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groups will improve the solubility. It would therefore, in many
cases, be desirable to have a protecting group containing additional
amino groups that increase the solubility of the peptide followed
by fast formation of the desired peptide at physiological pH.
A well-known reaction in the peptide chemistry is diketopiper-
azine formation. The most studied examples being of dipeptide es-
ters that can undergo a cyclization–elimination reaction resulting
in a cyclic diketopiperazine dipeptide and the alcohol.8 This reac-
tion can occur as a side reaction during solid-phase peptide syn-
thesis at the dipeptide stage, but it has also been used to design
the cyclization-activated prodrugs for vinca alkaloids and purine
and pyrimidine analogues.9–11 Another interesting study showed
that derivatization of the sparsely soluble immunosuppressive
peptide cyclosporine A with different dipeptides resulted in cyclo-
sporine A analogues with greatly improved solubility, and that
cyclosporine A could be generated when the peptide was exposed
to physiological pH.12 In all these examples the dipeptide moieties
were attached to a hydroxyl group but other studies have shown
that even amide bonds in peptides can be cleaved slowly by the
same reaction mechanism.13
used sucessfully.6,16–18 The synthesis of Fmoc-Trp(Boc-Sar-Sar)-
OH is described in the Supplementary data but requires some addi-
tional comments.
During the first step when Z-Trp-OBlz (1) is acylated by Boc-
Sar-Sar-ONp (2) it is important to have high concentrations of
the reactants. We found that when the reaction was performed
with Z-Trp-OBzl (4 g) and dry THF (50 ml) was used as the solvent,
only small amounts of the product were formed. Therefore, Z-Trp-
OBzl, Boc-Sar-Sar-ONp and 18-crown-6 were dissolved in a very
small amount of dry THF by gentle heating, N,N0-diisopropylethyl-
amine (DIEA) was added, the solution was placed in an ice-bath
followed by the addition of anhydrous KF. After 90 min at room
temperature all the Boc-Sar-Sar-ONp had been consumed and pep-
tide 3 could be readily purified by dry flash chromatography. After
the catalytic hydrogenation of 3 we encountered problems sepa-
rating 4 from the catalyst both by filtration and centrifugation.
These problems could be eliminated if the use of a Teflon stirring
bar was avoided during the reaction. The progress of the hydroge-
nation of 3 should also be carefully monitored by HPLC as a small
amount of by-product starts to accumulate at the end of the reac-
tion. This side-reaction could be minimized if the hydrogenation
was interrupted just prior to completion. The small amounts of
partly hydrogenated material could be removed by extraction of
the solid material with diethyl ether.
During the synthesis of peptides with Fmoc-Trp(Boc-Sar-Sar)-
OH there is a possibility that the Boc-Sar-Sar moiety could be
cleaved by the nucleophile piperidine. To what extent this reaction
will restrict the use of this protecting group is difficult to predict
from the literature. It was shown by Arai and co-workers who used
indole as protecting groups for carboxylic acids that a complete
cleavage required 3 M NaOH in methanol for 3.5–35 h depending
on the carboxylic acid used in the experiment.19 On the other hand,
it is well known that the Nin-formyl group is readily cleaved by
nucleophiles. Previous results from this laboratory have shown
that the Boc-4-(N-methylamino)butanoyl group was stable to
20% piperidine for up to 24 h.6 In order to investigate the stability
of the Boc-Sar-Sar group to piperidine, the model peptide Arg-
Trp(Boc-Sar-Sar)-Ala-Arg-Tyr-Ala-OH attached to a 2-chlorotrityl
resin was treated with 20% piperidine in NMP overnight followed
by cleavage of the peptide from the resin with TFA (Fig. 1). This re-
sulted in the partial cleavage of the Boc-Sar-Sar group, 14.4% of the
Boc-Sar-Sar group was cleaved, as measured by absorption at
280 nm. This rate of cleavage by piperidine corresponds to the loss
of 0.1% of the protecting group during a Fmoc group removal over
10 min (Fig. 1B). We, therefore, conclude that the Boc-Sar-Sar
group is sufficiently stable to be used in the synthesis of most pep-
tides using Fmoc chemistry.
In a project related to the synthesis of antimicrobial peptides
with tryptophan residues we encountered problems in purification
of the peptides and they were also difficult to dissolve for biologi-
cal testing. We, therefore, became interested in the possibility that
the indole nitrogen in tryptophan residues could be protected dur-
ing the synthesis of the peptide with N-protected dipeptides. We
hypothesized that such protecting groups would enhance the sol-
ubility of the peptide both during purification by HPLC and in the
preparation of the sample for biological testing, yet would generate
the active peptide rapidly when exposed to neutral pH in biological
experiments (Scheme 1). The dipeptide for the diketopiperazine
reaction is highly dependent on the dipeptide sequence and the
rate of the reaction can vary by several orders of magnitude.12 In
general the reaction is fast for dipeptides with an N-alkylated pep-
tide bond, probably as a result of a more favoured formation of cis-
peptide bonds.14 Therefore, we selected sarcosine (N-methylgly-
cine) as the C-terminal moiety in the dipeptide. Sarcosine was also
chosen as the N-terminal residue as it has been showed that the Z-
Gly-Pro-ONp dipeptides can cyclize to the N-acyl diketopipera-
zine.15 p-Nitrophenol is several orders of magnitude better as a
leaving group than an indole but we could not exclude that the
N-acylated nitrogen in primary amino groups could act as a nucle-
ophile during the synthesis of the peptide when they are exposed
to piperidine. Moreover, the attachment of dipeptides with N-ter-
minal primary amino groups to the indole nitrogen would also re-
quire more complex synthetic procedures. The synthesis of Fmoc-
tryptophan protected with the Boc-Sar-Sar group, Fmoc-Trp(Boc-
Sar-Sar)-OH (5), on the indole nitrogen is outlined in Scheme 2.
The most direct, but not the most general, route to introduce an
N-protected dipeptide onto the indole nitrogen on tryptophan in-
volves acylation of the indole nitrogen with the activated dipep-
tide. If a proton is abstracted from the indole nucleus the
nitrogen will become nucleophilic and can attack the electrophilic
In order to investigate the rate of cleavage of the Nin-Sar-Sar
group at physiological pH the purified model peptide Arg-
Trp(Sar-Sar)-Ala-Arg-Tyr-Ala-OH isolated as the TFA salt was ex-
posed to HKR buffer at pH 7.4 followed by the acidification with
TFA (Fig. 2). When the peptide was injected into the HPLC appara-
tus two peaks were observed corresponding to the original peptide
with the Nin-Sar-Sar group and a second peptide with a 142 amu
lower molecular mass corresponding to a peptide where the Nin-
Sar-Sar group had been lost. The reaction was fast, after 2 min at
room temperature 41% of the starting material had been converted
into the fully deprotected peptide as measured by the absorption
at 280 nm. Longer periods of exposure to physiological pH resulted
in a decreased relative area of the protected peptide and after
20 min only one peak was observed in the HPLC elution profile,
that is, the peptide in which the indole group had been deprotec-
ted. It is noteworthy that the deprotected peptide showed in-
creased retention on the column indicating that the protected
peptide was more polar and water soluble than the deprotected
peptide (Fig. 2).
a
C-terminal carbonyl group in N -protected dipeptides with a good
leaving group. This requires a strong base and in previous studies,
including our own synthesis of Fmoc-Trp(Nmbu)-OH, the naked
fluoride ion generated from solid KF and crown ethers has been
Scheme 1. Proposed result during the removal of the H-Sar-Sar group from the
indole nitrogen of tryptophan at pH 7.4.
Danielsson, Marie
