The P-TSTU (9a) and P-HSTU (9b) prepared were used as solid-supported peptide coupling reagents. Thus, differently N-protected amino acids reacted with amino acid ester hydrochlorides in the presence of 1.1 equiv of P-TSTU (9a) or P-HSTU (9b) and pyridine as organic base for 24 h (TLC) at 50 ºC in MeCN as solvent, yielding the corresponding peptides (Table 1), which were pure after workup according to their 1H NMR spectra. The use of other organic bases such as triethylamine or room temperature gave place to lower yields.
Table 1. Peptides prepared using P-TSTU (9a) and P-HSTU (9b) as coupling reagents
Entry
Reagent
Peptide
Yield (%)a
Mp (ºC)b
[a]D25 b (solvent)
1
9a
BocGly-PheOEt
65
oil
-24 (c 1, EtOH)
2
9b
BocGly-PheOEt
41
oil
-24 (c 1, EtOH)
3
9a
BocAla-PheOEt
63
100-101
-8 (c 1, EtOH)
4
9b
BocAla-PheOEt
35
100-101
-8 (c 1, EtOH)
5
9a
BocVal-PheOEt
59
117-118
-23 (c 1, EtOH)
6
9b
BocVal-PheOEt
31
117-118
-23 (c 1, EtOH)
7
9a
CbzVal-PheOEt
50
157-158
-9 (c 0.7, EtOH)
8
9a
BocAib-ValOMe
23
110-111
+19 (c 1, EtOH)
9
9a
CbzGlyPhe-ValOMec
55
-40 (c 1, MeOH)
10
9b
CbzGlyPhe-ValOMec
32
-40 (c 1, MeOH)
a Isolated yield based on the starting amino acids. b Measured from the crude peptide. c Anteunis’ test16: 50:1 diastereomer ratio (1H NMR, 500 MHz).
In all cases, the isolated yields of the peptides obtained were higher using P-TSTU (9a) than P-HSTU (9b) (Table 1). The coupling of an sterically hindered residue such as BocValOH afforded moderate yields with P-TSTU (9a) (Table 1, entry 5). However, when the coupling reaction was performed with both sterically hindered amino acid residues, the yield was rather low (Table 1, entry 8). The extent of possible racemization was checked by measuring the epimerization degree on the tripeptide generated by coupling CbzGlyPheOH and ValOMe (Anteunis’s test)16 (Table 1, entries 9 and 10). The epimerization was low, the final tripeptide obtained as a 10:0.2 mixture of CbzGly-L-Phe-ValOMe and CbzGly-D-Phe-ValOMe diastereoisomers detected by 1H NMR (500 MHz) analysis, both for P-TSTU (9a) or P-HSTU (9b). This value was considerably lower than the 26% of the D-isomer reported when using TSTU.5a
P-HOSu (9) was quantitatively recovered pure, once the coupling reaction was finished, by precipitation with hexane, filtration and washing with diluted HCl. The recovered P-HOSu (7) was employed for the preparation of new reagents 9a and 9b.
We conclude that P-TSTU (9a) and P-HSTU (9b) are new promising polymeric N- hydroxysuccinimide (P-HOSu)-derived uronium salts which can be employed, especially P- TSTU (9a), as solid-supported peptide coupling reagents, the P-HOSu liberated being easily separated and reused.
