1. Solid-phase synthesis of tyrosyl H-phosphonopeptides and methylphosphonopeptides
A Tholey, R Hoffmann J Pept Sci. 1997 May-Jun;3(3):186-92. doi: 10.1002/(SICI)1099-1387(199705)3:3%3C186::AID-PSC99%3E3.0.CO;2-9.
Phosphopeptides are a useful tool for the investigation of phosphorylation as a reversible posttranslational modification. There is a growing interest in using mimics of phosphoamino acids involved in phosphorylation in order to study the enzymes concerned in these processes. These mimics should contain a non-hydrolysable or isoelectrically modified phosphate moiety to be used as a specific inhibitor of phosphatases and kinases. We introduce sold-phase synthesis of H- and methylphosphonopeptides as a new class of mimics of phosphotyrosyl peptides. The peptides were synthesized on solid phase using the standard fluorenyl-methyloxycarbonyl (Fmoc) strategy. Tyrosine residues were incorporated as allyl-protected derivatives, which were selectively deprotected on the resin by treatment with Pd(PPh3)4. The peptide resin carrying the side-chain unprotected tyrosine of the model peptide Gly-Gly-Tyr-Ala was phosphonylated with di-tert-butyl-N,N-diethyl-phosphoramidite in the presence of 1H-tetrazole, yielding H-phosphonopeptides after trifluoroacetic acid (TFA) cleavage. Alternatively, phosphonylation of the unprotected tyrosine with O-tert-butyl-N,N-diethyl-P-methylphosphonamidite catalysed by 1H-tetrazole and followed by oxidation led to the methyphosphonopeptides after TFA cleavage. We obtained both the H-phosphonopeptides and the methylphosphonopeptides of the tetrapeptide in high yields and purities above 90%, according to reversed-phase high-performance liquid chromatography (RP-HPLC). To investigate the general applicability of our new methodology, we synthesized phosphonopeptides up to 13 amino acids long, corresponding to recognition sequences of tyrosine kinases. After cleavage and deprotection, all phosphonopeptides were obtained in high yields and purities of about 90%, as shown by mass spectrometry. The only by-product found was the unmodified peptide.
2. Short-chain peptide analysis by high-performance liquid chromatography coupled to electrospray ionization mass spectrometer after derivatization with 9-fluorenylmethyl chloroformate
K Gartenmann, S Kochhar J Agric Food Chem. 1999 Dec;47(12):5068-71. doi: 10.1021/jf990710s.
Resolution and characterization of short-chain peptides (M(r) = 200-1000) and free amino acids were demonstrated by the use of precolumn derivatization with 9-fluorenylmethyl chloroformate (Fmoc) followed by reverse-phase high-performance liquid chromatography (RP-HPLC) interfaced with an electrospray ionization mass spectrometer (ESI-MS). At pH 10, in addition to derivatization at the N terminus, epsilon-NH(2) and OH groups of lysine and tyrosine residues, respectively, were also derivatized. Fmoc derivatives showed at least 2 orders of magnitude higher ionization potential in the presence of trifluoroacetic acid. The detection levels for both the free amino acid and peptide derivatives were in a few hundred picomoles compared to 10-50 nmol for the underivatized samples. The mass spectra of the peptides before or after derivatization showed the presence of only singly charged ions. However, collision-induced dissociation of the derivatized peptides showed predominance of b-type ions that are relatively less complicated in assigning the peptide sequence.
3. L-O-(2-malonyl)tyrosine: a new phosphotyrosyl mimetic for the preparation of Src homology 2 domain inhibitory peptides
B Ye, M Akamatsu, S E Shoelson, G Wolf, S Giorgetti-Peraldi, X Yan, P P Roller, T R Burke Jr J Med Chem. 1995 Oct 13;38(21):4270-5. doi: 10.1021/jm00021a016.
Inhibition of Src homology 2 (SH2) domain-binding interactions affords one potential means of modulating protein-tyrosine kinase-dependent signaling. Small phosphotyrosyl (pTyr)-containing peptides are able to bind to SH2 domains and compete with larger pTyr peptides or native pTyr-containing protein ligands. Such pTyr-containing peptides are limited in their utility as SH2 domain inhibitors in vivo due to their hydrolytic lability to protein-tyrosine phosphatases (PTPs) and the poor cellular penetration of the ionized phosphate moiety. An important aspect of SH2 domain inhibitor design is the creation of pTyr mimetics which are stable to PTPs and have reasonable bioavailability. To date, most PTP-resistant pTyr mimetics which bind to SH2 domains are phosphonates such as (phosphonomethyl)phenylalanine (Pmp, 2), [(monofluorophosphono)methyl]phenylalanine (FPmp, 3) or [(difluorophosphono)methyl]-phenylalanine (F2Pmp, 4). Herein we report the incorporation of a new non-phosphorus-containing pTyr mimetic, L-O-(2-malonyl)tyrosine (L-OMT, 5), into SH2 domain inhibitory peptides using the protected analogue L-N alpha-Fmoc-O'-(O",O"-di-tert-butyl-2-malonyl)tyrosine (6) and solid-phase peptide synthesis techniques. Five OMT-containing peptides were prepared against the following SH2 domains: the PI-3 kinase C-terminal p85 SH2 domain (Ac-D-(L-OMT)-V-P-M-L-amide, 10, IC50 = 14.2 microM), the Src SH2 domain (Ac-Q-(L-OMT)-E-E-I-P-amide, 11, IC50 = 25 microM, and Ac-Q-(L-OMT)-(L-OMT)-E-I-P-amide, 14, IC50 = 23 microM), the Grb2 SH2 domain (Ac-N-(L-OMT)-V-N-I-E-amide, 12, IC50 = 120 microM), and the N-terminal SH-PTP2 SH2 domain (Ac-L-N-(L-OMT)-I-D-L-D-L-V-amide, 13, IC50 = 22.0 microM). These results show that peptides 10, 11, 13, and 14 have reasonable affinity for their respective SH2 domains, with the IC50 value for the SH-PTP2 SH2 domain-directed peptide 13 being equivalent to that previously observed for the corresponding F2Pmp-containing peptide. OMT may afford a new structural starting point for the development of novel and useful SH2 domain inhibitors.
