# US 10,500,221 B2 · Crystalline difucosyllactose

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 10,500,221 B2
- Document ID: US10500221B2
- Status: Granted
- Kind: B2
- Application number: 15/532,747
- Filed: 2015-12-04
- Published: 2017-11-30
- Granted: 2019-12-10
- Earliest claimed: 2014-12-05
- Assignees: Glycom A/S
- Applicants: Glycom A/S
- Inventors: Pierre Chassagne; Martin Matwiejuk; Markus Hederos; Györgyi Osztrovszky; Gyula Dekany
- Web page: https://patentgrep.com/patent/US10500221B2

## CPC

- A61K 31/702: Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
- A61K 31/7024: Esters of saccharides
- C07H 1/06: Separation; Purification
- C07H 3/06: Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
- C07H 15/18: Acyclic radicals, substituted by carbocyclic rings
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

Crystalline difucosyllactose, useful in a pharmaceutical composition and a nutritional composition, is disclosed.

## Claims

### Claim 1

Crystalline difucosyllactose characterized by X-ray powder diffraction reflections, based on a measurement using CuKα radiation, at 11.10±0.20 2Θ, 15.80±0.20 2Θ and 18.38±0.20 2Θ.

### Claim 2 (depends on 1)

The crystalline difucosyllactose according to claim 1, wherein the crystalline difucosyllactose contains 2-5 mols of water of hydration per mol of difucosyllactose.

### Claim 3 (depends on 1)

A pharmaceutical composition comprising the crystalline difucosyllactose of claim 1 as a pharmaceutically active ingredient, together with a pharmaceutically acceptable carrier.

### Claim 4 (depends on 1)

A nutritional composition comprising the crystalline difucosyllactose of claim 1 as a nutritionally active ingredient, in an infant formula or a food supplement.

### Claim 5 (depends on 1)

The crystalline difucosyllactose according to claim 1, wherein the crystalline difucosyllactose displays X-ray powder diffraction reflections, based on a measurement using CuKα radiation, at 11.10±0.20 2Θ, 15.80±0.20 2Θ, 18.38±0.20 2Θ and 14.46±0.20 2Θ.

### Claim 6 (depends on 1)

The crystalline difucosyllactose according to claim 1, wherein the crystalline difucosyllactose displays X-ray powder diffraction reflections, based on a measurement using CuKα radiation, at 11.10±0.20 2Θ, 15.80±0.20 2Θ, 18.38±0.20 2Θ, 14.46±0.20 2Θ and 16.54±0.20 2Θ.

### Claim 7 (depends on 1)

A method for producing crystalline difucosyllactose according to claim 1 comprising the steps of:
a) dissolving non-crystalline difucosyllactose in water or a solvent system containing one or more C1-C4 alcohols and water between room temperature and 80° C. to form a mixture, or providing a difucosyllactose solution in water or a solvent system containing one or more C1-C4 alcohols and water between room temperature and 80° C.,
b) stirring the mixture or solution from step a) and allowing the mixture or solution to cool to room temperature if step a) is performed above room temperature,
c) optionally adding one or more C1-C4 alcohols to the mixture during step b), and
d) collecting and drying precipitated difucosyllactose crystals from the mixture of step b) or c).

### Claim 8 (depends on 7)

The method according to claim 7, wherein 2.5-8 volumes C1-C4 alcohol and water per g of difucosyllactose to be crystallized are used, and the C1-C4 alcohol/water volume ratio is between 1 and 6.

### Claim 9 (depends on 7)

The method according to claim 7, wherein the C1-C4 alcohol is ethanol or isopropanol.

### Claim 10 (depends on 7)

The method according to claim 7 comprising the steps of:
dissolving non-crystalline difucosyllactose at 50-70° C., in 2.5-6.5 volumes of aqueous ethanol, wherein the ethanol-water ratio is 1.5-3, or first adding 1-2.5 volumes of water then 1.5-4 volumes of ethanol, to form the mixture in step a),
cooling the mixture to room temperature in step b),
optionally adding further 1-3.5 volumes of ethanol to the mixture during step c).

### Claim 11 (depends on 10)

The method according to claim 10, wherein the non-crystalline difucosyllactose is made by chemical fucosylation of a suitably protected 3,2′-dihydroxy-lactose acceptor followed by complete deprotection.

### Claim 12 (depends on 11)

The method according to claim 11, wherein the chemical fucosylation of a suitably protected 3,2′-dihydroxy-lactose acceptor followed by complete deprotection comprises the hydrogenolysis of a compound of formula 1
wherein R1 and R2 are independently a group removable by hydrogenolysis; and R3 is a group removable by hydrogenolysis or H.

### Claim 13 (depends on 11)

The method according to claim 11 comprising the steps of:
dissolving the non-crystalline difucosyllactose at 50-70° C., in 2.5-4 volumes of aqueous ethanol, wherein the ethanol-water ratio is 1.8-2.8, or first adding around 1 volume of water then 1.5-3 volumes of ethanol in step a),
stirring the mixture and allowing the mixture to cool to room temperature in step b),
adding 2-3.5 volumes of ethanol to the mixture in step c).

### Claim 14 (depends on 7)

The method according to claim 7 comprising the steps of:
dissolving non-crystalline difucosyllactose at room temperature, in 3-4 volumes of aqueous isopropanol, wherein the isopropanol-water ratio is about 1, or first adding 1.5-2 volumes of water then 1.5-2 volumes of isopropanol, to form the mixture in step a),
stirring the mixture for 12-24 hours in step b).

### Claim 15 (depends on 14)

The method according to claim 14, wherein the non-crystalline difucosyllactose is made by chemical fucosylation of a suitably protected 3,2′-dihydroxy-lactose acceptor followed by complete deprotection.

### Claim 16 (depends on 15)

The method according to claim 15 comprising the steps of:
dissolving the non-crystalline difucosyllactose at room temperature, in 3-4 volumes of aqueous isopropanol, wherein the isopropanol-water ratio is about 1, or first adding 1.5-2 volumes of water then 1.5-2 volumes of isopropanol, to form the mixture in step a),
stirring the mixture from step a) for 12-24 hours in step b).

### Claim 17 (depends on 15)

The method according to claim 15, wherein the chemical fucosylation of a suitably protected 3,2′-dihydroxy-lactose acceptor followed by complete deprotection comprises the hydrogenolysis of a compound of formula 1
wherein R1 and R2 are independently a group removable by hydrogenolysis; and R3 is a group removable by hydrogenolysis or H.

### Claim 18 (depends on 7)

The method according to claim 7 comprising the steps of:
providing a difucosyllactose solution in 2-3 volumes of water at room temperature in step a), and
6-13 volumes of the one or more C1-C4 alcohols are added in step c).

### Claim 19 (depends on 7)

The method according to claim 7 comprising the steps of:
providing a difucosyllactose solution in 3-6 volumes of aqueous C1-C2 alcohol at room temperature in step a), and
5-10 volumes of the one or more C1-C4 alcohols are added in step c), wherein the one or more C1-C4 alcohols is C3-C4 alcohol.

### Claim 20 (depends on 7)

The method according to claim 7, wherein the difucosyllactose of step a) is made by a biotechnological method comprising the steps of:
culturing, in an aqueous fermentation broth or culture medium containing lactose, a genetically modified LacZ−Y+E. coli containing a recombinant gene that encodes a 1,2-fucosyl transferase, to produce difucosyllactose by fucosylating lactose, and separation of the aqueous carbohydrate fraction comprising difucosyllactose from non-carbohydrate particulates and contaminants of the fermentation broth.

### Claim 21 (depends on 20)

The method according to claim 20, wherein the aqueous carbohydrate fraction contains 2′-fucosyllactose and difucosyllactose in which difucosyllactose is at least 2.5% of the weight of 2′-fucosyllactose.

### Claim 22 (depends on 21)

The method according to claim 21, wherein the difucosyllactose of step a) is made by a method comprising the steps of:
i) selective crystallization of 2′-fucosyllactose,
ii) dissolving the concentrated mother liquor after step i), at 50-70° C., in 6-7 volumes of aqueous ethanol, wherein the ethanol-water ratio is 1.5-2, or first adding 2-2.5 volumes of water then 4.5-5.5 volumes of ethanol,
iii) stirring the mixture obtained in step ii) and allowing it to cool to room temperature,
iv) adding seeding crystal of DFL to the mixture during step iii) to assist crystallization,
v) optionally adding further 0.5-1.5 volumes of ethanol to the mixture, and
vi) collecting and drying the precipitated crystals from the mixture of step iii) or v).

### Claim 23 (depends on 1)

A method for producing crystalline difucosyllactose according to claim 1 comprising the steps of:
a) providing, at room temperature, a difucosyllactose solution in 3-7 volumes of aqueous C1-C4 alcohol not containing an organic solvent other than an alcohol,
b) adding 3-13 volumes of a ketone or an ester and mixing the difucosyllactose solution and the ketone or ester to form a mixture,
c) collecting and drying precipitated difucosyllactose crystals from the mixture.