Peptide-Based Drug Design

180 Jenssen and Aspmo
and peptide derivatives soluble for further analysis. To obtain reliable and reproducible
results, peptide degradation is performed under conditions where the
peptide concentration is not the rate-limiting concentration, but rather where
the reaction speed is linearly dependent on the serum concentration (e.g., at
25% serum), i.e., the enzyme concentration is rate limiting, not the substrate
concentration.
Peptide analysis is performed on the supernatant with either mass
spectrometry, e.g., MALDI-TOF (15), or with RP-HPLC under stability-specific
chromatography conditions. It should be noted that mass spectrometry is rarely
a valid quantitative measure without utilizing isotopic internal standards, while
RP-HPLC analysis of peptides is directly quantitative with a UV detector.
However, mass spectrometry of the degradation products will provide insight
into if or where the peptide is cleaved and/or if other modifications to the
peptide have occurred in the serum (glycosylations, phosporylations, or deglycosylations,
dephosphorylations, etc.). It should also be taken into account
that several factors may cause misleading stability results for the peptides (see
Note 4).
In vivo testing of peptide stability is obviously of more relevance than in vitro
testing. However, a better term would probably be peptide pharmacokinetics (in
mice), given that these measurements basically are also done in vitro.
3.1. Solid Phase Peptide Synthesis and Purification
1. Solid phase peptide synthesis has been described earlier in great detail (16).
But, in brief, a standard way of doing this is by using Fmoc-chemistry
(9-fluorenylmethoxycarbonyl), a baselabile protection group that easily can be
removed during peptide chain elongation.
2. The peptides can be synthesized automatically using Fmoc amino acids and PAL-
PEG resin on a Milligen 9050 PepSynthesizer (17).
3. The first amino acid is coupled onto the resin in the presence of DIPEA, using
HBTU as a coupling reagent.
4. The next amino acid in the sequence is coupled to the active N-terminal end of
the first amino acid, and the process can be repeated over and over until the entire
peptide is made.
5. When the peptide is finished it can be cleaved from the solid support (resin) using
TFA. This acid-based cleavage process also results in deprotection of the amino
acid side chains.
6. The peptide is then precipitated out in ether and washed extensively with
additional ether prior to drying.
7. The dried peptide is then weighed out and dissolved in water containing 1%
acetonitrile, to a concentration of 20 mg/mL of peptide.
8. The peptides are purified by preparative RP-HPLC on a C18 column with a loading
capacity of 20 mg.