Synthesis and conformational analysis of pseudosugar analogues of chitotriose

This article was downloaded by: [New York University]

On: 29 May 2015, At: 20:19

Publisher: Taylor & Francis

Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,

37-41 Mortimer Street, London W1T 3JH, UK

Journal of Carbohydrate Chemistry

Publication details, including instructions for authors and subscription information:

http://ww w .tandfonline.com/loi/lcar20

SYNTHESIS AND CONFORMATIONAL ANALYSIS OF

PSEUDOSUGAR ANALOGUES OF CHITOTRIOSE ^*

Gabriela Thiele ^a, Antje Rottmann ^a, Antje Germer ^a, Erich Kleinpeter ^a, Klaus-Dieter

Spindler ^b, Bjørnar Synstad ^c, Vincent G.H. Eijsink ^c& Martin G. Peter ^d

^aInstitut für Chemie , Universität Potsdam , Karl-Liebknecht-Str. 25, Golm, D-14476,

Germany

^bAllgemeine Zoologie und Endokrinologie , Universität Ulm , Albert-Einstein-Allee 11, Ulm,

D-89081, Germany

^cDepartment of Chemistry and Biotechnology , Agricultural University of Norway , P.O. Box

5040, Ås, N-1432, Norway

^dInstitut für Chemie , Universität Potsdam , Karl-Liebknecht-Str. 25, Golm, D-14476,

Germany

Published online: 16 Aug 2006.

To cite this article: Gabriela Thiele , Antje Rottmann , Antje Germer , Erich Kleinpeter , Klaus-Dieter Spindler , Bjørnar

Synstad , Vincent G.H. Eijsink & Martin G. Peter (2002) SYNTHESIS AND CONFORMATIONAL ANALYSIS OF PSEUDOSUGAR

ANALOGUES OF CHITOTRIOSE ^*, Journal of Carbohydrate Chemistry, 21:6, 471-489

To link to this article: http://dx.doi.org/10.1081/CAR-120016847

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained

in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no

representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the

Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and

are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and

should be independently verified with primary sources of information. Taylor and Francis shall not be liable for

any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever

or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of

the Content.

This article may be used for research, teaching, and private study purposes. Any substantial or systematic

reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any

form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://

www.tandfonline.com/page/terms-and-conditions

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

JOURNAL OF CARBOHYDRATE CHEMISTRY

Vol. 21, No. 6, pp. 471–489, 2002

SYNTHESIS AND CONFORMATIONAL ANALYSIS OF

PSEUDOSUGAR ANALOGUES OF CHITOTRIOSE^*

1,

1

Gabriela Thiele,^1,Antje Rottmann, Antje Germer,

y

Erich Kleinpeter,¹Klaus-Dieter Spindler,²Bjørnar Synstad,³

Vincent G. H. Eijsink,³and Martin G. Peter^1,

yy

¹Institut fu¨r Chemie, Universita¨t Potsdam, Karl-Liebknecht-Str. 25,

D-14476 Golm, Germany

²Allgemeine Zoologie und Endokrinologie, Universita¨t Ulm,

Albert-Einstein-Allee 11, D-89081 Ulm, Germany

³Department of Chemistry and Biotechnology, Agricultural University of

˚

Norway, P.O. Box 5040, N-1432 As, Norway

ABSTRACT

EDC mediated coupling of the 4-O-succinoyl glycosyl azide 2 with gly-

cosylamine 3 gave the protected glycosylazide 4. Hydrogenation of 4

afforded the glycosylamine 5. Chemoselective hydrolysis of the reducing end

glycosylamine, followed by hydrogenation afforded N,N@-diacetylpseudochi-

totriose 7. Coupling of 5 with heterocyclic or with Cbz protected aliphatic

amino acids yielded glycosyl amides 8–12. Deprotection of 11 and 12

afforded the N,N@-diacetylpseudochitotriosyl amides of b-alanine, 13, and ^L-

arginine, 14. Molecular modelling calculations revealed that the pseudo-

trisaccharides exist in low energy extended conformations which show si-

milar space filling as N,N’,N@-triacetylchitotriose.

Key Words: Chitotriose; Glycosyl amides; Molecular modelling;

Pseudooligosaccharides; Sugar mimics; Spacers; Succinoyl amides

*

y

yy

Dedicated to Professor Dr. Joachim Thiem on the occasion of his 60th birthday.

Current address: Scienion AG, Volmerstrasse 7b, D-12489 Berlin, Germany.

Corresponding author. E-mail: peter@chem.uni-potsdam.de

471

DOI: 10.1081/CAR-120016847

0732-8303 (Print); 1532-2327 (Online)

www.dekker.com

Copyright D 2002 by Marcel Dekker, Inc.

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

472

THIELE ET AL.

INTRODUCTION

Chitooligosaccharides are formed in nature by enzymatic degradation of chitin or

chitosan as well as by de novo biosynthesis in a variety of organisms, including hu-

mans. Due to their intriguing biological activities, chitooligosaccharides [(GlcNAc/

GlcN)_n, n> 2] attract increasing interest (for a review, see Ref. [1]). Current concepts in

carbohydrate chemistry and biology include sugar mimics as tools for investigations of

carbohydrate-receptor interactions (for recent reviews, see Refs. [2-4]). A variety of

spacer modified oligosaccharides are described as sugar mimics suitable for studies of

certain polysaccharide binding proteins, including enzymes.^[5,6]Within our work on the

synthesis of derivatives of chitooligosaccharides^[7–9]it appeared to be of interest to

prepare spacer containing pseudochitooligosaccharides that should resist hydrolysis

by chitinases. In this paper, we report on the synthesis of analogues of N,N’,N@-tri-

acetylchitotriose in which the central sugar residue was replaced by a succinic acid

unit. The non-reducing end GlcNAc residue was linked to one of the carboxy groups of

succinic acid via an N-glycosyl amide whereas the reducing end GlcNAc residue was

attached to the other as an ester via the 4-OH group, giving the sequence GlcNAc-1-N-

Succ-4-O-GlcNAc (Scheme 1, formula 7). This was based on the consideration that 7

could possibly bind to the ꢀ 2, ꢀ 1, and + 1 or to the ꢀ 3 to ꢀ 1 sites of chitinases

(for nomenclature of binding sites of glycosyl hydrolases, see Ref. [10]). In either case,

Scheme 1. Synthesis of pseudotrisaccharide 7. i:0.7 EDC, 0.5 HOBt, 0.6 NEt₃, CH₂Cl₂, rt, 10-14

d; ii:EDC, 0.2 HOBt, 0.4 NEt₃, CH₂Cl₂, rt, 30 h; iii: H₂, Pd/C, MeOH, 60 °C to rt, 35 min; iv:

MeOH/CH₂Cl₂, HCl/H₂O, rt, 20 h; v: H₂, Pd(OH)₂/C, MeOH, 50°C to rt, 5 h.

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

PSEUDOSUGAR ANALOGUES OF CHITOTRIOSE

473

Scheme 2. Synthesis of glycosylamides 8–12. i: CH₂Cl₂, Pyr, -35 °C to rt; ii: EEDQ, CH₂Cl₂, rt.

enzyme inhibition could be observed when the affinities of the pseudotrisaccharide and

the substrate are comparable.

Furthermore, in an extension of our earlier work on glycosyl amides as potential

chitinase inhibitors,^[7–9]we report on the synthesis of novel protected carboxylic acid

glycosylamides of 7 (Scheme 2, formulas 8, 9, and 10) as well as the glycosylamides

of b-alanine, 13, and of ^L-arginine, 14 (Scheme 3). Compound 13 would introduce a

basic primary aliphatic amino group at the catalytic site, when the pseudotrisaccharide

binds at sites ꢀ 4 to ꢀ 2. If compound 14 binds analogously, the positively charged

arginine residue would give an ion pair with the catalytic glutamate residue, leading

again to enzyme inhibition. However, very little is known on the mode of binding of

chitooligosaccharides to chitinases in terms of the sequences of binding sites and

contribution of individual sugar residues to binding affinity (cf. Refs. [11–13]).

Likewise, the roles of the amino acid side chains involved in substrate recognition

of these enzymes are largely unknown, though a few high resolution crystal

structures of enzymes belonging to these highly diverse families 18 and 19 of

glycosyl hydrolases have been published^[14–17](see also Ref. [1] for a review of the

earlier literature).

Scheme 3. Structures of glycosylamides 13 and 14.

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

474

THIELE ET AL.

None of the compounds 7, 13, or 14 showed appreciable competition with the

fluorogenic 4-methylumbelliferone glycosides of (GlcNAc)₂or (GlcNAc)₃in enzyme

assays, using chitinases from a bacterium (ChiA or ChiB from Serratia marcescens), a

plant (Hevea brasiliensis), or an insect (Chironomus tentans) cell line, respectively

(data not shown). Therefore, conformational analysis was of interest. Especially the

flexibility and the size of the succinoyl spacer group of the substrate analogue should

be compared with those of N,N’,N@-triacetylchitotriose. Because neither scalar (J) nor

dipolar coupling (NOE) information concerning the conformation of the oligosacchar-

ide analogues could be obtained from NMR routines, we report on molecular modelling

calculations of 7 and 14, introducing new sets of parameters for the spacer unit and for

the glycosyl amide linkage.

RESULTS AND DISCUSSION

Synthesis

Glycosyl azide 1 was prepared from 2-acetamido-3-O-benzyl-4,6-O-benzylidene-

2-deoxy-b-^D-glucopyranosyl azide^[18]by reductive opening of the benzylidene ring with

NaCNBH₃in THF (Scheme 1). The isolated yield of the crystalline compound 1 never

exceeded 71%, as partial decomposition occurred during flash chromatography of the

crude product. Reaction of 1 with succinic anhydride in the presence of EDC, HOBt

and NEt₃afforded hemisuccinate 2 in 90% yield. The exact ratio of the reagents was

crucial in this reaction (see Experimental).

O-Benzylation of 2-acetamido-2-deoxy-b-^D-glucopyranosyl azide^[19]gave 2-

acetamido-3,4,6-tri-O-benzyl-2-deoxy-b-^D-glucopyranosyl azide in 52% yield (cf.

Refs. [20–22]). Catalytic hydrogenation of this compound over Adam’s catalyst

yielded the novel tri-O-benzyl-N-acetamidoglucosylamine 3. EDC mediated coupling

of 2 with glycosylamine 3 gave, after trituration of the crude solid sequentially with

toluene, MeCN and MeOH, the protected pseudotrisaccharide 4 in 65% isolated yield.

Also in this reaction, the ratio of EDC, HOBt and NEt₃had to be adjusted carefully.

Hydrogenation of 4 over Pd/C (10%) afforded the protected glycosylamine 5. In

contrast to azide 4, the amine 5 is rather unstable and, therefore, was always freshly

prepared immediately before use in subsequent reactions.

Attempts to achieve complete debenzylation of 5 by catalytic hydrogenation

always resulted in a mixture of the corresponding amine with pseudotrisaccharide 7 as

the minor product. It is reasonable to assume that 7 was generated from traces of water

which could not be removed by the method of preparation. Alternatively, 7 was

prepared in 55% overall yield by chemoselective hydrolysis of glycosylamine 5,

followed by catalytic hydrogenation (Scheme 1).

The glycosyl carboxamides 9, 11, and 12 were prepared by EEDQ mediated

coupling of the corresponding carboxylic acid with glycosylamine 5 (Scheme 2).^[7–9]

Optimum yields were obtained after reaction of the carboxylic acid with EEDQ prior to

addition of freshly prepared 5. Synthesis of the glycosyl carboxamides 8 and 10 was

achieved by coupling of 5 with the corresponding dimethylaminooxazoline- and

dimethylaminopyridinecarboxylic acid chlorides, respectively.^[9]

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

PSEUDOSUGAR ANALOGUES OF CHITOTRIOSE

475

Catalytic hydrogenation of glycosyl amides 8, 9, and 10 over Pd or Ni catalysts

always resulted in partial reduction of the heterocyclic rings, as evidenced by MALDI-

TOF mass spectrometry, thus lowering the yields of fully deprotected products. De-O-

benzylation of 11 by hydrogenation over Pd(OH)₂/C (20%) in both, MeOH or EtOH,

gave always N^b-alkyl-b-alanine glycosyl amides as side products.^[23]

Due to the insolubility of 12 in most solvents, deprotection failed under standard

conditions, and complete decomposition ensued in many cases. Also, reductive N-

alkylation of the guanidino group moiety was observed after hydrogenation of 12 in the

presence of MeOH or EtOH. Attempts to suppress the N-alkylation by addition of small

amounts of H₂O to the reaction mixture resulted in partial hydrolysis of the O-

succinate. Finally, reproducible debenzylation of 11 and of 12 proceeded smoothly

when the hydrogenation was carried out at 40°C over 15 or 18 h, respectively, in a

rather large volume of MeOH, containing a few drops of H₂O, while maintaining a

pressure of 35 bar. The pseudochitotriosyl amides 13 and 14 were isolated in 95 and

64% yields, respectively.

Conformational Analysis

NMR spectroscopy (1D and 2D ¹H and ¹³C NMR routines as well as 1D

and 2D NOE/NOESY/ROESY experiments) did not yield conclusive information

about the conformations. In addition, the H,H coupling constants which could be

1

extracted from the H NMR spectra proved to be weighted averages of an unknown

number of conformations participating in conformational equilibria of the compounds

under study.

As force field calculations are useful alternatives to NMR for estimating global

minima conformations, compounds 7 and 14 were studied by molecular mechanical

calculations using the AMBER force field. In this approach, the molecule under study

is constructed from monomers. The relevant data for amino acid residues and various

linkages are available from appropriate data bases.^[24–27]The 3D geometry of the

molecule is defined by a z-matrix and each atom is defined by its type and net atomic

charge. Torsional angles, bond lengths and bond angles are specific for the atom type

selected. However, the available data sets do not contain the parameters for calculating

the succinoyl units in 7 and 14, and the arginine residue in 14. Using the AMBER force

field and based on the procedure described by Woods et al.,^[27]we have derived fully

compatible parameters for the carbohydrate residues as well as for the additional units

in 7 and 14 (see Experimental).

The principal approach towards analyzing the conformation of the pseudotri-

saccharides was dynamic simulation and systematic variation of dihedral angles,

followed by energy minimization. Dihedral angles are defined as: f (H1–C1–N1–C7);

f’ (C2’–C1’–O1’–C4); f@ (H1@–C1@–N1@–C4’); c’ (C1’–O4–C4–H4); w¹(N1@–

C4’–C3’–C2’); w²(C4’–C3’–C2’–C1’); w³(C3’–C2’–C1’–O1’); w⁴(N1–C7–C8–C9).

Some preselections were necessary, as follows: the bond angles C1@–N1@–C4’ and

4

C1’–O4–C4 in 7 and 14 were set to 117°; the pyranose rings were treated as rigid C₁

conformations; the dihedral angles o (O5–C5–C6–O6) and o@ (O5@–C5@–C6@–O6@)

were fixed at 60°, corresponding to the gt energy minimum conformations;^[28,29]the

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

476

THIELE ET AL.

Figure 1. Stereo-view of low energy conformation of 7, four each minimized for w¹and w³in two

gauche (A: w²= 60°; B: ꢀ 60°) and in the anti (C: w²= 180°) conformations.

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

PSEUDOSUGAR ANALOGUES OF CHITOTRIOSE

477

Figure 2. Stereo-view of the global minimum conformations of N,N’,N@-triacetyl-chitotriose

superimposed with the w²anti conformation of 7.

N-acetyl groups of the pyranose rings were set to the energetically favoured anti

orientation; and the amide group N1@–C4’–O4’ was treated as essentially planar.

Compound 7 was chosen as the b-anomer.

The results obtained with 7 are depicted in Figure 1. Conformations with the

lowest steric hindrance between the non-reducing end glycosyl amide and the succinoyl

spacer appeared with f@ = 10° and 170°. The dihedral angles w², describing

conformations of the succinoyl spacer, were 60°, ꢀ 60°, and 180°. Consequently,

two folded gauche (Figure 1, A and B) and one extended anti (Figure 1C) conformers

occupy the global minima in this part of the molecule, with anti being the most stable

one. In addition, each of the three conformers has four global minima, described by w¹

and w³(Figure 1).

When the extended conformer C is superimposed with triacetylchitotriose, it

appears that both compounds occupy similar space, leading to the conclusion that both

may a priori bind to chitinases in a similar fashion (Figure 2). However, with respect to

the dihedral angles f’ and c’, many conformations near the global minimum have been

found, suggesting a non-restricted torsional flexibilty in the ester moiety which is also

in accordance with the absence of any observable NOEs between the two GlcNAc

residues of 7.

The pseudotrisaccharide unit of 14 shows essentially the same conformational

behavior as 7. The C7 amide group in 14 favours the E-configuration. The four low

energy conformers of 14 which are found by energy minimization of f and w⁴are

shown in Figure 3. The conformation of the aliphatic arginine chain is not considered.

The fact that none of the compounds inhibits any of the chitinases tested in this

study leads to the conclusion that the high conformational flexibility of the pseu-

dotrisaccharides described in this work prevents high affinity binding, though the

spacer unit leaves the reducing end at nearly the same distance in the lowest energy

Figure 3. Stereo-view of the low energy conformers of 14 representing the energy minima of

f and w⁴(the non-reducing end GlcNAc and the succinamide are not shown in this figure).

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

478

THIELE ET AL.

conformation. Obviously, the decrease in entropy which must occur when 7 should

bind to the enzyme, cannot be compensated by the binding enthalpy. In addition, the

dipoles associated with the glycosyl amide and the ester group may cause unfavourable

interactions with amino acid residues flanking the binding site. We hope that future

synthesis of compounds which are designed on the basis of the results described in this

work, as well as on complementary knowledge will lead to more efficient pseudooli-

gosaccharides as chitinase inhibitors.

EXPERIMENTAL

General Methods. Solvents were purified by standard procedures. Solutions in

organic solvents were dried over anhydrous MgSO₄. Gel filtration was performed on

Biogel P2¹(BioRad) with water containing 2.5% BuOH. Evaporation of solvents:

Rotary evaporator (ca. 1.3 kPa), bath temp. at ꢁ 40°C. Chromatography: Silica gel

1

(Merck: 0.04–0.063 mm). NMR: Bruker AMX R 300 (300 MHz or 75 MHz, for H or

1

¹³C NMR, respectively), internal standards: TMS or solvent signals for H or ¹³C NMR

spectra, respectively. Assignments of chemical shifts (d) and coupling constants (J) of

compounds 7 and 14 are based on HMBC, HMQC, and NOE experiments. Optical

rotations: Jasco DIP-1000. EI-MS: Finnigan SSQ 710 (70 eV). MALDI-MS: Bruker

Franzen Reflex II MALDI-TOF, positive ion mode, matrix: THAP. FAB-HR-MS:

Fisons VG four sector tandem MS, ZAB T, positive ion mode, matrix: thioglycerol.

ESI-HR-MS: Finnigan MAT95, solvent: MeOH. Elemental analysis: LECO CHNS-932.

2-Acetamido-3,6-di-O-benzyl-2-deoxy-b-^D-glucopyranosyl Azide (1). To an

ice-cold solution of 2-acetamido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-b-^D-glucopyr-

anosyl azide^[18](500 mg, 1.2 mmol) and NaCNBH₃(700 mg, 11 mmol) in dry THF (100

mL), 12 mL of a 1M HCl solution in Et₂O was added dropwise during 45 min. The mixture

was stirred under reflux for 1.5 h. After cooling to rt, THF was evaporated, followed by

addition of CHCl₃(150 mL). The suspension was filtered through Celite, and the

remaining residue was repeatly washed with CHCl₃. The combined solutions were

concentrated to a small volume. Flash chromatography (CHCl₃/EtOH 20:1) and re-

crystallization from Et₂O afforded 1 (355 mg, 71%) as colourless needles, mp 127°C

25

(dec). [a]_Dꢀ 21.9 (c 1.0, CHCl₃). ¹H NMR (CDCl₃): d 1.90 (s, 3 H, NHCOMe), 2.95 (d,

1 H, OH, J_OH,42.1 Hz), 3.42 (dd, 1 H, H-2), 3.58 (m, 1 H, H-5), 3.66–3.76 (m, 3 H, H-4, H-

6ab), 3.82 (dd, 1 H, H-3), 4.58 (dd, 2 H, CH₂Ph, J_Ha,Hb12.0 Hz), 4.76 (dd, 2 H, CH₂Ph),

4.91 (d, 1 H, H-1, J_1,29.1 Hz), 5.63 (d, 1 H, NH, J_NH,28.0 Hz), 7.26–7.34 (m, 10 H, 2 Ph).

¹³C NMR (CDCl₃): d 23.4 (NHCOMe), 55.8 (C-2), 70.0 (C-6), 72.6 (C-4), 73.7, 74.2 (2

CH₂Ph), 76.0 (C-5), 80.1 (C-3), 87.8 (C-1), 127.8–138.2 (Ph), 170.7 (CO). MALDI-MS:

m/z 465.1 [M +K] ⁺, 449.1 [M +Na] ⁺, 427.1 [M + H] ⁺. FAB-HR-MS: m/z calcd for

[M +H] ⁺: 427.1952, found 427.1981.

2-Acetamido-3,6-di-O-benzyl-4-O-succinoyl-2-deoxy-b-^D-glucopyranosyl

Azide (2). To a solution of 1 (500 mg, 1.17 mmol) and succinic anhydride (250 mg,

2.5 mmol) in CH₂Cl₂(15 mL), EDC (157 mg, 0.82 mmol), HOBt (79 mg, 0.58 mmol),

and NEt₃(71 mg, 0.7 mmol) were added. The mixture was stirred at rt for 10–14 days,

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

PSEUDOSUGAR ANALOGUES OF CHITOTRIOSE

479

until TLC (CHCl₃/EtOH 19:1) indicated complete reaction. H₂O (15 mL) was added and

the mixture was stirred for 5 min. The organic layer was washed with 5% HCl and with

H₂O, followed by drying and solvent concentration. The remaining residue was

suspended in toluene (25 mL) under vigorous stirring overnight. Filtration gave 2 (559

18

1

mg, 90%) as a colourless solid. [a]_Dꢀ 17.5 (c 1.3, DMSO). H NMR (DMSO-d₆): d

1.80 (s, 3 H, NHCOMe), 2.46 (m, 4 H, H-2’, H-3’, J 12.3 Hz, J 1.5 Hz), 3.35 (b, 1 H,

COOH), 3.42–3.56 (2 dd, 2 H, H-6a, H-6b, J 11.5 Hz, J 12.1 Hz, J 5.2 Hz), 3.72–3.82

(m, 3 H, H-2, H-3, H-5), 4.44 (dd, 2 H, CH₂Ph, J 12.0 Hz, J 2.8 Hz), 4.53 (s, 2 H,

CH₂Ph), 4.69 (d, 1 H, H-1, J_1,28.5 Hz), 4.88 (dd, 1 H, H-4, J 9.3 Hz), 7.19–7.39 (m, 10

H, 2 Ph), 8.17 (d, 1 H, NH, J_NH,27.9 Hz). ¹³C NMR (DMSO-d₆): d 22.8 (NHCOMe),

28.6, 28.9 (C-2’, C-3’), 53.8 (C-2), 68.4 (C-6), 70.1 (C-4), 72.4, 73.4 (2 CH₂Ph), 74.6

(C-5), 79.4 (C-3), 87.9 (C-1), 127.5–138.3 (Ph), 169.5 (COO-1’), 171.2 (NHCOMe),

173.4 (COOH). MALDI-MS: m/z 565.8 [M+ K] ⁺, 549.8 [M + Na] ⁺, 527.7 [M +H] ⁺.

2-Acetamido-3,4,6-tri-O-benzyl-2-deoxy-b-^D-glucopyranosylamine (3). To an

ice-cold suspension of NaH (120 mg, 5 mmol) in dry DMF (30 mL), 2-acetamido-2-

deoxy-b-^D-glucopyranosyl azide^[20](600 mg, 2.4 mmol) was added under an atmo-

sphere of nitrogen, and the mixture was stirred for 15 min at 0°C. Benzyl bromide (1.5

mL, 12.6 mmol) was added, and the mixture was stirred for 30 min at rt. BaO (3.0 g,

19.6 mmol) and Ba(OH)₂ꢂ8 H₂O (1.1 g, 3.5 mmol) were added, and the mixture was

stirred overnight at rt. The suspension was diluted with dry CH₂Cl₂(150 mL) and

stirred under reflux for 4–5 h until TLC (toluene/EtOAc 1:1) indicated completeness of

the reaction. CH₂Cl₂(200 mL) was added, the mixture was filtered through Celite, and

the residue was repeatly washed with CH₂Cl₂. The combined solutions were

concentrated, and the remaining DMF and benzyl bromide were removed by distillation

at ca. 10 ^{ꢀ 4}kPa. Column chromatography (toluene/EtOAc 1:1) of the residue was

followed by recrystallization from EtOH to afford 2-acetamido-3,4,6-tri-O-benzyl-2-

deoxy-b-^D-glucopyranosyl azide^[20](660 mg, 52%) as colourless crystals, mp 176°C.

[a]_D²¹+3.8 (c 1.0, CHCl₃), [a]_Dꢀ 39.8 (c 0.4, MeOH) [lit.^[20]mp 171°C, [a]_Dꢀ 34

24

21

(c 0.4, MeOH)].

A solution of 2-acetamido-3,4,6-tri-O-benzyl-2-deoxy-b-^D-glucopyranosyl azide

(500 mg, 0.97 mmol) in boiling EtOH (150 mL) was cooled to rt, PtO₂ꢂnH₂O (50 mg)

was added, and the mixture was kept under hydrogen atmosphere at rt for 2.5 h. The

solution was filtered through Celite, and the filtrate was concentrated to a small

volume of EtOH. The solid was filtered off, affording 3 (474 mg, 100%) as colourless

crystals, mp 156–162°C (dec). [a]_D^22.5+21.8 (c 1.0, CHCl₃). ¹H NMR (CDCl₃): d

1.83 (s, H, NHCOMe), 2.01 (b, 2 H, NH₂), 3.48 (ddd, 1 H, H-5), 3.57–3.69 (m, 4 H,

H-3, H-4, H-6a, H-6b), 3.71 (dd, 1-H, H-2), 4.02 (d, 1 H, H-1, J_1,29.0 Hz), 4.51–4.86

(3 dd, 6 H, 3 CH₂Ph, J_Ha,Hb12.2 Hz, J_Ha,Hb11.6 Hz, J_Ha,Hb10.8 Hz), 5.19 (d, 1 H,

NH, J_NH,28.6 Hz), 7.16–7.35 (m, 15 H, 3 Ph). ¹³C NMR (CDCl₃): d 23.5 (NHCOMe),

56.0 (C-2), 68.9 (C-6), 73.3, 73.5, 74.3 (3 CH₂Ph), 74.8 (C-5), 78.8 (C-4), 82.0 (C-3),

85.8 (C-1), 127.7–138.2 (Ph), 170.6 (CO). MALDI-MS: m/z 529.1 [M + K] ⁺, 513.1

[M +Na] ⁺, 491.1 [M +H] ⁺. FAB-HR-MS: m/z calcd for [M +Na] ⁺: 513.23654; found

513.23610.

Anal. Calcd for C₂₉H₃₄N₂O₅: C 70.99, H 6.98, N 5.71%; Found: C 71.01, H 7.05,

N 5.55%.

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

480

THIELE ET AL.

2-Acetamido-4-O-[3-N-(2-acetamido-3,4,6-tri-O-benzyl-2-deoxy-b-D-gluco-

pyranosyl)carboxamidopropanoyl]-3,6-di-O-benzyl-2-deoxy-b-^D-glucopyranosyl

Azide (4). To a solution of 2 (500 mg, 0.95 mmol) and 3 (466 mg, 0.95 mmol) in dry

˚

CH₂Cl₂(10 mL), freshly activated, ground molecular sieves (4 A) were added, followed

by addition of EDC (183 mg, 0.95 mmol), HOBt (26 mg, 0.19 mmol), and NEt₃(38 mg,

0.38 mmol). After stirring at rt for 30 h, MeOH/CHCl₃(1:1) was added until a clear

solution was obtained (ca. 10 mL). After filtration through Celite, CH₂Cl₂(30 mL) was

added to the filtrate, and the solution was washed with 5% HCl and H₂O, dried, and the

solvent was removed. The residue was triturated with toluene (25 mL) for 1 h under

vigorous stirring and then filtered off. The solid was then briefly boiled with MeCN (25

mL), cooled to rt and filtered off. Finally, the solid was stirred with MeOH (15 mL) for

30 min. The suspension was filtered to afford 4 (615 mg, 65%) as a colourless solid, mp

23

1

190–221°C (dec). [a]_Dꢀ 6.9 (c 0.5, DMSO). H NMR (DMSO-d₆): d 1.79, 1.86 (2 s,

6 H, 2 NHCOMe), 2.30 (m, 4 H, H-2’, H-3’), 3.41–3.66 (m, 7 H, H-5@, H-4@, H-6ab,

H-6@ab, H-3@), 3.75–3.81 (m, 4 H, H-2, H-5, H-3, H-2@), 4.41–4.56 (m, 8 H, 4 CH₂Ph),

4.67–4.73 (m, 3 H, CH₂Ph, H-1), 4.88 (dd, 1 H, H-4, J 9.4 Hz), 4.97 (dd, 1 H, H-1@,

J

_1@,2@9.4 Hz), 7.16–7.37 (m, 25 H, 5 Ph), 8.01 (d, 1 H, NH-2@, J_NH,2@9.1 Hz), 8.18 (d, 1

H, NH-2, J_NH,28.0 Hz), 8.41 (d, 1 H, NH-1@, J_NH,1@9.2 Hz). ¹³C NMR (DMSO-d₆): d

22.9 (2 NHCOMe), 28.7, 29.8 (C-2’, C-3’), 53.9 (C-2, C-2@), 68.3, 68.6 (C-6, C-6@), 69.9

(C-4), 72.3, 72.4, 73.5, 74.0, 74.2 (5 CH₂Ph), 74.7 (C-5), 75.9 (C-5@), 77.7 (C-4@), 78.7

(C-1@), 79.4 (C-3), 83.0 (C-3@), 87.9 (C-1), 127.2–141.6 (Ph), 169.5, 169.6, 171.1,

171.3 (4 CO). MALDI-MS: m/z 1037.3 [M + K] ⁺, 1021.4 [M +Na] ⁺, 999.4 [M+ H] ⁺.

Anal. Calcd for C₅₅H₆₂N₆O₁₂: C 66.12, H 6.32, N 8.41%; Found: C 65.83, H

6.30, N 8.17%.

2-Acetamido-4-O-[3-N-(2-acetamido-3,4,6-tri-O-benzyl-2-deoxy-b-^D-gluco-

pyranosyl)carboxamidopropanoyl]-3,6-di-O-benzyl-2-deoxy-b-D-glucopyranosyla-

mine (5). A suspension of 4 (100 mg, 0.1 mmol) in dry MeOH was hydrogenated for

35 min over Pd/C (10%). The temp. was initially 60°C, and the mixture was allowed to

cool to rt during the reaction. Filtering through Celite was followed by evaporation of the

solvent at 25°C to dryness, yielding 5 (90 mg, 93%) as a colourless solid. The unstable

compound was immediately used for subsequent reactions. ¹H NMR (DMSO-d₆): d 1.79,

1.83 (2 s, 6 H, 2 NHCOMe), 2.38 (m, 4 H, H-2’, H-3’), 3.38–3.65 (m, 9 H, H-6ab,

H-6@ab, H-5, H-5@, H-4@, H-3, H-3@), 3.66 (dd, 1 H, H-2, J 8.7 Hz, J 9.5 Hz), 3.78 (dd, 1

H, H-2@, J 9.3 Hz), 4.02 (d, 1 H, H-1, J_1,28.7 Hz), 4.42–4.71 (m, 10 H, 5 CH₂Ph), 4.79

(dd, 1 H, H-4, J 9.3 Hz), 4.95 (dd, 1 H, H-1@, J 9.1 Hz), 6.55 (d, 1 H, NHaH, J_NHa,18.7

Hz), 7.17–7.32 (m, 25 H, 5 Ph), 7.95 (d, 1 H, NHbH, J_NHb,18.7 Hz), 8.04 (d, 2 H, 2 NH,

J

_NH,28.7 Hz), 8.41 (d, 1 H, 1@-NH, J_NH,1@9.0 Hz). ¹³C NMR (DMSO-d₆): d 22.9, 23.1 (2

NHCOMe), 28.8, 29.8 (C-2’, C-3’), 53.9, 55.6 (C-2, C-2@), 68.5, 69.0 (C-6, C-6@), 70.9

(C-4), 72.2, 72.4, 73.1, 74.1 (4 CH₂Ph), 73.2 (C-4@), 74.5 (CH₂Ph), 75.8, 77.7 (C-5,

C-5@), 78.6 (C-1@), 80.4, 82.9 (C-3, C-3@), 85.5 (C-1), 127.3–139.3 (Ph), 168.9, 169.1,

170.7, 171.0 ( 4 CO). MALDI-MS: m/z 1011.5 [M +K] ⁺, 995.5 [M + Na] ⁺, 973.5

[M +H] ⁺. FAB-HR-MS: m/z calcd for [M + H] ⁺: 973.461355; found 973.459899.

2-Acetamido-4-O-[3-N-(2-acetamido-3,4,6-tri-O-benzyl-2-deoxy-b-^D-gluco-

pyranosyl)carboxamidopropanoyl]-3,6-di-O-benzyl-2-deoxy-b-^D-glucopyranose

(6). To a suspension of freshly prepared 5 (100 mg, 0.1 mmol) in MeOH/CHCl₃(1:1,

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

PSEUDOSUGAR ANALOGUES OF CHITOTRIOSE

481

20 mL), H₂O (1 mL) and a 0.1 M HCl solution (1.3 mL) were added. The mixture was

stirred for 15–20 h, until TLC (CHCl₃/EtOH 19:1) indicated completeness of the

reaction. The solution was diluted with CH₂Cl₂, washed with H₂O, dried and eva-

porated. Flash chromatography (CHCl₃/EtOH 30:1) yielded 6 (68 mg, 70%) as a

1

colourless solid, mp 227°C. [a]_D²²+ 22.4 (c 1.0, DMSO). H NMR (DMSO-d₆): d 1.79,

1.86 (2 s, 6 H, NHCOM’), 2.36 (m, 4 H, H-2’, H-3’), 3.41–4.03 (m, 11 H, H-6ab,

H-6@ab, H-5, H-5@, H-4@, H-3, H-3@, H-2, H-2@), 4.41–4.98 (m, 13 H, 5 CH₂Ph, H-4,

H-1, H-1@), 7.02 (b, 1 H, OH), 7.16–7.31 (m, 25 H, 5 Ph), 8.03, 8.11 (2 d, 2 H, NH-2,

NH-2@, J_NH,2, J_NH,2@8.9 Hz), 8.42 (d, 1 H, NH-1@, J_NH,1@9.2 Hz). ¹³C NMR (DMSO-

d₆): d 22.6, 22.9 (2 NHCOMe), 28.8, 29.9 (C-2’, C-3’), 52.9, 53.9 (C-2, C-2@), 68.5,

68.8 (C-6, C-6@), 68.1, 70.9 (C-4, C-4@), 72.2, 72.3, 73.3, 73.9, 74.0 (5 CH₂Ph), 75.8,

76.9, 77.7, 82.9 (C-5, C-5@, C-3, C-3@), 78.6 (C-1@), 90.8 (C-1), 127.3–138.4 (Ph),

168.9, 169.1, 170.7, 171.0 (4 CO). MALDI-MS: m/z 1012.4 [M + K] ⁺, 996.4

[M +Na] ⁺, 974.4 [M + H] ⁺.

Anal. Calcd for C₅₅H₆₃N₃O₁₃ꢃ 3 H₂O: C 64.25, H 6.76, N 4.09%; Found: C

64.28, H 6.54, N 3.98%.

2-Acetamido-4-O-[3-N-(2-acetamido-2-deoxy-b-^D-glucopyranosyl)carbox-ami-

dopropanoyl]-2-deoxy-^D-glucopyranose (7). A suspension of 6 (68 mg, 0.07 mmol)

in MeOH (30 mL), containing H₂O (1 mL), was hydrogenated over Pd(OH)₂/C (20%)

for 5 h. The temp. was initially 50°C, and the mixture was allowed to cool to rt during

the reaction. The mixture was filtered through Celite, the residue was thoroughly

washed with MeOH/H₂O (1:1), and concentrated in vacuum at 25°C. Gel filtration and

lyophylization afforded 7 (28 mg, 78%) as a colourless solid, mp 109–110°C.

[a]_D²¹+23.8 (c 0.5, H₂O). H NMR (D₂O): d 2.02, 2.03 (2 s, 6 H, NHCOMe), 2.53,

1

2.61 (2 m, 4 H, H-2’, H-3’), 3.49 (m, 2 H, H-4@, H-5@), 3.54–4.15 (m, 9 H: 3.59 H-3@,

3.61 H-5, H-6ab, H-6@ab, 3.65 H-4b, 3.77 H-2b, 3.81 H-2@, 3.96 H-2a, 4.00 H-4a),

4.77 (d, 0.5 H, H-1b), 4.89 (m, 1 H, H-3a, H-3b), 5.06 (d, 1 H, H-1@, J_1@,2@9.7 Hz),

5.22 (d, 0.5 H, H-1a, J_1a,2a3.5 Hz). ¹³C NMR (D₂O): d 26.4, 26.6 (2 NHCOMe), 33.4,

34.6 (C-2’, C-3’), 58.4 (C-2a), 58.8 (C-2@), 60.9 (C-2b), 64.7, 65.0 (C-6, C-6@), 73.1

(C-4a), 74.0 (C-4b), 74.1 (C-4@), 76.2 (C-3a), 77.2 (C-3b), 78.8 (C-3@), 82.1 (C-5, C-

5@), 82.9 (C-1@), 95.3 (C-1a), 99.5 (C-1b), 178.2, 179.1, 179.4, 180.0 (4 CO). MALDI-

MS: m/z 562.3 [M +K] ⁺, 546.3 [M +Na] ⁺, 524.3 [M +H] ⁺. ESI-HR-MS m/z calcd for

[M +H] ⁺: 524.2092; found 524.2110.

N-{2-Acetamido-4-O-[3-N-(2-acetamido-3,4,6-tri-O-benzyl-2-deoxy-b-^D-gluco-

pyranosyl)carboxamidopropanoyl]-3,6-di-O-benzyl-2-deoxy-b-^D-glucopyranosyl}-

(4S,5R)-2-dimethylamino-5-methyl-D²-oxazoline-4-carboxylic Acid Amide (8). 2-

Dimethylamino-4-carboxy-5-methyl-D²-oxazoline^[9](40 mg, 0.23 mmol) was suspended

in oxalyl chloride (3 mL) under nitrogen and stirred for 12 h at rt. The excess of oxalyl

chloride was removed by evaporation. The residue was dissolved in dry CH₂Cl₂(5 mL)

under nitrogen and cooled to ꢀ 30°C. This solution was then added to a cooled ( ꢀ 30°C)

suspension of freshly prepared 5 (80 mg, 0.08 mmol) in CH₂Cl₂(10 mL), containing

˚

ground activated molecular sieves (4 A). The mixture was allowed to warm up to ꢀ 10°C,

and pyridine (1 mL) was added over a period of 45 min. Stirring was continued at rt

overnight. The mixture was diluted with CH₂Cl₂and filtered through Celite. The filtrate

was washed with sat. NaHSO₄(2 ꢃ) and H₂O, dried and concentrated. Flash chro-

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

482

THIELE ET AL.

matography (CHCl₃/EtOH 12:1) afforded 8 (30 mg, 40%) as a colourless solid, mp 212–

214°C. [a]_D¹⁸+12.4 (c 0.5, DMSO). ¹H NMR (DMSO-d₆): d 1.33 (d, 3 H, Me, J_Me,106.0

Hz), 1.80 (s, 6 H, 2 NHCOMe), 2.43 (m, 4 H, H-2’, H-3’), 2.85, 2.87 (2 s, 6 H, NMe₂),

3.42–3.66 (m, 9 H, H-3, H-3@, H-8, H-6ab, H-6@ab, H-5, H-5@), 3.75–3.88 (3 dd, 3 H, H-2,

H-2@, H-4@), 4.42–4.72 (m, 11 H, 5 CH₂Ph, H-10), 4.92, 4.96 (m, dd, 3 H, H-4, H-1, H-1@,

J 9.7 Hz), 7.17–7.32 (m, 25 H, 5 Ph), 8.01 (d, 2 H, NH-2, NH-2@), 8.42 (d, 2 H, NH-1, NH-

1@). ¹³C NMR (DMSO-d₆): d 21.5 (Me), 22.6, 22.8 (2 NHCOMe), 28.7, 29.8 (C-2’, C-3’),

37.0. 37.1 (NMe₂), 53.9 (C-2, C-2@), 68.6 (C-6, C-6@), 70.2 (C-4), 72.3, 72.4 (CH₂Ph), 74.3

(C-8), 74.0, 74.1 (CH₂Ph), 73.1 (C-4@), 75.9, 77.8 (C-5, C-5@), 78.7 (C-1, C-1@), 79.9, 80.0,

83.0 (C-10, C-3, C-3@), 127.5–138.6 (Ph), 166.5 (C-9), 169.4, 170.0, 171.1, 171.3, 173.6

(5 CO). MALDI-MS: m/z 1165.8 [M +K] ⁺, 1149.8 [M + Na] ⁺, 1127.8 [M + H] ⁺.

N-{2-Acetamido-4-O-[3-N-(2-acetamido-3,4,6-tri-O-benzyl-2-deoxy-b-^D-gluco-

pyranosyl)carboxamidopropanoyl]-3,6-di-O-benzyl-2-deoxy-b-^D-glucopyranosyl}-3-

pyridinecarboxylic Acid Amide (9). A solution of nicotinic acid (15 mg, 0.12 mmol)

and EEDQ (27 mg, 0.11 mmol) in dry CH₂Cl₂(10 mL) was stirred overnight and

subsequently added to freshly prepared 5 (50 mg, 0.05 mmol). The suspension was

vigorously stirred for 48 h. The solvent was evaporated to dryness, and the residue was

suspended in EtOH (20 mL), stirred for 30 min, and the suspension was filtered. Flash

chromatography (CHCl₃, then CHCl₃/EtOH 20:1) of the residue afforded 9 (22 mg,

23

1

41%), mp > 240°C (dec). [a]_D+ 3.9 (c 0.5, DMSO). H NMR (DMSO-d₆): d 1.83,

1.86 (2 s, 6 H, 2 NHCOMe), 2.53 (m, 4 H, H-2’, H-3’), 3.40–4.05 (m, 10 H, H-5, H-5@,

H-6ab, H-6@ab, H-4@, H-3@, H-2@, H-3), 4.15 (dd, 1 H, H-2), 4.46–4.78 (m, 10 H, 5

CH₂Ph), 5.01 (dd, 2 H, H-4, H-1@, J 7.6 Hz), 5.33 (dd, 1 H, H-1), 7.22–7.37 (m, 25 H,

5 Ph), 7.54 (dd, 1 H, H-10, J_10,117.9 Hz, J_9,104.8 Hz), 8.08 (d, 1 H, NH-2@, J_2@,NH9.0

Hz), 8.24 (d, 1 H, H-9), 8.18 (d, H, 1 H, NH-2, J_2,NH8.9 Hz), 8.45 (d, 1 H, NH-1@,

J_1@,NH9.2 Hz), 8.75 (dd, 1 H, H-11, J 1.4 Hz), 9.0 (d, 1 H, H-12, J 1.5 Hz), 9.25 (d, 1

H, NH-1, J_1,NH9.1 Hz). ¹³C NMR (DMSO-d₆): d 22.7, 22.8 (2 NHCOMe), 28.7, 29.8

(C-2’, C-3’), 53.0 (C-2, C-2@), 68.3, 68.6 (C-6, C-6@), 70.2 (C-4), 72.3, 72.5, 73.5, 73.9,

74.1 (5 CH₂Ph), 74.5 (C-4@), 75.9 (C-5), 77.8 (C-5@), 78.7, 79.3 (C-1, C-1@), 80.1, 83.0

(C-3, C-3@), 127.5–138.6 (Ph), 123.5 (C-8), 129.4 (C-10), 135.3 (C-9), 148.7 (C-11),

152.5 (C-12), 165.3, 169.4, 169.7, 171.1, 171.3, 175.2 (5 CO) . MALDI-MS: m/z

1116.3 [M + K] ⁺, 1100.3 [M +Na] ⁺, 1078.3 [M +H] ⁺.

N-{2-Acetamido-4-O-[3-N-(2-acetamido-3,4,6-tri-O-benzyl-2-deoxy-b-^D-gluco-

pyranosyl)carboxamidopropanoyl]-3,6-di-O-benzyl-2-deoxy-b-^D-glucopyranosyl}-2-

dimethylamino-5-pyridinecarboxylic Acid Amide (10). 2-Dimethylamino-5-pyridi-

necarboxylic acid^[9](20 mg, 0.12 mmol) was suspended in oxalyl chloride (3 mL)

under nitrogen and stirred for 12 h at rt. The excess of oxalyl chloride was removed by

evaporation. The residue was dissolved in dry CH₂Cl₂(5 mL) under nitrogen, cooled

to ꢀ 35°C and subsequently added to a cooled ( ꢀ 35°C) suspension of freshly prepared

5 (58 mg, 0.06 mmol) in CH₂Cl₂(8 mL), containing pyridine (3 mL) and ground

˚

activated molecular sieves (4 A). The cooling bath was removed and the mixture was

stirred for another 3 h. The mixture was then diluted with CH₂Cl₂and filtered through

Celite. The filtrate was washed with NaHSO₄(2 ꢃ) and with H₂O, dried and con-

centrated. Flash chromatography (CHCl₃/EtOH 10:1) afforded 10 (25 mg, 50%) as a

1

colourless solid. H NMR (CDCl₃): d 1.74, 1.76 (2 s, 6 H, 2 NHCOMe), 2.35–2.75 (m,

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

PSEUDOSUGAR ANALOGUES OF CHITOTRIOSE

483

4 H, H-2’, H-3’), 3.12, 3.16 (2 s, 6 H, NMe₂), 3.45–4.40 (4 m, 11 H, H-5, H-5@, H-6ab,

H-6@ab, H-3, H-3@, H-4@, H-2, H-2@), 4.42–4.79 (m, 10 H, 5 CH₂Ph), 4.91–5.12 (3 dd,

3 H, H-4, H-1, H-1@), 6.45–8.88 (m, 28 H, Ph, H-9, H-10, H-12). MALDI-MS: m/z

1159.3 [M +K] ⁺, 1143.4 [M +Na] ⁺, 1121.3 [M +H] ⁺.

N-{2-Acetamido-4-O-[3-N-(2-acetamido-3,4,6-tri-O-benzyl-2-deoxy-b-^D-gluco-

pyranosyl)carboxamidopropanoyl]-3,6-di-O-benzyl-2-deoxy-b-^D-glucopyranosyl}-

(3-benzyloxycarbonylamino)propanoic Acid Amide (11). A solution of N-benzyl-

oxycarbonyl-b-alanine (44 mg, 0.2 mmol) and EEDQ (47 mg, 0.19 mmol) in dry

CH₂Cl₂(10 mL) was stirred for 6 h and subsequently added to freshly prepared 5 (85

mg, 0.09 mmol). The suspension was vigorously stirred overnight. After concentration,

the remaining colourless solid was suspended in EtOH (20 mL) and stirred for 30 min

at rt. Filtration gave 60 mg (58%) of 11 as a colourless solid, mp 287°C. [a]_D²²+9.4 (c

1

0.5, DMSO). H NMR (DMSO-d₆): d 1.79 (s, 6 H, 2 NHCOMe), 2.28 (m, 2 H, H-8),

2.41 (m, 4 H, H-2’, H-3’), 3.18 (m, 2 H, H-9), 3.35–3.81 (m, 11 H, H-5, H-5@, H-6ab,

H-6@ab, H-4@, H-3, H-3@, H-2, H-2@), 4.40–4.71 (m, 10 H, 5 CH₂Ph), 4.87 (dd, 1 H,

H-4, J 9.2 Hz), 4.99 (m, 4 H, COOCH₂Ph, 1-H, 1@-H), 7.18–7.24 (m, 31 H, 6 Ph, NH-

9), 8.01, 8.06 (2 d, 2 H, NH-2, NH-2@, J 9.0 Hz, J 8.5 Hz), 8.42, 8.48 (2 d, 2 H, NH-1,

NH-1@, J 9.1 Hz, J 9.3 Hz). ¹³C NMR (DMSO-d₆): d 22.7, 22.8 (2 NHCOMe), 28.7,

29.8 (C-2’, C-3’), 35.6 (C-8), 36.8 (C-9), 53.7 (C-2, C-2@), 65.2 (COOCH₂Ph), 68.4,

68.6 (C-6, C-6@), 70.2 (C-4), 72.3, 72.4, 73.5, 73.9, 74.1 (5 CH₂Ph), 74.3 (C-4@), 75.9,

77.8 (C-5, C-5@), 78.4, 78.7 (C-1, C-1@), 80.3, 83.0 (C-3, C-3@), 127.4–138.6 (Ph),

155.9 (CO-3), 169.4, 170.7, 171.1, 171.3 (5 CO). MALDI-MS: m/z 1216.7 [M +K] ⁺,

1200.8 [M +Na] ⁺, 1178.7 [M +H] ⁺.

Anal. Calcd for C₆₆H₇₅N₅O₁₅ꢃ 1 H₂O: C 66.27, H 6.49, N 5.85%; Found: C

66.09, H 6.32, N 5.57%.

N-{2-Acetamido-4-O-[3-N-(2-acetamido-3,4,6-tri-O-benzyl-2-deoxy-b-^D-gluco-

pyranosyl)carboxamidopropanoyl]-3,6-di-O-benzyl-2-deoxy-b-^D-glucopyranosyl}-

(N^a,N^d,N^o-tris-benzyloxycarbonyl)-L-arginine Amide (12). A solution of N ^a,N ^d,N^o

-tri-benzyloxycarbonyl-L-arginine (119 mg, 0.21 mmol) and EEDQ (47 mg, 0.19 mmol)

in dry CH₂Cl₂(5 mL) was stirred for 6 h. This solution was added to 85 mg (0.09 mmol)

of freshly prepared 5. The suspension was vigorously stirred for 18 h, followed by

concentration. The residue was suspended in EtOH (15 mL) and stirred for 30 min.

Filtration gave 12 (100 mg, 75%) as a colourless solid, mp 249–250°C. [a]_D²²+10.5

1

(c 0.5, DMSO). H NMR (DMSO-d₆): d 1.45–1.60 (m, 6 H, H-9, H-10, H-11), 1.79

(s, 6 H, 2 NHCOMe), 2.35–2.49 (m, 4 H, H-2’, H-3’), 3.35–3.84 (m, 12 H, H-5, H-5@,

H-6ab, H-6@ab, H-4@, H-3, H-3@, H-2, H-2@, H-8), 4.37–4.71 (m, 10 H, 5 CH₂Ph),

4.75–5.19 (m, 9 H, H-4, H-1@, H-1, 3 CH₂COOPh), 7.16–7.44 (m, 42 H, NH-2, NH-12, 8

Ph), 8.02 (dd, 2 H, NH-2, NH-2@, J 9.6 Hz, J 11.2 Hz), 8.38 (dd, 2 H, NH-1, NH-1@, J 9.2

Hz, J 8.8 Hz), 9.15 (b, 1 H, CNH). ¹³C NMR (DMSO-d₆): d 22.7, 22.8 (2 NHCOMe), 25.1

(C-10), 28.7, 29.8 (C-2’, C-3’, C-9), 44.3 (C-11), 53.3, 53.9, 54.7 (C-2, C-2@, C-8), 65.4,

66.1, 68.1 (3 CH₂COOPh), 68.3, 68.6 (C-6, C-6@), 70.2 (C-4), 72.3, 72.4, 73.4, 73.9, 74.1

(5 CH₂Ph), 74.4 (C-4@), 75.9, 77.8 (C-5, C-5@), 78.7 (C-1, C-1@), 80.3, 83.0 (C-3, C-3@),

127.5–138.6 (Ph), 154.9, 155.9, 159.7 (3 CH₂COOPh), 162.9 (CN), 169.4, 169.5,

171.1, 171.2, 172.5 (5 CO). MALDI-MS: m/z 1569.5 [M +K] ⁺, 1553.5 [M +Na] ⁺,

1531.5 [M +H] ⁺.

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

484

THIELE ET AL.

Anal. Calcd for C₈₅H₉₄N₈O₁₉ꢃ 1 H₂O: C 65.88, H 6.24, N 7.23%; Found: C

65.62, H 6.29, N 6.95%.

N-{2-Acetamido-4-O-[3-N-(2-acetamido-2-deoxy-b-^D-glucopyranosyl)-car-

boxamidopropanoyl]-2-deoxy-b-^D-glucopyranosyl}-3-aminopropanoic Acid Amide

(13). A suspension of 11 (63 mg, 0.05 mmol) in MeOH (100 mL), containing of H₂O

(0.1 mL), was hydrogenated over Pd/C (10%) at 35 bar and 40°C overnight. The

mixture was filtered through Celite, washed with MeOH/H₂O (1:1), and the combined

solutions were concentrated at 25°C, followed by lyophylization to afford 13 (30 mg,

23

95%) as a colourless solid, mp 128–129°C. [a]_D+ 11.3 (c 0.5, H₂O). ¹H NMR

(D₂O): d 1.98, 1.99 (2 s, 6 H, NHCOMe), 2.58 (m, 2 H, H-2’), 2.60 (m, 4 H, H-3’, H-

8), 3.23 (m, 2 H, H-9), 3.44–3.89 (m, 11 H, H-3@, H-4@, H-5, H-6ab, H-6@ab, H-5@, H-

2@, H-3, H-2), 4.81 (m, 1 H, H-4), 5.03 (d, 1 H, H-1@, J_1@,2@9.6 Hz), 5.11 (d, 1 H, H-1,

J_1,28.8 Hz). ¹³C NMR (D₂O): d 26.5, 26.6 (2 NHCOMe), 33.4, 34.5 (C-2’, C-3’), 36.5

(C-8), 39.8 (C-9), 58.6, 58.8 (C-2, C-2@), 64.5, 65.0 (C-6, C-6), 74.0 (C-4@), 75.5 (C-4),

76.3 (C-3), 78.7 (C-3@), 79.9 (C-5), 82.1 (C-5@), 82.8, 82.9 (C-1, C-1@), 177.5, 178.1,

179.4, 179.9 (5 CO). MALDI-MS: m/z 632.2 [M +K] ⁺, 616.2 [M + Na] ⁺, 594.2

[M +H] ⁺. FAB-HR-MS: m/z calcd for [M + H] ⁺: 594.2591; found 594.2623.

N-{2-Acetamido-4-O-[3-N-(2-acetamido-2-deoxy-b-^D-glucopyranosyl)-car-

boxamidopropanoyl]-2-deoxy-b-^D-glucopyranosyl}-L-arginine Amide (14). A sus-

pension of 12 (100 mg, 0.065 mmol) in MeOH (80 mL), containing H₂O (0.1 mL), was

hydrogenated over Pd/C (10%) at 35 bar and 40°C for 18 h. The mixture was filtered

through Celite, washed with MeOH/H₂O (1:1). The combined solutions were con-

centrated at 25°C, followed by lyophylization to afford 14 (28 mg, 64%) as a colourless

solid, mp > 212°C (dec). [a]_D²²+ 12.7 (c 0.5, H₂O). H NMR (D₂O): d 1.67 (m, 2 H, H-

1

10), 1.93 (m, 2 H, H-9), 2.58, 2.73 (2 m, 4 H, H-2’, H-3’), 3.22 (dd, 2 H, H-11, J_10,116.7

Hz), 3.49 (m, 2 H, H-5@, H-4@), 3.59 (dd, 1 H, H-3@, J_3@,4@8.1 Hz, J_3@,2@10.6 Hz), 3.68–

3.95 (m, 8 H: 3.71 H-5, 3.57, 3.87 H-6a, H-6b, 3.73, 3.87 H-6@a, H-6@b, 3.81 H-2@, 3.86

H-3, 3.95 H-2), 3.99 (dd, 1 H, H-8, J_8,96.0 Hz, J_8,NH6.4 Hz), 4.87 (dd, 1 H, H-4, J 4.8

Hz), 5.05 (d, 1 H, H-1@, J_1@,2@9.7 Hz), 5.16 (d, 1 H, H-1, J_1,29.3 Hz). ¹³C NMR (D₂O):

d 26.7 (2 NHCOMe), 27.9 (C-10), 32.4 (C-9), 33.5, 34.6 (C-2’, C-3’), 44.9 (C-11), 57.4

(C-8), 58.6 (C-2), 58.9 (C-2@), 64.6 (C-6), 65.1 (C-6@), 74.1 (C-4@), 75.6 (C-4), 76.6

(C-3), 78.8 (C-3@), 80.1 (C-5), 82.2 (C-5@), 83.0 (C-1), 83.1 (C-1@), 161.4 (CN), 175.0

(C-7), 178.3, 179.4, 179.6, 180.0 (4 CO). MALDI-MS: m/z 717.3 [M +K] ⁺, 701.3

[M +Na] ⁺, 679.3 [M + H] ⁺. FAB-HR-MS: m/z calcd for [M + H] ⁺: 679.3269; found

679.3263.

Enzyme assays. Hevamine A was isolated from the latex of Hevea brasilien-

sis.^[30]ChiA and ChiB were isolated from Serratia marcescens.^[31]The enzymes from

Chironomus tentans were obtained from the cell cultures of an epithelial cell line.^[32]

Chitinase activities were determined by microfluorimetric assays,^[33]adapted for

a microtitre plate fluorimeter. The substrates (GlcNAc)₂-MU or (GlcNAc)₃-MU,

respectively, were used as described by McCreath and Gooday.^[34]The final amounts of

the enzymes in each assay mixture were 40 ng hevamine or ChiA, or 80 ng ChiB,

respectively. Solutions of the enzymes (20 mL) were incubated with a substrate solution

(50 mL, final concentration 125 mM) in 0.2 M Na-citrate-phosphate buffer (pH 5.5), and

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

PSEUDOSUGAR ANALOGUES OF CHITOTRIOSE

485

Scheme 4. Naming of the groups nob, nom, ohe, ber, and arp.

a solution of the test compound (30 mL), containing at least three different con-

centrations, in a 96-well microtitre plate for 30 min at 37°C. The reaction was termi-

nated by addition of a 1 M glycine-NaOH buffer, pH 10.6 (100 mL). Fluorescence

(excitation: 360 nm; emission: 460 nm) was measured by means of a microtitre

fluorimeter (Cytofluor 4000, Perseptive Biosystems). All measurements were performed

in triplicate. Standard deviations were <10%.

Conformational analysis. The carbohydrate parameter set of Woods et al.^[27]is

based on the AMBER force field of Cornell et al.;^[24]this force field is implemented

into the SYBYL^[35]program and was used for the molecular modelling throughout

this work.

For force field calculations, the parameters and charges for the GlcNAc moieties

(nom, nob; see Scheme 4) were taken from Ref. [27]. The parameter sets for the units

ber, arn, and ohe were derived from ab initio quantum molecular mechanical cal-

culations (HF/6-31G^*). The glycosidic linkages nob-ber, ber-nom, nom-ohe and nom-

arn of 7 and 14 were calculated from the monomers, taking the loss of water into

account. The atom types for the residues ber and arn were taken from the AMBER-

Cornell-P94 parameter sets.^[24]The bond lengths, bond angles and torsional angles of

the carbohydrate residues were again taken from Ref. [27] and supplemented by data

for the ber-nom linkage, as shown in Table 1. The parametrization of the atomic

Table 1. Parameters for Compounds 7 and 14

Atom Types

K

r_eq

Y_eq

V_n/2

0.00

g

n

3

C

OG Stretching

OG Bending

OG CT Bending

450.0

70

1.364

CT

C

117

126

O

C

OG Bending

OG X Torsion

80

X

0.00

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

486

THIELE ET AL.

Table 2. Atom Types and HF/6-31G^*RESP Charges of Residues nob, nom, ber, arp and ohe

Atom Type, Charges (au)

Atom

nob

nom

ber

arp

ohe

C1/N1/O1

C2

C3

EC, 0.476

CT, ꢀ 0.098

CT, 0.450

CT, 0.090

CT, 0.407

CT, 0.224

C, 0.962

EC, 0.476

CT, ꢀ 0.098

CT, 0.450

CT, 0.090

CT, 0.407

CT, 0.224

C, 0.962

N, ꢀ 0.833

C, 0.785

N, ꢀ 0.797

C, 0.764

OH, ꢀ 0.5349

CT, ꢀ 0.437

CT, ꢀ 0.411

C, 0.813

CT, ꢀ 0.082

CT, ꢀ 0.334

CT, ꢀ 0.364

CT, ꢀ 0.122

N2, ꢀ 0.825

CA, 1.068

C4

C5

C6

C7/N7

C8

H1/H1a

CT, ꢀ 0.676

H, 0.000

H, 0.385

HC, 0.228

H, 0.000

HO, 0.0000

HO, 0.3549

H2, 0.028

H1, 0.193

H1, ꢀ 0.017

H2, 0.028

H1, 0.193

H1, ꢀ 0.017

H1b

H2

H, 0.387

H3/H3a

H1, 0.193

HC, 0.220

H3b

H4/H4a

HC, 0.231

HC, 0.242

H1, 0.032

H4b

H5/H5a

HC, 0.244

HC, 0.199

HC, 0.249

H1, ꢀ 0.005

H5b

H6a

HC, 0.199

HC, 0.229

HC, 0.225

H1, ꢀ 0.017

H1, 0.006

HC, 0.189

H1, ꢀ 0.017

H1, 0.006

HC, 0.189

H6b

H8a

H8b

H8c

HC, 0.175

HC, 0.172

HC, 0.175

HC, 0.172

O2/N2

O3

O4

N, ꢀ 0.707

OH, ꢀ 0.728

OH, ꢀ 0.715

OS, ꢀ 0.535

OH, ꢀ 0.701

O, ꢀ 0.672

N, ꢀ 0.707

OH, ꢀ 0.728

OH, ꢀ 0.452

OS, ꢀ 0.535

OH, ꢀ 0.701

O, ꢀ 0.672

O, ꢀ 0.618

O, ꢀ 0.589

O, ꢀ 0.621

O5

O6

O7

N9

N2, ꢀ 0.925

N, ꢀ 0.847

N10

N11

(HO)3

(HO)4

(HO)6

(HN)2

(HN)7

(HaN)9

(HbN)9

(HaN)10

(HbN)10

(HaN)11

(HbN)11

Total

HO, 0.440

HO, 0.443

HO, 0.437

H, 0.327

HO, 0.440

HO, 0.00

HO, 0.437

H, 0.327

H, 0.428

H, 0.453

H, 0.440

H, 0.439

H, 0.373

H, 0.356

0.82

0.18

0

ꢀ 0.18

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

PSEUDOSUGAR ANALOGUES OF CHITOTRIOSE

487

charges of the oligosaccharides 7 and 14 proved to depend on the conformation of the

molecule and were defined as average values for each atom, as obtained from the ab

initio HF/6-31G^*RESP charges. In order to maintain neutrality of the molecules 7 and

the positive charge of 14, small adjustments for each atom were necessary, as shown in

Table 2. Hydrogen bonds were simulated adequately without selecting a special

hydrogen bonding term. The van der Waals terms employed in the present calculations

were taken from Refs. [24,27].

ACKNOWLEDGMENTS

This work was supported by EU grant no. BIO4-CT-960670. MGP acknowledges

partial support by the Fonds der Chemischen Industrie. AG thanks the ‘‘Studienstiftung

des Deutschen Volkes’’ for a PhD scholarship.

REFERENCES

1. Peter, M.G. Chitin and Chitosan from Animal Sources. In Biopolymers; Stein-

bu¨chel, A., Ed.; Wiley-VCH: Weinheim, 2002; Vol. 6, 481–574.

2. Skrydstrup, T.; Vauzeilles, B.; Beau, J.-M. Synthesis of C-Oligosaccharides. In

Carbohydrates in Chemistry and Biology; Ernst, B., Hart, G.W., Sinay¨, P., Eds.;

Wiley-VCH: Weinheim, 2000; Vol. 1, 495–530.

3. Fairweather, J.K.; Driguez, H. Synthesis of Oligosaccharide Mimics: S-Analogs. In

Carbohydrates in Chemistry and Biology; Ernst, B., Hart, G.W., Sinay¨, P., Eds.;

Wiley-VCH: Weinheim, 2000; Vol. 1, 531–564.

4. Wessel, H.P. Saccharide-Peptide Hybrids. In Carbohydrates in Chemistry and

Biology; Ernst, B., Hart, G.W., Sinay¨, P., Eds.; Wiley-VCH: Weinheim, 2000; Vol.

1, 565–585.

5. Lehmann, J.; Petry, S.; Schmidt-Schuchardt, M. Photolabile, spacer-modified oli-

gosaccharides for regioselective chemical modification and probing of receptor-

binding sites. ACS Symp. Ser. 1994, 560, 198–232.

6. Lehmann, J.; Schmidt-Schuchardt, M. Spacer-modified oligosaccharides as pho-

toaffinity probes for porcine pancreatic a-amylase. Methods Enzymol. 1994, 247,

265–288.

7. Ley, J.P.; Peter, M.G. Synthesis of N-(2-acetamido-2-deoxy-b-^D-glucopyranosyl)-

and of N-(N,N’-diacetylchitobiosyl)-amide of ^L-histidine. Synthesis 1994, 28–30.

8. Ley, J.P.; Peter, M.G. Synthesis of ^L-histidine and (ꢀ)-spinacine chitooligosyl

amides. J. Carbohydr. Chem. 1996, 15, 51–64.

9. Rottmann, A.; Synstad, B.; Eijsink, V.G.H.; Peter, M.G. Synthesis of N-acetyl-

glucosaminyl and diacetylchitobiosyl amides of heterocyclic carboxylic acids as

potential chitinase inhibitors. Eur. J. Org. Chem. 1999, 2293–2297.

10. Davies, G.J.; Wilson, K.S.; Henrissat, B. Nomenclature for sugar-binding subsites

in glycosyl hydrolases. Biochem. J. 1997, 321, 557–559.

11. Bokma, E.; Barends, T.; Terwisscha van Scheltinga, A.C.; Dijkstra, B.W.; Bein-

tema, J.J. Enzyme kinetics of hevamine, a chitinase from the rubber tree Hevea

brasiliensis. FEBS Lett. 2000, 478, 119–122.

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

488

THIELE ET AL.

12. Honda, Y.; Fukamizo, T. Substrate binding subsites of chitinase from barley

seeds and lysozyme from goose egg white. Biochim. Biophys. Acta 1998, 1388,

53–65.

13. Fukamizo, T.; Sasaki, C.; Schelp, E.; Bortone, K.; Robertus, J.D. Kinetic properties

of chitinase-1 from the fungal pathogen Coccidioides immitis. Biochemistry 2001,

40, 2448–2454.

˚

14. Terwisscha van Scheltinga, A.C.; Hennig, M.; Dijkstra, B.W. The 1.8 A resolution

structure of hevamine, a plant chitinase/lysozyme, and analysis of the conserved

sequence and structure motifs of glycosyl hydrolase Family 18. J. Mol. Biol. 1996,

262, 243–257.

15. van Aalten, D.M.F.; Synstad, B.; Brurberg, M.B.; Hough, E.; Riise, B.W.; Eijsink,

V.G.H.; Wierenga, R.K. Structure of a two-domain chitotriosidase from Serratia

˚

marcescens at 1.9-A resolution. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 5842–

5847.

16. van Aalten, D.M.F.; Komander, D.; Synstad, B.; Gaseidnes, S.; Peter, M.G.;

Eijsink, V.G.H. Structural insights into the catalytic mechanism of a Family 18 exo-

chitinase. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 8979–8984.

17. Papanikolau, Y.; Prag, G.; Tavlas, G.; Vorgias, C.E.; Oppenheim, A.B.; Petratos, K.

High resolution structural analyses of mutant chitinase a complexes with substrates

provide new insight into the mechanism of catalysis. Biochemistry 2001, 40,

11338–11343.

18. El-Shaban, M.A.; Jeanloz, R.W. The synthesis of antigenic glycopeptides, 2-

acetamido-N-(b-^L-aspartyl)-2-deoxy-4-O-b-(D-galactopyranosyl)-b-D-gluco-pyra-

nosylamine. (N-Acteyllactosaminyl-^L-asparagine). Bull. Chem. Soc. Jpn. 1981, 54,

3570–3576.

19. Szila´gyi, L.; Gyo¨rgydea´k, Z. A ¹³C-N.M.R. investigation of glycosyl azides and

other azido sugars: Stereochemical influences on the one-bond ¹³C-¹H coupling

constants. Carbohydr. Res. 1985, 143, 21–42.

`

20. Kere´kgya´rto´, J.; Agoston, K.; Batta, G.; Kamerling, J.P.; Vliegenthart, J.F.G.

Synthesis of fully and partially benzylated glycosyl azides via thioalkyl glycosides

as precursors for the preparation of N-glycopeptides. Tetrahedron Lett. 1998, 39,

7189–7192.

21. Mizuno, M.; Muramoto, I.; Kobayashi, K.; Yaginuma, H.; Inazu, T. A simple

method for the synthesis of N^b-glycosylated–asparagine and –glutamine deriva-

tives. Synthesis 1999, 162–165.

22. Mizuno, M.; Haneda, K.; Iguchi, R.; Muramoto, I.; Kawakami, T.; Aimoto, S.;

Yamamoto, K.; Inazu, T. Synthesis of a glycopeptide containing oligosaccharides:

Chemoenzymatic synthesis of eel calcitonin analogues having natural N-linked

oligosaccharides. J. Am. Chem. Soc. 1999, 121, 284–290.

23. Kondo, A.; Ando, K.; Ishida, H.; Kato, I.; Hasegawa, A.; Kiso, M. An efficient

structural analysis of N-methyl-deoxynojirimycin-containing sialo-oligosaccharides

by ion-spray mass spectrometry and the mechanism of palladium catalyzed N-

methylation in methanol. J. Carbohydr. Chem. 1994, 13, 545–554.

24. Cornell, W.D.; Cieplak, P.; Bayly, C.I.; Gould, I.R.; Merz, K.M.; Ferguson, D.M.;

Spellmeyer, D.C.; Fox, T.; Caldwell, J.W.; Kollman, P.A. A second generation

force field for the simulation of proteins, nucleic acids, and organic molecules. J.

Am. Chem. Soc. 1995, 117, 5179–5197.

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

PSEUDOSUGAR ANALOGUES OF CHITOTRIOSE

489

25. Homans, S. A molecular mechanical force field for the conformational analysis

of oligosaccharides: Comparison of theoretical and crystal structures of Mana1-

3Manb1-4GlcNAc. Biochemistry 1990, 29, 9110–9118.

26. Glennon, T.M.; Zheng, Y.; Le Grand, S.M.; Shutzberg, B.A.; Merz, K.M. A force

field for monosaccharides and linked polysaccharides. J. Comput. Chem. 1994, 15,

1019–1040.

27. Woods, R.J.; Dwek, R.A.; Edge, C.J.; Fraiser-Reid, B. Molecular mechanical

and molecular dynamical simulations of glycoproteins and oligosaccharides. 1.

GLYCAM-93 parameter development. J. Phys. Chem. 1995, 99, 3832–3846.

28. Dzakula, Z.; Westler, W.M.; Edison, A.S.; Marley, J.L. The CUPID method for

calculating the continuous probability distribution of rotamers from NMR data. J.

Am. Chem. Soc. 1992, 114, 6195–6199.

29. Poppe, L. Modeling carbohydrate conformations from NMR data: Maximum

entropy rotameric distribution about the C5 C6 bond in gentiobiose. J. Am. Chem.

Soc. 1993, 115, 8421–8426.

30. Subroto, T.; van Koningsveld, G.A.; Schreuder, H.A.; Soedjanaatmadja, U.M.S.;

Beintema, J.J. Chitinase and b-1,3-glucanase in the lutoid-body fraction of Hevea

latex. Phytochemistry 1996, 43, 29–37.

31. Brurberg, M.B.; Nes, I.F.; Eijsink, V.G.H. Comparative studies of chitinases A and

B from Serratia marcescens. Microbiology 1996, 142, 1581–1589.

32. Spindler, K.-D.; Spindler-Barth, M. Inhibition of chitinolytic enzymes from Strep-

tomyces griseus (bacteria), Artemia salina (crustacea), and a cell line from Chi-

ronomus tentans (insecta) by allosamidin and isoallosamidin. Pestic. Sci. 1994, 40,

113–120.

33. Spindler, K.-D. Chitinase and Chitosanase Assays. In Chitin Handbook; Muzzarelli,

R.A.A., Peter, M.G., Eds.; Atec: Grottammare, 1997; 229–235.

34. McCreath, K.J.; Gooday, G.W. A rapid and sensitive microassay for determination

of chitinolytic activity. J. Microbiol. Methods 1992, 14, 229–237.

35. SYBYL¹Molecular Modelling Software, Version 6.4; TRIPOS, Inc.: St. Louis,

MO, USA, 1997.

Received July 5, 2001

Accepted September 5, 2002

Article Doi

Synthesis and conformational analysis of pseudosugar analogues of chitotriose

DOI: 10.1081/CAR-120016847

Source and publish data:

Authors:

Article abstract of DOI:10.1081/CAR-120016847

Full text of DOI:10.1081/CAR-120016847

Products guided by the article

R&D Labs maybe for 522634-50-0

Relevant to this article

Hot Product