Carbohydrates are of fundamental importance to human skin. For instance, proteoglycans (PGs) and glycosaminoglycans (GAGs) are pivotal in dermal matrix structure that embeds and sustains collagen fibers network.¹  A decrease in the content of GAGs has been linked to changes in the mechanical properties of human skin with ageing and aged skin contains less GAGs than young skin.²  GAGs also play a basic role in structural arrangement of water supply at a molecular level, in cell adhesion and in signalling through their ability to interact with cells, growth factors and cytokines at both dermal and epidermal levels. The major way to maintain dermal matrix structure during ageing or to restore its functions following alteration is to stimulate GAGs synthesis. As a consequence, the discovery of a new class of molecules active on the stimulation of GAGs biosynthesis was a key-objective in the field of anti-ageing formulae. In most GAGs found in human skin, xylose is an essential carbohydrate unit. It is involved in their biosynthesis and in their linking to a protein core via a β-O-glycoside bond between xylose and the hydroxyl group of a specific serine amino acid of protein core to form PGs (Fig. 1 and Fig. 2).

Here, we report a brief description of the work of L'Oréal's Research group in the eco-design of a new class of activators of GAGs biosynthesis based on xylose and close carbohydrate units.³ 

For several years, the L'Oréal Research group has been implementing action plans for sustainable innovation, and has been reporting progresses of these actions annually.⁴  Among these actions, the commitment to green chemistry plays an essential role with respect of the green chemistry principles⁵  based on the following fundamental pillars:

•Use of renewable raw materials from plants.

•Development of eco-friendly processes.

•Launching of new ingredients with very low environmental impact.

In order to respect and deepen our commitment of eco-design, we have also set up green indicators:

•“Atom economy”⁶  evaluation.

•E-Factor⁷  for the evaluation of amount of waste generated by the processes.

•Rate of renewable carbon.

•Environmental risk assessment according to European guidelines.⁸ 

To ensure efficient energy, the use of processes which show too high energy demand was avoided such as:

•Temperature <−15°C or >150°C

•Duration>10 h

In compliance with this strong commitment, the known interest of C-glycosyl derivatives as carbohydrates biomimetics⁹  and the discovery of a new process of C-glycosylation invented by Pr. Lubineau and his team¹⁰  (Scheme 1) were instrumental in such strategy.

The new process, contrarily to other processes known to synthesize C-glycosyl compounds,¹¹  is in perfect agreement with green chemistry principles, notably avoiding the use of protecting groups and toxic reagents and solvents. It proceeds¹⁰  via a Knoevenagel's reaction between activated methylene and a naked aldose followed by a Michael-type intramolecular addition and a retro-Claisen aldol condensation leading to the β-C-glycosyl anomer (Scheme 2). The yield of the pure anomer β reinforced our interest in this reaction in complete agreement with the “biomimetic” approach.

In order to study the potential of this reaction in our research for GAGs biosynthesis activators, various C-glycosyl derivatives were synthesized from different carbohydrate units and β-diketones as described in Scheme 3.

Table 1 shows that the nature of the sugar has an impact on the reaction yield with, for instance, a limited interest for arabinose as compared to xylose, illustrating the importance of the stereochemistry. Moreover, the choice of activated methylenes is also restrictive since only the β-diketone with a simple methyl residue (2,4-pentanedione) gives quantitative yields.

In our hands, malonates, malonamide, malononitrile, Meldrum's acid, hexafluoroacetylaceton, 1,3-indanedione, ethyl cyanoacetate also failed to lead to C-glycosyl products. However, collaborating with Pr. Lubineau's team,¹²  we succeeded in replacing the 2,4-pentanedione by diketones bearing long alkyl chains as described in Scheme 4, but without quantitative yields.

End-products with a C–8 chain (total chain with n=5) in particular are obtained with 75% yields from d-glucose and 65% from d-maltose. As depicted in Scheme 4, the diketonic reagent should necessarily be symmetric to avoid the concomitant synthesis of a mixture of C-glycosyl ketones of various chain lengths, inevitable with asymmetrical diketones. Moreover, the synthesis of the C-maltosyl products clearly points out that these disaccharides also show reactive in the Lubineau's reaction.

In order to increase the yield of the respective C-glycosyl compounds, the conditions (nature of the base, time, temperature, solvent) of the reaction were modified. Xylose was chosen as a model carbohydrate (Scheme 5), due to its potential interest in the activation of GAGs biosynthesis, as seen before.

The nature of the base also has a very significant effect on the yield and on the reaction time as shown in Table 2. We confirmed on the model reaction using the 2,4-pentanedione that NaOH is the ablest base to quantitatively yield the β-C-xylosyl product with a decreasing reaction time at a lower temperature (50°C).

To allow further structure-activity relationships studies, we chose to enhance the structural diversity of this new class of C-glycosyl derivatives by subjecting the keto C-glycosyl compound to further transformations. For this objective, the reduced products 14–20 (mixture of diastereoisomers obtained, see list in Table 3) were synthesized after treatment with aqueous sodium borohydride for the first batch at a laboratory scale.¹³ 

In order to respect the principles of green chemistry and avoid complex procedure to remove borate salts, a catalytic hydrogenation was developed. Accordingly, Ru/C was used as catalyst as described in Scheme 6 on a model reaction based on the reduction of the keto C-xylose¹⁴  then confirming that the reduced C-xylosyl compound 18 is a 50/50 diastereoisomer mixture.

The interest of synthesized C-glycosyl derivatives as potential activators of the biosynthesis of GAGs was evaluated using human fibroblast cultures and assessed by the incorporation of the d-[6-H3]-glucosamine within the GAG fraction.¹⁵  The main results, reported in Table 4, highlight the better activity of compound 18.

This evaluation confirms the interest of xylose unit in the biosynthesis of GAGs.¹⁶  It also confirms that the C-xylosyl structure and the reduction of the exocyclic ketone are essential to obtain the best results. Moreover, further studies gave evidence that the β anomer was crucial to maintain activity, as compared to the α anomer.

These results show the interest of our approach based upon biomimicry and green chemistry to select a new active ingredient of high performance in skin anti-ageing strategy. Compound 18 selected as the best activator of the biosynthesis of GAGs in vitro, was further confirmed also very active in vivo in a clinical trial when topically applied. Introduced in cosmetic skin care products,¹⁷  it has been marketed under the trade name Pro-Xylane™.

This active ingredient respects the green chemistry principles including:

•Sustainable origin of d-xylose from beech trees originating from renewable forest certified by Forest Stewardship Council.

•A two-step process in water using Lubineau's C-glycosylation and a catalytic hydrogenation (Scheme 6). This new process avoids activation steps used in previously described syntheses.¹⁸ 

•Low environmental impact of Pro-Xylane™ since not ecotoxic.

The calculation of green indicators based on Pro-Xylane™ validated this respect of green chemistry principles (see Fig. 3). For example, we took into account the amount of water used in the reaction for the calculation of the E-Factor.

This compound is the first example of “green” chemical described in cosmetics, L'Oréal being the first company to develop a C-glycosyl derivative originating from Lubineau's reaction at an industrial scale.

In summary, the successful launch of a new ingredient of high performance counter-acting skin ageing stresses the high interest of the Lubineau's reaction for green innovation and new green building blocks. For such, illustrative examples are given in the literature.¹⁹ 

We dedicate this chapter to the memory of Professor Lubineau. We thank Pr. Lubineau's team for their essential contribution to the elaboration of the C-glycosylation reaction in water. Special thanks to all L'Oréal participants, being from Research or Industry for their valuable contribution.