The first N-terminal unprotected (Gly-Aib)_n peptide: H-Gly-Aib-Gly-Aib-OtBu

Glycine (Gly) is incorporated in roughly half of all known peptaibiotic (non­ribosomally biosynthesized anti­biotic peptides of fungal origin) sequences and is the residue with the greatest conformational flexibility. The conformational space of Aib (α-amino­isobutyric acid) is severely restricted by the second methyl group attached to the C_α atom. Most of the crystal structures containing Aib are N-terminal protected. Deprotection of the N- or C-terminus of peptides may alter the hydrogen-bonding scheme and/or the structure and may facilitate crystallization. The structure reported here for glycyl-α-amino­isobutyryl­glycyl-α-amino­isobutyric acid tert-butyl ester, C₁₆H₃₀N₄O₅, describes the first N-ter­minal-unprotected (Gly-Aib)_n peptide. The achiral peptide could form an intra­molecular hydrogen bond between the C=O group of Gly1 and the N—H group of Aib4. This hydrogen bond is found in all tetra­peptides and N-terminal-protected tripeptides containing Aib, apart from one exception. In the present work, this hydrogen bond is not observed (N...O = 5.88 Å). Instead, every mol­ecule is hydrogen bonded to six other symmetry-related mol­ecules with a total of eight hydrogen bonds per mol­ecule. The backbone conformation starts in the right-handed helical region (and the left-handed helical region for the inverted mol­ecule) and reverses the screw sense in the last two residues.

S1. Introduction

The presence of Aib (α-amino­isobutyric acid) and Gly (glycine) in peptides combines the residue with the greatest conformational flexibility (Gly) with a residue, the conformational space of which is severely restricted by the second methyl group attached to the Cα atom (Aib). This space available for Aib comprises the left- and right-handed helical regions of the Ramachandran plot (Ramachandran et al., 1963). Gly is incorporated in roughly half of all known peptaibiotic (i.e. nonribosomally biosynthesized anti­biotic peptides of fungal origin) sequences and frequently as the –Aib-Gly- dipeptide or as the –Aib-Gly-Aib- tripeptide unit (Stoppacher et al., 2013). On the contrary, the motifs –Gly-Aib-Gly-Aib- or –Gly-Aib-Gly- do not occur in any of the >1300 peptaibiotics known to date. Most of the crystal structures containing Aib are N-terminal protected. Deprotection of the N– or C-terminal of peptides may alter the hydrogen-bonding scheme from intra- to inter­molecular hydrogen bonds, may alter the structure and may facilitate crystallization.

S2. Experimental

S2.1. Synthesis and crystallization

Z-(Gly-Aib)₂—OtBu (1.40 g, 2.84 mmol; Gessmann et al., 2015) was dissolved in MeOH (20 ml) and hydrogeno­lysed in the presence of Pd (ca 140 mg, 5% on charcoal) by bubbling hydrogen gas through the solution. Qu­anti­tative deprotection was already completed within 1 h as revealed by thin-layer chromatography. After removal of the catalyst by filtration and evaporation of the organic phase, a colourless oil remained. The tetra­peptide was crystallized from 50% aqueous methanol by slow evaporation. Tiny crystals were detected after several weeks.

S2.2. Refinement

Crystal data, data collection and structure refinement details are summarized in Table 1. One single plate with the smallest dimension of about 40 µm was mounted on a cryoloop without cryoprote­ctant and kept in place with a minimal amount of vacuum grease. Diffraction data were collected at 100 K on the microfocus beamline I24 (Evans et al., 2011) of the Diamond Light Source in Didcot, England, using a Pilatus 6M detector (Dectris Ltd, Baden, Switzerland). A data set of 1800 images covering 360° of rotation was collected in the resolution range 26.07–0.7 Å. 54850 reflections were recorded in total, including 3303 systematic absences by space-group symmetry. Of these observed reflections, 5235 were unique including 549 systematic absences. The data were integrated using the software package XDS (Kabsch, 2010). and scaled with AIMLESS (Evans & Murshudov, 2013) implemented in the CCP4 suite (Winn et al., 2011). All non-H peptide atoms were detected by direct methods with the program SHELXS97 (Sheldrick, 2008) as the highest 25 peaks. Anisotropic refinement was performed without any constraints or restraints. All H atoms could be detected in a difference Fourier map and each one of their four parameters was freely refined. No cocrystallized solvent molecule could be detected. The hydrogen-bond distances to N atoms are in the range 0.835 (19)–1.0 (2) Å, with an average of 0.89 (2) Å, and to C atoms are in the range 0.895 (17)–1.07 (3) Å, with an average of 0.94 (2) Å.

S3. Results and discussion

The tetra­peptide ester H-Gly-Aib-Gly-Aib-OtBu adopts a very unusual backbone conformation (Table 2). In the molecule taken as the asymmetric unit, the torsion angles ψ (Gly1) and φ/ψ (Aib2) lie in the right-handed helical region of the Ramachandran plot. In residues Gly3 and Aib4, the conformation is turned to lie in the left-handed helical region. There exist no intra­molecular hydrogen bonds, even though a hydrogen bond of type 1←4 would be possible between the C═O group of Gly1 and the N—H group of Aib4. The peptide adopts the usual trans-planar conformation, with significant deviations from planarity (ω = 180°, Table 2). The valency around the C_α atom is asymmetric for the Aib residues (Table 3). If one designates as C_L and C_R the atoms which occupy the same position as C_β and α-hydrogen in L-amino acids, respectively, the bond angles N—C_α—C_L and C—C_α—C_L are significantly smaller than the respective N—C_α—C_R and C—C_α—C_R angles in Aib2, while they are significantly greater in Aib4 (Table 3). This is in excellent agreement with the findings for other Aib residues with torsion angles φ/ψ in the right-handed 3₁₀-helical regions, which is the case of Aib2 and for other Aib residues with torsion angles φ/ψ in the left-handed 3₁₀-helical regions of the Ramachabdran plot, which hold true for the Aib4 residue (Table 2) (Gessmann et al., 2014). In the crystal, there is a network of hydrogen bonds between each molecule and the six surrounding molecules, forming a total of eight hydrogen bonds (Fig. 2). The four hydrogen bonds in which the chosen central molecule acts as hydrogen-bond donor are listed in Table 4. In the other four hydrogen bonds, the central molecule is a hydrogen-bond acceptor from the molecules which are symmetry related by (x − 1/2, y, −z + 1/2), (−x + 1, y + 1/2, −z + 1/2) and (−x + 3/2, y + 1/2, z). These hydrogen bonds connect molecules of both handednesses in the a and b directions, while a pairwise connection is established in the c direction (Fig. 3), leading to hydrogen-bonded slices parallel to the ab plane which stack in the c direction via apolar contacts. The recently determined structure of Z-Gly-Aib-Gly-Aib-OtBu (Gessmann et al., 2015) possesses instead of the free N-terminus of the title compound a benzyl­oxycarbonyl protecting group. This difference leads to two 1←4 intra­molecular hydrogen bonds, a semi-extended conformation for Gly1 and left- or right-handed 3₁₀-helical values for the Aib2, Gly3 and Aib4. Another difference between these two peptide structures is that in the case of Z-Gly-Aib-Gly-Aib-OtBu the crystallization agent, ethyl acetate, was cocrystallized along with the peptide. Among the peptide structures examined, N-terminal-unprotected Tyr-Aib-Tyr-Val (Das et al., 2005) is the only Aib-containing short peptide, which could form one intra­molecular 4→1 hydrogen bond based on its length. Nevertheless, no hydrogen bond was found in this tetra­peptide.

Computing details

Cell refinement: XDS (Kabsch, 2010); data reduction: AIMLESS (Evans & Murshudov, 2013); program(s) used to solve structure: SHELXS86 (Sheldrick, 2008); program(s) used to refine structure: SHELXL2014 (Sheldrick, 2015); molecular graphics: XTALVIEW (McRee, 1999) and SwissPDBViewer (Guex & Peitsch, 1997); software used to prepare material for publication: CHEMDRAW (Mills, 2006), ORTEPIII (Burnett & Johnson, 1996), ORTEP-3 for Windows (Farrugia, 2012) and POVRAY (Persistence of Vision, 2004).

Figures

	Fig. 1. The molecular structure of H-Gly-Aib-Gly-Aib-OtBu, showing 50% probability displacement ellipsoids. The atom names can be constructed by adding the residue number in each case, e.g. N of the GLY3 residue has the atom label N_3.
	Fig. 2. Intermolecular hydrogen bonding of H-Gly-Aib-Gly-Aib-OtBu. Atom colours in the central reference molecule have been chosen according to the element type, whereas hydrogen-bonded molecules have been assigned arbitrary colours. H atoms have been omitted for clarity.
	Fig. 3. Crystal packing of H-Gly-Aib-Gly-Aib-OtBu, viewed down the a axis. The molecules with the handedness chosen as asymmetric unit are shown in different shades of red and the molecules with the opposite handedness in shades of blue. H atoms have been omitted for clarity.

Glycyl-α-aminoisobutyryl-glycyl-α-aminoisobutyric acid tert-butyl ester

Crystal data

C16H30N4O5	Dx = 1.225 Mg m^−3
Mr = 358.44	Synchrotron radiation, λ = 0.65253 Å
Orthorhombic, Pbca	Cell parameters from 4686 reflections
a = 9.3500 (19) Å	θ = 1.4–27.7°
b = 15.950 (3) Å	µ = 0.05 mm^−1
c = 26.070 (5) Å	T = 100 K
V = 3887.9 (14) Å^3	Plate, colourless
Z = 8	0.08 × 0.07 × 0.04 mm
F(000) = 1552

Data collection

Synchrotron diffractometer	Rint = 0.099
f–scans	θmax = 27.7°, θmin = 1.4°
51547 measured reflections	h = −11→11
4686 independent reflections	k = −22→21
4379 reflections with I > 2σ(I)	l = −36→37

Refinement

Refinement on F^2	0 restraints
Least-squares matrix: full	Hydrogen site location: difference Fourier map
R[F^2 > 2σ(F^2)] = 0.059	All H-atom parameters refined
wR(F^2) = 0.151	w = 1/[σ^2(Fo^2) + (0.0955P)^2 + 1.0581P] where P = (Fo^2 + 2Fc^2)/3
S = 1.11	(Δ/σ)max < 0.001
4686 reflections	Δρmax = 0.49 e Å^−3
346 parameters	Δρmin = −0.51 e Å^−3

Special details

Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes.

Refinement. One single plate with the smallest dimension of about 40 µm was mounted on a cryoloop without cryoprotectant and kept in place with a minimal amount of vacuum grease. Diffraction data were collected at 100 K on the microfocus beamline I24 (Evans et al., 2011) of the Diamond Light Source in Didcot, England, using a Pilatus 6M detector (Dectris Ltd, Baden, Switzerland). A data set of 1800 images covering 360° of rotation was collected in the resolution range 26.07–0.7 Å. 54850 reflections were recorded in total, including 3303 systematic absences by space-group symmetry. Of these observed reflections, 5235 were unique including 549 systematic absences. The data were integrated using the software package XDS (Kabsch, 2010). and scaled with AIMLESS (Evans & Murshudov, 2013) implemented in the CCP4 suite (Winn et al., 2011). All non-H peptide atoms were detected by direct methods with the program SHELXS97 (Sheldrick, 2008) as the highest 25 peaks. Anisotropic refinement was performed without any constraints or restraints. All H atoms could be detected in a difference Fourier map and each one of their four parameters was freely refined. No cocrystallized solvent molecule could be detected. The hydrogen-bond distances to N atoms are in the range 0.835 (19)–1.0 (2) Å, with an average of 0.89 (2) Å, and to C atoms are in the range 0.895 (17)–1.07 (3) Å, with an average of 0.94 (2) Å.

Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Ų)

	x	y	z	Uiso*/Ueq
N_1	0.61076 (13)	−0.26950 (7)	0.20859 (5)	0.0230 (3)
H1_1	0.591 (2)	−0.3237 (15)	0.1909 (9)	0.039 (5)*
H2_1	0.703 (3)	−0.2496 (15)	0.2023 (9)	0.046 (6)*
CA_1	0.51123 (13)	−0.20546 (8)	0.19305 (5)	0.0196 (3)
HA1_1	0.540 (2)	−0.1719 (12)	0.1617 (8)	0.027 (4)*
HA2_1	0.423 (2)	−0.2313 (13)	0.1823 (8)	0.029 (5)*
C_1	0.47834 (12)	−0.14172 (7)	0.23400 (5)	0.0156 (2)
O_1	0.40238 (9)	−0.08063 (6)	0.22329 (4)	0.0203 (2)
N_2	0.53398 (11)	−0.15262 (6)	0.28108 (4)	0.0158 (2)
H_2	0.579 (2)	−0.1977 (14)	0.2880 (8)	0.034 (5)*
CA_2	0.50131 (12)	−0.09512 (7)	0.32365 (5)	0.0151 (2)
CL_2	0.60190 (14)	−0.11646 (8)	0.36828 (5)	0.0205 (3)
HL1_2	0.5870 (16)	−0.0789 (10)	0.3932 (6)	0.012 (3)*
HL2_2	0.5806 (19)	−0.1711 (12)	0.3834 (7)	0.024 (4)*
HL3_2	0.702 (2)	−0.1169 (12)	0.3564 (7)	0.025 (4)*
CR_2	0.34621 (13)	−0.10395 (9)	0.34067 (5)	0.0208 (3)
HR1_2	0.3253 (18)	−0.0618 (12)	0.3682 (7)	0.023 (4)*
HR2_2	0.334 (2)	−0.1593 (13)	0.3527 (7)	0.026 (4)*
HR3_2	0.281 (2)	−0.0922 (12)	0.3125 (7)	0.025 (4)*
C_2	0.53213 (12)	−0.00347 (7)	0.30872 (4)	0.0137 (2)
O_2	0.45775 (10)	0.05424 (6)	0.32470 (4)	0.0201 (2)
N_3	0.65058 (10)	0.00881 (6)	0.28071 (4)	0.0150 (2)
H_3	0.7018 (19)	−0.0322 (12)	0.2731 (7)	0.020 (4)*
CA_3	0.70985 (13)	0.09161 (7)	0.27268 (5)	0.0153 (2)
HA1_3	0.7652 (18)	0.0899 (10)	0.2434 (7)	0.017 (4)*
HA2_3	0.6353 (19)	0.1327 (11)	0.2657 (7)	0.019 (4)*
C_3	0.79998 (12)	0.12386 (7)	0.31742 (4)	0.0145 (2)
O_3	0.80326 (10)	0.19905 (6)	0.32858 (4)	0.0190 (2)
N_4	0.87403 (11)	0.06554 (7)	0.34303 (4)	0.0170 (2)
H_4	0.8780 (19)	0.0165 (13)	0.3313 (7)	0.021 (4)*
CA_4	0.96427 (13)	0.08592 (8)	0.38700 (5)	0.0185 (3)
CL_4	1.10210 (14)	0.12845 (11)	0.36957 (6)	0.0271 (3)
HL1_4	1.080 (2)	0.1774 (15)	0.3505 (8)	0.036 (5)*
HL2_4	1.156 (2)	0.0906 (13)	0.3470 (7)	0.028 (5)*
HL3_4	1.161 (2)	0.1461 (14)	0.3985 (9)	0.043 (6)*
CR_4	0.99587 (17)	0.00400 (10)	0.41545 (6)	0.0286 (3)
HR1_4	1.043 (2)	−0.0383 (15)	0.3936 (9)	0.039 (5)*
HR2_4	0.911 (2)	−0.0231 (13)	0.4275 (8)	0.031 (5)*
HR3_4	1.053 (2)	0.0187 (15)	0.4461 (9)	0.043 (6)*
C_4	0.88318 (13)	0.14365 (8)	0.42425 (5)	0.0188 (3)
O_4	0.94003 (11)	0.20061 (7)	0.44673 (4)	0.0266 (2)
O_5	0.74945 (10)	0.11792 (6)	0.43058 (4)	0.0221 (2)
C1_5	0.65102 (15)	0.16443 (10)	0.46480 (5)	0.0256 (3)
C2_5	0.64146 (19)	0.25583 (12)	0.44851 (6)	0.0348 (4)
H21_5	0.620 (3)	0.2558 (16)	0.4135 (11)	0.051 (6)*
H22_5	0.566 (3)	0.2812 (16)	0.4657 (10)	0.053 (7)*
H23_5	0.737 (3)	0.2887 (16)	0.4550 (9)	0.050 (6)*
C3_5	0.69906 (19)	0.15469 (13)	0.51999 (6)	0.0350 (4)
H31_5	0.708 (2)	0.0922 (14)	0.5293 (8)	0.036 (5)*
H32_5	0.630 (3)	0.1817 (16)	0.5444 (9)	0.047 (6)*
H33_5	0.785 (3)	0.1844 (16)	0.5253 (9)	0.047 (6)*
C4_5	0.51065 (18)	0.11932 (16)	0.45571 (8)	0.0458 (5)
H41_5	0.521 (3)	0.0577 (18)	0.4651 (10)	0.051 (7)*
H42_5	0.483 (3)	0.1303 (19)	0.4164 (12)	0.066 (8)*
H43_5	0.433 (3)	0.1429 (18)	0.4794 (11)	0.059 (7)*

Atomic displacement parameters (Ų)

	U^11	U^22	U^33	U^12	U^13	U^23
N_1	0.0265 (6)	0.0154 (5)	0.0272 (6)	0.0029 (4)	−0.0038 (4)	−0.0011 (5)
CA_1	0.0197 (6)	0.0158 (6)	0.0233 (6)	−0.0007 (4)	−0.0049 (4)	−0.0001 (5)
C_1	0.0117 (5)	0.0124 (5)	0.0226 (6)	−0.0029 (4)	−0.0011 (4)	0.0002 (5)
O_1	0.0169 (5)	0.0195 (4)	0.0244 (5)	0.0031 (3)	−0.0041 (3)	0.0025 (4)
N_2	0.0154 (5)	0.0096 (4)	0.0224 (5)	0.0022 (3)	−0.0017 (3)	0.0004 (4)
CA_2	0.0137 (5)	0.0130 (5)	0.0187 (5)	0.0009 (4)	−0.0004 (4)	0.0020 (5)
CL_2	0.0234 (6)	0.0182 (6)	0.0200 (6)	0.0008 (4)	−0.0058 (4)	0.0031 (5)
CR_2	0.0165 (6)	0.0221 (6)	0.0237 (6)	−0.0028 (4)	0.0037 (4)	0.0043 (5)
C_2	0.0136 (5)	0.0116 (5)	0.0159 (5)	0.0011 (4)	−0.0026 (4)	0.0004 (4)
O_2	0.0198 (4)	0.0155 (4)	0.0251 (5)	0.0058 (3)	0.0040 (3)	0.0007 (4)
N_3	0.0134 (5)	0.0102 (4)	0.0214 (5)	−0.0004 (3)	0.0012 (3)	−0.0020 (4)
CA_3	0.0168 (6)	0.0121 (5)	0.0170 (5)	−0.0023 (4)	0.0002 (4)	0.0006 (4)
C_3	0.0136 (5)	0.0139 (5)	0.0161 (5)	−0.0019 (4)	0.0037 (4)	−0.0007 (4)
O_3	0.0222 (4)	0.0123 (4)	0.0224 (4)	−0.0018 (3)	−0.0006 (3)	−0.0011 (3)
N_4	0.0186 (5)	0.0136 (5)	0.0187 (5)	0.0017 (3)	−0.0013 (3)	−0.0042 (4)
CA_4	0.0156 (5)	0.0217 (6)	0.0182 (5)	0.0020 (4)	−0.0009 (4)	−0.0024 (5)
CL_4	0.0153 (6)	0.0370 (8)	0.0290 (7)	−0.0017 (5)	0.0023 (5)	−0.0064 (7)
CR_4	0.0297 (7)	0.0278 (7)	0.0283 (7)	0.0091 (5)	−0.0065 (5)	0.0011 (6)
C_4	0.0172 (6)	0.0231 (6)	0.0161 (5)	0.0016 (4)	0.0000 (4)	0.0004 (5)
O_4	0.0247 (5)	0.0317 (6)	0.0236 (5)	−0.0020 (4)	−0.0024 (4)	−0.0104 (4)
O_5	0.0180 (5)	0.0277 (5)	0.0207 (4)	−0.0011 (3)	0.0052 (3)	−0.0032 (4)
C1_5	0.0219 (6)	0.0356 (8)	0.0192 (6)	0.0052 (5)	0.0076 (5)	0.0000 (6)
C2_5	0.0377 (9)	0.0408 (9)	0.0259 (7)	0.0173 (7)	0.0115 (6)	0.0066 (7)
C3_5	0.0386 (9)	0.0481 (10)	0.0185 (6)	0.0122 (7)	0.0061 (5)	0.0050 (7)
C4_5	0.0238 (8)	0.0686 (14)	0.0449 (10)	−0.0061 (7)	0.0141 (7)	−0.0090 (10)

Geometric parameters (Å, º)

N_1—CA_1	1.4400 (16)	C_3—N_4	1.3382 (16)
N_1—H1_1	1.00 (2)	N_4—CA_4	1.4599 (16)
N_1—H2_1	0.93 (2)	N_4—H_4	0.84 (2)
CA_1—C_1	1.5060 (18)	CA_4—CR_4	1.5313 (19)
CA_1—HA1_1	1.01 (2)	CA_4—CL_4	1.5256 (18)
CA_1—HA2_1	0.97 (2)	CA_4—C_4	1.5380 (17)
C_1—O_1	1.2377 (15)	CL_4—HL1_4	0.95 (2)
C_1—N_2	1.3443 (16)	CL_4—HL2_4	0.98 (2)
N_2—CA_2	1.4717 (16)	CL_4—HL3_4	0.98 (2)
N_2—H_2	0.85 (2)	CR_4—HR1_4	0.99 (2)
CA_2—CR_2	1.5231 (16)	CR_4—HR2_4	0.96 (2)
CA_2—CL_2	1.5344 (17)	CR_4—HR3_4	0.99 (2)
CA_2—C_2	1.5400 (16)	C_4—O_4	1.2048 (17)
CL_2—HL1_2	0.895 (17)	C_4—O_5	1.3263 (16)
CL_2—HL2_2	0.98 (2)	O_5—C1_5	1.4810 (16)
CL_2—HL3_2	0.982 (19)	C1_5—C3_5	1.515 (2)
CR_2—HR1_2	1.003 (19)	C1_5—C4_5	1.515 (2)
CR_2—HR2_2	0.94 (2)	C1_5—C2_5	1.521 (2)
CR_2—HR3_2	0.972 (19)	C2_5—H21_5	0.94 (3)
C_2—O_2	1.2267 (14)	C2_5—H22_5	0.93 (3)
C_2—N_3	1.3409 (15)	C2_5—H23_5	1.05 (3)
N_3—CA_3	1.4475 (15)	C3_5—H31_5	1.03 (2)
N_3—H_3	0.835 (19)	C3_5—H32_5	1.01 (2)
CA_3—C_3	1.5280 (16)	C3_5—H33_5	0.94 (3)
CA_3—HA1_3	0.923 (17)	C4_5—H41_5	1.02 (3)
CA_3—HA2_3	0.974 (18)	C4_5—H42_5	1.07 (3)
C_3—O_3	1.2343 (15)	C4_5—H43_5	1.02 (3)
CA_1—N_1—H1_1	111.6 (13)	O_3—C_3—N_4	123.02 (11)
CA_1—N_1—H2_1	107.8 (15)	O_3—C_3—CA_3	121.37 (11)
H1_1—N_1—H2_1	112 (2)	N_4—C_3—CA_3	115.61 (10)
N_1—CA_1—C_1	114.29 (11)	C_3—N_4—CA_4	122.41 (11)
N_1—CA_1—HA1_1	115.5 (11)	C_3—N_4—H_4	119.2 (12)
C_1—CA_1—HA1_1	105.6 (11)	CA_4—N_4—H_4	117.9 (12)
N_1—CA_1—HA2_1	109.4 (12)	CR_4—CA_4—CL_4	111.14 (11)
C_1—CA_1—HA2_1	108.6 (12)	N_4—CA_4—C_4	110.13 (10)
HA1_1—CA_1—HA2_1	102.6 (16)	CA_4—CL_4—HL1_4	109.8 (13)
O_1—C_1—N_2	122.00 (11)	CA_4—CL_4—HL2_4	110.0 (12)
O_1—C_1—CA_1	119.25 (11)	HL1_4—CL_4—HL2_4	107.7 (17)
N_2—C_1—CA_1	118.75 (10)	CA_4—CL_4—HL3_4	112.0 (14)
C_1—N_2—CA_2	121.84 (10)	HL1_4—CL_4—HL3_4	106.9 (19)
C_1—N_2—H_2	119.6 (14)	HL2_4—CL_4—HL3_4	110.3 (18)
CA_2—N_2—H_2	117.9 (14)	CA_4—CR_4—HR1_4	112.9 (13)
N_2—CA_2—CL_2	107.84 (10)	CA_4—CR_4—HR2_4	112.5 (12)
C_2—CA_2—CL_2	106.71 (10)	HR1_4—CR_4—HR2_4	104.5 (18)
N_4—CA_4—CL_4	110.70 (11)	CA_4—CR_4—HR3_4	107.0 (14)
C_4—CA_4—CL_4	109.78 (11)	HR1_4—CR_4—HR3_4	112.7 (19)
N_2—CA_2—CR_2	111.08 (10)	HR2_4—CR_4—HR3_4	107.0 (18)
C_2—CA_2—CR_2	109.85 (10)	O_4—C_4—O_5	126.07 (12)
N_4—CA_4—CR_4	107.56 (11)	O_4—C_4—CA_4	122.76 (11)
C_4—CA_4—CR_4	107.46 (11)	O_5—C_4—CA_4	110.98 (11)
CR_2—CA_2—CL_2	110.01 (10)	C_4—O_5—C1_5	120.39 (11)
N_2—CA_2—C_2	111.23 (10)	O_5—C1_5—C3_5	109.66 (11)
CA_2—CL_2—HL1_2	107.9 (10)	O_5—C1_5—C4_5	101.90 (12)
CA_2—CL_2—HL2_2	112.3 (11)	C3_5—C1_5—C4_5	110.89 (14)
HL1_2—CL_2—HL2_2	105.7 (15)	O_5—C1_5—C2_5	110.38 (11)
CA_2—CL_2—HL3_2	110.2 (11)	C3_5—C1_5—C2_5	112.38 (14)
HL1_2—CL_2—HL3_2	112.5 (15)	C4_5—C1_5—C2_5	111.13 (15)
HL2_2—CL_2—HL3_2	108.3 (15)	C1_5—C2_5—H21_5	106.6 (16)
CA_2—CR_2—HR1_2	109.4 (10)	C1_5—C2_5—H22_5	109.1 (16)
CA_2—CR_2—HR2_2	107.3 (11)	H21_5—C2_5—H22_5	108 (2)
HR1_2—CR_2—HR2_2	111.5 (16)	C1_5—C2_5—H23_5	112.5 (14)
CA_2—CR_2—HR3_2	111.0 (11)	H21_5—C2_5—H23_5	110 (2)
HR1_2—CR_2—HR3_2	106.8 (15)	H22_5—C2_5—H23_5	111 (2)
HR2_2—CR_2—HR3_2	110.9 (16)	C1_5—C3_5—H31_5	110.2 (12)
O_2—C_2—N_3	122.93 (11)	C1_5—C3_5—H32_5	111.4 (14)
O_2—C_2—CA_2	121.36 (10)	H31_5—C3_5—H32_5	108.5 (18)
N_3—C_2—CA_2	115.52 (10)	C1_5—C3_5—H33_5	109.9 (15)
C_2—N_3—CA_3	121.89 (10)	H31_5—C3_5—H33_5	112.5 (19)
C_2—N_3—H_3	119.2 (12)	H32_5—C3_5—H33_5	104 (2)
CA_3—N_3—H_3	117.5 (12)	C1_5—C4_5—H41_5	109.7 (14)
N_3—CA_3—C_3	114.07 (10)	C1_5—C4_5—H42_5	106.4 (16)
N_3—CA_3—HA1_3	107.9 (10)	H41_5—C4_5—H42_5	114 (2)
C_3—CA_3—HA1_3	109.4 (10)	C1_5—C4_5—H43_5	110.0 (16)
N_3—CA_3—HA2_3	111.5 (10)	H41_5—C4_5—H43_5	106 (2)
C_3—CA_3—HA2_3	108.1 (10)	H42_5—C4_5—H43_5	110 (2)
HA1_3—CA_3—HA2_3	105.5 (15)

Hydrogen-bond geometry (Å, º)

D—H···A	D—H	H···A	D···A	D—H···A
N_1—H_1···O_2^i	1.00 (2)	2.04 (2)	3.0111 (16)	164 (2)
N_2—H_2···O_3^ii	0.85 (2)	2.25 (2)	3.0735 (14)	164 (2)
N_3—H_3···O_1^iii	0.84 (2)	2.030 (19)	2.7548 (14)	145 (2)
N_4—H_4···O_1^iii	0.84 (2)	2.12 (2)	2.9146 (15)	158 (2)

Symmetry codes: (i) −x+1, y−1/2, −z+1/2; (ii) −x+3/2, y−1/2, z; (iii) x+1/2, y, −z+1/2.