Synthesis and Purification of Highly Hydrophobic Peptides Derived from the C-terminus of Amyloid -protein

Some biotechnological inventions involve expensive, sophisticated machines. Others are relatively simple innovations that nevertheless address, and solve difficult problems. Synthesis and purification of highly hydrophobic pep-tides can be a difficult and challenging task, particularly when these peptides have low solubility in both aqueous and organic solvents. Here we describe the synthesis and purification of a series of peptides derived from the hydrophobic C-terminus of the 42-residue form of amyloid-protein (A42), a peptide believed to be the primary cause for Alzheimer's disease (AD). The series of C-terminal fragments (CTFs) had the general formula A(x-42), x=28-39, which potentially can be used as inhibitors of A42 assembly and neurotoxicity. Synthesis and purification of peptides containing 8-residues or less were straightforward. However, HPLC purification of longer peptides was problematic and provided <1% yield in particularly difficult cases due to very poor solubility in the solvent systems used both in reverse-and in normal phase chromatography. Modification of the purification protocol using water precipitation followed by removal of scavengers by washing with diethyl ether circumvented the need for HPLC purification and provided these peptides with purity as high as HPLC-purified peptides and substantially increased yield.


INTRODUCTION
Amyloid -protein (A ) is a small protein of unknown function whose accumulation and self-assembly are believed to be the seminal events causing Alzheimer's disease (AD) [1,2].A is produced from the amyloid -protein precursor (APP), a type 1 transmembrane protein, in two predominant forms comprising 40 or 42 amino acids, and termed A 40 and A 42, respectively.Aging-related imbalance between production and clearance of A leads to elevation in its concentration, which in turn causes self-assembly of A into neurotoxic oligomers.The oligomers injure susceptible neurons by mechanisms that are not fully understood, aggregate further into larger assemblies, and eventually form polymers that deposit in the brain in the form of amyloid plaques, one of the pathological hallmarks of AD.Thus, inhibition of A assembly is a promising strategy for prevention of, and therapy for AD.
Multiple lines of evidence point at A 42 as the form of A that predominantly causes AD.A 40 and A 42 exist in the plasma and cerebrospinal fluid (CSF) at a concentration ratio of ~10:1, respectively, yet A 42 is deposited first during the development of AD [3], is the predominant component in parenchymal plaques [4], and is more neurotoxic than A 40 [5].An increase in the A 42/A 40 concentration ratio is associated with early onset familial AD [6], whereas treatments that decrease this ratio reduce the risk for AD [7].
A 40 and A 42 form distinct oligomer populations in vitro [8].Several kinds of oligomers formed only by A 42 have been shown to be highly neurotoxic [9][10][11].Direct *Address correspondence to this author at the Department of Neurology, David Geffen School of Medicine, University of California at Los Angeles, USA; E-mail: gbitan@mednet.ucla.educomparison of A 40 and A 42 oligomers formed under similar conditions demonstrate substantially higher toxicity of the A 42 oligomers [12,13].Because the only structural difference between A 40 and A 42 is the absence or presence of the last two amino acids, Ile 41 -Ala 42 , respectively, it is reasonable to hypothesize that the C-terminus plays an important role in the assembly and/or toxicity of A 42.In support of this hypothesis, increased toxicity of A 42 relative to A 40 correlates not only with distinct oligomer populations, but also with increased conformational stability [14][15][16][17] and a putative quasi-stable turn conformation [18][19][20] in the C-terminal region of A 42 but not A 40.
The C-terminal region (last 14 residues) of A 42 is highly hydrophobic and believed to reside within the membrane before A is cleaved from APP.Thus, we hypothesized that intermolecular association of the C-termini of several A 42 monomers, leading to formation of a core in which hydrophobic residues are sequestered from the aqueous environment, may be a major driving force for formation of A oligomers.If that is the case, we reasoned that Cterminal fragments (CTFs) of A 42 might compete with the C-terminus of full-length A 42 and disrupt oligomer formation (Fig. 1).To test this hypothesis, we set up to synthesize a series of A 42-derived CTFs [A (x-42), x=28-39] and test their ability to inhibit A 42 self-assembly and toxicity.Here we report on the synthetic challenges associated with obtaining long CTFs and ways to overcome these challenges.Results of biological and structural characterization of the CTFs will be reported elsewhere.
Synthesis and purification of peptides comprising mainly hydrophobic residues is challenging because of the low solubility of these peptides in both hydrophilic and hydrophobic solvents and their high tendency to aggregate [21][22][23].A and its derivatives are known to be difficult to synthesize and obtain in high purity [24,25].In fact, an early solid-state NMR investigation of A (34)(35)(36)(37)(38)(39)(40)(41)(42) has reported that this peptide readily aggregated into amyloid fibrils [26].Nevertheless, the potential of A 42-derived CTFs as inhibitors of A 42 toxicity made them attractive for us to synthesize and characterize.

Peptide Synthesis
All the peptides were synthesized by 9-fluorenylmethoxycarbonyl (FMOC) chemistry using automated Applied Biosystems 433A synthesizers.The synthesis scale was between 0.20-0.25 mmol.The coupling and deprotection cycles were extended from the manufacturer recommended times, 30 and 10 min, respectively, to 60 and 30 min, respectively.Coupling cycles were performed using 4 eq. of incoming amino acid, HBTU, and DIPEA in NMP.FMOC deprotection was done using 20% piperidine in NMP.The -NH 2 group of Lys was protected by tert-butoxycarbonyl (BOC).Cleavage of the peptide from the resin and side-chain deprotection (where appropriate) was performed using 10 mL of the following mixtures: A) 9.5:0.5 -TFA:H 2 O; B) 9.25:0.5:0.25 -TFA:H 2 O:EDT; C) 8.75:0.5:0.5:0.25 -TFA:H 2 O:thioanisole: EDT; or D) 9.5:0.25:0.25 -TFA:EDT:TIPS.After filtration of the resin, several methods were used for isolation of the crude peptide from the cleavage mixture (see Results).The integrity of the final products was verified by MS and AAA (Table 1).

RESULTS
With exception of the longest CTF we prepared, A (28-42) (Lys-Gly-Ala 30 -Ile-Ile-Gly -Leu -Met -Val -Gly -Gly -Val-Val 40 -Ile-Ala, full-length A numbering is used throughout the manuscript), all other CTFs comprised only amino acids with non-charged side-chains.The Gly residues in positions 29, 33, 37, and 38 were the only hydrophilic residues in the sequence, whereas all other residues were hydrophobic.Previous work suggested that A (34-42) readily formed -sheetrich fibrillar aggregates [26].Therefore, we anticipated that the synthesis and purification of the CTFs would become difficult above a certain length.Encouragingly, using the protocol we routinely use for the synthesis of full-length A 42, which includes extended coupling and deprotection times (see Methods and Materials) we could synthesize all peptide sequences and did not encounter special problems in any coupling/deprotection cycle as determined by the synthesizer's online UV monitoring of deprotection products.
The type of resin used was an important determinant of synthesis success.Initially, peptides were synthesized using a 200-400 mesh, 0.4-0.6 mmol/g Wang resin from Novabiochem.This resin worked well in all the cases it was used, but unfortunately, during the period of the project the company discontinued this particular resin.We therefore switched to a 100-200 mesh Wang resin from Novabiochem and found out that with this resin, substitution level had to be kept under 0.5 mmol/g for the synthesis to be successful.A (39-42) through A (33)(34)(35)(36)(37)(38)(39)(40)(41)(42) were synthesized successfully with this resin but longer CTFs produced negligible yields.Therefore, A (32-42) and longer peptides were synthesized using a 100-200 mesh TGA resin (Novabiochem), for which we also had to use low substitution levels.Towards the end of the project period, we also have used successfully a 200-400 mesh, 0.7 mmol/g Wang resin from Bachem.
Isolation of the CTFs from the crude cleavage mixture was relatively straightforward for A (39-42) through A (35-42) but became difficult for A (34-42) and longer peptides.
When attempting to use the same purification protocol for A (34-42) through A (29-42), we realized that these peptides did not precipitate by dilution of the concentrated cleavage mixture in ice-cold ether.Therefore, we tried to purify the crude peptides by loading the cleavage mixture directly onto an HPLC column.To simplify the cleavage mixture, we attempted excluding all the scavengers except water, taking into account that none of the amino acids used for the synthesis contained a side-chain protecting group and that the only moiety predicted to be sensitive to carbocations was the thioether in the side-chain of Met 35 .Ten mL cleavage mixture A were used to cleave each peptide from the resin for 120 min.After filtration of the resin, the volume was reduced to 1-2 mL and loaded directly onto an HPLC column.Because the mixture contained high concentration of TFA, which could partially dissolve the resin in silicabased columns, we used in these experiments a polymerbased, 250 21 mm polyhydroxyethyl A column (The Nest Group, Southborough, MA).However, we could not recover meaningful quantities of peptide from the column using this protocol.
In subsequent experiments, we therefore attempted to go back to the silica-based C4 column used for purification of shorter CTFs.In order to avoid harming the silica-based resin, after reducing the volume of the cleavage mixture to 1-2 mL, it was diluted in a mixture containing milli-Q water, ACN, and in some cases 2,2,2-trifluoroethanol (TFE) or 1,1,1,3,3,3-hexafluoroisopropanol (HFIP).These fluorinated alcohols are known to induce -helical conformation and help disaggregate -sheet-rich amyloid protein aggregates.This step was adjusted empirically for each peptide and each synthesis batch with the goal to keep most of the crude peptide in solution.It was impossible to measure precisely the relative quantity of each solvent in each experiment.The final volume loaded onto the C4 column was ~10 mL.Using this procedure, small amount of product could be purified (see Table 1).However, all the peptides contained a sideproduct that eluted with highly similar retention time to the  d) For these peptides the purity could not be determined using UV monitoring of HPLC.Therefore, the assessment is based on a chromatogram for which the total ion current was measured continuously by a mass-spectrometer.
desired product and displayed a molecular mass increase of 164 mass units.We interpreted this result to signify that during the cleavage reaction, some of the 4-hydroxymethylphenoxyacetamide linker connecting the peptide to the TGA resin, got cleaved and formed this adduct, presumably by reaction with the thioether side-chain of Met.The close proximity in retention time of the adduct and the pure peptide compromised the yield substantially, in some cases to <1%.In addition, the absorbance peak of the adduct was considerably larger than that of the pure peptide (Fig. 2), either because of higher extinction coefficient at 214 nm or due to higher solubility of the side product containing the adduct relative to the desired peptide that did not contain the adduct.To reduce this side product, we attempted using cleavage mixtures 3 or 4.However, although the scavengers reduced the amount of the side product, its apparent relative abundance in HPLC analysis did not change significantly because of the difficulty to detect the desired product.We also attempted to use 2-chlorotrityl resin instead of TGA resin, to eliminate the presence of the 4-hydroxymethylphenoxyacetamide linker altogether.However, these experiments yielded negligible amounts of desired products.
The low solubility of the CTFs, A (34-42) through A (29-42), in the HPLC solvent system and the need to separate them from closely migrating impurities caused very low recovery of the peptides from the HPLC columns used.Although some of the material loaded onto the column was eluted, during the course of the work we realized that the bulk of these peptides could not be recovered, likely because they precipitated out of solution during the early steps of the elution and did not re-dissolve in later stages or during wash steps.The recovery decreased with increasing peptide length.Peptides longer than A (33-42) necessitated analyzing fractions collected during semi-preparative HPLC purification by MS because the amount of peptide soluble in the mobile phase was too low to detect by UV absorbance (Fig. 2).
UV trace followed by multiple narrow peaks that probably represent negligible impurities and/or system "noise."The total ion current (TIC) trace of the MS detector shows a ~3 min broad peak followed by a ~30 min broad peak, both likely corresponding to the desired product.The combined mass-spectrum of the entire chromatogram (Fig. 3, gray box) shows A (31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42) as the only product.In addition, the average AAA error (Table 1) is similar to that of the CTFs purified by HPLC, suggesting similar degree of purity.

DISCUSSION
Despite decades of research, AD is a disease that currently cannot be cured or prevented.Currently approved drugs for AD treat the symptoms, rather than the causes of the disease and provide only moderate and temporary relief [27].AD is the leading cause of dementia and one of the leading causes of death among elderly people [28].A recent report by the Alzheimer Association indicated that in 2007, the prevalence of AD in the US has exceeded 5 million and may increase up to 16 million by the middle of the century if no cure is found [29].As the population ages, this situation may lead to an epidemic.Current estimates of cost of care for patients with AD in the US are over $148 billion a year [29].Numbers in other countries also are highly alarming [30][31][32].Thus, there is an urgent need for disease-modifying therapeutic strategies for AD.Following the modified Amyloid Cascade Hypothesis [2], leading strategies are focusing on A as a primary cause of AD and therefore target inhibition of A production, enhancement of A clearance, or disruption of A assembly [33].The normal physiologic function of A is unknown, thus inhibiting its production or increasing its clearance may lead to adverse side effects.In contrast, A self-association into oligomers and polymers is purely a pathologic phenomenon and therefore is an attractive target for development of inhibitors.
Multiple examples of small molecule inhibitors of A fibrillization exist in the literature [34].Recently, several groups reported inhibitors of A oligomer formation [35][36][37][38], following a paradigm shift in the amyloid field that identified pre-fibrillar oligomers as the primary cause of cytotoxicity [39,40].Inhibitor selection in these studies was based on empirical findings, rather than on systematic rational design.Therefore, little can be deduced about their mechanism of action.In contrast, structure-based inhibitor design approaches can provide mechanistic data that can be used to improve inhibitor efficacy and pharmacokinetics.
The main driving force for A self-assembly likely is intermolecular hydrophobic interactions that lead to sequestering hydrophobic patches from the aqueous environment [18].Thus, a reasonable rational design approach to inhibiting the assembly is to use short peptides derived from hydrophobic regions in A itself, which can compete with the same region in the full-length A .Following this rationale, a large body of work has focused on peptides derived from the central hydrophobic cluster (A (17-21)) [41,42], which had been shown to be important for A fibrillogenesis [43], though not necessarily for the formation of early oligomers.Because oligomers of A 42 likely are the main culprit causing neurotoxicity in AD, we decided to use peptides derived from the C-terminus of A 42 as potential inhibitors of oligomerization and toxicity of full-length A 42.Although this seems like an attractive strategy, presumably it has not been attempted previously because of the difficulty in preparing and solubilizing these highly hydrophobic peptides, particularly of longer CTFs.
The systematic study reported here shows that the synthesis and purification of A 42 CTFs longer than 8-amino acids indeed are difficult.Although no particular problems were encountered during the synthesis itself, the low solubility of these CTFs in both aqueous and organic solvents made their purification by common RP-HPLC techniques difficult.Although the bulk of the crude preparation presumably contained the desired peptides, RP-HPLC analysis, particularly of the long CTFs, was substantially skewed, over-representing impurities with higher solubility and/or higher absorbance at the detection wavelength (Figs. 2 and 3).For the same reasons, recovery of the pure peptides following preparative or semi-preparative HPLC purification was very low.To solve this difficulty, we adopted a protocol that circumvented the need for HPLC purification by taking advantage of the same properties that make HPLC purification difficult -low solubility in both hydrophilic and hydrophobic solvents.By precipitating the peptides in water followed by resuspending and washing with diethyl ether, we obtained some of the most difficult sequences in the CTF series with high purity and moderate yield (Fig. 3 and Table 1).
The solubility of the purified CTFs in aqueous buffers ranged from a few micromolar to a few hundred micromolar [44].Somewhat surprisingly, although A (28-42) could be purified by HPLC at acidic pH with reasonable yield, it was the least soluble CTF at pH 7.4 [44].The solubility of all other CTFs was sufficient for biological and biophysical studies, the details of which will be published elsewhere.These studies show that the CTFs are capable of inhibiting A 42-induced neurotoxicity and therefore are good lead candidates for developing peptidomimetic inhibitors with improved pharmacokinetic characteristics, for prevention and treatment of AD.

Fig. ( 1 )
Fig. (1).Schematic representation of putative mechanism of action of A 42-derived CTF.A) The C-termini of several A 42 molecules are hypothesized to form the hydrophobic core of the oligomer.B) CTFs derived from the C-terminus of A 42 may displace the Cterminus of the full-length peptide, leading to disruption of oligomerization.

Table 1 .
a) Average % error for each amino acid type divided by number of amino acids.b) Purified by semi-preparative HPLC.c) Precipitated in water and washed with diethyl ether.