Functionalized Arginine Building Blocks
Scheme 5. Synthesis of the arginine building blocks 7.5, 7.13 and 7.14 from building block 7.12
Reagents and conditions: (a) (1) CDI, NEt3, CH2Cl2, rt, 20 h; (2) 10 % Pd/C, H2, MeOH, rt, 15 min, 48 % (7.5), 30 % (7.13); (b) triphosgene, diisopropylethylamine, CH2Cl2, rt, 2.5 h; (2) 10 % Pd/C, H2, MeOH, rt, 30 min, 20 % (7.14). *Raw yields.
Chapter 7
Figure 3. Panel A: HPLC analysis of the crude product 7.5 obtained after cleavage of the benzyl ester (Scheme 5). Conditions: column: Eurospher-100 C18 (250 × 4 mm, 5 µm), eluent: acetonitrile (A) and water (B), gradient: 0 to 30 min: A/B 20/80 to 95/5, 30 to 40 min: 95/5, flow rate: 0.8 mL/min, UV-detection: 220 nm (A1) and 254 nm (A2). 7.5: tR = 29.2 min, II: 9-methylene-9H-fluorene (Cleavage of N -Fmoc!). B: HPLC analysis of 7.5 purified with preparative HPLC. Conditions: the same as in A, UV-detection: 220 nm. 7.5: tR = 28.4 min, 97 %. C: HPLC analysis of Boc-deprotected 7.5 (7.5-B). Conditions:
the same as in A, but with 0.05 % aq. TFA instead of water, UV-detection: 220 nm. 7.5-B: tR = 11.0 min, 7.16 (N-Fmoc-arginine): tR = 13.1 min.
An attempt to purify arginine building block 7.13 with column chromatography using methylene chloride and methanol as eluent (column packed with 1 % triethylamine) resulted in an extensive degradation of the compound. Therefore building blocks 7.5, 7.13 and 7.14 were isolated with preparative HPLC. Because of the carboxyl group an acidification of the mobile phase is advantageous to improve the separation, but under these conditions a significant cleavage of the N-Boc group occurred during removal of acetonitrile from the eluate under reduced pressure (35 °C). Consequently, acetonitrile and pure water were used as eluent components (pH ≈ 5 – 6) to purify compounds 7.5 and 7.13, although this led to broadening of the peaks, large volumes of product fractions and substantial increase in time necessary (> 60 min) for removal of the organic solvent at 35 °C prior to lyophilization. Under these conditions the N-acyl substituents were cleaved off, as analyzed with electrospray mass spectrometry
Functionalized Arginine Building Blocks 179 (ES-MS) and confirmed by HPLC of the Boc-deprotected compounds (Figure 3C and 4B). To exclude fragmentation during MS analysis a small amount of pure product 7.13 (1 mL of the eluate from preparative HPLC) was treated under mild conditions to remove the solvent (temp.
< 10 °C); ES-MS analysis of the residue revealed no degradation.
Whereas degradation of compound 7.5 to N-Boc-N-Fmoc-arginine (7.17) during evaporation of acetonitrile at 35 °C was moderate (Figure 3B/C), about 40 % of the linker in 7.13 was cleaved off under the same conditions (Figure 4B). The mixture of arginine building block 7.13 and its degradation product N-Boc-N-Fmoc-arginine (7.17) was not separated when analyzed with HPLC using acetonitrile and water (both compounds were “co-eluted” with a fronting;
Figure 4A). By contrast the Boc-deprotected compounds were readily separated as shown in Figure 4B.
Figure 4. A: HPLC analysis of the arginine building block 7.13 purified with preparative HPLC.
Conditions: column: Eurospher-100 C18 (250 × 4 mm, 5 µm), eluent: acetonitrile (A) and water (B), gradient: 0 to 30 min: A/B 20/80 to 95/5, 30 to 40 min: 95/5, flow rate: 0.8 mL/min, UV-detection: 210 nm.
III: co-elution of 7.13 and its degradation product N-Boc-N-Fmoc-arginine 7.17, tR = 23.4 min (peak-fronting). The coexistence of 7.13 and 7.17 becomes obvious from B and was confirmed with ES-MS. B:
HPLC analysis of Boc-deprotected 7.13 (7.13-B). Conditions: the same as in A, but with 0.05 % aq. TFA instead of water, UV-detection: 220 nm. 7.13-B: tR = 10.5 min, 61 %; 7.16 (N-Fmoc-arginine): tR = 13.0 min, 37 %. C: HPLC analysis of 7.14 purified with preparative HPLC. Conditions: the same as in A, UV-detection: 220 nm. 7.14: tR = 25.8 min, 74 %. D: HPLC analysis of Boc-deprotected 7.14 (7.14-B).
Conditions: the same as in B. 7.14-B: tR = 10.7 min. N-Fmoc-arginine (7.16) (tR ≈ 13 min) was not detected, i.e. the N-carbamoyl-substituent was not cleaved during the preparative work-up.
Chapter 7 180
Carbamoylation of precursor 7.12 with the isocyanate generated from N-Boc-butane-1,4-diamine gave a considerable amount of the N-substituted isomer of 7.14 (Scheme 5). This was also observed for the carbamoylation of N-Boc, O-t-butyl protected BIBP 3226 (2.7) using the same isocyanate (discussed in chapter 4). Purification of the arginine building block 7.14 with preparative HPLC using acetonitrile and 0.025 % aqueous TFA obviously resulted in fractional cleavage of the N-Boc group whereas the N-carbamoyl substituent was retained (Figure 4C/D).
Thus, the synthetic route using building block 7.12 (Scheme 3) is very efficient including the N -acylation step, but subsequent hydrogenolytic debenzylation results in a fractional cleavage of the N-Fmoc group, and due to instability of the desired compounds the separation from by-products is complicated. The N-Boc analog of the N-Cbz-protected compound 7.6 (Scheme 2) as a universal building block for the introduction of aminoalkanoyl substituents is considered a promising alternative, but preventing decomposition during purification of the products remains challenging anyway. Separation of the products from by-products formed by N-Fmoc cleavage during hydrogenolytic debenzylation under water free conditions taking advantage of different solubilities may be feasible.
7.2.2 Application of the Modified Arginine Building Blocks in Peptide Synthesis
The octapeptide angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-OH) was selected as a model compound to investigate the suitability of the arginine building blocks for solid phase peptide synthesis. Since the peptide chain is built from C-terminus to N-terminus in the case of angiotensin II only one additional amino acid (Asp) had to be coupled after the introduction of the modified arginine. In a first approach the N-Cbz protected building block 7.7 was introduced. Modified angiotensin II was obtained in acceptable yield after cleavage from the resin under neutral conditions. However, hydrogenolytic cleavage of Cbz in MeOH gave considerable amounts of C-terminal methyl ester, presumably due to catalysis by a small amount of acid (TFA, AcOH) in the reaction mixture. After Cbz-deprotection the acid sensitive protecting groups were removed. Unfortunately, purification of the target compound turned out to be too difficult because of nearly identical retention times of both the desired product and the C-terminally esterified peptide. When the peptide mixture was treated with lithium hydroxide extensive cleavage of the N-acyl linker occured.
In a second attempt building block 7.5 was employed. For coupling 2.5 equivalents of 7.5 were used as well as 2.5 equivalents of HOBt/HBTU and 5 equivalents of DIPEA (single coupling, 8 h). The resulting angiotensin II derivative 7.15 (Scheme 6) was cleaved from the trityl resin under acidic conditions. Since the N-Boc group was simultaneously removed in this step the
Functionalized Arginine Building Blocks 181 N-acyl substituent gained better stability (discussed in the beginning). HPLC-MS analysis revealed two main products, the designated one (7.15) and the heptapeptide lacking the arginine. Thus, coupling of the solid phase bound angiotensin 1 - 6 with arginine building block 7.5 was incomplete although a Kaiser-test had been negative. Only a very small fraction of peptide was lacking the N-acyl linker. The angiotensin II derivative 7.15 was obtained with 95
% purity (210 nm) after isolation with preparative HPLC (Figure 5A).
The aminohexanoyl substituted angiotensin II (7.15) was allowed to react with the pyrylium dye Py-17, 8 (Scheme 6, cf. also chapter 4) to yield the fluorescence labeled peptide 7.5-Py1 with about 20 % yield and 94 % purity (220 nm, Figure 5B).