Molecular Salts of l-Carnosine: Combining a Natural

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Molecular salts of L-carnosine: Combining a natural antioxidant and geroprotector with “Generally Regarded As Safe” (GRAS) organic acids. Oleksii Shemchuk, Vânia André, Maria Teresa Duarte, Paola Taddei, Katia Rubini, Dario Braga, and Fabrizia Grepioni Cryst. Growth Des., Just Accepted Manuscript • Publication Date (Web): 08 May 2017 Downloaded from http://pubs.acs.org on May 10, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Crystal Growth & Design is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Crystal Growth & Design

Molecular salts of L-carnosine: Combining a natural antioxidant and geroprotector with “Generally Regarded As Safe” (GRAS) organic acids. Oleksii Shemchuk,† Vânia André,‡ M. Teresa Duarte,*‡ Paola Taddei,*§ Katia Rubini,† Dario Braga,† Fabrizia Grepioni†* † Dipartimento di Chimica “G. Ciamician”, Università degli Studi di Bologna, via Selmi 2 40126 Bologna, Italy ‡

Centro de Química Estrutural, Instituto Superior Técnico Universidade de Lisboa, Av Rovisco

Pais, 1049-001 Lisbon, Portugal § Dipartimento di Scienze Biomediche e Neuromotorie, Università di Bologna, Via Belmeloro 8/2, 40126 Bologna, Italy

Abstract The potent natural hydrophilic antioxidant L-carnosine has been reacted with a number of organic acids belonging to the “Generally Regarded As Safe” (GRAS) group, resulting in the preparation of eleven new crystalline salts, which were characterized by X-ray powder diffraction and thermal analysis; the structure of six of them was determined from powder data, and one was fully characterized via single crystal data. The reaction with folic acid resulted in an

ACS Paragon Plus Environment

1


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 27

amorphous folate salt, which was characterized by a combination of thermal methods and Raman spectroscopy; the solubility of the folate salt was also measured, and found to be comparable to that of the commercial sodium folate.

Introduction The issue of “Healthy Ageing” has become a significant challenge in Europe because of the continuous population ageing.1 Increasing life expectancy is accompanied by a higher danger of development of age-associated diseases (e.g. coronary heart disease, cerebrovascular disease, cancer, arthritis, dementia, cataract, osteoporosis, type II diabetes, hypertension, Alzheimer's disease, Parkinson’s disease, etc.).2 Natural antioxidants such as L-carnosine have been successfully applied as geroprotectors.3-4 They reduce the risk of developing ageing-related diseases, 5-9 which is one the main aspects of healthy ageing. Carnosine has been the subject of increasing investigation, as it is witnessed by the number of scientific publications in the period 1950-2017 containing the word carnosine in their titles: 1508, of which 68 in January 2016-January 2017) (source: Web of ScienceTM). L-carnosine plays an essential role in human healthy life; in general, a shortage of antioxidants may result into oxidative damage of DNA, lipids and proteins: potential risks are accelerated senescence and development of ageing-related diseases. Therefore, the increasing interest in the consumption of antioxidants, also in nutraceutical preparations, is not surprising. L-carnosine (see Scheme 1) is a dipeptide of the amino acids β-alanine and L-histidine. The numerous studies of L-carnosine biological activity show that it is a potent natural hydrophilic antioxidant, which preserves human tissues from oxidative stress and is extremely useful in brain function protection.10 L-carnosine works as a scavenger of hydroxyl and superoxide radicals and,


2

Page 3 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


even more efficiently, of the singlet oxygen molecule.11 It shows the ability to inhibit some of the biochemical changes that might be responsible for ageing and ageing-associated diseases (protein oxidation, glycation).11

Scheme 1. L-carnosine in its neutral (left) and zwitterionic (right) forms. The nitrogen marked in red is the target atom for protonation by the organic acids used in the present work.

Nowadays design, formulation and characterization of multiple crystal forms, e.g. polymorphs, solvates, co-crystals and molecular salts, are of great interest for solid-state chemists, especially in the pharmaceutical field, where Active Pharmaceutical Ingredients (APIs) properties can be modified and improved by careful selection of crystals coformers or (de)protonable systems.12 In this work we report on the reactivity of zwitterionic L-carnosine with carboxylic acids of the GRAS type (GRAS = Generally Regarded As Safe) accepted by the pharmacopeia,13 resulting in the molecular salts listed in Table 1. Characterization of the reaction products was not a routine task, as most molecular salts could only be obtained, and not without difficulties, as crystalline powders. In the case of L-carnosine salts with adipic, pimelic, and suberic acid only partial characterization of the resulting solids could be achieved (see Supporting Information). The reaction with folic acid invariably yielded an amorphous solid: in this case Raman spectroscopy was instrumental in the identification of the reaction product. The solubility of the folate salt


3


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 27

turned out to be comparable to that of sodium folate, the form of folic acid commonly administered due to folic acid low solubility in water:14 the folate salt of carnosine, therefore, could represent a new, possible way of introducing folic acid as a dietary supplement in cases when the intake of sodium ions – especially in an aged population – has to be limited to a low dosage.

Table 1. Molecular salts of L-carnosine (L-car) described in the present work. Monohydrated L-carnosine salts COOH

GRAS acid

Product

COOH

HOOC

fumaric acid, H2fum

[L-Hcar][Hfum]⋅H2O

COOH

HOOC

HOOC

maleic acid, H2mal [L-Hcar][Hmal]⋅H2O

COOH

succinic acid, H2suc [L-Hcar][Hsuc]⋅H2O

OH

salicylic acid, Hsal [L-Hcar][sal]⋅H2O

Anhydrous L-carnosine salts GRAS acid

Product

HOOC

OH

glycolic acid, Hgly [L-Hcar][gly]

HOOC

COOH

glutaric acid, H2glu [L-Hcar][Hglu]

azelaic acid, H2aze [L-Hcar][Haze]

folic acid, H2fol [L-Hcar]2[fol]

Partially characterized L-carnosine salts GRAS acid Adipic acid Product

-

Pimelic acid [L-Hcar][Hpim]

Suberic acid a

-

(a) cell parameters from powder data (see below).

Experimental section Materials and instrumentation All reagents and solvents used in this work were purchased from Sigma-Aldrich and used without further purification.


4

Page 5 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Solution synthesis L-carnosine (0.1 mmol) and the selected organic acid (0.1 mmol) were dissolved in 1 to 3 mL of water or water/EtOH mixture; the solution was left to evaporate at room temperature. The crystallization invariably resulted in the formation of transparent oils, from which the molecular salts precipitated in 1-2 weeks as microcrystalline powders; following this procedure it was possible to prepare monohydrated L-carnosine salts of fumaric, maleic, salicylic and succinic acids, and anhydrous L-carnosine salts of glutaric, glycolic and azelaic acids. Carnosine salts with adipic, pimelic, and suberic acid were also synthetized using the same approach: unfortunately, all our attempts at structurally characterize them have yet been unsuccessful. The anhydrous L-carnosine salt of folic acid was obtained when L-carnosine and folic acid were reacted in a 2:1 stoichiometric ratio. Vapour diffusion synthesis L-carnosine salt with azelaic acid was also obtained via slow evaporation of ethanol into the saturated water solution of equimolar quantities of the reagents. Needle-like crystals suitable for single crystal X-ray diffraction were formed in three weeks. Solid state synthesis The use of mechanochemistry in our work with L-carnosine encountered serious limitations: first, grinding only yielded physical mixtures of the starting materials as ascertained by X-ray powder diffraction. Second, the use of drops of water in kneading experiments usually led to formation of sticky substances. The use of ethanol as a solvent for the kneading experiments looked more promising. By kneading with ethanol we managed to obtain the same product with fumaric acid as we did from solution. In the case of salicylic acid, however, the X-ray powder


5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 27

diffraction pattern recorded after ball milling for 30 minutes with L-carnosine showed that the reaction was not complete. Similar behaviour was observed with pimelic and azelaic acids. X-ray powder diffraction measurements Room temperature X-ray powder diffraction (XRPD) patterns were collected on two different diffractometers: (i) on a Bruker D2 phaser X-ray diffractometer in the 2θ range from 3° to 37° using a Cu-Kα (λ=1.54 Å) source equipped with a LinxEye detector, nickel filter and operated at 30 kV and 10 mA, and (ii) on a PANalytical X’Pert PRO automated diffractometer equipped with an X’celerator detector in the 2θ range 2.5–60° (step size 0.011, time/step 50 s, VxA 40x40). Data analyses were carried out using the Panalytical X’pert Highscore Plus program. The identity between the bulk material obtained via solution and solid-state processes was always verified by comparing calculated and observed powder diffraction patterns. Structural characterization from powder data Powder diffraction data were analysed with the software X’Pert HighScore Plus. 15-25 peaks were chosen in the 2θ range 2.5-42.5°, and unit cell parameters were found using DICVOL4 or DICVOL algorithms. The procedure for the structure solution of the L-carnosine salt with fumaric acid is described here as an example: analogous procedures were used for all the systems whose structure was solved from powder diffraction data. This salt is characterized by a triclinic unit cell with a volume of 418.74 Å3, compatible with the presence of one L-carnosine cation, one hydrogen fumarate anion and one water molecule in the asymmetric unit. The structure was solved in the space group P1 by simulated annealing, performed with EXPO201415 using protonated L-carnosine, hydrogen fumarate and water molecules. Ten runs for simulated annealing trials were set, and a cooling rate (defined as the ratio Tn/Tn-1) of 0.95 was used. The best solutions were selected and used for Rietveld refinement, which was performed with the


6

Page 7 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


software TOPAS5.0.16 A shifted Chebyshev function with 16 parameters and a Pseudo-Voigt function were used to fit background and peak shape, respectively. Soft constraints were applied for all bond distances and angles of the fumarate moiety, and a planar group restraint was applied to the aromatic ring. An overall thermal parameter was adopted for all atoms of the L-carnosine and fumarate ions. All the hydrogen atoms were fixed in calculated positions. Refinement converged with χ2 = 3.718 and R_wp = 4.760. Rietveld refinements for all structures solved from powder data are collected in the Electronic Supplementary Information. Structural data for all compounds investigated in this work are listed in Table SI-2. Single Crystal X-ray Diffraction Single-crystal data for [L-Hcar][Haze] were collected at RT on an Oxford X’Calibur S CCD diffractometer equipped with a graphite monochromator (Mo-Kα radiation, λ = 0.71073Å). Data collection and refinement details are listed in Table SI-2. All non-hydrogen atoms were refined anisotropically. HNH and HOH atoms were either directly located or added in calculated positions. SHELX97.17 was used for all structure solutions and refinements on F2. Mercury18 was used for molecular

graphics.

Crystal

data

can

be

obtained

free

of

charge

via

www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB21EZ, UK; fax: (+44)1223-336-033; or e-mail: [email protected]). CCDC numbers 1536199-1536205. Differential Scanning Calorimetry (DSC) DSC measurements were performed for L-carnosine salts with a Perkin–Elmer Diamond. Samples (3–5 mg) were placed in

hermetic aluminium pans. Heating was carried out at

5°C min-1 for all samples. Thermogravimetric Analysis (TGA)


7


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 27

TGA measurements of L-carnosine salts were performed using a Perkin-Elmer TGA7 in the temperature range 30-400 °C under an N2 gas flow, at a heating rate of 5 °C min-1. Raman spectroscopy Raman spectra were recorded on a Bruker MultiRam FT-Raman spectrometer equipped with a cooled Ge-diode detector. The excitation source was an Nd3+-YAG laser (1064 nm) in the backscattering (180°) configuration. The focused laser beam diameter was about 100 µm and the spectral resolution 4 cm-1. Laser power at the sample was about 40 mW. Solubility test for [L-Hcar]2[fol] A test tube containing 1 mL of water was immersed in a water bath of an Elmasonic S 15 (H) sonicator at 298 K. A small amount of [L-Hcar]2[fol] was weighed, and portions of it were added to the test tube and sonicated until the solid was completely dissolved. The procedure was repeated until no more [L-Hcar]2[fol] could be dissolved. The remaining salt was weighed and the difference with the initial quantity calculated as the amount of the dissolved [L-Hcar]2[fol]. The experiment was repeated 3 times and the average value was used.

Results and Discussion A range of organic acids as possible co-formers was used in the reaction with L-carnosine, yielding, in most cases, oily products; however, these oils behave differently upon standing at ambient conditions. Based on their behaviour they can be divided into three groups: Group 1 - Oils that remain stable at ambient conditions and do not change their appearance with time. This is the case of the reactions products with oxalic, citric, malic, aconitic, tricarboxylic, vanillic, gallic and tartaric acids. No modifications were observed after 6 months.


8

Page 9 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Group 2 - Oils that precipitate as a physical mixture of starting materials after 10-30 days (with isophthalic, trans-cinnamic, 5-hydroxyisophthalic, nicotinic and isonicotinic acids). Group 3 - Oils that precipitate as new salts in one-two weeks, as observed in the case of reaction with salicylic, maleic, azelaic and pimelic acids; when pimelic, azelaic and benzoic acids were used, though, the reaction was not complete, and starting material could also be detected. The L-carnosine nitrogen marked in red in Scheme 1 is the target atom for protonation by organic acids in this reaction. Thus, an important issue was to establish whether the protonation of this atom occurred and a salt or a co-crystal was formed via interaction with a carboxylic group of the chosen acid. The “empirical rule of two”20 was used: if [pKa (base) – pKa (acid)] ≥ 2, where pKa (base) is the dissociation constant of the protonated pyridine-type nitrogen and pKa (acid) is the dissociation constant of the organic acid, then the probability of proton transfer and salt formation is high, and the larger this difference, the more certain is the result. Given that pKa values for L-carnosine are 2.64, 6.83 (imidazole ring) and 9.51,21,22 in all products obtained the difference value ranges from ca. 2.5 to ca. 4. We could thus assume with some degree of confidence that proton transfer with NH+⋅⋅⋅COO- formation had occurred in all cases. This inference helped us in the structural determinations from powder data. The correctness of this approach was confirmed when the structure of carnosine with azelaic acid was solved from single crystal data. Encouraged by these preliminary results, we decided to explore the reactivity of Lcarnosine towards α,ω-dicarboxylic acids HOOC-(CH2)n-COOH (n = 0-8, 10). We found a marked relationship between the length of the aliphatic chain of the dicarboxylic acids


9


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 27

and the products of the reactions. The interaction of L-carnosine with oxalic and malonic acids (n=0 and n=1, respectively) resulted in the formation of quite stable oils. Succinic, glutaric, and adipic (n=2-4) reacted with L-carnosine producing oils which precipitated in 1-2 weeks into new substances. The acids with longer chains formed oils which precipitated as a mixture of new products together with unreacted starting materials. However, while the percentage of the starting materials in carnosine salts with pimelic, suberic and azelaic acids was rather low, it greatly increased when sebacic and 1,12dodecanedioic acids were used. Taking into account the fact that L-carnosine is an antioxidant, the following step was to test the possibility of its interaction with other acids, in addition to succinic, also possessing antioxidant properties. Ascorbic, glycolic and folic acids were chosen and made to react with L-carnosine in 1:1 stoichiometry via slow evaporation from solution. The reaction of L-carnosine with ascorbic acid resulted in an unstable oil. The reaction with folic acid in 1:1 stoichiometry was found to be incomplete, since the X-ray powder diffraction pattern showed the presence of peaks of folic acid. Consequently, we decided to repeat the experiment using folic acid:L-carnosine in 1:2 ratio; this time the resulting product was completely amorphous, as it had been observed upon reaction of folic acid with alkali hydroxides or carbonates.14 The salt formation was investigated and confirmed by Raman spectroscopy (see below). Reaction of L-carnosine with glycolic acid resulted in an unstable oil that precipitated into the anhydrous salt [L-Hcar][gly]. Attempts to grow single crystals, suitable for single crystal X-ray diffraction, have so far been unsuccessful. For this reason we decided to attempt structural determination from Xray powder diffraction data, still a non-routine approach if compared to structure


10

Page 11 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


determination from single crystal data. Preliminary investigation of the salts thermal behaviour via TGA (see Figure 1) and DSC was essential to collect information on the possible solvent content. Figures 1a and 1b show the TGA traces of [L-Hcar][Hsuc]⋅H2O and [L- Hcar][Hgly], respectively, chosen here as examples of hydrated and anhydrous salts (TGA traces for all compounds reported in this study are collected in the Supporting Information). The TGA trace for the hydrogen succinate salt shows at ca. 80°C a weight loss of approximately 2.5%, corresponding to the loss of one water molecule per formula unit. On the contrary, in the case of the glycolate salt no significant weight loss is observed before decomposition, which indicates that the salt is anhydrous.

(a)

(b)

Figure 1. TGA traces for [L-Hcar][Hsuc]⋅H2O (a) and [L-Hcar][gly] (b).


11


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 27

On the basis of TGA and DSC measurements we could determine that the salts obtained by reaction of L-carnosine with fumaric, maleic, succinic and salicylic acids, i.e. four salts out of seven, contained one water molecule per formula unit. Once the amount of water had been ascertained, we were able to structurally characterize the molecular salts from X-ray powder diffraction data. Unfortunately, the numerous attempts to fully characterize the products of the reactions with adipic, pimelic and suberic acids have so far been unsuccessful. However, reasonable assumptions on the structure of the adipic acid salt have been made on the basis of powder data (see below).

Monohydrated salts obtained from the reaction of L-carnosine with fumaric, maleic, succinic and salicylic acids. The monohydrated salts obtained by reaction of carnosine with fumaric and succinic acids are isomorphous, and their packings are compared in Figure 2. The [L-Hcar]+ cations are arranged in such a way as to form a porous 3D-network: infinite channels extend parallel to the crystallographic c-axis, and they are filled with hydrogen fumarate/hydrogen succinate anions and the water molecules. On passing to the maleate salt, it can be seen that the channel structure becomes more open and a loose stacking is observed (see Figure 3) involving the hydrogen maleate anion and the imidazole ring on the [L-Hcar]+ cation. In the case of the salicylate salt the packing is definitely dominated by the π-stacking of salicylate and imidazole rings (see Figure 4), reinforced by the opposite charge on the two moieties.


12

Page 13 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(a)

(b)

(c)

(d)

Figure 2. View along the c-axis of isomorphous [L-Hcar][Hfum]⋅H2O (a, b) and [LHcar][Hsuc]⋅H2O (c, d). The carnosine cations form a 3-D network characterized by channels (a, c), extending along the c-axis, filled (b, d) with anions and water molecules (these last represented by light-blue spheres).

(a)


13


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 27

(b) Figure 3. Crystalline [Hcar][Hmal]⋅H2O: (a) view of the cationic 3D-network down the c-axis. Light blue spheres represent the water molecules; (b) a “loose” stacking of hydrogen maleate anions and the imidazole ring on the [L-Hcar]+ cations is observed along the b-axis direction.

(a)

(b) Figure 4. Crystalline [Hcar][sal]⋅H2O: (a) view down the c-axis. (b) The packing is dominated by π-stacking, parallel to the c-axis, of the [sal] and [Hcar] six- and five-membered rings, respectively.


14

Page 15 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Anhydrous salts obtained from the reaction of L-carnosine with glycolic, glutaric, azelaic and adipic acids. The monocarboxylic glycolic acid forms a hydrated salt by reaction with L-carnosine, as is shown in Figure 5. As observed for the hydrated salts obtained by reaction with fumaric and maleic acids, the [L-Hcar]+ cations are arranged in a 3D-channelled framework, and interact via hydrogen bonds between –(NH3)+ / –(NH)+ and carboxylate groups. Stability is ensured by the presence of the small glycolate anions filling the channels parallel to the c-axis direction.

(a)

(b)

Figure 5. Crystalline [Hcar][gly]: view down the a-axis showing (a) the cationic 3D-framework and (b) the channel filling by glycolate anions.

Lengthening the aliphatic chain in the dicarboxylic acid affects the packing features of the resulting molecular salts, with loss of the channeled network: the packing of the anhydrous salts obtained by reaction of L-carnosine with glutaric and azelaic acids, which turned out to be isostructural, are characterized by an alternation of anionic and cationic sheets (see Figure 6).


15


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 27

(a)

(b) Figure 6. Crystalline [Hcar][Hglu]: view down the c-axis (a). Crystalline [Hcar][Haze]: view down the a-axis (b).

The structure of the salt obtained by reaction with adipic acid has not been fully characterized, but assumptions have been made on the basis of X-ray powder diffraction data. Unit cell parameters for the salt with adipic acid have been obtained in the monoclinic space group, and they compare well with those of [Hcar][Hglu] and [Hcar][Haze] (see Table SI-2). The major difference is observed for the length of the b-axis: this is related to the aliphatic chain length, and increases on increasing the number of –CH2 groups in the aliphatic chain, while the a- and c-axes lengths remain almost unchanged.


16

Page 17 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Raman investigation of [L-Hcar]2[fol] Figure 7 shows the Raman spectra of the molecular salt [L-Hcar]2[fol], obtained as an amorphous solid by ball milling of folic acid dihydrate and L-carnosine. For a correct comparison of its spectrum with that of folic acid, we ball-milled a sample of pure folic acid dihydrate to render it amorphous: as expected, the Raman spectrum measured on the amorphous acid was significantly different - in width, relative intensity and wavenumber position of several bands - if compared to that previously reported for a crystalline sample14 (Figure 7). The former appeared therefore to be the best reference spectrum for the [L-Hcar]2[fol] molecular salt. Table SI-1 reports the wavenumbers and assignments14,23-27 for the main Raman bands of crystalline and ball-milled folic acid dihydrate, L-carnosine and [L-Hcar]2[fol]. As it can be seen in Figure 7, the spectrum of [L-Hcar]2[fol] is dominated by the bands of folic acid, although it is not coincident with that of the ball-milled sample, and some spectral features assignable to L-carnosine were detected. As detailed in Table SI-1, some bands of [L-Hcar]2[fol] are assignable to only one component of the salt, while others to both of them. On passing from ball-milled folic acid dihydrate to the folate salt, the C=O stretching band shifted from 1702-1687-1677 cm-1 to 1684-1675 cm-1 and increased its relative intensity; the latter behaviour may be ascribed to the contribution of the C=O stretching mode of L-carnosine, which was observed at 1664-1645 cm-1 (Table SI-1). On the other hand, the shifts observed in this spectral range (which appear clear in the fourth derivative spectra, see Figure SI-1) suggest that the C=O groups of both the components underwent changes in hydrogen bond interactions; the weakening of the highest wavenumber


17


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 27

component (i.e. at about 1700 cm-1), due to the C=O stretching of the COOH group of the glutamic acid moieties14, suggests that they should be mainly present as deprotonated (i.e. as carboxylate groups); this hypothesis would be confirmed by the appearance of the components at 1520 and 1398 cm-1, assignable to glutamate, without excluding the possibility that the appearance of the former band could be ascribed to rearrangements in the folic acid pyrazine ring23 (see Table SI-1).

Figure 7. Raman spectra of the molecular salt [L-Hcar]2[fol], crystalline and ball-milled folic acid dihydrate, and L-carnosine.

In the spectrum of [L-Hcar]2[fol], the main contributions assignable to the incorporation of L-carnosine were identified at about 3140, 2970-2927, 1435, 1300, 995 and 290 cm-1.


18

Page 19 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


In most of these spectral ranges the bands wavenumber position and intensity were different from those observed in the spectrum of pure L-carnosine reported in Figure 7, as well as in the spectra obtained for L-carnosine at pH 5 and 723; this behaviour would suggest that the molecule interacts with folic acid and is not present in a detectable amount as free L-carnosine. Most of the observed differences may be ascribed to the shift in the equilibria involving the imidazole ring of L-carnosine. Actually, it is well known that two tautomers (Scheme 2) exist for its neutral form, i.e. the Nτ protonated (tautomer I; Nτ = pyrrole-like nitrogen) and the Nπ protonated (tautomer II; Nπ = imine-like nitrogen):22

Scheme 2. Tautomers of imidazole

Moreover, depending on the pH, the nitrogen atom may be protonated (pKa = 6.8322) giving rise to a positively charged imidazole group. All these forms have been successfully identified by Raman spectroscopy and discriminated through their characteristic spectral features,23 as detailed in Table SI-1. In spite of the superposition of the folic acid bands in most of these diagnostic spectral ranges, the marker bands of imidazole were tentatively identified in the Raman spectrum of the salt; the presence of the positively charged imidazole group may be indicated by the appearance of its marker components23 at 1631 cm-1 (fourth-derivative spectrum of the salt, see Figure SI-1), 1478 and 1182 cm-1 (Figure 7), without excluding the possibility that these bands could also be


19


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 27

ascribed to folic acid rearrangements (mainly involving the C=O groups, the pyrazine ring, the aliphatic and NH groups, see assignments in Table SI-1) upon interaction with L-carnosine. Going from the spectrum of ball-milled folic acid to that of the salt, the band at about 1300 cm-1 strengthened; this behaviour could be ascribed to the incorporation of tautomer I. However, the appearance of the bands at 1435, 1270 and 995 cm-1 would suggest that also tautomer II should be present. As detailed in Table SI-1, also the bands assignable to folic acid underwent wavenumber shifts and changes in relative intensity upon interaction with L-carnosine; this is the case of the components observed in the spectrum of the ball-milled sample at 1598, 1529, 1343, 1210 and 968 cm-1. The bands observed in the spectrum of the salt at 1571 and 895867 cm-1 had contributions from both folic acid and carnosine, as reported in Table SI-1; their wavenumbers and intensities were different with respect to both these components (Figure 7). In summary, the Raman results are consistent with the presence in the salt of the protonated form of carnosine and deprotonated COO- glutamate groups of folic acid; therefore, in agreement with the “empirical rule of two”, proton transfer with NH+⋅⋅⋅COOformation had occurred; the wavenumber shifts and the changes in relative intensities of several Raman bands suggested that both folic acid and L-carnosine rearranged upon their interaction.


20

Page 21 of 27

Solubility test for [L-Hcar]2[fol] The solubility of the molecular salt [L-Hcar]2[fol] was measured (see Experimental) and compared to those of folic acid,28 sodium folate29 and pure L-carnosine30. It can be appreciated from Figure 8 that the [L-Hcar]2[fol] solubility in water is comparable to that of sodium folate, which is the salt usually commercialized in nutraceutical formulations. 400

solubility / g L

-1

384

350

59.5 50.0

50

0.0016 0

L-carnosine [L-Hcar]2[fol] Na2[fol]

folic acid

1.697

-1

1.7

solubility / mol L

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1.6

0.1

0.103 0.067 0.0036

0.0

L-carnosine [L-Hcar]2[fol] Na2[fol]

folic acid

Figure 8. Comparison of solubilities in water, given in g L-1 (top) and mol L-1 (bottom), for L-carnosine,30 [L-Hcar]2[fol], Na2[fol]29 and folic acid.28 (The experimental value for [L-Hcar]2[fol] is 59.5(5) g L-1; all other values taken from the literature.)


21


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 27

The use of L-carnosine as a co-former in the formation of [L-Hcar]2[fol] resulted into a significant increase in the solubility of folic acid: the molar amount of folic acid released by the salt into the same quantity of solvent is more than 18 times higher than that of pure folic acid. Moreover, this value is only a third lower than that of sodium folate (figure 8, bottom); in addition to this, it can be noted that [L-Hcar]2[fol] solutions are stable, as no precipitate formation was observed after a matter of days. Therefore, [L-Hcar]2[fol] could be considered a valid alternative for the administration of folic acid.

Conclusions In this paper we have reported the results of a systematic exploration of L-carnosine salts formation with a number of co-formers containing at least one carboxylic group. Eleven new salts of L-carnosine have been obtained, of which six have been fully structurally characterized via X-ray powder diffraction. All the obtained salts are potentially useful as drugs, since the acidic co-formers belong to the GRAS group. The L-carnosine salts with glycolic and succinic acids might be of higher interest, since both of them also possess antioxidant activity. This pharmaceutical formulation may be beneficial since L-carnosine is used as a nutraceutical usually in combination with other antioxidants. Furthermore, there is some evidence that glycolic acid31 and some derivatives of succinic acid32 can act synergistically with other antioxidants; for these reasons investigation of the biological activity of these salts might be an interesting development. The L-carnosine salt obtained by interaction with folic acid could be of particular interest. Folic acid dihydrate is quite insoluble in water at room temperature, and for this reason it is generally administered as its sodium salt; as the salt obtained by


22

Page 23 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


reaction with L-carnosine is characterized by a molar solubility comparable to that of sodium folate, and as both species possess antioxidant activity, their association could represent a way of introducing folic acid as a dietary supplement, at the same time reducing the daily intake of sodium ions, especially in an aged population. Another relevant issue is the stability of all the obtained salts: as far as X-ray powder patterns are concerned, they do not show changes after two months storage at ambient conditions. This might be beneficial since, according to Sigma Aldrich requirements,33 pure L-carnosine must be stored at a temperature of -20 °C. ASSOCIATED CONTENT

Supporting information Crystal structure details, TGA and DSC analyses, XRPD and Rietveld refinements are supplied as Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. Accession Codes CCDC 1506017-1506020 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033. AUTHOR INFORMATION

Corresponding Author *E-mail: [email protected] *E-mail: [email protected] *E-mail: [email protected]


23


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 27

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interests. ACKNOWLEDGMENT The University of Bologna and MIUR are acknowledged (OS, KR, DB and FG). TD and VA acknowledge Fundação para a Ciência e a Tecnologia for funding (PEst-OE/QUI/UI0100/2013, RECI/QEQ-QIN/0189/2012 and SFRH/BPD/78854/2011).

References 1.

http://www.healthyageing.eu\

2.

Masoro, E. J.; Austad, S. N. Handbook of the Biology of Aging. Academic Press., 2011.

3.

Boldyrev, A. A. Ann. N.Y. Acad. Sci., 2005, 1057, 193-205.

4.

Anisimov, V. N.; Popovich, I. G.; Zabezhinski, M. A. Biochim. Biophys. Acta, 2006 1757, 573-589.

5.

Medvedev, Z. A. Biol. Rev., 1990, 65, 375 – 398.

6.

Cardona, B. Med Health Care and Philos, 2008, 11, 475–483.

7.

Harman, D. J. Gerontol., 1956, 11, 298-300.

8.

Fuente, M. D. Eur J Clin Nutr., 2002, 56, 5–8.

9.

Ames, B. N.; Shigenaga, M. K.; Hagen, T. M. Proc. Natl. Acad. Sci., 1993, 90, 7915-7922.


24

Page 25 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


10. Boldyrev, A. A.; Stvolinsky, S. L.; Fedorova, Z T.; Suslina, A. N. Rejuvenation Res., 2010, 13, 156-158. 11. Hipkiss, A. R. Ann. N.Y. Acad. Sci., 2006, 1067, 369–374. 12. Jones, W. Isr. J. Chem., 2017, 57, 117-123, and references therein. 13. http://www.fda.gov/Food/IngredientsPackagingLabeling/GRAS/ 14. Braga, D.; Chelazzi, L.; Grepioni, F.; Maschio L.; Nanna, S.; Taddei, P. Cryst. Growth Des., 2016, 16.4, 2218-2224. 15. Altomare, A.; Cuocci, C.; Giacovazzo, C.; Moliterni, A.; Rizzi, R.; Corriero N.; Falcicchio, A. J. Appl. Crystallogr., 2013, 46, 1231–1235. 16. Cohelo, A., TOPAS-Academic, Coelho Software, Brisbane, Australia, 2007. 17. Sheldrick G. M. SHELXL97: Program for Crystal Structure Determination, University of Göttingen, Germany, 1997. 18. Macrae, C. F.; Bruno, I. J.; Chisholm, J. A.; Edgington, P. R.; McCabe, P.; Pidcock, E.; Rodriguez-Monge, L.; Taylor, R.; van de Streek, J.; Wood, P. A. J. Appl. Crystallogr. 2008, 41, 466−470. 19. If stable oils obtained by reaction of L-carnosine with malic and citric acids are touched with a metal pin, an amorphous solid is slowly formed. If the oil is thermally quenched, it solidifies as a glass, which shows a glass transformation temperature of ca. 0°C upon heating via DSC (see Figure SI-23). Oil formation is a well known phenomenon: see for example the comment on page 15 in Gavezzotti, A. Isr. J. Chem., 2017, 57, 1323. 20. Wouters, J.; Quéré, L. Pharmaceutical salts and co-crystals; Royal Society of Chemistry, 2011. 21. Jozanović, M.; Sakač, N.; Jakobović, D.; Sak-Bosnar, M. Int. J. Electrochem. Sci, 2015, 10, 5787-5799. 22. Tanokura, M.; Tasumi, M. , Myazawa, T. Biopolymers, 1976, 15, 393–401. 23. Torreggiani, A.; Tamba, M.; Fini, G. Biopolymers 2000, 57, 149-159.


25


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 27

24. Moore, J.; Wood, J. M.; Schallreuter, K. U. Biochemistry, 1999, 38, 15317-15324. 25. Ashikawa, I.; Itoh, K. Biopolymers, 1979, 18, 1859–1876. 26. Austin, J. C.; Fitzhugh, A. ; Villafranca, J. E. ; Spiro, T. G. Biochemistry, 1995, 34, 7678-7685. 27. Hurst, J. K.; Wormell, P.; Bacskay, B.; Lacey, A. R. J. Phys. Chem. A, 2000, 104, 7386-7397. 28. O'Neil, M. J.; Smith, A.; Heckelman, P. E.; Budavari, S. The Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals, 13 édition. Whitehouse Station (NJ): Merck & Co, 2001. 29. https://www.sigmaaldrich.com/content/dam/sigmaaldrich/docs/Sigma/Product_Information_Sheet/f8758pis.pdf [Na2fol] 30. http://www.hmdb.ca/metabolites/HMDB00033 31. Morreale, M.; Livrea, M. A. Biochem Mol Biol Int., 1997, 42, 1093–102. 32. V. Kokilavani, M. A. Devi, K. Sivarajan, C. Panneerselvam, Toxicol. Lett., 2005, 160, 1-7. 33. http://www.sigmaaldrich.com/catalog/product/sigma/c9625?lang=it®ion=IT


26

Page 27 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


For Table of Contents Use Only Molecular salts of L-carnosine: Combining a natural antioxidant and geroprotector with “Generally Regarded As Safe” (GRAS) organic acids. Oleksii Shemchuk, Vânia André, M. Teresa Duarte,* Paola Taddei,* Katia Rubini, Dario Braga, Fabrizia Grepioni*

The natural antioxidant L-carnosine has been reacted with a number of organic acids belonging to the “Generally Regarded As Safe” (GRAS) group; eleven new crystalline salts were obtained and characterized by X-ray powder/single crystal diffraction and thermal analysis. The reaction with folic acid resulted in an amorphous folate salt, characterized by thermal methods and Raman spectroscopy, the solubility of which is comparable to that of the commercial sodium folate.


27

Molecular Salts of l-Carnosine: Combining a Natural

Recommend Documents