Deprotection of the indole (Nind)-formyl (for) group on tryptophan employing a new reagent, N,N′-dimethylethylendiamine (DMEDA) in an aqueous solution

Article abstract of DOI:10.1248/cpb.57.211

The deprotection of the indole (N ^ind)-formyl (For) group on Trp was achieved in a 95% yield using N,N′-di- methylethylendiamine (DMEDA) (1.5, 2.0, 3.0 eq) in water at room temperature. A new reagent was successfully applied to the deprotection

Full text of DOI:10.1248/cpb.57.211

February 2009
Notes
Chem. Pharm. Bull. 57(2) 211—213 (2009)
211
Deprotection of the Indole (Nind)-Formyl (For) Group on Tryptophan
Employing a New Reagent, N,Nꢀ-Dimethylethylendiamine (DMEDA)
in an Aqueous Solution
a
b
Takenao ODAGAMI, Yuko TSUDA, ,b,c Yuji KOGAMI,a Hiroyuki KOUJI,a and Yoshio OKADA
*
a Research Institute, PRISM Bio Lab; 4259–3 Nagatsuta-cho, Midori-ku, Yokohama 226–8510, Japan: b Faculty of
Pharmaceutical Sciences, Kobe Gakuin University; and c Cooperative Research Center of Life Sciences, Kobe Gakuin
University; Chuo-ku, Kobe 650–8586, Japan.
Received October 1, 2008; accepted November 28, 2008; published online December 1, 2008
The deprotection of the indole (Nind)-formyl (For) group on Trp was achieved in a 95% yield using N,Nꢀ-di-
methylethylendiamine (DMEDA) (1.5, 2.0, 3.0 eq) in water at room temperature. A new reagent was successfully
applied to the deprotection of a model peptide, H-Phe-Trp(Nind-For)-Lys-Tyr-OH, to give H-Phe-Trp-Lys-Tyr-
OH in a 91% yield.
Key words deprotection; N,Nꢀ-dimethylethylendiamine; indole-formyl group; peptide synthesis; tryptophan
In peptide synthesis, it is well known that tryptophan in (DMEDA) could remove the Nind-For group in aqueous
the growing peptide chain undergoes oxidation during treat- medium. In light of not only commercial manufacturing
ment with HCl in organic solvent necessary for the cleavage practices, but also especially keeping mind “green chem-
of the a-amino protected Boc (tert-Butoxycarbonyl) group. istry,” a new Nind-For deprotecting method with equivalently
Furthermore, the sequential mild acid treatments to remove controlled reactions under aqueous conditions and without
the a-amino protecting group often lead to electrophilic using irritant reagents would be beneficial. In this communi-
alkylation on the unprotected tryptophan residue. It is ideal cation, we describe the deprotection of the Nind-formyl group
to prevent these side reactions by protecting the indole moi- on Trp employing a new reagent, DMEDA, in an aqueous so-
ety during the synthesis. This has been achieved by using the lution.
indole-formyl (Nind-For) protecting group in Boc chemistry
of peptide synthesis.
Results and Discussion
To date, a number of Nind-For deprotection methods have
We investigated Nind-For in the deprotecting reaction of H-
been developed, including most well known method using Trp(Nind-For)-OH (1) under aqueous conditions using both
low-high concentration hydrogen fluoride (HF) treatment.1) DPEDA and DMEDA. Of these compounds, the former was
However, this method is accompanied with treatment of large inactive in the reaction due to its low solubility in water. In
quantities of a reagent with considerable stench, such as 1,2- contrast, DMEDA was highly active in this regard. There-
ethanedithiol and use of special equipment. On the other fore, our studies were directed to the deprotection of Nind-For
hand, the Nind-For group can be removed in a weak alkaline by DMEDA and we set out to evaluate the reaction condi-
medium, such as 1 M NH2OH,2) 1 M NaHCO3,3) or hydrazine tions that allowed easy conversion of H-Trp(Nind-For)-OH (1)
hydrate (20—30 eq) in dimethylformamide (DMF).4) How- to H-Trp-OH (2) and to optimize the conditions for prepara-
ever, these procedures required excess amounts of alkaline tion of the model peptide.
reagent and the Nind-For group migration to an a-amino
As shown in Fig. 1A, the treatment with 1.0 eq DMEDA
group of the peptide was reported.3)
did not completely remove the Nind-For group even after 2 h.
We were intrigued by a previous report showing that N,Nꢀ- However, as the number of equivalents of DMEDA increased
diphenylethylendiamine (DPEDA) reacted with aldehyde to from 1.0 to 1.5, 2.0 and 3.0, the rate of the conversion
form a tetrahydroimidazole.5) It hinted that N-alkylated di- became substantially faster. Complete deprotection was
amines, such as DPEDA and N,Nꢀ-dimethylethylendiamine achieved after 1.0 h with 1.5 eq of DMEDA or in 20 min with
Fig. 1. The Effect of N,Nꢀ-Dimethylethylendiamine (DMEDA) on the Deprotection of Trp(Nind-For) in Water
(A) The deprotecion of H-Trp(Nind-For)-OH with DMEDA in water. ∗ Both conversion yield of Trp and recovery yield of Trp(N ind-For) were quantified by RP-HPLC. ꢀ: 1.0 eq
of N,Nꢀ-dimethylethylendiamine (DMEDA), ꢁ: 1.5 eq of DMEDA, ꢂ: 2.0 eq of DMEDA, ꢃ: 3.0 eq of DMEDA, ——: H-Trp-OH, ------: H-Trp(N ind-For)-OH. (B) Comparison
of conversion yields for the model peptide with various reagents. ∗∗ Conversion yield was assessed by PDA integration on LC/MS/MS analysis. ꢂ: 3 eq of N,Nꢀ-dimethylethylendi-
amine (DMEDA), ꢃ: 30 eq of hydrazine, ꢁ: 1 M NaHCO3, ꢀ: 1 M NH2OH.
∗ To whom correspondence should be addressed. e-mail: tsuda@pharm.kobegakuin.ac.jp
© 2009 Pharmaceutical Society of Japan

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Vol. 57, No. 2
2.0 eq of DMEDA, respectively; the addition of 3.0 eq of one of side products was identified as peptide 6, arising from
DMEDA led to a complete conversion after 2 min. Concern- migration of the Nind-For group to the e-amino group of Lys
ing side reactions, the migration of the Nind-For group to a- residue. The migration of the Nind-For group to an a-amino
amino group of Trp was not observed under any of the condi- group was not detected in our conditions. The results ob-
tions investigated using RP-HPLC. LC/MS analysis revealed tained are summarized in Fig. 3. Those findings suggest that
that the Nind-For group underwent migration on one amino the e-amino group of Lys might have higher nucleophilicity
group of DMEDA to form 3 (Fig. 2), rather than tetrahy- than the a-amino group and subsequently acted as a nucle-
droimidazole. Thus, DMEDA can serve as a nucleophile for ophile under these conditions.
the removal of the Nind-For group.
We further examined the preparation of 4 by DMEDA
We applied this deprotecting reagent to the preparation of method. Peptide 5 (5.00 mg) was treated with DMEDA
a model peptide. We chose a sequence of Phe-Trp-Lys-Tyr (3.0 eq) in water at room temperature for 2 h, and the result-
(4), which is a fragment of urotensin II.6) First, we prepared a ing peptide was purified with RP-HPLC8) to give 4 (4.37 mg)
formyl derivative of 4, namely H-Phe-Trp(Nind-For)-Lys-Tyr- in 91% yield. Identification of 4 was assessed by spectral
OH (5). Synthesis of 5 was carried out by solution methods data (HR-ESI-MS, LC/MS/MS). The deprotection of the
using Boc chemistry, where Boc-Phe-Trp(Nind-For)-Lys(Ne-
N
ind-For group by DMEDA in water was successfully applied
2-chlorobenzyloxycarbonyl)-Tyr(O-2,6-dichlorobenzyl)bezyl to the preparation of the peptide.
ester was treated with TMSOTf (trimethylsilyl trifluoro-
Finally, we compared the efficiency of the DMEDA depro-
methanesulfonate)-thioanisole/TFA (trifluoroacetic acid)7—9) tection method with other basic conditions using 5 as a
to give H-Phe-Trp(Nind-For)-Lys-Tyr-OH. The crude peptide model peptide. As shown in Fig. 1B, the conversion yield by
was purified with RP-HPLC10) to isolate H-Phe-Trp(Nind
-
our method using DMEDA (3.0 eq) for 2 h at room tempera-
3)
For)-Lys-Tyr-OH (5). Identification and purity of 5 were es- ture was more than 95%. Treatment with 1 M NaHCO3
tablished by spectral data (HR-ESI-MS, LC/MS/MS). And (150 eq) and 1 M NH2OH2) (150 eq) revealed conversion
then, 5 was subjected to deprotection using DMEDA. After yields at room temperature of 95% after 24 h and 90% after
3)
treatment of 5 with DMEDA (3.0 eq) in water for 1 h, LC/MS 6 h, respectively. Treatment with 1 M NaHCO3 (150 eq) and
analysis revealed a major peptide along with very few side 1 M NH2OH2) (150 eq) led to the migrations of Nind-For group
products in the reaction mixture (Fig. 3). Characterization of to the e-amino group of Lys residue, and to an a-amino
the products of DMEDA treatment was performed by group of Phe residue, respectively. Hydrazine hydrate4) treat-
LC/MS/MS analysis without prior isolation: the main prod- ment (30 eq) exhibited a conversion yield of 90% after 2 h at
uct was identified as the desired peptide 4 (yieldꢁ95%) and room temperature; however, 4 was found to be unstable in
30 eq hydrazine hydrate. DMEDA treatment proved to be su-
perior to other basic reagents in terms of its quick reaction
with higher purity.
In summary, a new method for the Nind-For group depro-
tection has been developed. Trp(Nind-For) was quantitatively
converted to Trp in the presence of 3.0 eq of DMEDA under
aqueous condition. This new reagent was successfully ap-
plied to the deprotection of a model peptide, H-Phe-Trp(Nind
For)-Lys-Tyr-OH, to give H-Phe-Trp-Lys-Tyr-OH in excel-
lent yield. The procedure would be practical and environ-
-
Fig. 2. Summary of the Deprotection of Trp(N ind-For) with N,Nꢀ-Di-
methylethylendiamine (DMEDA) in Water
Fig. 3. Summary of the Deprotection of the Model Peptide with N,Nꢀ-Dimethylethylendiamine (DMEDA) in Water and HPLC Profile for Compounds 4—
6
HPLC profile for 4—6. 4 (H-Phe-Trp-Lys-Tyr-OH): tRꢁ14.7 min, 5 [H-Phe-Trp(Nind-For)-Lys-Tyr-OH]: tRꢁ15.4 min, 6 [H-Phe-Trp-Lys(Ne-For)-Tyr-OH]: tRꢁ18.3 min.

February 2009
213
(Mꢃ1)ꢃ (Calcd for C36H43N6O7: 671.3191). LC/MS/MS m/z: 490.2 (M)ꢃ
[Calcd for C27H32N5O4/(H-Phe-Trp(Nind-For)-Lys-OH)-OH: 490.5], 310.2
(M)ꢃ [Calcd for C15H23N3O4/H-Lys-Tyr-OH: 310.3], 129.1 (M)ꢃ [Calcd for
C6H13N2O1/(H-Lys-OH)-OH: 129.1]. tRꢁ15.4 min.
mentally advantageous over the previously reported methods,
especially from the current views of peptide manufacturing
processes and application of the principles of green chem-
istry.
H-Phe-Trp-Lys(Ne-For)-Tyr-OH (6): HR-ESI-MS m/z: 671.2402 (Mꢃ1)ꢃ
(Calcd for C36H43N6O7: 671.3191). LC/MS/MS m/z: 524.2 (Mꢃ1)ꢃ [Calcd
for C27H34N5O6/(H-Trp-Lys(Ne-For)-Tyr-OH)ꢃH: 524.5], 490.2 (M)ꢃ
[Calcd for C27H32N5O4/(H-Phe-Trp-Lys(Ne-For)-OH)-OH: 490.5], 338.2
(Mꢃ1)ꢃ [Calcd for C16H24N3O5/(H-Lys(Ne-For)-Tyr-OH)ꢃH: 338.3], 334.1
(M)ꢃ [Calcd for C20H20N3O2/(H-Phe-Trp-OH)-OH: 334.2]. tRꢁ18.3 min.
Experimental
General RP-HPLC Analyses for the Determination of 1—3 In a typi-
cal experiment, H-Trp(Nind-For)-OH·HCl (26.8 mg) was dissolved in water
(0.4 ml) and 1.0 to 3.0 eq of DMEDA was added. The mixture was stirred
under ambient temperature for the appropriate time. Quantification of H-
Trp-OH and H-Trp(Nind-For)-OH was performed on Shimadzu CR4A HPLC
system (YMCpack C18, 4.6ꢂ50 mm) in 0.1% acetic acid/water with a linear
gradient from 5 to 98% acetonitrile over 5 min at a flow rate of 1.0 ml/min.
Peak areas were integrated with Shimadzu SPD-6AV UV VIS detector at
280 nm.
Acknowledgements The authors greatfully acknowledge Dr. Kiyoshi
Ogawa at the Asahi Kasei Pharma Corp. for helpful analysis of RP-HPLC,
LC/MS and LC/MS/MS, and Dr. Lawrence H. Lazarus at NIEHS, NC,
U.S.A., for his invaluable advice during preparation of the manuscript.
H-Trp(Nind-For)-OH (1): ESI-MS m/z: 233.13 (Mꢃ1)ꢃ (Calcd for
C12H13N2O3: 233.23). tRꢁ2.98 min.
References and Note
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Romanic A. M., Louden C. S., Foley J. J., Sauermelch C. F., Coatney
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7) Fujii N., Ikemura O., Funakoshi S., Matsuo H., Segawa T., Nakata Y.,
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10) The desired peptide 5 was purified by RP-HPLC performed using De-
velosil C30-UG-5 (20ꢂ150 mm).
H-Trp-OH (2): ESI-MS m/z: 205.18 (Mꢃ1)ꢃ (Calcd for C11H13N2O2:
205.22). tRꢁ1.73 min.
N-Formyl-N,Nꢀ-dimethylethylendiamine (3): ESI-MS m/z: 117.04
(Mꢃ1)ꢃ (Calcd for C5H13N2O1: 117.16 ). tRꢁ0.35 min.
General LC/MS and LC/MS/MS Analyses for the Determination of
4—6 A 1.5 mg of H-Phe-Trp(Nind-For)-Lys-Tyr-OH·2AcOH was dis-
solved in water (0.10 ml) and treated with various reagents including
DMEDA (3.0 eq). LC/MS spectra of the reaction mixtures were obtained on
Micromass ZMD (ESI) mass spectrometers and a Waters 600 HPLC system
(Develosil C30-UG-5, 4.6ꢂ50 mm) in 0.1% acetic acid/water with a linear
gradient from 5 to 98% acetonitrile over 4 min at 1.0 ml. Peak areas were in-
tegrated with Waters 2996 photodiode array detector. LC/MS/MS spectra of
compounds were obtained by ESI (ABI, QSYAR pulsar-i) and a Hewlett
Packard 1100 series (CAPCELL PAK C18, 1.5ꢂ150 mm) with a linear gra-
dient of 5% acetonitrile containing 0.05% acetic acid/water containing 0.1%
acetic acid to 100% acetoritrile containing 0.05% acetic acid over 40 min at
0.1 ml.
H-Phe-Trp-Lys-Tyr-OH (4): HR-ESI-MS m/z: 643.2416 (Mꢃ1)ꢃ (Calcd
for C35H43N6O6: 643.3244). LC/MS/MS m/z: 462.3 (M)ꢃ [Calcd for
C26H32N5O3/(H-Phe-Trp-Lys-OH)-OH: 462.5], 334.2 (M)ꢃ [Calcd for
C20H20N3O2/(H-Phe-Trp-OH)-OH: 334.3], 310.2 (M)ꢃ [Calcd for
C15H23N3O4/H-Lys-Tyr-OH: 310.3], 129.1 (M)ꢃ [Calcd for C6H13N2O1/(H-
Lys-OH)-OH: 129.1]. tRꢁ14.7 min.
H-Phe-Trp(Nind-For)-Lys-Tyr-OH (5): HR-ESI-MS m/z: 671.2835