Invited Review Chemoenzymatic Synthesis of Lipidated Peptides

Summary.  This review highlights the use of enzymatic protecting group techniques in the synthesis of lipidated peptides. Lipidated proteins play key roles in signal transduction processes. Moreover, structurally well-defined peptides containing the characteristic linkage region of the peptide backbone with the lipid can provide valuable tools for the study of biological phenomena associated with these protein conjugates. The multifunctionality and pronounced lability towards acids and bases of such compounds render their synthesis a formidable challenge. However, the recent development of enzymatic protection groups provides an efficient access to these sensitive and biologically relevant peptide conjugates under particular mild conditions and with high selectivity. Received December 12, 1999. Accepted January 26, 2000     

The synthesis of acid- and base-labile lipopeptides on solid support

Lipidated peptides, including characteristic partial structures of human Ras proteins, were synthesized by means of a new solid-phase technique in 22-68 % yield. This technique gives access to farnesylated, palmitoylated, and doubly lipidated peptides as methyl esters or carboxylic acids carrying a fluorescent tag or a maleimide moiety for coupling to proteins. The peptide backbones were built up on the resin by using 9-fluorenylmethoxycarbonyl chemistry together with the oxidatively cleavable hydrazide linker. As a key step, the acid-labile farnesyl and basic-labile palmitoyl lipid groups were introduced onto the resin after the cleavage of appropriate acid- or reduction-sensitive protecting groups from the cysteine residues. Optional introduction of different fluorescent tags or a maleimide group into the peptide was followed by release of the resin-bound target peptide as the methyl ester or carboxylic acid by very mild copper(II)-mediated oxidation in slightly acidic or basic media. This new methodology should substantially facilitate the access to lipidated peptides for the study of important biological phenomena like biological signal transduction, localization, and vesicular transport.     

脂蛋白合成新进展

生物体内的信号传导蛋白在膜上的定位与其生物功能的发挥依赖于特定脂肪链的修饰,然而传统的基因表达法合成脂蛋白,得到的纯品产率很低.在近10年中,逐渐发展起来一种新的合成方法,即将化学合成脂修饰的多肽与基因表达培养蛋白相结合,可以合成出具有多条脂肪链修饰的蛋白缀合物,并且整个合成过程在非常温和的环境中进行,产品能保持较高的纯度和活性.采用该方法合成的脂蛋白用于体外的实验中,其结果与生物体内的现象非常接近.脂蛋白合成方法的发展对研究细胞中的信号传导过程具有重要的意义,并在药物合成和提高药效方面都有很多应用,这对于研究恶性肿瘤等疾病的发病机理起到了重要的推动作用.同时该脂蛋白合成的成功是采用化学法合成生物大分子解释生物体内的现象一个重大的突破,是化学生物学发展重要的一步.     

Acid-labile protecting groups for the synthesis of lipidated peptides

Lipidated peptides and their neolipoprotein derivatives are efficient tools for the investigation of biological processes in molecular detail. These compounds are often acid- and base-labile, and their synthesis requires the use of a combination of blocking groups that can be removed under very mild conditions. In this article we demonstrate that the Boc urethane and different trityl-type protecting groups can be cleaved selectively under acidic conditions that are mild enough to be compatible with the demands of lipopeptide synthesis. Thus, the Boc group was cleaved with TMS triflate in the presence of lutidine, and the methyltrityl (Mtt) and the methoxytrityl (Mmt) group were removed with 1% TFA in dichloromethane in the presence of triethylsilane as cation scavenger. Removal of the phenylfluorenyl group was achieved with up to 3% TFA in dichloromethane in the presence of triethylsilane at 0 degrees C. These protecting-group techniques were successfully applied in the synthesis of differently lipidated H-Ras peptides.     

Convergent synthesis of a fully lipidated glycosylphosphatidylinositol anchor of Plasmodium falciparum

A highly convergent strategy for the synthesis of fully lipidated GPI anchors of malarial origin is reported. This strategy utilized three orthogonal protecting groups, which can be chemoselectively deprotected and functionalized in the late stage of the synthesis. Rapid access to the target GPIs in a highly efficient manner in sufficient quantities for the biological studies has been achieved.     

Solid-phase synthesis of asymmetrically branched sequence-defined poly/oligo(amidoamines)

We present for the first time the synthesis of asymmetrically branched sequence-defined poly/oligo(amidoamines) (PAAs) using solid-phase synthesis with the capability of introducing diversity at the side chains. We introduce two new versatile (diethylenetriamine) building blocks for solid-phase synthesis bearing Fmoc/Boc and Fmoc/Alloc protecting groups expanding recently used Fmoc/Boc protecting group strategy for linear PAAs to an Fmoc/Alloc/Boc strategy. This allows for orthogonal on-resin cleavage of Fmoc and Alloc protecting groups during solid-phase synthesis of PAAs with backbones differing in chain length and sequence. With these structures we then demonstrate the potential for generating asymmetrical branching by automated multiple on-resin cleavage of Alloc protecting groups as well as the introduction of side chains varying in length and number. Such systems have high potential as nonviral vectors for gene delivery and will allow for more detailed studies on the correlation between the degree of branching of PAAs and their resulting biological properties.     

Synthesis of Sulfotyrosine‐Containing Peptides by Incorporating Fluorosulfated Tyrosine Using an Fmoc‐Based Solid‐Phase Strategy

Tyrosine O‐sulfation is a common protein post‐translational modification that regulates many biological processes, including leukocyte adhesion and chemotaxis. Many peptides with therapeutic potential contain one or more sulfotyrosine residues. We report a one‐step synthesis for Fmoc‐fluorosulfated tyrosine. An efficient Fmoc‐based solid‐phase peptide synthetic strategy is then introduced for incorporating the fluorosulfated tyrosine residue into peptides of interest. Standard simultaneous peptide‐resin cleavage and removal of the acid‐labile side‐chain protecting groups affords the crude peptides containing fluorosulfated tyrosine. Basic ethylene glycol, serving both as solvent and reactant, transforms the fluorosulfated tyrosine peptides into sulfotyrosine peptides in high yield.     

Solid-phase synthesis of lipidated peptides

A new flexible and efficient methodology for the solid-phase synthesis of lipidated peptides has been developed. The approach is based on the use of previously synthesized building blocks and overcomes the limitations of previously reported methods, since long doubly lipidated peptides can be synthesized by using this route. Furthermore, it was thus possible to prepare a large number of N- and H-Ras peptides bearing a wide range of reporter and/or linking groups--efficient tools for the investigation of biological processes. In terms of efficiency and flexibility this solid-phase method is superior to the solution-phase synthesis. It gives pure peptides in multimilligram amounts within a much shorter time and with superior overall yield.     

Aryldithioethyloxycarbonyl (Ardec): a new family of amine protecting groups removable under mild reducing conditions and their applications to peptide synthesis

The development of phenyldithioethyloxycarbonyl (Phdec) and 2-pyridyldithioethyloxycarbonyl (Pydec) protecting groups, which are thiol-labile urethanes, is described. These new disulfide-based protecting groups were introduced onto the epsilon-amino group of L-lysine; the resulting amino acid derivatives were easily converted into N alpha-Fmoc building blocks suitable for both solid- and solution-phase peptide synthesis. Model dipeptide(Ardec)s were prepared by using classical peptide couplings followed by standard deprotection protocols. They were used to optimize the conditions for complete thiolytic removal of the Ardec groups both in aqueous and organic media. Phdec and Pydec were found to be cleaved within 15 to 30 min under mild reducing conditions: i) by treatment with dithiothreitol or beta-mercaptoethanol in Tris.HCl buffer (pH 8.5-9.0) for deprotection in water and ii) by treatment with beta-mercaptoethanol and 1,8-diazobicyclo[5.4.0]undec-7-ene (DBU) in N-methylpyrrolidinone for deprotection in an organic medium. Successful solid-phase synthesis of hexapeptides Ac-Lys-Asp-Glu-Val-Asp-Lys(Ardec)-NH2 has clearly demonstrated the full orthogonality of these new amino protecting groups with Fmoc and Boc protections. The utility of the Ardec orthogonal deprotection strategy for site-specific chemical modification of peptides bearing several amino groups was illustrated firstly by the preparation of a fluorogenic substrate for caspase-3 protease containing the cyanine dyes Cy 3.0 and Cy 5.0 as FRET donor/acceptor pair, and by solid-phase synthesis of an hexapeptide bearing a single biotin reporter group.     

An efficient synthesis of N-methyl amino acids by way of intermediate 5-oxazolidinones

N-Methyl amino acids occur in many natural products. Experimental strategies are presented for a unified approach to the synthesis of N-methyl derivatives through 5-oxazolidinones of the 20 common l-amino acids. The amino acids with reactive side chains that required protecting groups or devoted syntheses for side chain construction for N-methylation to proceed included serine, threonine, tyrosine, cysteine, methionine, tryptophan, asparagine, histidine, and arginine. The studies have provided improved methods for the preparation of N-methyl serine, threonine, and tyrosine. All 20 of the common l-amino acids are now available in suitable forms for solid or solution-phase peptide synthesis.     

Ethoxyethylidene protecting group prevents N-overacylation in aminooxy peptide synthesis

We report herein an improved synthetic route for the preparation of homogenous aminooxy peptides suitable for oxime ligation. Aminooxyacetic acid (Aoa) was protected with 1-ethoxyethylidene group (Eei) then incorporated either using PyBOP or as N-hydroxysuccinimidyl ester at N-terminal end or at a lysine side chain into model peptides in solution and on solid support. Due to the Eei protecting group, these new reagents prevent the N-overacylation side reaction in comparison with Boc-Aoa derivatives. Subsequent deprotection under mild acidic conditions gave the corresponding pure aminooxylated peptides.     

Synthesis and biological activity of photoactivatable N-ras peptides and proteins

A modular strategy for the assembly of farnesylated N-Ras heptapeptides carrying a photoactivatable benzophenone (BP) group within the lipid residue is described. This strategy is based on the fragment condensation of a N-terminal hexapeptide synthesized on the solid support with a cysteine methyl ester which is modified with different farnesyl analogues, incorporating the photophor. At the N-terminus of the peptides different functional groups can be attached, e.g., biotin for product enrichment and detection after photoactivation or a maleimido (MIC) linker, allowing for the coupling to proteins carrying a C-terminal free cysteine. Using this strategy, 24 peptides were synthesized, incorporating farnesyl analogues with four different chain lengths. Two of these photoactivatable conjugates were ligated to oncogenic human N-RasG12V Delta 181. A cellular transformation assay revealed that the semisynthetic proteins retain their biological activity despite the photolabel. The first photolabeling experiments with a geranyl-BP-labeled N-Ras construct and the farnesyl-sensitive guanine nucleotide exchange factor hSos1 indicate that this photoaffinity labeling system can be particularly useful for studying protein-protein interactions, e.g., the participation of the farnesyl group in Ras signaling, which is still discussed with controversy.     

AJIPHASE®: A Highly Efficient Synthetic Method for One‐Pot Peptide Elongation in the Solution Phase by an Fmoc Strategy

We previously reported an efficient peptide synthesis method, AJIPHASE®, that comprises repeated reactions and isolations by precipitation. This method utilizes an anchor molecule with long‐chain alkyl groups as a protecting group for the C‐terminus. To further improve this method, we developed a one‐pot synthesis of a peptide sequence wherein the synthetic intermediates were isolated by solvent extraction instead of precipitation. A branched‐chain anchor molecule was used in the new process, significantly enhancing the solubility of long peptides and the operational efficiency compared with the previous method, which employed precipitation for isolation and a straight‐chain aliphatic group. Another prerequisite for this solvent‐extraction‐based strategy was the use of thiomalic acid and DBU for Fmoc deprotection, which facilitates the removal of byproducts, such as the fulvene adduct.     

The lipidated membrane anchor of full length N-Ras protein shows an extensive dynamics as revealed by solid-state NMR spectroscopy

Many proteins involved in signal transduction are equipped with covalently attached lipid chains providing a hydrophobic anchor targeting these molecules to membranes. Despite the considerable biological significance of this membrane binding mechanism for 5-10% of all cellular proteins, to date very little is known about structural and dynamical features of lipidated membrane binding domains. Here we report the first comprehensive study of the molecular dynamics of the C-terminus of membrane-associated full-length lipidated Ras protein determined by solid-state NMR. Fully functional lipid-modified N-Ras protein was obtained by chemical-biological synthesis ligating the expressed water soluble N-terminus with a chemically synthesized (2)H or (13)C labeled lipidated heptapeptide. Dynamical parameters for the lipid chain modification at Cys 181 were determined from static (2)H NMR order parameter and relaxation measurements. Order parameters describing the amplitude of motion in the protein backbone and the side chain were determined from site-specific measurements of (1)H-(13)C dipolar couplings for all seven amino acids in the membrane anchor of Ras. Finally, the correlation times of motion were determined from temperature dependent relaxation time measurements and analyzed using a modified Lipari Szabo approach. Overall, the C-terminus of Ras shows a versatile dynamics with segmental fluctuations and axially symmetric overall motions on the membrane surface. In particular, the lipid chain modifications are highly flexible in the membrane.     

Synthesis of a family of cyclic peptide-based anion receptors

We report here the design and synthesis of a family of novel backbone modified cyclic peptides, bearing dipicolylamine side chains for metal complexation and subsequent anion binding studies. Two approaches to the cyclic peptides were investigated. Initially, a stepwise approach was employed, involving solid-phase assembly of oxazole-based building blocks, followed by solution-phase macrolactamisation of the resulting linear precursor. The alternative strategy involved the formation of linear bisoxazole fragments in solution-phase, followed by a cyclodimerisation reaction. The zinc(II) complexes of these receptors bind selectively to di- and tri-phosphate ions over hydrogenphosphate.     

Multiple Peptide Synthesis Methods and Their Applications. New Synthetic Methods (87)

Rapid developments in the biotechnology of new proteins, as well as advances in immunology and the development of pharmaceuticals based on inhibitors and antagonists, have led to immense demands for synthetic peptides. Simultaneous preparation of 100–150 completely different peptides, having chain lengths of up to 20 amino acids can nowadays be achieved using multiple synthesis methods. The yields and qualities of the peptides so obtained are high enough to permit reliable in vivo and in vitro screening for biological activities. Moreover, it is possible to optimize synthetic conditions and to carry out comparative studies on the secondary structures and conformational mapping of proteins. Special multiple synthesis methods facilitate the epitope mapping of larger peptides for diagnostic purposes and for the development of vaccines based on a few hundreds of free or rod-bound peptides that are useful for immunoassays. Multiple methods of peptide synthesis also enable the preparation of so-called peptide libraries which could comprise hundreds of thousands of peptides, and by which new perspectives for the screening of lead structures will be opened up. Peptide synthesis using a combination of photolabile protecting groups and photolithographic procedures enables the assembling of peptide libraries on small plates for use in miniature immunoassays. Furthermore, lipopeptide-antigen conjugates allow both the preparation of peptide-specific and monoclonal antibodies as well as a complete screening of epitopes of B-, T-helper and T-killer cells. Applications in the areas of AIDS diagnosis, the development of vaccines, and screening for the hormone analogues, demonstrate just some of the possibilities that have been opened up by multiple peptide synthesis methods.     

Synthesis of functionalized rab GTPases by a combination of solution- or solid-phase lipopeptide synthesis with expressed protein ligation

Prenylated proteins with non-native functionalities are generally very difficult to obtain by recombinant or enzymatic means. The semisynthesis of preparative amounts of prenylated Rab guanosine triphosphatases (GTPases) from recombinant proteins and synthetic prenylated peptides depends largely on the availability of functionalised prenylated peptides corresponding to the proteins' native structure or modifications thereof. Here, we describe and compare solution-phase and solid-phase strategies for the generation of peptides corresponding to the prenylated C terminus of Rab7 GTPase. The solid-phase with utilisation of a hydrazide linker emerges as the more favourable approach. It allows a fast and practical synthesis of pure peptides and gives a high degree of flexibility in their modification. To facilitate the analysis of semisynthetic proteins, the synthesised peptides were equipped with a fluorescent group. Using the described approach, we introduced fluorophores at several different positions of the Rab7 C terminus. The position of the incorporated fluorescent groups in the peptides did not influence the protein-ligation reaction, as the generated peptides could be ligated onto thioester-tagged Rab7. However, it was found that the positioning of the fluorescent group had an influence on the functionality of the Rab7 proteins; analysis of the interaction of the semisynthetic Rab7 proteins with REP (Rab escort protein) and GDI (guanosine diphosphate dissociation inhibitor) molecules revealed that modification of the peptide side chains or of the C-terminal isoprenoid did not significantly interfere with complex formation. However, functionalisation of the C terminus was found to have an adverse effect on complex formation and stability, possibly reflecting low structural flexibility of the Rab GDI/REP molecules in the vicinity of the lipid-binding site.     

N-methyl-phenacyloxycarbamidomethyl (Pocam) group: a novel thiol protecting group for solid-phase peptide synthesis and peptide condensation reactions

In the so-called thioester method for the condensation of peptide segments, protecting groups for amino and thiol groups are required for chemoselective ligation. In this study, we developed a novel thiol protecting group, N-methyl-phenacyloxycarbamidomethyl (Pocam). We used it for protection of cysteine side chains, and synthesized Pocam-containing peptides and peptide thioesters. These were condensed by the thioester method. After the condensation reaction, Pocam groups were cleaved by Zn/AcOH treatment. At the same time, the azido group, which was used for the protection of lysine side chains, was also converted to an amino group, demonstrating that this protecting group strategy simplified the deprotecting reaction after the peptide condensation reaction to only one step.