Hmb protection

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). 

However, it can be difficult to couple to the hindered Hmb-protected amino acids so in cases where the peptide chain contains Ser or Thr, oxazolidine dipeptide derivatives 3 have been employed (Scheme 7). These pseudoproline derivatives induce a turn in the peptide chain disrupting P-sheet formation. 

In a study examining the effect of Hmb protection of the C-terminal amino acid on fragment condensation, the formed 4,5-dihydro-8-methoxy-... 

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. 

With peptides that have become aggregated it is frequently not possible to obtain complete reaction, even after carrying out multiple acylation reactions. In such cases it is often necessary to repeat the synthesis, incorporating an Hmb-protected residue (Chapter 5) or an oxazolidine-dipeptide (26-28) two to four residues before the region prone to aggregation. 

Rgure4. Scheme for the incorporation of /V,Obis(Fmoc)-Af-(Hmb)-protected pentafluoro-phenyl esters (a) into a peptide chain. Key hydrogen-bonded tautomer (c) catalyses esterification to (e) which undergoes 0 - N acyl transfer. 

The incorporation of (Hmb)amino acid residues is conveniently achieved through 1-hydroxybenzotriazole activation of the Af,0-bis(Fmoc)-N-(Hmb)-protected pentafluorophenyl esters (Figure 4a). A number of groups have recently described the successful use of the N -Fmoc-iV-(Hmb)-amino acid derivatives (54, 55) in the synthesis of diflicult peptides. However, we have previously found that the monoFmoc derivatives of residues other than glycine or alanine couple slowly to resin-bound peptide chains (56). The slow coupling of these species is attributed to the formation of a relatively low... 

The choice of site for Hmb protection is based on the application of a set of standard considerations of the difhcult peptide being synthesized ... 

Given a choice of position for Hmb protection, dependent on the desired sequence, the chemically easiest bond to synthesize is protected (see following example). 

The details of synthetic improvements upon the introduction of Hmb protection into a wide range of difficult peptides have been published, illustrating the decisions (55,60,61,63-66). 

Lys(Boc), then one would expect to see a significant desAsn product. This later position for aggregation onset, at residue 3 or 4, is consistent with the inhibiting effect exhibited by Arg(Pmc) (c/. Figure 4d, Arg(Pmc) behaves in a similar manner to Arg(Mtr)). Thus Hmb protection needs to be implemented between Lys(Boc) and Ala in order to be effective for the remainder of the synthesis. The three available options are between Asn(Trt) and Lys(Boc) , Lys(Boc) and He , or Arg(Pmc) and Ala . Each of these options is reasonable, thus the decision point is simply based on the bond with the least steric constraints and the easiest (and therefore least precious) Hmb residue to prepare. 

Incorporate a /V-Hmb-protected amino acid prior to addition of Asp... 

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