Protective Group Chemistry

4.1. Arginine Side Chain Protection In general the nucleophilic character of the arginine side chain has to be masked in order to avoid unwanted side reactions during activation and coupling steps. One of the possible complications is the intramolecular δ-lactam formation resulting from nucleo-philic attack of the N ^δ-nitrogen at the

activated carboxylic group (cf. Scheme 9). This side reaction is impending whenever the N ^δ-nitrogen is unsubstituted.

Scheme 10: N ^ω-Acylation of arginine derivatives and subsequent degradation to ornithine derivatives.

Another risk is the intermolecular N ^ω-acylation of insufficiently protected arginine residues by activated amino acid derivatives. Subsequently, N ^ω

-(α-aminoalkanoyl)-RHN

Scheme 9: δ-Lactam formation of carboxy-acti-vated arginine derivatives.

substituted arginines degrade under formation of the parent ornithines and 2-amino-4H-imidazol-4(5H)-ones (cf. Scheme 10).

4.1.1. PROTONATION

Under common coupling conditions acylation of the arginine side chain is largely prevented by protonation of the guanidino function. Chain elongation steps of peptide fragments with N-terminal, protonated arginine residues have been performed successfully (cf. synthesis of secretin^[17]). However, the poor solubility of arginine salts in organic solvents limits the practicability of this approach.

For the original synthesis of BIIE 0246, described in the patent specification^[18], Cbz-protected arginine hydrochloride was used as arginine building block. The reported yield for the coupling of Z-Arg(HCl)-OH with HCl salt of 4-(2-aminoethyl)-1,2-diphenyl-1,2,4-triazolidine-3,5-dione was 45 % using CDI in DMF. Our own attempts to couple N ^α-protected arginine hydrochlorides gave only poor results, due to the limited solubility, incomplete conversion and problems with the isolation and purification of the product.

4.1.2. N^G-NITRO-PROTECTED ARGININES

Boc-Arg(NO₂)-OH and Z-Arg(NO₂)-OH are commercially available, inexpensive arginine building blocks. The N^G-nitro group is stable against TFA, HBr/CH₃CO₂H or alkaline conditions. Liquid HF, Zn/CH₃CO₂H, or — most favorable — catalytic hydrogenolysis are suitable cleavage conditions. Still, N ^ω’-nitro protection does not completely exclude δ-lactam formation in the coupling step^[19]. Sometimes deprotection under reductive methods (Zn/CH₃CO₂H or H₂/Pd) is incomplete and leads to only partially reduced by-products (e.g. N^G-aminoarginines)^[7].

4.1.3. N^ω-ALKOXYCARBONYL-PROTECTED ARGININES

N ^ω,ω’- or N ^δ,ω-bis(alkoxycarbonyl)-substituted arginine derivatives are sufficiently protected against acylation. Among the urethane-type protective groups, which were applied in peptide chemistry to block the arginine side chain, are 1-Adoc, Alloc, Boc, and Cbz (= Z).

While Z-Arg(δ,ω-Adoc2)-OH, Boc-Arg(δ,ω-Alloc2)-OH, and Z-Arg(δ,ω-Z2)-OH can be prepared from X-Arg-OH (X = Boc, Cbz) and the alkyl haloformates Adoc-F, Alloc-Cl, and Cbz-Alloc-Cl, respectively^[7], the reaction of X-Arg-OH with excess Boc₂O leads to a mixture of the ω,ω’- and the δ,ω-bis(Boc) regio-isomers, the latter being unstable^[20]. X-Arg(ω,ω’-Boc2)-OH (X = Boc, Cbz) is best prepared by gu-anidinylation of the corresponding ornithine precursors (cf. section 4.2).

Z-Arg(Z₂)-OH or its active esters are excellent building blocks for the incorporation of arginine residues. After subsequent, simultaneous cleavage of the Cbz-groups by catalytic hydrogenation the guanidine residue is shielded by protonation during the acylation of N ^α.

We adapted this approach for the synthesis of N^G-unsubstituted BIIE 0246 and analogs. The coupling of Z-Arg(Z₂)-OSu with 4-(2-aminoethyl)-1,2-diphenyl-1,2,4-triazolidine-3,5-dione proceeded smoothly and yielded a pure, crystalline product.

After removal of the Cbz groups by catalytic transfer hydrogenation using formic acid in the presence of Pd on carbon, the subsequent N ^α-acylation yielded BIIE 0246.

Unfortunately, HPLC analysis of the final product revealed the presence of a closely related by-product with a molecular mass corresponding to that of BIIE 0246 plus 2 units. Analysis of the ESI mass spectra showed that the intensity of the MH+2 peak exceeds the expected value for a normal isotope distribution. This phenomenon was observed for all isolated intermediate products after the hydrogenation step.

Obviously, the 1,2-diphenylurazole moiety is not inert towards catalytic hydrogenation. Therefore, protective groups which require hydrogenolytic cleavage can not be applied for the preparation of BIIE 0246 analogs with the 1,2-diphenyl urazole substructure.

Fig. 4: Relative intensities of ESI-MS peaks (in brackets: calculated values for isotope peaks of pure compound). After hydrogenolytic deprotection of A the MH + 2 peaks of the following products B and C are too intensive, which indicates the presence of a hydrogenated by-product. A: N ^α,N ^δ,N ^ω -tris-(benzyloxycarbonyl)-N-(2-(1,2-diphenyl-1,2,4-triazolidine-3,5-dione-4-yl)ethyl)argininamide; B: N-(2-(1,2-diphenyl-1,2,4-triazolidine-3,5-dione-4-yl)ethyl)argininamide; C: contaminated BIIE 0246 (2); D:

pure BIIE 0246 (2) (control).

4.1.4. N^ω-ARENESULFONYL-PROTECTED ARGININES

The 4-toluenesulfonyl group (Tos) was largely used for the protection of the arginine side chain in peptide synthesis in solution or on solid support. Though, the N^G -p-toluenesulfonyl substitution almost completely masks the basic and nucleophilic character of the guanidino function, the harsh conditions required for its removal (HF/anisole or Na/NH₃) distract chemist from the application of the Tos protective group. In recent years many electron-rich arylsulfonyl protective groups have been developed for the protection of the arginine side chain. Mtr, Pmc, and Pbf (cf.

[MH]⁺

Scheme 11) can be cleaved off using TFA and have become standard arginine side chain protective groups in Fmoc/^tBu chemistry.

S

Scheme 11: Arenesulfonyl-based arginine side chain protective groups: toluenesulfonyl (Tos), 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr), 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc), and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf).

Fmoc-Arg(Pbf)-OH was successfully used in our lab to prepare BIIE 0246 and analogs (cf. chapter 7).

4.2. Orthogonal N^α-/ Side Chain Protection (Tactics)

Our synthetic scheme required the temporary protection of the α-amino group. N ^α -Protection had to be orthogonal to the groups used for the protection of the side chain function. Furthermore, cleavage conditions had to be compatible with functional groups in other portions of the molecule.

The protective groups used for the preparation of N^G-unsubstituted BIIE 0246 analogs are described in section 4.1. For the synthesis of substituted argininamides we had to start with orthogonally protected ornithine.

In Table 1 some properties of commonly used amino protective groups are shown.

The protective groups applicable for our purpose not only had to be orthogonal to each other, but also they had to be compatible with other functional groups present in the molecule. The Cbz group could not be used for the synthesis of BIIE 0246 (2) analogs, because the 1,2-diphenyl-1,2,4-triazolidine-3,5-dione moiety is sensitive to hydrogenolysis (vide infra). Moreover, the diarylmethylpiperazino substructure is assumed to be sensitive towards strong acids and catalytic hydrogenolysis. However, 50 % TFA in CH₂Cl₂ was tolerated. Therefore, the Boc group was chosen as

temporary N ^α-protection; as acid resistant N ^δ-protective group we successfully utilized the phthaloyl (Pht) group.

Table 1: Properties of some conventional amino protective groups.

Boc Cbz Fmoc Alloc Pht

Cbz-OSu [162] Fmoc–Cl [357]

Fmoc-OSu [870] Alloc-Cl [56]

Alloc2O [2718] EtOC(O)NPht