Addition of a PeptideFragment on an �-HelicalDepsipeptide Induces �/310-Conjugated Helix: Synthesis,Crystal Structure, and CDSpectra of Boc–Leu–Leu–Ala–(Leu–Leu–Lac)3–Leu–Leu–OEt

Hiroyuki Oku1

Takafumi Ohyama1

Akihiro Hiroki1

Keiichi Yamada1

Keiichi Fukuyama2

Hiroyuki Kawaguchi3

Ryoichi Katakai11 Department of Chemistry,

Gunma University,Kiryu,

Gunma 376-8515,Japan

2 Department of Biology,Graduate School of Science,

Osaka University,Toyonaka,

Osaka 560-0043,Japan

3 Institute for MolecularScience, Okazaki,

Aichi 444-8585, Japan

Received 23 September 2003;accepted 23 June 2004

Published online 12 August 2004 in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/bip.20117

Abstract: The depsipeptide Boc1–Leu2–Leu3–Ala4–Leu5–Leu6–Lac7–Leu8–Leu9–Lac10–Leu11–Leu12–Lac13–Leu14–Leu15–OEt16 (1) (Boc � tert-butyloxycarbonyl, Lac � L-lactic acid residue)has been synthesized from the peptide Boc–Leu–Leu–Ala–OEt (2) and a depsipeptide, Boc–(Leu–Leu–Lac)3–Leu–Leu–OEt (3). Single crystals of 1 were successfully obtained and the structure hasbeen solved by direct methods (such as Sir2002 and Shake-and-Bake). Interestingly, 1 adopts an�/310-conjugated helix containing a kink at the junction of peptide and depsipeptide segments,Leu3–Lac7. This is significantly different from the conformation of 3, which has a straight �-helicalstructure with standard � and � angles. Microcrystalline CD spectra were also studied to comparestructural properties of 1 and 3. The differences between �/310- and �-helices appear in these CDspectra. © 2004 Wiley Periodicals, Inc. Biopolymers 75: 242–254, 2004

Keywords: depsipeptide; �-helix; 310-helix; leucine; lactic acid; nanomaterial; crystal structure;liquid-phase peptide synthesis; segment condensation

INTRODUCTION

Depsipeptides are oligomers and polymers composedof hydroxy acids and amino acids linked by amide and

ester bonds. In nature, depsipeptides are found inantibacterials. In some cases, depsipeptides are ap-plied in cancer therapeutics such as actinomycin D,FK-228, and FR 901228.1–3

Correspondence to: Ryoichi Katakai; email: katakai@chem.gunma-u.ac.jp

Contract grant sponsor: Ministry of Education, Culture, Sports,Science and Technology of Japan

Contract grant number: 15036212 and 16033211Biopolymers, Vol. 75, 242–254 (2004)© 2004 Wiley Periodicals, Inc.

242

Except for naturally occurring compounds, Good-man et al. first reported the usefulness of the dep-sipeptides for conformational studies.4–8 They haveprepared sequential polymers, such as poly(Ala–Ala–Lac) (Lac: L-lactic acid) and poly(Val–Pro–Hea–Val–Gly) (Hea: 1-hydroxy ethanoic acid). Until today,various depsipepides have been reported from theviewpoint of structural interest: (1) stabilization anddestabilization of �-turn conformation,9,10 (2) the sta-bility of helical structures consisting of �-amino ac-ids,11,12 (3) peptide compounds having 2-hydroxy-2,3-dimethylbutanoic acid residues,13 and (4) replace-ment with a Lac residue in –Val–Ala–Leu–Aib–based helices (Aib: �-aminoisobutyric acid).14,15 Wehave also studied sequential oligo- and polydepsipep-tides composed of nonpolar amino acid residues (Leu,Val, Ala, and Gly) and the corresponding hydroxyacid residues {Hmp [L-leucic acid, (S)-2-hydroxy-4-methyl-pentanoic acid], Hmb [(S)-2-hydroxy-3-meth-yl-butanoic acid], Lac, and Hea}.16–30 Hydroxy acidmutation is also useful in the study of native pro-teins.31–38 For example, a coiled-coil structure inGCN4 has been tested for the difference of thermaldenaturation energy.31 As a model of amyloidgenesis,depsipeptides have been applied to prevent �-sheetaggregation.32

A practical application of polydepsipeptides is forbiodegradable and bioabsorbable materials.39–44 Inthose polymers, ester linkages can be hydrolyzed byesterases at each implanted site and can be decom-posed into oligomers. As an example, we have devel-oped a material for a drug delivery system42 where wecan modulate the degradation period from days to ayear depending on their hydrophobicity.

Recently we have found that a series of Leu- andLac-based depsipeptides are suitable for nanostruc-tural materials due to the solubility,17 conformationalstability,21,22,24,25 and crystallinity.18–23,27 For exam-ple, by the crystallographic analysis we have realizedthat Boc–(Leu–Leu–Lac)3–Leu–Leu—OEt (3)19

(Boc: tert-butyloxycarbonyl) still have an �-helixconformation even with three Lac residues. In thehelical structure of 3, a slight distortion was observedat the level of the Lac residues. We have also prepareda composite sequence, Boc–(Leu–Leu–Ala)2–(Leu–Leu–Lac)3–OEt (4)27,18 which is made of two units–Leu–Leu–Ala– and –Leu–Leu–Lac–. The crystalstructure of 4 is an �-helix with a 310-helical part atthe connective region between the peptide andthe depsipeptide moieties, –Ala7–Leu8–Leu9–Lac10–Leu11–Leu12–. These two examples have indicatedthat the local structure around Lac residues is proba-bly flexible compared with those of Leu residues.

In this article, we describe the synthesis and geo-metrical parameters of a novel sequence, Boc–Leu–Leu–Ala–(Leu–Leu–Lac)3–Leu–Leu–OEt (1), whichis a block oligomer prepared from a peptide, Boc–Leu–Leu–Ala–OEt (2) and a depsipeptide Boc–(Leu–Leu–Lac)3–Leu–Leu–OEt (3).

EXPERIMENTAL

Peptide Synthesis

The depsipeptide 1 was synthesized by a condensation of apeptide and depsipeptide fragments. These fragments weresynthesized by stepwise elongation of peptide chains. Cou-plings between the segments were mediated by dicyclo-hexylcarbodiimide-N-hydroxysuccinimide (DCC/HONSu)in CHCl3.

HCl � H–(Leu–Leu–Lac)3–Leu–Leu–OEt (5). Boc–(Leu–Leu–Lac)3–Leu–Leu–OEt19 (37.2 g, 0.1 mol) was dissolvedin 2.5 N HCl/dioxane (100 mL). The solution was allowedto stand for 1 h at room temperature, and then the solventwas evaporated under reduced pressure. Addition of diethylether and hexane gave crystals of the hydrochloride salt 5,which were collected on a glass filter and washed withhexane. Yield, 29.4 g (95%).

Boc–Leu–Leu–Ala–(Leu–Leu–Lac)3–Leu–Leu–OEt (1).5 (29.4 g, 0.095 mol) was dissolved in CHCl3. To thesolution were added N-methylmorpholine (NMM) (10.45mL, 0.095 mol), Boc–Leu–Leu–Ala–OH (39.6 g, 0.095mol), and HONSu (21.9 g, 0.19 mol). The solution wascooled at 0°C, and DCC (19.6 g, 0.095 mol) was added. Thesolution was stirred for 1 h at 0°C and allowed to stand atroom temperature for 12 h. The solution was diluted withethyl acetate and the crystals of dicyclohexylurea wereremoved by filtration. The filtrate was concentrated in vacuoto give a solid, which was dissolved in ethyl acetate. Thesolution was washed with 10% citric acid, water, saturatedNaHCO3, water, and saturated NaCl solution, and driedover Na2SO4, successively. The solvent was then evapo-rated again. Addition of hexane into the residue gave acolorless solid. The crude product was purified by a silicagel column chromatography, eluting with a solvent system,chloroform–ethyl acetate [2:1 (� v/v)]. The product wasrecrystallized from ethyl acetate to give 42.0 g (65 % yield)of 1. Melting point (mp) 216–217°C; [�]D

20 � �40.5° (c� 0.1, CHCl3); ir (KBr) 3305, 2959, 2872, 1745, 1685,1661, 1648, 1541, 1470, 1388, 1369, 1283, 1241, 1166,1101, 1036, 949, 865, 701, 666, 588, 468 cm�1; 1H-NMR(CDCl3, 500 MHz) � 7.67 (d, J � 5.49 Hz 1H, NH), 7.57 (d,J � 6.53 Hz 1H, NH), 7.50 (d, J � 6.41 Hz 1H, NH), 7.49(d, J � 7.94 Hz 1H, NH), 7.46 (d, J � 5.19 Hz 1H, NH),7.41 (d, J � 5.49 Hz 1H, NH), 7.38 (d, J � 6.41 Hz 1H,NH), 7.37 (d, J � 5.80 Hz 1H, NH), 7.24 (d, J � 7.33 Hz1H, NH), 6.77 (d, J � 3.36 Hz 1H, NH), 5.38 (br, 1H, NH),5.10 (q, J � 7.02 Hz 1H, C�H), 5.01 (q, J � 7.02 Hz 1H,

Addition of a Peptide Fragment 243

C�H), 4.94 (q, J � 7.02 Hz 2H, C�H), 4.49 (m, 1H, C�H),4.43 (m, 1H, C�H), 4.24 (m, 2H, C�H), 4.16 (m, 2H, C�H),4.13 (q, J � 7.02 Hz 2H, CH3CH2), 4.07 (m, 1H, C�H),3.88 (m, 1H, C�H), 1.98–1.56 (m, 30H, C�H, C�H), 1.53(d, J � 7.02 Hz 3H, C�H), 1.52 (d, J � 7.02 Hz 3H, C�H),1.52 (d, J � 7.02 Hz 3H, C�H), 1.47 (d, J � 7.02 Hz 3H,C�H), 1.47 (s, 9H, Boc), 1.23 (t, J � 7.02 Hz 3H, CH3CH2),1.02–0.83 (m, 60H, C�H); electrospray ionization–massspectroscopy (ESI-MS) m/z (M�) calcd for C79H141N11O20

1564.0, obsd 1587.2 (M � Na�); Anal. calcd forC79H141N11O20: C, 60.63; H, 9.08; N, 9.84. Found: C,60.34; H, 8.94; N, 9.39.

X-Ray Crystallography

To obtain single crystals suitable for crystallographic anal-ysis, 10 mg of 1 was dissolved in 1 mL of methanol(MeOH) and equilibrated via vapor phase diffusion against1 mL of water, resulting in fine needle crystals after 1–2weeks at 15°C. X-ray diffraction data were collected on aRigaku RAXIS-RAPID imaging plate area detector withgraphite monochromated Cu-K� radiation. The data collec-tion conditions and crystallographic parameters are listed inTable I. We have successfully solved the structure of 1 bydirect methods for macromolecular crystals (such as Shake-and-Bake45 and SIR200246). An empirical absorption col-lection program, DIFABS,47 was applied which resulted intransmission factors ranging from 0.72 to 1.10. Nitrogenand oxygen atoms were refined anisotropically. Carbonatoms were refined with isotropic displacement parameters

due to the limited numbers reflections. Hydrogen atomswere placed in calculated positions and refined with a ridingmodel, and with Uiso constrained to be 1.2 times Uiso of thecarrier atom. The final cycles of full-matrix least-squaresrefinement was based on 8232 observed reflections, and 737variable parameters and converged with R1 � 0.081 andwR2 � 0.192. Refinement calculations were performed onthe Crystal Structure 3.50 (RIGAKU/MSC, 2003). The finalatomic parameters are deposited into the Cambridge Crys-tallographic Data Centre (CCDC) database (no. 236273).

Fourier Transform Infrared (FTIR), CD, and1H-NMR Spectroscopic Measurements

FTIR spectra were taken on a JASCO FT/IR-660-plus spec-trometer equipped with a TGS detector at 273 K.

CD spectra were recorded on a JASCO J-720 spectropo-larimeter at 273 K. Cylindrical fused quartz cells with pathlengths of 0.01 cm was used for all the experiments. Sampleconcentrations for CD experiments were 1.00 mg/mL.

Microcrystalline CD spectra were taken under the samecondition as in solution. The nujol–mull sample was pre-pared from 1 mL of spectrograde nujol and 1.00 mg ofpeptide crystals, which were carefully mixed and ground onan agate mortar and by a pestle.

1H-NMR studies were carried out on JEOL �-500 and�-500 spectrometers. Peptide concentrations were in 5 mMand the probe temperature was maintained at 308 or 303 K.

RESULTS

Crystal Structure of 1

A stereo view of the molecular structure and a pack-ing diagram are shown in Figures 1 and 2, respec-tively.

The torsion angles of main and side chains arelisted in Table II. The � and � torsional angles of thehelical residues from Leu2 to Leu14 have shownvarious pairs of angles ranging from �49 to �93° and�18 to �44°, respectively. We cannot simply classifyeach pair of � and � data to either �- or 310-helicalconformations.46–48 In these cases, the hydrogen-bond pattern is suitable to assign helical classes asdiscussed below. For the side chains of Leu residues,the torsion angle data have suggested a stable orien-tation, g�(t, g�),50–54 except Leu2 and Leu11 whichadopt the other stable form, t(g�, t).

The intramolecular and intermolecular hydrogenbonds are listed in Tables III and IV, respectively. Themolecules are packed into infinite chains by the threeintermolecular bonds. The connecting residues arefound at the head-to-tail positions, Lac13 � � � Leu2,Leu14 � � � Leu2, and Leu14 � � � Leu3. The dotted linesin Figure 2 show interactions by three hydrogen

Table I Crystal and Diffraction Parameters of Boc–Leu–Leu–Ala–(Leu–Leu–Lac)3–Leu–Leu–OEt (1)

Empirical formula C79H141N11O20

Crystal color, habit Colorless, needleCrystal size (mm) 0.70 � 0.10 � 0.10Formula weight 1565.04Crystal system, Space group Monoclinic, P2 (#4)a (A) 11.504(6)b (A) 20.43(1)c (A) 20.726(9)� (deg) 97.54(1)Volume (A3) 4829.3(4)Z 2Density (g/cm3) (calcd) 1.076F(000) 1704.00� (Cu-K�), (cm�1) 6.30temp, (°C) �1002max (deg) 135.7Measured reflections 39337Unique reflections 8232 (Rint � 0.057)R1a 0.081wR2b 0.192Goodness of fit 0.92

a R1 � (¥�F0� � �Fc�) / ¥�F0�.b wR2 � [(¥w�F0�2 ��Fc�2�2)/¥w(F0

2)2]1/2

244 Oku et al.

bonds, NH � � � OAC, although the Leu14–Leu3 hy-drogen bond cannot be seen clearly due to the over-lapping of atoms.

For intramolecular interactions, a total of 13 hy-drogen bonds are found for carbonyl oxygens andamide NHs. Among them, eight interactions are of[i, i�4] types, which form �-helical segments. Theobserved pairs are O12(Boc1) � � � N51(Leu5),O21(Leu2) � � � N61(Leu6), O41(Ala4) � � � N81(Leu8),O51(Leu5) � � � N91(Leu5), O72(Lac7) � � � N111(Leu11),O81(Leu8) � � � N121(Leu12), O102(Lac10) � � � N141-(Leu14), and O111(Leu11) � � � N151(Leu15). The restof five interactions are of [i, i�3] types, which forms310-helical segments. These are found in the pairs ofO12(Boc1) � � � N41(Leu4), O21(Leu2) � � � N51(Leu5),O51(Leu5) � � � N81(Leu8), O81(Leu8) � � � N111(Leu11),and O111(Leu11) � � � N141(Leu14). The N � � � O lengths ofNH � � � CAO hydrogen bonds are in the range of3.02(2)–3.31(1) Å. All [i, i�3] hydrogen bonds areexpected to have very weak interactions except one,which have a bond length of 3.02(2) Å. These valuescorresponded well to the reported data (mean �3.0 Å)observed in the peptide structures.55–58

The connections between Leu6–Lac7, Leu9–Lac10, and Leu12–Lac13 residues are ester linkagesand do not have NHs. Therefore, both �- and 310-helical interactions cannot be expected for the carbon-yls of Leu3, Leu6, and Leu9 (�-type) and for those ofAla4, Lac7, and Lac9 (310-type). Interestingly, anyester CAOs do not contribute to the hydrogen bond-ing. For example, O91 � � � N121 distance is 4.00(2) Å.This length is far beyond the range of bonding inter-action.

The other oxygen atoms in those ester moieties(OOA) are found to contribute to the hydrogenbonding with amide NHs. The bonding pairs areO71 � � � N81, O101 � � � N111, O131 � � � N141, andO161 � � � N151.

CD Spectra of 1 and 3

Solution Spectra. Solution conformations of the dep-sipeptides 1 (Figure 3a) and 3 (Figure 3b) were ex-amined in n-BuOH (n-butanol), 1,1,1-trifluoroethanol(TFE), and MeOH by using CD spectra. The spectrumof 1 in n-BuOH is characterized by a positive band at

FIGURE 1 Stereo drawing of Boc–Leu–Leu–Ala–(Leu–Leu–Lac)3–Leu–Leu–OEt (1). Displace-ment spheres are drawn at the 20% probability level. Hydrogen atoms except NHs are omitted forclarity.

Addition of a Peptide Fragment 245

192 nm and a negative band at 203 nm, accompaniedby an indistinct shoulder near 220 nm with meanresidue ellipticity []203 � �20,500 and []220

� �4800 deg � cm2 � dmol�1, respectively. Theellipticity of amide n3* transition at 220 nm59 wassmaller than that of 3* at 203 nm. This spectralpattern is often observed for short helical peptides anddepsipeptides having 14–16 residues.17,60 In the case

of a typical �-helix, the spectrum is characterized bya positive ellipticity at 190 nm and a double minimumat about 208 and 222 nm with equal intensity.60–64 Inpolar solvents, TFE and MeOH, the peptide haveshown CD spectra with a negative ellipticity at around190–203 nm, which is characteristic of unorderedstructure in oligopeptides.61,62,65–67

The spectra of 3 show weaker negative bands at

Table II Torsion Anglesa (deg) for Boc–Leu–Leu–Ala–(Leu–Leu–Lac)3–Leu–Leu–OEt (1)

Residues

Angles

� � � �1 �2

Boc(1) �173.3 (8)b

Leu(2) �58 (1)c �41 (1) �173.4 (7) 174.9 (8) 62 (1)�174.4 (8)

Leu(3) �71 (1) �24 (1) 171.5 (8) �59 (1) �57 (1)�179.7 (7)

Ala(4) �80 (1) �33 (1) �179.7 (7)Leu(5) �67 (1) �39 (1) 178.3 (8) �67 (1) �71 (1)

168.7 (9)Leu(6) �49 (1) �41 (1)d �177.5 (7)e �71 (1) �71 (1)

162.4 (9)Lac(7) �84.1 (9)f �44 (1)g �171.4 (7)Leu(8) �76 (1) �38 (1) 175.9 (8) �64 (1) �62 (1)

171.9 (9)Leu(9) �59 (1) �33 (1)h �178.2 (7)i �65 (1) �64 (1)

174 (1)Lac(10) �80.9 (9)j �37 (1)k �179.7 (6)Leu(11) �66 (1) �41 (1) �179.4 (8) 179.7 (7) 56 (1)

176.9 (9)Leu(12) �59 (1) �27 (1)l �177.2 (7)m �71 (1) �72 (1)

166.4 (9)Lac(13) �88.2 (9)n �18 (1)o �176.2 (8)Leu(14) �93 (1) �35 (1) �179.5 (9) �68 (1) �68 (1)

169 (1)Leu(15) �143.2 (2) �19.4 (2)p �179.2 (6)q �73 (1) 74 (1)

�174 (1)

a The torsion angles for rotation about bonds of the peptide backbone (�, �, and �) and about bonds of the amino acid side chains (�1,�2) are described in Ref. 51.

b O(1)—C� (1)—N(2)—C�(2).c C�(1)—N(2)—C�(2)—C�(2).d N(6)—C�(6)—C�(6)—O(7).e C�(6)—C�(6)—O(7)—C�(7).f C�(6)—O(7)—C�(7)—C�(7).g O(7)—C�(7)—C�(7)—N(8).h N(9)—C�(9)—C�(9)—O(10).i C�(9)—C�(9)—O(10)—C�(10).j C�(9)—O(10)—C�(10)—C�(10).k O(10)—C�(10)—C�(10)—N(11).l N(12)—C�(12)—C�(12)—O(13).m C�(12)—C�(12)—O(13)—C�(13).n C�(12)—O(13)—C�(13)—C�(13).o O(13)—C�(13)—C�(13)—N(14).p N(15)—C�(15)—C�(15)—O(16).q C�(15)—C�(15)—O(16)—C1(16).

246 Oku et al.

around 190–203 nm even in n-BuOH solvent, whichis less polar than TFE and MeOH and can stabilize thehelical structure. These spectra are corresponding tounordered structure61,62,65–67 that is often observedfor the sequences having short chain length (�12residues).

The CD spectra of 1 and 3 have indicated that thesolution conformation of 1 was significantly stabilizedby the addition of a peptide fragment, –Leu–Leu–Ala–, into the depsipeptide sequence of 3. The stabil-ity is probably attributable to the difference of (1)chain length (14 and 11 residues for 1 and 3, respec-tively) and (2) the number of possible intramolecular

NOH � � � OAC hydrogen bonds. Actually, in thecrystalline state of 1, there are 13 intramolecular hy-drogen bonds as revealed by the X-ray diffractionstudy. On the contrary, there are only six hydrogenbonds in that of 3. Therefore we can estimate that theincreased number of hydrogen bonds also stabilizesthe helical conformation in solution states.

Microcrystalline Spectra. Figure 4 shows micro-crystalline CD spectra of 1 and 3 by using thenujol–mulls. The ellipticity was normalized by re-sidual molar concentration to compare band inten-sity. Full scale of the ordinate axis is formally

Table III Comparison of NH—OA Geometries (A, deg) for Boc–Leu–Leu–Ala–(Leu–Leu–Lac)3–Leu–Leu–OEt (1)a

Acceptor CAO or —O— Donor NH O- - -N H- - -O N—H- -O N—H- -O Type

O12 CAO (Boc1) N41 (Ala4) 3.15 (1) 2.46 129.8 129.8 i � 3N51 (Leu5) 3.062 (9) 2.20 150.9 151.1 i � 4

O21 CAO (Leu2) N51 (Leu5) 3.15 (1) 2.60 117.7 117.9 i � 3N61 (Leu6) 3.019 (9) 2.10 161.7 161.6 i � 4

O31 CAO (Leu3) No pairs foundO41 CAO (Ala4) N81 (Leu8) 2.897 (9) 2.04 149.6 150.1 i � 4O51 CAO (Leu5) N81 (Leu8) 3.312 (9) 2.83 112.2 112.2 i � 3

N91 (Leu9) 2.95 (1) 2.04 159.7 158.7 i � 4O61 CAO (Leu6) No pairs foundO71 —O— (Lac7) N81 (Leu8) 2.830 (9) 2.49 101.2 101.4 Ester–amideO72 CAO (Lac7) N111 (Leu11) 2.924 (9) 2.11 143.3 144.0 i � 4O81 CAO (Leu8) N111 (Leu11) 3.30 (2) 2.80 113.7 113.9 i � 3

N121 (Leu12) 2.930 (9) 1.99 169.9 169.4 i � 4O91 CAO (Leu9) No pairs foundO101 —O— (Lac10) N111 (Leu11) 2.801 (9) 2.47 100.5 100.0 Ester–amideO102 CAO (Lac10) N141 (Leu14) 3.131 (9) 2.46 127.5 126.8 i � 4O111 CAO (Leu11) N141 (Leu14) 3.05 (1) 2.27 138.8 138.2 i � 3

N151 (Leu15) 2.95(1) 2.04 159.8 159.6 i � 4O121 CAO (Leu12) No pairs foundO131 —O— (Leu13) N141 (Leu14) 2.70 (1) 2.30 45.6 104.0 Ester–amideO132 CAO (Leu13) No pairs foundO141 CAO (Leu14) No pairs foundO151 CAO (Leu15) No pairs foundO161 —O— (OEt16) N151 (Leu15) 2.74 (1) 2.45 97.9 97.6 Ester–amide

a Hydrogen atoms were placed at idealized positions with N—H � 0.95 A.

Table IV Intermolecular Hydrogen Bonds (A, deg) for Boc–Leu–Leu–Ala–(Leu–Leu–Lac)3–Leu–Leu–OEt (1)a,b

Acceptor (Type) —O— Donor NH O- - -N H- - -O N—H- -O Codec

O132 (main chain) N21 2.88 (1) 1.96 160.7 (i)O141 (main chain) N21 3.32 (1) 3.17 90.8 (i)O141 (main chain) N31 2.87 (1) 1.98 156.6 (i)

a Hydrogen bonds were picked up within 3.5 A.b Hydrogen atoms were placed at idealized positions with N—H � 0.95 A.c Symmetrical operators: (i) 1 � x, �y, 1 � z.

Addition of a Peptide Fragment 247

corresponding to �12,000 through �12,000 mdeg �cm�2 � mol�1.

Solid-state CD using microcrystalline and thin-filmsamples has been widely used in the field of inorganiccomplexes and small organic molecules, and pep-tides.68–76 In particular, the microcrystalline tech-nique is powerful to obtain crystalline-specific infor-mation. The measurements can be done either in nujolor in a KBr disk form where grounded crystal parti-cles are dispersed homogeneously in each matrix.Although the spectral measurement is quite simple,we should treat the spectra very carefully. The mostimportant artifact is the scattering effect. The incidentpolarized light can be partially reflected by the sus-pended peptide crystals, and probably broaden thebandwidth and weaken the band intensity comparedwith an idealized spectrum. Many good examples areavailable in the published papers such as by Kuroda etal.73–76 and Bosnich et al.68 In our case, all the sam-ples were prepared very thoroughly to achieve homo-geneous states. Each spectrum was confirmed by us-

FIGURE 2 A packing view of Boc–Leu–Leu–Ala–(Leu–Leu–Lac)3–Leu–Leu–OEt (1). Intermo-lecular hydrogen bonds are indicated as dotted lines (�).

FIGURE 3 Solution CD spectra of (a) Boc–Leu–Leu–Ala–(Leu–Leu–Lac)3–Leu–Leu–OEt (1) and (b) Boc–(Leu–Leu–Lac)3–Leu–Leu–OEt (3). [peptide] � 1 mg/1 mL at293 K in n-BuOH (� � � � �), TFE (——), and MeOH ( ).

248 Oku et al.

ing four different samples to test reproducibility of thespectral shape.

The spectrum of 1 has a positive band at 193 nm anda negative shape consisting of broad bands at 211 and219 nm with similar intensity. The broad spectra prob-ably come from the scattering effect of the incident lightin the spectrometer, although the samples were preparedwith careful treatment and no distinct crystals can beseen in the nujol suspensions. The spectrum of 3 has apositive band at 193 nm, a negative shoulder near 211nm, and a negative peak at 223 nm. The band at 223 nmshows stronger intensity than that at 211 nm. Both spec-tra are similar to the �-helical pattern having a positiveband at 190 nm and double negative bands at about 208and 222 nm.61–64 A minor dissimilarity, the intensitydifference at 223 nm, is attributable to the conforma-tional properties of �-helix (3) and �/310-conjugatedhelix (1) in crystal states.

Spectral Difference Between Solution and Microc-rystalline States. CD spectroscopy is particularlyuseful to examine structural changes. By the spectralcomparison between solution and crystalline states,we have realized that the spectral shapes are signifi-cantly dissimilar for compounds 1 and 3. In the crys-talline states, both depsipeptides have shown singlepositive (at 193 nm) and double negative (at 208 and220 nm) bands, which are a typical pattern for helicalstructure. On the contrary, in the solution states, wehave observed a positive (193 nm)–negative (203 nm)shape for 1 and a weak-negative signal (200 nm) for3 in n-BuOH. In the case of 3, the observed spectraldifferences between both solution and microcrystal-line states have suggested that the helical structurewill be loosened when dissolving the depsipeptideeven in n-BuOH, which is known as a helix-promot-ing solvent. In the case of an n-BuOH solution of 1, a

negative band (at 220 nm) disappeared, although thehelical property (positive–negative spectrum) still re-mained. One possible explanation is the structuralchange from �/310 (crystalline) to 310 (solution). Byusing CD spectroscopy, many attempts have beendone to distinguish 310- and �-helical conforma-tions.61,77,78 A criterion proposed by Manning andWoody61 is that 310-helices would have weaker222-nm bands relative to their 208-nm bands based onthe CNDO/S calculations of N-methylacetamide.Therefore in our case, the disappearing of the 220-nmband of 1 suggests that a slight conformational tran-sition occurs in an n-BuOH solution. The structuralchange is probably an energetically favorable processwhere a helical compound accommodates to the sol-vent environment from the crystal packing state.

DISCUSSION

Figures 5 and 6 show the side and top views of 1 and3 to compare the folding geometries. The structure of1 contains both 310- and �-helical interactions. Thewhole helical chain is not straight but is slightly bentat the junction of the peptide and depsipeptide seg-ments, Leu3–Lac7. This is significantly different fromthe conformation of 3, which has a straight �-helicalstructure with standard � and � angles.

Here we discuss three interesting points by thecomparison of folding geometries of 1 and 3: (1) Doesester groups stabilize or destabilize the helical struc-ture? (2) What makes the �/310-conjugated helicalstructure? (3) What kind of force bends (approxi-mately 40°) the overall helical fold of 1? For the firstquestion, the answer is both. In the crystal structure ofdepsipeptides, we have observed evidence that repre-sent the stabilization and/or destabilization of helicesby the ester groups as discussed below.

The destabilization effect is found in the repulsiveinteraction between the ester oxygen,OOO, and thecarbonyl oxygen, CAO. The repulsion has been de-duced from the interatomic distances observed both in1 and 3 as shown in Table V. The distances are in therange of 3.11(1)–3.52(1) Å for [i, i�3] pairs, and3.61(1)–4.19(1) Å for [i, i�4] pairs as shown in TableV. The angles, OOO � � � OAC, are in the range of100.5(2)–111.1(2)° for [i, i�3] pairs, and 114.4(2)–125.9(2)° for [i, i�4] pairs. The [i, i�3] pairs haveshorter O � � � O distances and smaller angles ofO � � � OAC than those of the [i, i�4] pairs. If there isno interaction between two oxygen atoms, the inter-atomic length is expected to be in the range of 2.8–3.1Å, which is corresponding to the N � � � O length in an�-helix. Therefore, the O � � � O distance values are

FIGURE 4 Microcrystalline CD spectra of Boc–Leu–Leu–Ala–(Leu–Leu–Lac)3–Leu–Leu–OEt (1) (–––) andBoc–(Leu–Leu–Lac)3–Leu–Leu–OEt (3) ( ) using nu-jol–mulls (1 mg/1 mL) at 293 K.

Addition of a Peptide Fragment 249

significantly greater and clearly suggest the repulsiveforce between the electronegative oxygen atoms. Thelong O � � � O distances have been found in our helicaldepsipeptide, Boc–(Leu–Leu–Ala)2–(Leu–Leu–Lac)3–OEt (4) as listed in Table V.18 The distance values in4 are 3.24(2) and 3.30(2) Å for [i, i�3], and 3.80(2)and 3.87(2) Å for [i, i�4] interactions.18 Similarphenomena have been observed in the compounds15

Boc–Val–Val–Ala–Leu–Val–Lac–Leu–Aib–Val–Ala–Leu–OMe and Boc–Val–Ala–Leu–Aib–Val–Ala–Leu–Val–Lac–Leu–Aib–Val–Ala–Leu–OMe. Inboth helices, the O � � � O distances of [i, i�3] typesare 3.47 and 3.24 Å, respectively. These of [i, i�4]types are 3.81 and 3.87 Å, respectively.15 A relativelyshort distance is found in the crystal structure ofBoc–Val–Ala–Leu–Aib–Val–Lac–Leu–Aib–Val–Ala–Leu–OMe. In this case, the ester OOO residesin the helix with an OOO � � � OAC distance of 3.1Å.14 Two Aib residues probably prevent the distortionat around the Lac moiety.

Not only destabilization, the ester group also sta-bilizes helical structure when it works as a �-sheetbreaker. The formation of sheet or helix for 1 and 3can be considered from two governing factors. One isthe critical length to stabilize helices in a solid state.

The other is the repulsive force between an esteroxygen, OOO, and a carbonyl oxygen, CAO. Ac-cording to the conformational study of oligopeptides,there is a critical size to fold helices in the solidstates.79–88 Under the critical length, they invariablyexist in the sheet form. For Leu- and Ala-based se-quences, 10–14 residues are at least required to forma helical structure. In the case of pentadecapeptides,far-ir spectra have detected the transformation fromsheet to helix promoted by shear stress.81 Therefore,the depsipeptides could take either conformation de-pending on the chain length.

Antiparallel �-sheet structure is expected to bevery unstable due to the close contact and the repul-sive force between ester groups for the depsipeptidescontaining a –(Leu–Leu–Lac)n– sequence. In fact, theX-ray crystal analyses of Boc–Leu–Lac–OEt and Boc–(Leu–Leu–Lac)––OEt (n � 1 and 2) have revealed theexpelling interaction between oxygen atoms for their�-sheet arrangements.89

As a result of two governing factors, chain lengthand repulsion, helical conformations are probablychosen for 1 and 3 by nature. Therefore, we haveconcluded that the ester connections can prevent�-sheet formation and thus promote helix folding.

FIGURE 5 Helix bending at the junction of peptide and depsipeptide segments, Leu3–Lac7,observed for Boc–Leu–Leu–Ala–(Leu–Leu–Lac)3–Leu–Leu–OEt (1). The �- and 310-helical inter-actions are indicated as thin (- - - -) and bold (�) dotted lines, respectively.

250 Oku et al.

For the second question, a key to the answer isfound in the repulsive interaction betweenLeu3(CAO) and Lac7(OOO). To compensate forthe absence of the [i, i�4] hydrogen bond at the CAOof Leu3, there is a suitable NH at Leu6 closely locatedto this CAO. Then there appears the other hydrogenbond by an [i, i�3] interaction that forms a 310-helicalsegment between Leu3(CAO) and Leu6(NH). Fol-lowing the formation of the 310-helical segment, thereadjustment of hydrogen-bonding pattern probablyhappens throughout the helix. This is the answer tothe second question. The �/310-conjugated conforma-tion is thus the result of rearrangement from an ideal�-helix by the expelling force between electronega-tive oxygen atoms. A similar explanation is also ap-plicable for the formation of 310-type segments in 4.18

In this case, the repulsive interaction is observedbetween Leu6(CAO) and Lac10(OOO). The asso-ciative interaction is also found between Leu6(CAO)and Leu9(NH).

We have not yet clarified the reason why the dep-sipeptide 3 does not adopt a �/310-conjugated helix as

found in 1. At present we have estimated that thereason is in the sequence uniformity of 3, whichconsists of a simple repetition, –Leu–Leu–Lac–Leu–Leu–. To find out the mechanism of 310-helix forma-tion, we are now continuing the further study forvarious depsipeptide sequences.

To the third question, an answer is found in therepulsive and attractive force at the CAO of Leu3 asdiscussed above. This push-and-pull effect hindersfolding into a straight helix. Thus the overall structureof 1 is divided into two helices centered at Lac7.These domains can be classified as a peptide (Boc1–Leu6) and a depsipeptide (Lac7–Leu14) fragments.The conjugation of two different segments appears toreadjust 1 into the bent-helical conformation. Thebent angle between two helical domains is approxi-mately 140°. A bent conformation was also found inthe longer sequence, 4.18 In this structure, one [i, i�3]type of interaction was found at the junction of apeptide (Boc1–Leu9) and a depsipeptide (Lac10–Leu14) helix. This 310-helical segment located at thekink undoubtedly comes from the repulsive interac-

FIGURE 6 Straight helical structure observed for Boc–(Leu–Leu–Lac)3–Leu–Leu–OEt (3).�-Helical interactions are indicated as dotted lines (- - - -).

Addition of a Peptide Fragment 251

tion between oxygen atoms (Leu5 CAO and Lac9

OOO),18 as shown in Table V.The structural distortion is expected for the engi-

neered proteins, if an amino acid residue is replacedwith a corresponding hydroxy acid residue. For ex-ample, the helix-inducing effect by the depsipeptidescould prevent aggregation of model sequences of hu-man amylin.32

�/310-conjugated helices are often observed in var-ious enzymes and proteins such as in a �-class car-bonic anhydrase,90 a neutrophil gelatinase,91 a ubiq-uitin-conjugating enzyme Rad6,91 and a PII-homo-logue protein GlnK.93 In those cases, the helices seemto be independent and have no strong interactionswith other domains. Therefore to stabilize the localsegments by themselves, the helices probably choosethe �/310-conformation by forming both [i, i�4] and[i, i�3] interactions and thus maximizing the numberof hydrogen bonds in their structure.

CONCLUSION

We have successfully prepared a single crystal of 1 bythe segment condensation between a peptide fragment,Boc–Leu–Leu–Ala–OH, and a depsipeptide sequence,H–(Leu–Leu–Lac)3–Leu–Leu–OEt. From the crystallo-

graphic results, 1 adopts an �/310-conjugated helix witha kink at the junction of peptide and depsipeptide seg-ments, Leu3–Lac7. The bent structure is derived fromthe repulsive and the attractive interactions at Leu3

CAO with Lac7 OOO and Leu6 HN, respectively.Microcrystalline CD spectra were also useful to comparehelical properties of 1 (�/310) and 3 (�).

Supplementary Material: CCDC 236273, 236274, and236275 contain the supplementary crystallographic data for1, 3, and 4, respectively, in this article. These data can beobtained free of charge via www.ccdc.cam.ac.uk/data_re-quest/cif, by emailing data_request@ccdc.cam.ac.uk, or bycontacting The Cambridge Crystallographic Data Centre,12, Union Road, Cambridge CB2 1EZ, UK; fax: �44 1223336033.

This research was supported in part by a Grant-in-Aidfor Scientific Research on Priority Areas (HO, no.15036212 and 16033211, Reaction Control of DynamicComplexes) from the Ministry of Education, Culture,Sports, Science and Technology of Japan.

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Table V Intramolecular O- - -O Atomic Distances (A, deg) for Boc–Leu–Leu–Ala–(Leu–Leu–Lac)3–Leu–Leu–OEt(1), Boc–(Leu–Leu–Lac)3–Leu–Leu–OEt (3), Boc–(Leu–Leu–Ala)2–(Leu–Leu–Lac)3–OEt (4)a

Compound Ester —O— Ester or Amide OAC O- - -O O- - -OAC Type

1 O71 (Lac7) O31 (Leu3) 4.071 (9) 123.1 (6) i � 4O41 (Ala4) 3.40 (1) 104.4 (6) i � 3

O101 (Lac10) O61 (Leu6) 4.19 (1) 114.4 (2) i � 4O72 (Lac7) 3.508 (9) 99.1 (6) i � 3

O131 (Lac13) O91 (Leu9) 3.63 (1) 128.0 (8) i � 4O102 (Lac10) 3.150 (9) 110.2 (6) i � 3

3b O41 (Lac4) O11 (Boc1) 3.29 (2) 117 (1) i � 3O71 (Lac7) O31 (Leu3) 3.47 (3) 131 (1) i � 4

O42 (Lac4) 3.17 (2) 103 (1) i � 3O101 (Lac10) O61 (Leu6) 3.24 (3) 141 (1) i � 4

O72 (Lac7) 3.40 (3) 102 (1) i � 3

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a Inter atomic distances were picked up from the helical segments, not from the chain ends, such as —OEt.b Residue numbering scheme for 3: Boc1–Leu2–Leu3–Lac4–Leu5–Leu6–Lac7–Leu8–Leu9–Lac10–Leu11–Leu12–OEt13.c Residue numbering scheme for 4: Boc1–Leu2–Leu3–Ala4–Leu5–Leu6–Ala7–Leu8–Leu9–Lac10–Leu11–Leu12–Lac13–Leu14–Leu15–Lac16–

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Reviewing Editor: Dr. Murray Goodman

254 Oku et al.