Conformational properties of oxazoline-amino acids

Journal of Molecular Structure 1109 (2016) 192e200

Contents lists available at ScienceDirect

Journal of Molecular Structure journal homepage: http://www.elsevier.com/locate/molstruc

Conformational properties of oxazoline-amino acids Monika Sta
s, Małgorzata A. Broda, Dawid Siodłak* Faculty of Chemistry, University of Opole, Opole 45-052, Poland

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 September 2015 Received in revised form 17 December 2015 Accepted 2 January 2016 Available online 6 January 2016

Oxazoline-amino acids (Xaa-Ozn) occur in natural peptides of potentially important bioactivity. The conformations of the model compounds: Ac-(S)-Ala-Ozn(4R-Me), Ac-(S)-Ala-Ozn(4SeMe), and (gaucheþ, gauche, anti) Ac-(S)-Val-Ozn(4R-Me) were studied at meta-hybrid M06-2X/6e311þþG(d,p) method including solvent effect. Boc-L-Ala-L-Ozn-4-COOMe and Boc-L-Val-L-Ozn-4-COOMe were synthesized and studied by FT-IR and NMR-NOE methods. The conformations in crystal state were gathered from the Cambridge Structural Data Base. The main conformational feature of the oxazoline amino acids is the conformation b2 (f,j ~ 161, 6 ), which predominates in weakly polar environment and still is accessible in polar surrounding. The changes of the conformational preferences towards the conformations aR (f,j ~ 70 , 15 ) and then b (f,j ~ 57, 155 ) are observed with increase of the environment polarity. © 2016 Elsevier B.V. All rights reserved.

Keywords: Oxazoline Non-standard amino acids Ramachandran map Conformational analysis DFT

1. Introduction Non-standard amino acids residues containing heterocyclic rings can be found in numerous macrocyclic compounds produced by microorganisms [1,2]. Oxazoline-amino acids, Xaa-Ozn (Fig. 1), in which the oxazoline ring is placed between the a-carbon atom and C-terminal amide bond, are constituents of cyclamides [3] and cyanobactins [4] isolated mainly from ascidian or sponges [5]. These peptides exhibit significant biological activities e.g. cytotoxicity [6] or activity against the malaria parasites [7,8]. Literature survey reveals that in nature exist the following oxazoline-amino acids: alanine [3,9e11], valine [11e17], isoleucine [10,13,14,18,19], leucine [13], phenylalanine [3,11,20e24], histidine [14], and cysteine [13]. Non-standard amino acid residues as well as main chain modification, e.g. introduction of N-methyldehydroamino acids [25] or ester instead of amide [26e28], usually results in specific properties, which have influence on adopted conformation, and in a consequence, on bioactivity of peptides. The conformational properties of the oxazoline-amino acids were not investigated to date. The oxazoline-amino acids are closely related to the recently studied oxazole-amino acids [29]. In nature, the oxazole ring is created by microorganism in post-translational modifications from serine or threonine, and oxazoline ring is intermediate product of

* Corresponding author. E-mail address: [email protected] (D. Siodłak). http://dx.doi.org/10.1016/j.molstruc.2016.01.001 0022-2860/© 2016 Elsevier B.V. All rights reserved.

this reaction [30]. The oxazoline ring has no aromatic character and contains asymmetric tetrahedral carbon atom. Thus, despite of structural similarity, it should show different features from oxazole. This study describes the conformational properties of oxazolinealanine and oxazoline-valine, the simplest and the most commonly occurring natural representatives of this group (Fig. 2). The results obtained using theoretical methods were supported by FTIR and NMR-NOE studies of the synthesized model compounds as well as analysis of the conformations found in the crystal state.

2. Experimental section 2.1. Theoretical calculations The conformational properties of the oxazoline-amino acid were calculated for the following molecules: Ac-(S)-Ala-Ozn(4RMe) (1), Ac-(S)-Ala-Ozn(4SeMe) (2), and (gaucheþ, gauche, anti) Ac-(S)-Val-Ozn(4R-Me) (3e5). Only proteinogenic S-amino acids were selected (denoted as L according the Fisher convention). In case of oxazoline ring, the change in notation should be explained. In the studied model compounds (1e5), the C-terminal part of the main chain at the oxazoline ring is mimicked by methyl group. This influences the order of numbering of substituents at the chiral centre, and thus, the proper position in space is denoted as R. However, the influence of both optic isomers R and S of the oxazoline ring was considered. In case of valine, the rotation of the side chain around the Ca-Cb bond was described by three most adopted

M. Sta
s et al. / Journal of Molecular Structure 1109 (2016) 192e200

Fig. 1. Schematic formula of oxazoline-amino acids.

configuration anti, gauchee and gaucheþ according to Newman projection [31]. The torsion angle c was defined as Ha-Ca-Cb-Hb according to ref. [31]. The Gaussian 09 Package was used [32]. Calculations were performed on the trans-amide bond (u0 z 180 ). The torsion angles 4, j potential energy surfaces of Ac-(S)-Ala-

193

Ozn(4SeMe) were created on the basis of 169 points calculated at meta-hybrid M06-2X/6e311þþG(d,p) method [33] with a 30 increment for the 4, j main-chain dihedral angle, within a range from 180 to 180 . In each of the calculated structures the geometrical parameters were fully relaxed, expect for the constrained torsion angles 4 and j. To estimate the solvation effects on the conformations, single-point calculations were also conducted in each grid point using a self-consistent reaction field (SCRF), the SMD method was used following its successful application to the 4metoxyproline diamide model [34,35]. The presence of chloroform and water were mimicked. The energy surfaces were obtained using the Surfer8 program [36] with the radial basis function as a gridding method. Possible energy minima of every low-energy region on the map were fully optimized at the M06-2X/ 6e311þþG(d,p) using SMD model. Frequency analyses was carried out to verify the nature of the minimum state of all stationary points obtained and calculate the zero-point vibrational energies (ZPVEs). Minima for other studied compounds were calculated on the basis of the maps for Ac-(S)-Ala-Ozn(4R-Me). The kind and number of atoms were modified using standard GaussView tools, and then, the obtained structures were subjected for full geometry optimization. According literature [37,38] expected population (prel ¼ pany conformer/pglobal minimum) of the conformers at a temperature of 300 K (where RT ¼ 0.595371 kcal/mol) was calculated Eg (1): prel ¼ 100%exp(-DE/RT). The conformational composition P was estimated using Eq. (2): p ¼ prel/ conformersprel. 2.2. Synthesis

Fig. 2. General formula and the Newmann projections for the model compounds (1e7) studied in this work.

N-tert-Butoxycarbonyl-L-alanine (Boc-L-Ala-OH) was applied as purchased (Fluka). N-tert-Butoxycarbonyl-L-valine (Boc-L-Val-OH) was synthesized according to the ref. [39]. The corresponding dipeptides: Boc-L-Ala-L-Ser-OMe and Boc-L-Val-L-Ser-OMe were obtained by coupling with serine methyl ester hydrochloride using N,N’-dicyclohexylcarbodiimide [40]. The studied oxazoline compounds were synthesized on the basis of the general procedure described by Philips et al. [41]. N-tert-Butoxycarbonyl-L-valine (Boc-L-Val-OH) C10H19NO4 (217.26). Yield 90%, oil. 1H NMR (400 MHz, CDCl3) d 8.86 (1H, s, COOeH), 5.05 (1H, d, J ¼ 9.2, NeH), 4.28 (1H, q, J ¼ 8.4, 4.8, Ca-H), 2.24 (1H, m, J ¼ 6.4, 6.8, Cb-H), 1.45 (9H, s, t-Bu), 1.01 (3H, d, J ¼ 2.8, CgH3), 0.98 (3H, m, CgH3). 13C NMR (400 MHz, CDCl3) d 177.19 (s), 155.83 (s), 80.07 (s), 58.39 (s), 34.65 (s), 34.52 (s), 31.59 (s), 31.06 (d), 28.29 (t), 26.90 (s), 25.26 (s), 22.65 (s), 20.69 (s), 19.04 (s), 17.43 (s), 14.13 (s). N-(tert-Butoxycarbonyl)-L-alanyl-L-serine methyl ester (Boc-LAla-L-Ser-OMe) C11H22N2O5; (262.30). Yield 80%, white oil. 1H NMR (400 MHz, DMSO-d6) d 8.03 (1H, dd, J ¼ 7.6, 8.0, NeH), 7.00 (1H, q, J ¼ 8.0, 7.2, 7.6, NeH), 5.10 (1H, q, J ¼ 5.6, 6.0, Ca-H (Ser)), 4.36 (1H, m, Ca-H (Ala)), 4.06 (1H, m, OeH), 3.75 (2H, m, Cb-H2), 3.63 (3H, s, OeCH3), 1.38 (9H, s, t-Bu), 1.20 (3H, m, Cb-H3). N-(tert-Butoxycarbonyl)-L-valinyl-L-serine methyl ester (Boc-LVal-L-Ser-OMe) C14H26N2O6 (318.37). Yield 50%, white oil. 1H NMR (400 MHz, CDCl3) d 7.00 (1H, d, J ¼ 4, NeH), 5.28 (1H, d, J ¼ 8.4, NeH), 4.69 (1H, t, J ¼ 4.0, 3.6, OeH), 3.96 (2H, m, Cb-H2), 3.93 (1H, m, (Val) Ca-H), 3.79 (3H, s, OeCH3), 2.11 (2H, m, Cb-H, (Ser) Ca-H), 1.44 (9H, s, t-Bu), 1.00 (6H, dd, J ¼ 6.8, 4.4, (CgH3)2). 1H NMR (400 MHz, DMSO-d6) d 8.15 (1H, d, J ¼ 7.2, NeH), 6.69 (1H, d, J ¼ 9.2, NeH), 5.06 (1H, t, J ¼ 5.6, Ca-H (Ser)), 4.37 (1H, m, Ca-H (Val)), 3.91 (1H, m, OeH), 3.73 (1H, m, Cb-H2), 3.64 (4H, m, OeCH3, Cb-H2), 1.99 (1H, m, Cb-H), 1.38 (9H, s, t-Bu), 0.87 (6H, dd, J ¼ 6.8, 13.2, (CgH3)2). 13 C NMR (400 MHz, CDCl3) d 172.11 (s), 170.77 (s), 156.28 (s), 80.26 (s), 62.73 (s), 60.36 (s), 54.61 (s), 52.69 (s), 30.82 (s), 28.28 (s), 19.18 (s), 18.08 (s). Methyl (S,S)-2-[1-(((tert-butoxy)carbonyl)amino)et-1-yl]-1,3-

194

M. Sta
s et al. / Journal of Molecular Structure 1109 (2016) 192e200

oxazoline-4-carboxylate (Boc-L-Ala-L-Ozn-4-COOMe) (6) C12H20N2O5 (272.29). Yield 62%, yellow oil, purity 93% (HPLC). 1H NMR (400 MHz, CDCl3) d 5.28 (1H, d, NeH), 4.79 (1H, m, Ozn-H), 4.59 (2H, m, Ozn-CH2), 4.51 (1H, m, Ca-H), 3.80 (3H, s, OCH3), 1.44 (13H, m, t-Bu, CbH3). 1H NMR (400 MHz, DMSO-d6) d 7.26 (1H, d, J ¼ 8.4, NeH), 4.75 (1H, m, Ozn-H), 4.45 (2H, m, Oxn-CH2), 4.27 (1H, m, Ca-H), 3.67 (3H, s, OCH3), 1.38 (9H, s, t-Bu), 1.25 (3H, d, J ¼ 7.2, CbH3). 13C NMR (400 MHz, CDCl3) d 171.28 (s), 171,20 (s), 154.91 (s), 79.76 (s), 70.22 (d), 67.77 (d), 52.75 (s), 44.75 (d), 28.31 (s), 19.56 (d). 13C NMR (400 MHz, DMSO-d6) d 171.78 (d), 170.65 (d), 155.31 (s), 78.48 (s), 70.09 (d), 67.94 (s), 52.58 (s), 44.45 (d), 28.64 (d), 18.74 (d). Methyl (S,S)-2-[1-(((tert-butoxy)carbonyl)amino)-2-metylprop-1yl]-1,3-oxazoline-4-carboxy-late (Boc-L-Val-L-Ozn-4-COOMe) (7) C14H24N2O5 (300.35). Yield 25%, yellow syrup. 1H NMR (400 MHz, CDCl3) d 5.19 (1H, d, J ¼ 9.2, NeH), 4.79 (1H, m, Ozn-H), 4.56 (2H, m, Ozn-CH2), 4.38 (1H, m, CaH), 3.79 (3H, s, OCH3), 2.13 (1H, m, CbH3), 1.44 (9H, s, t-Bu), 0.98 (6H, dd, (CgH3)2). 13C NMR (400 MHz, CDCl3) d 171.46 (s), 170.11 (s), 155.50 (s), 79.64 (s), 70.08 (s), 67.66 (s), 53.64 (s), 52.69 (s), 31.73 (s), 28.28 (s), 18.75 (s), 17.22 (s).

bands were obtained by Fourier self-deconvolution technique and by means of the second derivative as an “initial guess”. To determine the number of component bands, the results of the quantummechanical calculations were also taken into account. Then, the accurate band positions were determined by the curve-fitting procedure with a mixed (Gauss-Lorentz) profile. 2.4. NMR spectroscopy The 1H and 13C NMR spectra were recorded in CDCl3 or DMSO-d6 solution, with internal TMS standard, on Bruker NMR Spectrometer Ultrashield 400 MHz (2005) at room temperature. Data acquisition and processing were performed using standard Bruker TopSpin version 1.3 software. The assignment of the conformations was achieved using the NOE difference method (mixing time 300 ms) within the standard programs. The NOE spectra were recorded using 6000 scans. 3. Results and discussion 3.1. Theoretical calculations

2.3. FTIR spectroscopy The IR spectra were recorded at room temperature using a Nicolet Nexus spectrometer equipped with DTGS detector and flushed with dry nitrogen during the measurements. The thickness of the KBr liquid cell was 2.86 mm. The concentration was between 8.6 and 12.8  103 mol/L. All spectra were recorded at the 1 cm1 resolution and averaged using 256 scans. The solvent spectra obtained under the same conditions were subtracted from sample spectra. The spectra were analysed using GRAMS/A1 version 9 (Thermo Fisher Scientific). The number and position of component

3.1.1. Ac-(S)-Ala-Ozn(4R-Me) (1) Fig. 3 presents the Ramachandran diagrams calculated for Ac(S)-Ala-Ozn(4R-Me) (1). Seven energy minima were found, regardless of the simulated environment. The values of the torsion angles of the corresponding conformations with their relative energy are presented in Table 1. The energy order for the isolated molecules in gas phase is as follows: b2 < aR < C5 < b < aD < a’ < aL. The lowest in energy is the semi-extended conformation b2 (4, j ~ 161, 5 ) and the gap in energy DE to the second righthanded helical conformation aR (4, j ~ 78 , 10 ) equals

Fig. 3. Potential energy surfaces E ¼ f(4,j) and local minima for Ac-(S)-Ala-Ozn-(4R-Me) (1). Energy contours are plotted every 1 kcal/mol. Conformers calculated at the M062X/ 6e311þþG(d,p) in chloroform.

M. Sta
s et al. / Journal of Molecular Structure 1109 (2016) 192e200

195

Table 1 Selected Torsion Angles (deg) of Local Minima for Ac-(S)-Ala-Ozn(4R-Me) (1) and Ac-(S)-Ala-Ozn(4SeMe) (2) and their relative energies (kcal/mol). Ac-(S)-Ala-Ozn(4R-Me) (1) conformer

Ac-(S)-Ala-Ozn(4SeMe) (2)

4

j

E (hartrees)

DE

%

conformer

161.0 78.0 159.3 54.6 48.4 142.1 55.6

5.3 10.0 141.8 151.3 147.7 112.7 25.6

573.39373 573.39014 573.38897 573.38819 573.38767 573.38714 573.38707

0.00 2.25 2.99 3.48 3.80 4.14 4.18

96.5 2.2 0.6 0.3 0.2 0.1 0.1

b2 aR

160.1 74.0 56.4 152.0 50.4 108.7 54.4

4.7 10.2 149.1 136.3 146.1 118.2 45.3

573.41056 573.40869 573.40754 573.40734 573.40718 573.40708 573.40448

0.00 1.18 1.89 2.02 2.12 2.19 3.82

78.8 10.9 3.3 2.7 2.2 2.0 0.1

55.5 66.2 161.9 50.5 93.0 154.0 52.8

143.2 26.5 3.9 143.6 120.0 132.9 57.2

573.41620 573.41605 573.41514 573.41471 573.41468 573.41400 573.41318

0.00 0.09 0.67 0.93 0.95 1.38 1.89

36.7 31.3 12.0 7.6 7.4 3.6 1.5

Gas Phase

b2 aR C5

b aD a0 aL Chloroform b2 aR

b C5 aD

a0 aL

C5 aL

j

E (hartrees)

DE

%

161.1 69.6 159.9 57.0 48.3 141.2 58.5

5.9 15.3 141.7 155.4 146.5 109.8 20.2

573.39379 573.39028 573.38955 573.38822 573.38767 573.38766 573.38682

0.00 2.20 2.66 3.49 3.84 3.84 4.37

95.9 2.4 1.1 0.3 0.2 0.2 0.1

160.4 68.0 57.0 107.0 154.8 50.4 56.9

6.9 21.0 150.5 122.2 138.0 143.8 36.7

573.41003 573.40889 573.40818 573.40750 573.40746 573.40708 573.40496

0.00 0.72 1.16 1.59 1.62 1.86 3.19

61.5 18.4 8.7 4.3 4.1 2.7 0.3

66.0 55.5 49.0 159.6 159.3 93.1 54.3

27.0 143.1 141.1 10.4 134.3 123.9 53.7

573.41682 571.41677 573.41488 573.41487 573.41472 573.41376 573.41271

0.00 0.03 1.22 1.23 1.32 1.92 2.58

42.2 40.1 5.5 5.4 4.6 1.7 0.6

Gas Phase

Water

b aR b2 aD a0

4

2.3 kcal/mol. The energy difference increase gradually through the third fully-extended conformation C5 (4, j ~ 159 , 142 ), the bturn conformation b (4, j ~ 55 , 151 ), aD (4, j ~ 48 , 148 ), a’ (4, j ~ 142 , 113 ), and the left-handed helical conformation aL (4, j ~ 56 , 26 ). In the weakly polar environment, mimicked by chloroform, the energy difference between conformations is reduced, and the energy order is b2 < aR < b < C5 < aD < a’ < aL. As can be seen, the conformations b2 and aR maintain to be the lowest in energy. A considerably increase of the stability of the conformations b is observed. The geometry of the conformations are generally maintained within Df, Dj ~ ±7, except for the highest in energy conformation aL as well as the conformation a0 , which are placed in the shallow region of the map, where even considerable changes in torsion angles values occur at relatively small energy input. Increase the polarity of environment simulated by water results in changes in energy order as follows: b < aR < b2 < aD < a’ < C5 < aL. Two lowest conformations, b and aR, have almost the same energy. The energy difference further decreases. The geometrical changes are moderate, again the biggest changes are observable for the highest in energy conformations a0 and aL. In order to have confidence in the stability order of conformations, the calculations were also performed at MP2/6e311þþG(d,p) (Table 1S). As can be seen, the MP2 level of theory does not predict the conformations aD and a’. Also, the differences in relative energies are smaller than those predicted at M06-2X level. However, the energy order of the conformations predicted by both level of theory does not change, regardless of the environment simulated. 3.1.2. Ac-(S)-Ala-Ozn(4SeMe) (2) The influence of the optical isomer at the oxazoline ring on the conformational preferences was studied by analysis of Ac-(S)-AlaOzn(4SeMe) (2). As can be seen in Table 1 and Fig. 4, seven conformations are present, regardless of the environment simulated. In gas phase, the energy order is maintained as for (1). Differences in energy for analogous conformations of (1) and (2) do not exceed 0.3 kcal/mol. The geometry changes Df, Dj are within ±3 , except the conformations aR and aL. Simulation of the weakly polar

C5

b aD a0 aL Chloroform b2 aR

b a0 C5 aD aL Water aR

b aD b2 C5

a0 aL

environment by chloroform, maintains the conformation b2 as the lowest in energy, and the conformations aR and b as the second and third. Again, the differences in energy for analogous conformations of (1) and (2) do not exceed 0.6 kcal/mol and the geometry changes Df, Dj ~ ±3 , except the conformations aR and aL. For more polar water mimicked environment, the lowest in energy are the

Fig. 4. Conformers for Ac-(S)-Ala-Ozn(4SeMe) (2) calculated at the M062X/ 6e311þþG(d,p) in chloroform.

(Anti)-Ac-(S)-Val-Ozn(4R-Me) (3) conformer Gas Phase b2 aR C5

b aD aL

4

j

(Gauche-)-Ac-(S)-Val-Ozn(4R-Me) (4)

c

E (hartrees)

DE

%

33.3 32.9 127.6 145.5 143.7 21.2

174.5 173.5 177.9 177.0 159.9 156

652.05816 652.05590 652.05580 652.05304 652.05206 652.05107

1.53 2.95 3.01 4.75 5.36 5.98

75.4 7.0 6.3 0.3 0.1 0.0

134.0 63.8 132.1 129.7 53.4 56.1

35.9 40.0 122.5 88.7 142.3 53.0

173.9 174.0 175.5 178.3 159.0 164.2

652.07657 652.07618 652.07583 652.07384 652.07163 652.07040

1.09 1.34 1.55 2.80 4.19 4.96

45.8 30.3 21.1 2.6 0.3 0.1

61.7 137.1 67.8 138.0 136.0 60.3 58.1

42.7 116.8 127.9 39.5 87.9 66.8 139.9

175.2 175.2 174.4 174.8 176.9 168.0 156.5

652.08227 652.08169 652.08127 652.08016 652.07723 652.07587 652.07551

0.00 0.36 0.63 1.33 3.16 4.02 4.24

49.9 271 17.3 5.4 0.3 0.1 0.0

Chloroform

b2 aR C5

a0 aD aL Water

aR C5

b b2 a0 aL aD

Gas Phase b2 C5

b aD aL

4 137.6 137.5 63.1 36.3 39.5

Chloroform b2 136.4 C5 131.8 b 60.8 a0 121.4 aD 38.6 aL 40.7 Water b 58.6 b2 138.0 C5 136.7 aR 76.2 aD 42.9 0 a 126.8 aL 43.0

j

c

(Gaucheþ)-Ac-(S)-Val-Ozn(4R-Me) (5) E (hartrees)

DE

%

5.9 158.1 164.7 139.3 48.9

58.6 58.9 60.5 67.6 67.6

652.06061 652.05667 652.05408 652.05383 652.04976

0.00 2.47 4.10 4.25 6.81

98.3 1.6 0.1 0.1 0.0

4.9 154.4 157.4 144.1 140.8 52.4

58.5 62.0 57.0 68.5 67.3 67.9

652.07831 652.07610 652.07506 652.07376 652.07328 652.06980

0.00 1.39 2.04 2.85 3.16 5.34

87.5 8.5 2.9 0.7 0.4 0.0

152.4 3.0 151.0 8.0 140.8 136.6 63.3

53.8 57.2 57.5 55.8 67.3 69.2 70.9

652.08166 652.08132 652.08052 652.07992 652.07763 652.07702 652.07489

0.38 0.60 1.10 1.48 2.91 3.30 4.63

45.9 32.0 13.8 7.3 0.7 0.4 0.0

conformer Gas Phase b2 C5 aR

b aD aL

4

j

c

E (hartrees)

DE

%

157.7 157.0 63.8 55.7 35.6 38.0

8.4 151.6 29.9 154.2 136.4 63.6

76.2 69.1 56.5 73.0 63.0 57.0

652.05976 652.05650 652.05506 652.05403 652.05148 652.04804

0.53 2.58 3.48 4.13 5.73 7.89

96.0 3.1 0.7 0.2 0.0 0.0

10.4 144.3 150.7 106.5 137.8 63.2

75.4 65.8 71.4 56.8 63.3 58.6

652.07652 652.07510 652.07465 652.07108 652.07005 652.06702

1.12 2.01 2.301 4.54 5.18 7.08

73.1 16.4 10.2 0.2 0.1 0.0

145.3 34.9 139.6 7.8 138.1 73.4 96.2

67.2 62.0 65.0 76.7 62.1 55.0 57.5

652.08121 652.08034 652.07948 652.07894 652.07439 652.07352 652.07344

0.67 1.21 1.75 2.09 4.95 5.49 5.54

60.4 24.2 9.8 5.5 0.1 0.0 0.0

Chloroform b2 157.6 C5 152.9 b 56.0 a0 135.9 aD 38.0 aL 38.4 Water b 55.9 aR 63.3 C5 150.6 b2 157.9 aD 41.0 aL 41.4 a0 139.3

conformations aR and b, with the following conformations aD and b2. The energy differences are little higher, up to 0.6 kcal/mol, with the greatest changes in geometry for the conformation b2 and a0 , but do not exceed Df, Dj ~ ±6 .

3.1.3. Ac-(S)-Val-Ozn(4R-Me) (3e5) The influence of the bulky and asymmetric side chain on the conformational preferences was studied by analysis of Ac-(S)-ValOzn(4R-Me) (3e5). The results presented in Table 2 show that the types of the conformations are maintained as for alanine analogue (1), although their number is smaller for weakly polar environment

Fig. 5. Region nS(NeH) of FTIR spectra for the compounds Boc-L-Ala-L-Ozn-4-COOMe (6) and Boc-L-Val-L-Ozn-4-COOMe (7) recorded in CCl4 and CHCl3. The component band (dotted line) was obtained by a curve-fitting procedure.

M. Sta
s et al. / Journal of Molecular Structure 1109 (2016) 192e200

136.6 61.9 136.0 54.6 51.3 60.2

conformer

196

Table 2 Selected Torsion Angles (deg) of Local Minima for (Anti)-Ac-(S)-Val-Ozn(4R-Me) (3), (Gauche-)-Ac-(S)-Val-Ozn(4R-Me) (4), and (Gaucheþ)-Ac-(S)-Val-Ozn(4R-Me) (5), and their relative energies (kcal/mol).

M. Sta
s et al. / Journal of Molecular Structure 1109 (2016) 192e200

and for isolated molecules. Regardless of the rotation of the side chain, the conformation b2 is the most stable in the gas phase and weakly polar environment. In more polar environment, mimicked by water, the most stable is the conformation aR. Both these results are similar as for alanine analogue (1). It should be pointed, however, that the gap in energy to the second conformation in energy order is greater. There are considerable changes in the values of the torsion angles f and j depending on the value of the torsion angle c, which usually are in range of Df, Dj ~ ±20e30 , but even reach higher differences, up to 55 , as in the case of the conformation aL for (5) in the gas phase, the conformation a0 for (4) in chloroform environment, or the conformations aR, C5, b2, and a0 for (3) in the mimicked water. Generally, the closest geometry to the conformations of (1), has (5) (gaucheþ) for the lower in energy conformations b2, aR, C5, and b, and (3) (anti) for the higher in energy conformations aD and aL. To get more detailed insight in the energy order of the conformations, Tables 2e5S and Fig. 1S contain parameters of the main stabilizing forces [42,43], which can be present within the oxazoline-amino acid residue. As can be seen, the conformation C5 and b2 are stabilized primarily by the hydrogen bond/contacts C5type created by the NeH group of the N-terminal amide bond and the oxygen or nitrogen atom of the oxazoline ring. It should be noted that the nitrogen atom creates double bond in the oxazoline

197

ring, with the lone pair in the plane of the oxazoline ring. Thus, the nitrogen atom has higher proton affinity in comparison to the oxygen atom, as in case of the oxazole ring [29,44,45], which explains lower energy of the conformation b2 in comparison to the conformation C5. The remaining conformations, mainly aR and b, are stabilised by dipoleedipole attractions between carbonyl group of the N-terminal amide bond and oxazoline ring. It can be predicted that increase of the environment polarity will favour the conformations not involved with internal hydrogen bond, but instead those with the C]O and NeH groups opened for interaction with environment, which enables better stabilization. Thus the helical conformations b and aR should prevail, still stabilized by the carbonylecarbonyl dipole attractions [43].

3.2. FTIR analysis The model compounds, Boc-L-Ala-L-Ozn-4-COOMe (6) and Boc(7) were synthesized and the Fourier transform infrared spectra (FTIR) were analyzed in the nS (NeH) stretching mode region in non-polar CCl4 and weakly polar CHCl3 solutions (Fig. 5). In the CCl4 solution, the observed broad halfwidth of the bands indicate the presence of conformational equilibrium. The spectra after deconvolution show bands at 3456, 3438, and 3419 cm1 for Ala-Ozn (6) and at 3467, 3441, and 3423 cm1 for L-Val-L-Ozn-4-COOMe

Fig. 6. NOE difference spectra for the compounds Boc-L-Ala-L-Ozn-4-COOMe (6) and Boc-L-Val-L-Ozn-4-COOMe (7) recorded in CDCL3 and DMSO-d6.

198

M. Sta
s et al. / Journal of Molecular Structure 1109 (2016) 192e200

Fig. 7. Potential energy surfaces E ¼ f(4,j) for Ac-(S)-Ala-Ozn(4R-Me) (1) and local minima for B Ac-(S)-Ala-Ozn(4SeMe) (1), , (Anti)-Ac-(S)-Val-Ozn(4R-Me) (3), ◊ (Gaushe-)-Ac-(S)-Val-Ozn(4R-Me) (4), and D (Gausheþ)-Ac-(S)-Val-Ozn(4R-Me) (5). Energy contours are plotted every 1 kcal/mol. Conformations retrieved from the Cambridge Structural Data Base (crosses) for oxazoline-amino acids.

Val-Ozn (7). Stability of the conformations presented in Tables 1 and 2, their calculated populations, together with analysis of the theoretical frequencies shown in Table 6S enable to assign the bands, respectively, to the conformations C5, b2, and aR. In the nonpolar environment the molecules adopt mostly the conformation b2. In the CHCl3 solution only two bands can be separated for each compound. The main bands at 3440 and 3442 cm1 correspond to the conformation b2. The second smaller bands, at 3417 and 3415 cm1 for Ala-Ozn and Val-Ozn, respectively, can be assigned to the conformation aR. As can be seen, when the environment polarity increases, the conformation C5 disappears. In contrast, population of the conformation aR increases. 3.3. NMR-NOE analysis NOE experiments were applied to study the conformational preferences in weakly polar CDCl3 and more polar DMSO-d6 environment (Fig. 6). The hydrogen atoms of the N-terminal amide group and oxazoline ring were selected for irradiation because the changes of distances depend on the adopted conformation, as is presented in Table 7S. As can be seen in the spectrum for Boc-L-Ala-L-Ozn-4-COOMe recorded in CDCl3, irradiation of the amide hydrogen atom (dH 5.28) gives enhancement of the signal at dH 4.77 assigned to the hydrogen atom at the position 4 of the oxazoline ring. Proximity of these hydrogen atoms indicates the presence of the conformation b2. Enhancement of the signal at dH 4.45, assigned to the hydrogen atom at the position a, can be perceived as reference, because the distance (2.9 Å) is almost the same in the low energy conformations predicted by calculations, thus it can be assumed that this signal comes from the whole population of molecules, regardless of the conformation adopted. The low intensity of the signal of the oxazoline hydrogen atom (dH

4.77) results from longer distance (4.3 Å) as well as smaller number of molecules, which adopt the conformation b2. Similar dependence is found for the valine analogue, Boc-L-Val-L-Ozn-4-COOMe. Additionally, the signal at dH 2.10 assigned to the hydrogen atom at the position b was separated from t-butyl group. Comparison of the distance presented in Table 7S with the theoretical results as well as FTIR analysis, suggest that intensity of this signal results from the presence of the conformation aR. It should be also noted that irradiation of the signal at dH 4.79 assigned to the oxazoline hydrogen atom at the position 4, enhances the signal of amide hydrogen (dH 5.20) with distance 4.3 Å, but not the hydrogen at the position b (dH 2.10) with distances 4.7 and 5.2 Å, respectively, for the conformations b2 and aR. Analysis of the NOE spectra recorded in DMSO environment show again proximity of the amide and 4-oxazoline hydrogen atoms, which indicates the presence of the conformation b2 for both studied oxazoline-amino acid residues. In contrast, a significant enhancement of the signal assigned to the hydrogen atom b (dH 1.24 for (6) and 1.94 for (7)) is observed when the amide hydrogen atom is irradiated. When the signal of the hydrogen atom a (dH 4.23 for (6) and 3.90 for (7)) is used as reference, it indicates an increase of the population of the conformations, in which the distance HN … Hb is smaller. According to the calculated energy order this can be the conformation aR. Furthermore, when the signal of the 4-oxazoline hydrogen atom is irradiated (dH 4.73 for (6)) the signal assigned for the hydrogen atom b can be seen, roughly of the same intensity as the signal of amide hydrogen atom. This can be explained by appearance of the conformation b. 3.4. Solid state conformations Fig. 7 and Table 8S show the solid-state conformations of the oxazoline-amino acids [12,46e68] in various crystal structures retrieved from the Cambridge Structural Data Base [42,67]. The conformations correspond to the calculated minima on the conformational maps. As can be seen, the most adopted is the conformation b. The considerable population of the conformation b2 should be also noted. In majority cases the choice of the conformation b2 results from the geometric requirement of the cyclic compounds, which crystal structure were determined. However, it is still accessible, and thus can be perceived as the intrinsic feature of the oxazoline-amino acids. 4. Conclusions The presented conformational study of the two amino acids, alanine and valine, with the oxazoline ring in place of the C-terminal amide bond shows changes in conformational preferences depending on environment polarity. In non-polar or weakly polar environment the oxazoline-amino acids reveal strong tendency toward the semi-extended conformation denoted as b2, which for the alanine residue has the value of torsion angles f and j about 161 and 6 . Increase of the polarity favours other conformations, in particular the helical conformations aR (f,j ~ 70 , 15 ), and then, the conformation b (f,j ~ 57, 155 ). But still, the conformation b2 can be adopted. This is also the feature of the oxazole-amino acids. However, the non-aromatic oxazoline ring has carbon atoms having sp3 hybridisation and tetragonal geometry of bonds. In consequence, the oxazoline-amino acid residues have specific conformational properties, which could influence on conformations of peptide molecules, and potentially biological activity. The carbon atom of the oxazoline ring is also asymmetric centre. It seems not to have a considerable influence on the adopted conformations of the oxazoline-amino acids, but it will have on the conformation of the

M. Sta
s et al. / Journal of Molecular Structure 1109 (2016) 192e200

molecules they constitute. The presence of more bulky side chain as well as its rotation influence on the value of the main chain torsion angles f and j, but interestingly, it seems to increase tendency toward the conformation b2. These studies can be helpful in understanding the bioactive conformations of the natural peptides with antibacterial activity containing the oxazoline-amino acid residues. Acknowledgement This research was supported in part by PL-Grid Infrastructure. The authors gratefully acknowledge the Academic Computer Centre
w for the calculation facilities and CYFRONET AGH in Krako software. Appendix A. Supplementary data

[20] [21]

[22]

[23]

[24] [25]

[26]

[27]

Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.molstruc.2016.01. 001. These data include MOL files and InChiKeys of the most important compounds described in this article.

[29]

References

[30]

[1] M.C. Bagley, J.W. Dale, E.A. Merritt, X. Xiong, Thiopeptide antibiotics, Chem. Rev. 105 (2005) 685e714. [2] Z. Jin, Muscarine, imidazole, oxazole and thiazole alkaloids, Nat. Product. Rep. 26 (2009) 382e445. [3] C. Portmann, J.F. Blom, M. Kaiser, R. Brun, F. Jüttner, K. Gademann, Isolation of aerucyclamides C and D and structure revision of microcyclamide 7806A: heterocyclic ribosomal peptides from Microcystis aeruginosa PCC 7806 and their antiparasite evaluation, J. Nat. Prod. 71 (2008) 1891e1896. [4] M.S. Donia, J. Ravel, E.W. Schmidt, A global assembly line for cyanobactins, Nat. Chem. Biol. 4 (2008) 341e343. [5] Y. Kashman, A. Bishara, M. Aknin, Recent N-atom containing compounds from Indo-Pacific invertebrates, Mar. Drugs 8 (2010) 2810e2836. [6] K. Sivonen, N. Leikoski, D. Fewer, J. Jokela, Cyanobactinsdribosomal cyclic peptides produced by cyanobacteria, Appl. Microbiol. Biotechnol. 86 (2010) 1213e1225. [7] D. Davyt, G. Serra, Thiazole and oxazole alkaloids: isolation and synthesis, Mar. Drugs 8 (2010) 2755e2780. [8] K. Gademann, J. Kobylinska, Antimalarial natural products of marine and freshwater origin, Chem. Rec. 9 (2009) 187e198. [9] N. Ziemert, K. Ishida, P. Quillardet, C. Bouchier, C. Hertweck, N.T. De Marsac, E. Dittmann, Microcyclamide biosynthesis in two strains of Microcystis aeruginosa: From structure to genes and vice versa, Appl. Environ. Microbiol. 74 (2008) 1791e1797. [10] C. Portmann, J.F. Blom, K. Gademann, F. Jüttner, Aerucyclamides A and B: isolation and synthesis of toxic ribosomal heterocyclic peptides from the cyanobacterium Microcystis aeruginosa PCC 7806, J. Nat. Prod. 71 (2008) 1193e1196. [11] C. Portmann, S. Sieber, S. Wirthensohn, J.F. Blom, L. Da Silva, E. Baudat, M. Kaiser, R. Brun, K. Gademann, Balgacyclamides, antiplasmodial heterocyclic peptides from Microcystis aeruguinosa EAWAG 251, J. Nat. Prod. 77 (2014) 557e562. [12] T.W. Hambley, C.J. Hawkins, M.F. Lavin, A. van den Brenk, D.J. Watters, Cycloxazoline: a cytotoxic cyclic hexapeptide from the ascidian lissoclinum bistratum, Tetrahedron 48 (1992) 341e348. [13] X. Fu, T. Do, F.J. Schmitz, V. Andrusevich, M.H. Engel, New cyclic peptides from the Ascidian Lissoclinum patella, J. Nat. Prod. 61 (1998) 1547e1551. [14] B.M. Degnan, C.J. Hawkins, M.F. Lavin, E.J. McCaffrey, D.L. Parry, A.L. Van den Brenk, D.J. Watters, New cyclic peptides with cytotoxic activity from the ascidian Lissoclinum patella, J. Med. Chem. 32 (1989) 1349e1354. [15] B.M. Degnan, C.J. Hawkins, M.F. Lavin, E.J. McCaffrey, D.L. Parry, D.J. Watters, Novel cytotoxic compounds from the ascidian Lissoclinum bistratum, J. Med. Chem. 32 (1989) 1354e1359. [16] L.J. Perez, D.J. Faulkner, W. Fenical, Bistratamides E-J, modified cyclic hexapeptides from the Philippines ascidian Lissoclinum bistratum, J. Nat. Prod. 66 (2003) 247e250. [17] H. Sone, H. Kigoshi, K. Yamada, Isolation and stereostructure of dolastatin I, a cytotoxic cyclic hexapeptide from the Japanese sea hare Dolabella auricularia, Tetrahedron 53 (1997) 8149e8154. [18] Y. Hamamoto, M. Endo, M. Nakagawa, T. Nakanishi, K. Mizukawa, A new cyclic peptide, ascidiacyclamide, isolated from ascidian, J. Chem. Soc. Chem. Commun. (1983) 323e324. [19] L.T. Tan, R.T. Williamson, W.H. Gerwick, K.S. Watts, K. McGough, R. Jacobs, cis,cis- and trans,trans-Ceratospongamide, new bioactive cyclic heptapeptides

[31]

[28]

[32]

[33]

[34] [35]

[36] [37] [38]

[39] [40] [41]

[42] [43]

[44] [45]

[46] [47] [48]

199

from the Indonesian red alga Ceratodictyon spongiosum and symbiotic sponge sigmadocia symbiotica, J. Org. Chem. 65 (2000) 419e425. S.G. Toske, W. Fenical, Cyclodidemnamide: a new cyclic heptapeptide from the marine ascidian Didemnum Molle, Tetrahedron Lett. 36 (1995) 8355e8358. A. Rudi, M. Aknin, E.M. Gaydou, Y. Kashman, Four new cytotoxic cyclic hexaand heptapeptides from the marine ascidian Didemnum molle, Tetrahedron 54 (1998) 13203e13210. A. Rudi, L. Chill, M. Aknin, Y. Kashman, A. Didmolamide, B, Two New, Cyclic Hexapeptides from the Marine Ascidian Didemnum molle, J. Nat. Prod. 66 (2003) 575e577. V. Admi, U. Afek, S. Carmeli, Raocyclamides A and B, Novel Cyclic Hexapeptides Isolated from the Cyanobacterium Oscillatoria raoi, J. Nat. Prod. 59 (1996) 396e399. A. Ploutno, S. Carmeli, Modified peptides from a water bloom of the cyanobacterium Nostoc sp, Tetrahedron 58 (2002) 9949e9957. D. Siodłak, A. Macedowska-Capiga, M.A. Broda, A.E. Kozioł, T. Lis, The cis-trans isomerization of N-methyl-a,b-dehydroamino acids, Biopolymers 98 (2012) 466e478. D. Siodłak, M. Bujak, M. Sta
s, Intra-and intermolecular forces dependent main chain conformations of esters of a,b-dehydroamino acids, J. Mol. Struct. 1047 (2013) 229e236. D. Siodłak, J. Grondys, M.A. Broda, The conformational properties of a,bdehydroamino acids with a C-terminal ester group, J. Pept. Sci. 17 (2011) 690e699. D. Siodłak, A. Macedowska-Capiga, J. Grondys, K. Paczkowska, B. Rzeszotarska, N-Methyldehydroamino acids promote a configuration cis of N-methylamide bond, J. Mol. Struct. THEOCHEM 851 (2008) 100e108. D. Siodłak, M. Sta
s, M.A. Broda, M. Bujak, T. Lis, Conformational properties of oxazole-amino acids: Effect of the intramolecular N-H,,,N hydrogen bond, J. Phys. Chem. B 118 (2014) 2340e2350. V.S.C. Yeh, Recent advances in the total syntheses of oxazole-containing natural products, Tetrahedron 60 (2004) 11995e12042. W. Viviani, J.L. Rivail, A. Perczel, I.G. Csizmadia, Peptide models. 3. Conformational potential energy hypersurface of formyl-L-valinamide, J. Am. Chem. Soc. 115 (1993) 8321e8329. M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G.A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H.P. Hratchian, A.F. Izmaylov, J. Bloino, G. Zheng, J.L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J.A. Montgomery Jr., J.E. Peralta, F. Ogliaro, M.J. Bearpark, J. Heyd, E.N. Brothers, K.N. Kudin, V.N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A.P. Rendell, J.C. Burant, S.S. Iyengar, J. Tomasi, M. Cossi, N. Rega, N.J. Millam, M. Klene, J.E. Knox, J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, R.L. Martin, K. Morokuma, V.G. Zakrzewski, G.A. Voth, P. Salvador, € Farkas, J.B. Foresman, J.V. Ortiz, J.J. Dannenberg, S. Dapprich, A.D. Daniels, O. J. Cioslowski, D.J. Fox, Gaussian 09, Gaussian, Inc., Wallingford, CT, USA, 2009. Y. Zhao, D. Truhlar, The M06 suite of density functionals for main group thermochemistry, thermochemical kinetics, noncovalent interactions, excited states, and transition elements: two new functionals and systematic testing of four M06-class functionals and 12 other functionals, Theor. Chem. Acc. 120 (2008) 215e241. Y.K. Kang, B.J. Byun, H.S. Park, Conformational preference and cisetrans isomerization of 4-methylproline residues, Biopolymers 95 (2011) 51e61. Y.K. Kang, H.S. Park, Conformational preferences of the 2-methylproline residue and its role in stabilizing b-turn and polyproline II structures of peptides, New J. Chem. 38 (2014) 2831e2840. Surfer 8, Golden Software, Inc., Golden, CO, 2002.
ky, A. Perczel, Prolylproline unit in model peptides and in fragments I. Huda from databases, proteins: structure, Funct. Genet. 70 (2008) 1389e1407. V.J. Hruby, G. Li, C. Haskell-Luevano, M. Shenderovich, Design of peptides, proteins, and peptidomimetics in chi space, Biopolymers - Peptide Sci. Sect. 43 (1997) 219e266. J. Houben-Weyl, Methods of Organic Chemistry, Thieme Stuttgart, New York, 2003. G.M. Bilcer, J.C. Lilly, L.M. Swanson, Comentis, Inc. Patent, 2011. A.J. Phillips, Y. Uto, P. Wipf, M.J. Reno, D.R. Williams, Synthesis of functionalized oxazolines and oxazoles with DAST and Deoxo-fluor, Org. Lett. 2 (2000) 1165e1168. R. Vargas, J. Garza, B.P. Hay, D.A. Dixon, Conformational study of the alanine dipeptide at the MP2 and DFT levels, J. Phys. Chem. A 106 (2002) 3213e3218. F.H. Allen, C.A. Baalham, J.P.M. Lommerse, P.R. Raithby, Carbonyl-carbonyl interactions can be competitive with hydrogen bonds, Acta Crystallogr. Sect. B Struct. Sci. 54 (1998) 320e329. D. Kaur, S. Khanna, Intermolecular hydrogen bonding interactions of furan, isoxazole and oxazole with water, Comput. Theor. Chem. 963 (2011) 71e75. S. Khanna, D. Kaur, R. Kaur, The saturated five-membered heterocyclic molecules as organic hydride donors: a computational study, J. Phys. Org. Chem. 27 (2014) 747e755. M. Doi, A. Asano, Acta Crystallogr. Sect. Sect. E Struct. Rep. Online 58 (2002). P. Wipf, C. Wang, Synthesis and Ag(I) complexation studies of Tethered Westiellamide, Org. Lett. 8 (2006) 2381e2384. F.J. Schmitz, M.B. Ksebati, J.S. Chang, J.L. Wang, M.B. Hossain, D. Van der Helm, M.H. Engel, A. Serban, J.A. Silfer, Cyclic peptides from the ascidian Lissoclinum

200

[49]

[50]

[51]

[52] [53]

[54]

[55] [56]

[57] [58]

M. Sta
s et al. / Journal of Molecular Structure 1109 (2016) 192e200 patella: conformational analysis of patellamide D by x-ray analysis and molecular modeling, J. Org. Chem. 54 (1989) 3463e3472. P. Wipf, S. Venkatraman, C.P. Miller, S.J. Geib, Metal complexes of marine peptide metabolites: a novel Ag4 cluster, Angewandte Chemie Int. Ed. Engl. 33 (1994) 1516e1518. A. Asano, T. Yamada, Y. Katsuya, T. Taniguchi, M. Sasaki, M. Doi, Conformational restraints induced by modification of configuration of threonine and oxazoline residues in ascidiacyclamide analogues, J. Peptide Res. 66 (2005) 90e98. A. Asano, T. Yamada, A. Numata, Y. Katsuya, M. Sasaki, T. Taniguchi, M. Doi, A flat squared conformation of an ascidiacyclamide derivative caused by chiral modification of an oxazoline residue, Biochem. Biophys. Res. Commun. 297 (2002) 143e147. G. Haberhauer, F. Rominger, Syntheses and structures of imidazole analogues of Lissoclinum Cyclopeptides, Eur. J. Org. Chem. 2003 (2003) 3209e3218. T. Ishida, M. Inoue, Y. Hamada, X-Ray crystal structure of ascidiacyclamide, a cytotoxic cyclic peptide from ascidian, J. Chem. Soc. - Ser. Chem. Commun. 5 (1987) 370e371. T. Ishida, M. Tanaka, M. Nabae, M. Inoue, S. Kato, Y. Hamada, T. Shioiri, Solution and solid-state conformations of ascidiacyclamide, a cytotoxic cyclic peptide from ascidian, J. Org. Chem. 53 (1988) 107e112. M. Doi, A. Asano, Private Communication, 2002. A. Asano, M. Doi, Turn-over of an oxazoline ring induced by chiral change of a folded ascidiacyclamide analogue: Cyclo(IIe-D-aThr-D-Val-Thz-IIe-D-Oxz-DVal-Thz) N,N-dimethylformamide disolvate, Acta Crystallogr. Sect. Sect. E Struct. Rep. Online 60 (2004) o2449eo2451. M. Doi, A. Yumiba, A. Asano, Acta Crystallogr.,Sect.E Struct.Rep.Online 58 (2002) 62. A. Asano, T. Yamada, M. Doi, The square conformation of phenylglycineincorporated ascidiacyclamide is stabilized by CH/p interactions between amino acid side chains, Biorg. Med. Chem. 19 (2011) 3372e3377.

[59] M. Doi, A. Asano, K. Yoza, Acta Crystallogr. Sect. C. Cryst. Struct. Commun. 59 (2003) 323. [60] T. Ishida, Y. In, M. Doi, M. Inoue, Y. Hamada, T. Shioiri, Molecular conformation of ascidiacyclamide, a cytotoxic cyclic peptide from Ascidian: X-ray analyses of its free form and solvate crystals, Biopolymers 32 (1992) 131e143. [61] M. Doi, A. Asano, Acta Crystallogr.,Sect.E Struct.Rep.Online 58 (2002). [62] A. Asano, K. Minoura, T. Yamada, A. Numata, T. Ishida, M. Doi, Y. Katsuya, Y. Mezaki, M. Sasaki, T. Taniguchi, M. Nakai, H. Hasegawa, A. Terashima, Effect of asymmetric modification on the conformation of ascidiacyclamide analogs, J. Peptide Res. 60 (2002) 10e22. [63] Y. In, M. Doi, M. Inoue, T. Ishida, Y. Hamada, T. Shioiri, Structure of ascidiacyclamide as the ethanol water solvate, a cytotoxic cyclic peptide from Ascidian, Acta Crystallogr. Sect. C. Cryst. Struct. Commun. 50 (1994). Pt 12/. [64] M. Doi, F. Shinozaki, Y. In, T. Ishida, D. Yamamoto, M. Kamigauchi, M. Sugiura, Y. Hamada, K. Kohda, T. Shioiri, Conformational change of ascidiacyclamide caused by asymmetric modification for an isoleucine residue: structural analyses of [Gly], [Leu], and [Phe]ascidiacyclamides by X-ray diffraction and NMR spectroscopy, Biopolymers 49 (1999) 459e469. [65] F. Yokokawa, H. Sameshima, Y. In, K. Minoura, T. Ishida, T. Shioiri, Total synthesis and conformational studies of ceratospongamide, a bioactive cyclic heptapeptide from marine origin, Tetrahedron 58 (2002) 8127e8143. [66] Y. In, M. Doi, M. Inoue, T. Ishida, Y. Hamada, T. Shioiri, Molecular conformation of patellamide A, a cytotoxic cyclic peptide from the ascidian Lissoclinum patella, by X-ray crystal analysis, Chem. Pharm. Bull. 41 (1993) 1686e1690. [67] A. Asano, T. Yamada, A. Numata, M. Doi, Cyclo(-Cha-Oxz-D-Val-Thz-Ile-OxzD-Val-Thz-) N,N-dimethylacetamide dihydrate: a square form of cyclohexylalanine-incorporated ascidiacyclamide having the strongest cytotoxicity, Acta crystallogr. Sect. C, Cryst. Struct. Commun. 50 (2002) 488e490. [68] T. Ishida, Y. In, M. Doi, M. Inoue, Y. Hamada, T. Shiori, Molecular conformation of ascidiacyclamide, a cytotoxic cyclic peptide from Ascidian: X-ray analyses of its free form and solvate crystals, Biopolymers 32 (1992) 131e143.