The Impact of Trifluoroacetic Acid on Peptide ... - ACS Publications

Article Cite This: Cryst. Growth Des. XXXX, XXX, XXX−XXX

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The Impact of Trifluoroacetic Acid on Peptide Cocrystallization: Multicomponent Crystals of L‑Leu‑L‑Leu Dipeptides P. Lucaioli,† E. Nauha,‡ I. Singh,*,† and N. Blagden*,† †

School of Pharmacy and ‡School of Chemistry, University of Lincoln, Joseph Banks Laboratories, Green Lane, LN6 7DL Lincoln, United Kingdom

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S Supporting Information *

ABSTRACT: In a previous contribution, entitled “First Steps for the Direct Purification of L-Leu-L-Leu Dipeptide through Cocrystallization”, we reported and evidenced cocrystallization as a route to purify freshly synthesized trifluoroacetic acid (TFA)-contaminated Leu-Leu peptides. In this contribution ternary and quaternary crystal forms, isolated as transient phases to the previously reported pure cocrystals, are presented. This contribution and the one previously reported present the outcomes of large cocrystallization screening undertaken on the LLeu-L-Leu dipeptide, where a competing range of multiple crystal forms from target cocrystal to TFA salt is accessible. As a key point for this contribution we report the isolation of transient phases that consist of Leu-Leu peptide (both with amidic and carboxylic C-terminals) either with pyridazine, 1H-pyrazole, pyridine N-oxide, or pyrazine plus TFA, or solvent alcohol or water with TFA. Such a diversity of multicomponent phases add further to the understanding of the differential crystallization process established when cocrystallization is used to purify the crude synthesized peptide product contaminated with TFA. A description of the overall packing landscape was undertaken from a crystal engineering point of view to illustrate how the presence of trifluoroacetate anions, strong hydrogen bond acceptors, affect the crystal packing. Typically, the Leu-Leu peptide forms a multilayered structure with guest in cavities between the layers; however, alternative novel arrangements were also seen. The structures are part of a wide landscape of possible complexes that are difficult to isolate, as they often represent transient phases that are hard to reproduce. Nevertheless, the reported phases give further insight into the purification through cocrystallization pathway and, critically, help to understand the interplay between crystal growth and the packing landscape.

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INTRODUCTION The direct purification of freshly synthesized and crude LeuLeu dipeptide through cocrystallization was previously reported by us;1 in that contribution we showed a possible alternative route for the removal of residual trifluoroacetic acid (TFA) by a differential crystallization approach. Besides successfully isolating the cocrystal forms, a series of multicomponent intermediate phases containing TFA, solvent, and/ or conformer were also isolated, and these are the subject of this contribution. These multicomponent intermediate phases are transient to the targeted TFA-free cocrystals isolated, and herein we report a new class of ternary and quaternary complexes involving the leucine dipeptide. TFA is extensively used for a wide variety of reactions. This is in part due to its strong acidity (pKa = 0.23), high volatility, and miscibility with water and most of the common organic solvents. It represents one of the main reagents for solid-phase peptide synthesis (SPPS) protocols,2 as most of the cocktails for resin cleavage and final deprotection contain TFA. Despite its fundamental role in the synthetic process, this acid represents an undesired pollutant generating a strong ion pair with the positively charged amino terminals and side © XXXX American Chemical Society

chains (e.g., arginine, lysine, histidine). The presence of a trifluoroacetate counterion affects both the biological activity3−5 and physiochemical6 properties of the peptidic product interfering with analytical procedures. Most of the unbound TFA can be eliminated through freeze-drying, but additional purification steps through chromatographic techniques followed by ion-exchange reactions are required to obtain highly pure products. These steps are necessary for pharmaceutical application, but they determine a significant loss in the final yield. For the first time, TFA-contaminated L-Leu-L-Leucine dipeptides (Figure 1) in the lyophilized state have been used for cocrystallization experiments as a possible alternative purification method. The differential crystallization processes involving the different chemical species lead to two types of possible outcomes (Figure 2): (i) trifluoroacetate-free cocrystals (reported in our previous paper “First Steps for the Direct Purification of L-Leu-L-Leu Dipeptide through CoReceived: May 1, 2018 Revised: June 19, 2018 Published: June 22, 2018 A

DOI: 10.1021/acs.cgd.8b00665 Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

Article

Figure 1. Molecular structures of the synthesized L-Leu-L-Leu dipeptides. Both carboxylic (a) and amide (b) C-terminals were used in the present work. The red line in (a) represents the θ torsion angle, a useful parameter to describe the relative orientation of the two isobutyl side chains with respect to the plane of the hydrophilic backbone of the molecule (see Figure S1).

Figure 2. Some of the possible multicomponent crystals observed during the cocrystallization experiments using TFA-contaminated L-Leu-L-Leu dipeptides.

crystallization”1) and (ii) multicomponent systems containing TFA (described in this paper). The latter are transient phases representing solid-state intermediates between trifluoroacetate salts and cocrystals of the dipeptide. Insight on the crystal chemistry was aided by a Cambridge Structural Database (CSD) search for structures containing both short peptides and trifluoroacetate anions. Results show that most of the time the role of the latter is not considered in the description of the final crystal packing. Structures previously reported in literature7−10 show that L-Leu-L-Leu dipeptides self-assemble in two-dimensional sheets that stack on top of each other generating a multilayered structure with an alternation of hydrophilic and hydrophobic layers. An important aspect of such examples is the constant presence of guest molecules of solvents or coformers.1 The propensity to include a second component in the three-dimensional architecture arises from the presence of empty channels and voids between the isobutyl side chains in the crystal. Wide vacant spaces would usually undermine the stability of the crystal structure,11 and for this reason a suitable cocrystal former is required by the system to form a stable architecture. Furthermore, the inclusion of a second component represents a solution for a crystal packing issue involving the amino H atoms of the N-terminals of the peptidic molecules. While two of them are used for the formation of the sheet (through headto-tail interactions with the C-terminal),12 the third one usually interacts with the chemical functionalities contained in the side chains.13,14 Nevertheless, hydrophobic peptides do not contain any acceptor for such an interaction, and a cocrystal former is required (solvent or coformer).15 In the present work, structures containing the Leu-Leu dipeptide with both amidic and carboxylic C-terminals were examined. The use of nonpurified dipeptides (with the

consequent involvement of TFA) makes the difficult cocrystallization process of such molecules even more complicated. Numerous crystallization attempts resulted in the formation of residues not suitable for crystallographic analysis. In this situation, typically, small, poor-quality and fragile crystals were recovered from a sticky/oily material obtained at the end of the cocrystallization. The X-ray analysis of such samples showed often weak reflections with subsequent difficult solutions and refinements. Nevertheless, the ternary and quaternary systems obtained during our cocrystallization screening are presented in this paper as multicomponent crystals that must be contemplated as a tool for the understanding and analysis of such a complicated differential crystallization process. LLNH 2 :MeOH, LLNH 2 :H 2 O, LLNH2:EtOH, LLNH2:Pyd, LLNH2:Pyd(x4), LLNH2:1HPyz, LLNH2:PyNOx, LL:Pya, LL:Pya2, LL:Pyd and LL:Imi are described from a crystal engineering point of view to underline how the presence of trifluoroacetate anions, strong hydrogen-bond acceptors, affect the crystal packing. The structures generate a wide landscape of complexes that are difficult to isolate, as they often represent transient phases that are hard to reproduce. Nevertheless, the reported phases give further insight into the purification through cocrystallization pathway and, critically, help to understand the interplay between crystal growth and the packing landscape.

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EXPERIMENTAL SECTION

Dipeptide Synthesis. The L-Leu-L-Leu-OH dipeptide (Figure 1a) was synthesized through manual Fmoc SPPS using Fmoc-NH Leu-OH protected amino acid (Merck Millipore) and 2-chlorotrityl chloride resin (Iris Biotech GmBH). The initial manufacturer loading was 1.60 mmol/g. The resin initial loading was performed using 4 equiv of protected amino acid and 8 equiv of N,N-diisopropylethylB



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63 450 reflections measured (6.802° ≤ 2Θ ≤ 145.418°), 16 959 unique (Rint = 0.0615, Rsigma = 0.0564), which were used in all calculations. The final R1 was 0.0626 (I > 2σ(I)), and wR2 was 0.1754 (all data). LLNH2:H2O: C84H156F18N18O26.4 (M = 2182.66 g/mol): monoclinic, space group P21 (No. 4), a = 12.5942(4) Å, b = 34.6483(9) Å, c = 13.6522(3) Å, β = 97.1140(10)°, V = 5911.5(3) Å3, Z = 2, T = 173(2) K, μ(Cu Kα) = 0.939 mm−1, Dcalc = 1.226 g/cm3, 100 847 reflections measured (5.1° ≤ 2Θ ≤ 145.106°), 22 724 unique (Rint = 0.0759, Rsigma = 0.0634), which were used in all calculations. The final R1 was 0.1001 (I > 2σ(I)), and wR2 was 0.2933 (all data). LLNH2:Pyd(x4): C14.67H26.67F3N3.33O4.33 (M = 376.06 g/mol): monoclinic, space group P21 (No. 4), a = 12.6634(8) Å, b = 34.874(2) Å, c = 13.7343(8) Å, β = 95.426(3)°, V = 6038.2(6) Å3, Z = 12, T = 173(2) K, μ(Cu Kα) = 0.939 mm−1, Dcalc = 1.241 g/cm3, 66 065 reflections measured (5.068° ≤ 2Θ ≤ 133.952°), 20 716 unique (Rint = 0.0919, Rsigma = 0.0881), which were used in all calculations. The final R1 was 0.1401 (I > 2σ(I)), and wR2 was 0.3711 (all data). LLNH2:Pyd: C15.33H27.33F3N3.67O4 (M = 384.07 g/mol): triclinic, space group P1 (No. 1), a = 12.0432(8) Å, b = 13.5737(8) Å, c = 19.2018(11) Å, α = 84.825(3)°, β = 83.917(3)°, γ = 82.024(3)°, V = 3082.0(3) Å3, Z = 6, T = 173(2) K, μ(Cu Kα) = 0.923 mm−1, Dcalc = 1.242 g/cm3, 45 222 reflections measured (4.642° ≤ 2Θ ≤ 144.894°), 22 162 unique (Rint = 0.0618, Rsigma = 0.0763), which were used in all calculations. The final R1 was 0.0687 (I > 2σ(I)), and wR2 was 0.1904 (all data). LL:Pya: C28H56F3N5O10.5 (M = 687.77 g/mol): triclinic, space group P1 (No. 1), a = 11.5314(9) Å, b = 13.1759(11) Å, c = 13.3477(12) Å, α = 86.714(5)°, β = 76.660(4)°, γ = 89.821(4)°, V = 1969.9(3) Å3, Z = 2, T = 173(2) K, μ(Cu Kα) = 0.823 mm−1, Dcalc = 1.160 g/cm3, 33 264 reflections measured (6.72° ≤ 2Θ ≤ 139.458°), 13 405 unique (Rint = 0.0775, Rsigma = 0.0815), which were used in all calculations. The final R1 was 0.1318 (I > 2σ(I)), and wR2 was 0.3242 (all data). LL:Pya2: C26.5H49F3N4O11.5 (M = 664.69 g/mol): monoclinic, space group P21 (No. 4), a = 11.5466(5) Å, b = 24.4207(11) Å, c = 13.1200(6) Å, β = 90.149(2)°, V = 3699.5(3) Å3, Z = 4, T = 173(2) K, μ(Cu Kα) = 0.875 mm−1, Dcalc = 1.193 g/cm3, 19 459 reflections measured (7.648° ≤ 2Θ ≤ 140.234°), 12 224 unique (Rint = 0.0612, Rsigma = 0.0703), which were used in all calculations. The final R1 was 0.1416 (I > 2σ(I)), and wR2 was 0.3475 (all data). LL:Pyd: C18H29F3N4O5 (M = 438.45 g/mol): orthorhombic, space group P212121 (No. 19), a = 5.9150(3) Å, b = 14.8931(7) Å, c = 26.1139(10) Å, V = 2300.44(18) Å3, Z = 4, T = 173(2) K, μ(Cu Kα) = 0.929 mm−1, Dcalc = 1.266 g/cm3, 36 402 reflections measured (6.832° ≤ 2Θ ≤ 136.914°), 4206 unique (Rint = 0.1457, Rsigma = 0.0620), which were used in all calculations. The final R1 was 0.0630 (I > 2σ(I)), and wR2 was 0.1622 (all data). LL:Imi: C83H159F3N18O21 (M = 1802.27 g/mol): monoclinic, space group P21 (No. 4), a = 14.7545(8) Å, b = 24.4333(13) Å, c = 15.5378(8) Å, β = 110.756(3)°, V = 5237.9(5) Å3, Z = 2, T = 173(2) K, μ(Cu Kα) = 0.709 mm−1, Dcalc = 1.143 g/cm3, 66 795 reflections measured (6.082° ≤ 2Θ ≤ 147.354°), 19 966 unique (Rint = 0.0837, Rsigma = 0.0798), which were used in all calculations. The final R1 was 0.0737 (I > 2σ(I)), and wR2 was 0.2062 (all data). TFA:DMAP: C9H11F3N2O2 (M = 236.20 g/mol): triclinic, space group P1̅ (No. 2), a = 8.3158(5) Å, b = 8.3852(5) Å, c = 8.5193(4) Å, α = 100.183(3)°, β = 93.691(3)°, γ = 115.846(3)°, V = 519.45(5) Å3, Z = 2, T = 173(2) K, μ(Cu Kα) = 1.257 mm−1, Dcalc = 1.510 g/cm3, 6596 reflections measured (10.688° ≤ 2Θ ≤ 144.81°), 2042 unique (Rint = 0.0389, Rsigma = 0.0384), which were used in all calculations. The final R1 was 0.0369 (I > 2σ(I)), and wR2 was 0.1001 (all data). TFA:Imi: C5H5F3N2O2 (M = 182.11 g/mol): orthorhombic, space group I212121 (No. 24), a = 9.1391(7) Å, b = 8.9067(5) Å, c = 9.0834(5) Å, V = 739.38(8) Å3, Z = 4, T = 173(2) K, μ(Cu Kα) = 1.569 mm−1, Dcalc = 1.636 g/cm3, 3486 reflections measured (13.744° ≤ 2Θ ≤ 152.218°), 743 unique (Rint = 0.0208, Rsigma = 0.0182), which were used in all calculations. The final R1 was 0.0285 (I > 2σ(I)), and wR2 was 0.0772 (all data). Additional crystallographic information is presented in the Supporting Information. Structure solutions were performed by direct methods, and refinement with SHELXL16 was finished using the ShelXle17 software.

amine (DIPEA) in dichloromethane (DCM). The Fmoc deprotection steps were performed using a 20% solution of piperidine in dimethylformamide (DMF), while protected amino acid (4 equiv), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU; 4 equiv), and N,Ndiisopropylethylamine (8 equiv) were used for the coupling reactions in DMF. The peptide was cleaved from resin using a 20:5:75 mixture of TFA, triisopropylsilane, and DCM. The volatile components were evaporated with a rotavapor. The SPPS procedure for the L-Leu-LLeu-NH2 dipeptide (Figure 1b) was performed using a Fmoc-Rink Amide AM resin (Iris Biotech GmBH). The initial manufacturer loading was 0.74 mmol/g. Fmoc deprotection, coupling, and final cleavage steps were performed using the same procedure and parameters described for the L-Leu-L-Leu-OH. The final products were lyophilized to eliminate and avoid any possible moisture. These treatments led to the removal of most of the unbound TFA (boiling point: 72.4 °C), although it is still present in the final compound. Co-Crystallization Experiments. Pyridazine (Pyd), 1H-pyrazole (1H-Pyz), pyridine N-oxide (PyNOx), and pyrazine (Pya) were obtained from Alfa Aesar; imidazole (Imi) was obtained from SigmaAldrich. Such molecules were part of a list of 30 different coformers selected among the most commonly encountered cocrystal formers to have a wide variety of chemical functionalities (i.e., H-bond donors and acceptors), structural features (e.g., linear, aromatic, heterocyclic), and chemical properties (i.e., acid base). Coformers were used to prepare equimolar solution in MeOH (HPLC grade) along with the peptidic compound. LLNH2:EtOH and LLNH2:H2O represent the only exceptions in which a different solvent (ethanol and water, respectively) was used for the crystallization experiment. The LLNH2:Pyd(x4) represents the only sample sample in which the coformer has been used in eccess (peptide/pyridazine molar ratio = 1:4) with respect to L-Leu-L-Leu-NH2. 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 X-ray diffraction were collected from the solution (when possible) or recovered from the dry material. X-ray Diffraction. Single crystals of LLNH 2 :MeOH, LLNH 2 :H 2 O, LLNH 2 :EtOH, LLNH 2 :Pyd, LLNH 2 :Pyd(x4), LLNH2:1H-Pyz, LLNH2:PyNOx, LL:Pya, LL:Pya2, LL:Pyd, LL:Imi, TFA:DMAP, and TFA:Imi suitable for X-ray diffraction measurements were mounted on MiTeGen Dual-Thickness MicroMounts and analyzed 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 = 1.540 56 Å). Crystallographic data. LLNH2:MeOH: C15H30F3N3O5 (M = 389.42 g/mol): monoclinic, space group P21 (No. 4), a = 11.4724(7) Å, b = 7.9334(5) Å, c = 11.5608(8) Å, β = 103.120(4)°, V = 1024.74(12) Å3, Z = 2, T = 173(2) K, μ(Cu Kα) = 0.957 mm−1, Dcalc = 1.262 g/cm3, 13 699 reflections measured (7.852° ≤ 2Θ ≤ 139.48°), 3662 unique (Rint = 0.0766, Rsigma = 0.0598), which were used in all calculations. The final R1 was 0.1053 (I > 2σ(I)) and wR2 was 0.2910 (all data). LLNH2:EtOH: C16H32F3N3O5 (M = 403.44 g/ mol): monoclinic, space group P21 (No. 4), a = 11.0204(8) Å, b = 8.0498(7) Å, c = 12.4869(10) Å, β = 96.231(6)°, V = 1101.19(15) Å3, Z = 2, T = 173(2) K, μ(Cu Kα) = 0.907 mm−1, Dcalc = 1.217 g/cm3, 19 733 reflections measured (7.122° ≤ 2Θ ≤ 144.534°), 4226 unique (Rint = 0.0908, Rsigma = 0.0770), which were used in all calculations. The final R1 was 0.0694 (I > 2σ(I)), and wR2 was 0.1745 (all data). LLNH2:1H-Pyz: C15.5H29.5F3N4O4.75 (M = 404.93 g/mol): monoclinic, space group P21 (No. 4), a = 13.8019(5) Å, b = 24.0495(10) Å, c = 13.9270(5) Å, β = 111.988(2)°, V = 4286.5(3) Å3, Z = 8, T = 173(2) K, μ(Cu Kα) = 0.941 mm−1, Dcalc = 1.255 g/cm3, 61 209 reflections measured (6.844° ≤ 2Θ ≤ 144.378°), 16 699 unique (Rint = 0.0353, Rsigma = 0.0329), which were used in all calculations. The final R1 was 0.0422 (I > 2σ(I)), and wR2 was 0.1178 (all data). LLNH2:PyNOx: C63.5H117F12N13.5O19.5 (M = 1609.70 g/mol): monoclinic, space group P21 (No. 4), a = 13.5926(18) Å, b = 24.618(3) Å, c = 13.7987(17) Å, β = 109.635(4)°, V = 4348.9(10) Å3, Z = 2, T = 173(2) K, μ(Cu Kα) = 0.921 mm−1, Dcalc = 1.229 g/cm3, C



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groups that show similarities in the final crystal packing architectures. The first case is represented by structures (LLNH2:MeOH and LLNH2:EtOH) containing just one molecule of peptide in the asymmetric unit and crystallizing in P21. Leu-Leu-NH2 molecules use their amidic functionality (C-terminal) and the NH group of the internal peptide bond to generate straight ribbons running along the b-axis (Figure 3a). Parallel strands of dipeptides flank each other on the ab plane generating the typical two-dimensional sheet (Figure 3a): such aggregates stack on top of each other thanks to hydrophobic interactions between the isobutyl side chains. The final architecture is represented by the well-known multilayered scaffolding in which hydrophilic layers (peptidic backbones) and hydrophobic region (branched chains) alternate along the c-axis (Figure 3b). LLNH2:1H-Pyz and LLNH2:PyNOx crystallize in P21 and contain four independent molecules of Leu-Leu-NH2 in the asymmetric unit. As with the previous case, when only peptide−peptide interactions are considered, the resulting assembly is a straight ribbon running in one direction (Figure 4a). Parallel monodimensional strands lay close to each other on the ac plane to create the sheet of dipeptide molecules (Figure 4a). The multilayered structure resulting from the stacking of sheets (Figure 4b) is different with respect to those described above. The twofold screw axis runs in the same direction of the monodimensional ribbons in LLNH2:MeOH and LLNH2:EtOH, while in these packings it is perpendicular to the dipeptide sheets. Such variance results in a final stack in which each layer is rotated by 180° with respect to the previous one in both LLNH2:1H-Pyz and LLNH2:PyNOx (Figure S4). LLNH2:H2O and LLNH2:Pyd(x4) crystallize in P21, but in this case the asymmetric units of such structures contain six independent molecules of Leu-Leu-NH2. These components are characterized by a wide diversity of structural conformations, and this aspect can be highlighted considering the |θ| torsion angles that describe the relative orientation of the isobutyl side chains (Table 1). The dipeptides interact with each other through the same hydrogen-bonded network described above, but, in this case, the resulting arrangement is markedly different. When only peptide−peptide interactions are considered, the Leu-Leu-NH2 molecules generate a onedimensional strand with an undulated aspect (Figure 5a). Parallel ribbons laying on the two-dimensional plane results in the typical sheet of dipeptide (Figure 5a). The presence of one molecule with a low |θ| (105.79° in LLNH2:H2O and 110.16° in LLNH2:Pyd(x4)) along with the twofold screw axis running in the same direction of the monodimensional assemblies determine the particular zig-zagged aspect of both the ribbons and the sheets (Figure 5d). Because of such geometrical arrangement, the F molecule does not occupy the expected position (Figure S6), and the chain of nine-membered rings is disrupted. When the sheets stack on top of each other, the multilayered scaffolding is not planar (Figure 5b). Despite the same number of independent dipeptides in the asymmetric unit, LLNH2:Pyd crystallizes in P1 with a completely different packing. As seen for LLNH2:H2O and LLNH2:Pyd(x4), one of the Leu-Leu-NH2 molecules has a low torsion angle value (|θ| = 113.86°). Nevertheless, because of the only translational symmetry, such peptide does not represent a “kinking point” anymore and does not introduce the above-described zig-zagged arrangement (Figure 6a and

RESULTS AND DISCUSSION The involvement of trifluoroacetic acid in the cocrystallization of short peptides has a strong impact on different aspect of the process. First, the presence of such a strong acid often compromises the obtaining of good crystals with properties suitable for further X-ray analysis. Amorphous oily or rubbery phases were often obtained at the end of the slow solvent evaporation during our experimental screening. When obtained, the final crystals are often affected by physical defects (i.e., twinning, polycrystallinity) or disadvantageous mechanical properties (e.g., high fragility, low stability, degradation during the measurement time). The difficulty in handling such solid material is frequently accompanied by lowquality reflections and data with the unfeasibility to solve the final structure. The refinement process is another nontrivial aspect that is deeply affected by the complexity of the proposed systems. From a structural point of view, the CF3 group on the contaminating trifluoroacetate anions is one of the chemical entities most prone to be disordered due to the high symmetry and the low energetic barrier for the rotation around its threefold axis.18 Furthermore, as shown by the θ torsion angle values reported in this and in our previous paper, the dipeptides have a large degree of flexibility with many possible conformations. L-Leu-L-Leu-NH2 Peptide. The presence of the amidic functionality at the C-terminal of the L-Leu-L-Leu dipeptide determines a different kind of hydrogen-bonded network with respect to the one characterizing the Leu-Leu-COOH sequence. The absence of a negatively charged group results in the nonappearance of strong head-to-tail charge-assisted Hbonds between the amino- and carboxylic terminals. As confirmed by the only reported structure of a hydrophobic dipeptide (CSD refcode: BEJPEM;19 see Figure S3), when only peptide−peptide interactions are considered, monodimensional ribbons of L-Leu-L-Leu-NH2 molecules are generated through hydrogen-bonded interactions involving the amidic C-terminus and the NH group of the internal peptide bond (Figure 3). The crystal structures reported in the present paper are different in composition, content (i.e., Z′ of the peptide), and space groups (Table 1). They can be classified in four different

Figure 3. (a) Straight monodimensional ribbons of Leu-Leu-NH2 in LLNH2:EtOH. Such arrangements are generated through CO··· HN hydrogen bonds involving the amidic C-terminals (orange contacts) and interactions between the C-terminus and the NH group of the internal peptide bond (blue contacts). Two-dimensional sheet generated by flanking parallel strands. (b) Multilayered scaffolding with the typical alternation of hydrophilic (blue) and hydrophobic regions (pink). Isobutyl side chains are not shown in (a) for clarity. D



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Table 1. Crystal Structure of Leu-Leu-NH2a |Θ| torsion angle

sample (deposition no.)

composition

Z′ peptide

peptide/TFA

space group

LLNH2:MeOH (1840094)

peptide trifluoroacetate solvent (MeOH) peptide trifluoroacetate Solvent (EtOH) peptide trifluoroacetate 1H-pyrazole water peptide trifluoroacetate pyridine N-oxide

1

1:1

P21

147.6(7)°

1

1:1

P21

142.4(3)°

4

1:1

P21

4

1:1

P21

C9A-C2A···C4A-C5A: 121.9(3)° C9B−C2B···C4B−C5B: 153.2(3)° C9C−C2C···C4C−C5C: 115.4(2)° C9D-C2D···C4D-C5D: 141.9(3)° C9A-C2A···C4A-C5AB: 150.1(7)° C9A-C2A···C4A-C4AA: 128.1(5)° C9B−C2B···C4B−C5B: 142.7(4)° C9C−C2C···C4C−C5C: 114.2(4)° C9D-C2D···C4D-C5DA: 129.6(4)° C9D-C2D···C4D-C5DB: 159.4(8)° C9A-C2A···C4A-C5A:106.0(5)° C9B−C2B···C4B−C5B: 141.7(5)° C9C−C2C···C4C−C5C: 160.3(9)° C9D-C2D···C4D-C5D: 132.1(12)° C9P−C2D···C4D-C5D: 150.8(13)° C9E-C2E···C4E-C5E: 119.7(13)° C9E-C2E···C4Z-C5Z: 119.9(11)° C9F−C2F···C4F−C5F: 131.9(12)° C9F−C2F···C4F−C5W: 162.6(14)° C9W−C2F···C4F−C5F: 114.1(14)° C9W−C2F···C4F−C5W: 144.8(16)° C9A-C2A···C5A-C4A: 153.0(16)° C9B−C2B···C5B−C4B: 143.1(14)° C9P−C2B···C5B−C4B: 156.6(18)° C9C−C2C···C5C−C4C: 126.7(12)° C9D-C2D···C5D-C4D: 110.2(9)° C9E-C2E···C5E-C4E: 114.6(13)° C9F−C2F···C5F−C4F: 139.1(12)° C9F−C2F···C5F−C4L: 146.5(16)° C9A-C2A···C4A-C5A: 102.4(4)° C9B−C2B···C4B−C5B: 143.2(5)°

LLNH2:EtOH (1840095)

LLNH2:1H-Pyz (1840098)

LLNH2:PyNOx (1840100)

water LLNH2:H2O (1840104)

peptide trifluoroacetate solvent (H2O)

6

1:1

P21

LLNH2:Pyd(x4) (1840101)

peptide trifluoroacetate pyridazine water

6

1:1

P21

LLNH2:Pyd (1840096)

peptide trifluoroacetate pyridazine

6

1:1

P1

C9C−C2C···C4C−C5C: 131.6(4)° C9D-C2D···C4D-C5D: 128.1(4)° C9E-C2E···C4E-C5E: 145.3(4)° C9F−C2F···C4F−C5F: 152.5(5)° a

Torsion angle values in italic are representative of the less-frequent conformations.

Figure S5). Parallel straight strands lay on the bc plane and create planar sheets that stack on top of each other to generate the multilayered structure (Figure 6b). A common feature for all the structure is the location of the positively charged N-terminals at both sides of the monodimensional ribbons. Most of the amino H atoms are not used to generate peptide−peptide contacts, but they are involved in charge-assisted hydrogen bonds with the trifluoroacetate anions (Figure 7a). TFA also interacts with the hydrogens of the amidic C-terminals, and it acts as a crosslinker connecting peptides belonging to adjacent ribbons. Molecules of solvent or water are occasionally included in hydrogen-bonded networks and participate in the “bridge effect” (Figure 7b). Coformers use their H-bond donor and acceptors to interact with the chemical functionalities of the

peptides (Figure 7c) or the other nonpeptidic components (Figure 7d). Sheets of peptide stack on top of each other through van der Waals interactions between the isobutyl side chains of the Lleucine residues. Such arrangement results in an alternation of hydrophilic (dipeptide backbones) and hydrophobic layers (Figure 8a,b). The resultant peptide scaffolding is characterized by the presence of empty voids and channels generated by the relative orientation of the branched side chains. Such pockets host molecules of TFA, water, and coformers interacting only on one side of the layer without creating any connection between adjacent sheets. L-Leu-L-Leu-COOH Peptide. When the C-terminus is represented by the carboxylate, the Leu-Leu dipeptides are usually in a zwitterionic state, and this promotes the establishment of head-to-tail charge-assisted hydrogen bonds. E



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Figure 6. (a) Peptide molecules self-assemble through hydrogenbonded interactions involving the C-terminals and the NH group of the internal peptide bond in LLNH2:Pyd. The resulting strands flank to each other creating the typical two-dimensional sheet. The carbon atoms of the six independent peptides are differently colored by symmetry, and molecules are labeled for clarity. (b) Multilayered scaffolding with planar hydrophilic (blue) and hydrophobic (pink) layers. Isobutyl side chains are not shown in (a) for clarity.

Figure 4. (a) Peptide−peptide interactions generating the ribbon of dipeptide molecules in LLNH2:1H-Pyz. Flanking strands of dipeptide generate the typical two-dimensional sheet. The four independent molecules are labeled for clarity, and carbon atoms are differently colored by symmetry equivalence. (b) Multilayered structure generated by stacking of antiparallel sheets of dipeptide. Hydrophobic side chains are not shown in (a) for clarity.

Figure 7. (a) Typical bridge effect generated in LLNH2:MeOH by the trifluoroacetate anions interacting with both the N-terminals (magenta contacts) and the amidic C-termini (light green contacts). (b) Molecule of EtOH involved in the hydrogen-bonded network interconnecting different peptides in LLNH2:EtOH. Molecules of Leu-Leu-NH2 belonging to adjacent strand are differently colored in (a, b). (c) Molecules of pyridazine (coformer) interacting with two independent dipeptides in LLNH2:Pyd. (d) 1H-Pyrazole molecules interact with both peptidic and nonpeptidic components in LLNH2:1H-Pyz. Carbon atoms are shown in different colors by symmetry in (c, d). Isobutyl side chains are not shown for clarity. Figure 5. (a) Two-dimensional sheet generated by flanking undulated ribbons of dipeptide molecules in LLNH2:Pyd(x4). Carbon atoms of the six independent molecules of dipeptides are differently colored by symmetry equivalence. (b) Multilayered structure of LLNH2:Pyd(x4). Isobutyls side chains are not shown in (a) for clarity.

Previously reported cases (obtained using a crude TFA-free starting material) show that such hydrophobic peptides usually crystallize, generating solvates with the typical multilayered scaffolding or nanotube formations: such a trend was confirmed also by the trifluoroacetate-free multicomponent crystals described in our previous paper. The presence of an additional strong ionic species determines some important deviation from the classic self-assemblies with different packing solutions (Table 2). In LL:Pya and LL:Pya2, alternative peptide−peptide interactions are created, and the four independent Leu-LeuCOOH molecules interact forming tetramer with similar features (Figure 9a and Figure S7). Such an assembly is obtained through carboxylate−carboxylate (orange in Figure 9a), carboxylate−NH group (blue in Figure 9a), and amino

Figure 8. Scaffolding of LLNH2:Pyd (a) and LLNH2:Pyd(x4) (b) representative of both planar and undulated stacks of two-dimensional sheets. The nonpeptidic components (black molecules) are hosted in the cavities between the isobutyl side chains of the hydrophobic regions (pink).

terminal−carbonyl (light green in Figure 9a) interactions. Single tetramers are connected via C−O···H−N hydrogen bonds resulting in monodimensional ribbons running along one axis (Figure 9b). Such aggregates flank to each other creating the typical two-dimensional sheet. The parallel strands are not connected through peptide−peptide interactions in LL:Pya, while there are +N−H···OC H-bonds connecting F



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Table 2. Crystal Structures of Multicomponent Crystals of Leu-Leu-COOH Dipeptidesa samples (deposition no.) LL:Pya (1840099)

composition

Z′ peptide

peptide/TFA molar ratio

L-Leu-L-Leu-COOH

4

2:1

space group P1

4

2:1

P21

1

1:1

P212121

6

6:1

P21

trifluoroacetate pyrazine water LL:Pya2 (1840105)

L-Leu-L-Leu-COOH

trifluoroacetate methanol (solvent) water LL:Pyd (1840102)

LL:Imi (1840097)

Leu-Leu-COOH trifluoroacetate pyridazine Leu-Leu-COOH trifluoroacetate imidazole

torsion angle (|θ|) C9X-C2···C4−C5: 107.4(11)° C9Y−C2···C4−C5: 106.3(18)° C9B−C2B···C4B−C5B: 155.9(8)° C9C−C2C···C4C−C5C: 149.9(8)° C9D-C2D···C4D-C5D: 109.1(11)° C9A-C2A···C4A-C5A: 104.0(9)° C9B−C2B···C4B−C5B: 166.2(11)° C9B−C2B···C4B−C5P: 110.6(12)° C9C−C2C···C4C−C5C: 99.4(11)° C9C−C2C···C4C−C5Z: 150.2(12)° C9D-C2D···C4D-C5D: 101.6(9)° 103.5(4)°

C9A-C2A···C4A-C5A: 158.3(5)° C9B−C2B···C4B−C5B: 155.4(5)° C9C−C2C···C4C−C5C: 137.6(4)° C9D-C2D···C4D-C5D: 150.2(6)° C9E-C2E···C4E-C5E: 142.9(6)° C9F−C2F···C4F−C5F: 142.5(7)°

a

Torsion angle values in italic font are representative of the less-frequent conformations.

Figure 9. (a) Tetramer formed by the four independent molecules of dipeptide. Carbon atoms of the four independent molecules are colored by symmetry. Peptides are labeled for clarity. (b) Twodimensional sheet generated by flanking ribbons in LL:Pya2.

Figure 10. Multilayered stuctures of LL:Pya2 (a) and LL:Pya (b).The nonpeptidic molecules (black) are located in the hydrophobic pockets generated by the relative orientation of the isobutyl side chains. (c) Hydrogen-bonded network generated by the pyrazine molecule in LL:Pya.

molecules belonging to adjacent ribbons (Figure 9b) in LL:Pya2. The presence of many H-bond donors and acceptors on both sides of each strand determines the establishment of contacts between such functionalities and the nonpeptidic components. Trifluoroacetate anions, solvent, and water molecules act as cross-linkers between the different strands in LL:Pya and supply additional connections to the ribbons of LL:Pya2. As seen for the previous structures, such components are hosted in the voids of the hydrophobic regions of the multilayered peptidic scaffolding generated by the stacks of sheets (Figure 10a,b). The nitrogen atoms of the pyrazine molecule in LL:Pya are involved in two N···H−O contact with two molecules of water (Figure 10c). The resulting watercoformer-water hydrogen-bonded network represents an interconnection point between two subsequent sheets. Differently from the examples described above, the hydrogen-bonded network generating the sheet of peptide in LL:Imi is characterized by head-to-tail charge-assisted contacts (red in Figure 11a), typical when the C-terminal is represented by the carboxylate group. Two of the amino H atoms of each L-Leu-LLeu-COOH molecule are used to create H-bonds with two subsequent dipeptides. The resulting assembly is stabilized by

H-bonds involving the NH and CO groups of the internal peptide bonds of adjacent molecules (blue and light green in Figure 11a). The 21 screw axis is perpendicular to the ac plane, and the resulting multilayered structure is characterized by an alternation of antiparallel sheets of peptides along the b-axis (Figure 11b). Also in this case, the nonpeptidic components are contained in the hydrophobic regions and are involved in the formation of two types of interactions cross-linking two adjacent sheets. The protonated molecule of imidazole interacts with a second one (neutral) to create peptideimidazole+H-imidazole-peptide chains running along the b-axis. The third molecule of coformer interacts with both a dipeptide and the TFA creating a similar connection (Figure 11c). The arrangement of the Leu-Leu-COOH dipeptide in LL:Pyd is different from any other structure previously reported in literature. Neither the above-described multilayered scaffoldings nor the alternative hydrophilic nanotubes formation represent the final crystal packing. The only peptide contained in the asymmetric unit self-assemble generating monodimensional chains running through hydrogen bonds between the C-terminal and the NH group of the internal peptide bond along the a-axis (Figure 12a). The twofold screw axis running along the same direction creates an adjacent chain G



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obtained along with the above-described TFA-contaminated LL:Imi multicomponent crystal.

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CONCLUSION The results presented herein form part of an extensive screening of crystal forms on a freshly synthesized, nonpurified Leu dipeptide. Despite the crystallographic impediments, the structures obtained during our experiments and described in the present article represent an insight into the complex nature of differential crystallization pathways. Consideration of both the target cocrystal phase and transient intermediate forms when such experiments are performed are essential for success. The presence of an additional component with a strong ionic character (TFA), acting as a hydrogen-bonded acceptor, determines the establishment of a wide landscape of possible outcomes during cocrystallization screenings. The key point in this regard is the recognition that these structures highlight the competition between a series of pairwise interactions (peptide−TFA, peptide−coformer, TFA−coformer, etc.) and that the outcome dependsnot only on the crystallographic arguments but also on the steering contribution crystal growth imparts. Within the detail of competing interactions in the structures, the trifluoroacetate anions are strongly bonded to the positively charged amino terminals of the peptide. Such electrostatic interactions represent the strong ion pair involving the peptide and TFA when the latter is not removed from the synthesized material. When the Leu-Leu molecules have an amidic C-terminal, the contaminating agent does not influence the commonly seen hydrogen-bonded network between the peptides but generates contacts with multiple peptidic molecules cross-linking different ribbons and stabilizing the two-dimensional sheets. A switch was noted when the Leu-Leu molecules have a carboxylic C-terminal and when three different structural alternatives were observed. If LL:Imi is an example of a typical multilayered structure in which the peptides generate head-totail charge-assisted H-bonds between the amino- and carboxylic-terminals, LL:Pya and LL:Pya2 show that such an arrangement of Leu-Leu-COOH molecules is completely disrupted. An alternative peptide−peptide arrangement is then generated, in which the chemical functionalities of the internal peptide bond have a bigger role in the structural assembly. Lastly, LL:Pyd is a structure in which the title compound generates a three-dimensional scaffolding that has not been reported in literature before for the title compound. The wide range of possible crystal packing is followed by a large variety of composition, symmetries, and stoichiometric ratios between the peptide and the contaminating agent. Nevertheless, all the reported structures show the presence of channels and voids with the trifluoroacetate anions (along with the other nonpeptidic components) located inside these cavities and stabilizing the entire architecture. This situation has further implications with regard to competing interactions when a mixture of solvates and the transient TFA-contaminated phases of the peptide were isolated. For instance, LL:Pya and LL:Pya2 have been obtained from an equimolar solution of Leu-Leu-COOH and pyrazine, and they are related with the methanol solvate reported in our previous paper.1 In this case two different multicomponent crystals (the MeOH solvate and LL:Pya) have been recovered from the material of the same vial, while LL:Pya2 represents the recrystallization product. This

Figure 11. (a) Head-to-tail charge-assisted interactions (red contacts) between Leu-Leu-COOH molecules in LL:Imi. C−O···H−N (blue contacts) and + N−H···OC (light green contacts) hydrogen bonds stabilize the two-dimensional sheet. Carbons of the six independent dipeptides are colored by symmetry equivalence. Molecules are labeled for clarity. (b) Multilayered scaffolding of dipeptides: molecules of imidazole, water, and trifluoroacetate anions (black) are in the hydrophobic regions. (c) Hydrogen-bonded network involving the non-peptidic components. Isobutyl side chains are not shown in (a, c) for clarity.

Figure 12. (a) Monodimensional ribbon of Leu-Leu-COOH dipeptides in LL:Pyd. (b) Ribbons self-assemble generating columns containing a channel running along the a-axis. (c) Packing architecture with molecules of imidazole and trifluoroacetate anions (black) in the voids of the peptidic scaffolding. Isobutyl side chains are not shown in (a) for clarity.

connected to the first one though +N−H···OC contacts between the amino-terminal and the carbonyl (light green in Figure 12b). The P212121 space group leads the resulting ribbons of dipeptides to form columns held together by the van der Waals interactions between the isobutyl side chains (Figure 12c). The channel contains trifluoroacetate anions (interacting with the amino-terminals) and imidazole molecules (involved in bifurcated contacts with the C-terminals). Trifluoroacetate Salts of Coformers. During the large cocrystallization screening with the Leu-Leu-COOH dipeptide, two different salts containing only trifluoroacetate and coformer were obtained. Such crystals show that, in the wide landscape of possible outcomes, systems without peptidic molecules can be obtained. Blocks of TFA:DMAP were retrieved from the same crystallization vial containing needlelike crystals of LLhex, the hexagonal TFA-free nanotube structure described in our previous paper.1 When imidazole was used as a coformer, the TFA:Imi salt structure was H



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Notes

illustrates that the reported structures are transient forms that are often difficult to isolate during a cocrystallization experiment but must be considered, since they represent some of the possible combinations related to the differential crystallization process. The aim of the wider project is the purification through cocrystallization of unpurified synthesized peptides. Moving forward with this work, with insight from the previously reported cocrystal phases and the TFA-contaminated phases reported in this contribution, the upscale of the differential crystallization will be underpinned by the construction of the associated phase diagrams. Such investigation is fundamental for the understanding of the various conditions and parameters affecting the differential cocrystallization pathways involving nonpurified L-Leu-L-Leu dipeptides. The phase diagrams need to incorporate transient phases and associated equilibria between the target TFA-free peptidic phase and allied components. The presence of the trifluoroacetate salts of coformers shows that the coformers can compete with the peptide in the establishment of ionic interactions with TFA, generating a parallel crystallization pathway. This aspect can be regarded as a possible assisting process in the separation of TFA-free peptide forms. The findings to date on these transient phases reconfirm that care and appreciation is needed, as the outcome appears to be sensitive to the way in which the crystallization conditions impact the competing interactions.

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The authors declare no competing financial interest.

■

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.cgd.8b00665. Tables containing the crystallographic data of reported structures, carbon atoms labeling and θ torsion angle, crystal packing comparison of C-amidated structures with a previously reported hydrophobic peptide (BEJPEM), pictures showing the relative orientation of the two-dimensional sheets of peptides, similarities and impact of symmetry on the structures of multicomponent systems containing six molecules of Camidated Leu-Leu dipeptides, conformational comparison of tetramers of LL:Pya and LL:Pya2 (PDF) Accession Codes

CCDC 1840093−1840105 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.

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REFERENCES

(1) Lucaioli, P.; Nauha, E.; Singh, I.; Blagden, N. First Steps for the Direct Purification of L-Leu-L-Leu Dipeptide through Co-Crystallization. Cryst. Growth Des. 2018, 18, 1062−1069. (2) Chan, W. C.; White, P. D. Fmoc Solid Phase Peptide Synthesis: A Practical Approach; Oxford University Press, 2000. (3) Cornish, J.; Callon, K. E.; Lin, C. Q.-X.; Xiao, C. L.; Mulvey, T. B.; Cooper, G. J. S.; Reid, I. R. Trifluoroacetate, a Contaminant in Purified Proteins, Inhibits Proliferation of Osteoblasts and Chondrocytes. Am. J. Physiol. - Endocrinol. Metab. 1999, 277, E779−E783. (4) Ma, T. G.; Ling, Y. H.; McClure, G. D.; Tseng, M. T. Effects of Trifluoroacetic Acid, a Halothane Metabolite, on C6 Glioma Cells. J. Toxicol. Environ. Health 1990, 31, 147−158. (5) Tipps, M. E.; Iyer, S. V.; John Mihic, S. Trifluoroacetate Is an Allosteric Modulator with Selective Actions at the Glycine Receptor. Neuropharmacology 2012, 63, 368−373. (6) Shen, C. L.; Fitzgerald, M. C.; Murphy, R. M. Effect of Acid Predissolution on Fibril Size and Fibril Flexibility of Synthetic BetaAmyloid Peptide. Biophys. J. 1994, 67, 1238−1246. (7) Görbitz, C. H. L-Leucyl-L-Leucine 2-Methyl-1-Propanol Solvate. Acta Crystallogr., Sect. C: Cryst. Struct. Commun. 1999, 55, 670−672. (8) Gö rbitz, C. H. Nanotube Formation by Hydrophobic Dipeptides. Chem. - Eur. J. 2001, 7, 5153−5159. (9) Görbitz, C. H.; et al. Solvent Site Preferences in the Crystal Structures of L-Leucyl-L-Leucine Alcohol (1:1) Complexes. Acta Chem. Scand. 1998, 52, 1343−1349. (10) Mitra, S. N.; Subramanian, E. Observation of a Sterically Unfavorable Side-Chain Conformation in a Leucyl Residue: Crystal and Molecular Structure of L-Leucyl-L-Leucine · DMSO Solvate. Biopolymers 1994, 34, 1139−1143. (11) Kitaigorodskii, A. I. Organic Chemical Crystallography; Consultants Bureau: New York, 1961. (12) Prasad, G. S.; Vijayan, M. X-Ray Studies on Crystalline Complexes Involving Amino Acids and Peptides. XIX. Effects of Change in Chirality in the Complexes of Succinic Acid with DL- and L-Arginine. Int. J. Pept. Protein Res. 1990, 35, 357−364. (13) Eggleston, D. S. Conformation and Structure of L-Valyl-LGlutamic Acid, C10H18N2O5. Acta Crystallogr., Sect. C: Cryst. Struct. Commun. 1984, 40, 1250−1252. (14) Görbitz, C. H.; Backe, P. H. Structures of L-Valyl-L-Glutamine and L-Glutamyl-L-Valine. Acta Crystallogr., Sect. B: Struct. Sci. 1996, 52, 999−1006. (15) Görbitz, C. H. L-Alanyl-L-phenylalanine−2-Propanol (1/2) (αForm), L-Valyl-L-phenylalanine−2-Propanol (1/1) and L-Leucyl-Lphenylalanine−2-Propanol (1/1) (β-Form). Acta Crystallogr., Sect. C: Cryst. Struct. Commun. 1999, 55, 2171−2177. (16) Sheldrick, G. M. Crystal Structure Refinement with SHELXL. Acta Crystallogr., Sect. C: Struct. Chem. 2015, 71, 3−8. (17) Hübschle, C. B.; Sheldrick, G. M.; Dittrich, B. ShelXle: A Qt Graphical User Interface for SHELXL. J. Appl. Crystallogr. 2011, 44, 1281−1284. (18) Müller, P. Practical Suggestions for Better Crystal Structures. Crystallogr. Rev. 2009, 15, 57−83. (19) Doi, M.; Asano, A.; Yamamoto, D. Hydrogen Bond between Water and the Phenyl Ring in the Structure of a Dipeptide H−Phe− Leu−NH 2 at 90 K and the Structure-Based Energy Estimations. Chem. Lett. 2003, 32, 1102−1103.

AUTHOR INFORMATION

Corresponding Authors

*E-mail [email protected]. (N.B.) *E-mail: [email protected]. (I.S.) ORCID

E. Nauha: 0000-0001-9784-8988 I. Singh: 0000-0001-7822-1063 N. Blagden: 0000-0001-5363-6748 Funding

Funding for this project to P.L. was kindly provided by the Univ. of Lincoln. I


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