First Steps for the Direct Purification of l -Leu- l -Leu Dipeptide through Co-Crystallization

Article abstract of DOI:10.1021/acs.cgd.7b01516

Trifluoroacetate salt contamination of peptides represents a challenging issue related to solid phase peptide synthesis and purification because it affects the chemical and biological properties of peptides. Purification of such materials is typically performed through a two-step post-synthetic procedure based on chromatography followed by ion exchange. For the first time, co-crystallization is presented in this study as a possible alternative and advantageous single-step method for the obtaining of TFA-free crystals of a dipeptide. A trifluoroacetate-contaminated l-Leu-l-Leu dipeptide has been used for co-crystallization experiments along with different solid coformers. New multicomponent crystals containing only the title compound and the second co-crystal formers are described in this work. Such results represent a novelty in the field of peptide chemistry and a valid proof for the use of crystal engineering-based method for a combined purification and crystallization strategy.

Full text of DOI:10.1021/acs.cgd.7b01516

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Article
First steps for the direct purification of L-Leu-
L-Leu dipeptide through co-crystallisation
Paolo Lucaioli, Elisa Nauha, Ishwar Singh, and Nicholas Blagden
Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.7b01516 • Publication Date (Web): 07 Dec 2017
Downloaded from http://pubs.acs.org on December 11, 2017
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First steps for the direct purification of L-Leu-L-Leu dipeptide through co-
crystallisation
Paolo Lucaiolia, Elisa Nauhab, Ishwar Singh*a, and Nicholas Blagden*a
aUniversity of Lincoln, School of Pharmacy, Joseph Banks Laboratories, Green Lane, LN6 7DL, Lincoln, United Kingdom
bUniversity of Lincoln, School of Chemistry, Joseph Banks Laboratories, Green Lane, LN6 7DL, Lincoln, United Kingdom
Email: isingh@lincoln.ac.uk; nblagden@lincoln.ac.uk
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Abstract. Trifluoroacetate salts contamination of peptides represents a challenging issue related to Solid Phase
Peptide Synthesis and purifications because it affects the chemical and biological properties of peptides.
Purification of such materials is typically performed through a twoꢀstep postꢀsynthetic procedure based on
chromatography followed by ion exchange. For the first time, coꢀcrystallisation is presented in this study as a
possible alternative and advantageous singleꢀstep method for the obtaining of TFAꢀfree crystals of a dipeptide. A
trifluoroacetateꢀcontaminated LꢀLeuꢀLꢀLeu dipeptide has been used for coꢀcrystallisation experiments along with
different solid coꢀformers. New multicomponent crystals containing only the title compound and the second
cocrystal formers are described in this work. Such results represent a novelty in the field of peptide chemistry and
a valid proof for the use of crystal engineeringꢀbased strategy for a combined purification and crystallisation
strategy.
INTRODUCTION
Biologics might represent the only efficient way to treat some specific diseases but, in many cases, the solidꢀstate
form of these entities cannot be easily used in a dosage form due to their questionable physiochemical properties
(e.g. stability). Biologics typically exist in the amorphous state with consequent disadvantageous therapeutic and
preꢀformulation properties. A possible contemporary solution to this crystallisability issue is the preparation of
crystal forms of these molecules using and applying the concepts of crystal engineering and molecular
complexes1. Hydrophobic peptides can be considered as small prototypes of biologics with a nonꢀtoxic,
biocompatible, and ecological profile. From a chemical and crystal engineering point of view, their hydrophobic
and hydrophilic moieties are very attractive because of the possibility to generate both hydrogen bonded and Van
der Waals interactions leading to different types of crystal packing landscapes.
An innovative aspect presented in this work is the use of coꢀcrystallisation as an alternative method to overcome
the problematic removal of unwanted trifluoroacetic acid (TFA) from a synthesised peptide. The presence of such
chemical is a common challenge when peptides are synthesised through solid or solution phase peptide
synthesis. For example, the final step in the solid phase peptide synthesis (SPPS)2 is represented by the
detachment of the peptide from the resin: this operation is performed using a cleavage cocktail containing a large
excess of trifluoroacetic acid, a harmful and polluting strong acid, (pka=0.23), able to cleave the product from the
support. TFA binds to the free amino terminal as well as to any positively charged functionality on the side chains
of the peptide generating a strong ion pair. Trifluoroacetate salts negatively affect both the physiochemical3 and
the biological4–6 properties of the product. For this reason, additional purification steps through chromatographic
techniques followed by ionic exchange reactions are routinely used to remove this contaminant.
disadvantageous loss in the yield of the peptidic material is unavoidable.
A
To investigate the possible outcome of purification through cocrystallisation, a LꢀLeuꢀLꢀLeu dipeptide was not
completely purified after the Fmoc SPPS: the synthesis liquor (containing both the product and the TFA) has
been simply evaporated and the recovered material has been lyophilised to obtain a dehydrated peptide. Slow
solvent evaporation experiments conducted by using several coꢀcrystal formers along with the dipeptide product
show that there is a wide range of possible outcomes (Figure 1): this is due to the establishment of differential
crystallisation processes involving different chemical species and leading to the formation of adducts with distinct
compositions and solubilities. The novel TFAꢀfree multicomponent crystals presented in this paper have been
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obtained using the nonꢀpurified LeuꢀLeu dipeptide and 3ꢀaminobenzamide (LL:3-ABA), 1HꢀPyrazole (LL:1H-
Pyz), and methanol (LL:MeOH) confirming the hypothesis of coꢀcrystallisation as an alternative protocol for the
removal of trifluoroacetic acid. Crystal products containing TFA will be described in a following paper7 to highlight
the variety of outcomes.
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Figure 1 ꢀ Different multicomponent systems obtained during the coꢀcrystallisation screening experiments using
the TFAꢀcontaminated LꢀLeuꢀLꢀLeu dipeptide.
With regard to the crystal science of dipeptides, they typically crystallise in multiꢀlayered structures with
alternating hydrophobic and hydrophilic layers8: the latter are represented by twoꢀdimensional sheets generated
through headꢀtoꢀtail hydrogen bonds involving two of the three amino H atoms and the carboxylate functionality at
the other terminal9, while the third one is generally involved in an interaction with an acceptor contained in one of
the side chains of a peptide of an adjacent layer10,11. A crystal packing problem arises when dipeptides are
constituted of two hydrophobic residues: from a crystallographic point of view, amino acids are considered
hydrophobic when they have no chemical functionalities involved in strong hydrogen bonds interactions except
for the amino and carboxylate groups in the polar head12. In this case, the sideꢀchains do not contain any Hꢀbond
acceptor able to accept the third H atom on the aminoꢀterminal of the dipeptide. With the exception of the LꢀMetꢀ
LꢀMet peptide (in which the third amino H atom is not used for the crystal structure construction)13, the hydrogen
not involved in the creation of the planar sheet is usually accepted by a molecule of crystallisation solvent
included in the crystal packing14.
The crystal structures of LꢀLeuꢀLꢀVal and LꢀLeuꢀLꢀLeu peptides represent significant examples of such
arrangements: differently from other hydrophobic dipeptides, these compounds can generate good quality large
crystals (instead of thin needles or plates) that can be easily analysed. Solvates of both LꢀLeuꢀLꢀVal with
methanol (CSD refcode15: SUWLIF), ethanol (SWLOL), 2ꢀpropanol (JUCSEF01)16 and LꢀLeuꢀLꢀLeu with
isopropanol (HIQWAF)17, ethanol (JUQQUIV), propanol (JUQQUB), 2ꢀpropanol (JUQQUM), a 1ꢀpropanol:2ꢀ
propanol mixture (JUQRAO)18 and DMSO (YORPEA)19 have been already reported in literature. The inclusion of
such organic solvent molecules in the crystal packing is fundamental for two reasons: firstly, the hydroxyl group
are involved in the hydrogen bonded networks and represent a valid solution for the problem related with the third
amino H atom. On the other hand, these solvents are contained inside channels running in the hydrophobic
layers: structures with such empty spaces would not be stable20 and the presence of a second compound is
needed to fill this voids, creating solid architectures. Inclusion compounds of water21 (RELWIO), pyridine
(YIMWOH) and three different methylpyridine22 (YIMWUN, YIMXAU, YIMXEI) displaying a similar layered
structure have been obtained using the LꢀLeucine tripeptide: this results confirm that the presence of solvents is
fundamental for the crystal packing.
Despite the importance of their role, the presence of a second component that is liquid at room temperature is
usually considered as a source of practical problems (e.g. molecular disorder, characterisation issues,
polymorphism). An additional disadvantage is the questionable stability of solvate forms. This aspect is vital when
biological molecules are used for the formulation of pharmaceutical products that must maintain the same
properties under different environmental factors such as temperature, pressure, humidity, etc. Coꢀcrystallisation
experiments using various solid coꢀformers at room temperature have been performed to investigate the
possibility to obtain similar stable crystal structures and to analyse the influence of these molecules in the
packing architectures.
Interesting cases of hydrophobic dipeptides crystallised without solvent or as hydrates are represented by
nanotube formations with different space groups and with hydrophobic23–25 or hydrophilic26,27 channels. A novel
structure (LLhex) belonging to the latter type has been obtained while trying to coꢀcrystallise the title compound
with 4ꢀdimethylamino pyridine (DMAP) and will be discussed in this article.
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EXPERIMENTAL
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Dipeptide synthesis. The LꢀLeuꢀLꢀLeu dipeptide (Figure S1) was synthesized through manual Fmoc solid phase
peptide synthesis using FmocꢀNH LeuꢀOH protected amino acid (Merck Millipore) and 2ꢀChlorotrityl chloride resin
(Iris Biotech GmBH). The initial manufacturer loading was 1.60 mmol/g. The resin initial loading has been carried
out using 4 eq. of protected amino acid and 8 eq. of N,Nꢀdiisopropylethylamine (DIPEA) in dichloromethane. The
Fmoc deprotection steps were performed using a 20% solution of Piperidine in dimethylformamide (DMF) while
protected amino acid (4 eq.), HATU (4 eq.), and N,Nꢀdiisopropylethylamine (8 eq.) have been used for the
coupling reactions in DMF; a 20:5:75 mixture of trifluoroacetic acid, triisopropylsilane and DCM was the cleavage
cocktail (evaporated with a rotavapor). The final product was lyophilised to eliminate and avoid any possible
moisture. These treatments lead to the elimination of most of the excess of TFA (boiling point: 72.4 °C) although
it is still present in the final compound.
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Co-crystallisation experiments. 3ꢀaminobenzamide, 1HꢀPyrazole, Pyrazine and 4ꢀdimethylamino pyridine were
obtained from Alfa Aesar and used to prepare equimolar solution of dipeptide and coꢀformer, using methanol
(HPLC purity) as solvent. Thirty different coꢀformers have been used for a complete screening. Such molecules
have been chosen among the most common coꢀformers widely encountered, and have a wide variety of
structural features (e.g., linear, aromatic, heterocyclic), chemical functionalities (i.e. hydrogen bond donors and
acceptor) and chemical properties (i.e., acids and bases). The vials containing the filtered solutions were capped
with perforated parafilm to allow slow solvent evaporation at a controlled temperature (20°C) in an incubator.
Crystals with dimensions suitable for single crystal Xꢀray diffraction were collected from the solution (when
possible) or recovered from the dry material. The methanol solvate (LL:MeOH) has been obtained when trying to
coꢀcrystallise the dipeptide with pyrazine as a coꢀformer while the hexagonal nanotube structure LLhex was
crystallised using a 1:1 solution with 4ꢀdimethylamino pyridine.
X-ray diffraction. Single crystals suitable for Xꢀray diffraction measurements were mounted on MiTeGen Dualꢀ
Thickness MicroMounts and analysed using a Bruker D8 Venture diffractometer with a Photon detection system.
Unit cell measurements and data collections were performed at 173 K using a Cu Kα radiation (λ = 1.54056 Å).
Crystal data and refinement parameters are presented in Table 1. Structure solutions were carried out by direct
methods and refinement with SHELXL28 was finished using the ShelXle29 software. All nonꢀhydrogen atoms were
found from electron density map and refined with fixed bond distances and thermal parameter riding on the
parent atom. Highly disordered electron density inside the channel of LLhex was processed using Platon
Squeeze30.
Asymmetric Units. The asymmetric units of the four multicomponent systems are shown in Figure 2. LL:3-ABA,
LL:1H-Py, and LL:MeOH crystallise in the P21 space group with a variable number of components. Structures
containing 3ꢀaminobenzamide (Figure 2a) and methanol (Figure 2b) are composed of two independent molecules
of dipeptide along with two and three molecules of cocrystal former respectively. The dipeptide molecule in
LL:1H-Py shows one disordered isobutyl side chain with partial occupancies of 83:17%; disorder has also been
found in one of the methanol in LL:MeOH (occupancies: 65:35%). LL:(DMAP) crystallises in the hexagonal
crystal system with a P61 space group. The asymmetric unit shown in the present work (figure 2d) contains just
one molecule of dipeptide although additional electron density has been found inside the channel: the structure
was squeezed during the refinement process to improve the quality of the final result.
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Figure 2 ꢀ asymmetric unit of LL:3ꢀABA (a), LL:1hꢀPy (b), LL:MeOH (3), and LLhex (d)
The molecular conformation of the dipeptide molecules in the different structures can be described using the θ=
β
α
α
β
C1 ꢀC1 ——— C2 ꢀ C2 torsion angle (Figure 3). This parameter is a useful descriptor of the relative position of the
side chains with respect to the plane generated by the peptide bond in the molecule backbone. As reported by
Görbitz27, most of the dipeptides contained in the Cambridge Structural Database are characterised by a |θ|>90°:
this means that the side chains are usually pointing in almost opposite directions. A comparison of |θ|(Figure 4) of
the title compounds reveal a significant difference between the here reported structures: molecules in LL:3ꢀABA,
LL:1HꢀPy and LL:MeOH show values that are consistent with the general tendency, while LL:(DMAP) stands out
for a remarkably lower angle: this represent a completely different conformation of the dipeptide with the two
isobutyl chains positioned on the same side of the peptide bond plane.
Figure 3 ꢀ graphical illustration of θ torsion angle in a dipeptide molecule
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Figure 4 ꢀ |θ | torsion angle values [°] of the different dipeptide molecules in the asymmetric units. The grey plane
is the one generated by the amidic peptide bond.
Table 1 ꢀ Experimental details
(LL:3-ABA)
(LL:1H-Pyz)
(LL:MeOH)
(LLhex)
Chemical formula
C12H24N2O3—C7H8N2O C12H24N2O3—C3H4N2 C12H24N2O3—1.5(CH4O) C12H24N2O3[+solvent]
Mr
380.48
312.41
292.39
244.33
Crystal system, space group
Monoclinic, P21
5.5139 (3)
22.4965 (13)
16.3133 (9)
90
Monoclinic, P21
5.3441 (2)
13.8666 (6)
12.1977 (5)
90
Monoclinic, P21
10.5689 (5)
13.6495 (6)
12.4721 (5)
90
Hexagonal, P61
23.2661 (13)
23.2661 (13)
5.3295 (3)
90
a (Å)
b (Å)
c (Å)
α (°)
β (°)
92.344 (4)
90
90.514 (2)
90
107.003 (2)
90
90
γ (°)
120
V (Å3)
2021.9 (2)
4
903.87 (6)
2
1720.58 (13)
4
2498.4 (3)
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Z
m (mmꢀ1)
Crystal size (mm)
Tmin, Tmax
0.72
0.66
0.69
0.56
0.14 × 0.04 × 0.04
0.676, 0.753
0.49 × 0.27 × 0.08
0.542, 0.754
26545
0.36 × 0.19 × 0.11
0.606, 0.753
17122
0.61 × 0.10 × 0.06
0.531, 0.753
11658
No. of measured reflections 14809
measured
No. of independent reflections 7019
3559
3395
2942
2109
0.081
0.603
0.071
0.187
1.10
No. with [I > 2s(I)]
5347
0.058
0.596
0.050
0.106
1.03
3395
6054
Rint
0.054
0.617
0.032
0.079
1.05
0.039
0.603
0.045
0.122
1.07
(sin q/l)max (Åꢀ1)
R[F2 > 2s(F2)]
wR(F2)
S
No. of reflections
No. of parameters
No. of restraints
Dρmax, Dρmin (e Åꢀ3)
7019
559
3559
6185
2942
170
245
422
17
6
11
5
0.17, ꢀ0.19
0.13, ꢀ0.16
ꢀ0.07 (8)
0.27, ꢀ0.21
0.10 (6)
0.15, ꢀ0.19
0.1 (3)
Absolute structure parameter ꢀ0.06 (19)
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RESULTS AND DISCUSSION
Multi-layered structures
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The dipeptides molecules in the structures LL:3-ABA, LL:1H-Pyz and LL:MeOH selfꢀassemble in the acꢀ or ab
ꢀ
plane to generate twoꢀdimensional sheets with varying features that are related to the specific hydrogen bonded
networks created in each structure. The two isobutyl side chains of each leucine residue extend in opposite
directions above and below the plane. Neighbouring sheets stack on top of each other creating multiꢀlayered
architectures in which there is an alternation of hydrophilic layers represented by the dipeptide backbones and
hydrophobic regions formed by the side chains of two flanking sheets. The above described orientation of the
nonꢀpolar side chains creates channels of different sizes on both sides of each sheet: these empty spaces host a
variable number of coꢀformer (or solvent) molecules (figure 5).
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Figure 5 ꢀ General packing scheme for LL:3ꢀABA, LL:1HꢀPyz and LL:MeOH.
Two-dimensional sheets. The twoꢀdimensional sheet of LL:3-ABA in the acꢀplane is generated exclusively via
headꢀtoꢀtail interactions between the amino and the carboxylate groups of the two independent dipeptide
molecules: the internal amidic groups of the peptide bonds are not involved in conventional hydrogen bonds.
Only two of the three amino H atoms are involved in the contacts between the neighbouring peptide molecules:
the first one creates a single Hꢀbond with one of the carboxylic oxygens of a neighbouring dipeptide while the
second one forms a bifurcated contact with the COOꢀ group of a third molecule (Figure 6a).
The selfꢀassembly of the dipeptide molecules for the LL:1H-Pyz structure is different in that the functionalities of
the internal peptide bonds are involved in the formation of the sheet. Two of the three amino H atoms generate
strong headꢀtoꢀtail hydrogen bonds by interacting with the Oꢀ atoms of the Cꢀtermini of two neighbouring peptides
(red in Figure 6b). The internal peptide bond functional groups hydrogen bond with the charged terminals: the Nꢀ
H group generates a NꢀH———Oꢀ Hꢀbond with a carboxylate O atom (blue in Figure 6b), while the carbonyl acts as
an acceptor for an amino H atom of a neighbouring aminoꢀterminal (orange in Figure 6b).
Figure 6c shows the sheet of the LL:MeOH structure generated through a hydrogen bonded network that is an
intermediate between the previous two. The amino terminals of the two independent dipeptide molecules in the
asymmetric unit act as hydrogen bond donors for headꢀtoꢀtail chargeꢀassisted Hꢀbonds with the carboxylate
groups of the neighbouring molecules (red contacts in Figure 6c). One of the two dipeptides (yellow in Figure 6c)
generates the same interactions described for the LL:3-ABA, while the other one (magenta in Figure 3c) uses
the Nꢀterminus to create two single NꢀH———Oꢀ interactions with the Cꢀterminals of two adjacent dipeptides. In this
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case just the NꢀH group of the internal peptide bond of each dipeptide participates in a NꢀH———Oꢀ hydrogen bond
(blue in Figure 6c) with one of the carboxylate O atoms of a neighbouring molecule.
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Figure 6 – (a) Twoꢀdimensional sheet of LL:3ꢀABA on the acꢀplane. (b) Hydrogen bonded network generating the
LL:1ꢀPyz sheet. (c) Hydrogen bonded network generating the sheet of LL:MeOH on the abꢀplane. The two
independent dipeptide molecules of the asymmetric unit are shown in different colours. Isobutyl side chains are
not shown for clarity.
Three-dimensional stacking of sheets.The channels of LL:3-ABA can host 2 parallel chains of coꢀformer
molecules (Figure 7c), each one composed of only one type of the two molecules of coꢀformer in the asymmetric
unit: these strands are generated through a C=O———HꢀN interaction between the amide functional groups
(magenta in Figure 7c). The two parallel chains are connected through a weak (bond angle= 119.30°) NꢀH———O
hydrogen bond between the amino group of just one type of 3ꢀ aminobenzamide molecule and the carbonyl O
atom of the amidic groups on the flanking row (orange in Figure 7c). The metaꢀposition of the chemical
functionalities allows the coꢀformers to act as crossꢀlinkers between two adjacent sheets (Figure 7b). The amino
group generate a NH———Oꢀ with one of the oxygen atoms of the carboxylic terminal of a dipeptide molecule of one
sheet, while, on the other side, the amidic functionality generates two different hydrogen bonds: the NH2 is
involved in a NH———Oꢀ contact with one of the carboxylic O atoms of a peptide Cꢀterminus and the C=O act as a
hydrogen bond acceptor for the third amino H atom of a peptide Nꢀterminus (the only one not used to generate
the sheet).
Figure 7 ꢀ (a) Multiꢀlayered structure generated by stacks of dipeptides sheets along the bꢀaxis. Each channel
(green) can host two coꢀformer molecules that act as crossꢀlinkers between two parallel sheets (b and c).
Because of the different orientation of the dipeptide molecules in the sheet, the hydrophobic isobutyl chains are
closer in LL:1H-Pyz: this generates channels of smaller size (Figure 8a) that can host just one single row of 1Hꢀ
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Pyrazole molecules (not interacting between each other). The coꢀformer contains both an acceptor (N atom) and
a donor (NꢀH group) in its molecule and both are involved in the formation of hydrogen bonds: the acceptor
generates a N———HꢀN+ interaction with the amino H atom not included in the Hꢀbonded network creating the sheet;
the NꢀH functionality interact via single hydrogen bond with one of the O atoms of the Cꢀterminus of a second
adjacent dipeptide molecule (Figure 8b).
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Figure 8 – (a) Stacking of sheets for LL:1HꢀPyz and (b) interactions involving the coꢀformer.
The particular arrangement of the dipeptide molecules and the complex hydrogen bonded network in LL:MeOH
implicate a wavy conformation of the twoꢀdimensional sheets(Figure 9a) with a consequent closer approach
between the isobutyl side chains. This feature causes a reduction of the channels in the hydrophobic layers that,
in this case, only contain few small molecules of crystallisation solvent. Three different molecules of methanol
interact with the hydrophilic layers, acting as bridges between adjacent molecules: the disordered one (blue in
Figure 9b) acts as both an acceptor (for a N+ꢀH———O Hꢀbond with the amino terminal of a dipeptide) and a donor
(for a OꢀH———O=C interaction with the carbonyl of the peptide bond of a flanking molecule). The other two
molecules of solvent cooperate tighter to create a “bridge effect” between two facing LꢀleucineꢀLꢀleucine: also in
this case one of them (magenta in Figure 9b) act as both an acceptor (giving the same interaction of the
disordered one) and a donor (OꢀH———H hydrogen bond with the third molecule of MeOH, green in Figure 9b), while
the other one (green in Figure 9b) is involved in a bifurcated H———Oꢀ contact with the carboxyꢀterminal of a
dipeptide.
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Figure 9 – (a) Multiꢀlayered stacking of LL:MeOH with molecules of solvents (b).
Nanotube structure
The crystal packing of LL:hex can be included in the reported cases of nanotubes obtained using hydrophobic
dipeptides27. This structure crystallizes in P61. The dipeptides are connected into chains by strong bifurcated
head to tail hydrogen bonds (N+ꢀH———ꢀOꢀC, red in Figure 10b) resulting in helices contain six molecules per turn
(Figure 10a). Each carboxyꢀterminal act as a bridge connecting flanking parallel spirals generating two
interactions: a strong chargeꢀassisted Hꢀbond with the Nꢀterminus (orange in Figure 10b), and another with the
NꢀH group of the internal peptide bond (blue in Figure 10b). The hydrophilic channel runs along the c axis
surrounded by hydrophobic regions represented by the isobutyl chains.
Figure 10 ꢀ (a) Unit cell and molecular packing of LLhex. (b) Hydrogen bonded network: molecules of dipeptide
belonging to the same spiral chain are shown in different colours. Isobutyl side chains are not shown for clarity.
A LꢀLeuꢀLꢀLeu dipeptide nanotube formation has already been reported (CSD refcode: IDUZOW)27 but this
structure crystallizes in P212121 with four molecules per turn in the helices. The LꢀPheꢀLꢀPhe hexagonal structure
described by Görbitz in the same paper (IFABEW) is highly similar to LLhex (Figure S8). The hydrophilic channel
is around 10 Å in diameter comprising 15% of the unit cell volume and it likely contains methanol and water (SI
for details).
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CONCLUSION
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The success in obtaining TFAꢀfree multicomponent crystals represents a proof of concept for the use of crystal
engineering as an alternative purification method for freshly synthesised peptides. Further studies aiming to
probe the kinetics and thermodynamics behind the differential crystallisation mechanisms are the next step. Such
investigations will bring about the control of the process and allow a combined purification and crystallisation of
biologics in a single step, saving time and material.
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In the previously reported case of studies on the crystallisation of hydrophobic peptides, the impact of TFA was
not investigated since the dimers used for the experiments were obtained commercially with a high grade of
purity. However, structures reported in literature show the ability of such compounds to generate wellꢀdefined
crystal packings in which molecules of different solvents play a fundamental role for the stability of the dipeptide
scaffolding, leading to a crystal engineering solution.
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The structures of the present study demonstrate that the use of solid (at room temperature) coꢀformers
represents a valid alternative for the generation of crystal structures that are consistent with the already reported
ones, in which the solvent molecules have been completely replaced. The comparison of the different multiꢀ
layered packings shows the possibility to have channels of variable size according to the coꢀformers used in the
coꢀcrystallisation experiments. In LL:3-ABA the metaꢀ position of the two chemical functionalities in the 3ꢀ
aminobenzamide molecule leads the coꢀformer to act as a bridge connecting two parallel sheets, introducing an
additional anchoring point in the multiꢀlayered stacking. This leads to 3D Hꢀbonded architecture that is more
stable than an assembly of 2Dꢀsheets interacting through weaker interactions.
LLhex can be included in the family of hydrophilic nanotube structures of hydrophobic dipeptides (also known as
“the PheꢀPhe class”31). Such selfꢀassembling systems are considered useful models from both a biological and
chemical perspective. On one side they can be seen as valid models for ion channels or transmembrane pore
assemblies32. Also similar microporous materials attract attention and have been largely used to investigate the
relative sorption ability33–35 for gas storage and other applications.
AKNOWLEDGEMENT
Funding for PL provided by the University of Lincoln, School of Pharmacy.
Supplementary information. Pictures showing the different possible outcomes of the coꢀcrystallisation
screening, hydrogen bonded networks comparison between twoꢀdimensional sheet of LꢀLeuꢀLꢀLeu dipeptide and
LꢀLeucine, crystal packing analysis of LLhex, IFABEW and IDUZOW nanotubes. CCDC 1579695ꢀ1579698
contain the supplementary crystallographic data for this paper. These data are provided free of charge by The
Cambridge Crystallographic Data Centre.
References
(1)
(2)
Duggirala, N. K.; Perry, M. L.; Almarsson, Ö.; Zaworotko, M. J. Chem. Commun. 2016, 52, 640–655.
Chan, W. C.; White, P. D. Fmoc solid phase peptide synthesisꢁ: a practical approach; Oxford University
Press, 2000.
(3)
(4)
Shen, C. L.; Fitzgerald, M. C.; Murphy, R. M. Biophys. J. 1994, 67, 1238–1246.
Cornish, J.; Callon, K. E.; Lin, C. Q.ꢀX.; Xiao, C. L.; Mulvey, T. B.; Cooper, G. J. S.; Reid, I. R. Am. J.
Physiol. ꢀ Endocrinol. Metab. 1999, 277.
(5)
Tipps, M. E.; Iyer, S. V; John Mihic, S. Neuropharmacology 2012, 63, 368–373.
Ma, T. G.; Ling, Y. H.; McClure, G. D.; Tseng, M. T. J. Toxicol. Environ. Health 1990, 31, 147–158.
Lucaioli, P.; Nauha, E.; Singh, I.; Blagden, N. in preparation.
(6)
(7)
(8)
Görbitz, C. H.; Etter, M. C. Int. J. Pept. Protein Res. 1992, 39, 93–110.
Prasad, G. S.; Vijayan, M. Int. J. Pept. Protein Res. 2009, 35, 357–364.
Eggleston, D. S. Acta Crystallogr. Sect. C Cryst. Struct. Commun. 1984, 40, 1250–1252.
Görbitz, C. H.; Backe, P. H. Acta Crystallogr. Sect. B Struct. Sci. 1996, 52, 999–1006.
Görbitz, C. H. Crystallogr. Rev. 2015, 21, 160–212.
(9)
(10)
(11)
(12)
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(13)
Stenkamp, R. E.; Jensen, L. H. Acta Crystallogr. Sect. B Struct. Crystallogr. Cryst. Chem. 1975, 31, 857–
861.
(14)
(15)
Görbitz, C. H. Acta Crystallogr. Sect. C Cryst. Struct. Commun. 1999, 55, 2171–2177.
5
6
7
Groom, C. R.; Bruno, I. J.; Lightfoot, M. P.; Ward, S. C. Acta Crystallogr. Sect. B Struct. Sci. Cryst. Eng.
Mater. 2016, 72, 171–179.
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(16)
(17)
(18)
(19)
(20)
(21)
(22)
(23)
(24)
(25)
(26)
(27)
(28)
(29)
(30)
(31)
(32)
(33)
(34)
Görbitz, C. H.; Torgersen, E. Acta Crystallogr. Sect. B Struct. Sci. 1999, 55, 104–113.
Görbitz, C. H.; IUCr. Acta Crystallogr. Sect. C Cryst. Struct. Commun. 1999, 55, 670–672.
Görbitz, C. H. Acta Chem. Scand. 1998, 52, 1343–1349.
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55
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Mitra, S. N.; Subramanian, E. Biopolymers 1994, 34, 1139–1143.
Kitaigorodskii, A. I. Organic Chemical Crystallography; Consultants Bureau: New York, 1961.
Go, K.; Parthasarathy, R. Biopolymers 1995, 36, 607–614.
Burchell, T. J.; Soldatov, D. V.; Enright, G. D.; Ripmeester, J. A. CrystEngComm 2007, 9, 922.
Görbitz, C. H.; Gundersen, E. Acta Crystallogr. Sect. C Cryst. Struct. Commun. 1996, 52, 1764–1767.
Görbitz, C. H. Acta Crystallogr. Sect. B Struct. Sci. 2002, 58, 849–854.
Henrik Görbitz, C. New J. Chem. 2003, 27, 1789–1793.
Görbitz, C. H. Acta Crystallogr. Sect. E Struct. Reports Online 2004, 60, o626–o628.
Görbitz, C. H. Chem. ꢀ A Eur. J. 2001, 7, 5153–5159.
Sheldrick, G. M. Acta Crystallogr. Sect. C Struct. Chem. 2015, 71, 3–8.
Hübschle, C. B.; Sheldrick, G. M.; Dittrich, B. J. Appl. Crystallogr. 2011, 44, 1281–1284.
Spek, A. L. Acta Crystallogr. Sect. C Struct. Chem. 2015, 71, 9–18.
Görbitz, C. H. Chem. ꢀ A Eur. J. 2007, 13, 1022–1031.
Hartgerink, J. D.; Clark, T. D.; Ghadiri, M. R. Chem. ꢀ A Eur. J. 1998, 4, 1367–1372.
Soldatov, D. V.; Moudrakovski, I. L.; Ripmeester, J. A. Angew. Chemie Int. Ed. 2004, 43, 6308–6311.
Soldatov, D. V.; Moudrakovski, I. L.; Grachev, E. V.; Ripmeester, J. A. J. Am. Chem. Soc. 2006, 128,
6737–6744.
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Moggach, S. A.; Görbitz, C. H.; Warren, J. E.; Ruf, M.; Jameson, C. J.; Pulham, C. R.; Sawyer, L.; Taylor,
R.; Streek, J. van de; Wood, P. A. CrystEngComm 2010, 12, 2322.
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First steps for the direct purification of L-Leu-L-Leu dipeptide through co-
crystallisation
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Paolo Lucaiolia, Elisa Nauhab, Ishwar Singh*a, and Nicholas Blagden*a
aUniversity of Lincoln, School of Pharmacy, Joseph Banks Laboratories, Green Lane, LN6 7DL, Lincoln, United Kingdom
bUniversity of Lincoln, School of Chemistry, Joseph Banks Laboratories, Green Lane, LN6 7DL, Lincoln, United Kingdom
Email: isingh@lincoln.ac.uk ; nblagden@lincoln.ac.uk
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SYNOPSIS Coꢀcrystallisation as a possible singleꢀstep method for both crystallisation and purification of TFAꢀ
contaminated dipeptides synthesised through Fmoc Solid Phase Peptide Synthesis.
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113x23mm (300 x 300 DPI)
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