II.2 Chemical synthesis of Id2 protein fragments
II.2.1 The C-terminal fragments
The C-terminal domain of Id2 (product II.1, Table 1) consists of 58 residues, including a considerable number of the β-branched Leu (8) and Ile (4), as well as of Ser (9), Thr (5), Asx (6) and Glx (6). Peptide chain assembly starting from the C-end, Gly-134, using Fmoc chemistry and standard solid-phase procedures led to a crude peptide with very low homogeneity (Figure 1A), which prevented any attempt of purification. Instead, the shorter fragment 99-134 (II.2) was obtained as the major product (Figure 1B) and was then efficiently purified by preparative RP-HPLC. This suggests that the synthetic problems encountered in the preparation of the fragment 77-134 occurred during elongation from residue 98 to residue 77. However, the fact that the sequence 77-124 (II.3) was obtained with good homogeneity (Figure 1C) indicated that the difficulty of peptide chain assembly was dependent on the starting position. Indeed, when Glu-119 was chosen as the C-terminal residue, even after only 16 couplings a complex mixture of products was cleaved from the resin (product II.4, Table 1 and Figure 1D), which could not be further identified by mass spectrometry. The different accessibility of the last two Id2 fragments presumably reflected different aggregation propensity of the peptide chains; in fact, starting from Leu-124 rather than from Glu-119 was probably advantageous because the proline residue located four positions apart from the C-end, Pro-121, might have induced a backbone bending, thus reducing the aggregation of the growing sequence that was predicted to form preferentially β-sheets in the region 103-115 on the base of the Chou and Fasman analysis [5].
Synthesis of large Id2 protein fragments 22
Table 1: Amino acid sequence of the human Id2 protein and analytical data of the synthetic Id2 fragments.
Human Id2 a
M1KAFSPVRSVRKNSLSDHSLGISRSKTPVDDPMSL35LYNMNDCYSKLKELVP51SIPQNK KVS60KMEILQHVIDYILDLQ76IALDSHPTIVSLHHQRPGQNQASRTPLTTLNTDISILSLQA
a Underlined sequences: helix-1 (36-51) and helix-2 (61-76); double underlined sequence: loop (52-60). b The fragment is N-terminally acetylated. c The fragment is C-terminally amidated. d Determined by LC-ESI-MS (all others were determined by MALDI-ToF-MS).
Synthesis of large Id2 protein fragments 23
Figure 1: HPLC profiles of crude peptides II.1 (A), II.2 (B), II.3 (C), and II.4 (D).
It has been reported that solid-phase synthesis of peptides forming aggregates [6]
could be improved by using the pseudoproline dipeptides developed by Mutter and co-workers (Figure 2) [7].
Figure 2: The pseudoproline dipeptide developed by Mutter and co-workers [7].
Synthesis of large Id2 protein fragments 24 Pseudoproline dipeptides are masked serine or threonine residues that present some interesting features. First of all they allow the introduction of two residues in one step, and the side chain of serine or threonine can be regenerated by general cleavage conditions with TFA. Moreover, the introduction of a moiety that has a similar structure to proline helps disrupting secondary structure formation as well as aggregation. Additionally, peptides containing C-terminal pseudoproline dipeptide residues can be coupled without any risk of stereomutation. To obtain more effective results by using pseudoproline dipeptides, they must be introduced every four-six residues in the sequence, as it has been successfully done in the case of the human islet amyloid peptide (IAPP) [6].
As segment II.4 was a suitable candidate for the pseudoproline chemistry due to the presence of several Thr and Ser residues, the synthesis was repeated by introducing the pseudoproline at positions 111 and 114. However, also this approach led to a poorly homogeneous product.
Another strategy that can be applied in the case of difficult sequences containing native serine residues is the depsipeptide strategy (Figure 3) [8, 9]. Insertion of a depsipeptide unit in a sequence interrupts the regular pattern of amide bonds at the site of a Ser/Thr unit, as from that point the peptide chain is extended via the β-hydroxyl function and not via the α -amino function. Each depsipeptide unit provides an additional ionizable moiety, thereby increasing solubility and facilitating purification, as reported for the case of the amyloid peptide Aβ-(1-42) [10]. Conversion of the ester bonds to the target amide bonds is smoothly achieved through an O,N-acyl shift, which occurs quantitatively under mildly basic conditions over a short period of time.
Figure 3: Conversion of a depsipeptide to the amide form through a base-promoted O,N-acyl shift.
Synthesis of large Id2 protein fragments 25 This strategy was applied to assemble the Id2 peptide chain starting from residue 134, with the purpose of forming an ester bond between Ser-118 and Ala-117 or between Ser-114 and Leu-113 (Scheme 1). Unfortunately, in both cases the esterification reaction did not run to completion by using the conditions reported in the literature [8]. Therefore, further optimization of the reaction procedure is necessary to reach satisfactory results.
Scheme 1: Synthesis of Id2 C-terminal peptides by the depsipeptide strategy (the yellow sphere represents the Wang resin; the blue rectangles are the growing peptide chains; the dotted arrows represent the reactions that would have brought to the desired product after a successful esterification step).
In contrast to the synthetic difficulty of the Id2 sequence starting from Gly-134 (II.1), the Id2 sequence starting from Leu-124 (II.3) was chemically accessible by standard solid- phase methodology. However, this peptide was found to be only moderately soluble both in water and phosphate buffer, whereas it could be dissolved in methanol and in mixtures of methanol/water.
Another good starting point for the synthesis of C-terminally truncated Id2 analogs was Ile-110, as shown by the HPLC profiles of the crude products of peptides II.5-7 (Figure 4). For the synthesis of II.7 that contained the complete HLH motif with one mutation at position 42 (Cys was replaced with Ser), the pseudoproline dipeptide Asp(OtBu)-Ser(ΨMe,Mepro) was
Synthesis of large Id2 protein fragments 26 coupled instead of the single amino acids Asp-41 and Ser-42, to reduce the risk of chain aggregation during elongation [7].
Figure 4: HPLC profiles of crude peptides II.5 (A), II.6 (B), and II.7 (C).