Synthesis and conformation of a polyoxyethylene-bound undecapeptide of the alamethicin helix and (2-methylalanyl-L-alanine)1–7

The stepwise synthesis and conformational studies of the N-terminal helical partial sequence of the membrane-modifying polypeptide antibiotic alamethicin are described. The polyoxyethylen esters of the fragments N-t-Boc-L -Pro-Aib-Ala-Gln-Aib-Val-Aib-Gly-OH and N-Ac-Aib-L -Pro-Aib-Ala-Aib-Ala-Gln-Aib-Val-Aib-Gly-OH are synthesized using polyoxyethylene (molecular mass 10,000) as solubilizing support. CD spectra of each intermediate in ethanol show α-helix formation of the N-protected peptide polymers beginning with the nonapeptide and of the N-protonated sequences beginning with the decapeptide. Compared to the helix of alamethicin, temperature- and solvent-dependent CD measurements indicate analogous conformational behavior. The results suggest that in lipophilic media the alamethicin helix can extend the full length of the partial sequence between the two proline residues and that aqueous media favor an increase of random-coil conformation. For model studies of the particular lipid interaction of alamethicin, the stepwise synthesis of peptides with the alternating (Aib-L -Ala)_n sequence (n = 1–7) was carried out on a polyoxyethylene support (molecular mass 6000). CD and ORD studies in ethanol showed a change from the random coil to a right-handed α-helix with increasing peptide length. This change is observed for the N-protected peptides at a chain length of 8 residues and for the N-protonated peptides at a length of 9 residues. The comparison of the CD data of free and polyoxyethylene-bound peptides revealed that the solubilizing polymeric support cannot induce conformational changes. The intensities of the CD bands of t-Boc-(Aib-L -Ala)_n-OPOE (n ≥ 6) are higher than those of alamethicin, and these model peptides show similar temperature and solvent inducible changes of their helix contents.     

Crystal structure of Boc-Leu-Aib-Pro-Val-Aib-Aib-Glu(OBzl)-Gln-Phl × H2O,the C-terminal nonapeptide of the voltage-dependent ionophore alamethicin

Boc-L -Leu-Aib-Pro-Val-Aib-Aib-Glu(OBzl)-Gln-Phl (Boc = t-butyloxycarbonyl, Aib = α-aminoisobutyric acid, Bzl = benzyl, Phl = phenylalaninol), C₅₉H₉₀N₁₀O₁₄, the protected C-terminal nonapeptide with the sequence 12–20 of alamethicin, crystallizes in the orthorhombic space group P2₁2₁2₁ with a = 15.666, b = 16.192, c = 26.876 Å, and Z = 4. The molecular conformation is right-handed helical with three α-(5 → 1 hydrogen bonds) and three β-turns (4 → 1 hydrogen bonds). All but two of the hydrogen bonds are significantly longer than the usual value and show bifurcation to some extent. The α/3-helical nonapeptide molecules are arranged head-to-tail along the a direction. The resulting linear antiparallel chains are linked by a weak intermolecular hydrogen bridge, thus forming a two-dimensional layer structure in the ab plane. The conformation of this nonapeptide is almost identical with that of the corresponding C-terminal part found by x-ray crystallography of the eicosapeptide alamethicin.     

Structure of a Peptidal Antibiotic P168 Produced by Paecilomyces lilacinus (Thom) Samson

( + )-α-Kainic acid (1) was synthesized by starting from a building block, N-Boc-3-acetoxyallylglycine ethyl ester (2). The key intermediate, a methyl 4-[(tert-butoxycarbonyl)prenylamino]-5-hydroxy-2-pentenoate derivative (9), was prepared from 2 in eight synthetic steps. After converting 10 into a methyl ester (11), intramolecular ene-carbocyclization of 11 gave a pyrrolidine derivative (12), which was converted to 1 in a moderate yield.     

Synthesis and biological evaluation of 7-(aminoalkyl)pyrazolo[1,5-a]pyrimidine derivatives as cathepsin K inhibitors

A series of novel 7-aminoalkyl substituted pyrazolo[1,5-a]pyrimidine derivatives were synthesized and tested for inhibition of cathepsin K. The synthetic methodology comprises cyclization of 5-aminopyrazoles with N-Boc-α-amino acid-derived ynones followed by transformation of the ester and the Boc-amino functions. It allows for easy diversification of the pyrazolo[1,5-a]pyrimidine scaffold at various positions. Molecular docking studies with pyrazolo[1,5-a]pyrimidine derivatives were also performed to elucidate the binding mode in the active site of cathepsin K. The synthesized compounds exhibited moderate inhibition activity (K_i ≥ 77 μM).     

Stereochemistry of alpha-aminoisobutyric acid peptides in solution: helical conformations of protected decapeptides with repeating Aib-L-Ala and Aib-L-Val sequences

The decapeptides Boc-(Aib-L -Ala)₅-OMe and Boc-(Aib-L -Val)₅-OMe have been studied by 270-MHz ¹H-nmr in CDCl₃ and (CD₃)₂SO solutions. Intramolecular hydrogen-bonded NH groups have been delineated using the temperature and solvent dependence of the NH chemical shifts and differential broadening of the NH resonances, induced by addition of a nitroxide radical. Both peptides have eight solvent-shielded NH groups, suggesting that 3₁₀-helical conformations are maintained in the two solvents. In alternating Aib-X sequences, the Aib residues appear to play a dominant role in determining the preferred conformations, overriding the intrinsic stereochemical preferences of the X residues.     

Intra- and intermolecular charge-transfer interactions between terminal electron donor and acceptor attached to poly(γ-benzyl-L-glutamate) in solution

Poly(γ-benzyl-L -glutamate) having a terminal dimethylaminoanilide group as an electron donor (D) and a terminal 3,5-dinitrobenzoyl group as an electron acceptor (A) (A-[Glu(OBzl)]_n-D) was synthesized by the N-carboxyanhydride method. Polymer samples were fractionated by gel chromatography and their number-average degrees of polymerization n were determined by the absorbances of the terminal chromophores. These polymers in chloroform and dimethylformamide solutions showed a charge-transfer (CT) absorption band around 455 nm, and the fraction of the polymer forming the CT complex was evaluated as a function of the chain length. CT absorption for short chains (n = 5 ～ 20) was attributed to intramolecular CT complex in which the A-[Glu(OBzl)]_n-D chain takes cyclic conformations. An optimum chain length for the intramolecular CT was found to be n ? 10, where the [Glu(OBzl)]_n chain may most easily bend back to form cyclic conformations. Stronger CT absorption observed for longer chains than n = 20 was shown to be intermolecular, and an intermolecular head-to-tail aggregation was found to be a cause of the strong CT interaction. All helical A-[Glu(OBzl)]_n-D chains were found to form the head-to-tail dimers in chloroform solution.     

Cation-binding cyclic peptides with lipophilic tails

We have synthesized and characterized a series of cation-binding cyclic octapeptides which may function as potential ionophoric substances. The materials contain varying degrees of hydrophobic character, which was controlled systematically through the incorporation of N-alkylglycine residues where N-alkyl = methyl, n-hexyl, cyclohexyl, or n-decyl. The peptides reported include cyclo(Phe-Sar-Gly-Sar)₂, cyclo(Glu(OBzl)-Sar-Gly-Sar-Glu(OBzl)-Sar-Gly-(N-decyl)Gly), cyclo(Glu(OBzl)-Sar-Gly-(N-decyl)Gly)₂, cyclo(Glu(OBzl)-Sar-Gly-(N-hexyl)Gly)₂, cyclo(Glu(OBzl)-Sar-Gly-(N-cyclohexyl)Gly)₂, and the corresponding free diacid forms of the Glu-containing compounds. Using ¹³C- and ¹H-nmr spectra, we demonstrated that the mixture of cis/trans peptide bond-isomer conformers, characteristic of the free-peptide benzyl esters in solution, was converted to unique C₂-symmetric, presumably all-trans conformers on complexation with calcium ions. Cation-transport experiments, using the thick-liquid model of transport in a Pressman cell, established that these compounds transport a variety of cations and that one peptide examined in detail, cyclo(Glu(OBzl)-Sar-Gly-(N-decyl)Gly)₂ (selectivity Ca²⁺ > Na⁺ > K⁺ > Mn²⁺ > Cu²⁺ > Mg²⁺ > Co²⁺ > Zn²⁺), transports calcium about an order of magnitude more efficiently than magnesium.     

Solid-state conformation of copolymers of β-benzyl-L-aspartate with L-alanine,L-leucine,L-valine, γ-benzyl-L-glutamate,or ϵ-carbobenzoxy-L-lysine

The solid-state conformation of copolymers of β-benzyl-L -aspartate [L -Asp(OBzl)] with L -leucine (L -Leu), L -alanine (L -Ala), L -valine (L -Val), γ-benzyl-L -glutamate [L -Glu(OBzl)], or ?-carbobenzoxy-L -lysine (Cbz-L -Lys) has been studied by ir spectroscopy and circular dichroism (CD). The ir spectra in the region of the amide I and II bands and in the region of 700–250 cm^?1 have been determined. The results from the ir studies are in good agreement with data obtained by CD experiments. Incorporation of the amino acid residues mentioned above into poly[L -Asp(OBzl)] induces a change from the left-handed into the right-handed α-helix. This conformational change for the poly[L -Asp(OBzl)] copolymers was observed in the following composition ranges: L -Leu, 0–15 mol %; L -Ala, 0–32 mol %; L -Val, 0–8 mol %; L -Glu(OBzl), 3–10 mol %; and Cbz-L -Lys, 0–9 mol %.     

Synthesis,characterization and bioactivity of four novel trinuclear copper(II) and nickel(II) complexes with pentadentate ligands derived from N-acylsalicylhydrazide

Four novel trinuclear copper(II)/nickel(II) complexes with four trianionic pentadentate ligands, N-(3-t-butylbenzoyl)-5-nitrosalicylhydrazide (H₃3-t-bbznshz), N-(3,5-dimethylbenzoyl)salicylhydrazide (H₃3,5-dmbzshz), N-(phenylacetyl)-5-bromosalicylhydrazide (H₃pabshz) and N-(3-t-butylbenzoyl)salicylhydrazide (H₃3-t-bbzshz) have been synthesized and characterized by X-ray crystallography. These trinuclear compounds all have an M–N–N–M–N–N–M core formed by three metal ions and two ligands. The geometries of three Cu(II) ions in compound Cu₃(3-t-bbznshz)₂(H₂O)(DMF)(py)₂ · DMF (1) alternate between distorted square pyramidal and square planar, while in compound Cu₃(3,5-dmbzshz)₂(py)₂ (2), they are all square planar. Three Ni(II) ions in compound Ni₃(pabshz)₂(DMF)₂(py)₂ (3) and Ni₃(3-t-bbzshz)₂(py)₄ · 2H₂O (4) follow square-planar/octahedral/square-planar coordination geometry. Compounds 1, 2 and 4 are bent trinuclear, with the bend angles of 156.4°, 141.49° and 127.1°, respectively, while the three nickel ions in compound 3 are strictly linear, with an angle of 180°. Studies on the trinuclear Ni(II) complexes show that the β-branched N-acylsalicylhydrazide ligands with sterically flexible Cα methylene groups are easier to yield linear trinuclear Ni(II) complexes, while α-branched N-acylsalicylhydrazides ligands tend to form bent trinuclear Ni(II) complexes. Antibacterial screening data indicate that the trinuclear Cu(II) compound 2 is more active than 1 and mononuclear Cu(II) compound, bent trinuclear Ni(II) compound 4 is more active than linear compound 3 and less active than tetranuclear nickel compound in the previous study.     

Stabilizing effects of 2-methylalanine residues on β-turns and α-helices

¹³C-, ¹H-nmr, CD, and x-ray crystallography revealed β-turns of type III for Boc-Gly-L-Ala-Aib-OMe, Boc-L-Ala-Aib-L-Ala-OMe; the 3₁₀-helix for Boc-Aib-L-Ala-Aib-L-Ala-Aib-OMe; and antiparallel arranged α-helices for Boc-L-Ala-Aib-Ala-Aib-Ala-Glu(OBzl)-Ala-Aib-Ala-Aib-Ala-OMe. An N-terminal rigid α-helical segment is found in the polypeptide antibiotics alamethicin, suzukacillin, and trichotoxin. The α-helix dipole is essential for their voltage-dependent pore formation in lipid bilayer membranes, which is explained by a flip-flop gating mechanism based on dipole–dipole interactions of parallel and antiparallel arranged α-helices within oligomeric structures.     

Carrier design: Conformational studies of amino acid (X) and oligopeptide (X-DL-Alam) substituted poly(L-lysine)

The present study was undertaken to examine the influence of the reversal of the sidechain sequential order on the conformation of branched polypeptides. At the same time, the influence of the optically active amino acid joined directly to the poly (L -Lys) backbone and the DL -Ala oligomer grafted as chain-terminating fragment were separately analyzed. Therefore two sets of polypeptides were synthesized corresponding to the general formula poly [Lys-(X_i,)] (XK) and poly[Lys-(DL -Ala_m-X_i)] (AXK) when X = Ala, D -Ala, Leu, D -Leu, Phe, D -Phe, Ile, Pro, Glu.,D -Glu, or His. For coupling amino acid X to polylysine, three types of active ester methods were compared: the use of pentafluorophenyl or pentachlorophenyl ester, and the effect of the addition of an equimolar amount of 1-hydroxybenzotriazole. After cleavage of protecting groups, AXK polypeptides were synthesized by grafting short oligo (DL -Ala) chains to XK by using N-carboxy-DL -Ala anhydride. The CD measurements performed in water solutions of various pH values and ionic strengths were used for classification of the polypeptide conformations as either ordered (helical) or unordered. Different from what was observed with the unsubstituted poly (L -Lys), poly[Lys-(X_i)] type polypeptides can adopt ordered structure even under nearly physiological conditions (pH 7.3, 0.2M NaCl). These data suggest that the introduction of amino acid residue with either (ar) alkyl side chain (Ala, Leu, Phe) or negatively charged side chain (Glu) promotes markedly the formation of ordered structure. Comparison of chiroptical properties of poly [Lys- (DL -Ala_m-X_i)] and of poly [Lys- (X_i)] reveals that side-chain interactions play an important role in the stabilization of ordered solution conformation of AXK type branched polypeptides. The results give rather conclusive evidence that not only hydrophobic interactions, but also ionic attraction, can be involved in the formation and stabilization of helical conformation of branched polypeptides. © 1993 John Wiley & Sons, Inc.     

Reversing-pulse electric birefringence of poly (γ-benzyl-L-glutamate). II. Field-strength dependence of steady-state and decay signals of well-fractionated samples

The electric field dependence (up to 21 kV/cm) of the steady-state and decay signals has been examined on the four well-fractionated samples of poly(γ-benzyl-L -glutamate), [Glu(OBzl)]_n, in N,N-dimethylformamide at 535 nm and 20°C. Together with the data previously obtained from the reversing-pulse electric birefringence [Ueda, K., Nomura, M. & Yamaoka, K. (1983) Biopolymers 22 , 2077–2090], the steady-state birefringence and field-free relaxation time were analyzed by a method that takes into account the polydispersity of the chain length. The weight-average chain length, (l_w), permanent dipole moment, (μ_w), electric polarizability anisotropy, (Δα_w), and the length-independent optical anisotropy factor were evaluated. The axial translation per residue was calculated for the [Glu(OBzl)]_n helix, but the uncertainly involved in the weight-average molecular weights, determined from light scattering by different investigators, makes the determination of the exact conformation of [Glu(OBzl)]_n difficult. The contribution of Δα_w to electric field orientation was found to be significant, since Δα_w was approximately proportional to l_w. A linear relationship also exists between μ_w and l_w, when the [Glu(OBzl)]_n helix is shorter than about 1200 Å.     

Synthesis of Chlorodideoxynucleosides Using Tris(2,4,6-tribromophenoxy)-dichlorophosphorane (BDCP)

Abstract

5′-Chloro-5′-deoxy-N,3′-O-dibenzoylthymidine (3a), 5′-chloro-5′-deoxy-N⁴, 3′-O-dibenzoyldeoxycytidine(3b), 5′-chloro-5′-deoxy-N⁶,3′-O-dibenzoyldeoxyadenosine(3c), N-benzoyl-1-(3-chloro-2,3-dideoxy-5-O-trityl-ß-D-xylofuranosyl)thymine (5a) and N⁶-benzoyl-9-(3-chloro-2,3-dideoxy-5-O-trityl-ß-D-xylofuranosyl)adenine (5b) have been synthesized in very high yields using a new efficient reagent, tris(2,4,6-tribrom-ophenoxy)dichlorophosphorane (BDCP). The reaction time was greatly reduced to 5–8 min. NOE data suggested an inversion of configuration at C₃-position and thus an SN2 mechanism has been proposed for the chlorination reaction.

     

The crystal state conformation of Aib-rich segments of peptaibol antibiotics

Ac-(Aib-Ala)₃-OH (a protected segment of the peptaibols gliodeliquescin and paracelsin), Z-Leu-Aib-Val-Aib-Gly-OtBu (a segment of [Leu]⁷-gliodeliquescin), Z-Val-Aib-Aib-Gln-OtBu (a common segment of alamethicin, paracelsin, and hypelcin), and Ac-Aib-Pro-(Aib-Ala)₂-OMe and Z-Aib-Pro-(Aib-Ala)₂-OMe, which represent differently N^α-protected 1–6 segments of alamethicin and hypelcin, have been synthesized by solution methods. The crystal-state conformations of these five Aib-containing peptides have been determined by X-ray diffraction analysis. We have confirmed that the 3₁₀-helical structure is preferentially adopted by Aib-rich short peptides. An experimentally unambiguous proof for the 3₁₀→α-helix conversion has been provided by the two differently N-blocked -Aib-Pro-(Aib-Ala)₂-OMe hexapeptides. The β-bend ribbon conformation, commonly observed in the (Aib-Pro)_n sequential oligopeptides, is not found in the -Aib-Pro-Aib-Ala-Aib-Ala- sequence. As expected on the basis of the l -configuration of the C^α-monoalkylated residues, a right-handed helix screw sense was found in all peptides investigated. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

5-Hydroxytryptophan as a building block in oligopeptides using Fmoc/tBu SPPS

Summary One of the initial steps in the development of therapeutic agents is the identification of lead compounds that bind to the target of interest. Use of combinatorial libraries allows the screening of thousands to millions of compounds. To optimize the characteristics of the first generation of compounds, many of their analogues need to be synthesized. In this sense, 5-hydroxytryptophan closely resembles tryptophan. Both, however, are sensitive to oxidation and alkylation during solid-phase peptide synthesis. In an effort to overcome these side reactions during oligopeptide synthesis, we prepared N -Fmoc-N ⁱⁿ-Boc-5-O-benzyl-5-hydroxytryptophan. To evaluate its properties, this building block was incorporated in a pentagastrin analogue using Fmoc/tBu chemistry. After deprotection and cleavage from the support, the products were purified and identified. The fully deprotected peptide was the main compound, contaminated by its 5-O-benzylated analogue. The relative amount of the latter decreased using longer deprotection times.This work will be reported in more detail elsewhere (Lescrinier, Th. et al., paper in preparation).     

15N-nmr spectroscopy. 33. Assignments of isotactic and syndiotactic sequences in poly(D,L-amino acids)

¹⁵N-enriched (D ,L -Leu)_n, (γ-OMe-D ,L -Glu)_n, (D ,L -Val)_n, and (D ,L -Phe)_n were prepared, 40.55-MHz ¹⁵N-nmr spectra were measured in various solvents. The signal patterns depend strongly on the nature of the solvent, yet in most cases at least four signals are resolved, representing the four enantiomeric pairs of triads L -L -L (D -D -D ), L -D -L (D -L -D ), L -L -D (D -D -L ), and D -L -L (L -D -D ). Numerous copolypeptides of the general structure (A)_n-B*-(A)_m (the asterisk denotes 40–50% ¹⁵N enrichment) were synthesized and measured as models for syndiotactic sequences in the spectra of poly(D ,L -amino acids). In this way unambiguous assignments for both isotactic and syndiotactic trials were obtained. A spectroscopic rule was established: “isotactic sequences absorb downfield of syndiotactic ones.” Furthermore, the spectra of various types of stereocopolypeptides such as (L -Leu/L -Val)_n and (L -Leu/D -Val)_n were investigated, including the ternary systems (L -Leu/L -Ala/D -Ala)_n (L -Leu/L -Ala/Gly)_n, (L -Leu/D -Ala/Gly)_n, (L -Val/L -Ala/Gly)_n, and (L -Val/D -Ala/Gly)_n. All copolymerization of D - and L -amino acid NCAs investigated in this work showed a low degree of stereoselectivity.     

Modifications of cell surface sialic acids modulate cell adhesion mediated by sialoadhesin and CD22

An increasing number of mammalian cell adhesion molecules, including sialoadhesion, CD22 and the family of selectins, have been found to bind cell surface glycoconjugates containing sialic acids. Here we describe how the structural diversity of this sugar influences cell adhesion mediated by the related molecules sialoadhesin and CD22 in murine macrophages and B-cells respectively. We show that the 9-O-acetyl group of Neu5,9Ac₂ and theN-glycoloyl residue of Neu5Gc interfere with sialoadhesin binding. In contrast, CD22 binds more strongly to Neu5Gc compared to Neu5Ac. Of two synthetic sialic acids tested, only CD22 bound theN-formyl derivative, whereas aN-trifluoroacetyl residue was accepted by sialoadhesin. The potential significance for the regulation of sialic acid dependent cell adhesion phenomena is discussed.Dedicated to Professor Dr Gerhard Uhlenbruck on the occasion of his 65th birthday.     

Synthesis and matrix assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry study of phosphopeptide

The symmetry phosphoramidite reagents were synthesized via atwo-step route and used for the `one pot' synthesis of thephosphoserine building block Fmoc-Ser(PO(OBzl)OH)-OH. Theprocedure is simple and rapid, and product is obtained in highyield. When using this building block, the phosphopeptide(RKGS(PO₃H₂)SSNEPSSDSLSSPTLLAL) related to theC-terminal of c-Fos protein was synthesized manually byFmoc/t-Bu procedure and characterized by matrix assisted laserdesorption/ionization time of flight (MALDI-TOF) massspectrometry. In the fragment ion of phosphopeptide in theMALDI-TOF mass spectrometry with a curved field reflection, theacquired information can be used to identify the sequences andthe phosphorylation sites of the peptide.     

Synthesis of (+)-(R)- and (−)-(S)-trans-8-hydroxy-2-[N-n-propyl-N-(3′-iodo-2′-propenyl)] aminotetralin: New 5-HT1A receptor ligands

(R,S)-trans-8-Hydroxy-2-[N-n-propyl-N-(3′-iodo-2′-propenyl)amino]tetralin 7 , a new radioiodinated ligand based on 8-OH-DPAT, was reported as a potential ligand for 5-HT_1A receptors. The optically active (+)-(R)- and (?)-(S)- 7 were prepared to investigate the stereoselectivity of (R,S)- 7 . Racemic intermediate 8-methoxy-2-N-n-propyltetralin was reacted with the acyl chloride of (?)-(R)-O-methylmandelic acid to form a mixture of (S,R)- and (R,R)-diastereoisomers, which were separated by flash column chromatography. After removing the N-acyl group from the diastereoisomers, the desired (+)-(R)-or (?)-(S)- 7 was obtained by adding an N-iodopropenyl group. In vitro homogenate binding studies showed the stereoselectivity of this new compound for 5-HT_1A receptors. (+)-(R)- 7 isomer displayed 100-fold higher affinity than the (?)-(S)- 7 isomer. Biochemical study indicated that (+)-(R)- 7 potently inhibited forskolin-stimulated adenylyl cyclase activity in hippocampal membranes (E_max and EC₅₀ were 24.5% and 5.4 nM, respectively), while (?)-(S)- 7 showed no effect at 1 μM. The radioiodinated (+)-(R)- and (?)-(S)-[¹²⁵I] 7 were confirmed by coelution with the resolved unlabeled compound on HPLC (reverse phase column PRP-1, acetonitrile/pH 7.0 buffer, 80/20). The active isomer, (+)-(R)-[¹²⁵I] 7 , displayed high binding affinity to 5-HT_1A receptors (K_d = 0.09 ± 0.02 nM). In contrast, the (?)-(S)- 7 isomer displayed a significantly lower affinity to the 5-HT_1A receptor (K_d > 10 nM). Thus, (+)-(R)-[¹²⁵I]trans-8-OH-PIPAT, (+)-(R)- 7 , an iodinated stereoselective 5-HT_1A receptor agonist, is potentially useful for study of in vivo and in vitro function and pharmacology of 5-HT_1A receptors in the central nervous system. © 1995 Wiley-Liss, Inc.     

3-(1-Piperidinyl)alanine formation during the preparation ofC-terminal cysteine peptides with the Fmoc/t-Bu strategy

Summary Several side reactions have been detected for cysteine-containing peptides. During the synthesis ofC-terminal cysteine peptides, a base-catalyzed elimination of the sulfhydryl-protected side-chain to afford the dehydroalanine derivative followed by a nucleophilic addition to the alkene was observed. MALDI-TOF analysis was a useful analytical technique to determine this phenomenon.Abbreviations Acm acetamidomethyl - Boc tert-butyloxycarbonyl - t-Bu tert-butyl - DBU 1,8-diazabicyclo[5.4.0]undec-7-ene - DIEA N,N-diisopropylethylamine - DMF N,N-dimethylformamide - Fmoc 9-fluorenylmethyloxycarbonyl - HATU N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-yl-methylene]-N-methylmethanaminium hexafluorophosphateN-oxide - HBTU N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphateN-oxide - HOAt 1-hydroxy-7-azabenzotriazole - HOBt 1-hydroxybenzotriazole - HPLC high-performance liquid chromatography - MALDI-TOF matrix-assisted laser desorption/ionization time-of-flight mass spectrometry - PAC 4-hydroxymethylphenoxyacetic acid handle - PAL 5-(4-(9-fluorenylmethyloxycarbonyl)aminomethyl-3,5-dimethoxy-phenoxy)valeric acid handle - PEG-PS polyethylene glycol-polystyrene graft supports - PS polystyrene - Reagent R TFA/thioanisole/1,2-ethanedithiol/anisole (90:5:3:2) - S-t-Bu S-tert-butyl mercapto - TFA trifluoroacetic acid - Trt triphenylmethyl. Amino acid symbols denote thel-configuration, unless indicated otherwise