Peptide targets for synthesis are often desired with
C-terminal end groups other than the more usual
acid and amide functionalities. Relatively few routes exist for
synthesis of C-terminal-modified peptidesincluding
cyclic peptidesby either solution or solid-phase methods, and known
routes are often limited in terms of
ease and generality. We describe here a novel Backbone
Amide Linker (BAL) approach, whereby the
growing
peptide is anchored through a backbone nitrogen, thus allowing
considerable flexibility in management of the
termini. Initial efforts on BAL have adapted the chemistry of the
tris(alkoxy)benzylamide system exploited
previously with PAL anchors. Aldehyde precursors to PAL, e.g.
5-(4-formyl-3,5-dimethoxyphenoxy)valeric
acid, were reductively coupled to the α-amine of the prospective
C-terminal amino acid, which was blocked
as a tert-butyl, allyl, or methyl ester, or to the
appropriately protected C-terminal-modified amino acid
derivative.
These reductive aminations were carried out either in solution or
on the solid phase, and occurred without
racemization. The secondary amine intermediates resulting from
solution amination were converted to the
9-fluorenylmethoxycarbonyl (Fmoc)-protected preformed handle
derivatives, which were then attached to poly(ethylene glycol)−polystyrene (PEG-PS) graft or
copoly(styrene−1% divinylbenzene) (PS) supports and
used
to assemble peptides by standard Fmoc solid-phase chemistry.
Alternatively, BAL anchors formed by on-resin reductive amination were applied directly. Conditions were
optimized to achieve near-quantitative acylation
at the difficult step to introduce the penultimate residue, and a side
reaction involving diketopiperazine formation
under some circumstances was prevented by a modified protocol for
Nα-protection of the second residue/introduction of the third residue. Examples are provided for the
syntheses in high yields and purities of
representative peptide acids, alcohols, N,N-dialkylamides,
aldehydes, esters, and head-to-tail cyclic peptides.
These methodologies avoid postsynthetic solution-phase
transformations and are ripe for further extension.
A novel and general backbone amide linker (BAL) strategy has been devised for preparation of C-terminal modified peptides containing hindered, unreactive, and/or sensitive moieties, in concert with N(alpha)()-9-fluorenylmethoxycarbonyl (Fmoc) solid-phase synthesis protocols. This strategy comprises (i) start of peptide synthesis by anchoring the penultimate residue, with its carboxyl group orthogonally protected, through the backbone nitrogen, (ii) continuation with standard protocols for peptide chain elongation in the C --> N direction, (iii) selective orthogonal removal of the carboxyl protecting group, (iv) solid-phase activation of the pendant carboxyl and coupling with the desired C-terminal residue, and (v) final cleavage/deprotection to release the free peptide product into solution. To illustrate this approach, several model peptide p-nitroanilides and thioesters have been prepared in excellent yields and purities, with minimal racemization. Such compounds are very difficult to prepare by standard Fmoc chemistry, including the BAL strategy as originally envisaged.
Distributed Drug Discovery (D3) proposes solving large drug discovery problems by breaking them into smaller units for processing at multiple sites. A key component of the synthetic and computational stages of D3 is the global rehearsal of prospective reagents and their subsequent use in the creation of virtual catalogs of molecules accessible by simple, inexpensive combinatorial chemistry. The first section of this article documents the feasibility of the synthetic component of Distributed Drug Discovery. Twenty-four alkylating agents were rehearsed in the United States, Poland, Russia, and Spain, for their utility in the synthesis of resin-bound unnatural amino acids 1, key intermediates in many combinatorial chemistry procedures. This global reagent rehearsal, coupled to virtual library generation, increases the likelihood that any member of that virtual library can be made. It facilitates the realistic integration of worldwide virtual D3 catalog computational analysis with synthesis. The second part of this article describes the creation of the first virtual D3 catalog. It reports the enumeration of 24 416 acylated unnatural amino acids 5, assembled from lists of either rehearsed or well-precedented alkylating and acylating reagents, and describes how the resulting catalog can be freely accessed, searched, and downloaded by the scientific community.
This study details a series of conditions that may be applied to ensure 'safe' incorporation of cysteine with minimal racemization during automated or manual solid-phase peptide synthesis. Earlier studies from our laboratories [Han et al. (1997) J. Org. Chem. 62, 4307-4312] showed that several common coupling methods, including those exploiting in situ activating agents such as N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU), N-[1H-benzotriazol-1-yl)-(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HBTU), and (benzotriazol-1-yl-N-oxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) [all in the presence of N-methylmorpholine (NMM) or N,N-diisopropylethylamine (DIEA) as a tertiary amine base], give rise to unacceptable levels (i.e. 5-33%) of cysteine racemization. As demonstrated on the tripeptide model H-Gly-Cys-Phe-NH(2), and on the nonapeptide dihydrooxytocin, the following methods are recommended: O-pentafluorophenyl (O-Pfp) ester in DMF; O-Pfp ester/1-hydroxybenzotriazole (HOBt) in DMF; N,N'-diisopropylcarbodiimide (DIPCDI)/HOBt in DMF; HBTU/HOBt/2,4,6-trimethylpyridine (TMP) in DMF (preactivation time 3.5-7.0 min in all of these cases); and HBTU/HOBt/TMP in CH(2)Cl(2)/DMF (1:1) with no preactivation. In fact, several of the aforementioned methods are now used routinely in our laboratory during the automated synthesis of analogs of the 58-residue protein bovine pancreatic trypsin inhibitor (BPTI). In addition, several highly hindered bases such as 2,6-dimethylpyridine (lutidine), 2,3,5,6-tetramethylpyridine (TEMP), octahydroacridine (OHA), and 2,6-di-tert-butyl-4-(dimethylamino)pyridine (DB[DMAP]) may be used in place of the usual DIEA or NMM to minimize cysteine racemization even with the in situ coupling protocols.
Solid-phase synthesis of biomolecules, of which peptides are the principal example, is well established. However, synthetic peptides containing modifications at the carboxy termini are often desired because of their potential therapeutic properties. As a result, there is a necessity for effective solid-phase strategies for the preparation of peptides with C-terminal end groups other than the usual carboxylic acid and carboxamide functionalities. The present article primarily reviews literature reports on methods for solid-phase synthesis of C-terminal modified peptides. In addition, general information about biological activities and/or synthetic applications of each individual class of peptide is also provided.
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