EP3877537A1

EP3877537A1 - Biocatalytic method for producing 2h-hbo and beta-substituted analogues from lgo using a cyclohexanone monooxygenase

Info

Publication number: EP3877537A1
Application number: EP19818241.2A
Authority: EP
Inventors: Florent ALLAIS; Louis MOUTERDE; John Dale Stewart
Original assignee: Institut des Sciences et Industries du Vivant et de lEnvironnement AgroParisTech; University of Florida; University of Florida Research Foundation Inc
Current assignee: Institut des Sciences et Industries du Vivant et de lEnvironnement AgroParisTech; University of Florida; University of Florida Research Foundation Inc
Priority date: 2018-11-08
Filing date: 2019-11-08
Publication date: 2021-09-15
Also published as: US20210388403A1; AU2019375078A1; FR3088324A1; WO2020095008A1; FR3088324B1

Abstract

The invention relates to the field of producing 4-hydroxymethyl-γ-butyrolactone (2H-HBO) from levoglucosenone (LGO). More particularly, the invention concerns an eco-compatible method for synthesising 2H-HBO optionally substituted at the β-position of the lactone function from LGO or a saturated form of LGO such as dihydrolevoglucosenone (2H-LGO) or LGO hydrate (OH-LGO) via a biocatalytic reaction using a cyclohexanone monooxygenase (CHMO).

Description

BIOCATALYTIC PROCESS FOR THE PRODUCTION OF 2H-HBO AND B ANALOGS SUBTITUTED FROM LGO USING A CYCLOHEXANONE

MONOOXYGENASE

The invention relates to the field of the production of 4-hydroxymethyl-y-butyrolactone (2H-HBO) from levoglucosenone (LGO). More particularly, the invention relates to an eco-compatible process for the synthesis of 2H-HBO optionally substituted at b with the lactone function from LGO or from a saturated form of LGO such as dihydrolevoglucosenone (2H-LGO) or hydrate of LGO (OH-LGO) by biocatalytic reaction using a cyclohexanone monooxygenase (CHMO).

Introduction

In order to overcome the problems of dependence on fossil fuels, many processes for the production of chemical compounds of interest have been developed in recent decades from biomass. Lignocellulosic biomass is one of the most exploited green sources of carbon compounds.

Levoglucosenone (LGO) is one of the most interesting products that can be obtained from biomass, in particular by a cellulose pyrolysis flash technology. LGO is commonly used as a starting material for the synthesis of various chemical compounds of interest, in particular 4-hydroxymethyl-a, p-butenolide (HBO) and 4-hydroxymethyl-y-butyrolactone (2H-HBO ). These compounds constitute asymmetric chemical intermediaries (chirals) with high added value; they are in fact frequently used in the food industry, for the production of fragrances and flavors, or in the pharmaceutical industry, for the production of active principles of drugs, taking advantage of their lactonic nucleus and of their chiral center.

As a continuation of the process of using bio-based bio-based products obtained by green processes, it is desirable to develop industrial processes for transforming LGO into HBO and 2H-HBO or any other related molecule, the ecological impact of which is as low as possible. State of the art

Typical LGO enhancement routes in HBO and / or 2H-HBO are illustrated in Figure 1. These pathways use metals, on the one hand, and oxygenated water, on the other hand, two reagents whose undesirable effects are well known, both for the environment and in a context of industrial exploitation.

In order to propose a new, more eco-compatible 2H-HBO production route, the inventors sought to reduce the use of organic solvents and toxic reagents as much as possible.

Recent work by the inventors has made it possible to propose a new pathway for the hydrogenation of LGO into 2H-LGO and of HBO into 2H-HBO not using metals but an enzyme, the alkene reductase OYE 2.6 (WO2018 / 183706 ).

However, the overall process for producing 2H-HBO, although exempt from the use of metals, remains a consumer of hydrogen peroxide (H2O2), a molecule whose use is delicate at industrial level (danger during handling, pollution...).

Advantages of the invention

The inventors have developed a new process for the production of 2H-HBO from 2H-LGO using a natural enzyme, namely a cyclohexanone monooxygenase (CHMO), to replace hydrogen peroxide.

The CHMO activity can for example be carried by an enzyme derived from Acinetobacter sp. or Pseudomonas aeruginosa or any enzyme with the same activity.

This process has several advantages:

It is eco-compatible, due to the absence of organic solvent and metals;

It is industrially viable;

It does not present any risk of exothermicity and / or explosion unlike the synthetic route involving H2O2;

It can be implemented by biotransformation, in particular by using cells which express a cyclohexanone monooxygenase. DETAILED DESCRIPTION OF THE INVENTION

The inventors propose a biocatalytic process for the production of 4-hydroxymethyl-y-butyrolactone (2H-HBO) optionally substituted at b of the lactone function from levoglucosenone (LGO) in a saturated form represented by formula (I)

in which R represents an H or an OH group, NH2, SH, linear, cyclic or branched alkyl, OR1 (with Ri linear, cyclic or branched alkyl, silyl, acyl, benzyl, benzoyl), NHR1 (with Ri linear, cyclic alkyl or branched), NR1R2 (with Ri and R2 alkyl linear, cyclic or branched), SR1 (with Ri alkyl linear, cyclic or branched) or thioacetal, using a natural enzyme with CHMO activity.

Indeed, the inventors have previously shown that a prerequisite for the Baeyer-Villiger reaction by the LGO CHMO is that LGO is used in its saturated form (eg 2H-LGO also called Cyrene ^® ).

By “LGO in a saturated form”, is meant a levoglucosenone molecule whose carbons in positrons a and b of the ketone function are saturated. An example of a saturated molecule in positions a and b is the 2H-LGO (or Gyrene ^®). In its general definition, an LGO molecule in a saturated form is represented by the formula (I) as defined above.

In a preferred embodiment, the LGO in saturated form is 2H-LGO and OH-LGO.

In another preferred embodiment, the LGO is in a saturated diastereoisomerically pure form represented by formula (II). in which R represents an H or an OH group, NH2, SH, linear, cyclic or branched alkyl, OR1 (with Ri linear, cyclic or branched alkyl, silyl, acyl, benzyl, benzoyl), NHR1 (with Ri linear, cyclic alkyl or connected), NR1R2 (with Ri and R2 alkyl linear, cyclic or connected), SR1 (with Ri alkyl linear, cyclic or connected) or thioacetal.

Thus, the subject of the invention relates in particular to a process for the synthesis of 2H-HBO or of OH-HBO from 2H-LGO or of OH-LGO respectively, comprising a biocatalysis reaction using a CHMO followed by hydrolysis acid. More generally, this process allows the synthesis of 4-hydroxymethyl-y-butyrolactone (2H-HBO) optionally substituted at b of the lactone function such as (3S, 4R) -3-hydroxy-4-hydroxymethyl-y-butyrolactone, ( 3S, 4R) -3-methyl-4-hydroxymethyl-y-butyrolactone, (3S, 4R) -3-mercapto-4-hydroxymethyl-y- butyrolactone, (3S, 4R) -3-amino-4-hydroxymethyl-y -butyrolactone, etc.

Any enzyme exhibiting CHMO activity can be used in this process.

By "enzyme exhibiting CHMO activity" or "CHMO" is meant in particular the CHMO derived from Acinetobacter sp. or the Pseudomonas aeruginosa CHMO or any enzyme exhibiting the same activity, in particular variants thereof. By "variant" is meant any enzyme having CHMO activity and having a sequence homology of at least 85% with one of the sequences SEQ ID NO.1 and SEQ ID NO.3 defined below. This homology is preferably at least 90%, or even 95% or 98%.

Tables 1 and 2 show examples of such variants.

Table 1: Variants of the CHMO of Acinetobacter sp. NCIMB9871

Table 2: CHMO variants of Pseudomonas aeruginosa strain Pa1242

The nucleotide sequence of CHMO from strain NCIMB 9871 of Acinetobacter sp. is represented by the sequence SEQ ID NO.1; the corresponding protein sequence is represented by the sequence SEQ ID NO.2.

The nucleotide sequence of CHMO originating from the strain Pa1242 of Pseudomonas aeruginosa is represented by the sequence SEQ ID NO.3; the corresponding protein sequence is represented by the sequence SEQ ID NO.4. Thus, in a preferred embodiment of the invention, the cyclohexanone monooxygenase is an enzyme coded by the sequences SEQ ID NO.1 or SEQ ID NO.3 or a variant of one of these enzymes having a sequence homology d '' at least 85% with one of these sequences, provided that the CHMO activity is preserved.

In a particular embodiment, the synthesis reaction of 2H-HBO according to the invention firstly comprises the conversion of 2H-LGO into a relatively unstable intermediate, known as “cracked”, which gives rise to a second intermediate , formulated this one (2H-FBO). Acid hydrolysis then makes it possible to obtain 2H-HBO from 2H-FBO. Such a reaction is illustrated in its entirety in Figure 2.

The same type of conversion involving the formation of an unstable intermediate is observed during the reaction of synthesis of OH-HBO from OH-LGO and more generally during the synthesis of 4-hydroxymethyl-y-butyrolactone (2H- HBO) optionally substituted for b of the lactone function from levoglucosenone (LGO) in a saturated form.

The acid hydrolysis step can be carried out by any method known to a person skilled in the art, for example using hydrochloric acid, in particular in solution in methanol, acetic acid, sulfuric acid. , an Amberiyst® type resin or any acid zeolite.

In another particular embodiment, the method according to the invention further comprises a preliminary step of transformation of LGO into a saturated molecule of formula (I), such as 2H-LGO. This transformation step can be carried out using heavy metals such as palladium, nickel or platinum, and dihydrogen. In a preferred embodiment, this step is carried out by a hydrogenation reaction not using heavy metals or dihydrogen (H2). In the case where the process consists in converting LGO into 2H-LGO, such a reaction can be carried out using an alkene reductase enzyme such as OYE 2.6, as described in application WO2018 / 183706, or any enzyme having the same activity, including variants thereof.

In a particular embodiment of the invention, the transformation of LGO into a saturated molecule is carried out within a single reaction medium (so-called "one-pot" reaction), this reaction medium containing the catalysts or enzymes necessary for the transformation on the one hand of the LGO into a saturated molecule of formula (I), and on the other hand of the saturated molecule of formula (I) into a molecule of 4-hydroxymethyl-y-butyrolactone (2H-HBO) possibly substituted at b of the lactone function, such as 2H-HBO. So we can get 2H-HBO from LGO in one easy way. This reaction is shown in Figure 3.

In a preferred embodiment of this one-pot process, the transformation of LGO into 2H-LGO is obtained by the action of an alkene reductase, for example OYE 2.6.

The OYE 2.6 alkene reductase sequence. is represented by the nucleotide sequence SEQ ID NO. 5; the corresponding protein sequence is represented by the sequence SEQ ID NO.6.

In another preferred embodiment of this one-pot process, the transformation of 2H-LGO into 2H-HBO or of OH-LGO into OH-HBO is obtained by the action of a CHMO, in particular of the CHMO of Acinetobacter sp. or CHMO of Pseudomonas aeruginosa or a variant of these enzymes.

In an entirely preferred embodiment of this one-pot process, the transformation of LGO into 2H-LGO is obtained by the action of an alkene reductase and the transformation of 2H-LGO into 2H-HBO is obtained through the action of a CHMO.

Such a “one-pot” reaction can be carried out in a synthetic medium or in biotransformation using whole cells. For biotransformation, the cells used can be any type of cell suitable for this use, such as bacteria or animal or plant cells.

The various embodiments described above relate to the same general reaction and their combination is provided in the context of this disclosure.

The present invention will be better understood on reading the examples which follow, provided by way of illustration and should in no case be considered as limiting the scope of the present invention.

DESCRIPTION OF THE FIGURES

Figure 1: Classic ways of valuing LGO and / or 2H-LGO into 2H-HBO. Figure 2: Process for the synthesis of 2H-HBO from LGO and / or 2H-LGO by a biocatalysis reaction using CHMO.

Figure 3: Method of "one-pot" synthesis of 2H-HBO from LGO by a biocatalysis reaction using CHMO and the alkene reductase OYE 2.6.

Figure 4: Process for the synthesis of 4-hydroxymethyl-y-butyrolactone (2H-HBO) optionally substituted at b of the lactone function from levoglucosenone (LGO) in a saturated form.

EXAMPLES

EXAMPLE 1 Production of 2H-HBO from 2H-LGO in synthetic medium

In a solution containing 20 mM Cyrene®, 50 mM Glucose, 0.5 mM NADP ⁺ , 0.25 mM FADH2 and 62.5 U Glucose dehydrogenase (GDH) in a final volume of 25 mL of 0.1 M phosphate buffer pH 8, 0 was added 150 μL of purified CHMO. The solution was magnetically stirred (200 rpm) over a 12 hour period at 37 ° C.

The total consumption of Cyrène® was verified by thin layer chromatography. The analysis revealed that the Cyrène® was completely transformed into an intermediate being neither 2H-FBO nor 2H-HBO (intermediate called “Criégée”). The solution was then evaporated in vacuo and the raw material analyzed by NMR, confirming the analyzes by thin layer chromatography.

Acid hydrolysis (HCl, 2 hours at 45 ° C) makes it possible to convert this intermediate into 2H-FBO then 2H-HBO.

A negative control was carried out using the same conditions without adding CHMO. Under these conditions, no transformation of Cyrene® was observed (thin layer chromatography), which confirms the specific action of CHMO on the transformation of Cyrene®.

EXAMPLE 2 Bioproduction of 2H-HBO from 2H-LGO in a cellular system in bacteria expressing the CHMO of Acinetobacter sp.

Escherichia coli BL21 (DE3) bacteria containing a plasmid carrying an Ampicillin resistance gene and encoding the CHMO enzyme of Acinetobacter sp. were put in culture in 1 ml_ LB medium containing ampicillin at a concentration of 100 pg / L and incubated at 37 ° C / 200 rpm over a period of 12 hours. 500 ml of this solution were then transferred into 50 ml of medium LB containing ampicillin at a concentration of 100 pg / L, incubated at 37 ° C with shaking until reaching a ϋqboo = 0.9. The solution was then centrifuged at 4500 rpm at 4 ° C over a period of 15 minutes. The supernatant was removed and the cells resuspended in 50 mL of minimal M9 medium. IPTG and Cyrene® were then added at final concentrations of 0.15 mM and 10-40 mM respectively.

The total consumption of Cyrène® was verified by thin layer chromatography (maximum concentration of 5.2 g / L) (NB: the system can accept up to 10 g / L but at such a concentration the conversion of Cyrène® n 'is not total). Analysis by thin layer chromatography revealed that Cyrene® was completely transformed into a so-called “Criégée” intermediate different from 2H-FBO and 2H-HBO whereas at 10 g / L we have a mixture containing Cyrène®, the intermediary in question, 2H-FBO and 2H-HBO. Acid hydrolysis (HCl, 2 hours at 45 ° C) allows the transformation of the so-called "Criégée" intermediate into 2H-FBO and then into the molecule of interest, 2H-HBO (validated by NMR analysis).

EXAMPLE 3 Bioproduction of 2H-HBO from 2H-LGO in a cellular system in bacteria expressing CHMO of the strain Pa1242 of Pseudomonas aeruginosa

The same methodology as in Example 2 was used to transform an Escherichia coli BL21 strain using a plasmid carrying a sequence originating from the Pa1242 strain of Pseudomonas aeruginosa. The sequence introduced was described as potentially coding for an enzyme having NAD (P) / FAD-dependent oxidoreductase activity, therefore capable of carrying cyclohexanone monooxygenase activity.

The inventors have shown for the first time that this sequence actually has the suspected activity. Indeed, they tested its ability to convert Cyrene to 2H-HBO and thus showed that it does indeed carry cyclohexanone monooxygenase activity.

A total conversion is observed for concentrations of Cyrene of 1, 25, 2.5 and 5 g / L and a partial conversion for concentrations of 10 and 20 g / L.

NMR analysis confirmed a high purity, 2H-HBO, of the order of 95%.

Example 4 Bioproduction of OH-HBO from OH-LGO in a Cellular System in Bacteria Expressing CHMO

The experiments were carried out as described in Examples 2 and 3. A total conversion is observed for OH-LGO concentrations of 1 g / L. In addition, we observe the production of a single enantiomer, as shown in Figure 4.

Claims

CLAIMS 1. Process for the synthesis of 4-hydroxymethyl-y-butyrolactone (2H-HBO) optionally substituted at b of the lactone function from levoglucosenone (LGO) in a saturated form represented by formula (I)

in which R represents an H or an OH, NH ₂ , SH, linear, cyclic or branched alkyl group, OR ₁ (with Ri linear, cyclic or branched alkyl, silyl, acyl, benzyl, benzoyl), NHR ₁ (with Ri alkyl linear, cyclic or branched), NR ₁ R ₂ (with Ri and R ₂ alkyl linear, cyclic or branched), SR ₁ (with Ri alkyl linear, cyclic or branched) or thioacetal, which process comprises a biocatalysis reaction using a cyclohexanone monooxygenase followed by acid hydrolysis.

2. Method according to claim 1 wherein said levoglucosenone (LGO) in a saturated form is diastereoisomerically pure and represented by formula (II)

in which R represents an H or an OH, NH ₂ , SH, linear, cyclic or branched alkyl group, OR ₁ (with Ri linear, cyclic or branched alkyl, silyl, acyl, benzyl, benzoyl), NHR ₁ (with Ri alkyl linear, cyclic or connected), NR ₁ R ₂ (with Ri and R ₂ linear, cyclic or connected alkyl), SRi (with linear, cyclic or connected alkyl Ri) or thioacetal.

3. Method according to claim 1 or 2 wherein said 4-hydroxymethyl-y-butyrolactone (2H-HBO) optionally substituted at b of the lactone function is 4-hydroxymethyl-y-butyrolactone (2H-HBO) or hydrate of 4-hydroxymethyl-y-butenolide (OH-HBO).

4. Method according to one of claims 1 to 3 wherein said levoglucosenone in saturated form is dihydrolevoglucosenone (2H-LGO) or LGO hydrate (OH- LGO).

5. Method according to one of claims 1 to 4 comprising a prior step of converting LGO into a saturated molecule of formula (I) as defined in claim 1.

6. The method of claim 5 wherein the LGO is converted to 2H-LGO and said conversion of LGO to 2H-HBO is carried out by a reaction using an alkene reductase.

7. Process for the synthesis of 4-hydroxymethyl-y-butyrolactone (2H-HBO) from levoglucosenone (LGO) consisting in placing in an same reaction medium an enzyme allowing the conversion of LGO into 2H-LGO and an enzyme allowing the conversion of 2H-LGO into a molecule of 4-hydroxymethyl-y-butyrolactone (2H-HBO), said enzyme for converting 2H-LGO into 4-hydroxymethyl-y-butyrolactone (2H-HBO) being a cyclohexanone monooxygenase.

8. The method of claim 7 wherein said enzyme for converting LGO to 2H-LGO is an alkene reductase enzyme.

9. Method according to one of the preceding claims, in which said cyclohexanone monooxygenase enzyme is derived from a strain of Acinetobacter sp., Or from a strain of Pseudomonas aeruginosa.

10. The method of claim 9 wherein said cyclohexanone monooxygenase is an enzyme encoded by the sequences SEQ ID NO.1 or SEQ ID NO.3 or a variant of one of these enzymes having a sequence homology of at least 85 % with one of these sequences.

1 1. Method according to either of Claims 6 and 8, in which the said alkene reductase is the alkene reductase OYE 2.6 coded by the sequence SEQ ID NO. 5 or one of its variants exhibiting the same activity.

12. Method according to one of the preceding claims in which the synthesis reaction of 4-hydroxymethyl-y-butyrolactone (2H-HBO) optionally substituted at b of the lactone function is carried out in synthetic medium.

13. Method according to one of the preceding claims in which the synthesis reaction of 4-hydroxymethyl-y-butyrolactone (2H-HBO) optionally substituted at b of the lactone function is carried out by biotransformation using whole cells.