Side-chain Protection Research Articles

Combined solid-phase/solution synthesis of a 31-residue vasoactive intestinal peptide analog: general method for repetitive coupling of fragments without isolation and purification of intermediates.

A novel analog of vasoactive intestinal peptide (VIP) has been reported which exhibits high potency and enhanced duration of in vivo biological activity. This VIP analog, cyclo-(Lys21-Asp25)Ac[Glu8 Lys12 Nle17 Ala19, Asp25 Leu26,Lys27,28,Gly29,30,Thr31]-VIP, which also has a lactam bridge, has been reported to have relaxant effects that are significantly more potent than other beta-agonists such as salbutamol and salmeterol. Because it has potential use for the treatment of bronchial asthma in humans, various convergent syntheses were evaluated to enable the economic preparation of large quantities of this medium-sized hentriacontapeptide. From these studies we developed a combined solid-phase/solution synthesis which uses four protected fragments (each prepared by solid-phase synthesis with highly acid-labile resins) possessing Nalpha-9-fluorenylmethyloxycarbonyl and side-chain tert-butyl protection. Only equivalent amounts of each fragment were required to achieve near-quantitative coupling reactions using N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmeth anaminium hexafluorophosphate N-oxide/N-hydroxybenzotriazole. All reagents and side products were removed at each stage by simple extraction procedures. Final deprotection was carried out with 90% trifluoroacetic acid. Under these conditions only low levels of epimerization were observed (<2%). These diastereoisomers and other trace impurities were removed from the product in a single purification by preparative high-performance liquid chromatography. The procedure has been scaled up (10-g scale) and the final product obtained in an overall (nonoptimized) yield of 24%. This procedure for the repetitive coupling of fragments, without isolation of intermediates, may be generally applicable for the economic synthesis of other medium-sized and longer peptides.

Misacylated Transfer RNAs Having a Chemically Removable Protecting Group.

The 4-pentenoyl group and a number of derivatives have been studied as protecting groups for N(alpha) of the aminoacyl moiety in misacylated tRNAs. The unsubstituted 4-pentenoyl group itself was found to function as efficiently as any of the derivatives studied. Four different N-(4-pentenoyl)aminoacyl-tRNA(CUA)s were prepared and shown to undergo deprotection readily upon admixture of aqueous iodine; the derived misacylated tRNAs all functioned well as suppressors of a nonsense codon in an in vitro protein biosynthesizing system. Also prepared were four N(alpha)-(4-pentenoyl)aspartyl-tRNA(CUA)s that were protected on the side chain carboxylate as the nitroveratryl ester. Following treatment with aqueous iodine, the misacylated suppressor tRNAs incorporated the aspartate derivatives into position 27 of dihydrofolate reductase by suppression of a UAG codon in the mRNA. The suppression yields were significantly better than those obtained when side chain protection was absent. The resulting "caged proteins" were inactive, but full catalytic potential was restored by irradiation under conditions sufficient to effect deprotection of the side chain carboxylate moiety.

Solid phase synthesis of hydantoins by thermal cyclization and screening of reaction conditions using APOS 1200.

A novel strategy for solid-phase synthesis of hydantoins with high optical purity is described using a thermal pH-neutral cyclization and simultaneous release from resin. Hereby even hydantoins bearing a pH-sensitive side chain (protection) are available. The reaction conditions are well screened applying the parallel organic synthesizer APOS 1200.

Synthetic Rat V1a Vasopressin Receptor Fragments Interfere with Vasopressin Binding via Specific Interaction with the Receptor

To study the vasopressin receptor domains involved in the hormonal binding, we synthesized natural and modified fragments of V1a vasopressin receptor and tested their abilities to affect hormone-receptor interactions. Natural fragments mimicking the external loops one, two, and three were able to inhibit specific vasopressin binding to V1a receptor. In contrast, the natural N-terminal part of the V1a vasopressin receptor was found inactive. One fragment, derived from the external second loop and containing an additional C-terminal cysteine amide, was able to fully inhibit the specific binding of both labeled vasopressin agonist and antagonist to rat liver V1a vasopressin receptor and the vasopressin-sensitive phospholipase C of WRK1 cells. The peptide-mediated inhibition involved specific interactions between the V1a receptor and synthetic V1a vasopressin receptor fragment since 1) it was dependent upon the vasopressin receptor subtype tested (Ki(app) for the peptide: 3.7, 14.6, and 64.5 microM for displacing [3H]vasopressin from rat V1a, V1b, and V2 receptors, respectively; 2) it was specific and did not affect sarcosin 1-angiotensin II binding to rat liver membranes; 3) it was not mimicked by vasopressin receptor unrelated peptides exhibiting putative detergent properties; and 4) no direct interaction between [3H]vasopressin and synthetic peptide linked to an affinity chromatography column could be observed. Such an inhibition affected both the maximal binding capacity of the V1a vasopressin receptor and its affinity for the labeled hormone, depending upon the dose of synthetic peptide used and was partially irreversible. Structure-activity studies using a serie of synthetic fragments revealed the importance of their size and cysteinyl composition. These data indicate that some peptides mimicking extracellular loops of the V1a vasopressin receptor may interact with the vasopressin receptor itself and modify its coupling with phospholipase C.

Stepwise Solid-Phase Synthesis of Serine-, Tyrosine- and Homoserine-nucleopeptides

Hydroxylated amino acids can be introduced in nucleopeptides using the acetyl group for their side chain protection. Base-stable nucleopeptide analogues are obtained if homoserine is used as the linking residue.

Enzymatic semisynthesis of dicarba analogs of calcitonin.

The semisynthesis of eel[L-alpha-aminosuberic acid]calcitonin (elcatonin) was accomplished by alpha-chymotrypsin-catalyzed coupling of two peptide segments in a single reaction without the protection of any functional group. The eel calcitonin-(10-32)-peptide was prepared by a gene manipulation. The esters of cyclic desamino nonapeptide (segment 1-9) were synthesized by the conventional solution method including a thermolysin-mediated resolution of DL-alpha-aminosuberic acid via one-step tripeptide synthesis leading to the 7-9 sequence. The main aim of this work was to determine the conditions for protease-catalyzed segment condensation while avoiding a concurrent cleavage of other proteolytically labile peptide bonds in the hormone. The alpha-chymotrypsin condensation strategy under usual conditions led to a complicated mixture of split products with an insignificant amount of the required peptide. When the coupling reaction was carried out at 0 degrees C, the reaction resulted in a satisfactory yield of elcatonin with the complete conversion of the acyl donor (1-9 segment) accompanied by negligible concurrent peptide bond digestion. The same strategy was employed for the preparation of analogous dicarba salmon calcitonin using a synthetic elcatonin-(10-32)-peptide. Both calcitonin analogs exhibited hypocalcemic activity corresponding to the international standard of elcatonin. We demonstrate in this work a peptide synthesis based on the combination of genetic engineering, chemical synthesis and proteinase-catalyzed segment condensation. This approach enables effective incorporation of an unnatural amino acid into calcitonins without the side-chain protection.

Synthesis and pharmacology of novel analogues of oxytocin and deaminooxytocin: directed methods for the construction of disulfide and trisulfide bridges in peptides.

Using as models the neurohypophyseal nonapeptide hormone oxytocin and its analogue deaminooxytocin, several directed routes to formation of sulfur-sulfur bridges have been developed and evaluated. The linear sequences (through common octapeptide-resin intermediates) were assembled smoothly on tris(alkoxy)benzylamide (PAL) poly(ethylene glycol)-polystyrene (PEG-PS) graft supports, using stepwise Fmoc solid-phase chemistry. Side-chain protection of beta-mercaptopropionic acid (Mpa) and/or cysteine (Cys) was provided by S-2,4,6-trimethoxybenzyl (Tmob), S-acetamidomethyl (Acm), and/or a series of sulfenyl thiocarbonate and carbamoylsulfenyl protecting/activating groups: S-(methoxycarbonyl)sulfenyl (Scm), S-(methoxycarbonyl)disulfanyl (Sscm), S-(N-methyl-N-phenylcarbamoyl)sulfenyl (Snm), and S-(N-methyl-N-phenylcarbamoyl)disulfanyl (Ssnm). Thiolytic displacement of S-Snm (preferred) or S-Scm provided intramolecular cyclized peptide disulfides, and homologation of the chemistry with S-Ssnm (again preferred) and S-Sscm provided the corresponding trisulfides along with smaller amounts of disulfides and tetrasulfides. These chemistries could be implemented both in solution and in solid-phase modes. Various parameters were studied systematically and optimized, and the novel trisulfides of oxytocin and deaminooxytocin were synthesized and purified to homogeneity. The trisulfide compounds were evaluated in three assays: uterotonic in vitro, uterotonic in vivo, and pressor tests, and they showed substantial potencies, ranging from 5% to 40% of the parent (disulfide) activities, as well as protracted actions. The affinities of the peptide trisulfides to uterine membrane receptors were only 3.3-3.6-fold lower than those of the parent disulfides. Possible explanations of the biological results are discussed.

Synthetic studies on threonines. The preparation of protected derivatives of d- allo- and l- allo-threonine for peptide synthesis

N-Acetylated threonine derivatives, having tert-butyl or benzyl-based side-chain protection, form isolable 5(4H)-oxazolones on treatment with N-ethyl- N′-3-dimethylaminopropyl carbodiimide. N-chloroacetylated threonine derivatives, on the other hand, do not form oxazolones so readily. The N-acetylated oxazolones are easily epimerized and lead to diastereoisomeric mixtures of threonine derivatives on hydrolysis with dilute aqueous acid. The components of these mixtures can be separated chromatographically, but a useful alternative for the O-benzylated mixture is selective enzymatic hydrolysis using hog kidney acylase. These chemical transformations provide the basis for practical syntheses of protected derivatives of the non-proteinogenic allo-threonines, suitable for use in peptide synthesis.

Synthesis and NMR spectroscopy of peptides containing either phosphorylated or phosphonylated cis- or trans-4-hydroxy-L-proline.

Many proteins are regulated by reversible O-glycosylation and O-phosphorylation. Whereas O-glycosylation of hydroxy-L-proline is common and well investigated, phosphorylation has not been proved so far in vivo, but this post-translational modification is entirely possible. As a first step to identify this phosphoamino acid, we describe both the syntheses of peptides phosphorylated at 4-hydroxy-L-proline and the 1H and 31P NMR parameters of these phosphopeptides. The model peptides were synthesized on solid-phase using Fmoc-strategy. Both natural isomers of 4-hydroxy-L-proline (containing the hydroxyl group in either the cis or trans position) were introduced without side-chain protection. All peptides were globally phosphorylated with O,O'-tert-butyl-N,N-diethylphosphoramidite on the solid phase and cleaved with trifluoroacetic acid. Additionally, we synthesized two classes of phosphonopeptides that mimic phosphopeptides, namely H- and methylphosphonopeptides. The NMR data were based on the model peptide Gly-Gly-Hyp-Ala, which is regarded as a typical random-coil sequence. The NMR parameters showed a significant influence of the phosphate group on the cis-trans isomerization of the Gly-Hyp bond, which may reflect a possible regulation of proteins by changing their local conformations. The 1H and 31P NMR parameters differed for each isomer, and were distinct from the parameters of phosphorylated serine, threonine and tyrosine. These known shifts can be used to identify both cis- and trans-O-phospho-4-hydroxy-L-proline in vivo.

2,2-Difluoro-1,3,2-oxazaborolidin-5-ones: novel approach for selective side-chain protection of serine and threonine

2,2-Difluoro-1,3,2-oxazaborolidin-5-ones 1, which are synthesized from BF3 and salts of amino acids, are highly effective, convenient and, moreover, inexpensive intermediates for the simultaneous protection of both α-amino and α-carboxy groups in α-amino acids. The new method streamlines the hitherto tedious procedures for side-chain protection of Ser and Thr. Ser(But), Thr(But), Ser(Bzl) and Thr(Bzl) are obtained by this procedure in high yields and in pure form using highly reactive reagents.

4] Standard Fmoc protocols

This chapter discusses standard Fmoc protocols. This chapter describes standard protocols for Fmoc solid-phase peptide synthesis. It discusses Fmoc-amino acid synthesis, compatible side-chain protection, solid phases (and the derivatization of), linkage agents, assembly methods, cleavage from the solid support, and workup and purification protocols. The method that emerged is operationally simple and chemically less complex than the Boc procedure. It is a mild procedure, and because of the Fmoc base lability and orthogonal nature relative to the previously accepted acid-labile protecting groups, it has allowed an element of chemical versatility in solid-phase strategies. It is the method of choice for the solid-phase synthesis of most modified peptide species including phosphorylated, sulfated, and glycosylated peptides. Its use in combination with groups such as Boc, allyl, and Dde has allowed access to a new dimension of peptide species from solid-phase synthesis procedures.

Synthesis of N‐α‐(6‐Hydrazinonicotinoyl)‐Octreotide: A Precursor of a [99mTc] Complex

AbstractThe synthesis of the title compound 12 via [3 + 4] and [7 + 2] segment condensation, respectively, using minimal protection of amino acid side chains is presented. The need to include the 6‐hydrazinonicotinic acid into a complete peptide synthesis originates from the juxtaposition of two factors, namely from the poor regioselectivity of Boc2O towards the amino groups of octreotide 1 and secondly from the distinct chemical properties of N‐hydroxysuccinimide esters of 6‐hydrazinonicotinic acid derivatives in general. The structure of the ligated octreotide derivative 12 suitable for the preparation of a [99mTc]‐labelled metal complex was established by spectroscopic methods. The study is complemented by conformational considerations on the pharmacophore of 12 and the parent octapeptide 1, being based on circular dichroism spectroscopy and 2D‐NMR techniques.

Facile reduction of peptide oxime endothelin antagonist during trialkylsilane/TFA cleavage after solid-phase synthesis

We describe a new solid-phase strategy for the selective reduction of the C=N bond in peptide oximes using a trialkylsilane in trifluoroacetic acid. The reduction is performed directly on the resin-bound peptide, with concomitant cleavage of the peptide from the resin and deblocking of protected side chains.

A method for the facile solid-phase synthesis of gramicidin S and its analogs

A simple method is described for the facile synthesis of gramicidin S and six other analogs, using standard solidphase synthetic technology and a single solution-phase cyclization step. The peptides were purified to homogeneity and characterized by plasma desorption time-of-flight mass spectrometry and NMR spectroscopy. Complete 1H NMR assignments for all seven peptides (in aqueous solution) are presented. Unlike previous approaches, the presented method is simple, automatable, rapid (less than three days), high-yielding, requires no side-chain protection during cyclization, and appears to be generally applicable to the preparation of a variety of related head-to-tail cyclic peptides.

Synthesis of the very acid‐sensitive Fmoc‐Cys(Mmt)‐OH and its application in solid‐phase peptide synthesis

S-4-methoxytrityl cysteine was synthesized and converted into the corresponding Fmoc-Cys(Mmt)-OH by its reaction with Fmoc-OSu. As compared to the corresponding Fmoc-Cys(Trt)-OH, the S-Mmt-function was found to be considerably more acid labile. Quantitative S-Mmt-removal occurs selectively in the presence of groups of the tert butyl type and S-Trt by treatment with 0.5-1.0% TFA. The new derivative was successfully utilized in the SPPS of Tyr1-somatostatin on 2-chlorotrityl resin. In this synthesis groups of the Trt-type were exclusively used for amino acid side-chain protection. Quantitative cleavage from the resin and complete deprotection was performed by treatment with 3% TFA in DCM-TES (95:5) for 30 min at RT. We observed no reduction of tryptophan under these conditions.

Synthesis, Characterization and Binding Properties of Peptide-Oligonucleotide Conjugates Containing the Spkk Peptide Motif

Peptide-oligonucleotide hybrid molecules 6 and 7 have been synthesized by on-line solid phase synthesis using allyloxycarbonyl (Alloc) side chain protection for serine. The conjugates have been characterized by ma.trix-assisted laser desorption ionization time-of-flight mass spectrometry. The interaction of conjugates 6 and 7 with a complementary sequence has been studied by absorption spectroscopy.

Amino acids derived from ornithine

The use of copper complexes in the protection of lysine and tyrosine side chains has been extensively described. Based on this work and in order to produre a peptide library consisting of unnatural amino acids, three amino acids were prepared by acylation/sulfonation of the δ-amino function of ornithine and they were incorporated in a random sequence. Depending upon the acidity of the remaining proton on the δ-nitrogen, an intramolecular cyclization competed with the amide formation using standard coupling conditions (DIC/HOBt/DIEA). This side reaction could be suppressed by simple removal of the base from the condensation mixture. The obtained peptides were purified on a PLRP-s® column and their identity was confirmed by mass spectrometry.

Sequence dependence of aspartimide formation during 9-fluorenylmethoxycarbonyl solid-phase peptide synthesis

We have examined the sequence dependence of aspartimide formation during Fmoc-based solid-phase synthesis of the peptide Val-Lys-Asp-X-Tyr-Ile. The extent of aspartimide formation and subsequent conversion to the α- or β-piperidide was characterized and quantitated by analytical reversed-phase high-performance liquid chromatography and fast atom bombardment mass spectrometry. Aspartimide formation occurred for X=Arg(Pmc), Asn(Trt), Asp(OtBu), Cys(Acm), Gly, Ser, Thr and Thr(tBu). No single approach was found that could inhibit this side reaction for all sequences. The most effective combinations, in general, for minimization of aspartimide formation were (i) tert-butyl side-chain protection of aspartate, piperidine for removal of the Fmoc group, and either 1-hydroxybenzotriazole or 2,4-dinitrophenol as an additive to the piperidine solution; or (ii) 1-adamantyl side-chain protection of aspartate and 1,8-diazabicyclo[5.4.0]undec-7-ene for removal of the Fmoc group.

Synthesis and biological activity of locust AKH-I and its analogues with modifications at the threonine residues.

A convenient method of synthesis, using a combination of solid and liquid phase methodology, for locust Adipokinetic Hormone-I (AKH-I) and its analogues with modifications at the threonine residues are reported. The N-terminal nonapeptide acid of AKH-I is synthesized in the solid phase using the 2-chlorotrityl chloride resin and the Fmoc/t-Bu strategy. Quantitative cleavage of the nonapeptide acid from the resin, with the tert-butyl type side-chain protection intact, is achieved with a mixture of acetic acid/trifluoroethanol/dichloromethane. The nonapeptide acid is then coupled in solution to the threonine derivatives, H-Thr-NH2 or H-Thr(Bzl)-NH2, with the DCC/HOBt method. The efficiency of this approach in the synthesis of AKH-I is demonstrated by the high yields and purity of the synthesized peptides. All the synthesized peptides were tested in two ways: first, in a lipid mobilization assay in locusts in vivo; and second, in a novel assay in vitro concerned with the uptake of radiolabelled acetate into locust tissue. Replacement of the hydroxyl hydrogen in Thr5 of locust AKH-I by the bulky and highly lipophilic tert-butyl group reduced the potency markedly, whereas efficacy is unaffected, but when the hydroxyl hydrogen of Thr10 in AKH-I is replaced by a benzyl group, the activity of the resulting analogue is identical to that of the natural peptide. Structure-activity relationships are discussed.

Solid support-dependent alkylation of tryptophan residues in SPPS using a 2-methoxybenzyl alcohol-based linker

Several side reactions can be encountered in the synthesis of Trp-containing peptides, due to molecular species originating from side chain-protecting groups or from the linker during acidolytic cleavage of the peptide from the resin. The linker can be the source of both alkylation in solution of the indole moiety of the tryptophan side chain and permanent readdition of the cleaved peptide to the resin. We report that both these reactions occur at a high level during the synthesis of Trp-containing peptides on a PEG-PS resin containing a 2-methoxybenzyl alcohol-based linker, in spite of the presence of suitable scavengers in the TFA-based cleavage mixture. Both side reactions are efficiently prevented by the use of a protected analogue of tryptophan, namely Nim-Boc-Trp, previously reported for the synthesis of peptides containing tryptophan and arginine residues.