Base- and sugar-modified cytidine monophosphate N-acetylneuraminic acid (CMP-Neu5Ac) analogues - Synthesis and studies with α(2-6)-sialyltransferase from rat liver

FULL PAPER

Base- and Sugar-Modified Cytidine Monophosphate N-Acetylneuraminic Acid

(CMP-Neu5Ac) Analogues – Synthesis and Studies with α(2–6)-

Sialyltransferase from Rat Liver

Gesche Dufner,^[a]Ralf Schwörer,^[a]Bernd Müller,^[a]and Richard R. Schmidt*^[a]

Keywords: Glycosyl phosphates / Nucleosidephosphate sugars / Neuraminic acid / Glycosyltransferase / Carbohydrates

The reaction of sialyl phosphites 1, 22a–d, 28, 39, and 45 with

acyl-protected riboside 5-phosphorous acids 2a,b and 23 dir-

ectly furnished, without addition of a catalyst, under phos-

phite/phosphate exchange the corresponding β-configured

sialyl riboside monophosphates 3a,b, 24a–d, 29, 46, and 47.

The synthesis of the starting materials, formation of the prod-

anol and subsequent hydrolysis of the sialic acid ester moiety

to provide the unprotected target molecules 4a,b, 25a–d, 30,

48, and 49 is described. Investigations with α(2–6)-sialyl-

transferase from rat liver showed that base replacement in

CMP-Neu5Ac (4a,b) is not tolerated by the enzyme but that

modifications of the 5-, 8-, or 9-position of the neuraminic

ucts, and their treatment with sodium methanolate in meth- acid residue (25a–d, 30, 48, 49) are tolerated.

Introduction

Glycoside bond formation in nature is essentially based

on glycosyl donors having phosphates, pyrophosphates, or

their ribonucleoside and lipid monoester derivatives as leav-

ing groups, and glycosyltransferases as catalysts.^[1–3]For

instance, for aldoses and 3-deoxy-2-glyculosonates (Kdo,

Neu5Ac), nucleoside diphosphate sugars and nucleoside

monophosphate derivatives, respectively, are generally enco-

untered as glycosyl donors (Leloir pathway). The different

sugars are often linked to different nucleoside moieties as

part of the glycosyl donor leaving group. Therefore, glycos-

yltransferases seem to specifically recognize the sugar and

the nucleoside moieties, and generate glycosyl donor prop-

Scheme 2

A particularly gratifying property of O-glycosyl trichlo-

erties through activation of the phosphate residue. These roacetimidates as glycosyl donors D [Scheme 2, –OX ϭ

factors can be studied with the help of nucleoside phos- –OC(CCl₃)ϭNH] is their direct reaction with Brønsted ac-

phate sugars and especially with analogues that are often ids (HA) as glycosyl acceptors A under formation of glycos-

not accessible by enzymatic means.^[3–6]

ylation products P with trichloroacetamide [OϭXH ϭ Oϭ

A particularly interesting target is natural CMP-Neu5Ac C(CCl₃)NH₂]^[10]as the leaving group L. This reaction was

(Scheme 1) and base- and sugar-modified analogues successfully employed for the synthesis of nucleoside mono-

thereof, because CMP-Neu5Ac is a glycosyl donor for the and diphosphate sugars of aldoses.^[3,5,11]Similar reaction

enzymatic sialyltransfer to glycoconjugates, i.e. it is required behaviour can clearly be envisaged for glycosyl phosphites

for the biosynthesis of gangliosides and sialylated glycopro- as glycosyl donors D [–OX ϭ OP(OR)₂]. Such a process

teins. Thus, various biological processes are affected by this would provide, with HA as acceptor A, the product P upon

glycosyl donor, for which successful chemical and enzym- release of a phosphonate group [OϭXH ϭ OϭPH(OR)₂].

atic syntheses have been reported.^[4,6–9]

This direct phosphite/phosphate exchange reaction, starting

Scheme 1

from the diethyl phosphite derivative 1 of Neu5Ac and nuc-

leoside phosphorous acids, gave not only CMP-Neu5Ac

but also the corresponding nucleoside analogues UMP-

Neu5Ac, AMP-Neu5Ac, and GMP-Neu5Ac in our laborat-

[a]

Fakultät Chemie, Universität Konstanz,

Fach M 725, 78457 Konstanz, Germany

Eur. J. Org. Chem. 2000, 1467Ϫ1482

WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000

1434Ϫ193X/00/0408Ϫ1467 $ 17.50ϩ.50/0

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G. Dufner, R. Schwörer, B. Müller, R. R. Schmidt

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ory.^[6,7]Additionally, the N-glycolyl derivative CMP-

The required methyl 5-phosphoribofuranoside 2a was

Neu5Gc and new types of glycophospholipids could be pre- prepared from known methyl ribofuranoside^[15](Scheme 4)

pared using this reaction pathway.^[7]We have also employed by phosphitylation of this compound with dibenzyloxydi-

this methodology in the synthesis of various base- and isopropylaminophosphane, in the presence of tetrazole as a

sugar-modified analogues of CMP-Neu5Ac.^[12]These re- catalyst, and subsequent oxidation with tert-butylhydroper-

sults, along with the glycosyl donor properties of glycosyla- oxide to give the phosphorous triester derivative 5. Hydro-

tions with α(2–6)-sialyltransferase from rat liver,^[13]are re- genolytic O-debenzylation followed by triethylamine addi-

ported in this paper.

tion furnished triethylammonium salt 6, which upon ion

exchange led to the corresponding phosphorous acid mono-

ester 2a.

Results and Discussion

Investigations with the recently prepared nucleoside-

monophosphate Neu5Ac derivatives in sialyltransferase-ca-

talyzed reactions have shown that some base variations in

the glycosyl donor are tolerated.^[14]Therefore, more dra-

matic changes of the base moiety were envisaged in order

to ascertain whether the base was required at all or if a

structurally related compound could replace the cytosine

residue. To this end, it was planned to replace the cytosine

residue by a methoxy group and also by the structurally

related resorcinol moiety. The 5-phospho-ribofuranosides

2a,b (Scheme 3) were therefore required in which the hy-

droxy groups of the sugar and the resorcinol moieties were

protected by O-acetyl groups. On using these compounds

the phosphite/phosphate exchange reactions leading to in-

termediates 3a,b can be performed and, after deprotection,

the desired target molecules 4a,b would be readily

available.

Scheme 4

For the synthesis of 2b, ribofuranosylation of O-benzyl-

protected resorcinol with the known compound O-(2,3,5-

tri-O-benzylribofuranosyl)trichloroacetimidate (7)^[16]as a

donor was envisaged (Scheme 5). On using ZnCl₂–diethyl

ether complex as a catalyst, a 7:1 β/α mixture of C-glycoside

8 was obtained. However, hydrogenolytic O-debenzylation

in MeOH with palladium on carbon as a catalyst led not

only to O-debenzylation but also to cleavage of the benzylic

C–O bond of the tetrahydrofuran ring, thus furnishing 4-

tetrahydroxypentylresorcinol derivative 9, which was struc-

turally assigned as its O-acetyl derivative 9A. In order to

circumvent this problem a different strategy was followed.

The known tetra-O-benzyl--ribose 10^[17]was treated with

2,4-dibenzyloxyphenyllithium to afford the addition prod-

uct 11 as a 9:1 mixture of diastereoisomers; acetylation with

acetic anhydride in pyridine gave O-acetyl derivative 11A.

However, hydrogenolysis of 11 under the aforementioned

conditions gave undesired product 9. However, when the

same reaction was carried out in the presence of barium

carbonate, the completely O-debenzylated target compound

(12) was obtained and was transformed into per-O-acetyl

derivative 12A. Treatment of 12 with acetic acid in meth-

anol and acetic anhydride/pyridine led to the β--ribofur-

anoside, which was accompanied by some β--ribopyrano-

side. The desired product was isolated as per-O-acetyl deriv-

ative 13. Treatment of the O-unprotected intermediate with

tert-butyldimethylsilyl (TBDMS) chloride in the presence of

imidazole led to regioselective silylation of the primary hy-

droxy group of the furanose moiety. Subsequent O-

acetylation led to clean separation of the 5Ј-O-TBDMS de-

rivative 14 from the pyranosidic material. Treatment of 14

with aqueous acetic acid furnished 5Ј-O-unprotected C-ri-

bofuranosylresorcinol 15. With the help of the procedure

described above, 15 was readily transformed into phosphor-

Scheme 3

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Eur. J. Org. Chem. 2000, 1467Ϫ1482

Base- and Sugar-Modified CMP-Neu5Ac Analogues

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ous triester 16. Subsequent hydrogenolysis afforded phos- by sialyltransferases. Hence, our subsequent studies were

phorous acid derivative 2b.

directed to variations of the sugar moiety.

Table 1. Investigation of the substrate character of the CMP-

Neu5Ac analogues

K_M[µ]

4a

No substrate

Substrate^[a]

50 ± 10

4d

25a

25b

25c

Substrate^[a]

31 ± 5

25d

30

41 ± 5

48

Substrate^[a]

46 ± 5

49

CMP-Neu5Ac

[a]

Sialyltransfer was very slow and therefore the reaction was not

quantified.

Replacement of the sugar N-acetyl group seemed to be a

particularly promising approach given that the replacement

of the N-acetyl group of CMP-Neu5Ac by other N-acyl

groups is also found in nature.^[7,8]To this end, known

benzyl sialoside 17^[19]was transformed into N,O-deacylated

derivative 18 (Scheme 6). Attachment of an N-trifluoroace-

tyl group with ethyl trifluorothioacetate in the presence of

triethylamine, methyl ester formation with diazomethane,

and then O-acetylation led to known compound 20a.^[20]

Hydrogenolytic O-debenzylation afforded 2-O-unprotected

sialic acid ester 21a. An alternative procedure for N-de-

acetylation is based on treatment of 17 with trifluorome-

thanesulfonic acid anhydride (Tf₂O) in the presence of 2,6-

di-tert-butylpyridine (DTP) followed by addition of ethanol

in the presence of DTP to afford an O-ethyl acetimidate at

the 5-position. This compound gave, upon alcoholysis with

trifluoroacetic acid/methanol and ion exchange, 5-ammo-

nium derivative 19. Reaction of 19 with butyric acid and

trifluoroacetylamino acetic acid, along with water-soluble

carbodiimide (WSC) as a condensing agent, afforded N-

acyl derivatives 20b and 20c, respectively. Reaction of 19

with ethoxycarbonyl chloride in the presence of pyridine

furnished the urethane derivative 20d. Hydrogenolysis of

20b–d gave 21b–d. N-Modified neuraminic acid derivatives

21a–d were then treated with chlorodiethoxyphosphane in

Scheme 5

Both compounds 2a and 2b reacted directly with sialyl the presence of Hünig’s base to afford the β-phosphites

1

donor 1 in DMF/MeCN (2:1) as solvent to afford the de- 22a–d, as evidenced by the H-NMR data obtained for 3-

sired β-linked N-acetylneuraminyl phosphates 3a and 3b, H, 4-H, and 7-H.^[21,22]Reaction of 22a–d with known N,O-

respectively (Scheme 3). De-O-acetylation with NaOMe in acetyl-protected CMP derivative 23^[3]furnished, by phos-

methanol (Zemplen conditions^[18]) followed by methyl ester phite/phosphate exchange, the corresponding β-linked

´

saponification with NaOH in water afforded target molec- CMP-Neu5Ac analogues 24a–d. Removal of all protective

ules 4a and 4b. Their structures could be assigned by NMR groups under the conditions described above gave the 5"-

(¹H, ³¹P) and mass spectrometry. For the β-sialyl phos- amino-(25a), the 5"-N-pentanoylamino (25b), the 5"-glycyl-

phates a J_3a,Pcoupling constant of 5–6 Hz is character- amino (25c), and the 5"-ethoxycarbonylamino derivative

istic,^[7]and such values were found for 3a,b and 4a,b. Both (25d) (¹H NMR, J_3a,Pϭ 5.8 Hz).^[21,22]All compounds were

final products, 4a and 4b, showed no substrate character accepted as substrates by α(2–6)-sialyltransferase (Table 1).

in assays with α(2–6)-sialyltransferase from rat liver and p- Compounds 25b and 25d exhibited similar affinity to the

nitrophenyl N-acetyllactosamine as an acceptor^[13](see enzyme as CMP-Neu5Ac. Sialyltransfer with 25a and am-

Table 1). Therefore, it was concluded that only moderate ino acid derivative 25c was very slow and therefore the reac-

variations of the base moiety in CMP-Neu5Ac are tolerated tion was not quantified.

Eur. J. Org. Chem. 2000, 1467Ϫ1482

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G. Dufner, R. Schwörer, B. Müller, R. R. Schmidt

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Scheme 6

A further interesting example of 5"-modification of phane in the presence of Hünig’s base to give phosphite 28.

CMP-Neu5Ac is the replacement of the Neu5Ac residue by Reaction of 28 with 23 as described above afforded phos-

3-deoxy-2-nonulosonate (Kdn), a residue that lacks the 5"- phate 29, which on deprotection furnished CMP-Kdn 30

N-acetylamino functionality but has a hydroxy group in- (¹H NMR: J_3a,Pϭ 5.5 Hz). This compound exhibited a

stead (Scheme 7). Under standard conditions the known similar affinity to α(2–6)-sialyltransferase as the natural

Kdn methyl ester 26^[23]was transformed into 2-O-unprotec- substrate. Similar results have been reported only re-

ted derivative 27, which reacted with chlorodiethoxyphos- cently.^[24]These results demonstrate the tolerance of sialyl-

transferases to modifications of CMP-Neu5Ac at the 5"-po-

sition.

The tolerance of sialyltransferases to side-chain modifica-

tions was studied with two naturally occurring sialic acids,

namely the 8-O-methyl and the 9-O-phosphoryl derivatives

of Neu5Ac. To this end, compound 17^[19]was transformed

into known 8,9-O-isopropylidene derivative 31^[25]

(Scheme 8), which on regioselective 4-O-acetylation (Ǟ 32)

and then acid-catalyzed de-O-isopropylidenation led to

7,8,9-O-unprotected derivative 33. Benzylidenation with

benzaldehyde dimethylacetal, in the presence of p-tolu-

enesulfonic acid (pTsOH) as a catalyst, afforded mainly 7,9-

O-benzylidene derivative 34. 8-O-Methylation with methyl

iodide/silver oxide gave 35. Subsequent acid-catalyzed de-

benzylidenation (Ǟ 36), O-acetylation (Ǟ 37), and then hy-

drogenolysis under standard conditions gave 2-O-unprotec-

ted sialic acid derivative 38. Reaction with chlorodiethoxy-

phosphane, as described above, afforded the desired β-phos-

phite 39.

A modified synthetic strategy was required for the syn-

thesis of the 9"-O-phosphoryl derivative of CMP-Neu5Ac

Scheme 7

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because two differently linked phosphate residues are pre-

Eur. J. Org. Chem. 2000, 1467Ϫ1482

Base- and Sugar-Modified CMP-Neu5Ac Analogues

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Scheme 9

Scheme 8

sent in the final product. In order to cope with this situ-

ation, known allyl Neu5Ac derivative 40^[26](Scheme 9) was

selected as the starting material. Treatment of 40 with dime-

thoxytrityl chloride (DMTr-Cl) in pyridine in the presence

of 4-dimethylaminopyridine (DMAP, Steglich reagent) af-

forded the 9-O-DMTr derivative, which gave compound 41

upon O-acetylation. Acid-catalyzed removal of the DMTr

group led to 9-O-unprotected 42; immediate phosphoryl-

ation with dibenzyloxydiisopropylaminophosphane in the

presence of tetrazole as a catalyst, followed by oxidation

with tert-butylhydroperoxide afforded dibenzyl phosphate

43. The 2-O-allyl group was then removed with (1,5-cyclo-

octadiene)bis(methyldiphenylphosphane)iridium(I) hexa-

fluorophosphate^[27]as catalyst; subsequent cleavage of the

generated enol ether with iodine in THF/water furnished 2-

O-unprotected compound 44, which was transformed into

diethyl phosphite 45 as described above.

Both compounds 39 and 45 reacted with 23, thus re-

sulting in the desired phosphite/phosphate exchange reac-

tion to afford β-linked sialyl phosphate derivatives 46 and

47, respectively (Scheme 10). Deprotection of 46 was car-

ried out as described above with lithium hydroxide in the

saponification step, thus leading to CMP-8"-O-methyl-

Neu5Ac 48 as the dilithio salt. In the case of 47 hydro-

genolytic removal of the O-benzyl groups of the 9"-O-phos-

phoryl residue had to be performed first. The procedure

described above for de-O-acylation and methyl ester saponi-

Scheme 10

fication was then applied to give CMP-9"-O-phosphoryl- reactions. Thus, even N-deacetylated CMP-neuraminate

Neu5Ac 49. Compounds 48 and 49 were both substrates (25a) and CMP-9"-O-phosphoryl-Neu5Ac (49) could be

for α(2–6)-sialyltransferase from rat liver (Table 1), al- obtained. In the assay with α(2–6)-sialyltransferase from rat

though once again the transfer rate to the acceptor sub- liver, modification of the base residue turned out to be crit-

strate is low.

ical. However, modifications at the 5"-amino group of the

In conclusion, modified CMP-Neu5Ac derivatives are sialyl residue and modifications of the side chain of the sia-

readily accessible through phosphite/phosphate exchange lyl residue seem to be less critical; even the presence of

Eur. J. Org. Chem. 2000, 1467Ϫ1482

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G. Dufner, R. Schwörer, B. Müller, R. R. Schmidt

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(800 µL) was added by syringe. The reaction mixture was allowed

to warm up to room temperature during 12 h. Triethylamine (500

µL) was added and the solvents were evaporated under reduced

pressure. Flash chromatography (chloroform/methanol/triethylam-

ine, 8:1:0.1) of the residue afforded a colourless solid (143 mg,

52%). To a solution of this solid (100 mg, 0.111 mmol) in dry meth-

anol (3 mL) was added sodium methoxide in methanol (0.5 , 3

drops) and the solution was stirred at room temp. and the reaction

monitored by TLC. After completion of the reaction, the mixture

was neutralized with Amberlite IRC-50 (H^ϩ), (pH ϭ 8–9), filtered,

and concentrated. Flash chromatography through aminophase sil-

ica gel (ethanol/water, 4:1) yielded 3a (51 mg, 81%). – TLC, amino-

phase (ethanol/water, 3:7): R_fϭ 0.75. – ¹H NMR (250 MHz, D₂O):

charged groups in the sialyl residue of the modified glycosyl

donor substrate is tolerated by the enzyme.

Experimental Section

General: Solvents were purified according to standard procedures;

boiling range of petroleum ether: 35–60 °C. – Melting points are

uncorrected. – Optical rotations: Perkin–Elmer Polarimeter 241

MC; 1-dm cell, temperature 21 °C. – Thin-layer chromatography:

silica gel plastic plates 60 F₂₅₄(Merck); HPTLC glass plates NH₂

(Merck, layer thickness 0.2 mm); plastic plates Cellulose F (Merck,

layer thickness 0.2 mm). – Flash chromatography: Silica gel (J. T.

Baker, particle size 40 µm). – MPLC was performed at a pressure

of 5–10 bar; column: silica gel LiChroprep Si 60 (Merck, 15–25 µm,

28 ϫ 2.5 cm). – Preparative HPLC was performed with an Auto-

chrom system with a Shimadzu LC8A preparative pump and a Rainin

Dynamax UV 1 detector at 254 nm; column: LiChrosorb RP-18

(Knauer, 7 µm, 250 ϫ 16 mm); mobile phase: 0.0375 or 0.1  tri-

ethylammonium bicarbonate (TEAB) (pH ϭ 7.2–7.5); flow: 9 mL/

min. – Analytical HPLC was performed with a Merck–Hitachi sys-

tem with an L7200 autosampler and an L4000 UV detector; col-

umn: Eurospher 100-C18 (Knauer GmbH, 5 µm, 250 ϫ 4 mm);

flow: 0.8 mL/min. – Preparative paper chromatography: cellulose

δ ϭ 1.59 (ddd, J_3ЈЈa,Pϭ 5.2 Hz, J_3ЈЈa,4ЈЈϭ 11.0 Hz, J_{3ЈЈe,3ЈЈa}

13.2 Hz, 1 H, 3ЈЈa-H), 1.86 (s, 3 H, NCOCH₃), 2.33 (dd, J_3ЈЈe,4ЈЈ

ϭ

4.6 Hz, J_{3ЈЈe,3ЈЈa}ϭ 13.2 Hz, 1 H, 3ЈЈe-H), 3.24 (s, 1 H, OCH₃), 3.30

(d, J ϭ 9.7 Hz, 1 H, 7ЈЈ-H), 3.42 (dd, J_8ЈЈ,9ЈЈBϭ 6.6 Hz, J_{9ЈЈA,9ЈЈB}

11.8 Hz, 1 H, 9ЈЈB-H), 3.61–4.03 (m, 12 H, 2Ј-H, 4Ј-H, 5ЈA-H, 5ЈB-

H, 4ЈЈ-H, 5ЈЈ-H, 6ЈЈ-H, 8ЈЈ-H, 9ЈЈA-H, COOCH₃), 4.09 (dd, J_2Ј,3Ј

ϭ

4.7 Hz, J_4Ј,3Јϭ 6.5 Hz, 1 H, 3Ј-H), 4.74 (d, J_2Ј,1ЈϽ 1 Hz, 1 H, 1Ј-

H). – ³¹P NMR (161.7 MHz, D₂O): δ_Pϭ –1.28.

Triethylammonium [(1,3-Di-O-acetylresorcin-4-yl) (2,3-Di-O-acetyl-

1-deoxy-β-D-ribofuranosid-5-yl)] {[Methyl (5-Acetamido-4,7,8,9-

tetra-O-acetyl-3,5-dideoxy-

D

-glycero-β- -galacto-2-nonulopyrano-

D

1

paper (Schleicher Schuell, 2043 Bnyl 500 ϫ 600 nm). – H NMR:

syl)onate]} Phosphate (3b): A solution of 2b (100 mg, 0.204 mmol)

in N,N-dimethylformamide/acetonitrile (2:1, 0.5 mL) was cooled

to –20 °C. A solution of 1^[21](200 mg, 0.327 mmol) in dry aceto-

nitrile (400 µL) was added by syringe. The reaction mixture was

allowed to warm up to room temp. during 12 h. Triethylamine (300

µL) was added and the solvents were evaporated in vacuo. Flash

chromatography (chloroform/methanol/triethylamine, 9:1:0.1) of

the residue yielded 3b (110 mg, 50%) as a colourless solid. – ¹H

NMR (250 MHz, MeOD): δ ϭ1.21 (t, J ϭ 7.2 Hz, 9 H, 3 ϫ CH₃),

1.87 (s, 3 H, NCOCH₃), 1.99–2.35 (8 ϫ s, 25 H, 3ЈЈa-H, 8 ϫ

COCH₃), 2.78 (dd, J_3ЈЈe,4ЈЈϭ 4.8 Hz, J_{3ЈЈe,3ЈЈa}ϭ 13.2 Hz, 1 H, 3ЈЈe-

H), 3.13 (q, J ϭ 7.2 Hz, 6 H, 3 ϫ CH₂), 3.84 (s, 3 H, COOCH₃),

4.06 (dd, J_5ЈЈ,6ЈЈϭ J_4ЈЈ,5ЈЈϭ 10.5 Hz, 1 H, 5ЈЈ-H), 4.15–4.39 (m, 4

H, 4Ј-H, 5ЈA-H, 5ЈB-H, 9ЈЈA-H), 4.50 (dd, J_6ЈЈ,7ЈЈϭ 2.2 Hz,

J_5ЈЈ,6ЈЈϭ 10.6 Hz, 1 H, 6ЈЈ-H), 4.63 (dd, J_8ЈЈ,9ЈЈBϭ 2.5 Hz,

J_{9ЈЈA,9ЈЈB}ϭ 12.2 Hz, 1 H, 9ЈЈB-H), 5.19–5.41 (m, 4 H, 1Ј-H, 2Ј-H,

Bruker AC 250 (250 MHz) Cryospec, Bruker DRX 600 (600 MHz);

the resonance of the deuterated solvent was used as internal stand-

ard. – ³¹P NMR: Jeol JNM-GX 400; external standard 85% phos-

phoric acid. – FAB MS: Varian MAT 112. – MALDI MS: Kratos

Kompact Maldi 1. – Elemental analyses: Heraeus CHN-O-Rapid.

(Methyl 2,3-Di-O-acetyl-β-D-ribofuranosid-5-yl) Dihydrogen Phos-

phate (2a): A solution of compound 6 in water/methanol (1:1,

3 mL) was eluted from a column of Dowex 50 (H^ϩ) with water/

methanol (1:1, 20 mL). The eluate was lyophilized to yield 2a

(130 mg, 85%) as a colourless solid. – TLC (chloroform/methanol,

3:1): R_fϭ 0.11, [α]_Dϭ –11.2 (c ϭ 1.0, methanol). – ¹H NMR

(250 MHz, CD₃OD): δ ϭ 2.07 (s, 3 H, COCH₃), 2.13 (s, 3 H,

COCH₃), 3.43 (s, 3 H, OCH₃), 4.03–4.19 (m, 2 H, 5a-H, 5b-H),

3

4.29–4.36 (m, 1 H, 4-H), 4.96 (d, J_1,2Ͻ 1.0 Hz, 1 H, 1-H), 5.21

3

(dd, J_3,2ϭ 4.9 Hz, J_1,2Ͻ 1.0 Hz, 1 H, 2-H), 5.33 (dd, J_3,2

ϭ

4ЈЈ-H, 8ЈЈ-H), 5.42–5.52 (m, 2 H, 3Ј-H, 7ЈЈ-H), 6.98 (d, J_5,3

ϭ

4.9 Hz, J_3,4ϭ 6.7 Hz, 1 H, 3-H). – ³¹P NMR (161.7 MHz,

3

2.2 Hz, 1 H, 2-H), 7.14 (dd, J_6,5ϭ 8.5 Hz, J_2,6ϭ 2.2 Hz, 1 H, 6-

H), 7.85 (d, J_6,5ϭ 8.5 Hz, 1 H, 5-H). – DC (chloroform/methanol,

3:1): R_fϭ 0.34. – ³¹P NMR (242.5 MHz, D₂O): δ_Pϭ –3.39. – FAB

MS (negative Mode, matrix: methanol/nitrobenzyl alcohol, 1:1):

962 (M – H^ϩ)^–.

CD₃OD): δ_Pϭ 0.43.

[(1,3-Di-O-acetylresorcin-4-yl) (2,3-Di-O-acetyl-1-deoxy-β-D-ribofu-

ranosid-5-yl)] Dihydrogen Phosphate (2b): To a solution of 16

(300 mg, 0.447 mmol) in methanol/ethyl acetate (1:1, 50 mL) was

added palladium on charcoal (10%, 15 mg) and the mixture was

stirred at room temp. for 15 min under hydrogen (1 bar). After

filtration through Celite, the filtrate was concentrated under re-

duced pressure. The residue was lyophilized from water to yield 2b

(210 mg, 95%) as a colourless solid. – TLC (N-butyl alcohol/acetic

acid/water, 3:1:1): R_fϭ 0.27. – ¹H NMR (250 MHz, D₂O): δ ϭ

2.06–2.31 (4 ϫ s, 12 H, 4 ϫ COCH₃), 4.21–4.35 (m, 3 H, 4Ј-H,

Disodium [(5-Acetamido-3,5-dideoxy-d-glycero-β-d-galacto-2-non-

ulopyranosyl)onate] (Methyl-β- -ribofuranosid-5-yl) Phosphate (4a):

d

To a solution of 3a (50 mg, 87.5 µmol) in water/methanol (1:1,

1 mL) was added NaOH solution (1 , 1 mL) and the mixture was

stirred for 4 h at room temp. The solution was neutralized with

Amberlite IRC-50 (H^ϩ) (pH ϭ 8–9) and filtered. The filtrate was

lyophilized, dissolved in water and, after addition of acetone, 4a

(39 mg, 79%) precipitated as a colourless solid. – TLC, aminophase

5ЈA-H, 5ЈB-H), 5.12–5.22 (m, 2 H, 2Ј-H, 1Ј-H), 5.40 (dd, J_2Ј,3Ј

ϭ

J_4Ј,3Јϭ 4.8 Hz, 1 H, 3Ј-H), 6.91 (d, J_6,2ϭ 2.3 Hz, 1 H, 2-H), 6.99

(dd, J_6,5ϭ 8.5 Hz, J_2,6ϭ 2.3 Hz, 1 H, 6-H), 7.85 (d, J_6,5ϭ 8.5 Hz,

1 H, 5-H). – ³¹P NMR (161.7 MHz, D₂O): δ_Pϭ 3.89.

1

(ethanol/water, 1:1): R_fϭ 0.37. – H NMR (250 MHz, D₂O): δ ϭ

1.43 (ddd, J_3ЈЈa,Pϭ 6.0 Hz, J_3ЈЈa,4ЈЈϭ 11.2 Hz, J_{3ЈЈe,3ЈЈa}ϭ 12.9 Hz,

1 H, 3ЈЈa-H), 1.86 (s, 3 H, NCOCH₃), 2.29 (dd, J_3ЈЈe,4ЈЈϭ 4.7 Hz,

Sodium [(Methyl (5-Acetamido-3,5-dideoxy-

2-nonulopyranosyl)onate] (Methyl 2,3-Di-O-acetyl-β-

sid-5-yl) Phosphate (3a): A solution of 2a (100 mg, 0.305 mmol) in 3.81 (m, 3 H, 5ЈЈ-H, 6ЈЈ-H, 9ЈЈA-H), 4.82–3.98 (m, 7 H, 2Ј-H, 4Ј-

N,N-dimethylformamide/acetonitrile (2:1, 1.0 mL) was cooled to – H, 5ЈA-H, 5ЈB-H, 4ЈЈ-H, 8ЈЈ-H, 9ЈЈA-H), 4.11 (dd, J_2Ј,3Јϭ 4.9 Hz,

20 °C. A solution of 1^[21](240 mg, 0.392 mmol) in dry acetonitrile J_4Ј,3Јϭ 6.1 Hz, 1 H, 3Ј-H), 4.71 (d, J

Ͻ 1 Hz, 1 H, 1Ј-H). –

D-glycero-β-D-galacto-

J_{3ЈЈe,3ЈЈa}ϭ 13.0 Hz, 1 H, 3ЈЈe-H), 3.16–3.27 (m, 4 H, OCH₃, 7ЈЈ-H),

D-ribofurano-

3.42 (dd, J_8ЈЈ,9ЈЈBϭ 6.8 Hz, J_{9ЈЈA,9ЈЈB}ϭ 12.2 Hz, 1 H, 9ЈЈB-H), 3.67–

2Ј,1Ј

1472

Eur. J. Org. Chem. 2000, 1467Ϫ1482

Base- and Sugar-Modified CMP-Neu5Ac Analogues

FULL PAPER

³¹P NMR (161.7 MHz, D₂O): δ_Pϭ –3.83. – FAB MS (negative H), 4.87 (d, J_1.2Ͻ 1.0 Hz, 1 H, 1-H), 5.23 (dd, J_3.2ϭ 4.9 Hz, J_1.2

mode, matrix: glycerine/water, 1:1): 534 (M – H)^–.

Ͻ 1.0 Hz, 1 H, 2-H), 5.32 (dd, J_3.2ϭ 4.9 Hz, J_3.4ϭ 6.3 Hz, 1 H,

3-H).

Bis(triethylammonium) [(5-Acetamido-3,5-dideoxy-

D

-glycero-β-

D-

galacto-2-nonulopyranosyl)onate] [(Resorcin-4-yl) (1-Deoxy-β-

D-ri-

1,3-Di-O-benzyl-4-(2,3,5-tri-O-benzyl-α/β-D-ribofuranosyl)resorcinol

bofuranosid-5-yl)] Phosphate (4b): To a solution of 3b (55 mg, 51.7

µmol) in dry methanol (2 mL) was added sodium methoxide in

methanol (0.5 , 3 drops) and the mixture was stirred for 3 h at

room temp. The solution was neutralized with Amberlite IRC-50

(H^ϩ) (pH ϭ 8–9), filtered, concentrated under reduced pressure

and lyophilized from water. To a solution of the residue in water/

methanol (1:1, 2 mL) was added NaOH solution (1 , 1 mL) and

the mixture was stirred for 4 h at room temp. The solution was

neutralized with Amberlite IRC-50 (H^ϩ) (pH ϭ 8–9) and filtered.

Lyophilization and HPLC gave 4b (15 mg, 35%). HPLC: prep. RP-

18 column (flow: 9 mL min^–1, 0.05  triethylammonium bicarbon-

ate buffer). – TLC, aminophase (ethanol/water, 3:7): R_fϭ 0.75. –

¹H NMR (250 MHz, D₂O): δ ϭ 1.10 [t, J ϭ 7.1 Hz, 18 H, 2 ϫ

(CH₃)₃], 1.39–1.53 (m, 1 H, 3ЈЈa-H), 1.84 (s, 3 H, NHCOCH₃), 2.30

(dd, J_3ЈЈe,4ЈЈϭ 4.2 Hz, J_{3ЈЈe,3ЈЈa}ϭ 13.0 Hz, 1 H, 3ЈЈe-H), 2.81–2.93

(m, 1 H, 9ЈЈB-H), 2.96–3.09 [m, 12 H, 2 ϫ (CH₂)₃], 3.25 (d, J ϭ

9.7 Hz, 1 H, 7ЈЈ-H), 5.52–6.03 (m, 2 H, 5ЈA-H, 9ЈЈA-H), 6.04–6.11

(m, 1 H, 5ЈB-H), 6.18–6.36 (m, 2 H, 5ЈЈ-H, 6ЈЈ-H), 3.88 (dd,

J_3ЈЈe,4ЈЈϭ 4.3 Hz, J_3ЈЈa,4ЈЈϭ 11.5 Hz, 1 H, 4ЈЈ-H), 3.91–4.06 (m, 2

H, 4Ј-H, 8ЈЈ-H), 4.18–4.26 (m, J_2Ј,3Јϭ 4.9 Hz, 1 H, 2Ј-H), 4.31–

(8): To a solution of 1,3-dibenzylresorcinol (1.00 g, 3.45 mmol) in

dry dichloromethane/acetonitrile (3:1, 2.5 mL) was added zinc

chloride diethyl ether solution (2.2  in dry dichloromethane, 80

µL, 0.174 mmol). A solution of 7^[16](1.00 g, 1.77 mmol) in dry

dichloromethane (2.5 mL) was then added dropwise and the mix-

ture stirred for 2 h at room temp. The reaction mixture was poured

into half-saturated sodium bicarbonate solution and extracted with

dichloromethane. The organic layer was separated, dried with mag-

nesium sulfate, and concentrated. The residue was dissolved in eth-

anol (70 mL) and, after addition of sodium hydroxide solution

(700 mg in 10 mL water), refluxed for 2 h. The mixture was diluted

with water and extracted with ethyl acetate. The organic layer was

dried with magnesium sulfate and concentrated. Flash chromato-

graphy (toluene/acetone, 30:1) yielded 8 (490 mg, 41%) as a yellow

oil containing up to 7% of the α isomer. – TLC (toluene/acetone,

30:1): R_fϭ 0.32. – ¹H NMR (250 MHz, CDCl₃): δ ϭ 3.52 (dd,

J

ϭ 10.8 Hz, J_5ЈA,4Јϭ 2.5 Hz, 1 H, 5ЈA-H), 3.79 (dd,

5ЈA,5ЈB

J_5ЈA,5ЈBϭ 10.8 Hz, J_5ЈB,4Јϭ 4.0 Hz, 1 H, 5ЈB-H), 4.10–4.56 (m, 9

H, 2Ј-H, 3Ј-H, 4Ј-H, 3 ϫ CH₂Ph), 4.89–5.23 (m, 4 H, 2 ϫ CH₂Ph),

5.44 (d, J_1Ј,2Јϭ 2.4 Hz, 1 H, 1Ј-Hβ), 6.57 (d, J_2,6ϭ 2.1 Hz, 1 H,

2-H), 6.64 (dd, J_2,6ϭ 2.1 Hz, J_6,5ϭ 8.4 Hz, 1 H, 6-H), 6.93–7.46

(m, 25 H, 5 ϫ CH₂Ph), 7.58 (d, J_6,5ϭ 8.4 Hz, 1 H, 5-H). –

C₄₆H₄₄O₆(692.81): calcd. C 79.74, H 6.40; found C 79.32, H 6.42.

4.41 (m, 1 H, 3Ј-H), 7.71 (d, J

Ͻ 1 Hz, 1 H, 1Ј-H), 8.79 (d,

2Ј,1Ј

J_5,3ϭ 2.2 Hz, 1 H, 2-H), 8.88 (dd, J_6,5ϭ 8.5 Hz, J_2,6ϭ 2.2 Hz, 1

H, 6-H), 9.61 (d, J_6,5ϭ 8.5 Hz, 1 H, 5-H). – ³¹P NMR (161.7 MHz,

D₂O): δ_Pϭ –3.66. – FAB MS (negative mode, matrix: glycerine/

water, 1:1): 612 (M – H^ϩ)^–.

4-(1-Deoxy-D-ribit-1-yl)resorcinol (9). – (a) From Compound 8:

Compound 8 (2.80 g, 4.04 mmol) was dissolved in methanol

(150 mL) and palladium on charcoal (100 mg, 10% Pd) was added.

The mixture was stirred under hydrogen at normal pressure. After

5 h, the catalyst was filtered off and washed with methanol. Evap-

oration of the solvents afforded 9 (740 mg, 75%) as a yellow

Dibenzyl (Methyl 2,3-Di-O-acetyl-β-D-ribofuranosid-5-yl) Phos-

phate (5): To a solution of methyl ribofuranoside^[15](2.50 g, 10.1

mmol) and tetrazole (1.41 g. 20.2 mmol) in dry acetonitrile/di-

chloromethane (3:1, 20 mL) was added dibenzyloxydiisopropylami-

nophosphane (5.00 g, 14.5 mmol) in dry dichloromethane (10 mL)

under nitrogen. After 20 min, a solution of tert-butyl hydroperoxide

(3  in dry toluene, 4.0 mL, 0.012 mol) was added and stirring was

continued for a further 20 min. The reaction mixture was poured

into half-saturated sodium bicarbonate solution and extracted with

dichloromethane. The organic layer was separated, dried with mag-

nesium sulfate, and concentrated. Flash chromatography (toluene/

acetone, 4:1) yielded 5 (4.32 g, 85%) as a colourless foam. – DC

(toluene/acetone, 3:1): R_fϭ 0.58. – [α]_Dϭ –84.0 (c ϭ 1.0 in chloro-

syrup.

– (b) From Compound 11: Compound 11 (200 mg,

0.250 mmol) was dissolved in methanol (13 mL) and palladium on

charcoal (100 mg, 10% Pd) was added. The mixture was stirred

under hydrogen at normal pressure. After 3 h, the catalyst was fil-

tered off and washed with methanol. Evaporation of the solvents

afforded 9 (47 mg, 78%) as a yellow syrup. – TLC (chloroform/

methanol, 4:1): R_fϭ 0.19. – [α]_Dϭ –22.6 (c ϭ 1.0 in methanol). –

¹H NMR (250 MHz, D₂O): δ ϭ 2.43 (dd, J_1ЈA,1ЈBϭ 14.3 Hz,

J_1ЈB,2Јϭ 9.4 Hz, 1 H, 1ЈA-H), 2.47 (dd, J_1ЈA,1ЈBϭ 14.3 Hz, J_1ЈB,2Ј

ϭ

1

form). – H NMR (250 MHz, CDCl₃): δ ϭ 1.90 (s, 3 H, COCH₃),

2.9 Hz, 1 H, 1ЈB-H), 3.41–3.55 (m, 2 H, 3Ј-H, 5ЈA-H), 3.58–3.69

(m, 2 H, 4Ј-H, 5ЈB-H), 3.77–3.84 (m, 1 H, 2Ј-H), 6.24–6.26 (m, 2

H, 2-H, 6-H), 6.88 (d, J_5,6ϭ 8.3 Hz, 1 H, 5-H).

1.99 (s, 3 H, COCH₃), 3.22 (s, 3 H, OCH₃), 3.90–4.11 (m, 2 H, 5A-

H, 5B-H), 4.10–4.27 (m, 1 H, 4-H), 4.78 (d, J_1.2Ͻ 1.0 Hz, 1 H, 1-

H), 4.93 (2 ϫ d, J ϭ 11.6 Hz, 4 H, 2 ϫ CH₂Ph), 5.11 (dd, J_3.2

4.9 Hz, J_1.2Ͻ 1.0 Hz, 1 H, 2-H), 5.18 (dd, J_3.2ϭ 4.9 Hz, J_3.4

ϭ

1,3-Di-O-acetyl-4-(2,3,4,5-tetra-O-acetyl-D-ribit-1-yl)resorcinol

ϭ

(9A): Compound 9 (500 mg, 2.05 mmol) was dissolved in dry pyrid-

ine/acetic anhydride (15 mL, 2:1). After stirring for about 12 h, the

solution was concentrated in vacuo, coevaporated with toluene and

purified by flash chromatography (toluene/acetone, 5:1) to give 9A

(890 mg, 88%) as a colourless solid. – TLC (toluene/acetone, 4:1):

R_fϭ 0.35. – [α]_Dϭ ϩ34 (c ϭ 1.0 in chloroform). – ¹H NMR

(250 MHz, CDCl₃): δ ϭ 1.94–1.32 (6 ϫ s, 18 H, 6 ϫ COCH₃),

2.81–2.84 (m, 1 H, 1ЈA-H, 1ЈB-H), 4.12 (dd, J_5ЈA,5ЈBϭ 12.1 Hz,

6.7 Hz, 1 H, 3-H), 7.23–7.25 (m, 10 H, Ph). – C₂₄H₂₉O₁₀P (505.4):

calcd. C 56.69, H 5.72; found C 56.61, H 5.88.

Triethylammonium (Methyl 2,3-Di-O-acetyl-β-D-ribofuranosid-5-yl)

Hydrogen Phosphate (6): Compound 5 (800 mg, 1.57 mmol) was

dissolved in methanol (100 mL) and palladium on charcoal (80 mg,

10% Pd) was added. The mixture was stirred vigorously under hy-

drogen at normal pressure. After 15 min, the catalyst was filtered

off and washed with methanol. Triethylamine (1 mL) was added

and the filtrate was concentrated under reduced pressure. Purifica-

tion by flash chromatography (chloroform/methanol/triethylamine,

3:1:0.1) yielded 5 (546 mg, 81%) as a colourless solid. – TLC (chlo-

roform/methanol/water, 3:1:0.1): R_fϭ 0.15. – ¹H NMR (250 MHz,

J _5ЈA,4Јϭ 6.2 Hz, 1 H, 5ЈA-H), 4.33 (dd, J_5ЈA,5ЈBϭ 12.1 Hz, J_5ЈB,4Ј

ϭ

2.6 Hz, 1 H, 5ЈB-H), 5.21–5.32 (m, 3 H, 2Ј-H, 3Ј-H, 4Ј-H), 6.91–

6.97 (m, 2 H, 2-H, 6-H), 7.18 (d, J_5,6ϭ 8.3 Hz, 1 H, 5-H). –

C₂₄H₂₉O₁₀P (496.5): calcd. C 55.64, H 5.68; found C 55.34, H 5.65.

MeOD): δ ϭ 1.34 (t, J ϭ 7.2 Hz, 9 H, 3 CH₃), 2.06 (s, 3 H, 1,3-Di-O-benzyl-4-(2,3,4,5-tetra-O-benzyl-

D-allo/altro-pentit-1-

COCH₃), 2.12 (s, 3 H, COCH₃), 3.43 (q, 6 H, 3 ϫ CH₂), 3.42 (s, 3 yl)resorcinol (11): A solution of 10^[17](3.70 g, 0.10 mmol) in dry

H, OCH₃), 3.96–4.01 (m, 2 H, 5A-H, 5B-H), 4.29–4.34 (m, 1 H, 4-

THF (130 mL) was cooled to –70 °C under nitrogen. n-Butylli-

Eur. J. Org. Chem. 2000, 1467Ϫ1482

1473

G. Dufner, R. Schwörer, B. Müller, R. R. Schmidt

thium in n-hexane (1.6 , 6.0 mL) was added dropwise. After C₂₅H₃₀O₁₄.H₂O (572.51): calcd. C 52.44, H 5.28; found C 52.48,

FULL PAPER

15 min,

a

solution of 2,4-dibenzyloxybromobenzene (4.40 g,

H 5.44.

8.62 mmol) in dry THF (20 mL) was added dropwise and the reac-

tion mixture was warmed up to 0 °C over 2 h. The mixture was

poured into half-saturated ammonium chloride solution and ex-

tracted with ethyl acetate. The organic layer was dried with magnes-

ium sulfate and concentrated in vacuo. The crude product was puri-

fied by flash chromatography (petroleum ether/ethyl acetate, 7:1)

to give 11 (5.38 g, 78%) as a colourless solid that contained the two

diastereomers in a ratio of 9:1. – TLC (petroleum ether/ethyl acet-

ate, 5:1): R_fϭ 0.38. – ¹H NMR (250 MHz, CDCl₃): δ ϭ 3.62–3.65

(m, 1 H, 5ЈA-H), 3.68–3.81 (m, 1 H, 5ЈB-H), 3.92–4.01 (m, 2 H,

3Ј-H, 4Ј-H), 4.11–4.19 (m, 2 H, 2Ј-H, OH), 4.20–5.10 (m, 12 H, 6

ϫ CH₂Ph), 5.29 (dd, J_2Ј,1Јϭ 4.9 Hz, J_OH,1Јϭ 6.6 Hz, 1 H, 1Ј-H),

6.29–6.61 (m, 2 H, 2-H, 6-H), 6.94–7.49 (m, 31 H, 6 ϫ CH₂Ph, 5-

H). – FAB-MS (positive mode, matrix: acetone/nitrobenzyl alcohol,

1:1 and NaI): 823 (M ϩ Na^ϩ).

1,3-Di-O-acetyl-4-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)resorcinol

(13): Compound 12 (1 g, 3.84 mmol) was dissolved in acetic acid/

methanol (1:1, 10 mL) and the solution was stirred overnight. The

solution was concentrated and coevaporated with toluene. The res-

idue was dissolved in dry pyridine/acetic anhydride (15 mL, 2:1)

and kept overnight at room temp. The solution was concentrated

and coevaporated with toluene. The crude product was purified

by flash chromatography (petroleum ether/ethyl acetate, 1:1) and

separated from an isomeric side-product by MPLC (petroleum

ether/ethyl acetate, 1:1) to give 13 (1.06 g, 62%). – TLC (petroleum

ether/ethyl acetate, 1:1): R_fϭ 0.24. – [α]_Dϭ –5.7 (c ϭ 1.0 in chloro-

form). – ¹H NMR (250 MHz, CDCl₃): δ ϭ 2.07–2.29 (5 ϫ s, 15 H,

5 ϫ COCH₃), 4.29–4.32 (m, 2 H, 4Ј-H, 5ЈA-H), 4.42 (dd, J_5ЈA,5ЈBϭ

13.2 Hz, J_4Ј,5ЈBϭ 4.3 Hz, 1 H, 5ЈB-H), 5.09–5.16 (m, 2 H, 2Ј-H,

1Ј-Hβ), 5.25 (dd, J_2Ј,3Јϭ 4.9 Hz, J_4Ј,3Јϭ 6.2 Hz, 1 H, 3Ј-H), 6.96

(d, J_6,2ϭ 2.2 Hz, 1 H, 2-H), 7.04 (dd, J_6,5ϭ 8.5 Hz, 1 H, 6-H),

7.56 (d, J_6,5ϭ 8.5 Hz, 1 H, 5-H). – C₂₁H₂₄O₈₁(452.4): calcd. C

55.75, H 5.35; found C 55.61, H 5.44.

4-(1-O-Acetyl-2,3,4,5-tetra-O-benzyl-D-allo/altro-pentit-1-yl)-1,3-di-

O-benzylresorcinol (11A): Compound 11 (100 mg, 0.125 mmol) was

dissolved in dry pyridine/acetic anhydride (3 mL, 2:1). After stir-

ring overnight, the solution was concentrated in vacuo, coevapor-

ated with toluene and purified by flash chromatography (petroleum

ether/ethyl acetate, 6:1) to give 11A (84 mg, 83%) as a colourless

oil. The ratio of the two diastereomers was 9:1. – TLC (petroleum

ether/ethyl acetate, 7:1): R_fϭ 0.27. – ¹H NMR (250 MHz, CDCl₃):

1,3-Di-O-acetyl-4-(2,3-di-O-acetyl-5-O-tert-butyldimethylsilyl-β-D-

ribofuranosyl)resorcinol (14): Compound 12 (3.00 g, 0.01 mol) was

dissolved in acetic acid/methanol (1:1, 10 mL) and the solution

stirred for about 12 h at room temp. The solution was concentrated

and coevaporated with toluene. The residue was mixed with dry

N,N-dimethylformamide (30 mL) and treated in a ultrasonic bath

for 20 min. Imidazole (1.69 g, 0.02 mol) was added and, under

δ ϭ 1.98 (s, 3 H, COCH₃), 3.63 (dd, J_5ЈA,5ЈBϭ 10.5 Hz, J_4Ј,5ЈA

ϭ

6 Hz, 1 H, 5ЈA-H), 3.73 (dd, J_5ЈA,5ЈBϭ 10.5 Hz, J_4Ј,5ЈBϭ 2.6 Hz,1

H, 5ЈB-H), 3.86 (dd, J_2Ј,3Јϭ 4.5 Hz, 1 H, 3Ј-H), 3.95–4.01 (m, 1

H, 4Ј-H), 4.16 (dd, J_1Ј,2Јϭ J_3Ј,2Јϭ 4.5 Hz, 1 H, 2Ј-H), 4.30–5.11

(m, 12 H, 6 ϫ CH₂Ph), 6.29–6.61 (m, 2 H, 2-H, 6-H), 6.56 (d,

J_1Ј,2Јϭ 5.4 Hz, 1 H, 1Ј-H), 6.94–7.49 (m, 31 H, 6 ϫ CH₂Ph, 5-

H). – C₅₅H₅₄O₈(843.03): calcd. C 78.36, H 6.45; found C 78.29,

H 6.53.

vigorous stirring,

a solution of tert-butylchlorodimethylsilane

(2.05 g, 0.01 mol) in dry N,N-dimethylformamide (10 mL) was ad-

ded dropwise. Stirring was continued for ca. 12 h. The solvent was

evaporated in vacuo and the residue was dissolved in dry pyridine/

acetic anhydride (20 mL, 2:1). After stirring for ca. 12 h, the mix-

ture was concentrated and coevaporated with toluene. The crude

product was purified by flash chromatography (toluene/acetone,

5:1) to give 14 (2.96 g, 49%) as a colourless solid. – TLC (petroleum

ether/ethyl acetate, 1:1): R_fϭ 0.44. – [α]_Dϭ –11.6 (c ϭ 1.0 in

chloroform). – ¹H NMR (250 MHz, CDCl₃): δ ϭ 0.08 [s, 6 H,

4-(D-allo/altro-Pentit-1-yl)resorcinol (12): Compound 11 (2.50 g,

3.13 mmol) and barium carbonate (620 mg, 3.13 mmol) were dis-

solved in methanol (100 mL) and palladium on charcoal (80 mg,

10% Pd) was added. The mixture was stirred vigorously under hy-

drogen at normal pressure. After 2 d, the catalyst was filtered off Si(CH₃)₂]_,0.89 [s, 9 H, C(CH₃)₃], 1.99–2.27 (4 ϫ s, 12 H, 4 ϫ

and washed with methanol. Triethylamine (1 drop) was added and

the filtrate was concentrated under reduced pressure to yield 12

(51 mg, 63%, yellow oil) as diastereomeric mixture, 9:1. – TLC

(chloroform/methanol, 4:1): R_fϭ 0.15. – ¹H NMR (250 MHz,

COCH₃), 3.82 (dd, J_5ЈA,5ЈBϭ 11.2 Hz, J_4Ј,5ЈAϭ 2.6 Hz, 1 H, 5ЈA-

H), 3.89 (dd, J_5ЈA,5ЈBϭ 11.2 Hz, J_4Ј,5ЈBϭ 2.6 Hz, 1 H, 5ЈB-H),

4.09–4.15 (m, 1 H, 4Ј-H), 5.07 (dd, J_3Ј,2Јϭ 5.2 Hz, J_1Ј,2Јϭ 7.4 Hz,

1 H, 2Ј-H), 5.16 (d, J_1Ј,2Јϭ 7.4 Hz, 1 H, 1Ј-H), 5.34 (dd, J_3Ј,2Ј

5.2 Hz, J_4Ј,3Јϭ 3.8 Hz, 1 H, 3Ј-H), 6.91 (d, J_6,2ϭ 2.2 Hz, 1 H, 2-

11.8 Hz, J_4Ј,5ЈBϭ 3.1 Hz, 1 H, 5ЈB-H), 3.62–3.72 (m, 1 H, 4Ј-H), H), 6.98 (dd, J_6,5ϭ 8.5 Hz, J ϭ 2.2 Hz, 1 H, 6-H), 7.73 (d,

ϭ

D₂O): δ ϭ 3.31–3.50 (m, 2 H, 5ЈA-H, 3Ј-H), 3.59 (dd, J_5ЈA,5ЈB

ϭ

2,5

3.83 (dd, J_1Ј,2Јϭ 5.3 Hz, J_3Ј,2Јϭ 7.3 Hz, 1 H, 2Ј-H), 4.86 (d, J_1Ј,2Ј

ϭ

J_6,5ϭ 8.5 Hz, 1 H, 5-H). – C₂₅H₃₆O₁₀Si (524.6): calcd. C 57.23, H

5.4 Hz, 1 H, 1Ј-H), 6.18 (d, J ϭ 2.3 Hz, J_2,6ϭ 2.3 Hz, 1 H, 2-H), 6.91; found C 56.77, H 6.95.

6.23 (dd, J_2,6ϭ 2.3 Hz, J_5,6ϭ 8.3 Hz, 1 H, 6-H), 6.97 (d, J_5,6

ϭ

1,3-Di-O-acetyl-4-(2,3-di-O-acetyl-β-D-ribofuranosyl)resorcinol:

8.3 Hz, 1 H, 5-H).

(15): A solution of 14 (3.00 g, 5.72 mmol) in acetic acid/water (8:2,

30 mL) was refluxed for 5 h. The solvents were evaporated in vacuo

and the residue was coevaporated with toluene. Flash chromato-

graphy (toluene/acetone, 4:1) yielded 15 (1.64 g, 70%) as a colour-

less solid. – TLC (toluene/acetone, 2:1): R_fϭ 0.41. – [α]_Dϭ –26.5

(c ϭ 1.0 in chloroform). – ¹H NMR (250 MHz, CDCl₃): δ ϭ 2.04–

2.28 (4 ϫ s, 12 H, 4 ϫ COCH₃), 3.73 (dd, J_5ЈA,5ЈBϭ 12.3 Hz,

1,3-Di-O-acetyl-4-(2,3,4,5-penta-O-acetyl-D-allo/altro-pentit-1-yl)-

resorcinol (12A): Compound 12 (500 mg, 1.95 mmol) was dissolved

in dry pyridine/acetic anhydride (10 mL, 2:1). After stirring over-

night, the solution was concentrated in vacuo, coevaporated with

toluene and purified by flash chromatography (petroleum ether/

ethyl acetate, 1:1) to give 12A (910 mg, 85%) as a colourless solid.

The ratio of the two diastereomers was 9:1. – TLC (petroleum

ether/ethyl acetate, 1:1): R_fϭ 0.29. – ¹H NMR (250 MHz, CDCl₃):

J

_4Ј,5ЈAϭ 3.7 Hz, 1 H, 5ЈA-H), 3.88 (dd, J_5ЈA,5ЈBϭ 12.3 Hz, J_4Ј,5ЈBϭ

3.0 Hz, 1 H, 5ЈB-H), 4.05–4.11 (m, 1 H, 4Ј-H), 5.08 (d, J_1Ј,2Ј

5.6 Hz, 1 H, 1Ј-H), 5.21 (dd, J_2Ј,1Јϭ J_2Ј,3Јϭ 5.6 Hz, 1 H, 2Ј-H),

12.1 Hz, J_4Ј,5ЈAϭ 6.8 Hz, 1 H, 5ЈA-H), 4.32 (dd, J_5ЈA,5ЈBϭ 12.1 Hz, 5.28 (dd, J_3Ј,2Јϭ J_4Ј,3Јϭ 5.6 Hz, 1 H, 3Ј-H), 6.91 (d, J_6,2ϭ 2.3 Hz,

J_4Ј,5ЈBϭ 2.9 Hz, 1 H, 5ЈB-H), 5.26–5.39 (m, 1 H, 4Ј-H), 5.42–5.51 1 H, 2-H), 6.98 (dd, J_6,5ϭ 8.5 Hz, J ϭ 2.3 Hz, 1 H, 6-H), 7.73

(m, 2 H, 2Ј-H, 3Ј-H), 6.13 (d, J_1Ј,2Јϭ 5.5 Hz, 1 H, 1Ј-H), 6.91–6.95 (d, J_6,5ϭ 8.5 Hz, 1 H, 5-H). – C₁₉H₂₂O₁₀(410.38): calcd. C 55.61,

ϭ

δ ϭ 1.95–2.23 (7 ϫ s, 21 H, 7 ϫ COCH₃), 4.03 (dd, J_5ЈA,5ЈB

ϭ

2,6

(m, 2 H, 2-H, 6-H), 7.36 (d, J_5,6ϭ 8.1 Hz, 1 H, 5-H). –

H 5.40; found C 55.38, H 5.52.

1474

Eur. J. Org. Chem. 2000, 1467Ϫ1482

Base- and Sugar-Modified CMP-Neu5Ac Analogues

FULL PAPER

Dibenzyl [(1,3-Di-O-acetylresorcin-4-yl) (2,3-Di-O-acetyl-1-deoxy-

chloride 18^[20](3.20 g, 8.96 mmol) and triethylamine (1.40 mL)

β-D-ribofuranosid-5-yl)] Phosphate (16): Compound 15 (320 mg, were dissolved in dry methanol (60 mL) and S-ethyl trifluoro-

0.780 mmol) and 1H-tetrazole (80 mg, 1.14 mmol) were dissolved

in dry acetonitrile/dichloromethane (3:1, 10 mL) under nitrogen.

Bis(benzyloxy)(diisopropylamino)phosphane (426 mg, 1.24 mmol)

in dry dichloromethane (5 mL) was added by syringe. After 20 min

(TLC), a solution of tert-butyl hydroperoxide (3 ) in dry toluene

(310 µL, 0.935 mmol) was added and stirring was continued for a

further 20 min. The reaction mixture was treated with half-satur-

ated sodium bicarbonate solution and extracted with dichlorome-

thane. The organic layer was dried with magnesium sulfate and

concentrated in vacuo. Purification by flash chromatography (tolu-

thioacetate (1.40 mL, 0.011 mol) in dry methanol (13 mL) was ad-

ded. The solution was stirred for 3 h at room temp. and the reaction

monitored by TLC. The solvent was removed and the residue co-

evaporated with toluene. The residue was dissolved in methanol

(40 mL) and stirred for 30 min with Amberlite IR 120 (H^ϩ). The

mixture was filtered and to the filtrate was added a solution of

diazomethane (0.5  in ether) until the solution remained yellow.

After removal of the solvents, the residue was stirred overnight in

dry pyridine/acetic anhydride (2:1, 20 mL). Evaporation of the

solvents, codistillation with toluene and flash chromatography

ene/acetone, 4:1) yielded 16 (440 mg, 85%) as a colourless solid. – (toluene/acetone, 4:1) yielded 20a (3.6 g, 64%) as a colourless

TLC (toluene/acetone, 4:1): R_fϭ 0.17. – [α]_Dϭ ϩ4.60 (c ϭ 1.0 in

chloroform). – H NMR (250 MHz, CDCl₃): δ ϭ 2.03–2.26 (4 ϫ

solid. – TLC (toluene/acetone, 4:1): R_fϭ 0.41. NMR data were

1

identical with published data.^[20]

s, 12 H, 4 ϫ COCH₃), 4.16–4.27 (m, 3 H, 4Ј-H, 5ЈA-H, 5ЈB-H),

Methyl (Benzyl 4,7,8,9-Tetra-O-acetyl-3,5-dideoxy-5-valeroylamino-

4.98–5.14 (m, 6 H, 1Ј-H, 2Ј-H, 2 ϫ CH₂Ph), 5.72 (dd, J_2Ј,3Ј

ϭ

D

-glycero-α-D-galacto-2-nonulopyranosid)onate (20b): To a solution

J_4Ј,3Јϭ 5.0 Hz, 1 H, 3Ј-H), 6.83 (dd, J_5,6ϭ 8.5 Hz, J_2,6ϭ 2.2 Hz,

1 H, 6-H), 6.90 (d, J_6,2ϭ 2.2 Hz, 1 H, 2-H), 7.32–7.36 (m, 10 H,

2 ϫ Ph), 7.55 (d, J_6,5ϭ 8.5 Hz, 1 H, 5-H). – C₃₃H₃₅O₁₃P (670.60):

calcd. C 59.10, H 5.26; found C 59.00, H 5.46.

of 19 (400 mg, 0.694 mmol) and triethylamine (85 mg, 0.833 mmol)

in dry dichloromethane (10 mL) was added a solution of valeric

acid (133 mg, 1.30 mmol) in dry dichloromethane (3 mL). WSC

(water-soluble carbodiimide, 200 mg) was added in small portions

at 0 °C and the mixture was stirred overnight. The mixture was

diluted with dichloromethane (10 mL), washed with sodium bicar-

Methyl (Benzyl 4,7,8,9-Tetra-O-acetyl-5-amino-3,5-dideoxy-

D-gly-

cero-α- -galacto-2-nonulopyranosid)onate Hydrochloride (19): To a

D

solution of 17^[19](1.00 g, 1.72 mmol) and 2,6-di-tert-butylpyridine bonate solution and extracted with dichloromethane. The organic

(1.65 g, 8.60 mmol) in dry acetonitrile (6 mL) was added a solution layer was dried with magnesium sulfate and concentrated under

of Tf₂O (1.21 g, 4.30 mmol) in dry acetonitrile (5 mL) dropwise at – reduced pressure. Purification by flash chromatography (toluene/

25 °C. After stirring for 30 min, a solution of dry ethanol (1.56 g,

0.034 mol) and 2,6-di-tert-butylpyridine (1.65 g, 8.6 mmol) in ace-

tonitrile (4 mL) was added. The reaction mixture was treated with

half-saturated sodium bicarbonate solution and extracted with

dichloromethane. The organic layer was dried with magnesium

sulfate and concentrated in vacuo. Purification by flash chromato-

graphy (toluene/acetone 15:1) gave the imidate intermediate

(520 mg, 50%) as a colourless foam. – TLC (toluene/acetone, 15:1):

acetone, 6:1) yielded 20b (300 mg, 71%) as a colourless solid. –

TLC (toluene/acetone 3:1): R_fϭ 0.65. – [α]_Dϭ ϩ2.40 (c ϭ 1.0 in

chloroform). – ¹H NMR (250 MHz, CDCl₃): δ ϭ 0.90 (t, J ϭ

6.6 Hz, 3 H, CH₃), 1.26–1.37 (m, 2 H, CH₂CH₃), 1.48–1.58 (m, 2

H, CH₂CH₂CH₃), 1.96–2.17 (m, 15 H, 4 ϫ COCH₃, CH₂CO, 3a-

H), 2.66 (dd, J_3e,4ϭ 4.6 Hz, J_3e,3aϭ 13.1 Hz, 1 H, 3e-H), 3.67 (s,

3 H, COOCH₃), 4.08–4.16 (m, 3 H, 5-H, 6-H, 9B-H), 4.34 (dd,

J_8,9Aϭ 2.6 Hz, J_9A,9Bϭ 12.4 Hz, 1 H, 9A-H), 4.42 (d, J ϭ 11.9 Hz,

R_fϭ 0.58. – ¹H NMR (250 MHz, CDCl₃): δ ϭ 1.19 (t, J ϭ 7.1 Hz, 1 H, 0.5 ϫ CH₂Ph), 4.81 (d, J ϭ 11.9 Hz, 1 H, 0.5 CH₂Ph), 4.85–

3 H, CH₃), 1.73 (s, 3 H, CH₃), 1.83 (dd, J_3e,3aϭ 13.2 Hz, J_3a,45.28 (m, 1 H, 4-H), 5.20 (d, J_5,NHϭ 9.4 Hz, 1 H, NH), 5.33 (dd,

11.0 Hz, 1 H, 3a-H), 1.95–2.18 (4 ϫ s, 12 H, 4 ϫ COCH₃), 2.78 J_7,8ϭ 8.4 Hz, 1 H, 7-H), 5.45 (ddd, J_8,9aϭ 2.6 Hz, J_7,8ϭ 8.4 Hz,

ϭ

(dd, J_3e,3aϭ 13.2 Hz, J_3e,4ϭ 4.9 Hz, 1 H, 3e-H), 3.19 (dd, J_5,6

ϭ

1 H, 8-H), 7.23–7.34 (m, 5 H, Ph). – C₃₀H₄₁NO₁₃(623.65): calcd.

C 57.78, H 6.63; found C 57.52, H 6.64.

J_4,5ϭ 10.2 Hz, 1 H, 5-H), 3.69 (s, 3 H, COOCH₃), 3.89–4.22 (m,

4 H, 9A-H, 6-H, CH₂), 4.36 (dd, J_9A,9Bϭ 12.5 Hz, J_9B,8ϭ 1.9 Hz,

1 H, 9B-H), 4.42 (d, J ϭ 12.0 Hz, 1 H, 0.5 ϫ CH₂Ph), 4.7–4.85

(m, 2 H, 0.5 ϫ CH₂Ph, 4-H), 5.28 (dd, J_6.7ϭ 6.8 Hz, 1 H, 7-H),

5.49–5.50 (m, 1 H, 8-H), 7.21–7.38 (m, 5 H, Ph). To a solution of

the imidate intermediate (500 mg, 0.82 mmol) in methanol (50 mL)

was added trifluoracetic acid (2.5% in methanol, 7.0 mL) dropwise

at 0 °C and the mixture was stirred for 2 h. The solution was con-

centrated and coevaporated with toluene. The residue was dissolved

in methanol (20 mL) and stirred for 30 min with Amberlite IRA

420 (Cl^–). The ion-exchange resin was filtered off. The filtrate was

concentrated to dryness to yield 19 (450 mg, 95%), which was used

without further purification. – TLC (toluene/acetone, 3:1): R_fϭ

0.18. – ¹H NMR (250 MHz, CDCl₃): δ ϭ 1.72 (dd, J_3a,3eϭ J_4,3aϭ

11.0 Hz, 1 H, 3a-H), 2.01–2.21 (4 ϫ s, 12 H, 4 ϫ COCH₃), 2.82

(dd, J_3e,4ϭ 4.3 Hz, J_3e,3aϭ 11.0 Hz, 1 H, 3e-H), 2.91 (dd, J ϭ

10.3 Hz, 1 H, 5-H), 3.52–3.62 (m, 1 H, 9A-H), 3.71 (s, 3 H, CO-

OCH₃), 4.22–4.38 (m, 2 H, 6-H, 9B-H), 4.43 (d, J ϭ 11.9 Hz, 1 H,

0.5 CH₂Ph), 4.63 (d, J_5,NHϭ 10.3 Hz, 1 H, NH), 4.78 (d, J ϭ

11.9 Hz, 1 H, 0.5 CH₂Ph), 5.22–5.31 (m, 1 H, 8-H), 5.36 (ddd,

J_3a,4ϭ 11.0 Hz, J_3e,4ϭ 4.3 Hz,1 H, 4-H), 5.52 (dd, J_7,8ϭ 8.4 Hz,

1 H, 7-H), 7.23–7.34 (m, 5 H, Ph).

Methyl (Benzyl 4,7,8,9-Tetra-O-acetyl-3,5-dideoxy-5-(N-trifluor-

acetylglycylamido)-D-glycero-α-D-galacto-2-nonulopyranosid)onate

(20c): To a solution of 19 (250 mg, 0.335 mmol) and triethylamine

(53 mg, 0.521 mmol) in dry dichloromethane (6 mL) was added a

solution of (trifluoroacetylamino)acetic acid (110 mg, 0.652 mmol)

in dry dichloromethane (3 mL). WSC (125 mg) was added in small

portions at 0 °C and the mixture was stirred overnight. The mixture

was diluted with dichloromethane (10 mL), washed with sodium

bicarbonate solution and extracted with dichloromethane. The or-

ganic layer was dried with magnesium sulfate and concentrated un-

der reduced pressure. Purification by flash chromatography (tolu-

ene/acetone, 6:1) yielded 20c (180 mg, 75%) as a colourless solid. –

TLC (toluene/acetone 4:1): R_fϭ 0.52. – [α]_Dϭ ϩ2.4 (c ϭ 1.0 in

1

chloroform). – H NMR (250 MHz, CDCl₃): δ ϭ 1.99–2.15 (4 ϫ

s, 13 H, 4 ϫ COCH₃, 3a-H), 2.67 (dd, J_3e,4ϭ 4.6 Hz, J_3e,3a

12.8 Hz, 1 H, 3e-H), 3.65 (s, 3 H, COOCH₃), 3.87 (d, J_CHa,CHb

ϭ

J_NH,CHaϭ 5.4 Hz, 1 H, 0.5 ϫ COCH₂), 3.99–4.13 (m, 3 H, 5-H,

9A-H, 0.5 ϫ COCH₂), 4.18 (dd, J_7,6ϭ 2.1 Hz, J_8,6ϭ 10.7 Hz, 1

H, 6-H), 4.33 (dd, J_8,9Bϭ 2.6 Hz, J_9A,9Bϭ 12.5 Hz, 1 H, 9B-H),

4.40 (J ϭ 11.9 Hz, 1 H, 0.5 CH₂Ph), 4.78 (d, J ϭ 11.9 Hz, 1 H, 0.5

CH₂Ph), 4.84–4.91 (m, 1 H, 4-H), 5.29 (dϽ, J_6,7ϭ 2.1 Hz, J_7,8

ϭ

Methyl (Benzyl 4,7,8,9-Tetra-O-acetyl-3,5-dideoxy-5-trifluoracet-

8.6 Hz, 1 H, 7-H), 5.45 (ddd, J_8,9Bϭ 2.6 Hz, J_7,8ϭ 8.5 Hz, J_9A,8ϭ

5 Hz, 1 H, 8-H), 5.84 (d, J_5,NHϭ 9.8 Hz, 1 H, NH), 7.16–7.38 (m,

amido-D-glycero-α-D-galacto-2-nonulopyranosid)onate (20a): Hydro-

Eur. J. Org. Chem. 2000, 1467Ϫ1482

1475

G. Dufner, R. Schwörer, B. Müller, R. R. Schmidt

6 H, Ph, NH). – C₂₉H₃₅F₃N₂O₁₄(692.59): calcd. C 50.29, H 5.09, The catalyst was filtered off and washed with methanol. Evapora-

FULL PAPER

N 4.04; found C 49.92, H 5.24, N 3.96.

tion of the solvents and flash chromatography (toluene/acetone,

2:1) afforded 21c (150 mg, 86%) as a colourless solid. – DC (tolu-

ene/acetone, 4:1): R_fϭ 0.28. – [α]_Dϭ ϩ2.0 (c ϭ 1.0 in chloro-

form). – ¹H NMR (250 MHz, CDCl₃): δ ϭ 2.90–2.24 (4 ϫ s, 14 H,

4 ϫ COCH₃, 3a-H, 3e-H), 3.82 (s, 3 H, COOCH₃), 2.32–4.13 (m,

4 H, 5-H, 9A-H, CH₂), 4.30 (d, J_7,6ϭ 2.0 Hz, J_5,6ϭ 10.5 Hz, 1 H,

6-H), 4.55 (dd, J_9A,9Bϭ 12.2 Hz, J_8,9Bϭ 2.2 Hz, 1 H, 9B-H), 4.14–

5.27 (m, 2 H, 4-H, 8-H), 5.35 (dd, J_6,7ϭ 2.1 Hz, J_8,7ϭ 4.0 Hz, 1

H, 7-H), 6.82 (d, J ϭ 9.9 Hz, 1 H, NH), 7.44–7.51 (m, 1 H, NH). –

C₂₂H₂₉F₃N₂O₁₄(602.47): calcd. C 43.86, H 4.85, N 4.65; found C

43.5, H 4.98, N 4.67.

Methyl (Benzyl 4,7,8,9-Tetra-O-acetyl-3,5-dideoxy-5-ethoxycarbon-

ylamino-D-glycero-α-D-galacto-2-nonulopyranosid)onate (20d): To a

solution of 19 (170 mg, 0.296 mmol) in dry dichloromethane

(2.0 mL) was added dry pyridine (120 µL). The solution was cooled

to 0 °C and ethoxycarbonyl chloride (140 µL, 1.47 mmol) was ad-

ded dropwise. The resulting yellow solution was stirred for 30 min

at room temp., then poured into sodium bicarbonate solution and

extracted with dichloromethane. The organic layer was dried with

magnesium sulfate, concentrated and purified by flash chromato-

graphy (toluene/acetone, 5:1) to yield 20d (163 mg, 90%) as a col-

ourless foam. – TLC (toluene/acetone, 3:1): R_fϭ 0.50. – [α]_D

ϭ

Methyl 4,7,8,9-Tetra-O-acetyl-3,5-dideoxy-5-ethoxycarbonylamino-

ϩ8.2 (c ϭ 1.0 in chloroform). – ¹H NMR (250 MHz, CDCl₃): δ ϭ

D

-glycero-β-

1.20 (t, J ϭ 7.1 Hz, 3 H, CH₃), 1.91–2.15 (m, 13 H, 4 ϫ COCH₃, 20d (250 mg, 0.401 mmol) in methanol (20 mL) was added palla-

3a-H), 2.66 (dd, J_3e,4ϭ 4.6 Hz, J_3e,3aϭ 12.7 Hz, 1 H, 3e-H), 3.65 dium on charcoal (20 mg, 10% Pd). The mixture was stirred over-

D-galacto-2-nonulopyranosonate (21d): To a solution of

(s, 3 H, COOCH₃), 3.68–3.82 (m, 1 H, 0.5 ϫ OCH₂), 3.99–4.13 (m, night under hydrogen at normal pressure. The catalyst was filtered

4 H, 5-H, 6-H, 9B-H, 0.5 ϫ OCH₂), 4.32 (dd, J_8,9Aϭ 2.4 Hz, off and washed with methanol. Evaporation of the solvents and

J_9A,9Bϭ 12.5 Hz, 1 H, 9A-H), 4.39 (d, J ϭ 11.9 Hz, 2 H, 0.5 ϫ

CH₂Ph, NH), 4.79–4.92 (m, 2 H, 0.5 ϫ CH₂Ph, 4-H), 5.39–5.50

(m, 2 H, 7-H, 8-H), 7.20–7.36 (m, 5 H, Ph). – MS (MALDI): 636

(M ϩ Na^ϩϩ 1).

flash chromatography (toluene/acetone, 3:1) afforded 21d (168 mg,

79%) as a colourless solid. – TLC (toluene/acetone, 2:1): R_f

ϭ

0.43. – [α]_Dϭ ϩ0.5 (c ϭ 1.0 in chloroform). – ¹H NMR (250 MHz,

CDCl₃): δ ϭ 1.20 (t, J ϭ 7 Hz, 3 H, CH₃), 1.99–2.25 (m, 14 H, 4

ϫ COCH₃, 3a-H, 3e-H), 3.72–3.77 (m, 1 H, 0.5 ϫ OCH₂), 3.82 (s,

3 H, COOCH₃), 3.81–4.07 (m, 3 H, 5-H, 9A-H, 0.5 ϫ OCH₂), 4.13

(dd, J_7,6ϭ 1.8 Hz, J_5,6ϭ 10.5 Hz, 1 H, 6-H), 4.39–4.44 (m, 2 H,

OH, 9B-H), 4.78 (d, J ϭ 10.3 Hz, 1 H, NH), 5.12–5.27 (m, 2 H, 4-

H, 8-H), 5.37 (dd, J_6,7ϭ 1.8 Hz, J_8,7ϭ 5.8 Hz, 1 H, 7-H). – MS

(MALDI): 545 (M ϩ Na^ϩϩ 1).

Methyl 4,7,8,9-Tetra-O-acetyl-3,5-dideoxy-5-trifluoroacetamido-

D-

glycero-β- -galacto-2-nonulopyranosonate (21a): To a solution of

D

20a (1.40 g, 2.21 mmol) in methanol (60 mL) was added palladium

on charcoal (140 mg, 10% Pd). The mixture was stirred overnight

under hydrogen at normal pressure. The catalyst was filtered off

and washed with methanol. Evaporation of the solvents and flash

chromatography (toluene/acetone, 4:1) afforded 21a (980 mg, 81%)

Diethyl [Methyl (4,7,8,9-Tetra-O-acetyl-3,5-dideoxy-5-trifluoroacet-

as a colourless solid. – TLC (toluene/acetone, 4:1): R_fϭ 0.28. – amido-

[α]_Dϭ ϩ2.4 (c ϭ 1.0 in chloroform). – ¹H NMR (250 MHz,

(22a): To a solution of 21a (730 mg, 1.34 mmol) and ethyldiisopro-

CDCl₃): δ ϭ 1.95–2.32 (m, 13 H, 4 ϫ COCH₃, 3a-H), 3.81 (s, 3 H, pylamine (513 µL) in dry acetonitrile (10 mL) was added diethyl

COOCH₃), 4.01 (dd, J_8.9ϭ 6.8 Hz, J_9A,9Bϭ 12.3 Hz, 1 H, 9B-H), chlorophosphite (380 µL, 2.64 mmol) under nitrogen. After 10 min,

4.12 (dd, J_5,6ϭ J_5,4ϭ 10.2 Hz, 1 H, 5-H), 4.45 (dd, J_7,6ϭ 2.3 Hz, the solution was concentrated and coevaporated with toluene.

J_5,6ϭ 10.5 Hz, 1 H, 6-H), 4.68 (dd, J_9A,9Bϭ 12.3 Hz, J_8,9AFlash chromatography (toluene/acetone, 6:1) yielded 22a (783 mg,

2.3 Hz, 1 H, 9A-H), 5.13–5.27 (m, 1 H, 4-H), 5.28–5.35 (m, 2 H, 88%) as a colourless oil. – TLC (toluene/acetone): R_fϭ 0.64. – H

D-glycero-β-D-galacto-2-nonulopyranosyl)onate]

Phosphite

ϭ

1

7-H, 8-H), 5.49 (s, 1 H, OH), 7.75 (d, J ϭ 10 Hz, 1 H, NH). –

C₂₀H₂₆F₃NO₁₃(545.42): calcd. C 44.04, H 4.80, N 2.07; found C

44.11, H 4.84, N 2.51.

NMR (250 MHz, CDCl₃): δ ϭ 1.16–1.26 (m, 6 H, 2 ϫ CH₃), 1.89–

2.08 (m, 13 H, 4 ϫ COCH₃, 3a-H), 2.50 (dd, J_3e,4ϭ 4.8 Hz, J_3a,3eϭ

13.0 Hz, 1 H, 3e-H), 3.76 (s, 3 H, COOCH₃), 3.78–3.96 (m, 4 H, 2

ϫ CH₂), 4.05 (dd, J_6,5ϭ J_4,5ϭ 10.2 Hz, 1 H, 5-H), 4.16 (dd,

Methyl 4,7,8,9-Tetra-O-acetyl-3,5-dideoxy-5-valeroylamino-

D-gly-

J_8,9Bϭ 7.5 Hz, J_9A,9Bϭ 12.5 Hz, 1 H, 9B-H), 4.40 (dd, J_7,6

ϭ

cero-β- -galacto-2-nonulopyranosonate (21b): To a solution of 20b

D

1.8 Hz, J_5,6ϭ 10.5 Hz, 1 H, 6-H), 4.60 (dd, J_8,9Aϭ 1.9 Hz, J_9B,9Aϭ

12.5 Hz, 1 H, 9A-H), 5.08–5.11 (m, 1 H, 8-H), 5.34–5.42 (m, 2 H,

4-H, 7-H), 7.14 (d, J ϭ 9.9 Hz, 1 H, NH). – ³¹P NMR (167.7 MHz,

CDCl₃): δ_Pϭ 137.94. – FAB-MS (positive mode, matrix: acetone/

nitrobenzyl alcohol, 1:1 and NaI): 688 (M ϩ Na^ϩ).

(250 mg, 0.401 mmol) in methanol (20 mL) was added palladium

on charcoal (30 mg, 10% Pd). The mixture was stirred overnight

under hydrogen at normal pressure. The catalyst was filtered off

and washed with methanol. Evaporation of the solvents and flash

chromatography (toluene/acetone, 3:1) afforded 21b (180 mg, 85%)

as a colourless solid. – DC (toluene/acetone, 4:1): R_fϭ 0.24. – Diethyl [Methyl (4,7,8,9-Tetra-O-acetyl-3,5-dideoxy-5-valeroylam-

[α]_Dϭ ϩ2.9 (c ϭ 1.0 in chloroform). – ¹H NMR (250 MHz,

CDCl₃): δ ϭ 0.89 (t, J ϭ 7.2 Hz, 3 H, CH₃), 1.23–1.34 (m, 2 H,

CH₂CH₃), 1.37–1.59 (m, 2 H, CH₂CH₂CH₃), 1.76–2.35 (m, 16 H,

ino-

D

-glycero-α/β-

D

-galacto-2-nonulopyranosyl)onate]

Phosphite

(22b): To a solution of 21b (180 mg, 0.338 mmol) and ethyldiiso-

propylamine (134 µL) in dry acetonitrile (10 mL) was added diethyl

4 ϫ COCH₃, 3a-H, 3e-H, COCH₂), 3.86 (s, 3 H, COOCH₃), 4.00 chlorophosphite (100 µL, 0.696 mmol) under nitrogen. After

(dd, J_9aA9Bϭ 12.2 Hz, J_8,9Bϭ 7.4 Hz, 1 H, 9B-H), 4.13–4.19 (m, 10 min, the solution was concentrated and coevaporated with tolu-

2 H, 5-H, 6-H), 4.42 (dd, J_9A,9Bϭ 12.1 Hz, 2 H, 9A-H, OH), 5.19– ene. Flash chromatography (toluene/acetone, 3:1) yielded 22b

5.26 (m, 1 H, 4-H, 8-H), 5.30 (dd, J_6,7ϭ 6.5 Hz, 1 H, 7-H), 5.5– (187 mg, 85%) as an anomeric mixture. – TLC (toluene/acetone,

5.63 (m, 1 H, NH). – C₂₃H₃₅NO₁₃(533.53): calcd. C 51.78, H 6.61, 3:1): R_fϭ 0.36.

N 2.62; found C 51.58, H 6.49, N 2.78.

Diethyl [Methyl (4,7,8,9-Tetra-O-acetyl-3,5-dideoxy-5-N-trifluo-

Methyl 4,7,8,9-Tetra-O-acetyl-3,5-dideoxy-5-(N-trifluoracetylgly-

cylamido)- -glycero-β- -galacto-2-nonulopyranosonate

(21c): To a solution of 20c (200 mg, 0.289 mmol) in methanol

(10 mL) was added palladium on charcoal (20 mg, 10% Pd). The

mixture was stirred overnight under hydrogen at normal pressure.

roacetylglycylamido-D-glycero-β-D-galacto-2-nonulopyranosyl)onate]

D

Phosphite (22c): To a solution of 21c (100 mg, 0.166 mmol) and

ethyldiisopropylamine (62 µL) in dry acetonitrile (1.5 mL) was ad-

ded diethyl chlorophosphite (50 µL, 0.332 mmol) under nitrogen.

After 10 min, the solution was concentrated and coevaporated with

1476

Eur. J. Org. Chem. 2000, 1467Ϫ1482

Base- and Sugar-Modified CMP-Neu5Ac Analogues

FULL PAPER

toluene. Flash chromatography (toluene/acetone, 4:1) yielded 22c

1.28–1.43 (m, 11 H, CH₂CH₃, 3 ϫ CH₃), 1.50–1.60 (m, 2 H,

CH₂CH₂CH₃), 1.98–2.22 (m, 24 H, 6 ϫ COCH₃, NHCH₃,

(100 mg, 83%) as a colourless oil. – TLC (toluene/acetone, 4:1):

R_fϭ 0.30. – ¹H NMR (250 MHz, CDCl₃): δ ϭ 1.18–1.41 (m, 6 H, COCH₂, 3a-H), 2.76 (dd, J_3ЈЈe,4ЈЈϭ 4.8 Hz, J_{3ЈЈe,3ЈЈa}ϭ 13.2 Hz, 1

2 CH₃), 1.95–2.19 (m, 13 H, 4 ϫ COCH₃, 3a-H), 2.50 (dd, J_3e,4

4.9 Hz, J_3a,3eϭ 13.1 Hz, 1 H, 3e-H), 3.79 (s, 3 H, COOCH₃), 3.81–

4.19 (m, 8 H, 2 ϫ OCH₂, 5-H, 9A-H, COCH₂), 4.30 (dd, J_6,7

ϭ

H, 3ЈЈe-H), 3.19–3.34 (m, 6 H, 3 ϫ CH₂), 3.83 (s, 3 H, COOCH₃),

4.06–4.12 (m, 1 H, 5ЈЈ-H), 4.23–4.35 (m, 3 H, 5ЈA-H, 5ЈB-H, 9ЈЈB-

H), 4.46–4.52 (m, 2 H, 4Ј-H, 6ЈЈ-H), 4.63 (dd, J_9ЈЈA9ЈЈBϭ 12.1 Hz,

ϭ

2.1 Hz, J_5,6ϭ 10.7 Hz, 1 H, 6-H), 4.59 (dd, J_8,9Bϭ 2.4 Hz, J_9A,9Bϭ J_8ЈЈ,9ЈЈAϭ 2.6 Hz, 1 H, 9ЈЈA-H), 5.26–5.42 (m, 2 H, 4ЈЈ-H, 8ЈЈ-H),

12.3 Hz, 1 H, 9B-H), 5.10 (ddd, J_8,9Aϭ 7.3 Hz, 1 H, 8-H), 5.23 5.46 (dd, J_6ZЈ,7ЈЈϭ 2.0 Hz, J_8ЈЈ,7ЈЈϭ 4.7 Hz, 1 H, 7ЈЈ-H), 5.02–5.11

(ddd, J_{3e,4 ϭ}4.9 Hz, 1 H, 4-H), 5.38 (dd, J_7,8ϭ 2.4 Hz, 1 H, 7-H), (m, 2 H, 2Ј-H, 3Ј-H), 6.24 (d, J_2Ј,1Јϭ 4.4 Hz, 1 H, 1Ј-H), 7.58 (d,

6.05 (m, 1 H, NH), 7.10–7.21 (d, J ϭ 9.8 Hz, 1 H, NH).

J ϭ 7.5 Hz, 1 H, 5-H), 7.82 (d, J ϭ 9.8 Hz, 1 H, NH), 8.52 (d, J ϭ

7.5 Hz, 1 H, 6-H). – ³¹P NMR (242.5 MHz, D₂O): δ_Pϭ –2.23. –

FAB MS (negative mode, matrix: methanol/nitrobenzyl alcohol):

963 (M – H)^–.

Diethyl [Methyl (4,7,8,9-Tetra-O-acetyl-3,5-dideoxy-5-ethoxycar-

bonylamino-D-glycero-β-D-galacto-2-nonulopyranosyl)onate] Phos-

phite (22d): To a solution of 21d (170 mg, 0.336 mmol) and ethyldi-

isopropylamine (120 µL) in dry acetonitrile (3 mL) was added di-

ethyl chlorophosphite (90 µL, 0.626 mmol) under nitrogen. After

10 min, the solution was concentrated and coevaporated with tolu-

ene. Flash chromatography (toluene/acetone, 4:1) yielded 22d

(188 mg, 90%) as a colourless oil. – TLC (toluene/acetone, 4:1):

Triethylammonium (N-Acetyl-2Ј,3Ј-di-O-acetylcytidin-5Ј-yl) {Meth-

yl

[4,7,8,9-Tetra-O-acetyl-3,5-dideoxy-5-(N-trifluoroacetylglycyl-

-glycero-β- -galacto-2-nonulopyranosyl]onate)} Phosphate

amido)-

D

(24c): A solution of 23 (94 mg, 0.208 mmol) in dry N,N-dimethyl-

formamide/acetonitrile (2:1, 1.0 mL) was cooled to –20 °C. A solu-

R_fϭ 0.32. – [α]_Dϭ –19.2 (c ϭ 1.0 in chloroform). – ¹H NMR tion of 22c (100 mg, 0.138 mmol) in dry acetonitrile (0.4 mL) was

(250 MHz, CDCl₃): δ ϭ 1.10–1.29 (m, 9 H, 3 ϫ CH₃), 1.92–2.12 added by syringe. The reaction mixture was allowed to warm up

(m, 13 H, 4 ϫ COCH₃, 3a-H), 2.48 (dd, J_3e,4ϭ 4.9 Hz, J_3a,3e

13.1 Hz, 1 H, 3e-H), 3.77 (s, 3 H, COOCH₃), 3.81–4.06 (m, 6 H, 2 ded and the solvents were evaporated in vacuo. Flash chromato-

ϫ OCH₂, COOCH₂), 4.11 (dd, J ϭ 10.4 Hz, 1 H, 5-H), 4.12–4.20 graphy (chloroform/methanol/triethylamine, 7:1:0.1) yielded 24c

(m, 2 H, 6-H, 9A-H), 4.42 (d, J ϭ 9.9 Hz, 1 H, NH), 4.58 (dd, (70 mg, 45%) as a colourless solid. – TLC (chloroform/methanol,

ϭ

to room temperature during 12 h. Triethylamine (500 µL) was ad-

J_8,9Bϭ 2.2 Hz, J_9A,9Bϭ 12.3 Hz, 1 H, 9B-H), 5.15 (ddd, J_8,9A

7.9 Hz, J_8,9Bϭ 2.2 Hz, J_7,8ϭ 2.8 Hz, 1 H, 8-H), 5.22 (ddd, J_3e,4

4.9 Hz, J_4,5ϭ 10.4 Hz, J_3a,4ϭ 11.1 Hz, 1 H, 4-H), 5.48 (dd, J_7,8

ϭ

3:1): R_fϭ 0.51. – ¹H NMR (250 MHz, D₂O): δ ϭ 1.79–2.03 (m,

22 H, 7 ϫ COCH₃, 3a-H), 2.44 (dd, J_3ЈЈe,4ЈЈϭ 4.9 Hz, J_{3ЈЈe,3ЈЈa}

13.3 Hz, 1 H, 3ЈЈe-H), 3.63 (s, 3 H, COOCH₃), 3.70–3.71 (m, 2 H,

COCH₂), 3.79 (dd, J_4ЈЈ,5ЈЈϭ J_6ЈЈ,5ЈЈϭ 10.5 Hz, 1 H, 5ЈЈ-H), 3.98–

ϭ

2.8 Hz, 1 H, 7-H). – ³¹P NMR (161.7 MHz, CDCl₃): δ_Pϭ 137.28.

4.07 (m, 2 H, 5ЈA-H, 5ЈB-H), 4.15 (dd, J_8ЈЈ.9ЈЈBϭ 6 Hz, J_{9ЈЈA,9ЈЈB}

12.3 Hz, 1 H, 9ЈЈB-H), 4.27–4.36 (m, 3 H, 4Ј-H, 6ЈЈ-H, 9ЈЈA-H),

ϭ

Triethylammonium (N-Acetyl-2Ј,3Ј-di-O-acetylcytidin-5Ј-yl) [Meth-

yl (4,7,8,9-Tetra-O-acetyl-3,5-dideoxy-5-trifluoroacetamido-

D-gly-

5.01–5.12 (m, 2 H, 4ЈЈ-H, 8ЈЈ-H), 5.20 (dd, J_6ЈЈ,7ЈЈϭ 1.2 Hz, J_8ЈЈ,7ЈЈ

4.8 Hz, 1 H, 7ЈЈ-H), 5.28–5.36 (m, 2 H, 2Ј-H, 3Ј-H), 6.00 (d, J_2Ј,1Ј

4.1 Hz, 1 H, 1Ј-H), 7.22 (d, J ϭ 7.5 Hz, 1 H, 5-H), 8.17 (d, J ϭ

7.5 Hz, 1 H, 6-H). – ³¹P NMR (242.5 MHz, D₂O): δ_Pϭ –1.91. –

FAB MS (negative mode, matrix: glycerol/nitrobenzyl alcohol, 1:1):

1032 (M – H)^–.

ϭ

cero-β- -galacto-2-nonulopyranosyl)onate] Phosphate (24a): A solu-

D

tion of 23^[3](840 mg, 1.88 mmol) in dry N,N-dimethylformamide/

acetonitrile (2:1, 6.0 mL) was cooled to –20 °C. A solution of 22a

(780 mg, 1.17 mmol) in dry acetonitrile (3 mL) was added by syr-

inge. The reaction mixture was allowed to warm up to room tem-

perature during 12 h. Triethylamine (500 µL) was added and the

solvents were evaporated in vacuo. Flash chromatography (chloro-

form/methanol/triethylamine, 8:1:0.1) yielded 24a (706 mg, 56%) as

a colourless solid. – TLC (chloroform/methanol, 6:1): R_fϭ 0.32. –

¹H NMR (250 MHz, D₂O): δ ϭ 1.33 (t, J ϭ 7.2 Hz, 9 H, 3 ϫ

Triethylammonium (N-Acetyl-2Ј,3Ј-di-O-acetylcytidin-5Ј-yl) [Meth-

yl

(4,7,8,9-Tetra-O-acetyl-3,5-dideoxy-5-ethoxycarbonylamino-

D-

glycero-β-D-galacto-2-nonulopyranosyl)onate] Phosphate (24d): A

solution of 23 (180 mg, 0.400 mmol) in dry N,N-dimethylformam-

ide/acetonitrile (2:1, 1.5 mL) was cooled to –20 °C. A solution of

CH₃), 1.95–2.22 (m, 22 H, 7 ϫ COCH₃, 3a-H), 2.80 (dd, J_3ЈЈe,4ЈЈ

ϭ

5.0 Hz, J_{3ЈЈe,3ЈЈa}ϭ 13.2 Hz, 1 H, 3ЈЈe-H), 3.15–3.30 (m, 6 H, 3 ϫ 22d (150 mg, 0.234 mmol) in dry acetonitrile (0.5 mL) was added

CH₂), 3.85 (s, 3 H, COOCH₃), 4.08 (dd, J_4ЈЈ,5ЈЈϭ J_6ЈЈ,5ЈЈϭ 10.5 Hz, by syringe. The reaction mixture was allowed to warm up to room

1 H, 5ЈЈ-H), 4.19–4.39 (m, 3 H, 5ЈA-H, 5ЈB-H, 9ЈЈ-B-H), 4.40–4.45 temperature during 12 h. Triethylamine (500 µL) was added and

(m, 1 H, 4Ј-H), 4.56–4.67 (m, 2 H, 6ЈЈ-H, 9ЈЈA-H), 5.29–5.34 (m,

the solvents were evaporated in vacuo. Flash chromatography

1 H, 8ЈЈ-H), 5.40–5.49 (m, 2 H, 4ЈЈ-H, 8ЈЈ-H), 5.51–5.57 (m, 2 H, (chloroform/methanol/triethylamine, 7:1:0.1) yielded 24d (150 mg,

2Ј-H, 3Ј-H), 6.24 (d, J_2Ј,1Јϭ 4.3 Hz, 1 H, 1Ј-H), 7.58 (d, J ϭ 7.5 Hz, 61%) as a colourless solid. – TLC (chloroform/methanol, 3:1): R_fϭ

1 H, 5-H), 8.50 (d, J ϭ 7.5 Hz, 1 H, 6-H). – ³¹P NMR (161.7 MHz,

0.55. – ¹H NMR (250 MHz, MeOD): δ ϭ 1.18–1.40 (m, 12 H, 4 ϫ

MeOD): δ_Pϭ –5.50. – FAB MS (negative mode matrix: methanol/ CH₃), 1.98–2.22 (m, 22 H, 7 ϫ COCH₃, 3aЈЈ-H), 2.74 (dd, J_3ЈЈe,4ЈЈ

ϭ

nitrobenzyl alcohol, 1:1): 975 (M – H^ϩ)^–.

4.8 Hz, J_{3ЈЈe,3ЈЈa}ϭ 13.2 Hz, 1 H, 3ЈЈe-H), 3.25 (q, J ϭ 7.3 Hz, 6 H,

3 ϫ CH₂), 3.65–3.78 (m, 1 H, 5-H), 3.83 (s, 3 H, COOCH₃), 4.00–

4.10 (m, 2 H, COCH₂), 4.17–4.49 (m, 3 H, 5ЈA-H, 5ЈB-H, 9ЈЈB-H),

4.44–4.49 (m, 2 H, 4Ј-H, 6ЈЈ-H), 4.63 (dd, J_8ЈЈ,9ЈЈAϭ 2.8 Hz,

J_{9ЈЈA,9ЈЈB}ϭ 12.2 Hz, 1 H, 9ЈЈA-H), 5.23–5.38 (m, 2 H, 4ЈЈ-H, 8ЈЈ-

H), 5.53–5.59 (m, 3 H, 2Ј-H, 3Ј-H, 7ЈЈ-H), 6.24 (d, J_2Ј,1Јϭ 4.1 Hz,

1 H, 1Ј-H), 6.70 (d, J ϭ 10.2 Hz, 1 H, NH), 7.59 (d, J ϭ 7.5 Hz,

1 H, 5-H), 8.56 (d, J ϭ 7.5 Hz, 1 H, 6-H). – ³¹P NMR (161.7 MHz,

D₂O): δ_Pϭ –5.44. – FAB MS (negative mode, matrix: methanol,

glycerol/nitrobenzyl alcohol, 5:1): 951 (M – H)^–.

Triethylammonium (N-Acetyl-2Ј,3Ј-di-O-acetylcytidin-5Ј-yl) [Meth-

yl (4,7,8,9-Tetra-O-acetyl-3,5-dideoxy-5-valeroylamino-

D-glycero-β-

D-galacto-2-nonulopyranosyl)onate] Phosphate (24b): A solution of

23 (200 mg, 0.445 mmol) in dry N,N-dimethylformamide/acetonitr-

ile (2:1, 1.5 mL) was cooled to –20 °C. A solution of 22b (180 mg,

0.276 mmol) in dry acetonitrile (0.5 mL) was added by syringe. The

reaction mixture was allowed to warm up to room temperature

during 12 h. Triethylamine (500 µL) was added and the solvents

were evaporated in vacuo. Flash chromatography (chloroform/

methanol/triethylamine, 8:1:0.1) yielded 24b (143 mg, 49%) as a Lithium [(5-Amino-3,5-dideoxy-

colourless solid. – TLC (chloroform/methanol, 8:1): R_fϭ 0.26. – anosyl)onate] (Cytidin-5Ј-yl) Phosphate (25a): To a solution of 24a

¹H NMR (250 MHz, D₂O): δ ϭ 0.95 (t, J ϭ 7.2 Hz, 3 H, CH₃), (400 mg, 0.371 mmol) in dry methanol (10 mL) was added sodium

D-glycero-β-D-galacto-2-nonulopyr-

Eur. J. Org. Chem. 2000, 1467Ϫ1482

1477

G. Dufner, R. Schwörer, B. Müller, R. R. Schmidt

FULL PAPER

methoxide solution (0.5 , 5 drops) and the mixture was stirred for

3 h at room temp. The mixture was neutralized with Amberlite

IRC-50 (H^ϩ) (pH ϭ 8–9), filtered, and concentrated. The residue

was dissolved in water/methanol (1:1, 7 mL) and lithium hydroxide

solution (1 , 2 mL) was added. After stirring for 4 h at room

Disodium (Cytidin-5Ј-yl) [(3,5-Dideoxy-5-ethoxycarbonylamino-

D-

glycero-β- -galacto-2-nonulopyranosyl)onate] Phosphate (25d): To a

D

solution of 24d (125 mg, 0.119 mmol) in dry methanol (3 mL) was

added sodium methanolate solution (0.5 , 3 drops) and the mix-

ture was stirred for 3 h at room temp. The mixture was neutralized

temp., the solution was neutralized with Amberlite IRC-50 (H^ϩ) with Amberlite IRC-50 (H^ϩ) (pH ϭ 8–9), filtered and concen-

(pH ϭ 8–9) and filtered. Lyophilization of the filtrate and preparat-

ive HPLC (RP-18, 0.05  NEt₃H₂CO₃) yielded 25a (110 mg,

trated. The residue was dissolved in water/methanol (1:1, 1 mL)

and sodium hydroxide solution (1 , 1 mL) was added. After stir-

50%). – TLC, aminophase (ethanol/water, 1:1): R_fϭ 0.39. – ¹H ring for 4 h at room temp., the solution was neutralized with Am-

NMR (250 MHz, D₂O): δ ϭ 1.45 (ddd, J_3ЈЈa,Pϭ 5.8 Hz, J _{3ЈЈa,3ЈЈe}ϭ berlite IRC-50 (H^ϩ) (pH ϭ 8–9) and filtered. The filtrate was lyo-

13.2 Hz, J_3ЈЈa,4ЈЈϭ 11.1 Hz, 1 H, 3ЈЈa-H), 2.28 (dd, J_3ЈЈe,4ЈЈϭ 4.8 Hz, philized, redissolved in acetone and precipitated by addition of

J_{3ЈЈe,3ЈЈa}ϭ 13.2 Hz, 1 H, 3ЈЈe-H), 2.80 (d, J ϭ 10.2 Hz, 1 H, 5ЈЈ-H),

water. Centrifugation and drying under reduced pressure afforded

3.42 (dd, J ϭ 9.5 Hz, 1 H, 7ЈЈ-H), 3.40–3.51 (m, 1 H, 9ЈЈA-H). – 25b (47 mg, 60%). – TLC, aminophase (acetonitrile/water, 2:1):

1

³¹P NMR (161.7 MHz, D₂O): δ ϭ –4.26. – FAB MS (negative R_fϭ 0.19. – H NMR (250 MHz, D₂O): δ ϭ 1.04 (t, J ϭ 7.0 Hz,

mode matrix: DMSO/glycerol, 1:1): 571 (M – H^ϩ)^–.

3 H, CH₃), 1.42 (ddd, J_{3ЈЈa,3ЈЈe}ϭ 13.2 Hz, 1 H, 3ЈЈa-H), 2.24 (dd,

J_3ЈЈe,4ЈЈϭ 4.8 Hz, J_{3ЈЈe,3ЈЈa}ϭ 13.2 Hz, 1 H, 3ЈЈe-H), 3.32 (dd, J ϭ

9.5 Hz, 1 H, 7ЈЈ-H), 3.37–3.52 (m, 2 H, 5ЈЈ-H, 9ЈЈA-H), 3.65–3.77

Disodium (Cytidin-5Ј-yl) [(3,5-Dideoxy-5-valeroylamino-D-glycero-

β-D-galacto-2-nonulopyranosyl)onate] Phosphate (25b): To a solu-

(m, 2 H, 6ЈЈ-H, 9ЈЈB-H), 3.82 (dd, J_3ЈЈe,4ЈЈϭ 4.8 Hz, J_4ЈЈ,5ЈЈ

ϭ

10.7 Hz, 1 H, 4ЈЈ-H), 3.88–3.99 (m, 3 H, CH₂, 8ЈЈ-H), 4.00–4.10

(m, 5 H, 2Ј-H, 3Ј-H, 4Ј-H, 5ЈA-H, 5ЈB-H), 5.79 (d, J_2Ј,1Јϭ 4.2 Hz,

1 H, 1Ј-H), 5.91 (d, J ϭ 7.5 Hz, 1 H, 5-H), 7.78 (d, J ϭ 7.5 Hz, 1

H, 6-H). – ³¹P NMR (161.7 MHz, D₂O): δ_Pϭ –4.17. – FAB MS

(negative mode, matrix: DMSO/glycerol/acetic acid, 1:1:1): 665 (M

ϩ Na)^–.

tion of 24b (100 mg, 0.11 mmol) in dry methanol (3 mL) was added

sodium methoxide solution (0.5 , 3 drops) and the mixture was

stirred for 3 h at room temp. The mixture was neutralized with

Amberlite IRC-50 (H^ϩ) (pH ϭ 8–9), filtered and concentrated. The

residue was dissolved in water/methanol (1:1, 1 mL) and sodium

hydroxide solution (1 , 1 mL) was added. After stirring for 4 h at

room temp., the solution was neutralized with Amberlite IRC-50

(H^ϩ) (pH 8–9) and filtered. The filtrate was lyophilized, redissolved

in water and precipitated by addition of acetone. Centrifugation

and drying under reduced pressure afforded 25b (39 mg, 61%). –

Methyl 4,5,7,8,9-Penta-O-acetyl-3-deoxy-D-glycero-β-D-galacto-2-

nonulopyranosonate (27): Compound 26^[23](500 mg, 1.77 mmol)

was dissolved in acetyl chloride (12 mL) and the mixture was

stirred for 6 h at room temp. The solution was concentrated and

coevaporated with toluene. The residue was redissolved in aceton-

itrile/water (10 mL, 1:1) and stirred for 3 h at room temp. The reac-

tion mixture was diluted with half-saturated sodium bicarbonate

solution and extracted with ethyl acetate. The aqueous layer was

concentrated to a half of its original volume and extracted again

with ethyl acetate. The combined organic layers were dried with

magnesium sulfate and concentrated. Flash chromatography (tolu-

ene/acetone, 3:1) afforded 27 (565 mg, 65%) as a colourless solid. –

TLC (toluene/acetone 4:1): R_fϭ 0.40. NMR data were identical

with published data.

1

TLC, aminophase (acetonitrile/water, 2:1): R_fϭ 0.35. – H NMR

(250 MHz, D₂O): δ ϭ 0.69 (t, J ϭ 7.3 Hz, 3 H, CH₃), 1.11–1.20

(m, 2 H, CH₂CH₃), 1.31–1.50 (m, 3 H, CH₂CH₂CH_3,3ЈЈa-H),

2.05–2.11 (m, 2 H, COCH₂), 2.26 (dd, J_3ЈЈe,4ЈЈϭ 4.3 Hz, J_{3ЈЈe,3ЈЈa}

13.2 Hz, 1 H, 3ЈЈe-H), 3.22 (dd, J ϭ 9.5 Hz, 1 H, 7ЈЈ-H), 3.38 (dd,

_8ЈЈ,9ЈЈBϭ 9.7 Hz, J_{9ЈЈA,9ЈЈB}ϭ 12.0 Hz, 1 H, 9ЈЈ-B-H), 3.61–3.78 (m,

ϭ

J

2 H, 9ЈЈA-H, 8ЈЈ-H), 3.78 (d, J_5ЈЈ.6ЈЈϭ 10.0 Hz, 1 H, 5ЈЈ-H), 3.82

(dd, J_3ЈЈe,4ЈЈϭ 4.3 Hz, J_4ЈЈ,5ЈЈϭ 10.2 Hz, 1 H, 4ЈЈ-H), 3.93 (dd,

J_5ЈЈ,6ЈЈϭ 10 Hz, 1 H, 6ЈЈ-H), 3.99–4.18 (m, 5 H, 2Ј-H, 3Ј-H, 4Ј-H,

5ЈA-H, 5ЈB-H), 5.79 (d, J_2Ј,1Јϭ 4.2 Hz, 1 H, 1Ј-H), 5.90 (d, J ϭ

7.4 Hz, 1 H, 5-H), 7.76 (d, J ϭ 7.4 Hz, 1 H, 6-H). – ³¹P NMR

(242.5 MHz, D₂O): δ_Pϭ –0.78. – FAB MS (negative mode, matrix:

glycerol): 676 [(M ϩ Na^ϩ)^–].

Diethyl [Methyl (4,5,7,8,9-Penta-O-acetyl-3-deoxy-D-glycero-β-D-

galacto-2-nonulopyranosyl)onate] Phosphite (28): To a solution of

27 (360 mg, 0.732 mmol) and ethyldiisopropylamine (270 µL) in

dry acetonitrile (8.0 mL) was added diethyl chlorophosphite (216

µL, 1.50 mmol) under nitrogen. After 10 min, the solution was con-

centrated and coevaporated with toluene. Flash chromatography

(toluene/acetone, 6:1) yielded 28 (390 mg, 87%) as a colourless

oil. – TLC (toluene/acetone, 6:1): R_fϭ 0.48. – [α]_Dϭ –8.9 (c ϭ 1.0

Potassium (Cytidin-5Ј-yl) [(3,5-Dideoxy-5-glycylamido-

D-glycero-β-

D-galacto-2-nonulopyranosyl)onate] Phosphate (25c): Compound

24c (30 mg, 26.4 µmol) was dissolved in water/methanol (1:9,

0.5 mL) and potassium carbonate (3 mg, 21.7 µmol) was added.

After stirring for 20 h at room temp, the solution was neutralized

with Amberlite IRC-50 (H^ϩ) (pH ϭ 8–9) and filtered. The filtrate

was lyophilized, filtered on a column of P₂-Biogel and concen-

trated. The residue was dissolved in water and precipitated by addi-

tion of acetone. Centrifugation and drying under reduced pressure

afforded 25c (14 mg, 71%). – TLC, aminophase (ethanol/water,

1:1): R_fϭ 0.23. – ¹H NMR (250 MHz, D₂O): δ ϭ 1.45 (ddd,

1

in chloroform). – H NMR (250 MHz, CDCl₃): δ ϭ 1.20–1.29 (m,

6 H, 2 ϫ CH₃), 1.89–2.13 (m, 16 H, 5 ϫ COCH₃, 3a-H), 2.53 (dd,

J_3e,4ϭ 5.1 Hz, J_3a,3eϭ 13.1 Hz, 1 H, 3e-H), 3.76 (s, 3 H, CO-

OCH₃), 3.76–3.99 (m, 4 H, 2 ϫ CH₂), 4.15 (dd, J_8,9Bϭ 6.5 Hz,

J_9A,9Bϭ 12.4 Hz, 1 H, 9B-H), 4.33 (dd, J_7,6ϭ 2.1 Hz, J_5,6

ϭ

10.2 Hz, 1 H, 6-H), 4.50 (dd, J_8,9Aϭ 2.3 Hz, J_9B,9Aϭ 12.4 Hz, 1

H, 9A-H), 4.90 (dd, J_6,5ϭ J_4,5ϭ 9.9 Hz, 1 H, 5-H), 5.14–5.19 (m,

J

ϭ 13.0 Hz, 1 H, 3ЈЈa-H), 2.26 (dd, J_3ЈЈe,4ЈЈϭ 4.8 Hz,

_{3ЈЈe,3ЈЈa}ϭ 13.0 Hz, 1 H, 3ЈЈe-H), 3.22 (dd, J ϭ 9.5 Hz, 1 H, 7ЈЈ-H),

3ЈЈa,3ЈЈe

1 H, 4-H), 5.20–5.31 (m, 1 H, 8-H), 5.38 (dd, J_6,7ϭ 2.1 Hz, J_8,7

ϭ

5.1 Hz, 1 H, 7-H). – ³¹P NMR (161.7 MHz, CDCl₃): δ_Pϭ 136.65. –

FAB-MS (positive mode, matrix: acetone/nitrobenzyl alcohol, 1:1,

and NaI): 613 (M ϩ H^ϩ).

3.41 (dd, J_8ЈЈ,9ЈЈBϭ 6.3 Hz, J_{9ЈЈA,9ЈЈB}ϭ 11.8 Hz, 1 H, 9ЈЈB-H), 3.58

(d, J ϭ 3.3 Hz, 1 H, 0.5 ϫ COCH₂), 3.64 (d, J ϭ 3.3 Hz, 1 H, 0.5

ϫ COCH₂), 3.67–3.78 (m, 2 H, 8ЈЈ-H, 9ЈЈB-H), 3.78 (d, J_5ЈЈ,6ЈЈ

ϭ

10.1 Hz, 1 H, 5ЈЈ-H), 3.82 (dd, J_3ЈЈe,4ЈЈϭ 4.8 Hz, J_4ЈЈ,5ЈЈϭ 10.1 Hz,

Sodium (N-Acetyl-2Ј,3Ј-di-O-acetylcytidin-5Ј-yl) [Methyl (4,5,7,8,9-

1 H, 4ЈЈ-H), 3.88–3.99 (d, J_5ЈЈ,6ЈЈϭ 10.1 Hz, 1 H, 6ЈЈ-H), 4.00–4.19 Penta-O-acetyl-3-deoxy-D-glycero-β-D-galacto-2-nonulopyranosyl)-

(m, 5 H, 2Ј-H, 3Ј-H, 4Ј-H, 5ЈA-H, 5ЈB-H), 5.78 (d, J_2Ј,1Јϭ 4.2 Hz, onate] Phosphate (29): A solution of 23 (256 mg, 0.570 mmol) in

1 H, 1Ј-H), 5.91 (d, J ϭ 7.5 Hz, 1 H, 5-H), 7.76 (d, J ϭ 7.5 Hz, 1 dry N,N-dimethylformamide/acetonitrile (2:1, 3 mL) was cooled

H, 6-H). – ³¹P NMR (242.5 MHz, D₂O): δ_Pϭ –0.85.

to –20 °C. A solution of 28 (218 mg, 0.357 mmol) in dry acetoni-

1478

Eur. J. Org. Chem. 2000, 1467Ϫ1482

Base- and Sugar-Modified CMP-Neu5Ac Analogues

trile (1 mL) was added by syringe. The reaction mixture was allowed 1.69 mmol) was dissolved in acetic acid/water (4:1, 15 mL) and the

FULL PAPER

to warm up to room temperature during 12 h. Triethylamine (500

µL) was added and the solvents were evaporated in vacuo. Flash

chromatography (chloroform/methanol/triethylamine, 7:1:0.1) af-

forded a colourless solid, which was redissolved in dry methanol

(10 mL). Sodium methoxide solution (0.5 , 5 drops) was added

and the mixture was stirred for 1 h at room temp. The mixture was

neutralized with Amberlite IRC-50 (H^ϩ) (pH ϭ 8–9), filtered and

concentrated. Flash chromatography over aminophase silica gel

mixture was heated to 80 °C. After 2 h, the solution was concen-

trated and purified by flash chromatography (toluene/acetone, 1:1),

to yield 33 (654 mg, 85%) as a colourless solid. – TLC (toluene/

acetone, 1:1): R_fϭ 0.17. – [α]_Dϭ –12.4 (c ϭ 1.0 in chloroform). –

¹H NMR (250 MHz, CDCl₃): δ ϭ 1.97 (s, 3 H, NHCOCH₃), 2.04–

2.15 (m, 4 H, 3a-H, COCH₃), 2.48 (br. s, 1 H, OH), 2.65 (dd, J_3e,4ϭ

4.9 Hz, J_3e,3aϭ 12.9 Hz, 1 H, 3e-H), 3.46 (dd, J_6,7ϭ 1.6 Hz, J_5,6ϭ

10.4 Hz, 1 H, 6-H), 3.51–3.57 (m, 1 H, 7-H), 3.74 (s, 3 H, CO-

OCH₃), 3.75–3.79 (m, 2 H, 9A-H, OH), 3.85–4.04 (m, 3 H, 5-H, 8-

(ethanol/water, 3:1) yielded 29 (82 mg, 38%). – TLC, aminophase

1

(ethanol/water, 2:1): R_fϭ 0.37. – H NMR (250 MHz, D₂O): δ ϭ H, 9B-H), 4.46 (d, J ϭ 11.6 Hz, 1 H, 0.5 ϫ CH₂Ph), 4.84 (d, J ϭ

1.50 (ddd, J_3ЈЈa,Pϭ 5.7 Hz, J_3ЈЈa,4ЈЈϭ 11.5 Hz, J_{3ЈЈe,3ЈЈa}ϭ 13.4 Hz,

11.6 Hz, 1 H, 0.5 ϫ CH₂Ph), 4.77 (m, 1 H, OH), 4.90 (ddd, J_4,5ϭ

1 H, 3ЈЈa-H), 2.24 (dd, J_3ЈЈe,4ЈЈϭ 4.8 Hz, J_{3ЈЈe,3ЈЈa}ϭ 13.4 Hz, 1 H, 10.5 Hz, J_3e,4ϭ 4.9 Hz, 1 H, 4-H), 5.95 (d, J_5,NHϭ 7.6 Hz, 1 H,

3ЈЈe-H), 3.42 (dd, J_6ЈЈ,5ЈЈϭ J_4ЈЈ,5ЈЈϭ 9.5 Hz, 1 H, 5ЈЈ-H), 3.34–3.49

(m, 1 H, 9ЈЈB-H), 3.55 (dd, J ϭ 9.6 Hz, 1 H, 7ЈЈ-H), 3.63 (s, 3 H,

NH), 7.19–7.30 (m, 5H, Ph). – C₂₁H_29NO₁₀0.5 H₂O (464.47):

calcd. C 54.30, H 6.51, N 3.00; found C 54.20, H 6.59, N 3.00.

COOCH₃), 3.69–3.79 (m, 2 H, 8ЈЈ-H, 9ЈЈA-H), 3.81 (ddd, J_3ЈЈa,4ЈЈ

ϭ

Methyl (Benzyl 5-Acetamido-4-O-acetyl-7,9-O-benzylidene-3,5-di-

deoxy-D-glycero-α-D-galacto-2-nonulopyranosid)onate (34): To a so-

11.5 Hz, J_3ЈЈe,4ЈЈϭ 4.8 Hz, 1 H, 4ЈЈ-H), 3.89 (dd, J_6ЈЈ,5ЈЈϭ 9.5 Hz,

1 H, 6ЈЈ-H), 3.95–4.15 (m, 5 H, 2Ј-H, 3Ј-H, 4Ј-H, 5ЈA-H, 5ЈB-H),

5.80 (d, J_2Ј,1Јϭ 3.7 Hz, 1 H, 1Ј-H), 5.89 (d, J ϭ 7.5 Hz, 1 H, 5-H),

7.73 (d, J ϭ 7.5 Hz, 1 H, 6-H). – ³¹P NMR (161.7 MHz, D₂O):

δ_Pϭ –1.13. – FAB MS (negative mode, matrix: glycerol/water, 1:1):

586 [(M – H^ϩ)^–].

lution of 33 (590 mg, 1.30 mmol) in dry acetonitrile (8.0 mL) was

added p-toluenesulfonic acid monohydrate (8.0 mg) and benzal-

dehyde dimethylacetal (590 µL, 3.93 mmol). After stirring for 15 h,

the reaction mixture was poured into half-saturated sodium bicar-

bonate solution and extracted with ethyl acetate. The organic layer

was dried with magnesium sulfate and concentrated. Flash chroma-

tography (toluene/acetone, 1:1) yielded 34 (590 mg, 84%) as a col-

Diammonium (Cytidin-5Ј-yl) [(3-Deoxy-

D-glycero-β-D-galacto-2-

nonulopyranosyl)onate] Phosphate (30): To a solution of 29 (40 mg,

65.5 µmol) in water/methanol (1:1, 2 mL) was added sodium hy- ourless foam. – TLC (toluene/acetone, 1:1): R_fϭ 0.36 – [α]_Dϭ –

1

droxide solution (1 , 1 mL) and the mixture was stirred for 3 h at

room temp. The solution was neutralized with Amberlite IRC-50

(H^ϩ) (pH ϭ 8–9) and filtered. The filtrate was lyophilized and puri-

fied by preparative paper chromatography (isopropyl alcohol/water/

54.2 (c ϭ 1.0 in chloroform). – H NMR (250 MHz, CDCl₃): δ ϭ

1.93 (s, 3 H, NHCOCH₃), 1.95–2.05 (m, 4 H, 3a-H, COCH₃), 2.65

(dd, J_3e,4ϭ 5.0 Hz, J_3e,3aϭ 12.6 Hz, 1 H, 3e-H), 3.95 (d, J ϭ

4.8 Hz, 1 H, OH), 3.59–3.71 (m, 2 H, 7-H, 9A-H), 3.76 (s, 3 H,

ammonia, 55:35:10) to give 30 (30 mg, 79%). – TLC, aminophase COOCH₃), 3.99 (dd, J_6,7ϭ 1.1 Hz, J_5,6ϭ 10.4 Hz, 1 H, 6-H), 4.15–

1

(ethanol/water, 1:1): R_fϭ 0.31. – H NMR (250 MHz, D₂O): δ ϭ 4.40 (m, 3 H, 5-H, 8-H, 9B-H), 4.41 (d, J ϭ 11.6 Hz, 1 H, 0.5 ϫ

1.42 (ddd, J_3ЈЈa,Pϭ 5.5 Hz, J_3ЈЈa,4ЈЈϭ 11.6 Hz, J_{3ЈЈe,3ЈЈa}ϭ 12.8 Hz,

1 H, 3ЈЈa-H), 2.24 (dd, J_3ЈЈe,4ЈЈϭ 4.8 Hz, J_{3ЈЈe,3ЈЈa}ϭ 12.8 Hz, 1 H, 10.5 Hz, J_3e,4ϭ 5.0 Hz, 1 H, 4-H), 5.28 (d, J_5,NHϭ 7.6 Hz, 1 H,

3ЈЈe-H), 3.39 (d, J_5ЈЈ.6ЈЈϭ 9.8 Hz, 1 H, 5ЈЈ-H), 3.49 (dd, J_8ЈЈ,9ЈЈBNH), 5.45 (s, 1 H, CHPh), 7.19–7.61 (m, 10 H, 2 ϫ Ph). –

CH₂Ph), 4.80 (d, J ϭ 11.6 Hz, 1 H, 0.5 ϫ CH₂Ph), 4.99 (ddd, J_4,5ϭ

ϭ

5.4 Hz, J_{9ЈЈA,9ЈЈB}ϭ 12.3 Hz, 1 H, 9ЈЈ-B-H), 3.59 (dd, J ϭ 7.5 Hz, 1 C₂₈H₃₃NO₁₀(543.57): calcd. C 61.87, H 6.12, N 2.58; found C

H, 7ЈЈ-H), 3.70–3.86 (m, 3 H, 4ЈЈ-H, 9ЈЈA-H, 8ЈЈ-H), 3.91 (dd, 61.85, H 6.14, N 2.64.

J_5ЈЈ,6ЈЈϭ 9.8 Hz, 1 H, 6ЈЈ-H), 3.99–4.18 (m, 5 H, 2Ј-H, 3Ј-H, 4Ј-H,

Methyl (Benzyl 5-Acetamido-4-O-acetyl-7,9-O-benzylidene-3,5-di-

5ЈA-H, 5ЈB-H), 5.79 (d, J_2Ј,1Јϭ 4.2 Hz, 1 H, 1Ј-H), 5.92 (d, J ϭ

deoxy-8-O-methyl-D-glycero-α-D-galacto-2-nonulopyranosid)onate

7.4 Hz, 1 H, 5-H), 7.79 (d, J ϭ 7.4 Hz, 1 H, 6-H). – ³¹P NMR

(161.7 MHz, D₂O): δ_Pϭ –4.07. – FAB MS (negative mode, matrix:

water/glycerol): 572 [(M – H)^–].

(35): Alcohol 34 (370 mg, 0.681 mmol) was dissolved in methyl iod-

ide (3 mL). Drierite (330 mg, 2.43 mmol) and freshly prepared sil-

ver oxide (260 mg, 1.13 mmol) were added and the mixture was

stirred overnight at room temp. The reaction mixture was filtered

Methyl (Benzyl 5-Acetamido-4-O-acetyl-3,5-dideoxy-8,9-O-isoprop-

ylidene-D-glycero-α-D-galacto-2-nonulopyranosid)onate (32): To a through Celite. The filtrate was treated with half-saturated sodium

solution of 31^[23](1.10 g, 2.43 mmol) in dry dichloromethane

(12.0 mL) was added dry pyridine (600 µL) and acetic anhydride

(570 µL). After stirring overnight the solution was concentrated

and coevaporated with toluene. Flash chromatography (toluene/

bicarbonate solution and extracted with ethyl acetate. The organic

layer was dried with magnesium sulfate and concentrated in vacuo.

Flash chromatography (toluene/acetone, 3:1) yielded 35 (338 mg,

89%) as a colourless foam. – TLC (toluene/acetone, 2:1): R_fϭ

acetone, 3:1) afforded 32 (1.26 g, 88%) as a colourless solid. – TLC 0.33. – [α]_Dϭ –59.8 (c ϭ 1.0 in chloroform). – ¹H NMR (250 MHz,

1

(toluene/acetone): R_fϭ 0.51. – H NMR (250 MHz, CDCl₃): δ ϭ

CDCl₃): δ ϭ 1.93 (s, 3 H, NHCOCH₃), 1.95–2.05 (m, 4 H, 3a-H,

1.39 (s, 3 H, CH₃)_,1.36 (s, 3 H, CH₃), 1.93 (s, 3 H, NHCOCH₃), COCH₃), 2.59 (dd, J_3e,4ϭ 4.7 Hz, J_3e,3aϭ 12.8 Hz, 1 H, 3e-H),

1.94–2.07 (m, 4 H, 3a-H, COCH₃), 2.65 (dd, J_3e,4ϭ 4.9 Hz, J_3e,3aϭ 3.50–3.59 (m, 2 H, 7-H, 9A-H), 3.52 (s, 3 H, OCH₃), 3.69 (dd,

12.8 Hz, 1 H, 3e-H), 3.42–3.54 (m, 2 H, 6-H, 7-H), 3.70 (s, 3 H,

COOCH₃), 3.92 (dd, J_6,5ϭ 10.3 Hz, J_NH,5ϭ 7.9 Hz, 1 H, 5-H),

J_8,9Bϭ 6.5 Hz, J_9A,9Bϭ 11.8 Hz, 1 H, 9B-H), 3.77 (s, 3 H, CO-

OCH₃), 3.99 (dd, J_6,7ϭ 1.1 Hz, J_5,6ϭ 10.5 Hz, 1 H, 6-H), 4.35

(dd, J_5,6ϭ 10.5 Hz, 1 H, 5-H), 4.35–4.5 (m, 1 H, 8-H), 4.51 (d,

3.98–4.13 (m, 2 H, 8-H, 9A-H), 4.29 (dd, J_8,9Bϭ 6.3 Hz, J_9A,9B

ϭ

12.5 Hz, 1 H, 9B-H), 4.35 (d, J ϭ 4.5 Hz, 1 H, OH), 4.55 (d, J ϭ J ϭ 11.6 Hz, 1 H, 0.5 ϫ CH₂Ph), 4.86 (d, J ϭ 11.6 Hz, 1 H, 0.5

11.6 Hz, 1 H, 0.5 ϫ CH₂Ph), 4.80 (d, J ϭ 11.6 Hz, 1 H, 0.5 ϫ ϫ CH₂Ph), 4.99 (ddd, J_4,5ϭ 10.5 Hz, J_3e,4ϭ 4.7 Hz, 1 H, 4-H),

CH₂Ph), 4.90 (ddd, J_4,5ϭ 10.5 Hz, J_3e,4ϭ 4.9 Hz, 1 H, 4-H), 5.95 5.28 (d, J_5,NHϭ 7.6 Hz, 1 H, NH), 5.42 (s, 1 H, CHPh), 7.19–7.62

(d, J_5,NHϭ 7.8 Hz, 1 H, NH), 7.19–7.28 (m, 5 H, Ph). – (m, 10 H, 2 ϫ Ph). – C₂₉H₃₅NO₁₀(557.61): calcd. C 62.47, H 6.33,

C₂₄H₃₃NO₁₀0.5 H₂O (511.97): calcd. C 57.14, H 6.79, N 2.77; N 2.51; found C 62.22, H 6.45, N 2.36.

found C 57.35, H 6.84, N 2.99.

Methyl (Benzyl 5-Acetamido-4-O-acetyl-3,5-dideoxy-8-O-methyl-

glycero-α- -galacto-2-nonulopyranosid)onate (36): A solution of 35

galacto-2-nonulopyranosid)onate (33): Compound 32 (1.0 g, (200 mg, 0.359 mmol) in acetic acid/water (4:1, 6 mL) was heated

D-

Methyl (Benzyl 5-Acetamido-4-O-acetyl-3,5-dideoxy-

D-glycero-α-

D-

D

Eur. J. Org. Chem. 2000, 1467Ϫ1482

1479

G. Dufner, R. Schwörer, B. Müller, R. R. Schmidt

FULL PAPER

to 80 °C. After 3 h, the solvents were evaporated in vacuo. Flash

chromatography (toluene/acetone, 1:2) yielded 36 (146 mg, 87%) as

a colourless solid. – TLC (toluene/acetone, 1:2): R_fϭ 0.24. –

OCH₃), 3.66 (ddd, J_7,8ϭ 8.5 Hz, J_8,9Bϭ 7.9 Hz, 1 H, 8-H), 3.79

(s, 3 H, COOCH₃), 3.80–4.18 (m, 6 H, 2 ϫ OCH₂, 5-H, 9B-H),

4.24 (dd, J_8,9Aϭ 2.8 Hz, J_9A,9Bϭ 11.2 Hz, 1 H, 9A-H), 4.30 (dd,

[α]_Dϭ –32.2 (c ϭ 1.0 in chloroform). – ¹H NMR (250 MHz, J_6,5ϭ 10.6 Hz, J_7,6ϭ 2.1 Hz, 1 H, 6-H), 5.18–5.20 (m, 2 H, 7-H,

CDCl₃): δ ϭ 1.90 (s, 3 H, NHCOCH₃), 1.93–2.07 (m, 4 H, COCH₃,

3a-H), 2.65 (dd, J_3e,4ϭ 4.8 Hz, J_3e,3aϭ 12.8 Hz, 2 H, 3e-H, OH),

NH), 5.30 (dd, 5.31, J_3e,4ϭ 4.8 Hz, J_{5,4 ϭ}10.5 Hz, 1 H, 4-H). –

FAB MS (positive mode, matrix: dichloromethane/nitrobenzyl al-

3.42 (s, 3 H, OCH₃), 3.42–3.51 (m, 2 H, 7-H, 9A-H), 3.62 (dd, cohol and NaI): 664 [(M ϩ Na^ϩ)].

J_5,6ϭ 10.6 Hz, 1 H, 6-H), 3.67 (s, 3 H, COOCH₃), 3.70–3.78 (m,

Methyl (Allyl 5-Acetamido-4,7,8-tri-O-acetyl-3,5-dideoxy-9-O-di-

methoxytrityl- -glycero-α- -galacto-2-nonulopyranosid)onate (41):

2 H, 8-H, 9B-H), 4.10 (dd, J_NH,5ϭ 8.1 Hz, J_5,6ϭ 10.6 Hz, 1 H, 5-

H), 4.52 (d, J ϭ 11.7 Hz, 1 H, 0.5 ϫ CH₂Ph), 4.68 (d, J ϭ 3.1 Hz,

1 H, OH), 4.99 (ddd, J_4,5ϭ 10.6 Hz, J_3e,4ϭ 4.8 Hz, 1 H, 4-H),

4.80 (d, J ϭ 11.7 Hz, 1 H, 0.5 ϫ CH₂Ph), 5.98 (d, J_5,NHϭ 8.1 Hz,

1 H, NH), 7.20–7.27 (m, 5 H, Ph). – MALDI: 492 [(M ϩ Na)^ϩ].

D

To a solution of 40^[26](1.4 g, 3.86 mmol) in dry pyridine (14 mL)

was added 4-dimethylaminopyridine (94 mg, 0.771 mmol) and di-

methoxytrityl chloride (1.56 g, 4.63 mmol) and the mixture was

stirred overnight. Acetic anhydride (6 mL) was added and the mix-

ture was stirred for a further 12 h. The mixture was concentrated

and coevaporated with toluene. Flash chromatography (toluene/

acetone, 3:1) afforded 41 (2.07 g, 69%) as a colourless solid. – TLC

Methyl (Benzyl 5-Acetamido-4,7,8-tri-O-acetyl-3,5-dideoxy-8-O-

methyl-D-glycero-α-D-galacto-2-nonulopyranosid)onate (37): A solu-

tion of 36 (180 mg, 0.384 mmol) in dry pyridine/acetic anhydride

(2:1, 10 mL) was stirred overnight at room temp. The solution was

concentrated and coevaporated with toluene. Flash chromato-

graphy (toluene/acetone, 2:1) afforded 37 (187 mg, 88%) as a col-

1

(toluene/acetone, 3:1): R_fϭ 0.33. – H NMR (250 MHz, CDCl₃):

δ ϭ 1.81–2.22 (m, 13 H, 3 ϫ COCH₃, NHCOCH₃, 3a-H), 2.58

(dd, J_3e,4ϭ 4.5 Hz, J_3e,3aϭ 12.7 Hz, 1 H, 3e-H), 3.04 (dd, J_9A,9Bϭ

ourless solid. – TLC (toluene/acetone, 2:1): R_fϭ 0.38. – [α]_Dϭ – 12.3 Hz, J_8,9Bϭ 4.1 Hz, 1 H, 9B-H), 3.19 (dd, J_9A,9Bϭ 12.3 Hz, 1

1

12.7 (c ϭ 1.0 in chloroform). – H NMR (250 MHz, CDCl₃): δ ϭ

H, 9A-H), 3.78 (s, 9 H, 2 ϫ OCH₃, COOCH₃), 3.81–3.92 (m, 2 H,

1.85 (s, 3 H, NHCOCH₃), 1.94–2.08 (m, 10 H, 3 ϫ COCH₃, 3a- 5-H, 0.5 ϫ CH₂O), 4.19 (d, J_5,6ϭ 10.3 Hz, 1 H, 6-H), 4.20–4.29

H), 2.67 (dd, J_3e,4ϭ 4.8 Hz, J_3e,3aϭ 12.8 Hz, 1 H, 3e-H), 3.50 (s, (m, 1 H, 0.5 ϫ CH₂O), 4.95 (ddd, J_4,3aϭ 12.4 Hz, J_3e,4ϭ 4.5 Hz,

3 H, OCH₃), 3.67–3.73 (m, 1 H, 8-H), 3.75 (s, 3 H, COOCH₃), J_5,4ϭ 10.3 Hz, 1 H, 4-H), 5.13 (dd, J ϭ 1.2 Hz, 1 H, –CHϭCH₂),

3.75–4.08 (m, 2 H, 5-H, 9B-H), 4.13 (dd, J_6,7ϭ 1.8 Hz, J_5,6

10.7 Hz, 1 H, 6-H), 4.23 (dd, J_8,9Bϭ 3.0 Hz, J_9A,9Bϭ 12.3 Hz, 1

ϭ

5.20–5.30 (m, 2 H, NH, –CHϭCH₂), 5.40–5.49 (m, 2 H, 7-H, 8-

H), 5.77–5.91 (m, 1 H, CHϭCH₂), 6.78–7.43 (m, 13 H, Ph, 2 ϫ

H, 9A-H), 4.54 (d, J ϭ 11.7 Hz, 1 H, 0.5 ϫ CH₂Ph), 4.84 (d, J ϭ OMePh).

11.7 Hz, 1 H, 0.5 ϫ CH₂Ph), 4.99 (ddd, J_4,5ϭ 10.6 Hz, J_3e,4

ϭ

Methyl (Allyl 5-Acetamido-4,7,8-tri-O-acetyl-3,5-dideoxy-

D-gly-

4.8 Hz, 1 H, 4-H), 5.12 (dd, J_6,7ϭ 1.8 Hz, J_8,7ϭ 9.0 Hz, 1 H, 7-

H), 5.18 (d, J_5,NHϭ 9.9 Hz, 1 H, NH), 7.20–7.27 (m, 5 H, Ph). –

C₂₆H₃₅NO₁₂(553.56): calcd. C 56.41, H 6.37, N 2.53; found C

56.53, H 6.47, N 2.43.

cero-α- -galacto-2-nonulopyranosid)onate (42): A solution of 41

D

(1.3 g, 1.67 mmol) in acetic acid/water (4:1, 15 mL) was stirred for

1 h at room temp. Evaporation of the solvents in vacuo and puri-

fication by flash chromatography (toluene/acetone, 1:1) yielded 42

(645 mg, 79%) as a colourless solid. – TLC (toluene/acetone, 1:1):

R_fϭ 0.20. – [α]_Dϭ –27.5 (c ϭ 1.0 in chloroform). – ¹H NMR

Methyl 5-Acetamido-4,7,9-tri-O-acetyl-3,5-dideoxy-8-O-methyl-

D-

glycero-β- -galacto-2-nonulopyranosonate (38): To a solution of 37

D

(140 mg, 0.253 mmol) in methanol (10 mL) was added palladium (250 MHz, CDCl₃): δ ϭ 1.62 (s, 3 H, NHCOCH₃), 1.86–2.21 (m,

on charcoal (10%, 10 mg) and the mixture was stirred overnight

under hydrogen at normal pressure. After filtration through Celite,

the filtrate was concentrated under reduced pressure. Flash chro-

matography (toluene/acetone, 1:1) afforded 38 (115 mg, 84%) as a

colourless solid. – TLC (toluene/acetone 2:1): R_fϭ 0.17. – [α]_Dϭ –

10 H, 3 ϫ COCH₃, 3a-H), 2.58 (dd, J_3e,4ϭ 4.5 Hz, J_3e,3aϭ 12.7 Hz,

1 H, 3e-H), 2.97 (dd, J_9A,OHϭ 10.2 Hz, J_9B,OHϭ 4.8 Hz, 1 H,

OH), 3.49 (dd, J_9A,9Bϭ 12.3 Hz, J_8,9Bϭ 4.1 Hz, 1 H, 9B-H), 3.75

(s, 3 H, COOCH₃), 3.77–3 86 (m, 2 H, 9A-H, 0.5 ϫ CH₂O), 4.02

(d, J_5,6ϭ 10.3 Hz, 1 H, 6-H), 4.15 (dd, J_6,5ϭ 10.3 Hz, 1 H, 5-H),

4.21–4.28 (m, 1 H, 0.5 ϫ CH₂O), 4.80 (ddd, J_4,3aϭ 12.4 Hz, J_3e,4ϭ

1

31.8 (c ϭ 1.0 in chloroform). – H NMR (250 MHz, CDCl₃): δ ϭ

1.84 (s, 3 H, NHCOCH₃), 2.00–2.06 (3s, 9 H, 3 ϫ COCH₃), 2.15 4.5 Hz, J_5,4ϭ 10.3 Hz, 1 H, 4-H), 5.11–5.19 (m, 3 H, NH, 7-H, 8-

(dd, J_3e,4ϭ 5.2 Hz, J_3e,3aϭ 12.8 Hz, 1 H, 3e-H), 2.28 (dd, J_3e,3aH), 5.21–5.23 (m, 1 H, –CHϭCH₂), 5.28–5.30 (m, 1 H, –CHϭ

12.8 Hz, 1 H, 3a-H), 3.41 (s, 3 H, OCH₃), 3.52 (ddd, J_9B,8ϭ 4.9 Hz, CH₂), 5.77–5.28 (m, 1 H, CHϭCH₂). – MALDI: 512 (M ϩ Na). –

ϭ

J

_8,7ϭ 9.1 Hz, 1 H, 8-H), 3.85 (s, 3 H, COOCH₃), 3.95 (dd, J_9B,8ϭ

C₂₁H₃₁NO₁₂0.5 H₂O (498.17): calcd. C 50.63, H 6.47, N 2.81;

found C 50.55, H 6.67, N 2.71.

4.9 Hz, J_9A,9Bϭ 12.3 Hz, 1 H, 9B-H), 4.09–4.22 (m, 3 H, 5-H, 9A-

H, OH), 4.35 (dd, J_7,6ϭ 2.2 Hz, J_5,6ϭ 10.8 Hz, 1 H, 6-H), 5.17

(dd, J_6,7ϭ 2.2 Hz, J_8,7ϭ 9.1 Hz, 1 H, 7-H), 5.26 (ddd, J_3e,4

5.2 Hz, J_4,5ϭ 10.5 Hz, 1 H, 4-H), 5.36 (d, J ϭ 12.2 Hz, NH). –

C₁₉H₂₉NO₁₂(463.44): calcd. C 49.24, H 6.31, N 3.02; found C

49.28, H 6.42, N 3.27.

Methyl (Allyl 5-Acetamido-4,7,8-tri-O-acetyl-9-O-dibenzylphospho-

ryl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosid)onate (43):

ϭ

Alcohol 42 (300 mg, 0.613 mmol) and 1H-tetrazole (84 mg,

1.20 mmol) were dissolved in dry acetonitrile/dichloromethane (3:1,

3 mL) under nitrogen. Bis(benzyloxy)(diisopropylamino)phos-

phane (340 mg, 0.988 mmol) in dry dichloromethane (2 mL) was

added by syringe. After 20 min (TLC), a solution of tert-butyl hy-

droperoxide in dry toluene (3 , 300 µL, 0.920 mmol) was added

and the mixture was stirred for a further 20 min. The reaction mix-

ture was treated with half-saturated sodium bicarbonate solution

and extracted with dichloromethane. The organic layer was dried

with magnesium sulfate and concentrated in vacuo. Purification by

flash chromatography (toluene/acetone, 2:1) yielded 43 (460 mg,

Diethyl [Methyl (5-Acetamido-4,7,9-tri-O-acetyl-3,5-dideoxy-8-O-

methyl-D-glycero-β-D-galacto-2-nonulopyranosyl)onate] Phosphite

(39): To a solution of 38 (100 mg, 0.216 mmol) and ethyldiisoprop-

ylamine (81 µL) in dry acetonitrile (3.0 mL) was added diethyl

chlorophosphite (62 µL, 0.431 mmol) under nitrogen. After 10 min,

the solution was concentrated and coevaporated with toluene.

Flash chromatography (toluene/acetone, 4:1) yielded 39 (100 mg,

93%) as a colourless oil. – TLC (toluene/acetone, 6:1): R_fϭ 0.39. –

¹H NMR (250 MHz, CDCl₃): δ ϭ 1.12–1.35 (m, 6 H, 2 ϫ CH₃), 82%) as a colourless foam. – TLC (toluene/acetone, 1:1): R_fϭ

1.85 (s, 3 H, NHCOCH₃) 1.95–2.12 (m, 10 H, 3 ϫ COCH₃, 3a-H),

0.41. – [α]_Dϭ –17.5 (c ϭ 1.0 in chloroform). – ¹H NMR (250 MHz,

2.48 (dd, J_3e,4ϭ 4.8 Hz, J_3a,3eϭ 13.1 Hz, 1 H, 3e-H), 3.42 (s, 3 H, CDCl₃): δ ϭ 1.75–2.10 (m, 13 H, 4 ϫ COCH₃, NHCOCH₃, 3a-

1480

Eur. J. Org. Chem. 2000, 1467Ϫ1482

Base- and Sugar-Modified CMP-Neu5Ac Analogues

FULL PAPER

H), 2.59 (dd, J_3e,4ϭ 4.6 Hz, J_3e,3aϭ 12.8 Hz, 1 H, 3e-H), 3.73 (s,

a colourless solid. – TLC (chloroform/methanol, 10:1): R_fϭ 0.26. –

3 H, COOCH₃), 3.78–3.88 (m, 1 H, 0.5 ϫ CH₂O), 3.93–4.12 (m, 3 ¹H NMR (250 MHz, MeOD): δ ϭ 1.34 (t, J ϭ 7.3 Hz, 9 H, 3 ϫ

H, 9A-H, 5-H, 6-H), 4.19–4.32 (m, 2 H, 9B-H, 0.5 ϫ CH₂O), 4.83

(ddd, J_4,3aϭ 12.4 Hz, J_3e,4ϭ 4.6 Hz, J_5,4ϭ 10.3 Hz, 1 H, 4-H), 4.8 Hz, J_{3ЈЈe,3ЈЈa}ϭ 13.2 Hz, 1 H, 3ЈЈe-H), 3.22 (q, J ϭ 7.3 Hz, 6 H,

4.98 (d, J ϭ 11.6 Hz, 2 H, CH₂Ph), 4.49 (d, J ϭ 11.6 Hz, 2 H, 3 ϫ CH₂), 3.51 (s, 3 H, OCH₃), 3.85 (s, 3 H, COOCH₃), 3.79–3.89

CH₃), 1.90–2.22 (m, 22 H, 7 ϫ COCH₃, 3aЈЈ-H), 2.80 (dd, J_3ЈЈe,4ЈЈϭ

CH₂Ph), 5.05–5.38 (m, 5 H, NH, 7-H, 8-H, –CHϭCH₂), 5.71–5.88 (m, 1 H, 9ЈB-H), 3.99–4.02 (m, 2 H, 5ЈЈ-H, 8ЈЈ-H), 4.21 (dd,

(m, 1 H, CHϭCH₂), 7.25–7.39 (m, 10 H, 2 ϫ Ph). – ³¹P NMR

(161.7 MHz, CDCl₃): δ_Pϭ –0.60.

J_5ЈA,5ЈBϭ 11.6 Hz, J_4Ј,5ЈBϭ 2.8 Hz, 1 H, 5ЈB-H), 4.25 (dd, J_{4Ј.5ЈA ϭ}

4.3 Hz, J_5ЈA,5ЈBϭ 11.6 Hz, 1 H, 5ЈA-H), 4.32 (dd, J_8ЈЈ,9ЈЈAϭ 2.6 Hz,

J_{9ЈЈA,9ЈЈB}ϭ 11.8 Hz, 1 H, 9ЈЈA-H), 4.42–4.48 (m, 1 H, 4Ј-H), 4.52

(dd, J_5ЈЈ,6ЈЈϭ 10.2 Hz, J_7ЈЈ,6ЈЈϭ 2.0 Hz, 1 H, 6ЈЈ-H), 5.29 (dd,

Methyl 5-Acetamido-4,7,8-tri-O-acetyl-9-O-dibenzylphosphoryl-3,5-

dideoxy-D-glycero-β-D-galacto-2-nonulopyranosonate (44): To a so-

J_6ЈЈ,7ЈЈϭ 2.0 Hz, J_8ЈЈ,7ЈЈϭ 8.7 Hz, 1 H, 7ЈЈ-H), 5.38 (ddd, J_3ЈЈe,4ЈЈ

ϭ

lution of 43 (270 mg, 0.360 mmol) in dry THF (5 mL) under nitro-

gen was added (1,5-cyclooctadiene)bis(methyldiphenylphos-

phane)iridium(I) hexafluorophosphate (1 mg). The red mixture was

degassed and set under nitrogen – this procedure was repeated three

times. The mixture was stirred under hydrogen for 5 min, until the

red colour vanished. The solution was degassed again and stirred

overnight under nitrogen. The solvents were evaporated in vacuo

and the residue was dissolved in THF/water (4:1, 10 mL). Iodine

(183 mg, 0.721 mmol) was added and the mixture was stirred for

1 h, then diluted with water and extracted with chloroform. The

organic layer was washed several times with sodium thiosulfate so-

lution, dried with magnesium sulfate, and concentrated. The res-

idue was first purified by flash chromatography (toluene/acetone,

2:1) and finally by MPLC (toluene/acetone, 3:2) to yield 44

(166 mg, 65%) as a colourless foam. – TLC (toluene/acetone, 2:1):

R_fϭ 0.41. – [α]_Dϭ –3.6 (c ϭ 1.0 in chloroform). –¹H NMR

(250 MHz, CDCl₃): δ ϭ 1.81–2.17 (m, 13 H, 4 ϫ COCH₃,

NHCOCH₃, 3a-H), 2.21 (dd, J_3e,4ϭ 4.6 Hz, J_3e,3aϭ 12.8 Hz, 1 H,

3e-H), 3.68 (s, 3 H, COOCH₃), 3.92–4.17 (m, 2 H, 5-H, 9B-H),

4.33 (dd, J_8,9ϭ 2.2 Hz, J_9A,9Bϭ 12.2 Hz, 1 H, 9A-H), 4.68–4.76

(ddd, J_6,7ϭ 2.2 Hz, J_5,6ϭ 10.1 Hz, 1 H, 6-H), 4.93–5.02 (m, 4 H,

4.8 Hz, J_3ЈЈa,4ЈЈϭ 11.0 Hz, 1 H, 4ЈЈ-H), 5.51–5.58 (m, 2 H, 2Ј-H,

3Ј-H), 6.27 (d, J_2Ј,1Јϭ 4.1 Hz, 1 H, 1Ј-H), 7.60 (d, J ϭ 7.5 Hz, 1

H, 5-H), 8.52 (d, J ϭ 7.5 Hz, 1 H, 6-H). – ³¹P NMR (161.7 MHz,

D₂O): δ_Pϭ –5.96. – FAB MS (negative mode, matrix: methanol/

nitrobenzyl alcohol, 1:1): 893 [(M – H^ϩ)^–].

Triethylammonium (N-Acetyl-2Ј,3Ј-di-O-acetylcytidin-5Ј-yl) [Meth-

yl (5-Acetamido-4,7,8-tri-O-acetyl-9-O-dibenzylphosphorono-3,5-di-

deoxy-D-glycero-β-D-galacto-2-nonulopyranosyl)onate]

Phosphate

(47): A solution of 23 (120 mg, 0.267 mmol) in dry N,N-dimethyl-

formamide/acetonitrile (2:1, 1.0 mL) was cooled to –20 °C. A solu-

tion of 45 (110 mg, 1.38 ϫ 10^–4mol) in dry acetonitrile (0.5 mL)

was added by syringe. The reaction mixture was allowed to warm

up to room temperature during 12 h. Triethylamine (500 µL) was

added and the solvents were evaporated in vacuo. Flash chromato-

graphy (chloroform/methanol/triethylamine, 9:1:0.1) yielded 47

(85 mg, 50%) as a colourless solid. – TLC (chloroform/methanol,

1

6:1): R_fϭ 0.54. – H NMR (250 MHz, MeOD): δ ϭ 1.33 (t, J ϭ

7.3 Hz, 9 H, 3 ϫ CH₃), 1.87–2.25 (m, 22 H, 7 ϫ COCH₃, 3aЈЈ-H),

2.78 (dd, J_3ЈЈe,4ЈЈϭ 4.8 Hz, J_{3ЈЈe,3ЈЈa}ϭ 13.0 Hz, 1 H, 3ЈЈe-H), 3.22

(q, J ϭ 7.3 Hz, 6 H, 3 ϫ CH₂), 3.80 (s, 3 H, COOCH₃), 4.06 (d,

J ϭ 10.5 Hz, 1 H, 5ЈЈ-H), 4.18–4.39 (m, 3 H, 9ЈЈB-H, 5ЈA-H, 5ЈB-

H), 4.41–4.45 (m, 1 H, 4Ј-H), 4.52 (dd, J_8ЈЈ,9ЈЈAϭ 2.2 Hz,

2 ϫ CH₂Ph), 4.18 (ddd, J_3e,4ϭ 4.6 Hz, J_4,3aϭ 12.4 Hz, J_5,4

ϭ

10.3 Hz, 1 H, 4-H), 5.31 (dd, J_8,7ϭ 8.7 Hz, J_6,7ϭ 2.1 Hz, 1 H, 7-

H), 5.43–5.48 (m, 1 H, 8-H), 6.33 (br. s, 1 H, OH), 6.42 (d, J ϭ

10.2 Hz, 1 H, NH), 7.24–7.36 (m, 10 H, 2 ϫ Ph). MALDI: 730 [M

ϩ Na].

J_{9ЈЈA,9ЈЈB}ϭ 10.7 Hz, 1 H, 9ЈЈA-H), 4.71–4.79 (m, 1 H, 6ЈЈ-H), 5.08–

5.13 (m, 4 H, 2 ϫ CH₂Ph), 5.29–5.40 (m, 2 H, 7ЈЈ-H, 8ЈЈ-H), 5.50–

5.59 (m, 2 H, 2Ј-H, 3Ј-H), 6.23 (d, J_2Ј,1Јϭ 4.1 Hz, 1 H, 1Ј-H), 7.35–

7.48 (m, 10 H, 2 ϫ Ph), 7.56 (d, J ϭ 7.5 Hz, 1 H, 5-H), 7.98 (d,

J ϭ 7.5 Hz, 1 H, NH), 8.52 (d, J ϭ 7.5 Hz, 1 H, 6-H). – ³¹P NMR

(161.7 MHz, MeOD): δ_Pϭ –0.82, –5.04. – FAB MS (negative

mode, matrix: nitrobenzyl alcohol): 1139 [(M – H^ϩ)^–].

[Methyl (5-Acetamido-4,7,8-tri-O-acetyl-9-O-dibenzylphosphoryl-

3,5-dideoxy-D-glycero-β-D-galacto-2-nonulopyranosyl)onate] Diethyl

Phosphite (45): To a solution of 44 (140 mg, 0.197 mmol) and ethyl-

diisopropylamine (74 µL) in dry acetonitrile (2.5 mL) was added

diethyl chlorophosphite (60 µL, 0.696 mmol) under nitrogen. After

10 min, the solution was concentrated and coevaporated with tolu-

ene. Flash chromatography (toluene/acetone, 3:1) yielded 45

(138 mg, 88%) as a colourless oil. – TLC (toluene/acetone, 3:1):

R_fϭ 0.37. – ¹H NMR (250 MHz, CDCl₃): δ ϭ 1.11–1.40 (m, 6 H,

2 ϫ CH₃), 1.72–2.12 (m, 13 H, 4 ϫ COCH₃, 3a-H), 2.44 (dd,

J_3e,4ϭ 4.8 Hz, J_3a,3eϭ 13.1 Hz, 1 H, 3e-H), 3.71 (s, 3 H, CO-

OCH₃), 3.27–4.29 (m, 7 H, 5-H, 9A-H, 9B-H, 2 ϫ CH₂), 4.62–4.73

(m, 1 H, 6-H), 4.93–5.07 (m, 4 H, 2 ϫ CH₂Ph), 5.08–5.18 (m, 1 H,

NH), 5.23 (ddd, J_3e,4ϭ 4.8 Hz, J_4,5ϭ 10.5 Hz, 1 H, 4-H), 5.32 (d,

J ϭ 9.9 Hz, 1 H, 7-H), 5.88–5.93 (m, 1 H, 8-H), 7.25–7.39 (m, 10

H, 2 ϫ Ph).

Dilithium [(5-Acetamido-3,5-dideoxy-8-O-methyl-D-glycero-β-D-gal-

acto-2-nonulopyranosyl)onate] (Cytidin-5Ј-yl) Phosphate (48): To a

solution of 46 (45 mg, 45.3 µmol) in dry methanol (2 mL) was ad-

ded sodium methoxide solution (0.5 , 3 drops) and the mixture

was stirred for 3 h at room temp. The mixture was neutralized with

Amberlite IRC-50 (H^ϩ) (pH ϭ 8–9), filtered and concentrated. The

residue was dissolved in water/methanol (1:1, 1 mL) and lithium

hydroxide solution (1 , 0.5 mL) was added. After stirring for 4 h

at room temp., the solution was neutralized with Amberlite IRC-

50 (H^ϩ), pH ϭ 8–9, and filtered. Lyophilization of the filtrate and

preparative HPLC (RP-18, 0.05  NEt₃H₂CO₃) yielded 48 (16 mg,

58%). – TLC, aminophase (ethanol/water, 1:1): R_fϭ 0.40. – ¹H

NMR (250 MHz, D₂O): δ ϭ 1.51 (ddd, J_{3ЈЈa,3ЈЈe}ϭ 13.2 Hz, 1 H,

3ЈЈa-H), 1.86 (s, 3 H, NHCOCH₃), 2.39 (dd, J_3ЈЈe,4ЈЈϭ 4.8 Hz,

Triethylammonium (N-Acetyl-2Ј,3Ј-di-O-acetylcytidin-5Ј-yl) [Meth-

yl (5-Acetamido-4,7,9-tri-O-acetyl-3,5-dideoxy-8-O-methyl-

D-gly-

cero-β- -galacto-2-nonulopyranosyl)onate] Phosphate (46): A solu-

D

tion of 23 (95 mg, 0.211 mmol) in dry N,N-dimethylformamide/ace- J_{3ЈЈe,3ЈЈa}ϭ 13.2 Hz, 1 H, 3ЈЈe-H), 3.27 (s, 3 H, OCH₃), 3.33–3.58

tonitrile (2:1, 1.0 mL) was cooled to –20 °C. A solution of 39

(70 mg, 0.120 mmol) in dry acetonitrile (0.2 mL) was added by syr-

inge. The reaction mixture was allowed to warm up to room tem-

(m, 3 H, 7ЈЈ-H, 5ЈЈ-H, 9ЈЈA-H), 3.68–3.99 (m, 4 H, 4ЈЈ-H, 6ЈЈ-H,

8ЈЈ-H, 9ЈЈB-H), 4.00–4.21 (m, 5 H, 2Ј-H, 3Ј-H, 4Ј-H, 5ЈA-H, 5ЈB-

H), 5.88 (d, J_2Ј,1Јϭ 3.1 Hz, 1 H, 1Ј-H), 5.91 (d, J ϭ 7.5 Hz, 1 H,

perature during 12 h. Triethylamine (200 µL) was added and the 5-H), 7.80 (d, J ϭ 7.5 Hz, 1 H, 6-H). – ³¹P NMR (161.7 MHz,

solvents were evaporated in vacuo. Flash chromatography (chloro-

form/methanol/triethylamine, 10:1:0.1) yielded 46 (64 mg, 54%) as

D₂O): δ_Pϭ –5.47. – FAB MS (negative mode, matrix: glycerol/

DMSO, 1:1): 633 [(M ϩ Li^ϩ– 2 H^ϩ)^–].

Eur. J. Org. Chem. 2000, 1467Ϫ1482

1481

G. Dufner, R. Schwörer, B. Müller, R. R. Schmidt

FULL PAPER

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Trisodium [(5-Acetamido-4,7,8-tri-O-acetyl-3,5-dideoxy-9-O-phos-

phorono-D-glycero-β-D-galacto-2-nonulopyranosyl)onate] (Cytidin-

[8]

[9]

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in Carbohydrate Synthesis (Eds.: S. H. Khan, R. A. O’Neill),

Harwood Academic Publishers GmbH, Chur, Schweiz, 1996,

p. 20–54.

5Ј-yl) Phosphate (49): To a solution of 47 (30 mg, 26.3 µmol) in

dioxane (4 mL) was added sodium bicarbonate (10 mg) and palla-

dium on charcoal (10%, 3 mg) and the mixture was stirred for

12 min under hydrogen. Triethylamine (1 drop) was added and the

catalyst was filtered off and washed with methanol. Sodium meth-

anolate in methanol (0.5 , pH ഠ 13) was added to the filtrate and

the mixture was stirred for 3 h at room temp. The solution was

neutralized with Amberlite IRC-50 (H^ϩ) (pH ϭ 8–9) and filtered.

The filtrate was diluted with water (10 mL) and after lyophilization

it was redissolved in water (2 mL) and treated with sodium hydrox-

ide solution (1, 0.5 mL, pH ഠ 13) for 3 h. The solution was neut-

ralized with Amberlite IRC-50 (H^ϩ) (pH ϭ 8), filtered and lyophil-

ized. Purification by preparative HPLC (RP-18, 0.05



[10]

NEt₃H₂CO₃) and ion exchange (Na^ϩ), yielded 49 (11 mg, 64%). –

TLC, cellulose (2-propanol/water/ammonia, 55:35:5): R_fϭ 0.05. –

¹H NMR (250 MHz, D₂O): δ ϭ 1.48 (ddd, J

ϭ 13.2 Hz, 1

3ЈЈa,3ЈЈe

H, 3ЈЈa-H), 1.87 (s, 3 H, NHCOCH₃), 2.32 (dd, J_3ЈЈe,4ЈЈϭ 4.8 Hz,

_{3ЈЈe,3ЈЈa}ϭ 13.2 Hz, 1 H, 3ЈЈe-H), 3.45 (d, J_8ЈЈ,7ЈЈϭ 8.7 Hz, 1 H, 7ЈЈ-

J

[11]

[12]

[13]

H), 3.71–3.92 (m, 5 H, 4ЈЈ-H, 5ЈЈ-H, 8ЈЈ-H, 9ЈЈA-H, 9ЈЈB-H), 3.99

(d, J_5ЈЈ,6ЈЈϭ 10.2 Hz, 1 H, 6ЈЈ-H), 4.01–4.19 (m, 5 H, 2Ј-H, 3Ј-H,

4Ј-H, 5ЈA-H, 5ЈB-H), 5.81 (d, J_2Ј,1Јϭ 3.1 Hz, 1 H, 1Ј-H), 5.97 (d,

J ϭ 7.5 Hz, 1 H, 5-H), 7.79 (d, J ϭ 7.5 Hz, 1 H, 6-H). – ³¹P NMR

(161.7 MHz, D₂O): δ_Pϭ –4.17, ϩ5.29. – FAB MS (negative mode,

matrix: DMSO/nitrobenzyl alcohol/glycerol, 1:1:1): 705 [(M ϩ

2 Na – 2 H^ϩ)^–].

U. Schmelzer, Dissertation, Universität Konstanz, 1994.

G. Dufner, Dissertation, Universität Konstanz, 1997.

C. Schaub, B. Müller, R. R. Schmidt, Glycoconjugate J. 1998,

15, 345–354.

D. H. van den Eijnden, private communication: In sialyl-

transferase reactions UMP-Neu5Ac showed about 10% of the

activity compared with CMP-Neu5Ac.

[14]

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Acknowledgments

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G. Zemplen, Ber. Dtsch. Chem. Ges. 1927, 60, 1554–1564.

This work was supported by the Deutsche Forschungsgemeinschaft

and the Fonds der Chemischen Industrie. We are grateful to Dr. A.

Geyer for DQF-COSY, HMQC, and HMBC experiments. – We are

grateful to Dr. U. Kragl, Forschungszentrum Jülich, for providing

Kdn.

V. Eschenfelder, R. Brossmer, Carbohydr. Res. 1980, 78, 190–

194.

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1992, 237, 161–175.

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6126; T. J. Martin, R. Brescello, A. Toepfer, R. R. Schmidt,

Glyconjugate J. 1993, 10, 16–25.

H. Paulsen, H. Tietz, Carbohydr. Res. 1984, 125, 47–64; and

references cited therein.

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therein.

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277–284.

[3]

R. R. Schmidt in: Carbohydrates – Synthetic Methods and Ap-

plication in Medicinal Chemistry (Eds.: H. Ogura, A. Hase-

gawa, T. Suami), Kodanasha Ltd., Tokyo, 1992, 68–88; R. R.

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1585–1588.

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T. J. Martin, H. Braun, R. R. Schmidt, Bioorg. Med. Chem.

Received October 12, 1999

[7]

[O99565]

1482

Eur. J. Org. Chem. 2000, 1467Ϫ1482

Article Doi

Base- and sugar-modified cytidine monophosphate N-acetylneuraminic acid (CMP-Neu5Ac) analogues - Synthesis and studies with α(2-6)-sialyltransferase from rat liver

DOI: 10.1002/(SICI)1099-0690(200004)2000:8<1467::AID-EJOC1467>3.0.CO;2-E

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