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Alma Mater Studiorum - Università di Bologna

Dipartimento di Chimica Industriale “Toso Montanari”

Corso di Studio in

Chimica Industriale
Classe L-27- Scienze e Tecnologie Chimiche


Gabriele Leita Prof.ssa Cristina Femoni

Dr. Simon Lawrence

Anno Accademico 2018-2019

L-Alanyl-L-Glutamine(ALA-GLN) is a dipeptide which exhibits a lot of benefits for diarrhoea.
However, ALA-GLN applications are quite limited due to its low solubility in water and so this
issue makes ALA-GLN a suitable candidate for cocrystallizatyion processes.
Cocrystals can be divided in two main groups: molecular cocrystals and ionic cocrystals depending
on which kind of coformer the cocrystal is made of, and they are studied for improving the physical
proprieties of a molecule or for purifying it from some solvent residues.
In my case the cocrystallization process was used for improving the solubility of the ALA-GLN in
order to make the molecule more suitable for being ingested and assimilated by the body.
For this research project two crystallization techniques were used: the grinding technique, which
attempts to crystallize the molecule and the coformer by using a ball mill, and the crystallization
technique, which tries to crystallize the reactants by dissolving them in a solvent and leaving it to
evaporate for a few days.

L-Alanyl-L-Glutamine è un dipeptide che mostra molti benefici per la cura alla diarrea, tuttavia la
sua insufficiente solubilità in acqua gli impedisce di essere utilizzato come principio attivo per un
possibile farmaco.
La cocristallizzazione cerca di migliorare queste proprietà tramite l’aggiunta di un coformero ma
può anche essere utilizzata per la purificazione di una molecola dal solvente.
Esistono due tipi di cocristalli: molecolari e ionici, a seconda di che tipo di coformero che viene
usato, nel mio caso è stato creato un cocristallo ionico per cercare di migliorare la solubilità della
molecola di partenza.
Per questa ricerca sono state utilizzate due tecniche di cristallizzazione: tramite il grinding che cerca
di cristallizzare i reagenti usando una macchina Ball Mill e la cristallizzazione tramite solvente che
cerca di cristallizzare i reagenti sciogliendoli prima in un solvente e lasciandolo poi evaporare
all’aria per un paio di giorni.


 PXRD SPECTRA pag. 33
 REFERENCES pag. 41
In the last 20 years, the publications about cocrystals have increased due to renewed interest from
the pharmaceutical field, in particular within solid drugs. The term ‘’cocrystal’’ has been debated in
the literature; one of the most common definitions is that cocrystals are solids composed of
crystalline materials in a single phase, and they are not solvates or hydrates. Cocrystal drugs are
composed of two parts: the first and most important one is called API (active pharmaceutical
ingredient), which is the drug itself and is the active part of the medicine; the other part is normally
an inert material (coformer), which could increase the proprieties of the drug such as stability or
solubility. 1


Scientists differentiate these materials into generally two types: molecular and ionic, the
differentiation depending on the nature of the coformer. Molecular cocrystals (MCCs) contain two
or more neutral coformers, which are in general bonded by hydrogen bonds 1. ICC is usually defined
as a multicomponent crystal composed by a salt and another material, with the general formula A +B-
N, where AB is a salt and N is a neutral molecule. When one of these components is an API we
could have the opportunity to modify the characteristics of the drug 1. The nomenclature of MCCs
was inconsistent in early literature, they were just called “organic molecular compounds” by
Buheler and Heap to describe MCCs of 1,2-dinitrotoluene, 2,4-dinitrobenzene and 2,4-
dinitrophenol with amino derivates of naphthalene and aniline. Higughi and Ikeda found that
digoxin, indicated for the treatment of heart failure, had dissolution rate and its bioavailability was
very low1. These proprieties increased considerably in the presence of hydroquinone. These studies
revealed that the peak serum and the digoxin concentration form the MCCs were achieved faster
than digoxin tablets. Even in 1974, the potential use of MCCs to improve clinical performances was
envisioned by the authors who stated that ‘’the principle of complexing a drug with substances as
hydroquinone to enhance the dissolution might be applied to other medicines whose absorption is
erratic following poor in vivo dissolution’’1. Another term to describe MCCs is ‘’hydrogen bond
complex’’. Hoogsteen prepared such MCCs to provide evidence for the existence of purine-
pyrimidine base pairs in DNA. The MCC between 9-methyladenine and 1-methyltymine shows
what is known as a Hoogsteen base pair1. The crystal structure is sustained by two hydrogen bonds.
The term cocrystal was not popularized until the 1990’s, due to Etter. 5 Her research contributions in
cocrystal design include concepts that are still used for determining the propensity of hydrogen
bond interactions1. In summary, ICCs have been known as acid salts, complex salts, metal salt

complexes, conversely MCCs have been known as molecular organic compounds, addition
compounds and mixed crystals.

Figure 1: Types of Crystal

Figure 2: Formation of a Crystal


With the pharmaceutical cocrystals, as said before, we can synthesize new drugs with different
properties; however, developing a drug substance into a drug product is not a trivial task. In general
the process can be divided into eight steps. The first one is the design and coformer selection of the
drug1. A suitable coformer in a context of pharmaceutical cocrystals should be safe enough for a
drug product The PAFA (Priority-based Assessment of Food Activities) database contains chemical
and toxicological information on about ca. 2000 substances, including those with the GRAS
(Generally Recognised as Safe) designation, which could be applied directly to food. The section of
coformers that are likely to form cocrystals from a given API is often based upon supramolecular
compatibility. There are a variety of methods such as the molecular electrostatic potentials (MEPs),
Fabian’s method, lattice method or (COSMO-RS), each approach could be used independently or
coupled with the others. Once a library of coformer has been selected the next stage is discovery.
Generally, traditional methods to discover cocrystals include slow solvent evaporation, slurry
mediated transformation and mechanical grinding1. Other methods known to facilitate the formation
of cocrystals included ultrasound assisted solution cocrystallization, high throughput screening and
supercritical fluid technologies. Thermal and microscopic methods are generally used for the
identifications of new cocrystals. Computational studies have been demonstrated that stable
cocrystals may be predicted in advance, however, this does not mean that cocrystals are easily
prepared through traditional methods. In short, comprehensive screening including multiple
techniques should be conducted to enable the discovery of new cocrystals. 1 In the third step the
crystals are characterized: the primary techniques to describe novel crystals are usually single
crystal X-ray-diffraction (SC-XRD), powder X-ray diffraction (PXRD), or differential scanning
calorimetry (DSC)1. The former is the preferred characterization technique in determining whether a
cocrystalline material has been generated; however, suitable X-ray quality crystals cannot always be
produced. Even if single crystals can be grown of sufficient size and quality, the exact location of
hydrogen atoms could be ambiguous. Thus, it is advantageous to utilize a variety of solid-state and
spectroscopic techniques when attempting to characterize potentially new cocrystalline materials.
The major motivation for discovering new crystals is to improve physicochemical proprieties that
are of critical relevance to their performances as drug substance, including aqueous solubility and
physical stability. Jones’ research group discovered the cocrystal of caffeine with oxalic acid where
the physical stability was increased than the caffeine molecule.1

A recent review by Shan et al.6 addressed the effect of cocrystallization upon API pharmacokinetics
(PK): 64 cocrystals representing 21 APIs afforded 76 PK studies. Moreover, 80% of the APIs were
classified class II (low solubility, high permeability) according to the Biopharmaceutics
Classification System (BCS). This is unsurprising given that improving solubility has motivated the
study of pharmaceutical cocrystals. Analysis of the PK results suggests that solubility enhancements
generally result in increasing the area under the curve (AUC). The impact of cocrystals upon AUC
can be significant, ranging from ca. 10.2-fold decrease to ca. 28.4-fold increase. C max changes
ranging from a 4-fold decrease to a 44-fold increase have been observed. Most cocrystals, however,
were found to exhibit less significant changes in AUC and C max. Variations in PK after dosing
different drug substances can affect the safety, efficacy and clinical performance of a drug product.
Further, if the application for the API is fast onset of pain relief, then the ability to manipulate
specific PK parameters becomes critical1. For instance, to develop an API for acute pain relief, the
time required to reach the effective concentration must be short. Weyna et al. illustrated how
meloxicam, an NSAID indicated for rheumatoid arthritis and postoperative pain, is impacted by
cocrystallization1. Meloxicam is a BCS class II API with a 4–5 hour Tmax. MCCs of meloxicam were
synthesized and PK studies in rats revealed an earlier onset of action, suggesting that a T max of over
30 min is possible for some of the studied MCCs. Before introducing a cocrystal into a drug it is
necessary to formulate it. Usually cocrystals are bonded by hydrogen bonds, that means that their
stability in presence of excipients, which also contain hydrogen bond groups, becomes a risk.
Remenear and Alhalaweh highlighted the use of excipients to alter the rate of dissolution of
celecoxib-nicotinamide and indomethacin-saccharin MCCs. Huang and Rodriguez-Hornedo8
manipulated the micellar solubilization and stability in solution1. Abourahma9 studied the robustness
of theophylline p-hydroxybenzoic acid in the presence of additives during solvent drop grinding
experiments1. Their experiments demonstrated that cocrystal is robust in the presence of additives
containing carboxylic acid, amide and phenol functional groups, but not in the presence of
acetamide1. Therefore, formulation of cocrystals is a necessary stage in drug development. The
scale up is the choice of the right method for the distribution of the new drug. Traditional solution
method could present some challenges for large-scale manufacturing and, in fact, cocrystallization
from solution could be viewed as counterintuitive. However, if a cocrystal is thermodynamically
favoured versus single component crystal and the ternary diagram phase is also well delineated,
then solution crystallisation is suitable to process cocrystals. It is also possible that cocrystallization
can address problem with the purification of APIs. Furthermore, cocrystallization may be used for
the separation of stereoisomers. The last step in the development of a new drug is achieving
regulatory approval. In 2011 the FDA released draft guidance for industry on the subject of
pharmaceutical cocrystals. Within one of the latest guidance a cocrystal could be considered
dissociable ‘’API-excipient’’ molecular complex, an intermediate drug, and not a new API. It is

notable because an intermediate in a drug development process is treated in a different manner than
a different API. To obtain the approval FDA there are two requirements:

 The API and excipient must completely dissociate prior to reaching the pharmacologically
active site
 The API and the excipient are in neutral sites and do not interact by ionic bonds.

Responding to FDA guidance, a perspective article was published in 2012 where the authors opined
that cocrystals should not be treated any differently than salts. 10 The path of the commercialization
of cocrystals in USA remains unclear and the EMA (European Medicines Agency) published a
reflection paper summarizing their position about cocrystals. Currently EMA considers cocrystals as
homogeneous crystalline structures made up two or more components in a different stoichiometric
ratio. Other salient matter raised by the paper include:

 Cocrystals are considered eligible for generic drug product application in the same way as
 Cocrystals are not considered as NAS (new active substances)
 Cocrystals and salts share many conceptual similarities, and similar principles for
documentation should be applied.

Even though there are different positions between FDA and EMA there are position documents
from both agencies that provide guidance to industry.

Figure 3: Formulation of a drug

Figure 4: Specific stages in the formulation of a drug


The physical and chemical proprieties of cocrystals need to be investigated in the same manner as
solid form. These properties, such as melting point, stability, solubility, become very important
when moving a new compound through early development. A table can be used to analyse the
physicochemical proprieties that combine different types of priorities inherent to drug

1. Melting point

The melting point is one of the fundamental properties, which is determined by the temperature at
which the solid phase is at equilibrium with liquid phase. The differential scanning calorimetry
(DSC) is the preferred technique for obtaining melting point data. It is standard practice to
determine the melting point of a compound as means of characterization or purity identification,
however, melting point is very valuable also due to its connection with solubility and vapour
pressure. Therefore, the melting point of an API is easy to find before it is synthetized; moreover, it
would be very important also to know its aqueous solubility. Generally, the factors that contribute to
the melting point are; molecular arrangement within the crystal lattice, molecular symmetry,
intermolecular interactions and conformational degrees of freedom for a molecule.

2. Stability

Stability is a heavily studied parameter during the development of a new chemical entity, different
types of stability need to be considered depending on the structure of the molecule. Chemical and

physical stability data are commonly obtained in accelerated stability conditions to determine
development and shelf life. Water absorption is included from a handling and packaging point of
view. The amount of water present can also change the form and degrade the material if the effect of
the water uptake is not investigated. Thermal stress studies are also incorporated, and extra work
may be warranted for hydrates or thermally labile materials. In the case of cocrystals and salts,
solution stability may be a factor due to dissociation of the material resulting in precipitation of the
less soluble parent compound or a less soluble form. In general, there are four types of stability:

 Relative humidity stress.

It is the ratio of the partial pressure of water vapor to the equilibrium vapor pressure of water at
given temperature; it depends on the temperature and the pressure of the system2.

 Thermal stress

It is the resistance of the material at high temperature. Very few reports discuss thermal stress
experiments on cocrystals. These stresses can lead to fracture or plastic deformation, depending on
the other variables of heating, which include material types and constraints2.

 Chemical stability

Chemical stability is generally used to explain the thermodynamic stability of a chemical system
that occurs when a system is in its lowest energy state. It is commonly investigated early in the
development of a new compound and during formulation studies in order to minimize chemical
degradation that may occur2.

 Solution stability

Solution stability for the discussion is defined as the ability of the cocrystal components to stay in
solution and not readily crystallize. Solution stability can be an important parameter to assess
during development, not only for solutions or suspensions, but also for dosage forms2.

3. Solubility

One of the main reasons for investigating cocrystals is to increase solubility of a poorly soluble
compound. For neutral molecules, cocrystal can certainly expand the possible solid forms for
development. For a free acid or base, both salt and cocrystals could be used to increase solubility.
There are some points we ought to consider when discussing solubility data. Firstly, the equilibrium
versus kinetic solubility measurements. Kinetic solubility value are generally approximated values
based on one measurement at one time point. Usually, it is not possible to known if the equilibrium
is reached unless some further experiments have been performed, and a number of experiments is
usually taken to ensure that the equilibrium is reached. The time for reaching the equilibrium is also
an important factor for the drug development in the pharmaceutical field, due to the time off
residence of the drug on the stomach and the intestines. It is desirable that the drug dissolves when
it is in the gastrointestinal tract, and very long dissolution times may result in a lower absorption of
the drug. A second consideration regards changes during the experiment. When form changes occur,
the obtained solubility data may not be relevant to the starting compound. Form changes can be
suggested by solubility data collected at various time points by a precipitous drop in concentration
indicating crystallization of a less soluble form. In these cases, the maximum solubility observed
over the time profile may be reported along with the time it occurred. Another consideration
concerns the media used for the experiment. Acidic or basic media can certainly influence
molecules containing acid or basic groups. This shows the wide range of experimental variables that
can be used for solubility tests that can be tailored to obtain the desired information.

4. Bioavailability

In the pharmaceutical field, bioavailability is a subcategory of absorption and is the fraction of an

administered dose of unchanged drug that reaches the systemic circulation. It is also correlated to
the rate of the active drug that reaches the system. When preparing a new drug, it is important to
consider the animal bioavailability. A study with both the parent material and the cocrystal will give
a direct assessment of bioavailability improvements due to the cocrystal.

Figure 5: Example flowchart for choosing a cocrystal candidate.


Peptide drugs (e.g. Dalargin or Noopept) are very well described in the literature. 12 Physiologically,
active peptides responsible for global body regulation are important targets for the design of new-
generation drugs. The selection of the peptides is based on the chemical features of compounds. The
regulatory peptides interact with the receptors via a restricted area of the peptide chain of the
amino-acid, whose size is a compromise between the ligand-receptor complex dissociation rate and
the recognition accuracy. These regions, as a rule, correspond to β-turns peptide chains that have 4
amino acids, two of them at the centre of the chain. The interaction between receptor and ligand
plays a key role in the ligand-receptor link. It is important to specify that this statement is only a
theoretical base for the dipeptide drugs’ design. The advantages of peptide drugs over the
nonpeptide ones include high activity, absence of toxicity due to the metabolism to endogenous
amino acids, absence of severe adverse effects owing to the regulatory type of action, and lower
probability of tolerance or dependence development. Furthermore, dipeptide agents compare
favourably with oligopeptides in that they are orally administrable owing to higher stability and the
ability to penetrate biological barriers. Dipeptides are less polyfunctional and synthetically more


The crystallization technique can be used also for purifying a series of dipeptides. A research group
from the University of Lincoln has studied an innovative aspect for using crystallization as an
alternative method to overcome the problematic removal of unwanted trifluoroacetic acid (TFA)
from synthesized peptides, inserted in the last step of the synthesis. TFA negatively affects both the
physiochemical and biological proprieties of the product. The dipeptide picked out for the
experiment was the L-Leu-L-Leu, as it is easy to analyse after cocrystallization. 13 After carrying out
the experiment, the research team found that TFA-free multicomponent crystals were obtained.
Thus, this proves that the use of crystal engineering is an alternative purification method for freshly
synthesized peptides. The analysed structure demonstrated that the use of a solid as a conformer at
room temperature represents a valid alternative for the generation of crystal structure that are
consistent with the already reported ones, in which the solvent molecules have been completely


Alanylglutamine is a nutritional supplement containing a stable, water-soluble dipeptide comprised

of the amino acids L-glutamine and L-alanine, with potential protective and absorption enhancing
activities. Upon oral or enteral administration, alanylglutamine works locally in the gastrointestinal
(GI) tract to both protect the integrity of the intestinal mucosa and maintain intestinal barrier
functions. This reduces bacterial translocation, the risk of infection, infection-induced inflammatory
damage and infection-associated symptoms, such as diarrhoea, dehydration, malabsorption and
electrolyte imbalances. Alanylglutamine also increases absorption of other chemicals, enhances
epithelial repair, and inhibits apoptosis due to cellular damage, and stimulates cellular proliferation.
Altogether, this improves absorption of nutrients, decreases weight loss, reduces diarrhoea,
decreases the risk of GI-associated infections and improves recovery. Upon absorption,
alanylglutamine may also help inhibit muscle protein catabolism.

Figure 6: Structure of ALA-GLN

Figure 7: 3-D structure of ALA-GLN


One of the possible syntheses of ALA-GLN is related to a reaction between a reaction of -

halogenated with thionyl chloride and the Schotten-Baumann reaction, which is an organic
reaction used to convert an acyl halide or anhydride to an amide if reacted with an amine and base, or an
ester if reacted with an alcohol and base with L-glutamine followed by an ammonolysis reaction. This
reaction is very simple to carry out and for its simplicity it has been also scaled up for the
production in large quantity.

Figure 8: Reaction for obtaining ALA-GLN

The studies on the L-glutamine proved that the solubility in water of this molecule is low and in
addiction L-glutamine may decompose into toxic products, pyroglutaminic acid and ammonia.
Therefore, peptides with L-glutamine have taken the place of L-glutamine. L-Alanyl-L-glutamine
has higher solubility in water and high stability than the previous molecule. There are several
methods for developing this compound. The first one consists on the use of protecting groups. For
example the method which comprises condensing N-benzyloxylcarbonyl-L-alanine (Cbz-ALA)
with protected L-glutamine in the presence of N;N-dicyclohexanylcarbodiimide (DCC) and
removing the protecting group from the intermediate or the method which comprises condensing
Cbz-ALA with protecting γ-methyl L-glutamine in the presence of the DCC, removing the
protecting group from the intermediate compound, and furthering reacting the deprotected product
with ammonia. This method needs the extra step of removing the protecting group from the
intermediate compound and the operation is complicated and expensive.

Another possibility for producing ALA-GLN is using N-carboxyl anhydride but this process,
without involving protecting groups, presents a significant quantity of by-products such as
tripeptides and the yield of the intended product is very low. Furthermore, it is difficult to purify the
final product.

One more method may be the synthesis of the ALA-GLN via D-2-bromopropyonil chloride as
starting compound via an intermediate compound, D-2-bromocopyonil-L-glutamine. In this method,
since an acid chloride that has a high reactivity towards water is added to an aqueous solution of L-
glutamine, for example, in the reaction of D-2-bromopropionyl chloride, the method suffers from
partial hydrolysis of the acid chloride. Therefore, the method (3) yields by-products, and the yield
of the intended product is low. Since the produced d-2-bromopropionyl-L-glutamine is purified by
extraction with organic solvent, the yield of the product is low. In addition, in the method, since the

ammonolysis of D-2-bromopropionyl-L-glutamine is carried out at an elevated temperature, by-
products are considerably enhanced, and the optical purity of the produced ALA-GLN is often low.

Thus, the method via d-2-halopropyonil-L-glutamine has been selected for being scaled up. At the
beginning the reaction have been carried out in water but after having figured out that there were
problems associated with the decomposition of the acid chloride the problem has been overcome
under the Schotten-Baumann procedures.


Studies carried out by a group of Brazilian researchers, have analysed the effect of this active
substance on Brazilian children. In Brazil, growth deficits are common in children living in rural
areas, where 8.3% of children are underweight and the infant mortality is at 26/1000. One of the
reasons analysed have been the mucosal damage in malnourished children affected by diarrhoea and
malnutrition.15 It has been discovered that glutamine plays a central role in the healing of the
mucosa due to during the oxidation of glutamine by intestinal epithelia cells, energy sources have
been provided. During diarrhoea and malnutrition, the role of glutamine is even more important.
The major limitations of glutamine, however, are its limited solubility and its tendency to hydrolyse
to potentially toxic glutamate. Both drawbacks are solved by linking glutamine to alanine, and we
have found that alanyl-glutamine (AG) is stable, highly soluble, well tolerated, and at least as
effective in driving sodium co- transport and intestinal injury repair in vitro, in animals, and in
patients. It is hypothesized that AG would improve the intestinal barrier function and growth
deficits in undernourished children affected by diarrhoea and malnutrition. It has been found that
Alanyl-Glutamine produced a significant increase in weight for height and weight for age compared
with Glutamine placebo, suggesting that AG improves tissue mass and reduces wasting in children
living in an urban community in north-east of brazil. It has been also observed that AG
supplementation was associated with an improvement in intestinal barrier function, as measured by
the decreased percentage of lactulose excretion rate.

Analysing the structure of the L-Alanyl-L-Glutamine it is noted that it has a dipeptide form. In fact,
as cited before, one of the powerful sources for the design of novel effective medicines are
regulatory peptides and proteins. Peptide drugs have advantages over non-peptide drugs such as
high activity and low toxicity.

L-Alanyl-L-Glutamine may be defined also as a hydrophobic dipeptide. An ‘hydrophobic amino

acid residue’ is defined as one devoid of N and O atoms in its side chains. ‘Hydrophobic

dipeptides’, with two such residues, share the same set of strong hydrogen bond donors (amino +
amide groups) and acceptors (amide + carboxylate groups) in their main chains, but vary in terms of
the shape and size of the hydrophobic moieties in their side chains. 16 As said above, an important
propriety for the dipeptides is that they are usually absorbed faster than free amino acids, due to the
intact and efficient dipeptide transport carrier they employ. This carrier system is neither used by,
nor is it shared with, free amino acids, and appears to demonstrate a high affinity for absorption
sites. So, these physicochemical properties alone may provide greater effectiveness as compared to


The products usually have been analysed with different characterisation techniques. Single-Crystal-
X-ray Diffraction (SCXD) gives detailed information about the lattice of a crystals such as the
length of the pattern, the length of the bonds or the angle of the bond. The structure of a crystal is a
characteristic propriety that is the basis for understanding many of the proprieties of them.

Another technique for the determination of the unknown crystals is the Power X-ray Diffraction
(PXRD). The Bragg’s law relates the wavelength of a crystal to a 2α angle and a lattice space. By
scanning the sample through a range of 2θ angles, all possible diffraction directions of the lattice
should be attained due to the random orientation of the powdered material. Conversion of the
diffraction peaks to d-spacings allows identification of the mineral because each mineral has a set of
unique d-spacings. Typically, this is achieved by comparison of d-spacings with standard reference

Usually a combination of both techniques explained above is required for understanding better the
characteristic structure of the crystal.

The last technique used is the IR spectroscopy. Every chemical structure has its own spectrum that
allows its identification. Co-crystals have a very small shifting of the position of one or more
vibrational bonding that indicate the presence of intramolecular interactions that may refer to
conformer of the crystal. The IR spectra of the cocrystal may be identified comparing the spectra of
both the original compound and the cocrystal and looking at the shifting of the peaks. Big shifting
may indicate the presence of salts instead of cocrystals. The IR technique has to be a first analysis
for the determination of a cocrystal, the use of PXRD and SCXRD is required in order to have
better information about the compound.


The aim of the research was the discover of new types of conformer for the ALA-GLN using metal
nitrate, chloride and acetate salts. The study focused on the ionic cocrystals and so some salts were
used as ionic part and the ALA-GLN as neutral part. It ALA-GLN was selected for its easy
availability and for its beneficial proprieties. As discussed above, with the formation of new co-
crystals the increase of one or more proprieties of the molecule taken as sample was verified. Even
though ALA-GLN is very water-soluble, solubility is one of the major properties that co-
crystallization looks at. Two types of techniques were used for preparing the co-crystals. The first
one was the grinding technique that uses ball milling. The second one was the solvent technique,
where the compounds were dissolved together in the same solvent and then left to dry for a couple
of days at room temperature.


As said above, the aim of the experiment was to investigate cocrystal formation of ALA-GLN

A series of compounds shown in Table 1 were used as coformers. The compounds selected were
already in the salt form and so there was no need to react these compounds in order to form the salt.
For every compound a grinding test and a solvent test has been carried out.

Table 1: List of Coformers Used


ALA-GLN NaNO3 L-Alanyl-L-Glutamine Sodium nitrate

ALA-GLN KNO3 L-Alanyl-L-Glutamine Potassium nitrate

ALA-GLN Ca(NO3)2 L-Alanyl-L-Glutamine Calcium nitrate

ALA-GLN Mg(NO3)2 L-Alanyl-L-Glutamine Magnesium nitrate

ALA-GLN Zn(NO3)2 L-Alanyl-L-Glutamine Zinc nitrate

ALA-GLN NaOAc L-Alanyl-L-Glutamine Sodium acetate

ALA-GLN KOAc L-Alanyl-L-Glutamine Potassium acetate

ALA-GLN Mg(OAc)2 L-Alanyl-L-Glutamine Magnesium acetate

ALA-GLN Zn(OAc)2 L-Alanyl-L-Glutamine Zinc acetate

ALA-GLN NaCl L-Alanyl-L-Glutamine Sodium chloride

ALA-GLN KCl L-Alanyl-L-Glutamine Potassium chloride

ALA-GLN CaCl2 L-Alanyl-L-Glutamine Calcium chloride

ALA-GLN MgCl2 L-Alanyl-L-Glutamine Magnesium chloride

ALA-GLN ZnCl2 L-Alanyl-L-Glutamine Zinc chloride

As mentioned earlier, the crystallization was carried out using two techniques: the grinding
technique and the evaporation technique. In the first one the two coformers were weighted in a
stoichiometric ratio and put in an appropriate box for the grinding machine. The grinding was
carried out with a ball mill. The resulting powder was collected in a vial for further analysis.

For the second one, the coformers were weighted and put in a vial. Subsequently ethanol was used
as a solvent for the two coformers. The solubility of the coformers was not high in ethanol so it was
necessary to dissolve them as much as possible with a spatula. The resulting solution was left for
few days at room temperature to allow a slow evaporation of the solvent.

The study of the IR and PXRD spectra of the powders obtained from crystallisation and grinding
were carried out for determining a possible formation of a new cocrystal. As for the IR analysis, a
new cocrystal may be determined from little shifts of the peaks of the reacted compound comparing
with those of the pure compound. Thus, the IR analysis of the ALA-GLN was run before the others
to figure out better the presence or not of a cocrystal. Afterwards, other tests were carried out for
determining the real structure.


For identifying the presence of a cocrystal the ALA-GLN spectrum was compared with the spectra
of the other compounds. The IR spectrum of ALA-GLN is reported in Figure 11, the spectra of
ALA-GLN with other salts are illustrated in Figure 21.

By observing the ALA-GLN spectrum it is possible to identify some characteristic peaks. The one
at 3331.24 cm-1 may be possibly associated to the N-H stretching. The one at 2978.90 cm -1 is related
to the C-H stretching. Other relevant peaks are those at 1631.62 cm -1 and at 1378.97 cm-1. The first
is related to a carboxylic (C=O) stretching, while the second one is matched with a single bond
between carbon atom and an oxygen atom. With those 4 peaks the identification of possible
cocrystals may be easier.

Figure 9: IR spectrum of ALA-GLN

A. ALA-GLN with NaNO3

Figure 10: IR Spectrum for ALA-GLN + NaNO3 Grind Product.

It is possible to notice that most peaks are the same of the original molecule, while the C=O
stretching is slightly shifted. The conclusion is that this is not a cocrystal.


Figure 11: IR Spectrum for ALA-GLN + Potassium Nitrate Grind Product.

As the previous one, also in this spectrum it is not possible to notice a relevant peak shift from the
parent compound, so this is not a cocrystal.


Figure 12: IR Spectrum for ALA-GLN + Sodium Acetate Grind Product.

The peak shifts are overall the same as those observed for pure ALA-GLN. The presence of a new
peak in the N-H area was probably already present in the ALA-GLN spectrum but not identified, so
also this compound is not a cocrystal


Figure 13: IR Spectrum for ALA-GLN + Potassium Acetate Grind Product.

Here is not possible to define any shifting of the spectra from the original molecule so ALA-GLN
KOAc is not a cocrystal


Figure 14: IR Spectrum for ALA-GLN + Magnesium Acetate Grind Product.

There are no differences from the ALA-GLN spectrum and the product spectrum, so neither this
compound is a cocrystal

F. Zn(OAc)2

Figure 15: IR Spectrum for ALA-GLN + Zinc Acetate Grind Product.

In this spectrum the peaks are only slightly shifted, and the presence of a new peak around 2364.17
cm-1 can be detected, probably due to carbon monoxide. Despite this, it is possible to hypothesis
that the zinc and ALA-GLN compound is not a cocrystal.

G. NaCl

Figure 16: IR Spectrum for ALA-GLN + Sodium Chloride Grind Product.

The spectrum does not present particularly shifting from the original one, so it is not possible to
predict any presence of the formation of cocrystals.

H. KCl

Figure 17: IR Spectrum for ALA-GLN + Potassium Chloride Grind Product.

In this spectrum the disappearance of the N-H signal is probably due to the NaCl propriety of
absorbing water, and all the peaks are shifted. In particular, it is evident that the C=O peak has been
shifted to 1656.31 cm-1. Further analysis must be carried out for determining the composition of this
compound. From the IR spectrum the potassium chloride and ALA-GLN may in fact be a cocrystal.

I. MgCl2

Figure 18: IR Spectrum for ALA-GLN + Magnesium Chloride Grind Product.

The spectrum presents a signal at 2982.22 cm-1 and the peak at 3310 cm-1 is no longer present. Some
shifting can be observed, especially the one at 1662.37 cm-1. All is related to the presence of a
cocrystal but the possible explanation for the spectrum may be a degradation due to the initial
proprieties of the magnesium chloride. Further analysis must be carried out for allowing or
changing the first hypothesis. Another image of the overlapping of the two spectra (ALA-GLN and
ALA-GLN/MgCl2) has been attached below for allowing a better comprehension.

Figure 19: Overlapping of the ALA-GLN Spectrum (Blues) and the ALA-GLN + Magnesium Chloride Spectrum

J. ZnCl2

Figure 20: IR Spectrum for ALA-GLN + Zinc Chloride Grind Product.

Similarly to the previous two spectra, also in this one there is no signal at 3310 cm -1 and the peaks
are slightly shifted from the original spectrum. The possibilities are either the presence of a
cocrystal or the degradation of the ALA-GLN due to the corrosive proprieties of the zinc chloride.
Further analysis must be carried out for determining the real behaviour of this reaction. The
overlapping spectrum between ALA-GLN spectrum and ALA-GLN ZnCl 2 are attached below for
allowing a better comprehension.

Figure 21: Overlapping of the ALA-GLN Spectrum (Red) and the ALA-GLN + Magnesium Chloride Spectrum (Blue).


PXRD analysis was performed on the three possible cocrystals to figure out their real nature. The
ALA-GLN and each conformer were analysed individually to compare their PXRD patterns. As
said before, the only three compounds to be analysed were: ALA-GLN/KCl; ALA-GLN/NaCl and


The first product to be analysed was the ALA-GLN/NaCl compound. The spectrum illustrated in
the figure below shows three spectra. The first one in black is the ALA-GLN’s, the second one in
red is the ALA-GLN/NaCl’s and the one in blue is the conformer’s, in this case NaCl. The ALA-
GLN/NaCl’s spectrum shows a poor match with the peaks of the conformer and ALA-GLN,
therefore it can be concluded that this product is not a cocrystal.

Figure 22: PXRD Spectra for ALA-GLN (Black), ALA-GLN + Sodium Chloride Grind Product (Red), and Sodium
Chloride (Blue).


The second product to be analysed was the ALA-GLN/MgCl2 compound. For this spectrum, the
compound signal showed a high background noise, possibly because there was not enough product
for the analysis or due to a non-crystalline structure in the compound. The red spectrum signals
(ALA-GLN/MgCl2) do not match the conformer and ALA-GLN ones, so this compound is not a
cocrystal either.

Figure 23: PXRD Spectra for ALA-GLN (Black), ALA-GLN + Magnesium Chloride Grind Product (Red), and
Magnesium Chloride (Blue).


The last IR analysis which indicated the presence of a possible cocrystal was performed on the
ALA-GLN/KCl product. Again, this spectrum showed a high background noise and its peaks do not
completely match those of the other two compounds, so this product is not a cocrystal.

Figure 24: PXRD Spectra for ALA-GLN (Black), ALA-GLN + Potassium Chloride Grind Product (Red), and
Potassium Chloride (Blue).


For the grinding, a ball mill, with box 5 mL in inox steel and two steel balls with 2.5 mm diameter,
was used. The mill grinds the powder inside it with a frequency of 30 Hz for 30 min.

The compounds were characterized with IR spectroscopy with a Bruker Tensor ATR 37
spectrometer connected with the OPUS 7.2 programme putting the samples on a diamond probe. A
range of 4000-400 cm-1. An average of 16 scans was taken with a resolution of 4 cm-1.

The compounds were characterized with Powder X-Ray Diffractometry with a Stoe Stadi MP
diffractometer with Cu Kα radiation at 40 kV and 40 mA using a linear PSD over a 2 θ range of 3.5
– 45° with a step size of 2° and step time of 30 s.


For the crystallisation a 1:1 stoichiometric quantity of coformers (with respect to ALA-GLN) was
chosen. We decided to use a quantity of 0.092 mmol. ALA-GLN is only soluble in water (1 mg/ml)
or in EtOH/water mixtures, therefore the solvent for crystallization was a solution with a 60:40 ratio
of ethanol and water. After weighting the right quantity of each conformer, they were put in a vial
filled with the solvent solution. The solution was left to evaporate for a couple of days at room
temperature to allow the formation of single crystals. Subsequently, if crystals were obtained, each
coformer was reacted with ALA-GLN. A summary of the quantities of the used components is
provided in Table 2.

However, in my case, after few days of evaporation no crystals of coformers were recovered, due to
their high solubility in alcohols (20 mg/ml).

Table 2: Masses of Reagents used for Solution Crystallization.

SAMPLE FORMULA Mole (mmol) Molar Weight Mass (mg)

(g mol-1)
ALA-GLN 0.092 217.22 20
Sodium nitrate NaNO3 0.092 84.99 8
Potassium nitrate KNO3 0.092 101.10 9
Calcium nitrate Ca(NO3)2·4H2O 0.092 236.10 22
Magnesium nitrate Mg(NO3)2·6H2O 0.092 256.43 24
Zinc nitrate Zn(NO3)2·6H2O 0.092 297.48 27
Sodium acetate NaOAc 0.092 82.034 8
Potassium acetate KOAc 0.092 98.14 9
Magnesium acetate Mg(OAc)2·4H2O 0.092 214.46 20
Zinc acetate Zn(OAc)2·2 H2O 0.092 219.50 20
Sodium chloride NaCl 0.092 58.44 5
Potassium chloride KCl 0.092 74.551 7
Calcium chloride CaCl2·2H2O 0.092 147.0 14
Magnesium chloride MgCl2 0.092 95.211 9
Zinc chloride ZnCl2 0.092 136.286 13


For the grinding technique a stoichiometric quantity of ALA-GLN and the other conformer have
been put into a steel box contained a steel ball. The box has been placed in the ball mill instrument.
The experiments of grinding have been carried out into a ball mill. After running the ball mill and
grinding the solution for 30 m at the frequency of 30 Hz the final powder has been collected in a
vial. A summary of the grinding experiment is showed below, Table 3.

Table 3: Masses of Reagents used for Ball Milling Experiments.

SAMPLE FORMULA Mole (nmol) Molar Weight Weigh (g)

L-Analyn-L- ALA-GLN 0.2 217.22 0.043
Sodium nitrate NaNO3 0.2 84.99 0.017
Potassium nitrate KNO3 0.2 101.10 0.020
Calcium nitrate Ca(NO3)2·4H2O 0.2 236.10 0.047
Magnesium Mg(NO3)2·6H2O 0.2 256.43 0.051
Zinc nitrate Zn(NO3)2·6H2O 0.2 297.48 0.059
Sodium acetate NaOAc 0.2 82.034 0.016
Potassium KOAc 0.2 98.14 0.028
Magnesium Mg(OAc)2·4H2O 0.2 214.46 0.043
Zinc acetate Zn(OAc)2·2H2O 0.2 219.50 0.044
Sodium chloride NaCl 0.2 58.44 0.012
Potassium KCl 0.2 74.551 0.015
Calcium chloride CaCl2.2H2O 0.2 147.0 0.029
Magnesium MgCl2 0.2 95.211 0.019
Zinc chloride ZnCl2 0.2 136.286 0.027

All the powders collected were observed and a first impression of the product indicated the possible
presence of a crystal. Of course, just visual impression is not enough to determine whether a
cocrystal has been formed but may influence the choice of further analyses. Several products were
not recoverable due to bad characteristics of the product. A summary of the product appearance is
provided in Table 4.

Table 4: Description of the Physical State and Recovery of the Material Generated from the Ball


ALA-GLN NaNO3 Clean white powder COMPLETELY
ALA-GLN KNO3 Clean white powder COMPLETELY
ALA-GLN Ca(NO3)2 Sticky white paste, POOR RECOVERY
ALA-GLN Mg(NO3)2 Sticky white paste, NO RECOVERY
ALA-GLN Zn(NO3)2 Sticky white paste, NO RECOVERY
ALA-GLN NaOAc Clean white powder COMPLETELY
ALA-GLN KOAc White powder, slightly wet COMPLETELY
ALA-GLN Mg(OAc)2 Clean white powder COMPLETELY
ALA-GLN Zn(OAc)2 Clean white powder COMPLETELY
ALA-GLN NaCl Clean white powder COMPLETELY
ALA-GLN KCl Clean white powder COMPLETELY
ALA-GLN CaCl2 White powder, slightly wet COMPLETELY
ALA-GLN MgCl2 Clean white powder COMPLETELY
ALA-GLN ZnCl2 Clean white powder COMPLETELY


The importance of the dipeptide drugs in the latest years has focused the researches on new methods
for synthetizing cocrystals and developing them. As said before, ALA-GLN and dipeptide drugs
have a lot of benefits and advantages and with the formation of a cocrystal of ALA-GLN the
research team has tried to improve the water solubility of this compound. This research project
started by using some data obtained from previous experiments and tried to replicate those results
also with this reagent. Some salts and acetates have been mixed with the ALA-GLN in order to
obtain some suitable cocrystals that might have improved the solubility of the molecule.

The success rate of these types of experiments is quite low and this project is no exception, so it
ended without discovering any cocrystal. Design of the experiments can be difficult to do in
advance and so many experiments would need to be carried out to see impactful results.


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