Process Development of Biocatalytic Regioselective 5â²-O

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Process Development of Biocatalytic Regioselective 5’-O-Levulinylation of 2’-Deoxynucleosides Alejandro Carnero, Yogesh S. Sanghvi, Vicente Gotor, Susana Fernández, and Miguel Ferrero Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.5b00152 • Publication Date (Web): 16 Jun 2015 Downloaded from http://pubs.acs.org on June 19, 2015

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Organic Process Research & Development

Process Development of Biocatalytic Regioselective 5’-O-Levulinylation of 2’Deoxynucleosides Alejandro Carnero,a Yogesh S. Sanghvi,b Vicente Gotor,a Susana Fernándeza,* and Miguel Ferrero,a,* a

Departamento de Química Orgánica e Inorgánica, and Instituto Universitario de

Biotecnología de Asturias, Universidad de Oviedo, 33006-Oviedo (Asturias), Spain b

Rasayan Inc., 2802 Crystal Ridge Road, Encinitas, CA 92024-6615, USA

[email protected], [email protected]

ACS Paragon Plus Environment

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Table of Contents Graphic


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ABSTRACT. An enzymatic process was optimized for regioselective levulinylation of 5’hydroxyl group in 2’-deoxynucleosides. The results revealed that the nucleobase protecting group influenced the successful outcome of the enzymatic reaction. Enhanced solubility of 4-tert-butylphenoxyacetyl protected 2’-deoxynucleoside played a major role during acylation reaction enabling the starting nucleosides to be the best substrates for Candida antarctica lipase B. Furthermore, we have developed a packed column protocol as a superior alternative to batch process carried-out in a flask, particularly when scale-up is required. The industrial application of this method was demonstrated via the synthesis of 5’O-levulinylthymidine on 25 g scale.

KEYWORDS: 2’-Deoxynucleosides, Regioselective protection, Lipase, CAL-B.


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Introduction Industrial chemists have been trying to achieve sustainable procedures for many years to satisfy the regulatory concerns.1 Among these, the principles of green chemistry2 provide a framework for chemists to implement chemical processes that are environmentally friendly and economically feasible.3 It is recommended that chemists design atom efficient routes, which incorporates more benign reagents and generate less waste products. Biocatalytic transformations have met some of these attributes particularly in areas such as fine chemicals and pharmaceuticals.4 There are examples of a variety of industrially useful processes ranging from the manufacture of small chiral specialty chemicals to the synthesis of more complex pharmaceutical intermediates.5 Advances in molecular biology, bioinformatics and protein engineering has made possible to design an enzyme for a specific process.6 Biocatalysts are made from renewable sources and are capable of accepting a wide array of substrates, catalyzing reactions with enantio- regio- and chemoselectivities that contribute to shorter synthetic pathways. Furthermore, biocatalytic processes are less energy intensive and generate less waste than conventional organic syntheses. An important feature is the reusability of enzymes when they are immobilized. The immobilized enzymes maintain high selectivity and stability offering easy of handling during large-scale operations. Additionally, continuous flow (CF) processes are emerging as powerful tools to achieve multi-step synthesis of complex natural products and pharmaceuticals agents under sustainable conditions.7 CF chemistry results in reduced manual handling, increased process safety, higher control of the reaction variables, easier reproducibility and reuse of solidsupported catalyst.8 The CF approach has great synergy when coupled with


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biotransformation where enzyme recycling is possible, simplifying reaction work-up between cycles, reduced solvent usage, minimize waste generation and the possibility of automation. Recent publications on the enzyme-catalyzed processes carried out in CF systems are the resolution of 2-(1H-imidazolyl)cycloalkanols,9 1,3,6-tri-O-benzyl-myoinositol,10 2-methylene-substituted cycloalkanols11 or alkaloid calicotomine.12 To the best of our knowledge, there is only one example of continuous flow enzymatic strategy in the nucleoside area reported by Gallou et al.13 This work describes a practical synthesis of isatoribine prodrug via a regioselective hydrolysis catalyzed by Candida antarctica lipase B (CAL-B). Furthermore, application of biocatalysis in nucleoside chemistry has been well recognized as practical alternative to conventional organic synthesis.14 In our ongoing research related with the preparation of regioselectively protected nucleoside precursors for oligonucleotide synthesis, we have successfully executed enzymatic acylations15 and hydrolysis reactions.16 In 2’-deoxynucleosides, CAL-B and Pseudomonas cepacia lipase (PSL-C) have demonstrated excellent selectivity towards the primary hydroxyl group and secondary hydroxyl group, respectively. 5’-O-Levulinylated 2’deoxynucleosides were prepared in high yields via acylation reaction catalyzed by CAL-B with acetonoxime levulinate or hydrolysis reaction catalyzed by PSL-C of the corresponding dilevulinylated nucleosides. These experiments were originally carried out in a batch mode and with the advent of CF chemistry, we elected to apply the concepts of CF system for scale-up using a packed column. Herein, we describe for the first time a comparison of the enzymatic acylation of 2’deoxynucleosides in batch and packed column mode. The data obtained from batch mode has been applied to the column process. The key objective is to perform a comparative study


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of several commercial immobilized lipases to first identify the most efficient catalyst and then establish a scale-up protocol with the preferred lipase. We also evaluated the influence of the base protecting group on the exocyclic amine of 2’-deoxynucleosides (dC, dA and dG) and its impact on the activity and selectivity of the enzyme. It is of paramount importance to choose the correct protecting groups that ensures their stability during oligonucleotide synthesis and fast cleavage upon deprotection. Therefore, we studied the role of conventional base protecting groups (e.g. Bz on dC and dA and Ibu on dG) and included 4-tert-butylphenoxyacetyl (Tac) as a fast cleaving alternative for nucleo-bases.

Results and discussion The enzyme-catalyzed acylation of 2’-deoxynucleosides were performed with CAL-B and PSL-C immobilized on different commercial supports using acetonoxime levulinate as acylating agent and THF as solvent. We selected four CAL-B: first Novozym 435, from Novo Nordisk, adsorbed on Lewait E resin, and three CAL-B from SPRYNzymes; first adsorbed on a methacrylic esters matrix, hereinafter CAL-B1, second adsorbed on polystyrene resin, hereinafter CAL-B2, and third covalently immobilized preparation lipase on epoxy acrylic resin, hereinafter CAL-B3. With respect to the PSL-C, we have observed excellent results with PSL-C from Amano, immobilized on ceramic particles.15h However, this enzyme is not longer commercially available. It is therefore important to identify an alternative enzyme. We included three different immobilized PSL-C: PSL-IM from Amano, immobilized on diatomaceous earth; adsorbed preparation on polystyrene resin, hereinafter PSL1; and covalently immobilized preparation on epoxy acrylic resin, hereinafter PSL2. The last two are from SPRYNzymes.


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The general enzymatic acylation reaction of 2’-deoxynucleosides is outlined in Scheme 1. Scheme 1

First, we studied the acylation of thymidine with various Pseudomonas cepacia lipases in batch mode. Preliminary enzyme screening was performed at 30 °C and the reaction with 3 equiv of acetonoxime levulinate in THF. This screening exhibited high selectivity toward the acylation of the secondary hydroxyl group compared to the primary hydroxyl group, for the three lipases tested. The best conversions were observed with PSL-IM and PSL1. Whereas, the maximal conversion for PSL2 was only 29% after 52 h (entries 1-3, Table 1). In order to improve the conversion, higher reaction temperature was evaluated. The results with PSL1 at 45 °C and 60 °C exhibited similar conversions (69-75%) compared to 30 °C,


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although with shorter reaction times (entries 2, 5, and 8, Table 1). With PSL2, the conversion was improved to 60-61% at higher temperature (entries 6 and 9, Table 1). Best conversions and selectivity was achieved with PSL-IM (entries 4 and 7, Table 1); this lipase afforded 97% conversion after 19 h at 60 °C and exhibited excellent selectivity during formation of the 3’-O-levulinyl derivative 2a, only trace amounts of 5’-O-acyl and 3’,5’-Odiacyl products were detected. Table 1. Batch mode enzymatic levulinylation of thymidine (1a). Entry

Enzymea

T (ºC)

t (h)

Conv. (%)b

2a (%)b

3a (%)b

4a (%)b

1

PSL-IM

30

48

74

67

1

6

2

PSL1

30

52

74

67

1

6

3

PSL2

30

52

29

23

2

4

4

PSL-IM

45

48

97

92

1

4

5

PSL1

45

32

75

70

1

4

6

PSL2

45

48

60

53

3

4

7

PSL-IM

60

19

97

94

1

2

8

PSL1

60

26

69

67

1

1

9

PSL2

60

47

61

58

1

2

10

Novozym 435

30

2.5

>99

5

91

4

11

CAL-B1

30

4

85

6

75

4

12

CAL-B2

30

6

89

6

78

5

13

CAL-B3

30

21

97c

5

87

5

a

See text or General Experimental Section for detailed description of enzymes. Percentage of compounds calculated by HPLC from area peaks. cAfter 6 h, the reaction furnished 85% conversion with 77% formation of 3a.

b

The reaction of thymidine with CAL-B was also performed in batch mode under similar conditions. All enzymes tested demonstrated high conversions and selectivity in short reaction times at 30 °C. Novozym 435 catalyzed the acylation of the 5’-O-position with a


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total conversion in 2.5 h (entry 10, Table 1). CAL-B1 and CAL-B2 exhibited reduced activity with 85-89% conversion (entries 11 and 12, Table 1). CAL-B3 showed similar behavior compared to Novozym 435 but the reaction was relatively slower. After 6 h, the conversion was 85%, and subsequently increased to 97% in 21 h (entry 13, Table 1). Thus, Novozym 435 proved to be the most efficient among 4 enzymes tested furnishing 91% of the desired 5’-O-acyl nucleoside 3a. Next, the acylation of dCBz (1b) with Pseudomonas cepacia lipase was studied. As PSLIM proved to be the most efficient catalyst with thymidine, levulinylation of 1b was first investigated with this enzyme at 45 °C and 60 °C. The reaction at higher temperature resulted in an increase in the biocatalytic activity with 91% conversion after 27 h at 60 °C while maintaining high selectivity (entry 1 vs entry 2, Table 2). The acylation reaction with PSL1 and PSL2 were highly selectively furnishing exclusively 3’-O-Lev-dCBz (2b). Notably, a maximum conversion is reached with both lipases after 24 h, and longer reaction times led to the hydrolysis of the acylated product (entry 3 and 4, Table 2). Table 2. Batch mode enzymatic levulinylation of dCBz (1b). Entry

Enzymea

T (ºC)

t (h)

Conv. (%)b

2b (%)b

3b (%)b

4b (%)b

1

PSL-IM

45

24

64

62

2

0

2

PSL-IM

60

27

91

88

1

2

3

PSL1

60

24

59

59

0

0

4

PSL2

60

24

47

47

0

0

5

Novozym 435

30

8

96

14

77

5

6

CAL-B1

30

4

71

11

59

1

7

CAL-B2

30

8

71

12

58

1

8

CAL-B3

30

20

90c

15

72

3

9

Novozym 435

45

4

98

9

84

5


9


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a

See text or General Experimental Section for detailed description of enzymes. Percentage of compounds calculated by HPLC from area peaks. cAfter 6 h, the reaction furnished 71% conversion with 60% formation of 3b. b

On the other hand, all the Candida antarctica lipases B evaluation demonstrated modest regioselectivity during acylation of the 5’-hydroxyl group (Table 2). Higher conversion was obtained with Novozym 435, which catalyzed the acylation reaction of 1b with almost complete conversion after 8 h at 30 °C. To study the influence of the temperature on the selectivity of the enzyme, the experiment was conducted at 45 °C. Shorter reaction time was required to reach 98% conversion, and the percentage of 3b increased from 77% to 84% (entry 9, Table 2). To further assess this enzymatic process, nucleosides with purine bases were examined. N6-Benzoyl-2’-deoxyadenosine (1c) was treated with different enzymatic preparations from PSL. The enzyme PSL-IM exhibited total selectivity at 45 °C and afforded 83% maximum conversion after 7 h (entry 1, Table 3). An increase in temperature from 45 °C to 60 °C does not result in an increase in conversion rather decrease in reaction regioselectivity (entry 2, Table 3). PSL1 and PSL2 also showed excellent results in the levulinylation of the 3’hydroxyl of 1c (entries 3 and 4, Table 3); however, both enzymes are less active and lower conversions are obtained in comparison with PSL-IM. Table 3. Batch mode enzymatic levulinylation of dABz (1c). Entry

Enzymea

T (ºC)

t (h)

Conv. (%)b

2c (%)b

3c (%)b

4c (%)b

1

PSL-IM

45

7

83

83

0

0

2

PSL-IM

60

9

84

78

6

0

3

PSL1

45

7

31

31

0

0

4

PSL2

45

23

36

35

1

0

5

Novozym 435

30

4

97

4

92

1


10

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6

CAL-B1

30

6

41

4

37

0

7

CAL-B2

30

6

30

2

28

0

8

CAL-B3

30

2

68

4

64

0

a

See text or General Experimental Section for detailed description of enzymes. bPercentage of compounds calculated by HPLC from area peaks. All the enzymes from CAL-B demonstrated high selectivity during levulinylation of the

primary hydroxyl group of 1c. These experiments conclude that Novozym 435 resulted in higher conversion rates (97%) during shorter reaction time (entry 5, Table 3). In contrast, CAL-B1 and CAL-B2 reactions furnished 30-41% conversion (entries 6 and 7, Table 3). CAL-B3 increased the conversion until 68% in only 2 h, but the reaction does not proceed further with longer reaction times (entry 8, Table 3). When the acylation was performed on N2-isobutyryl-2’-deoxyguanosine (1d), the screening with all PSL was unsatisfactory, probably due to the low solubility of starting nucleoside in THF even at 60 °C. PSL-IM gave rise to 33% conversion with poor regioselectivity. In the case of PSL1 and PSL2 99

-

5

90

5

77

98


16

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4e

99

1

6

89

4

75

98

5e

97

3

5

87

5

72

99

a

The reaction was carried out at 30 °C as 0.1 M solution with 1 g of T in a ratio 1:3 of acylating agent and 1:1 of lipase. bPercentage of compounds calculated by HPLC from area peaks. cIsolated yields. dCalculated by HPLC from area peaks. eRecycled enzyme from the previous run. Encouraged by these results, the packed column protocol was tried for all 2’deoxynucleosides (1 g scale, Table 7). The reaction on 1g of dCBz (1b) furnished 86% of 5’O-Lev-dCBz after 18 h at 45 °C. In the case of dABz (1c) the reaction stopped after 1 h at 72% conversion and did not progress further (entry 2, Table 7). In order to drive the reaction to completion 1.5 equiv of acylating agent was added at the beginning of the experiment and another 1.5 equiv after 1 h of reaction. Under these conditions the product precipitated from the reaction mixture after 2 h (entry 3, Table 7). Gratifyingly, HPLC of the reaction mixture showed 94% conversion. The 5’-O-Lev derivative was easily isolated in 61% yield and 92% purity by simple filtration of the solid. On the other hand, high yield and excellent selectivity was observed during the acylation using column approach with Tac-protected nucleosides 1e–g (entries 4-6, Table 7). The data in Table 7 clearly indicates that Novozym 435 showed highest conversions offering 5’-regioselectivity in nucleosides carrying N-Tac group compared to nucleosides with N-benzoyl or N-isobutyryl groups. As illustrated in Figure 4, similar conversions were observed during batch process or during packed column system. The overall productivity was improved during column experiments given the ease of reaction work-up and ability to recycle enzyme. Table 7. Levulinylation of dN with Novozym 435 during packed column set-up.a Entry

1

T (°C)

t (h)

Conv. (%)b

1 (%)b

2 (%)b

3 (%)b

4 (%)b

Yield (%)c

Purity (%)d

1

dCBz

45

18

97

3

8

86

3

65

96


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2

dABz

30

1-4

72

28

Process Development of Biocatalytic Regioselective 5â²-O

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