Solution Structure Analysis using Circular Dichroism

The conformation of all synthesized peptides was further investigated in methanol by CD spectroscopy.³

Figure 90 shows the effect of the peptide chain length elongation on the dichroic properties of the oligopeptides. Peptides 167 (RS), 168 (RS)₂ and 169 (RS)₃ which have no helix character based on the NMR investigation, show two opposite CD bands near 210 nm (positive) and 222 nm (negative), with a crossover close to 215 nm. The intensity of the CD spectra decreases by increasing the peptide-chain length. Similarly, peptides 174 (SR)₂ and 175 (SR)₃ (no helix character based on NMR) are characterized by much less intense CD curves than dipeptide 173 (SR): they still show a broad minimum near 210 nm, whereas the positive shoulder of 173 at 230 nm disappears, until it becomes a weak negative shoulder for hexapeptide 175.

3 The interpretation of the measured data was accomplished with the help of Prof. Dr. C. Cabrele

Figure 90: CD-Spectra of compounds 167 - 169 (RS)_1-3 (panel A), 170 - 172 (SS)_1-3 (panel B), 173 - 175 (SR)1-3 (panel C) and 176 - 179 (RR)1-4 (panel D), recorded in methanol at a peptide concentration of 1mM.

Upon elongation of dipeptide 170 (SS) to tetrapeptide 171 (SS)2 and hexapeptide 172 (SS)₃ (right-handed 3₁₀-helix in the crystal), the minimum near 205 nm is slightly red-shifted and increases in intensity, whereas the positive CD contribution above 215 nm is substituted by a negative maximum near 222 nm, followed by a minimum near 234 nm.

The CD spectra of peptides 177 - 179 (RR)_2-4 (left-handed 3₁₀-helix in the crystal) are reminiscent of the one of dipeptide 176 (RR), but shifted towards positive ellipticity values, which leads to the appearance of a maximum near 210 nm and a positive valley near 222 nm. Moreover, a new positive band is detected near 233 nm, whose intensity increases when going from tetrapeptide 177 up to octapeptide 179. The corresponding enantiomers 171 (SS)2 and 172 (SS)3 also show this band, obviously opposite in sign, whereas the tetra- and hexapeptides from the (RS) and (SR) series do not (Figure 91): as only the peptides from the (RR) and (SS) series adopt a 310-helical structure in the crystal and show NOEs characteristic of helical structures, this band near 233 nm, together with the

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over 210-230 nm: 172 > 171, and 179 > 178 > 177).

Figure 91: Comparison of the CD-Spectra of the helical peptides 171, 172 (panel A) and 177 - 179 (panel B) with the non-helical oligomers 174, 175 and 168, 169, respectively.

We assume that the dipeptides contain the highest amount of disordered and irregular conformations. Therefore, the increase in the ordered peptide fraction of the larger peptides can be visualized by subtracting the CD spectrum of the dipeptides from the CD spectra of the larger peptides. This eliminates the CD contribution of the disordered fraction and possible CD contributions from the aromatic group of the TAA. The difference CD spectra for the four peptide series (SS, RR, SR, RS) are shown in Figure 92. The difference CD spectra obtained for the peptides (SS)_2-3 and (RR)_2-4 that adopt a right-handed and left-right-handed 310-helix, respectively, in the crystal are characterized by a red-shifted helix-like band pattern (panel A of Figure 92), with two minima for (171−170) and (172−170), or two maxima for (177−176), (178−176) and (179−176) near 210 nm and 230 nm.

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Figure 92: Difference CD spectra of oligopeptides (RR)_2-4 minus dipeptide (RR) and of oligopeptides (SS)2-3 minus dipeptide (SS) are shown in panel A. Difference CD spectra of oligopeptides (RS)_2-3 minus dipeptide (RS) and of oligopeptides (RS)_2-3 minus dipeptide (RS) are shown in panel B.

The remarkable red shift of the longer wavelength band can be due to the highly hydrophobic character of the peptides.²³⁷ The intensity of the spectra increases with the peptide length, suggesting an increase in ordered structure; moreover, for the tetrapeptides of both series the ratio between the ellipticity values at 230 nm and 210 nm is < 1, whereas for the larger peptides this ratio becomes > 1. This suggests that the tetrapeptides are likely to form both a 3₁₀- and an α-helical turn in methanol, whereas the larger peptides are able to build a stable short α-helix. The tendency of peptide chains with eight or more amino acids in length to form an preferred α-helical structure was previously discribed.^9a The difference CD spectra obtained for the peptides (SR)2-3 and (RS)_2-3 are completely different from those ones of the peptides (SS)_2-3 and (RR)_2-4, which excludes a helical conformation.

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2-1

3.4. Conclusion

In summary, 13 new peptides based on an alternated sequence of the S- or R-configured α-amino acid valine and the unnatural C^α-tetrasubstituted tetrahydrofuran α-amino acid rac-138 were prepared. Homo- and heterochiral stereoisomers with up to eight residues in length were systematically synthesized in good yields and high purity by solution phase chemistry. X-ray crystallography, NMR- and CD-measurements showed that all homochiral peptides, even the tetrapeptides, form helical structures in the solid state and in solution. The handedness of the helix is determined by the use of S-amino acids for right-handed or R-amino acids for left-handed peptide helices.

The stable and predictable secondary structure of the new peptides makes them suitable for applications as scaffolds and peptidomimetics. Additional moieties e.g. dyes, can be introduced by metal catalyzed functionalization of the brominated arene substituent (see Chapter 3).²¹⁸ Comparison of the crystallographic data of octamer 179 (RR)₄ with an idealized α-helix geometry reveals that the residues i, i+3 and i+6 of the octamer correspond to residues i, i+3 and i+7 of the natural α-helix. These positions are of importance for numerous protein-protein interactions, as the side chains of these residues are located on the same face of an amphipathic α–helical protein or peptide segment.²³⁸

Figure 93: Crystal structure of compound 179 (red ribbon, top) and of an α-helix (green ribbon, bottom). The i, i+3 and i+6 residues of peptide 179 are in close proximity to the i, i+3 and i+7 side chains of the natural α-helix (in this example alanine was used to indicate

3.5. Experimental Part

General

A Jasco Model J-710 spectropolarimeter was used. A 0.5 mm quartz cell was purchased from Hellma and Uvasol solvents from Merck. X-ray data collections were performed using an Oxford Gemini Ultra diffractometer. IR spectra were recorded on a Bio-Rad FT-IR FTS 155 and a Bio-Rad FTS 2000 MX FT-FT-IR using a Specac Golden Gate Mk II ATR accessory where stated. NMR spectrometers used were: Bruker Avance 600 (¹H: 600.1 MHz, ¹³C: 150.1 MHz, T = 300 K), Bruker Avance 400 (¹H: 400.1 MHz, ¹³C: 100.6 MHz, T

= 300 K) and Bruker Avance 300 (¹H: 300.1 MHz, ¹³C: 75.5 MHz, T = 300 K). The chemical shifts are reported in δ [ppm] relative to external standards (solvent residual peak). The spectra were analyzed by first order, the coupling constants are given in Hertz [Hz]. Characterization of the signals: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, bs = broad singlet, psq = pseudo quintet, dd = double doublet, dt = double triplet, ddd = double double doublet. Integration is determined as the relative number of atoms. Assignment of signals in ¹³C-spectra was determined with DEPT-technique (pulse angle: 135 °) and given as (+) for CH₃ or CH, (-) for CH₂ and (C_quat) for quaternary C. Error of reported values: chemical shift: 0.01 ppm for ¹H-NMR, 0.1 ppm for ¹³C-NMR and 0.1 Hz for coupling constants. The solvent used is reported for each spectrum. Mass spectra were recorded on Varian CH-5 (EI), Finnigan MAT 95 (CI; FAB and FD) and Finnigan MAT TSQ 7000 (ESI). Xenon served as the ionization gas for FAB. Melting Points were determined on a Büchi SMP-20 or Stanford Research System OpitMelt melting point apparatus and are uncorrected. Elemental analyses were carried out by the Center for Chemical Analysis of the Faculty of Natural Sciences of the University Regensburg.

All reagents and solvents used were of analytical grade purchased from commercial sources and were used without further purification. Unless stated otherwise, purification and drying of the solvents used was done according to accepted general procedures.²³⁹ All reactions were performed under an inert atmosphere of N2 using standard Schlenk techniques if not otherwise stated. TLC analyses were performed on silica gel 60 F-254 with a 0.2 mm layer thickness. Detection was via UV light at 254 nm / 366 nm or through discoloration with ninhydrin in EtOH. For preparative column-chromatography, Merck Geduran SI 60 (70-230 mesh) and Macherey-Nagel GmbH & Co. KG 60M (230–400 mesh) silica gels were used. For chromatography commercially available solvents of standard quality were used without further purification.

The unnatural amino acid Boc-TAA-OH rac-137 was synthesized according to a literature

(2S)-Methyl 2-(2-(4-bromophenyl)-3-(tert-butoxycarbonylamino)-tetrahydrofuran-3-carbox-amido)-3-methylbutanoate (167/170):

Under an atmosphere of nitrogen compound rac-138 (1.50 g, 3.88 mmol, 1 eq.) was dissolved in 3.9 ml DMF (1 ml/mmol) and cooled to 0 °C in an ice bath. To the solution DIPEA (1.99 ml, 11.7 mmol, 3 eq.), HOBt (793 mg, 5.83 mmol, 1.5 eq.) and EDC (1.03 ml, 5.83 mmol, 1.5 eq.) were added in this sequence. Then H-Val-OMe*HCl 180 (911 mg, 5.44 mmol, 1.4 eq.) was slowly added in portions. The mixture was allowed to warm to room temperature and stirred for 24 h. The reaction was quenched with 6 ml of water and 4 ml of 1M aqueous KHSO₄ and extracted with diethyl ether (3x10 ml). The combined organic layers were washed twice with brine. Afterwards the solution was dried over MgSO₄ and concentrated under reduced pressure. The crude product was then purified by column chromatography on flash silica gel (PE:diethyl ether 80:20) to give the product as two diasteriomers as colorless solids with an overall yield of 76 % (1.47 g, 2.94 mmol).

167: R_f (PE:diethyl ether 70:30) = 0.24

m/z (%) = 500.9 (100) [MH⁺], 518.0 (17) [MNH4+]. – Elemental analysis calcd. (%) for C₂₂H₃₁BrN₂O₆ (499.4): C 52.95, H 6.28, N 5.59; found: C 53.02, H 6.33, N 5.50. – IR (neat) [cm^-1]: ν^~ = 3384, 3271, 2959, 2518, 2361, 1725, 1673, 1527, 1491, 1437, 1373, 1211, 1147, 1072, 1009, 830, 802. – MF C₂₂H₃₁BrN₂O₆. – MW 499.4.