Peptide-Based Drug Design

220 Singer and Hoffmann
3. Whereas the scavenger mixture can be stored for one month, the reagent mixture
with TFA should be prepared fresh for each cleavage.
4. The three-letter code used for all amino acids refers always to the corresponding
l-amino acids. d-Enantiomers are abbreviated by d- followed by the corresponding
three-letter code, such as D-Ser for D-serine.
5. Store all amino acid derivatives and activation reagents dry at 4 ◦ C in the darkness
in aliquots of 25 g or less. Before opening a container, place the derivatives in
the laboratory for at least 2 h to reach room temperature. Thus the derivatives
stay dry even when opened several times at higher humidity. Dissolve all solid
compounds just before starting the synthesis without prolonged storage. It should
be noted that amines are formed slowly in DMF in the presence of water traces.
Therefore it is important to store DMF under nitrogen atmosphere or to consume
it within 1 wk. We use older DMF only to dilute piperidine.
6. It is important to use amidite reagent, 1 H-tetrazole, and all solvents for the
phosphitilation step of high purities and to keep all vials and solutions dry.
Thereby, many by-products described in the literature for the global amidite
approach (i.e., H-phosphonopeptides, dimers, etc.) can be omitted yielding the
corresponding peptides in high purities accompanied only by species with lower
phosphorylation degrees.
7. All buffers and solutions were prepared in desalted water with a conductivity
below 18.2 mS and a total organic content below 2 ppb (parts per billion) using
a ELGA PURELAB Ultra system (ELGA LabWater, Celle, Germany).
8. Eluents should be filtered through a 0.2-�m filter (GH Polypro, Hydrophilic
Polypropylene membrane filter, Pall Life Sciences, Ann Arbor, MI) and stored
in clean glass bottles for less than 1 mo. Solvents should be degassed by vacuum
(1 mbar) for 1–2 min if a low-pressure gradient mixture is used (do not use longer
times to minimize loss of TFA and acetonitrile). For high-pressure gradient
HPLC systems this degassing is not necessary if a backpressure valve is used
after the detector to prevent formation of air bubbles in the detector cell.
9. As acetonitrile has a higher absorption than water at 220 nm, the UV trace
slightly climbs with increasing acetonitrile content. Whereas this linear climb of
the background is a good measure for judging the reproducibility of the formed
gradient, it can be advantageous to compensate it by reducing the TFA content
in eluent B to 0.09%. If 100% acetonitrile is used as eluent B, the TFA content
should be further decreased to 0.085%. The resulting stable base line is especially
useful to analyze small peptide amounts with low signal intensities.
10. For most crude peptides a linear increase of 3% per min eluent B is sufficient to
separate the target peptide from all by-products. In order to obtain high purities
and load larger sample amounts on the preparative column use a linear increase
of only 1% per min eluent B at least 10 min before and after the targeted peptide
elutes. Calculate the retention time on the preparative column from the retention
time of the analytical separation using the known dead volumes of the system
and the different flow rates.
11. To distinguish peptides and partially protected peptidic by-products carrying an
aromatic protecting group (e.g., Fmoc, benzyl), it is advantageous to record the