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. 2025 Apr 22;16(22):9843-9853.
doi: 10.1039/d4sc07556k. eCollection 2025 Jun 4.

High-throughput encapsulated nanodroplet screening for accelerated co-crystal discovery

Jessica P Metherall  1 , Philip A Corner  2 , James F McCabe  2 , Michael R Probert  1 , Michael J Hall  1
Affiliations

High-throughput encapsulated nanodroplet screening for accelerated co-crystal discovery

Jessica P Metherall et al. Chem Sci. .

Abstract

Co-crystals are composed of two or more chemically inequivalent molecular species, excluding solvents, generally in a stoichiometric ratio. Co-crystals are particularly important in pharmaceutical development, where a suitable co-crystal can significantly improve the physiochemical and pharmacokinetic properties of an active pharmaceutical ingredient. However, co-crystal discovery remains both practically challenging and resource intensive, requiring the extensive searching of complex experimental space. Herein, we demonstrate a high-throughput (HTP) nanoscale co-crystallisation method for the rapid screening of large areas of co-crystallisation space with minimal sample requirements, based on Encapsulated Nanodroplet Crystallisation (ENaCt). HTP co-crystallisation screening by ENaCt allowed rapid access to all 18 possible binary co-crystal combinations of 3 small molecules and 6 co-formers (A/B), through the use of 3456 individual experiments exploring solvent, encapsulating oil and stoichiometry, including 10 novel binary co-crystal structures elucidated by single crystal X-ray diffraction (SCXRD). Higher-order co-crystal (HOC) discovery, accessing co-crystals containing three or more molecules, is one of the most challenging co-crystal research areas, due to the highly complex experimental landscape that must be navigated. Herein, we further exemplify the power of ENaCt co-crystallisation by application to HOC discovery. HTP ENaCt co-crystallisation screening of three component (A/B/C) and four component (A/B/C/D) combinations gave ready access to both ternary and quaternary HOCs, each containing three or four different molecular species respectively. In total, 13 056 individual ENaCt experiments are presented resulting in 54 co-crystal structures by SCXRD, including 17 novel binary co-crystals, 8 novel ternary co-crystals and 4 novel quaternary co-crystals. ENaCt co-crystallisation is thus demonstrated to be a highly impactful and efficient tool in the search for small molecule co-crystals, through the employment of parallelised HTP nanoscale experimental workflows.

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Conflict of interest statement

JPM, MJH and MRP and authors of a patent application related to this work.

Figures

Fig. 1
Fig. 1. Substrates and co-formers chosen for binary co-crystal screening, showing H-bond acceptor sites (red), H-bond donor sites (blue), and H-bond donor/acceptor sites (green).
Fig. 2
Fig. 2. Binary co-crystals accessed via ENaCt screening, including previously known co-crystals (white), new co-crystals (red), new co-crystal solvates, hydrates and solvate/hydrates (blue) and new co-crystals showing altered stoichiometry (green). CSD refcode (for known co-crystals), crystal composition ((A/B) : (solvent) : (water)), ENaCt crystallisation conditions (solvent, v/v ratio, oil) shown (MO = mineral oil, PDMSO = poly(dimethylsiloxane), FY = Fomblin YR-1800, and FC-40: Fluorinert FC-40).
Fig. 3
Fig. 3. SCXRD structures showing H-bonding networks in the 4,4′-bipyridine : methyl gallate co-crystal series. (A) 2 : 2 trihydrate, (B) 3 : 2 DMF solvate dihydrate and (C) 3 : 2 MeNO2 disolvate dihydrate.
Fig. 4
Fig. 4. Previously known ternary co-crystals obtained via HOC ENaCt screening. CSD refcode, crystal composition (A/B/C), and ENaCt crystallisation conditions (solvent, v/v/v ratio, oil) shown (MO = mineral oil, PDMSO = poly(dimethylsiloxane), FY = Fomblin YR-1800, and FC-40: Fluorinert FC-40).
Fig. 5
Fig. 5. Serendipitous discovery of a new ternary co-crystal tetramethylpyrazine : 2,2′-bipyridine : 2-chlororesorcinol (1 : 0.5 : 1) via HOC ENaCt screening.
Fig. 6
Fig. 6. HOC ENaCt screening outcomes for ternary co-crystal discovery via molecular replacement with shape-size mimics from the corresponding binary co-crystals, including parent binary systems (white) and new ternary co-crystals obtained (green). Crystal composition ratio (A/B/C) shown. Co-formers screened are given below each parent binary system, with successful (green) and unsuccessful (red) co-formers highlighted. Additional binary co-crystals obtained are indicated: anicotinamide : quinol (2 : 0.5) polymorph I, bnicotinamide : quinol (2 : 0.5) polymorph II, c4,4′-bipyridine : 3-hydroxy-2-naphthoic acid (0.5 : 1), d4,4′-bipyridine : 3-hydroxy-2-naphthoic acid (1.5 : 1), e3-hydroxy-2-naphthoic acid : 1,2-bis(4-pyridyl)ethane (1 : 0.5), fcaffeine : quinol (1 : 1.5), gquinol : tetramethylpyrazine (0.5 : 0.5), h4,4′-bipyridine : glutaric acid (2 : 2), i4,4′-bipyridine : 3,3′-thiodipropionoic acid (0.5 : 0.5).
Fig. 7
Fig. 7. Previously known quaternary co-crystals obtained via HOC ENaCt screening. CSD refcode, crystal composition ratio (A/B/C/D), and ENaCt crystallisation conditions (solvent, v/v/v/v ratio, oil) shown (MO = mineral oil, PDMSO = poly(dimethylsiloxane), FY = Fomblin YR-1800, and FC-40: Fluorinert FC-40).
Fig. 8
Fig. 8. HOC ENaCt screening outcomes for quaternary co-crystal discovery via molecular replacement with shape-size mimics from the corresponding ternary co-crystals, including parent ternary systems (white) and new quaternary co-crystals obtained (green). Crystal composition ratio (A/B/C/D) shown. Co-formers screened are given below each parent ternary system, with successful (green) and unsuccessful (red) co-formers highlighted. Additional binary and ternary co-crystals obtained are indicated: aorcinol : 3,5-dinitrobenzoic acid (4 : 4) hydrate, bcaffeine : oxalic acid (1 : 0.5), ccaffeine : 2-methylresorcinol : oxalic acid (1 : 1:0.5), dcaffeine : 3,5-dinitrobenzoic acid : 2-methylresorcinol (1 : 1 : 2) hydrate, e4,4′-bipyridine : methyl gallate : 2-chlororesorcinol (3 : 1 : 1), f4,4′-bipyridine : methyl gallate (3 : 2) MeNO2 disolvate dihydrate, g4,4′-bipyridine : methyl gallate : 2-chlororesorcinol (3 : 2 : 1) dihydrate, hmethyl gallate : [2,2′-bipyridine]-4,4′-diyldimethanol (1 : 0.5), i4,4′-bipyridine : methyl gallate (2 : 2) trihydrate, j4,4′-bipyridine : methyl gallate (3 : 2) DMF solvate dihydrate, k4,4′-bipyridine : propyl gallate (1 : 1), l2-bromoresorcinol : 4,4′-bipyridine (2 : 3), morcinol : 4,4′-bipyridine (1 : 1.5).
Fig. 9
Fig. 9. Serendipitous discovery of new ternary co-crystals (A) caffeine : 2-methylresorcinol : oxalic acid (1 : 1 : 1) and (B) 4,4′-bipyridine : methyl gallate : 2-chlororesorcinol (3 : 1 : 1).
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