NMR‐Structural Investigations of a β3‐Dodecapeptide with Proteinogenic Side Chains in Methanol and in Aqueous Solutions

The structural properties of an all‐β³‐dodecapeptide with the sequence H‐β‐HLys(N^ε‐CO(CH₂)₃‐S Acm)‐β‐HPhe‐β‐HTyr‐β‐HLeu‐β‐HLys‐β‐HSer‐β‐HLys‐β‐HPhe‐β‐HSer‐β‐HVal‐β‐HLys‐β‐HAla‐OH ( 1 ) have been studied by two‐dimensional homonuclear ¹H‐NMR and by CD spectroscopy. In MeOH solution, high‐resolution NMR spectroscopy showed that the β‐dodecapeptide forms an (M)‐3₁₄‐helix, and the CD spectrum corresponds to the pattern expected for an (M)‐3₁₄‐helical secondary structure. In aqueous solution, however, the peptide adopts a predominantly extended conformation without regular secondary‐structure elements, which is in agreement with the absence of the characteristic trough near 215 nm in the CD spectrum. The NMR and CD measurements with solutions of 1 in MeOH containing 3M urea further indicated that the peptide retains the regular secondary structural elements under these conditions, whereas, after addition of 40% (v/v) H₂O to the MeOH solution, the large ¹H‐chemical‐shift dispersion indicative of a defined spatial peptide fold was lost. The β³‐dodecapeptide is – so far – the longest β‐peptide shown to adopt a regular (M)‐3₁₄‐helix conformation in an organic solvent. The observation that the structure of this long β³‐peptide is not maintained in aqueous solution indicates that the (M)‐3₁₄‐fold is primarily stabilized by short‐range interactions.     

Pleated Sheets and Turns of β-Peptides with Proteinogenic Side Chains

Components of a toolbox with predictable secondary structural elements: β-peptides. The β-peptide shown here with proteinogenic side chains adopts a parallel pleated sheet structure in the solid state upon incorporation of suitably configured β-amino acids. When a β-dipeptide turn segment is incorporated in the center, a hairpin is formed in solution.     

Conformation and Circular Dichroism of β,β-Carotene Derivatives with Nitrogen-, Sulfur-, and Selenium-Containing Substituents

The CD spectra of several 3-substituted azido-, amino-, thio-, phenylseleno- and (phosphonoseleno)-β,β-carotene derivatives with one or two stereogenic centers are discussed with respect to conformational changes of the cyclohexene end group. N- and S-Substituents (N₃, NH₂, SH) have no influence on the conformer equilibrium compared to O-substituents, whereas Se-substituents (SePh, SePO(OC₃H₇)₂) slightly destabilize the preferred conformer of the β-end group.     

NMR‐Solution Structures in Methanol of an α‐Heptapeptide,of a β3/β2‐Nonapeptide,and of an all‐β3‐Icosapeptide Carrying the 20 Proteinogenic Side Chains

The NMR‐solution structure of an α‐heptapeptide with a central Aib residue was investigated in order to verify that, in contrast to β‐peptides, short α‐peptides do not form a helical structures in MeOH. Although the central Aib residue was found to induce a bend in the experimentally determined structure, no secondary structure typical for longer α‐peptides or proteins was found. A β²/β³‐nonapeptide with polar, positively charged side chains was subjected to NMR analysis in MeOH and H₂O. Whereas, in MeOH, it folds into a 10/12‐helix very similar to the structure determined for a corresponding β²/β³‐nonapeptide with only aliphatic side chains, no dominant conformation could be determined in H₂O. Finally, the NMR analysis of a β³‐icosapeptide containing the side chains of all 20 proteinogenic amino acids in MeOH is described. It revealed that this 20mer folds into a 3₁₄‐helix over its whole length forming six full turns, the longest 3₁₄‐helix found so far. Together, our findings confirm that, in contrast to α‐peptides, β‐peptides not only form helices with just six residues, but also form helices that are longer than helical sections usually observed in proteins or natural peptides. The higher helix‐forming propensity of long β‐peptides is attributed to the conformation‐stabilizing effect of the staggered ethane sections in β‐peptides which outweighs the detrimental effect of the increasing macrodipole.     

Electrophilic S‐Trifluoromethylation of Cysteine Side Chains in α‐ and β‐Peptides: Isolation of Trifluoro‐methylated Sandostatin® (Octreotide) Derivatives

The new electrophilic trifluoromethylating 1‐(trifluoromethyl)‐benziodoxole reagents A and B (Scheme 1) have been used to selectively attach CF₃ groups to the S‐atom of cysteine side chains of α‐ and β‐peptides (up to 13‐residues‐long; products 7 – 14 ). Other functional groups in the substrates (amino, amido, carbamate, carboxylate, hydroxy, phenyl) are not attacked by these soft reagents. Depending on the conditions, the indole ring of a Trp residue may also be trifluoromethylated (in the 2‐position). The products are purified by chromatography, and identified by ¹H‐, ¹³C‐, and ¹⁹F‐NMR spectroscopy, by CD spectroscopy, and by high‐resolution mass spectrometry. The CF₃ groups, thus introduced, may be replaced by H (Na/NH₃), an overall Cys/Ala conversion. The importance of trifluoromethylations in medicinal chemistry and possible applications of the method (spin‐labelling, imaging, PET) are discussed.     

Helix‐Forming Oligoureas: Temperature‐Dependent NMR,Structure Determination,and Circular Dichroism of a Nonamer with Functionalized Side Chains

To further investigate the degree of structural homology between γ‐peptides A and N,N′‐linked oligoureas B , we prepared oligourea nonamer 2 containing Ala, Val, Leu, Phe, Tyr and Lys side chains. Oligomer 2 was synthesized on solid support from activated monomers, i.e., from enantiomerically pure succinimidyl {2‐{[(9H‐fluoren‐9‐ylmethoxy)carbonyl]amino}ethyl}carbamates 3a – f that are further substituted at C(2) of the ethyl moiety. These precursors were conveniently prepared from N‐Fmoc‐protected β³‐amino acids with corresponding side chains. Detailed NMR studies (DQF‐COSY, TOCSY, and ROESY) in (D₅)pyridine revealed that 2 adopts a regular (P)‐2.5 helical secondary structure very similar to that previously determined for oligourea heptamer 1 and closely related to the (P)‐2.6₁₄ helix of γ‐peptides. Temperature‐dependent NMR further demonstrated the conformational homogeneity and remarkable stability of the structure of 2 in pyridine. The CD spectrum of 2 (0.2 mM ) was recorded in MeOH with the aim to gain more information about the conformation of oligoureas. In contrast to 2.6‐helical γ‐peptides, which display only a weak or no Cotton effect, oligourea 2 exhibits an intense positive Cotton effect at ca. 203 nm ([Θ]=+373000 deg cm² dmol⁻¹) that decreases only slowly upon increasing the temperature.     

Synthesis,and Helix or Hairpin‐Turn Secondary Structures of ‘Mixed’ α/β‐Peptides Consisting of Residues with Proteinogenic Side Chains and of 2‐Amino‐2‐methylpropanoic Acid (Aib)

Twelve peptides, 1 – 12 , have been synthesized, which consist of alternating sequences of α‐ and β‐amino acid residues carrying either proteinogenic side chains or geminal dimethyl groups (Aib). Two peptides, 13 and 14 , containing 2‐methyl‐3‐aminobutanoic acid residues or a ‘random mix’ of α‐, β²‐, and β³‐amino acid moieties were also prepared. The new compounds were fully characterized by CD (Figs. 1 and 2), and ¹H‐ and ¹³C‐NMR spectroscopy, and high‐resolution mass spectrometry (HR‐MS). In two cases, 3 and 14 , we discovered novel types of turn structures with nine‐ and ten‐membered H‐bonded rings forming the actual turns. In two other cases, 8 and 11 , we found 14/15‐helices, which had been previously disclosed in mixed α/β‐peptides containing unusual β‐amino acids with non‐proteinogenic side chains. The helices are formed by peptides containing the amino acid moiety Aib in every other position, and their backbones are primarily not held together by H‐bonds, but by the intrinsic conformations of the containing amino acid building blocks. The structures offer new possibilities of mimicking peptide–protein and protein–protein interactions (PPI).     

Investigation of the Interactions of β‐Peptides with DNA Duplexes by Circular Dichroism Spectroscopy

The interaction of β‐peptides with the DNA duplexes of dA₂₀dT₂₀ and a GCN4‐binding CRE sequence was examined. To gauge the factors that govern these interactions, two β‐pentadecapeptides, 1 and 2 , a β‐dodecapeptide, 3 , three β‐decapeptides, 4 – 6 , three β‐heptapeptides, 7 – 9 , and β‐octaarginine 10 were designed and synthesized. The β‐peptides were conceived to adopt a β‐peptide 3₁₄ helix, in which the side chains at position i and i + 3 are aligned vertically along one side of the helix. The side chains of Lys, Asn, and Arg were positioned such that potential H‐bonding sites were created for a helical conformation to interact with the base pairs of DNA. CD Analysis showed that β‐peptides 1, 2 , and 10 interacted with dA₂₀dT₂₀. In addition, β‐peptides 1 and 2 showed significant interaction with a DNA‐duplex 20mer containing the ATF/CREB recognition sequence for the regulatory protein GCN4. It is impossible, at this stage of the investigation, to make a safe proposal about the actual nature of the interaction of the structures(s) of the complexes, the formation of which is suggested by the CD spectra reported herein.     

Synthesis of β‐Peptides with β‐Helices from New C‐Linked Carbo‐β‐Amino Acids: Study on the Impact of Carbohydrate Side Chains

The design and synthesis of β‐peptides from new C‐linked carbo‐β‐amino acids (β‐Caa) presented here, provides an opportunity to understand the impact of carbohydrate side chains on the formation and stability of helical structures. The β‐amino acids, Boc‐(S)‐β‐Caa_(g)‐OMe 1 and Boc‐(R)‐β‐Caa_(g)‐OMe 2 , having a D ‐galactopyranoside side chain were prepared from D ‐galactose. Similarly, the homo C‐linked carbo‐β‐amino acids (β‐hCaa); Boc‐(S)‐β‐hCaa_(x)‐OMe 3 and Boc‐(R)‐β‐hCaa_(x)‐OMe 4 , were prepared from D ‐glucose. The peptides derived from the above monomers were investigated by NMR, CD, and MD studies. The β‐peptides, especially the shorter ones obtained from the epimeric (at the amine stereocenter C_β) 1 and 2 by the concept of alternating chirality, showed a much smaller propensity to form 10/12‐helices. This substantial destabilization of the helix could be attributed to the bulkier D ‐galactopyranoside side chain. Our efforts to prepare peptides with alternating 3 and 4 were unsuccessful. However, the β‐peptides derived from alternating geometrically heterochiral (at C_β) 4 and Boc‐(R)‐β‐Caa_(x)‐OMe 5 (D ‐xylose side chain) display robust right‐handed 10/12‐helices, while the mixed peptides with alternating 4 and Boc‐β‐hGly‐OMe 6 (β‐homoglycine), resulted in left‐handed β‐helices. These observations show a distinct influence of the side chains on helix formation as well as their stability.     

Efficient Access to Enantiopure γ4‐Amino Acids with Proteinogenic Side‐Chains and Structural Investigation of γ4‐Asn and γ4‐Ser in Hybrid Peptide Helices

Hybrid peptides composed of α‐ and β‐amino acids have recently emerged as new class of peptide foldamers. Comparatively, γ‐ and hybrid γ‐peptides composed of γ⁴‐amino acids are less studied than their β‐counterparts. However, recent investigations reveal that γ⁴‐amino acids have a higher propensity to fold into ordered helical structures. As amino acid side‐chain functional groups play a crucial role in the biological context, the objective of this study was to investigate efficient synthesis of γ⁴‐residues with functional proteinogenic side‐chains and their structural analysis in hybrid‐peptide sequences. Here, the efficient and enantiopure synthesis of various N‐ and C‐terminal free‐γ⁴‐residues, starting from the benzyl esters (COOBzl) of N‐Cbz‐protected (E)‐α,β‐unsaturated γ‐amino acids through multiple hydrogenolysis and double‐bond reduction in a single‐pot catalytic hydrogenation is reported. The crystal conformations of eight unprotected γ⁴‐amino acids (γ⁴‐Val, γ⁴‐Leu, γ⁴‐Ile, γ⁴‐Thr(OtBu), γ⁴‐Tyr, γ⁴‐Asp(OtBu), γ⁴‐Glu(OtBu), and γ‐Aib) reveals that these amino acids adopted a helix favoring gauche conformations along the central C^γ? C^β bond. To study the behavior of γ⁴‐residues with functional side chains in peptide sequences, two short hybrid γ‐peptides P1 (Ac‐Aib‐γ⁴‐Asn‐Aib‐γ⁴‐Leu‐Aib‐γ⁴‐Leu‐CONH₂) and P2 (Ac‐Aib‐γ⁴‐Ser‐Aib‐γ⁴‐Val‐Aib‐γ⁴‐Val‐CONH₂) were designed, synthesized on solid phase, and their 12‐helical conformation in single crystals were studied. Remarkably, the γ⁴‐Asn residue in P1 facilitates the tetrameric helical aggregations through interhelical H bonding between the side‐chain amide groups. Furthermore, the hydroxyl side‐chain of γ⁴‐Ser in P2 is involved in the interhelical H bonding with the backbone amide group. In addition, the analysis of 87 γ⁴‐residues in peptide single‐crystals reveal that the γ⁴‐residues in 12‐helices are more ordered as compared with the 10/12‐ and 12/14‐helices.     

Do Valine Side Chains Have an Influence on the Folding Behavior of β‐Substituted β‐Peptides?

The influence of valine side chains on the folding/unfolding equilibrium and, in particular, on the 3₁₄‐helical propensity of β³‐peptides were investigated by means of molecular‐dynamics (MD) simulation. To that end, the valine side chains in two different β³‐peptides were substituted by leucine side chains. The resulting four peptides, of which three have never been synthesized, were simulated for 150 to 200 ns at 298 and 340 K, starting from a fully extended conformation. The simulation trajectories obtained were compared with respect to structural preferences and folding behavior. All four peptides showed a similar folding behavior and were found to predominantly adopt 3₁₄‐helical conformations, irrespective of the presence of valine side chains. No other well‐defined conformation was observed at significant population in any of the simulations. Our results imply that β³‐peptides show a structural preference for 3₁₄‐helices independent of the branching nature of the side chains, in contrast to what has been previously proposed on the basis of circular‐dichroism (CD) measurements.     

Conformation Analysis of Ile-Ala-Val-Pro Peptide and Its Derivatives by Circular Dichroism

Ile-Ala-Val-Pro as a hypocholesterolemic peptide was isolated from soybean protein. We have synthesized four peptides, Ile-Ala-Val-Pro-Gly-Glu-Val-Ala, Leu-Ile-Ala-Val-Pro-Gly-Glu-Val-Ala, Ile-Ala-Val-Pro-Thr-Gly-Val-Ala, Leu-Ile-Ala-Val-Pro-Thr-Gly-Val-Ala, with a conserved Ile-Ala-Val-Pro amino acid sequence, for circular dichroism investigations. These four peptide sequences were also found in the amino acid sequence in soybean protein, which was defined from the genomic sequence. Additionally for a detailed analysis of conformation features of these peptides, the Ile-Ala-Val-Pro and Leu-Ile-Ala-Val-Pro were also synthesized. All peptides were prepared using standard fluorenylmethyloxycarbonyl methodology and the peptide yields ranged from 90 to 95% of the theoretical yields with purity after purification above 99%.     

苯甲酰基修饰环糊精衍生物的圆二色性与分子构象

环糊精(简称ＣＤ)分子的独特结构特点和性能使其成为超分子化学中重要的主体模型之一[1,2].ＣＤ及其衍生物在分子识别,模拟酶等领域的研究和应用受到广泛重视[3].光活性组分的超分子体系可通过光诱导能量传递进行选择性光化学反应[4].我们与日本Ｉｎｏｕｅ等以单(6ｏ苯甲酰基)βＣＤ为光增感剂,研究了顺式环辛烯的光异构化反应[5],但对于反应过程中ＣＤ衍生物构象有何变化,反应发生在ＣＤ空腔内还是空腔外等反应机理尚不清楚.为了研究环辛烯光异构化反应机理并提供新的光增感剂,我们合成并报导了一系列含发色团的ＣＤ衍生物[6,7].通过研究客…     

Preparation of β2‐Homotryptophan Derivatives for β‐Peptide Synthesis

In view of the prominent role of the 1H‐indol‐3‐yl side chain of tryptophan in peptides and proteins, it is important to have the appropriately protected homologs H‐β² HTrp OH and H‐β³ HTrp OH (Fig.) available for incorporation in β‐peptides. The β²‐HTrp building block is especially important, because β²‐amino acid residues cause β‐peptide chains to fold to the unusual 12/10 helix or to a hairpin turn. The preparation of Fmoc and Z β²‐HTrp(Boc) OH by Curtius degradation (Scheme 1) of a succinic acid derivative is described (Schemes 2–4). To this end, the (S)‐4‐isopropyl‐3‐[(N‐Boc‐indol‐3‐yl)propionyl]‐1,3‐oxazolidin‐2‐one enolate is alkylated with Br CH₂CO₂Bn (Scheme 3). Subsequent hydrogenolysis, Curtius degradation, and removal of the Evans auxiliary group gives the desired derivatives of (R)‐H β²‐HTrp OH (Scheme 4). Since the (R)‐form of the auxiliary is also available, access to (S)‐β²‐HTrp‐containing β‐peptides is provided as well.     

Enantioselective Preparation of β2‐Amino Acids with Aspartate,Glutamate, Asparagine,and Glutamine Side Chains

(S)‐β²‐Homoamino acids with the side chains of Asp, Glu, Asn, and Gln have been prepared and suitably protected (N‐Fmoc, CO₂^tBu, CONHTrt) for solid‐phase peptide syntheses. The key steps of the syntheses are: N‐acylation of 5,5‐diphenyl‐4‐isopropyl‐1,3‐oxazolidin‐2‐one (DIOZ) with succinic and glutaric anhydrides (Scheme 2), alkylation of the corresponding Li‐enolates with benzyl iodoacetate and Curtius degradation (Scheme 4), and removal of the chiral auxiliary (Scheme 5). In addition, numerous functional‐group manipulations (CO₂H?CO₂^tBu, CO₂Bn?CO₂H, CbzNH→FmocNH, CO₂H→CO₂NH₂→CONHTrt; Schemes 2, 4, 5, and 6) were necessary, in order to arrive at the four target structures. The configurational assignments were confirmed by X‐ray crystal‐structure determinations (Scheme 2 and Fig. 3). The enantiomeric purities of a β²hAsn and of a β²hGln derivative were determined by HPLC on a Chiralcel column to be 99.7 : 0.3 and >99 : 1, respectively (Fig. 4). Notably, it took up to twelve steps to prepare a suitably protected trifunctional product with a single stereogenic center (overall yield of 10% from DIOZ and succinic anhydride)!     

Enantioselective Palladium‐Catalyzed Oxidative β,β‐Fluoroarylation of α,β‐Unsaturated Carbonyl Derivatives

The site‐selective palladium‐catalyzed three‐component coupling of deactivated alkenes, arylboronic acids, and N‐fluorobenzenesulfonimide is disclosed herein. The developed methodology establishes a general, modular, and step‐economical approach to the stereoselective β‐fluorination of α,β‐unsaturated systems.