NEO-AMYLOPECTINYL MODELS SYNTHESIS OF COMPLEX BETA-BRANCHED MALTO-OLIGOSACCHARIDES IN SOLUBLE AND SOLID PHASE

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NEO-AMYLOPECTINYL MODELS SYNTHESIS OF COMPLEX BETA-BRANCHED

MALTO-OLIGOSACCHARIDES IN SOLUBLE AND SOLID PHASE

Christophe Bliard, Virginie Glaçon

To cite this version:

Christophe Bliard, Virginie Glaçon. NEO-AMYLOPECTINYL MODELS SYNTHESIS OF COM- PLEX BETA-BRANCHED MALTO-OLIGOSACCHARIDES IN SOLUBLE AND SOLID PHASE.

1st European Chemistry Congress, Aug 2006, Budapest, Hungary. 2006. �hal-02328669�

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Abstract:

Starch is the ubiquitous glucidic reserve compound in nature. Synthesized by most plants from solar energy it is easily produced in very large scale cultures (cereals, tubers, legumes…).

Besides its irreplaceable position in the food chain Starch is also a widely used commodity for its non-alimentary properties (paper, textile industry, adhesive, gels…) in almost all human activities¹. Though the polysaccharidic nature and the basic structure : polya(1-4) glucopyrannose of its minor constituent Amylose, and a(1-6) branched a(1-4) polyglucopyrannose of the major amylopectin has long been known, to date, the fine primary structure of the former still remains to be described ! The branching pattern found in amylopectin can reach extreme complexity. The determination of amylopectins’ primary structures from various botanical origins families can be a real challenge. Moreover, though the enzymes involved in starch synthesis have been well-described², no satisfactory in-vitro synthesis has been achieved to date, one of the reasons invoked being the lack of proper primer substrate³. In this paper we present an investigation of such structural diversity by re-building well-defined branched malto-oligosaccharidic model structures through chemical hemisynthesis, in order to obtain such substrates.

Several isoamylase resistant, beta-branched neo-amylopectinyl oligosaccharides having degrees of polymerisation (DP) 4 to 8, with well-defined structures, were obtained. The construction of these models was performed using chemically modified malto-oligosaccharides in solution. All structures were confirmed by long distance heteronuclear NMR spectroscopy. Using activated Wang resin, solid-phase supported oligosaccharides were synthesised and the structures analysed by HR-MAS NMR.

1- Sicard P.J. L’actualité chimique 11-12 (2002) 23-26

2 - Buléon A., Colonna P., Planchot V., Ball S., Int. J. Biol. Macromolecules, 23, (1998) 85-112.

3 - Ball S. et al., Cell, 86(1996)349-352

NEO-AMYLOPECTINYL MODELS

SYNTHESIS OF COMPLEX BETA-BRANCHED MALTO-OLIGOSACCHARIDES IN SOLUBLE AND SOLID PHASE.

Christophe BLIARD & Virginie GLAÇON

FRE 2715 CNRS, UFR Sciences, bâtiment 18 Europol’Agro, Moulin de la Housse, BP 1039, 51687 Reims cedex 2

Tel: 03 26 91 34 95 / Fax : 03 26 91 35 96 / christophe.bliard@univ-reims.fr Université de Reims

Amylose (25%)

linear

Amylopectin (75%)

branched

O O H O OH

O

O H O OH

O OH O OH

OH OH

O OH

H O H O OH OH

n OH

a1,4

Oligosaccharide Syntheses :

The model oligosaccharides, chosen as targets were the neo-amylopectines bbranched analogs. Retro-synthesis analysis showed the possibility a fast, flexible and easy access of a large variety of well defined specific branched structures of DP 3 to 9 from maltose and maltotriose in 7 to 11 steps. Oligosaccharides up to DP 8 branched neo-amylopectinyl models were synthesized from common activated substrates on selectively protected di- and trisaccharide derivatives. These derivatives are destined :

 to enzymatic test on starch synthetases and hydrolases in order to study biosynthesis and biodegradation

 to serve as RMN structure references in the analysis of limit dextrines from amylopectine enzymatic degradation

 to serve as reference in stuctural analysis (influence of parameters such as inter-branched chain distances).

 Synthesis of activated maltosyl derivative 5

 Synthesis of maltosyl acceptors 9 et 10

Synthesis of tera- penta, hexa and octasaccharides branched models 21, 22, 23, 24

RO O

RO OR

OR

O O

RO OR

OR

BzO O

BzO OBz

OBz

O O

BzO OBz

OBz

R

BzO O

BzO OBz

OBz

O O

BzO OBz

OBz

O C

CCl₃ NH

HBr , AcOH CCl₃CN, DBU

1 2 BzCl, Pyr. 5

98%

Ag₂CO₃,(CH₃)₂CO, H₂O

85%

R = H R = Bz

3 4

R = Br R = OH

80%

O

H O OH

O O H O OH O

OH OH

O O O

H O

OH OH

a1,4

a1,6 O

O H O OH

O O H O OH

OH

n' O

O O

H O

OH OH

a1,6

BnOH, TMS OTf

O BzOBzO

OBz OBz

O O BzO

OBz OBz

OBn

O BzOBzO

OBz OTr

O O

BzO OBz

OTr

OBn BzO O

BzO OBz

OTr

O O BzO

OBz OBz

88% OBn

6

1) MeONa, MeOH 2) TrCl, Pyr.

3) BzCl Pyr.

7 : 41% 8 : 15%

5 +

BuOH CF₃COOH

BzO O BzO

OBz OH

O O

BzO OBz

OH

OBn BzO O

BzO

OBz OH

O O

BzO OBz

OBz

OBn

9 10

+ 85%

(1- 4 maltosyl)

(1- 6 Branching)

Maltose

Maltotriose

2+2 Models

3+3 Models 2+3 Models

3+2 Models

O B zO OB z

OB z

O O B zO

OB z OB z B zO O

B zO OB z OB z

O

n O NH

CCl₃ n = 0 : 11

n = 1 : 12

O B zOB zO

OB z OH

O B zOO

OB z OH

OB n

19

n = 0

n = 1

O RO

RO OR

O ORO

OR OBn O

RORO

OR

RO O

ORO

OR

RO O

RO O RO

OR OR

O O RO

OR OR

O

O RO

RO OR

O OOR

OR OBn RO

RO O RO

OR OR

O O RO

OR OR

O

O OR

RO OR

O ORO

OR OBn O

RO

OR

RO O

ORO

OR

RO O

O RO

OR OR

O O RO

OR RO

O O

RO

RO OR

RO

O RORO OR

RO

O

O O

RO

RO OR

O ORO

OR OBn RO

O RO

OR OR

O O RO

OR OR

O O

RO

RO OR

RO

O 21 R = Bz : 76%

26 R = H

MeO Na, MeO H 22 R = Bz : 15%

27 R = H MeO Na, MeO H

24 R = Bz : 29 R = H

MeO Na, MeO H 25 R = Bz : 23%

+

+ +

ii) BzCl, pyr.

i) TM SOTf CH₂Cl₂

O BzOBzO

OBz OH

O O BzO

OBz OBz

OBn BzO O

BzO OBz

OBz

O O

BzO OBz

OBz

O C

CCl₃ NH

O RORO OR

O ORO OR

OBn O

RORO OR RO

O ORO OR

RO

OR O

48%

i) TM SOTf, CH₂Cl₂ ii) BzCl, DMAP, pyr.

+

11 18 23 R = Bz :

65%

I IV III

II

II I

III IV

V

I IV III

V

V I V II

V III

II II I

IV III

V V I

II I III IV

 Solid phase synthesis & HR-MAS nmr analysis

Attempts to transfer the same reaction protocol in solid phase supported

reaction using Wang resin failed. But the reaction of the trichloroacetimidate activated wang resin on the hemiacetalic maltose hepta-benzoate lead to the first maltose grafted wang resin. The structure was confirmed by HR-MAS

1H nmr in diffusion filter mode on the solvent swollen resin.

O O

O

OAc OAc

AcO AcO OAc

AcO AcO

O O CCl₃

NH

CH₂Cl₂ TMSTf Catalyseur

Température Ambiante

O

O AcO

AcO

OAc

OAc AcO

OAc

OH

+ 4 29

30

Diffusion HR-MAS proton nmr Spectrum of WANG resin

HR-MAS proton nmr Spectrum of trichloroacetimidate WANG resin

Diffusion HR-MAS proton nmr Spectrum of maltose grafted resin a

O

OH

TMSO Tf C H₂C l₂

O

1.8 mmol/g

BzO O BzO

OBz OB z

O O

B zO

OB z OBz

O C C Cl₃

NH

+

BzO O BzO

OBz OBz

O O

BzO

B zO OB z

O

5 : 1.5 éq.

B^- + CCl₃CN

First European Chemistry Congress ECC 27-31 aug.2006 Budapest Hungary

5

29

30

Wang

B

q_=57.4°

r^-3(3cos²θ-1) min

2.4 10⁵rpm 4 KHz

HR-MAS nmr probe

sample

I

II III

II III III II

IV I

1 2 1

1 2

2 1

3 nm6 nm 2

1

A B C D

Wheat Starch

Branching zone ?