Pseudopeptides Designed to Form Supramolecular Helixes: The Role

Dec 10, 2009 - E-mail: [email protected] (C.T.), [email protected] (G.F.). ... Boc-l-Phe-d-Oxd-(S,R)-β3-hPhg-OBn form crystals after e...
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DOI: 10.1021/cg9012558

Pseudopeptides Designed to Form Supramolecular Helixes: The Role of the Stereogenic Centers

2010, Vol. 10 923–929

Gaetano Angelici,† Nicola Castellucci,‡ Giuseppe Falini,*,‡ Daniel Huster,# Magda Monari,‡ and Claudia Tomasini*,‡ †

Department of Chemistry, University of Basel, St. Johanns-Ring 19 CH-4056 Basel, Switzerland, Dipartimento di Chimica “G. Ciamician” - Alma Mater Studiorum Universit a di Bologna - Via Selmi 2, I-40126 Bologna, Italy, and #Institute of Medical Physics and Biophysics - University of Leipzig - H€ artelstrasse 16-18, D-04107 Leipzig, Germany

‡

Received October 9, 2009; Revised Manuscript Received November 16, 2009

ABSTRACT: The two epimers Boc-L-Phe-D-Oxd-(S)-β3-hPhg-OBn (1) and Boc-L-Phe-D-Oxd-(R)-β3-hPhg-OBn (2) have been prepared by standard methods in solution, and their conformation was analyzed both in solution and in the solid state. While in solution 1 shows a random coil structure, 2 tends to assume a γ-turn conformation that is nearly retained in the solid state. On the other hand, in the solid state molecules of 1 associate generating a helix that involves the formation of elongated crystals with hexagonal cross-section. This effect is not observed in the crystals formed by Boc-L-Phe-D-Oxd-(R)-β3-hPhg-OBn 2.

Introduction In recent years, the self-assembly of small molecules is receiving a great deal of interest due to the possibility to form complex structures in a very simple and sustainable way.1 Applications in all fields of chemistry, materials science, and even pharmacological applications have already been demonstrated. In this context, the self-assembly of small peptides made of only 2-3 amino acids has been recently studied by Banerjee and co-workers, who demonstrated that one can obtain stable β-sheet or helix-like structures by interaction of small peptides that are held together by 2-3 hydrogen bonds.2 We have recently demonstrated that stable fibers can be obtained through self-assembly of small pseudopeptides that are connected by a single hydrogen bond, in a parallel or antiparallel structure.3 Moreover, other researchers demonstrated that very simple compounds such as L-Phe-L-Phe may produce complex structures such as helices or carbon nanotubes.4 Here we describe our recent results on the self-assembly of two epimers, Boc-L-Phe-D-Oxd-(S)-β3-hPhg-OBn (1) and Boc-L-Phe-D-Oxd-(R)-β3-hPhg-OBn (2). Both of these molecules contain the scaffold L-Phe-D-Oxd that we have recently demonstrated to be the basic pattern for the formation of fibers in the solid state, but differ for the introduction of the two enantiomers of β3-homophenylglycine. The reversal of the stereogenic center contained in this moiety has important effects on the preferred molecular conformation and, as a result, on the crystal morphology.

as white solids after purification by flash chromatography (cyclohexane/ethyl acetate 8/2 as eluants). The preferential conformation assumed by the two compounds was analyzed by FT-IR spectroscopy in diluted solution (3 mM) Figure 1. At this concentration, intermolecular interactions are negligible, so that the presence of intramolecular hydrogen bonds can be checked. Interestingly, 2 shows the presence of a chelate NH hydrogen bond, as demonstrated by the peaks centered at 3434 and 1670 cm-1, while 1 does not. This outcome suggests that only 2 forms a γ-turn structure. This outcome is confirmed by solution NMR spectroscopy, where we investigated the DMSO-d6 dependence of the NH proton chemical shifts. DMSO is a strong hydrogen bond acceptor and, if bound to a free NH proton, a considerable downfield shift of the proton signal can be expected. Both compounds have two NH groups, belonging to a carbamate group and an amide group, respectively. The results are reported in Figure S7, Supporting Information. While the chelation of the carbamate NH is uncertain for both compounds, we can notice that the Phe-NH group is poorly chelated for 1 and much more chelated for 2, thus confirming the outcome of the FT-IR spectra. The FT-IR spectra of 1 and 2 in the solid state are very similar and show that all the NH hydrogens are chelated, as all the stretching signals are below 3400 cm-1 (Figure 2). Scheme 1. (i) CF3CO2- þ3HN-(S)-β3-hPhg-OBn (1 equiv), HBTU (1.1 equiv), Et3N (3 equiv), dry CH3CN, 40 min, r.t.; (ii) CF3CO2- þ3HN-(R)-β3-hPhg-OBn (1 equiv), HBTU (1.1 equiv), Et3N (3 equiv), dry CH3CN, 40 min, r.t.

Results and Discussion Compounds 1 and 2 are easily prepared by coupling of the already described Boc-L-Phe-D-Oxd-OH moieties with (R)β3-hPhg-OBn or (S)-β3-hPhg-OBn, respectively, in high yield by standard methods in solution. The coupling steps are reported in Scheme 1. The compounds were obtained pure *To whom correspondence should be addressed. E-mail: claudia.tomasini@ unibo.it (C.T.), [email protected] (G.F.). r 2009 American Chemical Society

Published on Web 12/10/2009

pubs.acs.org/crystal

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Figure 1. FT-IR absorption spectra in the N-H (left) and CdO (right) stretching regions for 3 mM concentration samples of oligomers 1 and 2 in pure CH2Cl2 at room temperature.

Figure 2. FT-IR absorption spectra in the N-H (left) and CdO (right) stretching regions for a 1% solid mixture with KBr of oligomers 1 and 2 at room temperature.

Further interesting results are obtained by comparison of the 13C NMR spectra of 1 and 2 in solution and in the solid state (Table 1). As a reference, the chemical shifts of CR and Cβ of the phenylalanine residue in the peptide were measured in CDCl3 solution and furnished very similar outcomes (40.7 and 54.1 for 1 and 40.7 and 53.9 for 2), while significantly different chemical shifts were observed in the solid state. Two samples of each peptide, 10% 13C enriched for Phe, were prepared and analyzed by solid-state NMR. Both 1 and 2 have been evaporated under different conditions (1:1 mixture of cyclohexane/ethyl acetate and protonated solvents, methanol or ethanol). A significant difference in the chemical shifts of Phe in the tripeptides between solution and the solid state is observed. In the solid state, the chemical shifts of the Phe are indicative of random coil structures if compared to the standard secondary chemical shifts known from solution NMR data of proteins containing L-amino acids.5 The chemical shifts in solution indicate a tendency for β-sheet. However, having compounds featuring L- and D- as well as nonstandard amino acids, it is not straightforward to relate secondary chemical shift changes to secondary structure. From the analysis of the two compounds in solution and in the solid state, we can infer only that both compounds tend to aggregate in the solid state, without any information about how they do it. As 2 tends to form a γ-turn structure in solution, while 1 does not, we expect a different aggregation in the solid state. For this purpose, an analysis of the two compounds was carried out by means of microscopy and X-ray diffraction. A wide morphological variety of samples based on compounds 1 and 2 obtained by evaporation from different solvents were analyzed by optical and electronic microscopy.

Table 1. Comparison of the 13C NMR Results for the Phe Residue in Compounds 1 and 2 in Solution and in the Solid State sample δ Phe CR/ppm δ Phe Cβ/ppm 1

2

54.1 56.8

40.7 37.9

56.8 53.9 56.3

37.8 40.7 39.7

56.3

39.6

condition CDCl3 solution evaporated from cyclohexane/ ethyl acetate (1:1) evaporated from methanol CDCl3 solution evaporated from cyclohexane/ ethyl acetate (1:1) evaporated from methanol

The samples were precipitated by overnight evaporation of aliquots (20.0 mg each) from several solvents (methanol, ethanol, acetonitrile, cyclohexane/ethyl acetate, 1:1 mixture, or diethyl ether, 1.0 mL each). A fast precipitation was also achieved by rotary evaporation. The obtained solids showed different morphologies depending on the kind of solvent and the solvent evaporation rate. In any case, the precipitation of 1 provoked the formation of a solid material made of filamentous aggregates, in which the crystals showed a preferential direction of growth and had a strong tendency to aggregate laterally. This material always showed birefringence suggesting a high crystallinity (Figure 3a,c). Only when 1 was exsiccated by means of a rotary evaporator a powder-like material was obtained. Crystals of 1 suitable for a single crystal X-ray diffraction investigation were obtained only from methanol evaporation. An image of these crystals by scanning electron microscopy is reported in Figure 3c. They appear elongated in one direction with a hexagonal crosssection. Their length varied from one crystal to another in the range of several hundred of micrometers, while their thickness was almost constant around 20 μm. On the contrary, compound 2 did not always precipitate forming a filamentous

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Figure 3. (a) Optical micrographs of 1 precipitated from cyclohexane/ethyl acetate. The elongated growth of the crystal is observable together with their high lateral aggregation. (c) Single crystals of 1 precipitated from ethanol, they are birefringent and have a hexagonal cross-section. (b, d) crystals of 2 precipitated from ethanol observed by means of an optical and an electron microscope, respectively.

Figure 4. (Left) X-ray powder diffraction patterns of 1 precipitated from different solvents and exsiccated using a rotary evaporator. The calculated diffraction pattern from the structure of 1 obtained from crystals precipitated from methanol is shown as well. (Right) X-ray powder diffraction patterns of 2 precipitated from different solvents and exsiccated using a rotary evaporator. The calculated diffraction pattern from the structure of 2 obtained from crystals precipitated from ethanol is shown as well. Rotar indicate a material exsiccated by means of a rotary evaporator; MeOH, EtOH, MeCN, cHex/AcOEt, and Et2O indicate a material precipitated from methanol, ethanol, acetonitrile, cyclohexane/ ethyl acetate, and diethyl ether respectively; calc indicates the calculated diffraction pattern.

structure (data not shown). It precipitated as a mixture of powder and small (around 1 μm) elongated crystal aggregates from acetonitrile, cyclohexane/ethyl acetate, or diethyl ether, while by evaporation from protonated solvents, methanol or ethanol, elongated single crystals were obtained (Figure 3b,d). Of these, only the ones precipitated from ethanol were suitable for single crystal X-ray diffraction studies (see later). These crystals were also observed by scanning electron microscopy that showed an irregular morphology in which plate-like shapes are visible. The crystals of 1 and 2 that precipitated from different solvents were analyzed by X-ray powder diffraction. Compound 1 gave only one kind of crystals, irrespective of the solvent used (methanol, ethanol, acetonitrile, cyclohexane/ ethyl acetate, or diethyl ether) in the crystallization trials. Interestingly, the same kind of crystals precipitated even when the process was carried out in very harsh conditions, such as rotary evaporator (Figure 4a). This tendency to

precipitate in a unique conformation may indicate a high stability of this form, which may be already present in solution, before the precipitation process starts. Compound 2 showed a different behavior with respect to compound 1. It precipitated as a crystalline polymorph from methanol or acetonitrile, as a different polymorph from ethanol, and as a third polymorph from cyclohexane/ ethyl acetate or diethyl ether (Figure 4b). The exsiccation of 2 using a rotary evaporator provoked the formation of a low crystallinity material, which could be a new polymorph or a mixture of polymorphs already precipitated from other solvents. It is conceivable to consider that the precipitation of this latter polymorph is strongly affected by the polarity and protonation of the solvent, as well as by the rate of crystallization. It is also interesting to note that the material precipitated from ethanol contained traces of the polymorphs obtained after precipitation from methanol or acetonitrile (Figure 5b).

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Figure 5. X-ray molecular structures of the two epimers 1 (a) and 2 (b). Table 3. Most Relevant H Bond Parameters for 1 and 2

Table 2. Comparison of the Backbone Torsional Angles (°) for Molecules 1 and 2 torsion angles

1

2

C31-N2-C23-C22 (L-Phe1) (φ1) N2-C23-C22-N1 (ψ1) C22-N1-C3-C5 (φ2) N1-C3-C5-N3 (ψ2) C5-N3-C6-C13 (φ3) N3-C6-C13-C14 (ψ3)

-130.0(2) 154.6(2) 60.0(3) -126.5(2) 150.9 -67.6(3)

-132.6(2) 169.3(2) 74.2(3) -118.0(3) -105.9(3) 68.3(4)

Crystals suitable for an X-ray diffraction study were grown only by slow evaporation of a solution of 1 in methanol and of 2 in ethanol, both at room temperature. Any effort to grow from methanol crystals of 2, suitable for X-ray studies, was unsuccessful. The X-ray molecular structures of 1 and 2 are reported together in Figure 5, panels a and b, respectively, and relevant torsion angles for both are listed in Table 2. The two molecules are epimers and differ only for the configuration of the stereogenic center C6 which is S in 1 and R in 2. As a consequence of this different stereogenic center, 2 has a higher degree of folding than 1 (closer contact O8 3 3 3 H15A 3.05 A˚), but no classical intramolecular hydrogen bond is observed in both epimers, in contrast with the behavior of 2 in solution (Table 3). Interestingly, the crystal packing of 1 is completely different from those observed in the recently reported Boc-L-Phe-DOxd-OBn3a and Boc-(L-Phe-D-Oxd)2-OBn3b where single intermolecular NH 3 3 3 CO hydrogen bonds between neighbor units generate infinite parallel or antiparallel one-dimensional (1-D) H-bonded polymers, respectively. Each molecule of 1 is engaged in four intermolecular N-H 3 3 3 OdC hydrogen bonds of two types with the adjacent neighbors. Two interactions connect the amidic hydrogens of the side chain bearing the Boc group and the carbonylic oxygens of the amidic unit of the other side arm [N2-H2N 3 3 3 O30 , symm. op.: (I) -x þ y, -x, z þ 1/3; O3 3 3 3 H2N00 -N200 , symm. op.: (II) -y, x - y, -1/3 þ z], while the ones of the second type involve the amidic hydrogen H3N and the carbonylic oxygen of the D-Oxd ring [N3-H3N 3 3 3 O10 , O1 3 3 3 H3N00 -N300 ]. The crystal packing of 1 (Figure 6) therefore consists of parallel chains with a helical arrangement (Figure 7) running along the c axis. The formation of a ternary helix6 in the solid state is in agreement with the SEM analysis as the crystals of 1 from methanol appear elongated in one direction with a hexagonal crosssection (Figure 3c). A closer inspection of the crystal packing of 1 reveals the presence of weaker intermolecular non classical H bonds

D-H 3 3 3 A N3-H3N 3 3 3 O10 N2-H2N 3 3 3 O30

a

N2-H2N 3 3 3 O70 N3-H3N 3 3 3 O40

b

a

b

H 3 3 3 A, A˚

D 3 3 3 A, A˚

D-H 3 3 3 A, A˚

1 2.03 2.16

2.902(3) 2.964(3)

165(3) 151(3)

2 2.29(2) 2.44(2)

3.131(3) 3.230(4)

160(3) 149(3)

Symmetry operation for compound 1: (I) x þ y, -x, z þ 1/3. Symmetry operation for compound 2: (I) x - 1, y, z. a

b

Figure 6. View down the b axis of the crystal packing of 1 showing the N-H 3 3 3 OdC H bonds. Only the hydrogen atoms involved in H bonding are shown.

beside the already mentioned N-H 3 3 3 CO hydrogen bonds generating the 1-D network [Figure S1, Supporting Information]. In particular, two of these H bonds are established between the carbonylic oxygen O1 and the acidic proton of the 00 ˚, D-Oxd ring of an adjacent molecule [O1 3 3 3 H3 2.76 A 00 00 O1 3 3 3 H3 -C3 136°]. Other two weak H bonds are observed between the endocyclic oxygen of the D-Oxd ring and one proton of a phenyl ring [O2 3 3 3 H1200 2.65 A˚, O2 3 3 3 H1200 -C1200 128.5°]. All these intermolecular interactions further stabilize the 1-D network, being oriented almost parallel to the stronger N-H 3 3 3 CO hydrogen bonds. A completely different packing is observed for epimer 2 (Figure 8) which maintains one intermolecular NH 3 3 3 CO hydrogen bond between equivalent amidic groups

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[N2-H2N 3 3 3 O70 , symm. op.: x - 1, y, z] as observed in Boc3a L-Phe-D-Oxd-OBn and Boc-(L-Phe-D-Oxd)2-OBn.3b In addition, a second weaker hydrogen bond is observed between the amidic hydrogen of the other amide group and the uncoordinated carboxylic oxygen bearing the Bn group [N3-H3N 3 3 3 O40 ]. As a result, epimer 2 is engaged in four interactions that are weak in comparison with those found in Boc-L-Phe-D-Oxd-OBn3a and Boc-(L-Phe-D-Oxd)2-OBn,3b but the hydrogen bonding pattern in the crystal lattice shows the formation of infinite 1-D H-bonded polymer of tape running along the a axis.

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Figure 7. Space filling model showing the helical arrangement of one of the chains of 1: (a) top view, (b) view along the c axis. The molecules are represented in different colors for clarity. The hydrogen atoms involved in N-H 3 3 3 CO hydrogen bonds are represented as white cups.

The supramolecular assembly of 2 is completed by other hydrogen bonding interactions. In detail the 1-D supramolecular polymer of 2 is further stabilized by CH/π bonding between the acidic hydrogen (H6) of one molecule and one phenyl ring of the neighbors being 2.33 A˚ distance from the C7-C8-C9-C10-C11-C12 phenyl ring. As a difference with the crystal packing of 1, there are also weak interchain H bonds involving the endocyclic oxygen O2 of the D-Oxd ring and an aromatic hydrogen H19 [H19 3 3 3 O200 2.71 A˚, C19-H19 3 3 3 O200 ca. 150°]. To further investigate the effect of the absolute configuration variation of the β3-hPhg moiety on the properties of 1 and 2 in the solid state, single crystals of 1 and 2, obtained respectively, from methanol and ethanol, were also analyzed in their thermal properties by differential scanning calorimetry and thermogravimetry. Compound 1 undergoes an endothermic transition at a temperature of 170 °C, where it fuses converting to an amorphous material. After this first thermal event, 1 shows a successive broad endothermic peak having a temperature of onset at about 206 °C and maximum at about 222 °C (Figure 9). At 206 °C 1 starts to decompose, as shown by thermogravimetric analysis (TGA) (Figure S12, Supporting Information). Compound 2 shows a differential scanning calorimetry (DSC) profile similar to that of 1. First an endothermic peak appears at 167 °C, which is associated with a transition from the crystalline to an amorphous state

Figure 8. View down the c axis of the crystal packing of 2 showing the N-H 3 3 3 CO hydrogen bonds. Only the hydrogen atoms involved in N-H 3 3 3 CO hydrogen bonding are shown.

Figure 10. FT-IR absorption spectra in the CdO (right) stretching regions for oligomers 1 and 2 under different conditions. Black line: 1 in 3 mM solution in CH2Cl2; magenta line: 1 in 1% mixture with KBr; blue line: 1 crystallized from methanol, after heating at 180 °C (polymorph 1-II); red line: 2 in 3 mM solution of CH2Cl2; yellow line: 2 in 1% mixture with KBr; green line: 2 crystallized from ethanol, after heating at 180 °C (polymorph 2-II).

Figure 9. (Left) Differential scanning calorimetry profiles from single crystals of 1 and 2. (Right) X-ray powder diffraction patterns corresponding to different regions in the thermal profiles of 1 and 2.

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(Figure 9), and then a broad endothermic peak follows with an onset and maximum temperature of about 218 and 240 °C, respectively. The 1-II FT-IR spectrum in the CdO bands region presents a band at 1787 cm-1 and a broadband centered at about 1700 cm-1. A similar FT-IR spectral region is observed when 1 is present in a dilute solution of CH2Cl2 (Figure 10). This suggests that, as a consequence of the thermal treatment, after the first endothermic peak, compound 1 goes in an amorphous state, in which it assumes conformations similar to those in solution. In contrast, the IR analysis of a sample of 2 after thermal treatment does not show any peak at 1780 cm-1; thus, its amorphous state somehow still retains an ordered structure. Conclusions We reported the synthesis and the conformational analysis in solution and in the solid state of two pseudopeptides of the general formula Boc-L-Phe-D-Oxd-(S,R)-β3-hPhg-OBn. These two compounds form crystals after evaporation from protonated solvents, but the reversal of the absolute configuration of the stereogenic center of the hPhg moiety ends in a dramatic variation of the preferential conformation of the two compounds, that in turn induces a different crystal packing and consequently a different crystal morphology. Indeed, Boc-L-Phe-D-Oxd-(S)-β3-hPhg-OBn forms a ternary helix that crystallizes in hexagonal elongated crystals, while Boc-L-Phe3 D-Oxd-(R)-β -hPhg-OBn forms a 1-D H-bonded polymer of tape that crystallizes in different polymorphs, depending on the evaporation solvent. Experimental Section Synthesis. The melting points of the compounds were determined in open capillaries and are uncorrected. High quality infrared spectra (64 scans) were obtained at 2 cm-1 resolution using a 1 mm NaCl solution cell and a Nicolet 210 FT-infrared spectrometer. All spectra were obtained in 3 mM solutions in dry CH2Cl2 at 297 K or as a 1% solid mixture with dry KBr. All compounds were dried in vacuo, and all the sample preparations were performed in a nitrogen atmosphere. Routine NMR spectra were recorded with spectrometers at 400 or 300 MHz (1H NMR) and at 100 or 75 MHz (13C NMR). High quality 1H NMR spectra were recorded with a Varian Inova 600. The measurements were carried out in CDCl3 and in CD3OD. The proton signals were assigned by gCOSY spectra. Chemical shifts are reported in δ values relative to the solvent (CDCl3 or CD3OD) peak. The β-amino acids (S)-β3-hPhg (D-β3homophenylglycine) and (R)-β3-hPhg (L-β3-homophenylglycine) were prepared at DSM Research as described in PCT Patent Appl. WO 01/42173 and Eur. Patent Appl. No. 04075597.7 (patent pending), respectively. Boc-L-Phe-D-Oxd-(S)-β3-hPhg-OBn 1. A solution of Boc-L-Phe7 D-Oxd-OH (0.27 mmol, 0.10 g) and HBTU (0.3 mmol, 0.12 g) in dry acetonitrile (25 mL) was stirred under inert atmosphere for 10 min at room temperature. Then a mixture of H-(S)-β3-hPhg-OBn 3 TFA (0.27 mmol, 0.10 g) and Et3N (0.81 mmol, 0.12 mL) in dry acetonitrile (10 mL) was added at room temperature. The solution was stirred for 40 min under inert atmosphere, and then acetonitrile was removed under reduced pressure and replaced with ethyl acetate. The mixture was washed with brine, 1 N aqueous HCl (3  30 mL), and 5% aqueous NaHCO3 (1  30 mL), dried over sodium sulfate, and concentrated in vacuo. The product was obtained pure after silica gel chromatography (cyclohexane/ethyl acetate 8:2 as eluant) in 80% yield. mp = 168 °C; [R]D = þ14.0 (c = 1.0 in CH2Cl2); 1H NMR (CDCl3, 400 MHz; 25 °C): δ = 1.32 (d, 3H, 3J(H,H) = 7.2 Hz, Me-Oxd), 1.33 (s, 9H, t-Bu), 2.84-2.92 (m, 2H, CHH-Phe þ CHH-hPhg), 2.97 (dd, 1H, 3J(H,H) = 7.2, 15.6 Hz, CHH-Phe), 3.12 (dd, 1H, 3J(H,H) = 6.0, 13.6 Hz, CHH-hPhg),

Angelici et al. 4.32 (d, 1H, 3J(H,H) = 2.8 Hz, CHN-Oxd), 4.66 (dq, 1H, 3J(H,H) = 2.8, 7.2 Hz, CHO-Oxd), 5.03 (bs, 1H, NH-Boc), 5.05 (AB, 2H, 2 J(H,H) = 12.0 Hz, OCH2Ph), 5.46 (dt, 1H, 3J(H,H) = 7.2, 7.2 Hz, CHN-Phe), 5.64-5.67 (m, 1H, CHN-hPhg), 7.21-7.38 ppm (m, 16H, NH-hPhg þ 3 x Ph); 13C NMR (CDCl3, 100 MHz, 25 °C): δ = 21.1, 28.5, 40.7, 50.6, 54.1, 62.9, 66.8, 80.8, 126.8, 127.6, 128.0, 128.4, 128.5, 128.7, 129.0, 129.6, 151.83, 166.69 ppm; IR (CH2Cl2, 10 mM): ν = 3437 (N-H), 3366 (N-H), 1789 (CdO), 1716 (CdO), 1696 (CdO) cm-1; IR (1% in dry KBr): ν = 3358 (N-H), 3318 (N-H), 1785 (CdO), 1763 (CdO), 1735 (CdO), 1719 (CdO), 1701 (CdO), 1687 (CdO) cm-1; elemental analysis calcd. (%) for C35H39N3O8: C, 66.76; H, 6.24; N, 6.67; found: C 66.71, H 6.25, N 6.66. Boc-L-Phe-D-Oxd-(R)-β3-hPhg-OBn 2. A solution of Boc-L-Phe6 D-Oxd-OH (0.27 mmol, 0.10 g) and HBTU (0.3 mmol, 0.12 g) in dry acetonitrile (25 mL) was stirred under inert atmosphere for 10 min at room temperature. Then a mixture of H-(R)-β3-hPhg-OBn 3 TFA (0.27 mmol, 0.10 g) and Et3N (0.81 mmol, 0.12 mL) in dry acetonitrile (10 mL) was added at room temperature. The solution was stirred for 40 min under inert atmosphere, and then acetonitrile was removed under reduced pressure and replaced with ethyl acetate. The mixture was washed with brine, 1 N aqueous HCl (3  30 mL), and 5% aqueous NaHCO3 (1  30 mL), dried over sodium sulfate, and concentrated in vacuo. The product was obtained pure after silica gel chromatography (cyclohexane/ethyl acetate 8:2 as eluant) in 81% yield. mp = 160 °C; [R]D þ35.9 (c 1.0, CH2Cl2); 1H NMR (CDCl3, 400 MHz): δ = 1.23 (s, 9H, t-Bu), 1.31 (d, 3H, 3J(H,H) = 6.8 Hz, Me-Oxd), 2.85-2.92 (m, 2H, CHH-Phe þ CHH-hPhg), 2.99 (dd, 1H, 3J(H,H) = 8.4, 15.6 Hz, CHH-Phe), 3.15 (dd, 1H, 3J(H,H) = 6.0, 13.6 Hz, CHH-hPhg), 4.32 (d, 1H, 3 J(H,H) = 4.0 Hz, CHN-Oxd), 4.672 (dq, 1H, 3J(H,H) = 4.0, 6.8 Hz, CHO-Oxd), 5.00 (d, 1H, 3J(H,H) = 4 Hz, NH-Boc), 5.07 (s, 2H, OCH2Ph), 5.46 (dt, 1H, 3J(H,H) = 6.0, 8.4 Hz, CHN-Phe), 5.67-5.5.75 (m, 1H, CHN-hPhg), 7.20-7.36 (m, 15H, 3 x Ph), 7.54 ppm (d, 1H, 3J(H,H) = 7.6 Hz, NH-hPhg); 13C NMR (CDCl3, 100 MHz): δ = 21.1, 28.4, 29.9, 31.1, 37.6, 40.7, 50.9, 53.9, 62.7, 66.8, 75.3, 81.0, 126.4, 127.6, 127.7, 128.4, 128.5, 128.7, 128.8, 129.1, 129.6, 135.4, 135.7, 135.8, 140.5, 151.9, 167.1, 170.5, 173.6 ppm; IR (CH2Cl2, 10 mM): ν = 3436 (N-H), 3350 (N-H), 1788 (CdO), 1716 (CdO), 1693 (CdO), 1679 (CdO) cm-1; IR (1% in dry KBr): ν = 3381 (N-H), 3323 (N-H), 1764 (CdO), 1742 (CdO), 1719 (CdO), 1701 (CdO), 1691 (CdO), 1684 (CdO) cm-1; elemental analysis calcd. (%) for C35H39N3O8: C, 66.76; H, 6.24; N, 6.67; found: C 66.75, H 6.25, N 6.65. Crystal Precipitation. Several portions (20 mg each) of the purified compounds 1 and 2 dissolved in different solvents (ethanol, methanol, diethyl ether, acetonitrile, a 1:1 mixture of cyclohexane/ethyl acetate, 1 mL each) were crystallized by slow evaporation at room temperature. Samples suitable for single crystal X-ray diffraction were obtained from methanol (compound 1) and ethanol (compound 2). Microscopy. The precipitates were systematically observed by optical microscopy (OM) and scanning electron microscopy (SEM). The OM images were collected using a Leica optical microscope equipped with a CCD camera. Samples SEM images were collected on glass coverslip after coating with gold and observed using a Philips XL20 scanning electron microscope. The images were recorded using a CCD digital camera. Single Crystal X-ray Diffraction for 1 and 2. The X-ray intensity data for 1 and 2 were measured on a Bruker SMART Apex II CCD area detector diffractometer. Cell dimensions and the orientation matrix were initially determined from a least-squares refinement on reflections measured in three sets of 20 exposures, collected in three different ω regions, and eventually refined against all data. A full sphere of reciprocal space was scanned by 0.3° ω steps. The software SMART8 was used for collecting frames of data, indexing reflections, and determination of lattice parameters. The collected frames were then processed for integration by the SAINT program,9 and an empirical absorption correction was applied using SADABS.11 The structure was solved by direct methods (SIR 97)10 and subsequent Fourier syntheses and refined by full-matrix least-squares on F2 (SHELXTL),11 using anisotropic thermal parameters for all nonhydrogen atoms. All hydrogen atoms, except the amidic protons

Article and methine hydrogens, were added in calculated positions, included in the final stage of refinement with isotropic thermal parameters, U(H) = 1.2 Ueq(C) [U(H) = 1.5 Ueq(C-Me)], and allowed to ride on their carrier carbons. The absolute structure configuration was not determined from X-ray data, but was known from the synthetic route. Crystal data and details of the data collection for 1 and 2 are reported in Table S2, Supporting Information. X-ray Powder Diffraction Analysis. Powder X-ray diffraction patterns were collected using a PanAnalytical X’Pert Pro equipped with X’Celerator detector powder diffractometer using Cu KR radiation generated at 40 kV and 40 mA. The instrument was configured with a 1/32° divergence and 1/32° antiscattering slits. A standard quartz sample holder 1 mm deep, 20 mm high and 15 mm wide was used. The diffraction patterns were collected within the 2θ range from 3° to 40° with a step size (Δ 2θ) of 0.02° and a counting time of 300 s. Thermal Analysis. Calorimetric measurements were performed using a Perkin-Elmer DSC-7. Temperature and enthalphy calibrations were performed by using high purity standards (n-decane, benzene, and indium). The material (about 1.5 mg of sample) was sealed in aluminum pans. Heating was carried out at 3 °C min-1 in the temperature range 20-170 °C. Transition, denaturation, or desolvation temperature (TD) was determined as the maximum peak value of the corresponding endothermic phenomena. The value of denaturation enthalpy was calculated with respect to the mass of the sample. Weight loss during heating was evaluated by TGA using a calorimeter DSC-Q100 from TA Instruments Waters (USA). The temperature range was from 23 to 350 °C at a heating rate of 5 °C/min under dry nitrogen atmosphere. Solid-State NMR. For the solid-state NMR studies, powdered samples were filled into 4-mm MAS rotors with a Teflon insert providing a volume of approximately 50 μL. All 13C magic angle spinning (MAS) NMR experiments were carried out on a Bruker Avance 750 NMR (Bruker Biospin, Rheinstetten, Germany) spectrometer operating at a resonance frequency of 749.7 MHz for 1H and 188.5 MHz for 13C. A double-resonance MAS probe equipped with a 4 mm spinning module was used with a MAS frequency of 7 kHz at 30 °C. The 13C 90° pulse length was typically 5 μs. Crosspolarization (CP) MAS spectra were recorded with a 1H 90° pulse length of 4 μs and a CP contact time of 700 μs. The 1H radio frequency field strength during heteronuclear decoupling using TPPM12 was about 65 kHz. The 13C chemical shifts were referenced to the 13CdO signal of 13C-labeled Gly at 176.45 ppm as external standard. Thus, all ppm values are relative to TMS.13

Acknowledgment. G.A., N.C., G.F., M.M., and C.T. are grateful to Ministero dell’Universit a e della Ricerca Scientifica (PRIN 2006) and Universit a di Bologna (Funds for selected topics) for financial support. C.T. is grateful to Fondazione del Monte di Bologna e di Ravenna for financial support. D.H. thanks the DFG (SFB 610, A14) and the Experimental

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Physics Institutes of the University of Leipzig for measuring time the Avance 750 MHz NMR spectrometer. Furthermore, we thank DSM Research for a generous gift of (S)-β3-hPhg (D-β3-homophenylglycine) and (R)-β3-hPhg (L-β3-homophenylglycine). Supporting Information Available: Crystallographic data (cif files) for 1 and 2. IR and NMR spectra, supplemental crystallographic figures and thermogravimetric analysis of 1 and 2. This material is available free of charge via the Internet at http://pubs. acs.org.

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