A carbamoyl-protective group for tyrosine that facilitates purification of hydrophobic synthetic peptides

Article abstract of DOI:10.1016/j.tetlet.2008.04.014

The Boc-N-methyl-N-[2-(methylamino)ethyl]carbamoyl group (Boc-Nmec) is reported as a new side chain-protective group for tyrosine in Fmoc solid-phase peptide synthesis. Tyrosine is incorporated into the peptide as Fmoc-Tyr(Boc-Nmec)-OH by standard coupling methods. During the cleavage of the peptide from the resin with TFA the Boc group is simultaneously cleaved while the cationic N-methyl-N-[2-(methylamino)ethyl]carbamoyl group remains attached to the tyrosine residue, thereby increasing the solubility of the peptide. After purification of the peptide, the Nmec protective group can be cleaved under neutral or mild alkaline conditions via an intramolecular cyclization reaction.

Full text of DOI:10.1016/j.tetlet.2008.04.014

Available online at www.sciencedirect.com
Tetrahedron Letters 49 (2008) 3779–3781
A carbamoyl-protective group for tyrosine that facilitates
purification of hydrophobic synthetic peptides
*
´
Karolina Wahlstro¨m, Ove Planstedt, Anders Unden
Department of Neurochemistry, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden
Received 14 February 2008; revised 18 March 2008; accepted 2 April 2008
Available online 7 April 2008
Abstract
The Boc-N-methyl-N -[2-(methylamino)ethyl]carbamoyl group (Boc-Nmec) is reported as a new side chain-protective group for
tyrosine in Fmoc solid-phase peptide synthesis. Tyrosine is incorporated into the peptide as Fmoc-Tyr(Boc-Nmec)-OH by standard
coupling methods. During the cleavage of the peptide from the resin with TFA the Boc group is simultaneously cleaved while the
cationic N-methyl-N-[2-(methylamino)ethyl]carbamoyl group remains attached to the tyrosine residue, thereby increasing the solubility
of the peptide. After purification of the peptide, the Nmec protective group can be cleaved under neutral or mild alkaline conditions via
an intramolecular cyclization reaction.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Solid-phase peptide synthesis; Protective group; HPLC; Tyrosine; Fmoc amino acids
The solubility of synthetic peptides is often a major
problem in solid-phase peptide synthesis (SPPS). Poor sol-
ubility is often a result of the formation of b-sheet struc-
tures that favor aggregation of the peptide, a process that
occurs both during the assembly of the peptide chain and
during the purification steps.1,2
Purification of the synthetic peptides is routinely carried
out under aqueous acidic conditions using reversed phase
HPLC, and good solubility of the peptide in water or in
a water/acetonitrile mixture facilitates this step. Synthetic
peptides with histidine, lysine, or arginine residues gener-
ally increase the solubility of peptides in aqueous solution
as a result of the combined effect of increased hydration
and electrostatic repulsion between the peptide chains.
For synthetic peptides with few or no cationic charges,
however, the purification is usually hampered by aggrega-
tion and low solubility.
tyrosine residues, by employing a protective group that is
cleaved in a two-step procedure.
It has been shown that phenyl carbamates can be
hydrolyzed by hydroxyl ions by two different mechanisms.
N-Unsubstituted and N-monosubstituted carbamates
undergo an ElcB elimination reaction by proton abstrac-
tion and isocyanate formation while N-disubstituted carba-
mates are cleaved by the nucleophilic attack on the
carbonyl carbon.3 The latter mechanism proceeds much
more slowly, and it could therefore be expected that N-
disubstituted phenyl carbamates would be stable to the
reaction conditions in Fmoc SPPS where the weaker nucle-
ophile piperidine is used.4 N-Disubstituted phenyl carba-
mates could even be expected to be too stable to be used
in peptide synthesis, however, Saari et al. have shown that
N-disubstituted phenyl carbamates can be cleaved under
neutral or weakly basic conditions via an intramolecular
cyclization reaction.5 In that study, the use of N-methyl-
N-[2-(methylamino)ethyl]carbamates as potential prodrugs
for phenolic drugs was investigated, and it was found that
cleavage occurred relatively quickly even in aqueous solu-
tion at pH 7.4, with a half-life of 36 min. The cleavage
was shown to proceed via an intramolecular cyclization
In this Letter, we report a method in which these prob-
lems can be reduced to a significant extent for peptides with
*
Corresponding author. Tel.: +46 816 4117; fax: +46 816 13 71.
´
E-mail address: andersu@neurochem.su.se (A. Unden).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.04.014

3780
K. Wahlstro¨m et al. / Tetrahedron Letters 49 (2008) 3779–3781
resulting in the generation of N,N 0-dimethylimidazolidi-
none and the free phenol.
more than one Nmec protected tyrosine residue, it is
important that the peptide should remain in solution dur-
ing the cleavage as the solubility will be reduced when
the cationic Nmec group is cyclized to N,N0-dimethylimi-
dazolidinone. The selection of solvents for this reaction is
therefore highly dependent on the sequence of the peptide,
but in general we found that combinations of DMF and
water gave the most reliable results.
To demonstrate the potential value of the Nmec group
in the synthesis of hydrophobic peptides, the peptide H-
Ala-Tyr-Val-Leu-Tyr-Tyr-Ala-OH, was synthesized using
Boc-Nmec protection of all the tyrosine residues. The
HPLC elution profile of the crude product is shown in Fig-
ure 2.
The expected Nmec-protected peptide was obtained as
the major product. The purified Nmec- protected peptide
was dissolved in 30% DMF/water (1 mg/ml) and 10 equiv
of N-methyl morpholine was added. N-Methylmorpholine
is a weak non-nucleophilic base (pKa 7.41), and these mild
reaction conditions will minimize base-catalyzed side-reac-
tions. The cyclization reaction was monitored by removing
small samples at different time points, quenching with ace-
tic acid, and concentrating in vacuo. After being redis-
solved in 30% acetonitrile, the samples were analyzed by
HPLC. The cyclization reaction was complete after 4 h.
As expected, cleavage of the Nmec group resulted in signif-
Taken together, these reports suggest that tyrosine resi-
dues with a Boc-N-methyl-N-[2-(methylamino)ethyl]car-
bamoyl group (Boc-Nmec) as a side chain-protective
group for tyrosine residues could be expected to be stable
under the reaction conditions in Fmoc SPPS. After treat-
ment with TFA, the Boc group is cleaved but the proton-
ated Nmec group would still be attached to the peptide,
resulting in increased solubility. After purification, the
unprotected peptide can be obtained by short exposure to
a weak alkaline aqueous solution.
We therefore synthesized Fmoc-Tyr(Boc-N-methyl-
N-[2-(methylamino)ethyl]carbamoyl)-OH, Fmoc-Tyr(Boc-
Nmec)-OH (3), as shown in Figure 1. The phenacyl ester
of Fmoc-Tyr-OH (1) was treated with a slight excess of
p-nitrophenyl chloroformate and diisopropylethylamine
(DIPEA) in methylene chloride for 30 min followed by a
threefold excess of mono-Boc protected N,N0-dimethyleth-
ylenediamine and DIPEA. After aqueous work-up and dry
flash chromatography, the phenacyl ester 2 was cleaved
with zinc in acetic acid. Additional work-up and crystalli-
zation from ethyl acetate/petroleum ether gave the final
product with an overall yield of 89%.6
The product was characterized by 1H and 13C NMR and
the results were in agreement with the expected structure
(see Supplementary data).
The stability of the Boc-Nmec group in 20% piperidine
in DMF was studied by treating the peptide H-Ala-Tyr-
(Boc-Nmec)-Phe-Lys(Boc)-Lys(Boc)-OH attached to the
Wang resin for 24 h. After cleavage with TFA, the major
product was the Nmec-protected peptide. No significant
increase in the levels of by-products as compared to the
peptide not exposed to prolonged piperidine treatment
was observed by HPLC analysis and MALDI-TOF mass
spectrometry. These results are in agreement with those
obtained by Vontor et al. who determined the half-life of
phenyl N,N-dimethylcarbamate in 1 M aqueous NaOH at
40 °C to be 20 min.7 It can therefore be concluded that
the Boc-Nmec group will be stable to piperidine treatment
even during the synthesis of large peptides.
The rate of removal of the Nmec group will be depen-
dent on the peptide sequence, the pH, the temperature,
and the solvent. Saari et al. recorded a half-life of 36 min-
utes at pH 7.4 and 37 °C for Nmec-protected hydroxyani-
sole in aqueous solution.5 For peptides that are prone to
aggregation, especially very hydrophobic peptides with
Fig. 2. HPLC elution profile of the model peptide H-Ala-Tyr-Val-Leu-
Tyr-Tyr-Ala-OH synthesized by Boc-Nmec protection of the side chains
of all the tyrosine residues. (A) H-Ala-Tyr(Nmec)-Val-Leu-Tyr(Nmec)-
Tyr(Nmec)-Ala-OH after cleavage with TFA. (B) H-Ala-Tyr-Val-Leu-
Tyr-Tyr-Ala-OH after treatment of the purified peptide for 4 h with 30%
DMF/water and 10 equiv of N-methylmorpholine.
Fig. 1. Synthesis of Fmoc-Tyr(Boc-Nmec)-OH. Reagents: (a) (1) p-nitrophenyl chloroformate, DIPEA, DCM; (2) mono-Boc- N,N0-dimethylethylene-
diamine, DIPEA, DCM; (b) Zn/HOAc.

K. Wahlstro¨m et al. / Tetrahedron Letters 49 (2008) 3779–3781
3781
icantly longer retention times on HPLC as a result of loss
of the cationic Nmec group.
tected and purified peptide can be generated under mild
reaction conditions.
In MALDI-TOF mass spectrometry the Nmec group is
partly cleaved, and we always recorded molecular masses
corresponding to the loss of one or more Nmec groups
for peptides that eluted as a single peak on HPLC.
MALDI-TOF analysis of the Nmec-protected peptide gave
excellent signal-to-noise ratios, while removal of the Nmec
group resulted in weaker signals; and when redissolved, the
purified, lyophilized fully deprotected peptide gave a very
poor signal-to-noise ratio. This poor signal-to-noise ratio
in MALDI-TOF for the deprotected peptide is probably
the result of aggregation and of the reduced cationic char-
acter of the peptide leading to poor ionization.
Supplementary data
Supplementary data (1H NMR and 13C NMR
for Fmoc-Tyr(Boc-Nmec)-OH) associated with this article
can be found, in the online version, at doi:10.1016/
j.tetlet.2008.04.014.
References and notes
1. Coin, I.; Beyermann, M.; Bienert, M. Nat. Protocols 2007, 2, 3247–
3256.
In conclusion, for hydrophobic peptides containing
tyrosine residues, side chain protection using the Boc-
Nmec group is likely to be a valuable tool. The synthesis
of Fmoc-Tyr(Boc-Nmec)-OH can be carried out in a few
simple synthetic steps, the yield is good, and the product
is easy to crystallize. Once incorporated into a peptide, it
is stable to the reaction conditions in Fmoc chemistry.
After cleavage from the resin, the solubility of the peptide
is increased and usually the peptide gives stronger signals in
MALDI-TOF mass spectrometry. Finally, the fully depro-
2. Quibell, M.; Turnell, W. G.; Johnson, T. Tetrahedron Lett. 1994, 35,
2237–2238.
3. Dittert, L. W.; Higuchi, T. J. Pharm. Sci. 1963, 852–857.
4. Hegarty, A. F.; Frost, L. N.; Coy, J. H. J. Org. Chem. 1973, 39, 1089–
1093.
5. Saari, W. S.; Schwering, J. E.; Lyle, P. A.; Smith, S. J.; Engelhardt, E.
L. J. Med. Chem. 1989, 33, 97–101.
6. Mp = 181.7 °C.
7. Vontor, T.; Drobilic, V.; Socha, J.; Vecera, M. Collect. Czech. Chem.
Commun. 1974, 39, 281–286.