Linear,Peptidase‐Resistant β2/β3‐Di‐ and α/β3‐Tetrapeptide Derivatives with Nanomolar Affinities to a Human Somatostatin Receptor,Preliminary Communication

N‐Acyl‐β²/β³‐dipeptide‐amide somatostatin analogs, 5 – 8 , with β²‐HTrp‐β³‐HLys ('natural' sequence) and β²‐HLys‐β³‐HTrp (retro‐sequence) have been synthesized (in solution). Depending on their relative configurations and on the nature of the terminal N‐acyl and terminal C‐amino group, the linear β‐dipeptide derivatives have affinities for the human receptor hsst 4, ranging from 250 to >10000 nanomolar (Fig. 3). Also, N‐Ac‐tetrapeptide amides 9 and 10 , which contain one α‐ and three β‐amino acid residues (N‐β‐α‐β‐β‐C), have been prepared (solid‐phase synthesis), with the natural (Phe, Trp, Lys, Thr) and the retro‐sequence (Thr, Lys, Trp, Phe) of side chains and with two different configurations, each, of the two central amino acid residues. The novel ‘mixed', linear α/β‐peptides have affinities for the hsst 4 receptor ranging from 23 to >10000 nanomolar (Fig. 4), and, like ‘pure' β‐peptides, they are completely stable to a series of proteolytic enzymes. Thus, the peptidic turn of the cyclic tetradecapeptide somatostatin (Fig. 1) can be mimicked by simple linear di‐ and tetrapeptides. The tendency of β‐dipeptides for forming hydrogen‐bonded rings is confirmed by calculations at the B3LYP/6‐31G(d,p) level (Fig. 2). The reported results open new avenues for the design of low‐molecular‐weight peptidic drugs.     

Stereoselective Preparation of 3‐Amino‐2‐fluoro Carboxylic Acid Derivatives,and Their Incorporation in Tetrahydropyrimidin‐4(1H)‐ones,and in Open‐Chain and Cyclic β‐Peptides

The preparation of (2S,3S)‐ and (2R,3S)‐2‐fluoro and of (3S)‐2,2‐difluoro‐3‐amino carboxylic acid derivatives, 1 – 3 , from alanine, valine, leucine, threonine, and β³h‐alanine (Schemes 1 and 2, Table) is described. The stereochemical course of (diethylamino)sulfur trifluoride (DAST) reactions with N,N‐dibenzyl‐2‐amino‐3‐hydroxy and 3‐amino‐2‐hydroxy carboxylic acid esters is discussed (Fig. 1). The fluoro‐β‐amino acid residues have been incorporated into pyrimidinones ( 11 – 13 ; Fig. 2) and into cyclic β‐tri‐ and β‐tetrapeptides 17 – 19 and 21 – 23 (Scheme 3) with rigid skeletons, so that reliable structural data (bond lengths, bond angles, and Karplus parameters) can be obtained. β‐Hexapeptides Boc[(2S)‐β³hXaa(αF)]₆OBn and Boc[β³hXaa(α,αF₂)]₆‐OBn, 24 – 26 , with the side chains of Ala, Val, and Leu, have been synthesized (Scheme 4), and their CD spectra (Fig. 3) are discussed. Most compounds and many intermediates are fully characterized by IR‐ and ¹H‐, ¹³C‐ and ¹⁹F‐NMR spectroscopy, by MS spectrometry, and by elemental analyses, [α]_D and melting‐point values.     

Design,Synthesis, NMR‐Solution and X‐Ray Crystal Structure of N‐Acyl‐γ‐dipeptide Amides That Form a βII′‐Type Turn

Conformational analysis of γ‐amino acids with substituents in the 2‐position reveals that an N‐acyl‐γ‐dipeptide amide built of two enantiomeric residues of unlike configuration will form a 14‐membered H‐bonded ring, i.e., a γ‐peptidic turn (Figs. 1 – 3). The diastereoselective preparation of the required building blocks was achieved by alkylation of the doubly lithiated N‐Boc‐protected 4‐aminoalkanoates, which, in turn, are readily available from the corresponding (R)‐ or (S)‐α‐amino acids (Scheme 1). Coupling two such γ‐amino acid derivatives gave N‐acetyl and N‐[(tert‐butoxy)carbonyl] (Boc) dipeptide methyl amides ( 1 and 10 , resp.; Fig. 2, Scheme 2); both formed crystals suitable for X‐ray analysis, which confirmed the turn structures in the solid state (Fig. 4 and Table 4). NMR Analysis of the acetyl derivative 1 in CD₃OH, with full chemical‐shift and coupling assignments, and, including a 300‐ms ROESY measurement, revealed that the predicted turn structure is also present in solution (Fig. 5 and Tables 1 – 3). The results described here are yet another piece of evidence for the fact that more stable secondary structures are formed with a decreasing number of residues, and with increasing degree of predictability, as we go from α‐ to β‐ to γ‐peptides. Implications of the superimposable geometries of the actual turn segments (with amide bonds flanked by two quasi‐equatorial substituents) in α‐, β‐, and γ‐peptidic turns are discussed.     

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.     

Preparation of Protected β2‐ and β3‐Homocysteine, β2‐ and β3‐Homohistidine,and β2‐Homoserine for Solid‐Phase Syntheses

The Ser, Cys, and His side chains play decisive roles in the syntheses, structures, and functions of proteins and enzymes. For our structural and biomedical investigations of β‐peptides consisting of amino acids with proteinogenic side chains, we needed to have reliable preparative access to the title compounds. The two β³‐homoamino acid derivatives were obtained by Arndt–Eistert methodology from Boc‐His(Ts)‐OH and Fmoc‐Cys(PMB)‐OH (Schemes 2–4), with the side‐chain functional groups' reactivities requiring special precautions. The β²‐homoamino acids were prepared with the help of the chiral oxazolidinone auxiliary DIOZ by diastereoselective aldol additions of suitable Ti‐enolates to formaldehyde (generated in situ from trioxane) and subsequent functional‐group manipulations. These include OH→O^tBu etherification (for β²hSer; Schemes 5 and 6), OH→STrt replacement (for β²hCys; Scheme 7), and CH₂OH→CH₂N₃→CH₂NH₂ transformations (for β²hHis; Schemes 9–11). Including protection/deprotection/re‐protection reactions, it takes up to ten steps to obtain the enantiomerically pure target compounds from commercial precursors. Unsuccessful approaches, pitfalls, and optimization procedures are also discussed. The final products and the intermediate compounds are fully characterized by retention times (t_R), melting points, optical rotations, HPLC on chiral columns, IR, ¹H‐ and ¹³C‐NMR spectroscopy, mass spectrometry, elemental analyses, and (in some cases) by X‐ray crystal‐structure analysis.     

Solid‐Phase Synthesis of a β‐Dodecapeptide with Seven Functionalized Side Chains and CD‐Spectroscopic Evidence for a Dramatic Structural Switch When Going from Water to Methanol Solution

An all‐β³‐dodecapeptide with a protected N‐terminal thiol‐anchoring group and with seven side chains has been synthesized in multi‐mg amounts by the manual solid‐phase technique, applying Fmoc methodology and the Wang resin. The sequence is β‐HLys‐β‐HPhe‐β‐HTyr‐β‐HLeu‐β‐HLys‐β‐HSer‐β‐HLys‐β‐HPhe‐β‐HSer‐β‐HVal‐β‐HLys‐β‐HAla‐OH (from N‐ to C‐terminus; see 1 ). The functional groups in the side chains of the building blocks were Boc (β‐HLys) or t‐Bu ether (β‐HSer, β‐HTyr) protected to allow for simultaneous deprotection and detachment from the resin with trifluoroacetic acid. All coupling steps were achieved with HBTU (=O‐(1H‐benzotriazol‐1‐yl)‐1,1,3,3‐tetramethyl uronium hexafluorophosphate)/HOBt (=1‐hydroxy‐1H‐benzotriazole) in DMF. For Fmoc (=(9H‐fluoren‐9‐yl)methoxycarbonyl) deprotection, a protocol was developed to surmount the previously reported problems arising in solid‐phase synthesis of β‐peptides when the chain length exceeds seven or eight amino‐acid moieties: for up to seven amino acids, a 20% solution of piperidine in DMF was used for removal of Fmoc; for the subsequent five amino acids, DBU and piperidine were employed for complete deprotection. The crude product was purified by preparative reversed‐phase HPLC, and the yield of pure β‐dodecapeptide derivative ( 1 ) was 23%. As the compound is well‐soluble in H₂O, it was characterized by ¹H‐NMR (in MeOH and H₂O), ¹³C‐NMR (in MeOH), and CD spectroscopy (in MeOH and in H₂O at pH values ranging from 3.5 to 11), and its molecular weight and composition were confirmed by high‐resolution mass spectrometry (Figs. 1 – 4). In MeOH solution, the β‐dodecapeptide exhibits the expected CD pattern typical of an (M)‐3₁₄‐helical secondary structure. In H₂O, however, the characteristic trough near 215 nm is missing in the CD spectrum, only a strong positive Cotton effect at 202 nm was observed, indicating the presence of β‐peptidic secondary structures, containing ten‐membered H‐bonded rings, such as the 12/10 helix (Fig. 4, right) or the hairpin. Only a detailed NMR solution‐structure analysis will provide the clues necessary for understanding the effects leading to the observed dramatic structural change of the highly functionalized β‐dodecapeptide described.     

New Open‐Chain and Cyclic Tetrapeptides,Consisting of α‐, β2‐, and β3‐Amino‐Acid Residues,as Somatostatin Mimics – A Survey

Cyclo‐β‐tetrapeptides are known to adopt a conformation with an intramolecular transannular hydrogen bond in solution. Analysis of this structure reveals that incorporation of a β²‐amino‐acid residue should lead to mimics of ‘α‐peptidic β‐turns’ (cf. A, B, C ). It is also known that short‐chain mixed β/α‐peptides with appropriate side chains can be used to mimic interactions between α‐peptidic hairpin turns and G protein‐coupled receptors. Based on these facts, we have now prepared a number of cyclic and open‐chain tetrapeptides, 7 – 20 , consisting of α‐, β²‐, and β³‐amino‐acid residues, which bear the side chains of Trp and Lys, and possess backbone configurations such that they should be capable of mimicking somatostatin in its affinity for the human SRIF receptors (hsst_1–5). All peptides were prepared by solid‐phase coupling by the Fmoc strategy. For the cyclic peptides, the three‐dimensional orthogonal methodology (Scheme 3) was employed with best success. The new compounds were characterized by high‐resolution mass spectrometry, NMR and CD spectroscopy, and, in five cases, by a full NMR‐solution‐structure determination (in MeOH or H₂O; Fig. 4). The affinities of the new compounds for the receptors hsst_1–5 were determined by competition with [¹²⁵I]LTT‐SRIF₂₈ or [¹²⁵I] [Tyr¹⁰]‐CST₁₄. In Table 1, the data are listed, together with corresponding values of all β‐ and γ‐peptidic somatostatin/Sandostatin^® mimics measured previously by our groups. Submicromolar affinities have been achieved for most of the human SRIF receptors hsst_1–5. Especially high, specific binding affinities for receptor hsst₄ (which is highly expressed in lung and brain tissue, although still of unknown function!) was observed with some of the β‐peptidic mimics. In view of the fact that numerous peptide‐activated G protein‐coupled receptors (GPCRs) recognize ligands with turn structure (Table 2), the results reported herein are relevant far beyond the realm of somatostatin: many other peptide GPCRs should be ‘reached’ with β‐ and γ‐peptidic mimics as well, and these compounds are proteolytically and metabolically stable, and do not need to be cell‐penetrating for this purpose (Fig. 5).     

3β,7α,12α‐Tri­formyl­oxy‐24‐nor‐5β‐chol‐22‐ene

The title compound, alternatively called 24‐nor‐5β‐chol‐22‐ene‐3β,7α,12α‐triyl triformate, C₂₆H₃₈O₆, has a cis junction between two of the six‐membered rings. All three of the six‐membered rings have chair conformations that are slightly flattened and the five‐membered ring has a 13β,14α‐half‐chair conformation. The 3β, 7α and 12α ring substituents are axial and the 17β group is equatorial. The 3β‐formyl­oxy group is involved in one weak intermol­ecular C—H⋯O bond, which links the mol­ecules into dimers in a head‐to‐head fashion.     

Differentiation of Boc‐protected α,δ‐/δ,α‐ and β,δ‐/δ,β‐hybrid peptide positional isomers by electrospray ionization tandem mass spectrometry

Two new series of Boc‐N‐α,δ‐/δ,α‐ and β,δ‐/δ,β‐hybrid peptides containing repeats of L ‐Ala‐δ⁵‐Caa/δ⁵‐Caa‐L ‐Ala and β³‐Caa‐δ⁵‐Caa/δ⁵‐Caa‐β³‐Caa (L ‐Ala = L ‐alanine, Caa = C‐linked carbo amino acid derived from D ‐xylose) have been differentiated by both positive and negative ion electrospray ionization (ESI) ion trap tandem mass spectrometry (MS/MS). MSⁿ spectra of protonated isomeric peptides produce characteristic fragmentation involving the peptide backbone, the Boc‐group, and the side chain. The dipeptide positional isomers are differentiated by the collision‐induced dissociation (CID) of the protonated peptides. The loss of 2‐methylprop‐1‐ene is more pronounced for Boc‐NH‐L ‐Ala‐δ‐Caa‐OCH₃ (1), whereas it is totally absent for its positional isomer Boc‐NH‐δ‐Caa‐L ‐Ala‐OCH₃ (7), instead it shows significant loss of t‐butanol. On the other hand, second isomeric pair shows significant loss of t‐butanol and loss of acetone for Boc‐NH‐δ‐Caa‐β‐Caa‐OCH₃ (18), whereas these are insignificant for its positional isomer Boc‐NH‐β‐Caa‐δ‐Caa‐OCH₃ (13). The tetra‐ and hexapeptide positional isomers also show significant differences in MS² and MS³ CID spectra. It is observed that ‘b’ ions are abundant when oxazolone structures are formed through five‐membered cyclic transition state and cyclization process for larger ‘b’ ions led to its insignificant abundance. However, b₁⁺ ion is formed in case of δ,α‐dipeptide that may have a six‐membered substituted piperidone ion structure. Furthermore, ESI negative ion MS/MS has also been found to be useful for differentiating these isomeric peptide acids. Thus, the results of MS/MS of pairs of di‐, tetra‐, and hexapeptide positional isomers provide peptide sequencing information and distinguish the positional isomers. Copyright © 2010 John Wiley & Sons, Ltd.     

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.     

Preparation of the β3‐Homoselenocysteine Derivatives Fmoc‐β3hSec(PMB)‐OH and Boc‐β3hSec(PMB)‐OH for Solution and Solid‐Phase‐Peptide Synthesis and Selenoligation

The title compounds, 4 and 7 , have been prepared from the corresponding α‐amino acid derivative selenocystine ( 1 ) by the following sequence of steps: cleavage of the Se? Se bond with NaBH₄, p‐methoxybenzyl (PMB) protection of the SeH group, Fmoc or Boc protection at the N‐atom and Arndt–Eistert homologation (Schemes 1 and 2). A β³‐heptapeptide 8 with an N‐terminal β³‐hSec(PMB) residue was synthesized on Rink amide AM resin and deprotected (‘in air’) to give the corresponding diselenide 9 , which, in turn, was coupled with a β³‐tetrapeptide thiol ester 10 by a seleno‐ligation. The product β³‐undecapeptide was identified as its diselenide and its mixed selenosulfide with thiophenol (Scheme 3). The differences between α‐ and β‐Sec derivatives are discussed.     

Synthesis of β‐Hexa‐ and β‐Heptapeptides Containing Novel β2,3‐Amino Acids with Two Serine or Two Cysteine Side Chains – CD‐ and NMR‐Spectroscopic Evidence for 314‐Helical Secondary Structures in Water

Two representatives of a new type of β‐amino acids, carrying two functionalized side chains, one in the 2‐ and one in the 3‐position, have been prepared stereoselectively: a β‐Ser derivative with an additional CH₂OH group in the 2‐position (for β‐peptides with better water solubility; Scheme 2) and a β‐HCys derivative with an additional CH₂SBn group in the 2‐position (for disulfide formation and metal complexation with the derived β‐peptides; Scheme 3). Also, a simple method for the preparation of α‐methylidene‐β‐amino acids is presented (see Boc‐2‐methylidene‐β‐HLeu‐OH, 8 in Scheme 3). The two amino acids with two serine or two cysteine side chains are incorporated into a β‐hexa‐ and two β‐heptapeptides ( 18 and 23/24 , resp.), which carry up to four CH₂OH groups. Disulfide formation with the β‐peptides carrying two CH₂SH groups generates very stable 1,2‐dithiane rings in the centre of the β‐heptapeptides, and a cyclohexane analog was also prepared (cf. 27 in Scheme 6). The CD spectra in H₂O clearly indicate the presence of 3₁₄‐helical structures of those β‐peptides ( 18 , 23 , 24 , 27b ) having the `right' configurations at all stereogenic centers (Fig. 2). NMR Measurements (Tables 1 and 2, and Fig. 4) in aqueous solution of one of the new β‐peptides ( 24 ) are interpreted on the assumption that the predominant secondary structure is the 3₁₄‐helix, a conformation that has been found to be typical for β‐peptides in MeOH or pyridine solution, according to our previous NMR investigations.     

Preparation of the β2‐Homoselenocysteine Derivatives Fmoc‐(S)‐β2hSec(PMB)‐OH and Boc‐(S)‐β2hSec(PMB)‐OH for Solution and Solid‐Phase Peptide Synthesis

Fmoc‐β²hSer(^tBu)‐OH was converted to Fmoc‐β²hSec(PMB)‐OH in five steps. To avoid elimination of HSeR, the selenyl group was introduced in the second last step (Fmoc‐β²hSer(Ts)‐OAll→Fmoc‐β²hSec(PMB)‐OAll). In a similar way, the N‐Boc‐protected compound was prepared. With the β²hSe‐derivatives, 21 β²‐amino‐acid building blocks with proteinogenic side chains are now available for peptide synthesis.     

Isotopically Labelled and Unlabelled β‐Peptides with Geminal Dimethyl Substitution in 2‐Position of Each Residue: Synthesis and NMR Investigation in Solution and in the Solid State

The preparation of (S)‐β^2,2,3‐amino acids with two Me groups in the α‐position and the side chains of Ala, Val, and Leu in the β‐position (double methylation of Boc‐β‐HAla‐OMe, Boc‐β‐Val‐OMe, and Boc‐β‐Leu‐OMe, Scheme 2) is described. These β‐amino acids and unlabelled as well as specifically ¹³C‐ and ¹⁵N‐labelled 2,2‐dimethyl‐3‐amino acid (β^2,2‐HAib) derivatives have been coupled in solution (Schemes 1, 3 and 4) to give protected (N‐Boc, C‐OMe), partially protected (N‐Boc/C‐OH, N‐H/C‐OMe), and unprotected β^2,2‐ and β^2,2,3‐hexapeptides, and β^2,2‐ and β^2,2,3‐heptapeptides 1 – 7 . NMR Analyses in solution (Tables 1 and 2, and Figs. 2–4) and in the solid state (2D‐MAS NMR measurements of the fully labelled Boc‐(β^2,2‐HAib)₆‐OMe ([¹³C₃₀, ¹⁵N₆]‐ 1e ; Fig. 5), and TEDOR/REDOR NMR investigations of mixtures (Fig. 6) of the unlabelled Ac‐(β^2,2‐HAib)₇‐OMe ( 4 ) and of a labelled derivative ([¹³C₄,¹⁵N₂]‐ 5 ; Figs. 7–11, and 19), a molecular‐modeling study (Figs. 13–15), and a search in the Cambridge Crystallographic Data Base (Fig. 16) allow the following conclusions: i) there is no evidence for folding (helix or turn) or for aggregation to sheets of the geminally dimethyl substituted peptide chains in solution; ii) there are distinct conformational preferences of the individual β^2,2‐ and β^2,2,3‐amino acid residues: close to eclipsing around the C(O) C(Me₂(CHR)) bond (τ_1,2), almost perfect staggering around the C(2) C(3) ethane bond (τ_2,3), and antiperiplanar arrangement of H(C3) and H(N) (τ_3,N; Fig. 12) in the solid state; iii) the β^2,2‐peptides may be part of a turn structure with a ten‐membered H‐bonded ring; iv) the main structure present in the solid state of F₃CCO(β^2,2‐HAib)₇‐OMe is a nonfolded chain (>30 Å between the termini and >20 Å between the N‐terminus and the CH₂ group of residue 5) with all CO bonds in a parallel alignment (±10°). With these structural parameters, a simple modelling was performed producing three (maybe four) possible chain geometries: one fully extended, two with parallel peptide planes (with zick‐zack and crankshaft‐type arrangement of the peptide bonds), and (possibly) a fourth with meander‐like winding ( D – G in Figs. 17 and 18).     

β‐Hairpins Generated from Hybrid Peptide Sequences Containing both α‐ and β‐Amino Acids

The incorporation of the β‐amino acid residues into specific positions in the strands and β‐turn segments of peptide hairpins is being systematically explored. The presence of an additional torsion variable about the C(α) C(β) bond (θ) enhances the conformational repertoire in β‐residues. The conformational analysis of three designed peptide hairpins composed of α/β‐hybrid segments is described: Boc‐Leu‐Val‐Val‐^DPro‐β Phe ‐Leu‐Val‐Val‐OMe ( 1 ), Boc‐Leu‐Val‐β Val ‐^DPro‐Gly‐β Leu ‐Val‐Val‐OMe ( 2 ), and Boc‐Leu‐Val‐β Phe ‐Val‐^DPro‐Gly‐Leu‐β Phe ‐Val‐Val‐OMe ( 3 ). 500‐MHz ¹H‐NMR Analysis supports a preponderance of β‐hairpin conformation in solution for all three peptides, with critical cross‐strand NOEs providing evidence for the proposed structures. The crystal structure of peptide 2 reveals a β‐hairpin conformation with two β‐residues occupying facing, non‐H‐bonded positions in antiparallel β‐strands. Notably, βVal(3) adopts a gauche conformation about the C(α) C(β) bond (θ=+65°) without disturbing cross‐strand H‐bonding. The crystal structure of 2 , together with previously published crystal structures of peptides 3 and Boc‐β Phe ‐β Phe ‐^DPro‐Gly‐β Phe ‐β Phe ‐OMe, provide an opportunity to visualize the packing of peptide sheets with local ‘polar segments' formed as a consequence of reversal peptide‐bond orientation. The available structural evidence for hairpins suggests that β‐residues can be accommodated into nucleating turn segments and into both the H‐bonding and non‐H‐bonding positions on the strands.     

3β‐Acetoxy‐5α,6β‐di­hydroxybisnorcholanic acid 2216 lactone

In the title compound, C₂₄H₃₆O₆, the ester linkage in ring A is equatorial. The six‐membered rings A, B and C have chair conformations. The five‐membered ring D adopts a 13β,14α‐half‐chair conformation and the E ring adopts an envelope conformation. The A/B, B/C and C/D ring junctions are trans, whereas the D/E junction is cis.     

Mixed β2/β3‐Hexapeptides and β2/β3‐Nonapeptides Folding to (P)‐Helices with Alternating Twelve‐ and Ten‐Membered Hydrogen‐Bonded Rings

The structural properties of four mixed β‐peptides with alternating β²/β³‐ or β³/β²‐sequences have been analyzed by two‐dimensional homonuclear ¹H‐NMR‐ and CD spectroscopic measurements. All four β‐peptides fold into (P)‐helices with twelve‐ and ten‐membered H‐bonded rings (Figs. 3–6). CD Spectra (Fig. 2) of the mixed β³/β²‐hexapeptide 4a and β³/β²‐nonapeptide 5a , indicating that peptides of this type also adopt the 12/10‐helical conformation, were confirmed by NMR structural analysis. For the deprotected β³/β²‐nonapeptide 5d , NOEs not consistent with the 10/12 helix have been observed, showing that the stability of the helix decreases upon N‐terminal deprotection. From the NMR structures obtained, an idealized helical‐wheel representation was generated (Fig. 7), which will be used for the design of further 12/10 or 10/12 helices.     

Semisynthesis and absolute configuration of a novel rearranged 19,20‐δ‐lactone (9βH)‐pimarane diterpene

Annonalide (3β,20‐epoxy‐3α,16‐dihydroxy‐15‐oxo‐7‐pimaren‐19,6β‐olide, C₂₀H₂₆O₆, 1 ) is the major (9βH)‐pimarane diterpene isolated from tubers of Cassimirella ampla, and it exhibits cytotoxic properties upon interaction with ctDNA. We have prepared new derivatives of 1 by modification of the (9βH)‐pimarane backbone and report here the semisynthesis and absolute configuration of a novel rearranged 19,20‐δ‐lactone (9βH)‐pimarane. Our approach was the reduction of the carbonyl groups of 1 with sodium borohydride, at positions C15 (no stereoselectivity) and C3 (stereoselective reduction), followed by rearrangement of the 6,19‐γ‐lactone ring into the six‐membered 19,20‐δ‐lactone ring in 4a (3β,6β,16‐trihydroxy‐7‐pimaren‐19,20β‐olide monohydrate, C₂₀H₃₀O₆·H₂O). The absolute structure of the new compound, 4a , was determined unambiguously with a Flack parameter x of −0.01 (11), supporting the stereochemistry assignment of 1 redetermined here. Besides the changes in the pattern of covalent bonds caused by reduction and lactone rearrangement, the conformation of one of the three fused cyclohexane rings is profoundly different in 4a , adopting a chair conformation instead of the boat shape found in 1 . Furthermore, the intramolecular hydrogen bond present in 1 is lost in new compound 4a , due to hydrogen bonding between the 3‐OH group and the solvent water molecule.     

Synthesis and CD Spectra in MeCN,MeOH, and H2O of γ‐Oligopeptides with Hydroxy Groups on the Backbone,Preliminary Communication

γ⁴‐Tripeptides and γ⁴‐hexapeptides, 1 – 4 , with OH groups in the 2‐ or 3‐position on each residue have been prepared. The corresponding 2‐hydroxy amino acids were obtained by Si‐nitronate (3+2) cycloadditions to the acryloyl derivative of Oppolzer's sultam and Raney‐Ni reduction of the resulting 1,2‐oxazolidines (Scheme 1). The 3‐hydroxy amino acid derivatives were prepared by chain elongation via Claisen condensation of Boc‐Ala‐OH, Boc‐Val‐OH, and Boc‐Leu‐OH, and NaBH₄ reduction of the methyl 4‐amino 3‐oxo carboxylates formed (Scheme 2). The N‐Boc hydroxy amino acids were coupled in solution to give the γ‐peptides. CD Spectra of the new types of γ‐peptides were recorded and compared with those of simple γ²‐, γ³‐, γ⁴‐, and γ^2,3,4‐peptides (Figs. 3, 4, and 5). An intense Cotton effect at ca. 200 nm ([Θ]=−2⋅10⁵ deg⋅cm²⋅dmol⁻¹) indicates that the hexapeptide built of (3R,4S)‐4‐amino‐3‐hydroxy acids (with the side chains of Val, Ala, Leu) folds to a secondary structure so far unknown. The stability of peptides from β‐ and γ‐amino acids, which carry heteroatoms on their backbones is discussed (Fig. 1). Positions on the γ‐peptidic 2.6₁₄ helix are identified at which non‐H‐atoms are `allowed' (Fig. 2).     

The Effect of Backbone‐Heteroatom Substitution on the Folding of Peptides – A Single Fluorine Substituent Prevents a β‐Heptapeptide from Folding into a 314‐Helix (NMR Analysis)

The β‐heptapeptides H‐βhVal‐βhAla‐βhLeu‐βhAla(X_n)‐βhVal‐βhAla‐βhLeu‐OH 3 – 7 with central 3‐amino‐2‐fluoro‐, 3‐amino‐2,2‐difluoro‐, or 3‐amino‐2‐hydroxybutanoic acid residues (βhAla(X_n)) of like and unlike configuration were subjected to a detailed NMR analysis in MeOH solution. For the geminal difluoro and for the F‐ and OH‐substituted derivatives of u‐configuration (see 5, 4 , and 7 , resp.), 14‐helices were found, i.e., with axial disposition of the hetero atoms on the helix. The two compounds containing the central l‐configured β‐amino acid moieties (see 3 and 6 ) are not helical over the full lengths of the chains; they have ‘quasi‐helical’ termini and a central turn consisting of a ten‐membered H‐bonded ring (Fig. 2, d and e). Quantum‐mechanical calculations with l‐ and u‐AcNH‐CHMe‐CHF‐CONH₂ confirm the observed preference for a conformation with antiperiplanar arrangement of the F? C and the C?O bond. The calculated energy difference between the observed ‘non‐helical’ geometry of this moiety and a hypothetical helical one is 6.4 kcal/mol (Fig. 3).