Endo-linkers

Exo-linkers are composed of three units i) a group providing the site for enzyme catalyzed hydrolysis (R ) ii) a site for attachment of the target molecule (R ) and hi) a site for attachment to a further optional spacer (R ). 

By contrast, exo-linkers do not restrict the structure of the reactant and can be cleaved by more readily-available exo-enzymes, which act at the end of a chain or externally (Tab. 10.2). Furthermore exo-cleavable linkers yield untagged products upon cleavage from the soHd support 

For a better overview, examples of endo-linkers and the enzymes used for the cleavage of the product from the soHd phase which have been described in the literature so far are given in Tab. 10.1. 

Arrays of up to 1000 peptide nucleic acid (PNA) oligomers of different sequence were synthesized by Jensen et al. on polymer membranes (Fig. 10.2) [12]. 

The PNA chain was linked to the peptide spacer glutamic acid-(y-tert-butyl ester)-(fi-aminohexanoic acid)-(fi-aminohexanoic acid) (Glu [OtBuj-fiAhx-fiAhx) via an enzymatically cleavable Glu-Lys handle. The Glu [OtBuj-fiAhx-fiAhx spacer was coupled to the amino-functionalized membrane by standard Fmoc-Chemistry. Then the membranes were mounted in an ASP 222 Automated SPOT Robot and a grid of the desired format was dispensed at each position. The free amino groups outside the spotted areas were capped and further chain elongation was performed with Fmoc-protected PNA monomers to synthesize the desired PNA oligomers (18). After completion of the synthesis, the PNA oligomers were cleaved from the solid support by incubation with bovine trypsin solution in ammonium bicarbonate at 37 °C for 3 h. 

---

In principle, linker groups are polymer-enlarged versions of blocking functions used in regular solution-phase chemistry. Therefore, enzymatic transformations that may be employed for the removal of protecting groups in solution, in principle may also open up alternative opportunities for releasing compounds from polymeric supports. The linkers developed so far can be divided into exo- and endo-linkers (Fig. 10.1) cleavable by exo- respectively endo-enzymes, as proposed by Flitsch et al. [6]. 

Tab. 10.1 Examples of endo-linkers and respective cleavage enzymes. 

One of the very first papers reporting about endo-linkers was published by Elmore et al. (Scheme 10.4) [13]. They described a new linker containing a phos-phodiester group (19) for solid-phase peptide synthesis using a Pepsyn K (polyacrylamide) resin. After completion of coupling and deprotection cycles, the phos-phodiester (20) was cleaved with a phosphodiesterase. In this way / -casomorphin. Leu-enkephalin and a collagenase substrate (21) were synthesized in high yields. 

Table 18-5. Examples of endo-linkers and the appropriate cleavage enzymes.

Mechanical functions of cells require interactions between integral membrane proteins and the cyto-skeleton. These functions include organization of signaling cascades, formation of cell junctions and regulation of cell shape, motility, endo- and exocytosis. Several different families of membrane-associated proteins mediate specific interactions among integral membrane proteins, cytoskeletal proteins and contractile proteins. Many of these linker proteins consist largely of various combinations of conserved protein-association domains, which often occur in multiple variant copies. 

Sanders and coworkers96 97 catalyzed and directed the Diels-Alder reaction between 4-(maleimidomethyl)pyridine and 4-(3-furyl)pyridine using metalloporphyrin oligomers. When trimer 115a having three butadiyne linkers was used as the catalyst, the exo adduct was the exclusive product isolated at both 30 °C and 60 °C, whereas the uncatalyzed reaction provided an endo/exo ratio of 2/1 at 30 °C and a transient trace of endo adduct... 

Figure 1. The organization of catalytic and non-catalytic domains in cellulases from C. fimi and other bacteria. CfCenA, B and C, and CfCex are the endo- and exo-p- 1, 4-glucanases of C. fimi, ClfX is a translated open reading frame from Cellulomonas flavigena (29), CtEGD and PfEndA are endo-p-1, 4-glucanases from Clostridium thermocellum and Pseudomonas fluorescens, respectively (30,31), The primary structures are drawn approximately to scale and are numbered from the amino terminus of the mature protein ClfX is numbered from the start of the open reading frame. Unshaded areas represent catalytic domains, cross-hatched areas indicate cellulose-binding domains, repeated blocks of amino acids are stippled, and black areas represent linker regions.

The combination of the geometrical preference of the tether and the stereochemical preference of the dipolarophile substituent can be seen in the intramolecular cycloadditions of alkyl nitronates, (Scheme 2.6) (99). When the tether is restricted to two atoms, only the endo approach of the tether is observed in up to a 100 1 ratio, independent of the configuration of the disubstituted dipolarophile. However, in the case of a three-atom linker, there exists a matched and mismatched case with respect to the observed stereoselectivities. With a (Z)-configured dipolarophile, only the exo isomer was observed since the ester moiety also approaches on the exo to the nitronate. However, with an ( )-configured dipolarophile, the ester group is forced to approach in an endo manner to accommodate an exo approach of the tether, thus leading to lower selectivity. 

Surprisingly, with n = 2 or n = 3, the sense of stereoinduction is reversed. These results reflect the subtleties of the interactions among the DNA, the Cu(II) complex of the 9-aminoacridine derivative, and the diene and dienophile. The highest enantioselectivity was observed for the ligand having an ethylene linker (n = 2), R = 3,5-dimethoxybenzyl, and a methoxy substituent on the dienophile— with — 57% ee for the endo-isomer and —90% ee for the exo one (104,105). 

Iqbal and coworkers have reported another approach to cycUc di- and tripeptide mimics constrained with a disubstituted aromatic linker which exploits a free-radical-mediated macrocycHzation [8]. As an example, the tripeptide derivative 8 was promoted to undergo 7-endo ring closure under tin hydride conditions, providing the cyclic peptide 9 in 54% yield (Scheme 5). The reasonable yields obtained for these macrocycHzation processes is proposed by the authors to occur because of preorganization of the acycHc precursors, possibly owing to a reverse turn (y/ turn). 

Haberman Y, Grimberg E, Fukuda M, Sagi-Eisenberg R. Synap-totagmin IX, a possible linker between the perinuclear endo-c)4ic recycling compartment and the microtubules. J. Cell Sci. 2003 116 4307-4318. 

The structure of a hybrid DNA-RNA duplex containing a single MMI (3 -CH2N(CH3)-0-5 ) linker in the centre of the DNA strand has been studied by NMR. The lipophilic N-methyl group is peripheral to the duplex and the linker promotes a 3 -endo conformation for both adjacent sugar moieties. 

Two diastereoisomeric allylic silyl ethers 133 and 134 were first investigated and in both cases, the desired radical cyclization products were not observed. In the case of 133, cyclization proceeded exclusively in a 6-endo mode, affording diol 135 after oxidative cleavage of the silyl linker. The preference for 6-ring formation was attributed to the conformational rigidity of the allylic system and the less hindered nature of the distal sp -... 

A convergent monomer synthesis in which each base unit was attached to a preformed norbornene and linker unit was adopted. The linker chosen for initial work was 1,2-diaminoethane and the synthesis of the endo- and xo-linkers is shown in Scheme 6. Whilst the enJo-anhydride could be reacted directly with an excess of diaminoethane to give the desired mono-adduct, all attempts to carry out the same chemistry with the exo-anhydride were unsuccessful, giving only the product where the anhydride had reacted at both amino groups of the diamine. To circumvent this problem, the diaminoethane was first monoprotected, then allowed to react with the anhydride, then deprotected (Scheme 6). 

Of the nitrogen bases found in DNA / RNA, the simplest appeared to be thymine, so this was investigated first. Reaction of thymine with bromoacetic acid gave derivative 15, and this was coupled to the endo- and x -linkers to give monomers 16 and 17 (Scheme 7). It should be noted that the thymine in monomers 16 and 17 is substituted at the same location as in DNA, so that it is still capable of engaging in Watson-Crick... 

---