hz, 1H, CH HvCHCH₂),

5.49 (bs, 1H, 4^F-H), 5.56–5.66 (m, 2H, 1^E-H, 1^F-H), 5.79–

5.97 (m, 2H, 3^F-H, CH₂vCHCH₂), 7.24–7.56 (Ar–

Hs^benzyl), 7.77 (s, 1H, CONH), 8.46 (s, 1H, CONH), 9.10

(d, J_2E,NH¼7.3 Hz, 1H, NHCOCH₃). ¹³C NMR (100 MHz,

pyridine-d₅, H,C COSY, APT): d¼23.6 (2, NHCOC H₃),

58.4 (2, C-2^E), 68.9 (2, C-4^F), 69.0 (þ, CH₂vCHC H₂),

69.6 (þ, C-6^E), 73.0, 73.0 (2, C-5^F, C-3^F), 73.5 (þ,

CH₂^benzyl), 74.7, 74.8 (þ, 2£CH^b₂^enzyl), 75.0 (2, C-2^F, C-5^E),

79.2 (2, C-4^E), 81.6 (2, C-3^E), 99.3 (2, C-1^F), 102.0 (2,

C-1^E), 116.9 (þ, C H₂vCHCH₂), 127.7–128.7 (2, Ar–

CH^benzyl), 134.7 (2, CH₂vCHCH₂), 139.0, 139.2, 139.6

(þ, Ar–Cq^benzyl), 157.8 (þ, OCONH₂), 171.2, 172.1 (þ,

NHCOCH₃, C-6^F). C₃₉H₄₇N₃O₁₂(749.82, 749.32), FAB

MS: m/z¼772.3 [MþNa]^þ.

1113 cm²^{1 1}H NMR (200 MHz, CDCl₃, H,H COSY):

.

d¼1.59–1.85 (m, 4H, 2£CH₂CH₂O), 2.05–2.19 (m, 2H,

CH₂vCHCH₂), 3.26–3.53 (m, 6H, CH₂NHCbz, 2£CH₂O),

4.92–5.08 (m, 2H, CH₂vCHCH₂), 5.10 (bs, 2H, CH^C₂^bz),

5.27 (bs, 1H, NHCbz), 5.70–5.91 (m, 1H, CH₂vCHCH₂),

7.27–7.47 (m, 5H, Ar–Hs^Cbz). ¹³C NMR (50 MHz,

CDCl₃): d¼29.0, 29.8, 30.4 (2£C H₂CH₂O, CH₂v

CHC H₂), 39.6 (CH₂NHCbz), 66.6 (CH^C₂^bz), 69.4, 70.5

(2£CH₂O), 114.8 (C H₂vCHCH₂), 128.1–128.6 (Ar–

CH^Cbz), 136.9 (Ar–Cq^Cbz), 138.3 (CH₂vC HCH₂), 156.5

(CvO^Cbz). C₁₆H₂₃NO₃(277.36, 277.17), FAB MS:

m/z¼278.2 [MþH]^þ, ESI ICR MS: m/z¼278.1753 (calcd

278.17507) [MþH]^þ, 300.15724 (calcd 300.15701)

[MþNa]^þ.

3.6.4. N-{2-[2-(2-{2-Mercaptoacetamido}ethoxy)ethoxy]-

ethyl} tert-butyl carbamate (13c). To a stirred solution of

13b (600 mg, 2.42 mmol) and triethylamine (489 mg,

4.83 mmol) in dichloromethane (10 mL) at 208C a solution

of N-{2-[2-(2-aminoethoxy)ethoxy]ethyl} tert-butyl carba-

mate (10b)³²(491 mg, 2.42 mmol) in dichloromethane

(50 mL) was added slowly and the mixture was stirred at

208C for 2 h. Usual work-up and FC (cyclohexane–ethyl

acetate–methanol 10:10:1) provided 13c (763 mg, 94%) as

pale yellow oil. R_f¼0.13 (cyclohexane–ethyl acetate–

methanol 10:10:1). IR (KBr): 3338, 2972, 2931, 2870,

1707, 1655, 1535, 1452, 1363, 1275, 1252, 1171,

3.6.7. Allyl-2-O-(2-acetamido-2-deoxy-b-^D-glucopyrano-

syl)-3-O-carbamoyl-a-^D-galactopyranosiduronamide

(16b). A mixture (degassed by sonication while purging

with argon) of allyl-2-O-(2-acetamido-3,4,6-O-triacetyl-2-

deoxy-b-^D-glucopyranosyl)-3-O-carbamoyl-a-D-galacto-

pyranosiduronamide³⁵(200 mg, 330 mmol) and 2:1 metha-

nol–water (15 mL) was cooled to 08C, 0.3 M LiOH

(4.4 mL) was added and the reaction mixture was stirred

1111 cm²^{1 1}H NMR (300 MHz, CDCl₃, H,H COSY):

.

d¼1.44 (s, 9H, C(CH₃)₃), 1.60 (t, J_SH,CH2¼8.2 Hz, 1H, SH),

2.49 (t, J¼6.7 Hz, 2H, CH₂CO), 2.78 (dt, 2H, CH₂SH),

A. Buchynskyy et al. / Tetrahedron 58 (2002) 7741–7760

7753

at 08C for 30 min followed by 15 min at 208C. After

addition of Dowex W X2 (H^þ, 2.0 g) the mixture was stirred

at 208C for 30 min. The resin was ﬁltered off and washed

with 2:1 methanol–water and water. After solvent evapor-

ation the residue was adsorbed onto kieselguhr and puriﬁed

by FC (CHCl₃–methanol–water 9:6:1.4) to give after

lyophilization 135 mg (86%) 16b as a white solid.

R_f¼0.10 (CHCl₃–methanol–water 9:6:1.4). ¹H NMR

(300 MHz, D₂O, H,H COSY): d¼1.94 (s, 3H, NHCOCH₃),

3.32–3.48 (m, 3H, 3^E-H, 4^E-H, 5^E-H), 3.58–3.70 (m, 2H,

2^E-H, 6^E-H), 3.84 (d, 1H, 6^E-H⁰), 3.95 (dd, J_2F,3F¼10.4 Hz,

J_1F,2F¼3.8 Hz, 1H, 2^F-H), 4.02–4.19 (m, 2H, CH_2-

vCHCH₂), 4.31 (dd, J_3F,4F¼3.3 Hz, J_4F,5F¼1.3 Hz, 1H,

4^F-H), 4.38 (d, 1H, 5^F-H), 4.46 (d, J_1E,2E¼8.5 Hz, 1H, 1^E-

H), 4.89 (dd, 1H, 3^F-H), 5.15–5.21 (d, 1H, CHHvCHCH₂),

5.23–5.33 (m, 2H, 1^F-H, CH HvCHCH₂), 5.82–5.96 (m,

1H, CH₂vCHCH₂). ¹³C NMR (75 MHz, D₂O, H,C COSY):

d¼22.4 (NHCOCH₃), 55.6 (C-2^E), 61.0 (C-6^E), 68.1 (C-4^F),

69.2 (CH₂vCHC H₂), 70.0 (C-4^E), 70.3 (C-5^F), 71.3 (C-3^F),

73.8 (C-3^E), 75.1, (C-2^F), 75.9 (C-5^E), 97.6 (C-1^F), 102.9

(C-1^E), 119.1 (C H₂vCHCH₂), 133.3 (CH₂vC HCH₂),

157.9 (OCONH₂), 173.5, 174.9 (NHCOCH₃, C-6^F).

C₁₈H₂₉N₃O₁₂(479.44, 479.18), FAB MS: m/z¼480.2

[MþH]^þ, 486.2 [MþLi]^þ, 502.1 [MþNa]^þ.

(MeOH–H₂O 3:1), FC (CHCl₃–MeOH–H₂O 12:4.5:1),

another Sephadex LH 20 LC (MeOH–H₂O 3:1), and

lyophilization provided 15a (149 mg, 76%) as a white

solid. R_f¼0.29 (CHCl₃–methanol–water 12:4.5:1). ¹H

NMR (400 MHz, pyridine-d₅, H,H COSY): d¼2.32 (s,

3H, NHCOCH₃), 3.42–3.66 (m, 15H, including 5^E-H),

3.80–3.91 (m, 2H, 6^E-H, 4^E-H), 3.95–4.05 (3H, including

6^E-H⁰), 4.28–4.36 (m, 1H, 2^E-H), 4.65–4.72 (m, 2H,

including 3^E-H), 4.74–4.79 (m, 2H, including, 5^F-H),

4.91–5.00 (m, 2H, including 2^F-H), 5.40–5.45 (m, 2H,

1^E-H, 4^F-H), 5.53 (d, J_1F,2F¼3.6 Hz, 1H, 1^F-H), 5.60 (dd,

J_2F,3F¼10.6 Hz, J_3F,4F¼3.0 Hz, 1H, 3^F-H), 7.72 (bs, 1H,

CONH), 8.14 (bs, 1H, NHCbz), 8.43 (s, 1H, CONH), 9.63

(d, J_2E,NH¼8.9 Hz, 1H, NHCOCH₃). ¹³C NMR (100 MHz,

pyridine-d₅, HMQC): d¼23.6 (NHCOC H₃), 56.4 (C-2^E),

68.8 (C-4^F), 69.5 (C-6^E), 73.0 (C-3^F), 73.4, 73.5 (CH^b₂^enzyl

,

C-5^F), 74.8, 74.8, 75.3 (CH₂^benzyl, C-5^E, C-2^F), 78.93 (C-4^E),

82.72 (C-3^E), 100.2 (C-1^F), 103.7 (C-1^E), 157.4, 158.0

(CvO^Cbz, OCONH₂), 171.5, 171.9 (NHCOCH₃, C-6^F).

C₅₂H₆₇N₅O₁₆(1018.13, 1017.46), ESI ICR MS:

m/z¼1018.4632 (calcd 1018.4656) [MþH]^þ.

3.6.10. 2-O-(2-Acetamido-2-deoxy-b-^D-glucopyranosyl)-

1-O-(2-{2-[2-(2-aminoethoxy)ethoxy]ethylamino}ethyl)-

3-O-carbamoyl-a-^D-galactopyranuronamide (15b).

A

3.6.8. 2-Oxoethyl 2-O-(2-acetamido-3,4,6-O-tribenzyl-2-

deoxy-b-^D-glucopyranosyl)-3-O-carbamoyl-a-D-galac-

topyranosiduronamide (14). Through a solution of 16a

(160.0 mg, 213 mmol) in methanol (50 mL) O₃/O₂(Fischer

OZON 502, ﬂow rate 50 L/h¼2 g/h O₃) was passed at

2788C for 5 min. Then oxygen was bubbled through the

solution for 15 min and Ar for another 15 min. The mixture

was allowed to warm to ambient temperature. Dimethyl-

sulﬁde (200 mL) was added and the mixture was stirred at

208C for 2 h. TLC (CHCl₃–MeOH 3:1) showed the

formation of a single product which was obtained by

solvent evaporation and lyophilization. An aliquot was

submitted to FC (CHCl₃–MeOH 4:1) in order to remove

small amounts of DMSO to give a white solid (94%).

R_f¼0.23 (CHCl₃–methanol 4:1). ¹³C NMR (75 MHz,

pyridine-d₅, APT): characteristic signals at d¼23.6

(NHCOCH₃), 54.3 (CH(OC H₃)OH)), 58.1, 58.2 (C-2^E),

69.7 (C-6^E), 79.2, 79.3 (C-4^E), 81.9, 82.0 (C-3^E), 90.0

(CH(OH)₂), 97.0, 97.1 (C H(OCH₃)OH)), 100.4, 100.7,

101.0 (C-1^F), 102.4, 102.6 (C-1^E), 127.8–128.9 (Ar–

CH^benzyl), 139.0–139.8 (Ar–Cq^benzyl), 157.9–158.0

(OCONH₂), 171.3–172.4 (NHCOCH₃, C-6^F). C₃₈H₄₅N₃O₁₃

(751.79, 751.30, aldehyde), C₃₈H₄₇N₃O₁₄(769.80, 769.30,

aldehyde hydrate), C₃₉H₄₉N₃O₁₄(783.83, 783.32, hemi-

acetal), FAB MS: m/z¼752.3 [MþH]^þ(aldehyde), 774.3

[MþNa]^þ(aldehyde), 784.3 [MþH]^þ(hemiacetal), 792.3

[MþNa]^þ(aldehyde hydrate), 806.3 [MþNa]^þ(hemiacetal).

mixture of 15a (50 mg, 49.1 mmol) and 10% Pd/C

(25 mg) in 2:1:0.5 methanol–acetic acid–water (5 mL)

was stirred under hydrogen for 48 h at 208C.40 The catalyst

was removed by ﬁltration through Celite^w(elution with 2:1

methanol–water (5 mL)). Solvent evaporation, FC

(CHCl₃–methanol–water 10:10:2:0.1), Sephadex LH-20

LC (methanol–water 3:1), and lyophilization gave 15b

(30 mg, 61%) as a white solid. R_f¼0.05 (CHCl₃–metha-

nol–water–HCO₂H 10:10:2:0.1). ¹H NMR (400 MHz,

D₂O, H,H COSY): d¼2.00 (s, 3H, NHCOCH₃), 3.40–3.45

(m, 2H, 4^E-H, 5^E-H), 3.45–3.53 (m, 1H, 3^E-H), 3.66–3.84

(m, 13H, including 2^E-H, 6^E-H), 3.84 (1H, d, J¼12.2 Hz,

6^E-H⁰), 4.00–4.08 (m, 2H, including 2^F-H), 4.35–4.38 (m,

1H, 4^F-H), 4.44 (bs, 1H, 5^F-H), 4.52 (d, J_1E,2E¼8.3 Hz, 1H,

1^E-H), 4.96 (dd, J_2F,3F¼10.5 Hz, J_3F,4F¼3.2 Hz, 1H, 3^F-H),

5.28 (d, J_1F,2F¼3.4 Hz, 1H, 1^F-H). ¹³C NMR (100 MHz,

D₂O, HMQC): d¼22.9 (NHCOC H^E₃), 56.0 (C-2^E), 60.8

(C-6^E), 68.6 (C-4^F), 70.2, 70.2, 70.3 (C-7^SPA, C-8^SPA, C-4^E),

71.1, 71.2 (C-5^F, C-3^F), 74.5 (C-3^E), 76.2, 76.2 (C-2^F,

C-5^E), 99.2 (C-1^F), 103.6 (C-1^E), 158.4 (OCONH₂), 173.8,

175.1 (NHCOCH₃, C-6^F). C₂₃H₄₃N₅O₁₄(613.62, 613.28),

ESI ICR MS: m/z¼614.2885 (calcd 614.2879) [MþH]^þ,

636.2705 (calcd 636.2699) [MþNa]^þ.

3.6.11. 2-O-(2-Acetamido-3,4,6-O-tribenzyl-2-deoxy-b-

D-glucopyranosyl)-1-O-[(J)-8-(2-{2-[2-(benzyloxycarbo-

nylamino)ethoxy]ethoxy}ethylamino)-8-oxooct-2-enyl]-

3-O-carbamoyl-a-^D-galactopyranuronamide (17a).

A

3.6.9. 2-O-(2-Acetamido-3,4,6-tri-O-benzyl-2-deoxy-b-^D-

glucopyranosyl)-1-O-[2-(2-{2-[2-(benzyloxycarbonyl-

amino)ethoxy]ethoxy}ethylamino)ethyl]-3-O-carba-

moyl-a-^D-galactopyranuronamide (15a). A solution of

10a (82.6 mg, 293 mmol) in methanol (10 mL) was adjusted

to pH 7 (with acetic acid in methanol). Solutions of 14

(110 mg, 146 mmol) in methanol (30 mL) and sodium

cyanoborohydride (18.4 mg, 293 mmol) in methanol

(1 mL) were added successively and the mixture was stirred

at 208C for 18 h. Solvent evaporation, Sephadex LH 20 LC

suspension of 16a (84 mg, 112 mmol) and 11b (88 mg,

224 mmol) in ethanol-free chloroform (40 mL) was

degassed ﬂushing with argon and sonication. This solution

was transferred via a canula into a degassed solution

containing the Grubbs catalyst (catalytic amount) in

chloroform (5 mL). The mixture was left at 358C for 18 h

under Argon. Solvent evaporation and FC (petroleum

ether–ethyl acetate–methanol 2:2:1) furnished 43 mg

(49%) of not consumed 11b, 25 mg (29%) of the homo

metathesis product of 11b, 45 mg (54%) of 16a and a

7754

A. Buchynskyy et al. / Tetrahedron 58 (2002) 7741–7760

fraction containing 17a. The 17a fraction was adsorbed onto

kieselguhr and further puriﬁed by FC (CHCl₃–methanol

3:1) to give 26 mg (21%) of 17a as a white solid. R_f¼0.08

5^E-H), 3.37 (t, J¼9.0 Hz, 1H, 3^E-H), 3.43–3.63 (m, 12H,

including 2^E-H, 6^E-H), 3.77 (d, J¼12.0 Hz, 1H, 6^E-H⁰), 3.85

(dd, J_2F,3F¼10.4 Hz, J_1F,2F¼3.4 Hz, 1H, 2^F-H), 4.21–4.27

(m, 2H, 4^F-H, 5^F-H), 4.37 (d, J_1E,2E¼8.5 Hz, 1H, 1^E-H),

1

(petroleum ether–ethyl acetate–methanol 2:2:1). H NMR

(600 MHz, pyridine-d₅, H,H COSY): d¼2.17, 2.18 (2£s,

3H, NHCOCH₃), 3.53–3.70 (m, 13H, including 5^E-H),

3.78–3.96 (m, 4H, 2^E-H, 4^E-H, CH₂-6^E), 4.59–4.79 (Ar–

Hs^benzyl, 3^E-H), 4.85–5.01 (Ar–Hs^benzyl, 2^F-H, 5^F-H),

5.43–5.69 (m, 5H, 1^F-H, 1^E-H, 4^F-H, 2^SPA-H, 3^SPA-H),

5.79–5.85 (m, 1H, 3^F-H), 8.15 (s, 1H, NHCbz), 8.43 (s, 2H,

NH, CONH), 9.03–9.12 (m, 1H, NHCOCH₃). ¹³C NMR

(150 MHz, pyridine-d₅, HMQC): d¼23.6 (NHCOC H₃),

58.4 (C-2^E), 68.9 (C-4^F), 69.7, 69.8 (C-6^E, two isomers),

73.0, 73.1, 73.1, 73.2 (C-5^F, C-3^F, two isomers, respect-

ively), 75.0, 75.1 (C-5^E, C-2^F), 79.3 (C-4^E), 81.6 (C-3^E),

99.3, 99.6 (C-1^F, two isomers), 102.0, 102.1 (C-1^E, two

isomers), 126.2, 126.6 (C-2^SPA, two isomers), 133.6, 134.1

(C-3^SPA, two isomers), 157.4, 157.8 (CvO^Cbz, OCONH₂),

171.2, 172.1 and 172.2, 173.1 and 173.15 (NHCOCH₃,

C-6^F, C-8^SPA). C₅₈H₇₅N₅O₁₇(1114.26, 1113.52), ESI ICR

MS: m/z¼1114.5259 (calcd 1114.5231) [MþH]^þ,

1136.5080 (calcd 1136.5050) [MþNa]^þ.

4.79 (dd, J_3F,4F¼1.8 Hz, 1H, 3^F-H), 5.14 (d, 1H, 1^F-H). ¹³

C

NMR (100 MHz, D₂O, HMQC): characteristic signals at

d¼22.9 (NHCOC H₃), 36.3 (C-7^SPA), 39.6, 39.8 (C-10^SPA

,

C-17^SPA), 54.8 (C-2^E), 71.8 (C-3^F), 74.3 (C-3^E), 76.0

(C-2^F), 98.5 (C-1^F), 103.2 (C-1^E). C₂₄H₄₄N₄O₁₃(711.76,

711.35), ESI ICR MS: m/z¼712.3634 (calcd 712.3611)

[MþH]^þ, 734.3467 (calcd 734.3430) [MþNa]^þ.

3.6.14. 2-O-(2-Acetamido-2-deoxy-b-^D-glucopyranosyl)-

1-O-[6-(3-aminopropoxy)-hexyl]-3-O-carbamoyl-a-^D-

galactopyranuronamide (19b). Compound 17b (39 mg,

39 mmol) was hydrogenated as described for 17a. FC

(CHCl₃–methanol–water–HCO₂H 9:6:1.4:0.1), Sephadex

LH-20 LC (methanol–water 3:1) and lyophilization

furnished 21.8 mg (93%) 19b as a white solid. R_f¼0.08

1

(CHCl₃–methanol–water–HCO₂H 9:6:1.4:0.1). H NMR

(400 MHz, D₂O, H,H COSY): d¼2.01 (s, 3H, NHCOCH₃),

3.36–3.75 (m, 11H, including 2^E-H, 3^E-H, 4^E-H, 5^E-H, 6^E-

H), 3.84 (dd, J¼12.2 Hz, J_5E,6E¼1.9 Hz, 1H, 6^E-H⁰), 4.00

(dd, J_2F,3F¼10.6 Hz, J_1F,2F¼3.9 Hz, 1H, 2^F-H), 4.36–3.39

(m, 2H, 4^F-H, 5^F-H), 4.52 (d, J_1E,2E¼8.5 Hz, 1H, 1^E-H),

3.6.12. 2-O-(2-Acetamido-3,4,6-O-tribenzyl-2-deoxy-b-

D-glucopyranosyl)-1-O-{6-[3-(benzyloxy-carbonylami-

no)propoxy]-hexyl}-3-O-carbamoyl-a-^D-galactopy-

ranuronamide (17b). The CMR of 16a (95 mg, 127 mmol)

and 12 (70 mg, 253 mmol) was performed as described for

17a. FC (CHCl₃–methanol 6:1) gave 13 mg (18%) of 12,

37 mg (52%) of the homo metathesis product of 12, and

48 mg (38%) of 17b as a white solid. 53 mg (56%) of 16a

were recovered. R_f¼0.19 (CHCl₃–methanol 6:1). ¹H NMR

(400 MHz, pyridine-d₅, H,H COSY): d¼2.17, 2.18 (2s, 3H,

NHCOCH₃), 3.60–3.67 (m, 1H, 5^E-H), 3.80–3.96 (m, 4H,

2^E-H, 4^E-H, CH₂-6^E), 4.59–4.79 (CH^b₂^enzyl, 3^E-H), 4.90 (bs,

1H, 5^F-H), 4.93–5.07 (CH^b₂^enzyl, 2^F-H), 5.43–5.73 (m, 5H,

1^F-H, 1^E-H, 4^F-H, 2^SPA-H, 3^SPA-H), 5.81–5.87 (m, 1H, 3^F-

H), 7.76 (s, 1H, CONH), 7.90–8.00 (NHCbz), 8.47 (s, 1H,

CONH), 9.07 (d, J_2E,NH¼7.3 Hz, 1H, NHCOCH₃). ¹³C

NMR (100 MHz, pyridine-d₅, HMQC): d¼23.6

(NHCOC H₃), 58.3 (C-2^E), 68.9 (C-4^F), 69.6, 70.1 (C-6^E,

two isomers), 73.0–73.1 (C-5^F, C-3^F), 75.0–75.1 (C-5^E,

C-2^F), 79.2 (C-4^E), 81.6 (C-3^E), 99.3, 99.5 (C-1^F, two

isomers), 102.0, 102.1 (C-1^E, two isomers), 126.6, 126.7

(C-2^SPA, two isomers), 133.0, 133.8 (C-3^SPA, two isomers),

4.89 (dd, J_3F,4F¼3.2 Hz, 1H, 3^F-H), 5.27 (d, 1H, 1^F-H). ¹³

C

NMR (100 MHz, D₂O, HMQC): d¼22.8 (NHCOC H₃), 56.0

(C-2^E), 61.5 (C-6^E), 68.5 (C-4^F), 70.6, 70.7 (C-4^E, C-5^F),

71.8 (C-3^F), 74.3 (C-3^E), 75.6, (C-2^F), 76.3 (C-5^E), 98.7

(C-1^F), 103.4 (C-1^E), 158.4 (OCONH₂), 173.9, 175.4

(NHCOCH₃, C-6^F). C₂₄H₄₄N₄O₁₃(596.63, 596.29), ESI

ICR MS: m/z¼597.2973 (calcd 597.29776) [MþH]^þ.

3.6.15. 2-O-(2-Acetamido-2-deoxy-b-^D-glucopyranosyl)-

1-O-[3-(3-{2-[2-(2-aminoethoxy)ethoxy]ethylamino}-3-

oxopropylthio)propyl]-3-O-carbamoyl-a-^D-galactopy-

ranuronamide (18a). A solution of 16b (70 mg, 146 mmol)

and 13d (207 mg, 876 mmol) in ultrapure water (1 mL) was

degassed (sonication while purging with argon) and

irradiated at 108C in a quartz cuvette for 2 h at 254 nm

(Rayonet reactor, RPR-100). Solvent evaporation and FC

(CHCl₃–methanol–water–HCO₂H 9:6:1.4:0.1) followed

by Sephadex LH-20 chromatography (elution with metha-

nol–water 3:1) gave after lyophilization 18a (74 mg, 71%)

as a white solid. R_f¼0.10 (CHCl₃–methanol–water–

HCO₂H¼9:6:1.4:0.1). ¹H NMR (400 MHz, D₂O, H,H

COSY): d¼1.95 (s, 3H, NHCOCH₃), 3.30–3.39 (m, 4H,

4^E-H, 5^E-H, CH₂-9^SPA), 3.41–3.48 (m, 1H, 3^E-H), 3.59–

3.70 (m, ₀9H, including 2^E-H, 6^E-H), 3.84 (d, J¼12.4 Hz,

1H, 6^E-H ), 3.94 (dd, J_1,2¼10.6 Hz, J_2,3¼3.9 Hz, 1H, 2^F-H),

4.29–4.33 (m, 1H, 4^F-H), 4.36 (bs, 1H, 5^F-H), 4.46 (d,

J_1,2¼8.5 Hz, 1H, 1^E-H), 4.88 (dd, J_3,4¼3.2 Hz, 1H, 3^F-H),

5.21 (d, 1H, 1^F-H). ¹³C NMR (100 MHz, D₂O, HMQC):

d¼22.9 (NHCOC H^E₃), 56.1 (C-2^E), 61.5 (C-6^E), 68.6

(C-4^F), 70.6 (C-4^E), 70.8 (C-5^F), 71.7 (C-3^F), 74.3 (C-3^E),

75.6 (C-2^F), 76.4 (C-5^E), 98.6 (C-1^F), 103.4 (C-1^E), 158.4

(OCONH₂), 173.9, 175.3, 175.3 (NHCOCH₃, C-6^F,

C-7^SPA). C₂₇H₄₉N₅O₁₅S (715.77, 715.29), ESI ICR MS:

m/z¼716.3015 (calcd 716.3019) [MþH]^þ, 738.2838 (calcd

738.2838) [MþNa]^þ.

157.2, 157.7 (CvO^Cbz

,

OCONH₂), 171.2, 172.1

(NHCOCH₃, C-6^F). C₅₃H₆₆N₄O₁₅(999.12, 998.45), ESI

ICR MS: m/z¼999.4605 (calcd 999.4597) [MþH]^þ,

1021.44249 (calcd 1021.4417) [MþNa]^þ.

3.6.13. 2-O-(2-Acetamido-2-deoxy-b-^D-glucopyranosyl)-

1-O-(8-{2-[2-(2-aminoethoxy)ethoxy]-ethylamino}-8-

oxooctyl)-3-O-carbamoyl-a-^D-galactopyranuronamide

(19a). A mixture of 17a (17 mg, 15.3 mmol) and 10% Pd/C

(15 mg) in 4:1:0.5 methanol–acetic acid–water (5 mL) was

stirred under hydrogen for 18 h at 208C. The catalyst was

removed by ﬁltration through Celite^w(elution with 2:1

methanol–water (5 mL). FC (CHCl₃–methanol–water–

HCO₂H 5:5:1:0.1), Sephadex LH-20 LC (methanol–water

3:1) and lyophilzation provided 9.1 mg (84%) 19a as a

white solid. R_f¼0.14 (CHCl₃–methanol–water–HCO₂H

5:5:2:0.1). ¹H NMR (400 MHz, D₂O, H,H COSY): d¼1.87

(s, 3H, NHCOCH₃), 3.19–3.32 (m, 4H, CH₂-10^SPA, 4^E-H,

3.6.16. 2-O-(2-Acetamido-2-deoxy-b-^D-glucopyranosyl)-

3-O-carbamoyl-1-O-{3-[3-(2-{2-[2-(2-ethoxy-3,4-dioxo-

A. Buchynskyy et al. / Tetrahedron 58 (2002) 7741–7760

7755

cyclobut-1-en-1-ylamino)ethoxy]ethoxy}ethylamino)-3-

oxopropylthio]propyl}-a-^D-galactopyranuronamide

(18b). To a solution of 18a (50 mg, 69.8 mmol) in 0.5 M

phosphate buffer (pH 7.2, 3 mL) a solution of 3,4-diethoxy-

3-cyclobutene-1,2-dione (35.6 mg, 210 mmol) in ethanol

(0.5 mL) was added and the mixture was stirred at 208C for

18 h. After solvent evaporation buffer salts and the excess of

3,4-diethoxy-3-cyclobuten-1,2-dione were removed by

Sephadex LH-20 chromatography (elution with metha-

nol–water 3:1). The remaining material was adsorbed to

kieselguhr and placed on top of a FC column. Elution with

ethyl acetate–methanol–water 6:3:1) followed by Sepha-

dex LH-20 chromatography (elution with methanol–water

3:1) and lyophilization furnished 18b (39 mg, 66%) as a

white solid. R_f¼0.16 (ethyl acetate–methanol–water

1.44 (m, 3H, OCH₂CH₃^SA), 2.00 (s, 3H, NHCOCH^E₃), 3.38–

3.45 (m, 2H, 4^E-H, 5^E-H), 3.47–3.53 (m, 1H, 3^E-H), 3.61–

3.85 (m, 12H, including 2^E-H, 6^E-H), 3.90 (bd, J¼12.2 Hz,

1H, 6^E-H⁰), 4.00 (dd, J_1,2¼10.8 Hz, J_2,3¼3.9 Hz, 1H, 2^F-H),

4.35–4.38 (m, 1H, 4^F-H), 4.42 (d, 1H, 5^F-H), 4.52 (d,

J_1,2¼8.3 Hz, 1H, 1^E-H), 4.64–4.75 (m, 2H, OCH₂CH^S₃^A

partially hidden by the water signal), 4.94 (dd, J_3,4¼3.4 Hz,

1H, 3^F-H), 5.27 (d, 1H, 1^F-H). ¹³C NMR (100 MHz, D₂O,

HMQC): d¼15.7 (OCH₂CH^S₃^A), 22.9 (NHCOCH₃^E), 56.1

(C-2^E), 61.6 (C-6^E), 68.6 (C-4^F), 70.6 (C-4^E), 70.8 (C-5^F),

71.3 (OCH₂CH^S₃^A), 71.8 (C-3^F), 74.4 (C-3^E), 75.6 (C-2^F),

76.4 (C-5^E), 98.7 (C-1^F), 103.4 (C-1^E), 158.4 (OCONH₂),

173.9, 174.4, 175.2, 175.3 (NHCOCH₃, CvC^SA, C-6^F,

C-7^SPA), 177.8, 178.1 (CvC^SA), 184.0, 189.6 (2£CvO^SA).

C₃₃H₅₃N₅O₁₈S (839.87, 839.31), FAB MS: m/z¼840.3

[MþH]^þ, 862.3 [MþNa]^þ. ESI ICR MS: m/z¼840.3179

(calcd 840.3179) [MþH]^þ, 862.3003 (calcd 862.2998)

[MþNa]^þ.

20a (11.4 mg). IR (CHCl₃): 1740 (CvO), 1700 (CvC),

1380, 1220, 1160, 1080, 1030, 990 cm²^{1 1}H NMR

.

(300 MHz, CDCl₃, homo decoupling): d¼1.36, 1.51 (2s,

6H, C(CH₃)₂), 2.66–2.75 (m, 4H, CH₂-2^succ, CH₂-3^succ),

4.02 (dddd, 1H, 1^allyl1-H), 4.18 (dddd, 1H, 1⁰^allyl1-H), 4.41

(dd, J_3,4¼5.4 Hz, 1H, 3^F-H), 4.54 (d, 1H, 5^F-H), 4.60 (ddd,

J

¼3.8 Hz, 2H, CH₂-1^allyl-2), 4.63 (dd, J_4,5¼2.7 Hz, 1H,

allyl-2

1,2

4^F-H), 4.96 (dd, J_2,3¼7.8 Hz, 1H, 2^F-H), 5.09 (d, 1H,

J_1,2¼3.6 Hz, 1^F-H), 5.23, 5.24 (2dddd, J_cis¼10.4 Hz, 2H,

allyl-2

trans

allyl-2

cis

3^a_tr^l_a^ly_n^l_s^-1-H, 3

-H), 5.30, 5.32 (2dddd, J_trans¼17.3 Hz, 2H,

3^a_c^l_i^l_s^yl-1-H, 3

-H), 5.72, 6.50 (concentration dependent,

2s, b, 2H, CONH₂^F), 5.80–5.89 (m, 2H, 2^allyl-1-H, 2^allyl-2-H).

¹³C NMR (50 MHz, CDCl₃, C,H COSY, APT): d¼26.7,

28.3 (C(C H₃)^F₂), 29.5, 29.5 (C-2^succ

,

C-3^succ), 65.9

(C-1^allyl-2), 69.1 (C-5^F), 69.8 (C-1^allyl-1), 71.9 (C-2^F), 73.5

1

6:3:1). H NMR (400 MHz, D₂O, H,H COSY): d¼1.37–

(C-3^F), 73.9 (C-4^F), 95.8 (C-1^F), 110.6 (C(CH₃)₂), 118.7,

118.8 (C-3^allyl-1C-3^allyl-2), 132.5, 133.5 (C-2^allyl-1

, ,

C-2^allyl-2), 170.6, 172.1, 172.4 (CONH₂, 2CvO^succ). calcd

C 55.20, H 6.58, N 3.39, found C 55.65, H 6.20, N 3.57.

C₁₉H₂₇NO₉(413.42, 413.17), FAB MS: m/z¼436.1

[MþNa]^þ, 414.1 [MþH]^þ.

3.7.2. Allyl 2-O-[3-(allyloxycarbonyl)-propionyl]-a-^D-

galactopyranosiduronamide (21a). A mixture of 20b

(2.22 g, 5.4 mmol) and 20% acetic acid (97 mL) was stirred

at 508C for 8 h. Solvent evaporation (codestillation with

toluene), dissolving the residue in water and lyophilization,

followed by LC (petrol–CHCl₃–methanol 1:1:0.2)

furnished 21a (1.9695 g, 98%). IR (CHCl₃): 3550–3200,

3020, 2920, 1740, 1690, 1220, 1150, 1100, 1050,

1030 cm²¹

.

¹H NMR (300 MHz, CD₃OD): d¼4.03 (dd,

J_2,3¼10.4 Hz, J_3,4¼3.0 Hz, 1H, 3^F-H), 4.265 (s, 1H, 5^F-H),

4.269 (d, 1H, 4^F-H), 5.02 (dd, 1H, 2^F-H), 5.16 (d,

J_1,2¼3.6 Hz, 1H, 1^F-H), 7.31, 7.34 (2s, b, CONH^F₂). ¹³C

NMR (75 MHz, CD₃OD, C,H COSY): d¼68.6 (C-3^F), 71.3

(C-5^F), 72.4 (C-2^F), 72.8 (C-4^F), 97.0 (C-1^F), 173.7, 173.9,

174.1 (CONH^F₂, 2CvO^succ). C₁₆H₂₃NO₉(373.36, 373.14),

calcd C 51.47, H 6.21, N 3.75, found C 51.60, H 5.90, N

3.70, FAB MS: m/z¼769.2 [2MþNa]^þ, 747.2 [2MþH]^þ,

396.0 [MþNa]^þ, 374.0 MþH]^þ.

3.7. Conjugate 18c

A solution of 18b (5.7 mg, 6.7 mmol) in 0.1 M carbonate

buffer (pH 9.0, 0.5 mL) was added to a solution of BSA

(30 mg, 0.45 mmol) in 0.1 M carbonate buffer (pH 9.0,

1.2 mL) and the mixture was stirred at 208C for 40 h. Then

TLC (ethyl acetate–methanol–water 6:3:1, R_fof 18b: 0.16)

indicated the complete consumption of 18b. The conjugate

was puriﬁed by ultraﬁltration (30,000 cut off) to provide

after lyophilization 31 mg of the conjugate 18c. MALDI

TOF MS (BSA as internal standard, m/z around 66,590)

displayed for the conjugate a molecular peak maximum

around m/z¼76,560 corresponding to an average haptene–

BSA ratio of 12:13.⁴¹

3.8. Conversion of 21a to 21b

To a solution of 21a (1.45 g, 3.9 mmol) in CH₂Cl₂(300 mL)

trichloroacetyl isocyanate (560 mL, 4.7 mmol) was added at

268C. The mixture was stirred at 268C for 3.5 h. Excess

reagent was destroyed by addition of CH₃OH (16 mL) and

stirred at 208C for 1.5 h. After solvent evaporation the

residue was redissolved in CH₃OH (190 mL). Zn dust

(2.53 g, 38.8 mmol) was added and the mixture was

vigorously stirred at 208C for 12 h. Solids were removed

by ﬁltration and washed carefully with CH₃OH. The

combined ﬁltrates were evaporated. FC (CHCl₃–CH₃OH

9:1) provided 21b (1.1097 g, 69%), alongside with the

4-isomer (21c, 30.7 mg, 2%) and the 3,4-dicarbamoyl

derivative of 21d (179.9 mg, 10%).

3.7.1. Allyl 2-O-[3-(allyloxycarbonyl)-propionyl]-3,4-O-

isopropylidene-a-^D-galactopyranosiduronamide (20b).

To an ice-cold solution of allyl hydrogen succinate

(62.8 mg, 0.39 mmol) in 300:1 CH₂Cl₂–DMF (300 mL)

oxalyl chloride (38 mL, 54.6 mg, 0.43 mmol) was added

dropwise. The mixture was stirred at 08C for 1 h and 2 h at

208C. The solution of the acid chloride was then added to an

ice-cold solution of 20a (100.0 mg, 0.37 mmol) in CH₂Cl₂

(400 mL) and pyridine (700 mL). The mixture was stirred at

08C for 4 h and at 208C for 15 h. Excess acid chloride was

destroyed by addition of 2-propanol (500 mL). After 30 min

at 208C solvents were evaporated. LC (petrol–ethyl acetate

1.2:1!1:2) provided 20b (115.9 mg, 77%) and recovered

3.8.1. Allyl 2-O-[3-(allyloxycarbonyl)-propionyl]-3-O-

carbamoyl-a-^D-galactopyranosiduronamide (21b). IR

(KBr): 3650–3200, 2930, 1740, 1690, 1390, 1330, 1150,

1

1090, 1050, 1030 cm²¹. H NMR (300 MHz, pyridine-d₅,

homo decoupling): d¼4.88 (d, J_4,5¼1.1 Hz, 1H, 5^F-H), 5.48

(broad s, w_1–2<7 Hz, 1H, 4^F-H), 5.52 (d, J_1,2¼3.7 Hz, 1H,

7756

A. Buchynskyy et al. / Tetrahedron 58 (2002) 7741–7760

1^F-H), 5.94 (dd, J_2,3¼10.8 Hz, J_3,4¼3.2 Hz, 1H, 3^F-H), 6.13

7.08, 7.30 (2s, 2H, CONH₂^F), 11.7–12.7 (s, broad, 1H,

COOH^succ). ¹³C NMR (100 MHz, DMSO-d₆, C,H COSY,

APT): d¼67.5 (C-4^F), 68.2 (C-2^F), 69.4 (C-3^F), 71.5 (C-5^F),

95.1 (C-1^F), 156.3 (OCONH₂^F), 170.2, 172.1 (CONH^F₂,

C-1^succ), 173.4 (C-4^succ). C₁₄H₂₀N₂O₁₀, (376.32, 376.11),

FAB MS: m/z¼421.1 [Mþ2Na2H]^þ, 399.1 [MþNa]^þ,

377.1 [MþH]^þ.

(dd, 1H, 2^F-H), 7.60, 7.88, 7.96, 8.48 (4s, CONH^F₂,

1

OCONH^F₂). H NMR (400 MHz, DMSO-d₆, H,H COSY):

d¼4.08 (s, 1H, 5^F-H), 4.26 (dd, J_3,4¼2.9 Hz, J_4,OH¼6.2 Hz,

1H, 4^F-H), 4.91 (dd, J_2,3¼10.9 Hz, 1H, 3^F-H), 5.04 (d,

J_1,2¼3.7 Hz, 1H, 1^F-H), 5.10 (dd, 1H, 2^F-H), 5.46 (d, 1H,

4^F-OH), 6.57 (s, b, 2H, OCONH^F₂), 7.08, 7.31 (2s, 2H,

CONH^F₂). ¹³C NMR (50 MHz, pyridine-d₅, C,H COSY,

APT): d¼69.1 (C-4^F), 69.9 (C-2^F), 71.4 (C-3^F), 73.4 (C-5^F),

96.7 (C-1^F), 157.8 (OCONH^F₂), 172.1, 172.1, 172.8

(CONH^F₂, 2CvO^succ). C₁₇H₂₄N₂O₁₀, (416.38, 416.14),

FAB MS: m/z¼439.0 [MþNa]^þ, 417.0 [MþH]^þ.

3.8.5. Allyl 2-O-[3-(benzyloxycarbonylmethyl-carba-

moyl)-propionyl]-3-O-carbamoyl-a-^D-galactopyrano-

siduronamide (21f). To a solution of 21e (25.3 mg,

0.067 mmol) in DMF (.99.5%, 150 mL) a solution of

1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydro-

chloride (19.5 mg, 0.102 mmol) in bistilled water (22 mL)

and after 15 min a solution of N-hydroxysuccinimide (NHS,

11.9 mg, 0.100 mmol) in DMF (.99.5%, 13.5 mL) were

added. The reaction mixture was stirred at 208C for 20 h.

Then solutions of EDC·HCl (19.5 mg, 0.102 mmol) in

bidistilled water (22 mL) and NHS (11.9 mg, 0.100 mmol)

in DMF (.99.5%, 13.5 mL) were added. Stirring was

continued for another 3 h. The mixture was then cooled to

48C and a solution of benzyl glycinate hydrochloride

(20.4 mg, 0.101 mmol) in bidistilled water (110 mL, and

neutralized with 5% aq NaHCO₃) was added. The reaction

mixture was stirred at 48C for 24 h and then freeze-dried. FC

(CHCl₃–CH₃OH–AcOH 90:7:1) provided pure 21f

(4.7 mg, 13%), a fraction of slightly impure 21f (14.2 mg,

<40%), and 21e contaminated with benzyl glycinate

(16.7 mg). ¹H NMR (200 MHz, DMSO-d₆, homo

decoupling; H,H COSY): d¼3.91 (d, J_NH,CH¼5.9 Hz, 2H,

CO–NH–CH₂–COO–), 4.09 (s, 1H, 5^F-H), 4.28 (s, broad,

w_1–2<6.8 Hz, 1H, 4^F-H), 4.91 (dd, J_2,3¼10.7 Hz,

J_3,4¼2.6 Hz, 1H, 3^F-H), 5.03–5.12 (m, 2H, 1^F-H, 2^F-H),

5.40–5.60 (s, b, 1H, 4^F-OH), 6.61 (s, 2H, OCONH^F₂), 7.12

(s, 1H, CONH^F₂), the second CONH^F₂signal was hidden by

aromatic proton signals, 7.38 (m, 5H, Ar–H), 8.44 (t, 1H,

CO–NH–CH₂–COO). C₂₃H₂₉N₃O₁₁(523.50, 523.18),

FAB MS: m/z¼562.1 [MþK]^þ, 546.1 [MþNa]^þ, 524.1

[MþH]^þ.

3.8.2. Allyl 2-O-[3-(allyloxycarbonyl)-propionyl]-4-O-

carbamoyl-a-^D-galactopyranosiduronamide (21c). IR

(KBr): 3610–3180, 1720, 1710, 1640, 1380, 1320, 1210,

.

1150, 1040, 1020 cm²^{1 1}H NMR (200 MHz, pyridine-

d₅):⁴²d¼4.85 (dd, J_3,4¼3.4 Hz, 1H, 3^F-H), 4.94 (d,

J_4,5¼1.2 Hz, 1H, 5^F-H), 5.52 (d, J_1,2¼3.7 Hz, 1H, 1^F-H),

5.68 (dd, J_2,3¼10.5 Hz, 1H, 2^F-H), 6.50 (dd, 1H, 4^F-H), 7.52

(broad s, OCONH₂^F), 7.77, 8.49 (2s, CONH^F₂). ¹³C NMR

(50 MHz, pyridine-d₅): d¼67.1 (C-3^F), 69.4 (C-1^allyl-1),

71.9 (C-2^F), 72.9 (C-4^F), 73.1 (C-5^F), 96.7 (C-1^F), 158.13

(OCONH^F₂), 170.9, 172.2, 172.9 (CONH^F₂, 2CvO^succ).

C₁₇H₂₄N₂O₁₀, (416.38, 416.14), FAB MS: m/z¼855.3

[2MþNa]^þ, 833.3 [2MþH]^þ, 439.1 [MþNa]^þ, 417.1

[MþH]^þ.

3.8.3. Allyl 2-O-[3-(allyloxycarbonyl)-propionyl]-3,4-di-

O-carbamoyl-a-^D-galactopyranosiduronamide (21d). IR

(KBr): 3600–3140, 1730, 1670, 1380, 1320, 1140, 1060,

1

1030, 990 cm²¹. H NMR (300 MHz, pyridine-d₅, homo

decoupling): d¼4.94 (broad s, 1H, 5^F-H), 5.51 (d,

J_1,2¼3.5 Hz, 1H, 1^F-H), 5.73 (dd, J_2,3¼10.9 Hz, 1H, 2^F-

H), 6.07 (dd, J_3,4¼3.3 Hz, 1H, 3^F-H), 6.72 (s, 1H, 4^F-H),

7.40–7.80 (s, OCONH₂^F), 7.58, 7.65, 8.50 (3s, CONH^F₂,

OCONH^F₂). ¹³C NMR (75 MHz, pyridine-d₅, C,H COSY,

APT): d¼68.7 (C-3^F), 69.3 (C-2^F), 70.1 (C-4^F), 70.8 (C-5^F),

95.9 (C-1^F), 156.9, 157.1 (2OCONH^F₂), 170.0, 171.7, 172.2

(CONH^F₂, 2CvO^succ). C₁₈H₂₅N₃O₁₁(459.41, 459.15), FAB

MS: m/z¼941.2 [2MþNa]^þ, 482.1 [MþNa]^þ, 460.1

[MþH]^þ.

3.9. Coupling of 21e to BSA

To a solution of 21e (9.6 mg, 0.026 mmol) in analytical

grade DMF (60 mL) a solution of 1-ethyl-3-(3-dimethyl-

aminopropyl)-carbodiimide hydrochloride (EDC·HCl,

8.8 mg, 0.046 mmol) in water (15 mL) was added and

after 45 min a solution of N-hydroxysuccinimide (NHS,

4.5 mg, 0.038 mmol) in analytical grade DMF (5 mL). The

reaction mixture was stirred at 208C for 23 h. After this time

again solutions of EDC·HCl (8.8 mg, 0.046 mmol) in water

(15 mL) and after 20 min of NHS (4.5 mg, 0.038 mmol) in

DMF (.5 mL) were added. The mixture was stirred at 208C

for 2 h. Then, at 48C a cooled solution of BSA (5.6 mg/mL

50 mM PBS, pH 7.2, 2.8 mL) was added. The reaction

mixture was stirred at 48C for 12 d. Low-molecular

weight impurities were removed by ultraﬁltration at 28C.

In a second experiment puriﬁcation was performed by

dialysis. After lyophilization 18.8 mg of the conjugate 21g

were obtained. SDS-PAGE indicated the conversion of

BSA. The MALDI TOF MS (external calibration, mean of 8

spectra, each obtained from 100 scans) displayed

m/z¼72,569 [MþH]^þ^141 corresponding to hapten–

carrier ratio 17.

3.8.4. Allyl 2-O-succinyl-3-O-carbamoyl-a-^D-galacto-

pyranosiduronamide (21e). To

a solution of 21b

(219.0 mg, 0.53 mmol) in THF (5.3 mL) freshly prepared

Pd(PPh₃)₄(67.7 mg, 0.06 mmol), dissolved in THF

(5.7 mL), and morpholine (67 mL, 0.78 mmol) were

added. The yellow solution was stirred at 208C for 1 h.

During this time a white solid precipitated. After solvent

evaporation the residue was redissolved in CH₃OH and

Dowex 50 (H^þ) was added. The mixture was stirred. After

ﬁltration the resin was several times rinsed with CH₃OH.

The combined ﬁltrates were evaporated. The residue was

partitioned between CH₂Cl₂and water. The aqueous phase

was several times extracted with CH₂Cl₂and then freeze-

dried to give pure 21e (176.6 mg, 89%). IR (KBr): 3660–

3090, 1730, 1690, 1680, 1590, 1560, 1400, 1380, 1150,

1090, 1060, 1020, 990 cm²¹. ¹H NMR (400 MHz, DMSO-

d₆, H,H COSY): d¼4.08 (s, 1H, 5^F-H), 4.26 (m, 1H, 4^F-H),

4.90 (dd, J_2,3¼10.9 Hz, J_3,4¼3.1 Hz, 1H, 3^F-H), 5.04 (d,

J_1,2¼3.7 Hz, 1H, 1^F-H), 5.10 (dd, 1H, 2^F-H), 5.45 (d,

J_4,OH¼6.3 Hz, 1H, 4^F-OH), 6.56 (s, 2H, OCONH^F₂),

A. Buchynskyy et al. / Tetrahedron 58 (2002) 7741–7760

7757

3.9.1. 2-O-{2-Acetamido-4-O-[2-acetamido-4-O-((5R)-5-

{5-(3-carboxy-propionyl)-2-[4-nitro-3-(2-pyridin-2-

yldithio-ethylcarbamoyl)-phenyl]-2H-[1,2,4]triazol-3-

yl}-a-^L-arabinopyranosyl)-2,6-dideoxy-b-D-glucopy-

ranosyl]-2,6-dideoxy-b-^D-glucopyranosyl}-3-O-carba-

moyl-1-O-{[(R )-2-carboxy-2-((2Z,6E,13E )-3,8,8,14,18-

pentamethyl-11-methylene-nonodeca-2,6,13,17-tetra-

enyloxy)-ethoxy]-hydroxy-phosphoryl}-a-^D-galacto-

pyranuronamide (22a). Compound 22a was prepared as

described for 2a.¹⁸Lyophilization, followed by ultraﬁltra-

tion and FC (CHCl₃–MeOH–H₂O 18:11:2.7) provided 22a

in 65% yield. R_t: 20.1 min (RP-HPLC: ¹H NMR (400 MHz,

D₂O): (characteristic signals): d¼0.94 (s, CH₃-23^I, CH₃-

24^I), 1.22, 1.30 (2£d, J¼5.1, 5.9 Hz, CH₃-6^C, CH₃-6^E),

1.59, 1.60 (2£s, CH₃-20^I, CH₃-21^I), 1.66 (s, CH₃-19^I), 1.73

(s, CH₃-25^I), 1.85–2.15 (m, CH₂-10^I, CH₂-15^I, CH₂-16^I,

CH₂-5^I, CH₂-4^I), 1.98 (s, NHCOCH₃^E), 2.00 (s, NHCOCH^C₃),

2.73 (d, J¼7.3 Hz, CH₂-12^I), 3.10 (t, CH₂-2^Cys), 4.45–4.50

(m, H-5^F), 5.00 (bd, J¼8.8 Hz, 3^F-H), 5.10–5.20 (m, 13^I-H,

17^I-H), 5.30–5.50 (m, 2^I-H, 7^I-H, 6^I-H), 5.70–5.80 (m, 1^F-

H), 7.22–7.32 (m, 1H, 4^py-H), 7.75–7.90 (m, 2H, 3^py-H,

2^py-H), 7.94 (d, 1H, 2^Ar-H), 8.04 (bd, J¼8.8 Hz, 1H, 4^Ar-H),

8.35 (d, J¼4.4 Hz, 1H, 5^py-H), 8.51 (d, J¼8.8 Hz, 1H, 5^Ar-

H). C₇₆H₁₀₇N₁₀O₃₁S₂P (1751.82, 1750.63), FAB MS:

m/z¼1752.1 [MþH]^þ, 1774.3 [MþNa]^þ, 1795.6

[Mþ2Na2H]^þ.

(RP-HPLC). ¹H NMR (400 MHz, D₂O, H,H COSY):

(characteristic signals): d¼0.88 (s, CH₃-23^I, CH₃-24^I),

1.22, 1.30 (2£d, J¼6.5, 6.3 Hz, CH₃-6^C, CH₃-6^E), 1.27–

1.33 (m, 9^I-H), 1.53, 1.55 (2£s, CH₃-20^I, CH₃-21^I), 1.60 (s,

CH₃-19^I), 1.67 (s, CH₃-25^I), 1.82–1.87 (m, CH₂-10^I), 1.93

(s, NHCOCH^E₃), 1.96 (s, NHCOCH₃^C), 2.51–2.57 (m, CH₂-

3^ba), 2.65–2.70 (m, CH₂-6^BTR, CH₂-12^I), 2.89 (dd, J¼12.8,

4.8 Hz, 3a^BTR-H), 4.50 (dd, J¼8.2, 4.9 Hz, 6a^BTR-H), 4.92

(dd, J¼10.4, 3.6 Hz, 3^F-H), 5.07–5.12 (m, 13^I-H, 17^I-H),

5.23–5.38 (m, 2^I-H, 7^I-H, 6^I-H), 5.68–5.72 (m, 1^F-H), 7.94

(bs, 1H, 2^Ar-H), 7.97 (bd, J¼9.4 Hz, 1H, 4^Ar-H), 8.44 (d,

J¼8.6 Hz, 1H, 5^Ar-H). C₉₄H₁₃₇N₁₄O₃₈S₂P (2166.28,

2164.84), FAB MS: m/z¼2189.7 [MþNa]^þ, 2202.7

[MþK]^þ, 2210.8 [Mþ2Na2H]^þ.

0

5.6 Hz, CH₂-6^BTR), 3.08 (t, 2^Lys-H), 4.31 (dd, J¼8.2,

3.9.4. (R )-2-(16-Biotinamido-7,10,13-trioxa-3-azahexa-

decyloxy)-3-({b-^D-galactopyranuronamidosyl-(1!4)-2-

acetamido-2,6-dideoxy-b-^D-glucopyranosyl-(1!4)-[b-D-

glucopyranosyl-(1!6)]-2-acetamido-2-deoxy-b-^D-gluco-

pyranosyl-(1!2)-3-O-carbamoyl-4-C-methyl-a-^D-gluco-

pyranuronamidosyloxy}hydroxy-phosphoryloxy)-pro-

pionic acid (23). To a solution 9a (80 mg, 67 mmol) in

phosphate buffer (pH 7.0, 623 mL, 50 mM) and methanol

(1.250 mL) a solution of 24a (Pierce, 116.5 mg, 268 mmol)

in methanol (625 mL) was added. The pH was readjusted to

7.0 and a solution of NaBH₃CN (8.6 mg, 134 mmol) in

methanol (156 mL) was added. The mixture was stirred at

208C for 48 h and then directly ﬁltered through a Sephadex^w

LH-20 column (elution with H₂O–CH₃OH 1:4). All

fractions containing 23 were combined, the solvents were

evaporated and the residue was adsorbed on kieselguhr

(300 mg). This material was transferred to the top of a FC

column. Elution with FSC (ethyl acetate–i-propanol–H₂O

6:4:2) and subsequent removal of inorganic material via

Sephadex^wLH-20 chromatography (elution with H₂O–

CH₃OH 1:4) provided after lyophilization 26 mg (26%) of

23. ¹H NMR (400 MHz, D₂O, H,H COSY): d¼1.18 (s, CH₃-

4^F), 1.33 (d, J_5–6¼5.9 Hz, CH₃-6^C), 1.30–1.40 (m, CH₂-

4^L), 1.50–1.70 (m, CH₂-3^L, CH₂-5^L), 1.71–178 (m, CH₂-

9^K), 1.95–2.00 (m, CH₂-2^K), 1.99, 2.05 (s, NHCOCH^E₃, s,

NHCOCH^C₃), 2.21 (dd, J_2–3¼7.0 Hz, CH₂-2^L), 2.72–2.75,

2.93–2.97 (m, CH₂-6^M), 3.13 (dd, J_1–2¼7.5 Hz, CH₂-1^K),

3.19–3.23 (m, CH₂-10^K), 3.23–3.27 (m, 2^D-H), 3.52–3.54

(m, CH₂-8^K), 3.58–3.62 (m, CH₂-3^K), 3.60–3.70 (m, CH₂-

4^K-CH₂-7^K), 4.18 (s, H-4^B, H-5^B), 4.36–4.38 (m, 3a^M-H),

4.40 (s, 5^F-H), 4.47 (d, J_1–2¼7.8 Hz, 1^D-H), 4.57–4.58

(m, 6a^M-H), 4.96 (d, J_2–3¼10.8 Hz, 3^F-H), 5.77 (q,

J_1F–2F¼3.4 Hz, J_1F–P¼5.9 Hz, 1^F-H). ¹³C NMR (50 MHz,

D₂O, APT): d¼14.9 (CH₃-4^F), 16.8 (CH₃-6^C), 22.5, 22.6

(NHCOC H^E₃, NHCOC H^C₃), 25.3, 25.7, 27.9, 28.1, 28.4 (C-

2^K, C-9^K, C-3^L, C-4^L, C-5^L), 35.7, 36.5 (C-10^K, C-2^L), 39.9

(C-6^M), 45.4 (C-1^K), 47.5 (C-2^I), 55.3, 55.5, 55.7 (C-2^E,

C-2^C, C-4^M), 60.4 (C-6a^M), 61.0 (þ) (C-6^D), 62.3 (C-3a^M),

65.1 (þ) (C-1^I), 67.1 (C-1^H), 68.1–76.2 (C-5^C, C-4^D, C-4^B,

C-2^B, C-3^B, C-6^E, C-5^B, C-3^E, C-3^C, C-5^F, C-2^F, C-4^F, C-5^E,

C-2^D, C-3^F, C-5^D, C-3^D, C-3^K-C-8^K), 80.0 (C-4^E), 80.9

(C-2^H), 83.2 (C-4^C), 94.6 (C-1^F), 101.4, 102.3, 102.8, 103.4

(C-1^C, C-1^E, C-1^B, C-1^D), 158.3 (OCONH^F₂), 165.5 (C-2^M),

172.9, 173.4, 174.3, 174.7 (CONH^B₂, CONH₂^F, NHCOCH₃^E,

NHCOCH^C₃), 176.6, 176.9 (C-3^H, C-1^L). The signals in the

region 65–80 ppm are broad. ³¹P NMR (81 MHz, D₂O):

d¼2.19. C₆₁H₁₀₄N₉O₃₇PS (1618.57, 1617.59), ESI MS:

3.9.2. 2-O-{2-Acetamido-4-O-[2-acetamido-4-O-((5R)-5-

{5-(3-carboxy-propionyl)-2-[3-(2-mercapto-ethylcarba-

moyl)-4-nitro-phenyl]-2H-[1,2,4]triazol-3-yl}-a-^L-arabi-

nopyranosyl)-2,6-dideoxy -b-^D-glucopyranosyl]-2,6-

dideoxy-b-^D-glucopyranosyl}-3-O-carbamoyl-1-O-

{[(R )-2-carboxy-2-((2Z,6E,13E )-3,8,8,14,18-penta-

methyl-11-methylene-nonodeca-2,6,13,17-tetraenyloxy)-

ethoxy]-hydroxy-phosphoryl}-a-^D-galactopyranurona-

mide (22b). To a solution of dithiothreitol (24.6 mg,

0.16 mmol) in methanol (10 mL) a solution of 22a

(28.0 mg, 16 mmol) in methanol (5 mL) was added slowly.

The mixture was stirred at 208C for 5 h. Progress of the

reaction was controlled by RP-HPLC. Lyophilization

followed by removal of low molecular weight impurities

by ultraﬁltration furnished 22b (21.3 mg, 81%). R_t:

12.2 min

(RP-HPLC).

C₇₁H₁₀₄N₉O₃₁SP

(1642.68,

1641.63), FAB MS: m/z¼1665.6 [MþNa]^þ, 1687.7

[Mþ2Na2H]^þ.

3.9.3. 2-O-[2-Acetamido-4-O-(2-acetamido-4-O-{(5R)-5-

[2-(3-{2-[(RS )-1-(2-{(R )-1-carboxy-5-[5-((3aS )-2-oxo-

(3ar,6ac )-hexahydro-1H-thieno[3,4-d ]imidazol-4t-yl)-

pentanoylamino]-pentylcarbamoyl}-ethyl)-2,5-dioxo-

pyrrolidin-3-ylthio]-ethylcarbamoyl}-4-nitro-phenyl)-5-

(3-carboxy-propionyl)-2H-[1,2,4]triazol-3-yl]-a-^L-ara-

binopyranosyl}-2,6-dideoxy-b-^D-glucopyranosyl)-2,6-

dideoxy-b-^D-glucopyranosyl]-3-O-carbamoyl-1-O-

{[(R )-2-carboxy-2-((2Z,6E,13E )-3,8,8,14,18-penta-

methyl-11-methylene-nonodeca-2,6,13,17-tetraenyloxy)-

ethoxy]-hydroxy-phosphoryl}-a-^D-galactopyranurona-

mide (22c). A solution of 22b (15.0 mg, 9 mmol) and N-(3-

maleimidopropionyl)-biocytin (80% purity, Fluka, 4.6 mg,

7 mmol) in water (10 mL) was stirred 5 h at 208C. The

reaction was followed by RP-HPLC (diode array detector).

Lyophilization, followed by FC (CHCl₃–MeOH–H₂O

18:11:2.7) provided 11.1 mg (56%) of 22c. R_t: 10.3 min

7758

A. Buchynskyy et al. / Tetrahedron 58 (2002) 7741–7760

m/z¼1616.5989 (calcd 1616.5919) [M2H]², 807.7931

3.55 (m, 2H, 4^E-H, 5^E-H), 3.58–3.64 (m, 1H, 3^E-H), 3.71 (t,

(calcd 807.7923) [M22H]²²

.

J_10,11¼5.5 Hz, 2H, CH₂-10^SPA), 3.76–3.83 (m, 5H, CH₂-

7

^SPA, CH₂-8^SPA, 2^E-H), 3.85–3.94 (m, 4H, CH₂-5^SPA

,

3.9.5. 2-O-(2-Acetamido-3,4,6-O-tribenzyl-2-deoxy-b-^D-

glucopyranosyl)-1-O-(2-{2-[2-(2-biotinamidoethoxy)-

ethoxy]ethylamino}ethyl)-3-O-carbamoyl-a-^D-galacto-

pyranuronamide (25a). A solution of 14 (49.8 mg,

133 mmol) in methanol (10 mL) was adjusted to pH 7

with methanolic solution of acetic acid. Solutions of 24b

(Pierce, 50 mg, 66 mmol) in methanol (20 mL) and of

sodium cyanoborohydride (8.35 mg, 133 mmol) in methanol

(0.5 mL) were added successively and the mixture was

stirred at 208C for 18 h (Ar atmosphere). The reaction

mixture was adsorbed to kieselguhr and the material placed

on top of a FC column. Elution with CHCl₃–methanol–

HCO₂H 10:10:0.1, followed by Sephadex LH-20 chroma-

tography (elution with methanol–water 3:1) and lyophiliza-

tion provided 43 mg (58%) of 25a as a white powder.

R_f¼0.12 (CHCl₃–methanol–water 12:6:1). ¹H NMR

(400 MHz, D₂O, H,H COSY): d¼1.14–1.28 (m, 2H, CH₂-

CH H-1^SPA, 6^E-H), 4.00 (d, J¼12.0 Hz, 1H, 6^E-H⁰), 4.11–

4.19 (m, 2H, 2^F-H, CH H-1^SPA), 4.47–4.51 (m, 2H, 4^F-H,

3a^bio-H), 4.55 (d, J_4,5¼1.1 Hz, 1H, 5^F-H), 4.65–4.70 (m,

2H, 1^E-H, 6a^bio-H), 5.12 (dd, J_2,3¼10.6 Hz, J_3,4¼3.2 Hz,

1H, 3^F-H), 5.43 (d, J_1,2¼3.9 Hz, 1H, 1^F-H). ¹³C NMR

(100 MHz, D₂O, APT, HMQC): d¼22.9 (NHCOC H^E₃),

25.8, 28.3, 28.5 (C-3^bio, C-4^bio, C-5^bio), 36.1 (C-2^bio), 39.5

(C-11^SPA), 40.3 (C-6^bio), 47.3, 47.6 (C-2^SPA, C-4^SPA), 56.0

(C-2^E, C-6^bio), 60.9 (C-6^E, C-6a^bio), 62.7 (C-3a^bio), 64.5

(C-1^SPA), 66.3 (C-5^SPA), 68.6 (C-4^F), 69.6 (C-10^SPA), 70.1,

70.3, 70.3 (C-7^SPA, C-8^SPA, C-4^E), 71.1 (C-5^F), 71.3 (C-3^F),

74.5 (C-3^E), 76.1 (C-2^F), 76.2 (C-5^E), 99.2 (C-1^F), 103.6

(C-1^E), 158.4 (OCONH₂), 166.0 (C-2^bio), 173.8, 175.0

(NHCOCH₃, CONH₂), 177.6 (C-1^bio). C₃₃H₅₇N₇O₁₆S

(839.91, 839.36), ESI ICR MS: m/z¼840.3658 (calcd

840.3655) [MþH]^þ, 862.3486 (calcd 862.3475) [MþNa]^þ.

4

^bio), 1.31–1.61 (m, 4H, CH₂-3^bio, CH₂-5^bio), 1.89 (s, 3H,

3.10. Immunization procedure

NHCOCH₃), 2.07 (t, J_2,3¼7.1 Hz, 2H, CH₂-2^bio), 2.56 (d,

J¼13.2 Hz, 1H, CH₂-6^bio), 2.74 (dd, J_6,6a¼4.6 Hz, 1H,

CH₂-6^bio), 3.38–3.84 (m, 15H, 2^E-H, 3^E-H, 4^E-H, 5^E-H,

Rabbits were immunized by injection of moenomycin–

BSA conjugates to raise polyclonal antisera against

moenomycin. Approximately 500 mg of puriﬁed protein

(moenomycin–BSA 8) in phosphate buffered saline (PBS)

was emulsiﬁed with an equal volume of complete Freund’s

adjuvant and injected subcutaneously into the rabbit.

Subsequent injections (500 mg of 8 in PBS) were given in

14–28 d intervals. Last injections contained additionally

incomplete Freund’s adjuvant. Three days later, antiserum

was obtained and screened for anti-moenomycin antibodies.

For further experiments blood sera were incubated for 2 h at

room temperature, centrifuged (3000g, 10 min, 48C),

incubated again (568C, 45 min), and stored at 2708C.

CH₂-5^SPA, CH₂-7^SPA, CH₂-8^SPA, CH₂-10^SPA, CHH-1^SPA

,

CH₂-6^E), 3.93–4.03 (m, 2H, 2^F-H, CHH-1^SPA), 4.17 (m, 1H,

3a^bio-H), 4.30–3.66 (m, 10H, 1^E-H, 4^E-H, 5^E-H, 6a^bio-H,

CH^b₂^enzyl), 5.98 (dd, J_2,3¼10.5 Hz, J_3,4¼3.2 Hz, 1H, 3^F-H),

5.26 (d, J_1,2¼3.4 Hz, 1H, 1^F-H), 6.76–7.44 (m, 15H, Ar–

Hs^benzyl). ¹³C NMR (100 MHz, D₂O, APT, HMQC):

d¼22.8 (NHCOC H₃), 25.7, 28.2, 28.5 (C-3^bio, C-4^bio

,

C-5^bio), 35.9 (C-2^bio), 39.3 (C-11^SPA), 40.2 (C-6^bio), 47.0

(C-2^SPA, C-4^SPA), 55.4 (C-2^E), 55.8 (C-4^bio), 60.6 (C-6a^bio),

62.4 (C-3a^bio), 63.9 (C-1^SPA), 66.3 (C-5^SPA), 68.4 (C-4^F,

C-6^E), 69.4 (C-10^SPA), 70.0, 70.2 (C-7^SPA, C-8^SPA), 71.0,

71.1 (C-5^F, C-3^F), 73.6 (CH^b₂^enzyl), 73.9 (C-4^E), 75.3, 75.7

(2£CH₂^benzyl), 76.3 (C-2^F), 78.0, 82.6 (C-3^E, C-5^E), 99.0

(C-1^F), 103.3 (C-1^E), 128.7–129.4 (Ar–CH^benzyl), 137.5,

137.6, 138.2 (Ar–Cq^benzyl), 158.2 (OCONH₂), 165.6

(C-2^bio), 173.6, 174.2 (NHCOCH₃, C-6^F), 176.7 (C-1^bio).

C₅₄H₇₅N₇O₁₆S (1110.29, 1109.50), FAB MS: m/z¼1110.5

[MþH]^þ, 1124.5 [MþNa]^þ, ESI ICR MS: m/z¼1110.5065

(calcd 1110.5064) [MþH]^þ, 1132.4889 (calcd 1132.4883)

[MþNa]^þ.

3.11. Preparation of moenomycin–KLH

2 mg of 2a and 2 mg of maleimide–activated KLH (keyhole

limpet hemocyanin, Pierce) were mixed with 400 mL of

0.05 M sodium phosphate buffer, pH 7.2, 0.15 M NaCl,

0.1 M EDTA for 2 h. Non reacted maleimide groups were

blocked with cysteine for 1 h at room temperature. The

preparation was dialysed by ultraﬁltration. Quantitation of

coupling efﬁciency was performed by determination of thiol

groups with Ellmann’s reagent⁴³or, alternatively, by HPLC

analysis as described previously.²²

3.9.6. 2-O-(2-Acetamido-2-deoxy-b-^D-glucopyranosyl)-

1-O-(2-{2-[2-(2-biotinamidoethoxy)ethoxy]ethylami-

no}ethyl)-3-O-carbamoyl-a-^D-galactopyranuronamide

(25b). To a solution of 25a (19 mg, 17.1 mmol) in

methanol–acetic acid–water (2:1:0.5, 5 mL) 10% Pd/C

(20 mg) was added and the mixture was stirred under

hydrogen at 358C for 24 h. Filtration through celite and

washing with 2:1 methanol–water, solvent evaporation, FC

(CHCl₃–methanol–water–HCO₂H 9:6:1.4:0.1), Sephadex

LH-20 chromatography (methanol–water 3:1), and lyo-

philization furnished 9.3 mg (64%) of the biotinylated

disaccharide 25b as white solid. R_f¼0.09 (CHCl₃–metha-

nol–water–HCO₂H 9:6:1.4:0.1). ¹H NMR (400 MHz, D₂O,

H,H COSY): d¼1.43–1.55 (m, 2H, CH₂-4^bio), 1.59–1.86

(m, 4H, CH₂-3^bio, CH₂-5^bio), 2.11 (s, 3H, NHCOCH₃), 2.34

(t, J_2,3¼7.2 Hz, 2H, CH₂-2^bio), 2.85 (d, J¼12.7 Hz, 1H,

CH₂-6^bio), 3.06 (dd, J_6,6a¼5.3 Hz, 1H, CH₂-6^bio), 3.37–3.50

(m, 7H, 4^bio-H, CH₂-2^SPA, CH₂-4^SPA, CH₂-11^SPA), 3.51–

3.12. Screening of anti-moenomycin antibodies

Screening of the blood sera was performed in an enzyme-

linked immunosorbent assay (ELISA) and by Western

blotting. Polystyrene microtiter plates (Maxisorp or Poly-

sorp, NUNC) with high binding capacity were coated with

500 ng moenomycin–KLH/mL or 150 nM 8, or 10 mg

streptavidin/mL in 0.05 M sodium carbonat buffer, pH 9.6

and incubated overnight at 48C. Streptavidin-coated

microtiter plates were further incubated with 10 mg of a

the moenomycin A-biotin derivative reported in Ref. 18 in

the same buffer for 1 h. Control ELISA plates were coated

with KLH, BSA and streptavidin, respectively. The plates

were washed with 50 mM Tris–HCl, pH 10.2, 150 mM

NaCl, 0.05% (w/v) Tween 20 (washing buffer A), blocked

A. Buchynskyy et al. / Tetrahedron 58 (2002) 7741–7760

7759

with 1–2% BSA or Tween 20 in washing buffer A, and

washed again. Antisera were added in serial dilutions in

100 mL/well in washing buffer A for 2 h. For screening on 8

coated plates the antisera were preincubed with 2 mg/mL

BSA for neutralization of anti-BSA antibodies. After

washing (3£, washing buffer A) the plates were incubated

with 100 mL/well of pork anti-rabbit antibodies conjugated

with peroxidase (1:2000) for 1 h in washing buffer

A. Colorimetric analysis was performed by addition of

o-phenylene diamine (0.7 mg/mL) and 1% H₂O₂in 50 mM

Na₂HPO₄, pH 5.0, 20 mM citrate. The absorbance was

monitored at a wavelength of 492 nm. Antibodies that

reacted with control wells were considered to be of

undesired speciﬁty.

BC Biochemie, and the Fonds der Chemischen Industrie is

gratefully acknowledged.

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¨

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Synthesis of tools for raising antibodies against moenomycin epitopes and initial immunological studies

DOI: 10.1016/S0040-4020(02)00838-4

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Article abstract of DOI:10.1016/S0040-4020(02)00838-4

Full text of DOI:10.1016/S0040-4020(02)00838-4

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