Hmb backbone protection

2 Example 2. A myelin sheath-related dodecapepdde sequence 

The syntheses were performed on 0.2 mmol/g PEG-PS resin (Novabiochem) under standard conditions (7) using a Pioneer automated synthesizer (Perceptive Biosystems). The 12-mer target (III) was an in-house request for a rhetimatoid arthritis related project. 

This decapeptide sequence (IV) is a classic difficult peptide synthesis that has been used to assess the performance of new coupling procedures (8), additives (67), resin supports (35), backbone protection (60), etc. 

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The 2-hydroxybenzyl (Hbz) amide protecting group has been evaluated for use in Boc SPPS because it is stable to TFA but labile to TFSMA. To date, ACP (65-74) is the only reported synthesis using Hbz protection [214]. A comparison of sequences prepared using optimized Boc chemistry and Fmoc chemistry with Hmb backbone protection showed no significant difference between these two methods [205]. 

Piperidine in DMF 20% Piperidine with 0.1 m HOBt in DMF Hexamethyleneimine-JV-ethylpyrrolidine-HOBt in NMP-DMSO 20% Piperidine in DMF (with Hmb backbone protection) 10.90 5.55 1.49 Not detected... 

The retention of Hmb backbone protection on fully protected peptide segments in solution also gives extraordinarily soluble materials (71, 72). Backbone-protected segments are readily purified by standard reverse-phase HPLC and subsequently couple smoothly and cleanly in either DMF or DCM due to the highly concentrated coupling solutions prepared. 

O-Acylated Hmb is stable to acidolysis by TFA giving a useful additional feature to Hmb-containing peptides. Side-chain protection and the V -protection, if it is first replaced with Boc before Hmb O-acetylation, can be removed and backbone protection retained. After deacetylation of Hmb with aqueous hydrazine the peptide can be further purified before removal of the Hmb groups, useful when the product is poorly soluble. This feature is also useful for the purification of large polypeptides as the presence of backbone protection on the side-chain deprotected peptide prevents the formation of relatively stable folded structures that can complicate HPLC purification.1 11... 

Quibell, M., Owen, D., Packman, L. C., and Johnson, T. (1994) Suppression of piperidine-mediated side product formation for Asp(OBut)-containing peptides by the use of N-(2-hydroxy-4-methoxybenzyl)(Hmb) backbone amide protection. J. Chem. Soc. Chem. Commun. 20, 2343—2344. 

For a more appropriate adaptation of the benzyl-type backbone protection to the Fmoc/tBu strategy, the 2-hydroxy-4-methoxybenzyl (Hmb) group was proposed because incorporation of the 4-methoxy moiety leads to significantly enhanced acid lability.The Hmb amino acids are prepared analogously to the Hbz compounds (Section 2.3.2.1),P 1 and the AT -Fmoc-protected AT -Hmb amino acids can be obtained by reaction of the Hmb amino acids with Fmoc-OSut l or, upon silylation, with Fmoc-ClP in the presence of a base. 

The efficiency of postsynthetic acetylation of the Hmb groups is well demonstrated by the successful synthesis of the extremely insoluble P-amyloid-(1 3) peptide.P Additional examples of the advantageous use of this backbone protection are the recent syntheses of N-glycopeptidesf and phosphopeptides.t There are, however, also cases where even with multiple backbone protection only noinor improvements in solubility were obtained,and polar aprotic solvents with strong hydrogen-bond acceptor properties, such as DMSO, proved to be more efficient. ... 

N-2-hydroxy-4-methoxybenzyl (Hmb) for backbone protection, pseudoprolines and o-acyl isopeptides. 

The N-(2-hydroxy-4-methoxybenzyl) (Hmb) backbone amide-protecting group... 

Backbone protection need only be introduced a maximum of each sixth residue (51, 60, 61). As the synthesis extends this becomes more flexible. Thus for a 20 residue sequence containing no proline residues, a maximum of 2 or 3 Hmb residues are reqixired located approximately at residues 5-8 and 12-15. 

Figures. HPLC profiles of total crude products from the assembly of decadecapeptide in. (a) Standard assembly without backbone protection, (b) Introduction of (Hmb)Ala no aggregation during assembly. Conditions Phenomenex C4 (250 x 4.6 mm) 0-90% B in A linear gradient over 25 min, 1.5 ml/min, 215 nm UV detection, where solvent A = 0.1% aq. TFA and solvent B = 90% acetonitrile/10% solvent A.

Problems do, however, occur during cyclization with all-L- or all-D-residues (see Section 6.8.1.3.1). Cyclodimerization can be triggered by an extended backbone conformation of the linear precursor, and C-terminal epimerization derived from slow amide bond formation. These lead to dimers or epimerized side products. 73,241 Cyclization of such tetrapeptides are successful only in a few exceptional cases.1169,73 To overcome this problem, protection of the backbone amide with Boc has been proposed since this approach favors the c/s-amide bond configuration which induces one or more suitable conformations for cyclization. 69 As a consequence, the cyclization yield of c[-Ala-]4 242 improved from 1 to 27% 69 (see also the use of Al-Hmb protection in Section 6.8.1.3.1). 

The insertion of a proline or A -alkyl amino acid residue into a sequence is known to disrupt formation of p-sheets and other secondary structures thought to be responsible for the aggregation (73). The effects can be long range, with the outset of aggregation often being postponed for as many as six residues, or eliminated altogether. Recently, two independent approaches have been developed which exploit this principle. The approach by Sheppard and co-workers utilizes temporary N-2-hydroxy-4-methoxybenzyl (Hmb) protection of the peptide backbone amide and is discussed in detailed in Chapter 5. 

The following examples illustrate the dramatic improvement obtained in difficult peptide syntheses on the introduction of only one or two judiciously placed Hmb residues and exemplifies the decision processes undertaken when choosing the optimal backbone amide bond for protection. 

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