Towards the synthesis of mannose derivatives of natural phenolic compounds

0 50000 100000 150000 200000 In te n si ty ( m A U )

CM synthetized by the chemical route CM: chemical route CM: chemical MWA route

CM

CA

Towards the synthesis of mannose derivatives of natural phenolic compounds .

P. Sainvitu

a

_{, K. Nott}

b

_{, G. Richard}

b

_{, C. Blecker}

c

_{, C. Jérôme}

d

_{, J.-P. Wathelet}

b

_{, M. Paquot}

a

_{, M. Deleu}

a

_.

a_{Department of Industrial and Biological Chemistry, Gembloux Agro-Bio Tech, University of Liège, Belgium} b_{Department of General and Organic Chemistry, Gembloux Agro-Bio Tech, University of Liège, Belgium}

c_{Department of Food Industrial Technology, Gembloux Agro-Bio Tech, University of Liège, Belgium} d_{Center for Education and Research on Macromolecules, University of Liège, Belgium}

The aim of this project is to graft a sugar moiety onto polyfunctional natural phenolic

compounds (see examples in figure 1). This should enhance their water solubility and the

choice of an adequate sugar such as mannose could provide cellular recognition. The

synthesis route was first tested on cinnamyl alcohol which is structurally close to the base

pattern of the molecules in figure 1.

O H O OH OH H H O H OH H H H OH α-D-Mannopyranose M

+

Catalyst O OH H H O H OH H H H OH O Cinnamyl alcohol CA Cinnamyl mannoside CM O H OH p-Coumaryl alcohol O H OH OMe Coniferyl alcohol O H OH OMe MeO Sinapyl alcohol

Objectives

Figure 1: Structures of natural phenolic compounds

Synthesis of cinnamyl mannoside

Reactionnal Scheme

Comparison of the chemical and enzymatic synthesis routes

Experimental

Analysis conditions:

•HPLC Agilent 1200 Series, Agilent Eclipse XDB C18 column (5µm, 4.6*150mm), gradient elution with water and acetonitrile (start 5% -> end 100%) at 0.6ml/min

•ESI-MS data acquired with a Bruker spectrometer, N2 (30°C) as nebulization and drying gaz,scan range between 100 and 1000 m/z

•The enzymatic route seems to be more specific as only one product peak (CM) is observed (at least 5 by the chemical route) •MWA synthesis do not improve CM production in tested conditions. 0 5000 10000 15000 20000 10 11 12 13 14 15 16 17 In te n si ty ( m A U ) Time (min) CM: enzymatic route CM: enzymatic MWA route

CA CM a b a b 0 50000 100000 150000 200000 10 11 12 13 14 15 16 17 In te n si ty ( m A U ) Time (min) CM: chemical route CM: chemical MWA route

CM CA c d d c

MS data negative mode Detected m/z Attribution

294.9 [CM- H]

-341.0 [CM+ HCOO]

-cinnamyl

alcohol D-mannose catalyst water solvent

During, temperature Enzymatic -catalyzed synthesis conditions (Akita’s method, 2006) 0.8M 0.2M β -D-glucosidase from almond (12U/ml) 10% t-butanol 7 days, 50°C Chemical synthesis conditions (Richel’s method, 2010) 0.8M 0.2M Immobilized acid catalyst (35%, 8mg/ml)

none t-butanol 7 days, 50°C

Both synthesis were also performed in MicroWave assisted (MWA) conditions (45 min, 50°C, 200W)

We thank the belgian French Community – Concerted Research Actions

– Wallonia - Europe Academy for financial support. MD thanks the FNRS

for her position as research associate.

10 11 12 13 14 15 16 17

Time (min)

Akita, H. et al. Journal of Molecular Catalysis B: Enzymatic 2006, 40, 8–15

Richel, A. et al. Tetrahedron Letters 2010, 51,10, 1356-1360

Vic, G.; et al., Carbohydrate research 1995, 279, 315-319.

•

Results show that

β

-glucosidase is able to synthetize CM from M and

CA

•

Enzyme-catalyzed route lead to only one product and is so more

specific than the chemical route where several products are observed.

•

This reaction will be tested with more complex molecules (for example

coniferyl alcohol)

•

Obtention of only one product will be important for next fundamental

studies (Interaction of the product with model membranes by Isothermal

Titration Calorimetry and with the Langmuir Film Trough technique)

Conclusions

•Best CM production obtained when using the CA as solvent •However, tert-butanol is necessary to solubilize solid alcohol reagents •No activity was observed in the presence of Pyridine or DMSO.

0 0,55 5000000 10000000 15000000 0,40 0,1 0,2 _0,25 0,3 0,4 0,10 Aire/g 113h Eau Mannose

Comparison of two methods for the enzymatic synthesis

Initial concentration

conditions (Akita, 2006)

=> best CM production

Optimisation of mannose and water levels for the enzymatic route

0 0,55 5000000 10000000 15000000 0,40 0,1 0,2 _0,25 0,3 0,4 0,10 Aire/g 113h Eau Mannose Area/g Mannose Water

Optimum obtained with 0.2M mannose and

10% water

β-D-glucosidase from almond (12U/ml) + D-mannose (0.2M) + water (10%) +CA (0.8M) + t-BuOH (Akita’s method) [+co-solvent (pyridine or DMSO)] +CA (as solvent and reagent) ( Vic’s method )

Reaction for 7 days at 50°C

Acknowledgments

Area/g Mannose Water

CM enzymatic production

Perspectives

References

Time (min) Time (min)

Mannose

Water

15%

10%

5%

0.8M

0.4M

0.2M

Figure 2: CM synthetized by the enzymatic route Figure 3: CM synthetized by the chemical route

Journée Chimie Verte (Gembloux, 14 octobre 2010)

Design of experiment

0,E+00 2,E+04 4,E+04 6,E+04 8,E+04 0 2000 4000 6000 8000 10000 P e a k s a re a ( U V , 2 5 4 n m )

Reaction time (min)

Vic's method Akita's method with DMSO with pyridine