Hydrazine hydrate: A new reagent for Fmoc group removal in solid phase peptide synthesis

Article abstract of DOI:10.1016/j.tetlet.2018.11.054

In solid-phase peptide synthesis using the Fmoc/tBu strategy (SPPS-Fmoc/tBu), an orthogonal protection scheme of amino acids is used; specifically, the alpha-amine group is protected by the 9-fluorenylmethyloxycarbonyl (Fmoc) group, which is removed by weak bases, while side chains are protected by groups that are acid labile. We demonstrated that hydrazine hydrate is an efficient reagent for eliminating the Fmoc group in SPPS-Fmoc/tBu. First, experimental conditions were established for Fmoc group removal from Fmoc-Val-OH in solution. It was determined that the Fmoc group was completely removed with 16% hydrazine hydrate in DMF after 60 min at rt. Second, SPPS-Fmoc/tBu using hydrazine hydrate for Fmoc group removal was standardized. The Fmoc group removal was completed using 16% hydrazine hydrate in DMF for 10 min at rt (twice). When the reaction of Fmoc group removal was microwave-assisted, the reaction only required 30 s to efficiently remove the Fmoc group in SPPS-Fmoc/tBu. The method reported here can be routinely used, and it is equivalent to conventional SPPS-Fmoc/tBu methodologies where 4-methylpiperidine or piperidine is used.

Full text of DOI:10.1016/j.tetlet.2018.11.054

Accepted Manuscript
Hydrazine hydrate: a new reagent for Fmoc group removal in solid phase peptide
synthesis
Andrea V. Rodríguez, Julieth T. Roman, Ricardo Fierro, Zuly J. Rivera, Javier
E. García
PII:
S0040-4039(18)31401-1
https://doi.org/10.1016/j.tetlet.2018.11.054
TETL 50439
DOI:
Reference:
To appear in:
Tetrahedron Letters
Received Date:
Revised Date:
Accepted Date:
24 October 2018
16 November 2018
21 November 2018
Please cite this article as: Rodríguez, A.V., Roman, J.T., Fierro, R., Rivera, Z.J., García, J.E., Hydrazine hydrate: a
new reagent for Fmoc group removal in solid phase peptide synthesis, Tetrahedron Letters (2018), doi: https://
doi.org/10.1016/j.tetlet.2018.11.054
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Graphical Abstract
Hydrazine hydrate: a new reagent for Fmoc
group removal in solid phase peptide
synthesis
Leave this area blank for abstract info.
Andrea V. Rodríguez, Julieth T. Román, Ricardo Fierro, Zuly J. Rivera, Javier E. García

1
Tetrahedron Letters
Hydrazine hydrate: a new reagent for Fmoc group removal in solid phase peptide
synthesis
a
b
a
a
b,
Andrea V. Rodríguez , Julieth T. Roman , Ricardo Fierro , Zuly J. Rivera , Javier E. García *
a
b
Chemistry Department, Pharmacy Department, Universidad Nacional de Colombia, Bogotá Carrera 45 No 26-85, Building 450, office 203, Bogotá, Zip Code
1
*
1321, Colombia.
Corresponding author: jaegarciaca@unal.edu.co; Tel.: +57-320-241-0680
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
In solid-phase peptide synthesis using the Fmoc/tBu strategy (SPPS-Fmoc/tBu), an orthogonal
protection scheme of amino acids is used; specifically, the alpha-amine group is protected by the
9-fluorenylmethyloxycarbonyl (Fmoc) group, which is removed by weak bases, while side
chains are protected by groups that are acid labile. We demonstrated that hydrazine hydrate is an
efficient reagent for eliminating the Fmoc group in SPPS-Fmoc/tBu. First, experimental
conditions were established for Fmoc group removal from Fmoc-Val-OH in solution. It was
determined that the Fmoc group was completely removed with 16% hydrazine hydrate in DMF
after 60 min at rt. Second, SPPS-Fmoc/tBu using hydrazine hydrate for Fmoc group removal
was standardized. The Fmoc group removal was completed using 16% hydrazine hydrate in
DMF for 10 min at rt (twice). When the reaction of Fmoc group removal was microwave-
assisted, the reaction only required 30 s to efficiently remove the Fmoc group in SPPS-
Fmoc/tBu. The method reported here can be routinely used, and it is equivalent to conventional
SPPS-Fmoc/tBu methodologies where 4-methylpiperidine or piperidine is used.
Available online
Keywords:
Hydrazine Hydrate
Fmoc group removal
Solid phase peptide synthesis
Orthogonal protection
4
-methylpiperidine
2
009 Elsevier Ltd. All rights reserved.
1. Introduction
Synthetic peptides are now considered to be promising
dibenzofulvene
(DBF)
formation.
Afterward,
DBF
molecules for developing drugs: approximately 60 therapeutic
peptides have been approved in the United States, Europe and/or
Asia [1]. Currently, 150 peptides are being evaluated in clinical
development, and up to now 260 peptides have been tested in
human clinical trials [1]. Solid-phase peptide synthesis (SPPS) is
a valuable tool for designing and obtaining peptides with
therapeutic properties and enables peptide production with
pharmaceutical-grade purity [2-3]. In this context, it is necessary
to continuously improve the processes involved in peptide
synthesis [3]. The SPPS method requires orthogonal protection
schemes. The Fmoc/tBu strategy employs amino acids with the
alpha-amine group protected by the Fmoc group (weak base
labile), while the side chains are protected by acid labile groups
polymerization is avoided by the piperidine as well, since it acts
as a scavenger, leading to the formation of the DBF-piperidine
adduct, which is easily eliminated by filtration [5]. Efficient
Fmoc group removal in SPPS requires (i) abstraction of the
tertiary proton at the 9-position of the fluorenyl group, and (ii)
formation of the DBF-base adduct [5, 9-10].
DBF-base adduct formation mainly depends on the base
properties, e.g. basicity, nucleophilicity, and bulky structure
(
steric hindrance). Bases such as piperazine and its mono-n-
methyl and n-phenyl derivatives, piperidine, cis-3,5-
dimethylpiperidine, 4-methylpiperidine, and morpholine
efficiently form the DBF-base adduct [5, 9, 11]. Bases with
higher pKa values than piperidine can remove the Fmoc group,
but they do not form the DBF-base adduct. For example,
isonipecotic acid, 6-dimethyl, and 2,2,6,6-tetramethylpiperidine
do not form the DBF-base adduct. n-Propylamine and amino
methyl cyclohexane react easily with DBF, while ethanolamine,
ethylenediamine, and 2-methoxyethylamine require excessive
reaction times to induce DBF-base adduct formation [5].
[4-6]. After each Fmoc-amino acid is attached to the growing
peptide chain, the Fmoc group must be removed in order to
obtain the free alpha-amine group. In this process, the side chains
must remain protected. A free alpha-amine group is necessary in
order to attach the next Fmoc-amino acid through an amide bond.
Incomplete Fmoc group removal leads to obtaining truncated
species in the final product, which decreases the yield and
increases the costs, making the peptide synthesis inefficient.
Currently, commonly used reagents for Fmoc group removal in
SPPS are piperidine and its derivatives [7-8]. The reaction
mechanism, beta-elimination, involves abstraction of the tertiary
proton from the fluorenyl group at the 9-position, leading to
The aim of the present study was to explore a new alternative
for Fmoc group removal in SPPS-Fmoc/tBu. First, we evaluated
the use of hydrazine hydrate as a removal reagent of the Fmoc
group from Fmoc-Val-OH in solution. Second, optimal
experimental parameters for Fmoc group removal and their

2
Tetrahedron Letters
applicability in SPPS were established. In order to discover the
optimal conditions for the Fmoc group removal reaction from
Fmoc-Val-OH, different hydrazine hydrate concentrations (13,
16, 19, 26, 32, and 38% v/v) and reaction times (0, 5, 10, 20, 30,
and 60 min) were evaluated. Hydrazine hydrate (70%) aliquots
were diluted in DMF, and Fmoc-Val-OH (3.5 mg/mL) was added
to each solution.
Figure 1. Hydrazine hydrate as Fmoc group removal reagent. Reaction kinetics at different hydrazine hydrate concentrations (%v/v):
R
12.8% (Panel A), 16.0% (Panel B). Fmoc-Val-OH was used as a starting material. Fmoc-Val-OH (t = 6.5 min) and DBF-hydrazine
adduct (t = 4.3 min).
R
The reaction kinetics were monitored via RP-HPLC using a
Chromolith® C18 (50×4.6 mm) monolithic column. The elution
gradient was from 5 to 70% B, the gradient time was 10 min,
flow rate was 2.0 mL/min, and detection was at 210 nm. Solvent
A was TFA 0.05% in water and Solvent B was TFA 0.05% in
ACN. The Fmoc-Val-OH chromatographic profile, in the absence
of hydrazine hydrate, exhibited a main peak at t = 6.5 min. The
R
reaction chromatographic profile (Figure 1A), at time zero,
exhibited three peaks, corresponding to (1) DBF-hydrazine
0.21 mmol) were dissolved in DMF (2 mL), and the mixture was
shaken for 5 min at rt. Then the preactivated amino acid was
mixed with the resin (or resin-peptide) for 3h at rt. Incorporation
of each amino acid was monitored via the Ninhydrin test [12].
Side chain deprotection and peptide cleavage from the solid
support were carried out by means of treatment of the resin-
peptide with TFA/water/TIS/EDT (92.5/2.5/2.5/2.5% v/v), 6 h at
rt.
R R
hydrate adduct (t = 4.3 min), (2) Fmoc-Val-OH (t = 6.5 min),
and (3) DBF (t = 7.6 min), suggesting that the reaction proceeds
R
immediately. As can be seen, in the chromatographic profiles the
peak area of the signal at 4.3 min increases when the reaction
time increases, while the Fmoc-Val-OH peak area decreases.
Fmoc removal from the starting material leads to free valine
formation, which cannot be detected in the UV region. The Fmoc
group was not completely removed in 60 min when hydrazine
hydrate 12.8% was used. When the reaction was carried out with
1
3
1
6% hydrazine hydrate reagent or a higher concentration (16 to
8%), Fmoc group removal was completed in 60 min. (Figure
B). According to these results, we established that the optimal
conditions for removing the Fmoc group from Fmoc-Val-OH
were 16% hydrazine hydrate and a reaction time of 60 min at rt.
Then LfcinB (20-25), RRWQWR, and LfcinB (21-
25)Pal:RWQWRWQWR peptides were synthesized by means of
the SPPS-Fmoc/tBu method. For Fmoc group removal, (i)
hydrazine hydrate or (ii) 4-methylpiperidine was used. The
peptides were synthesized using the SPPS-Fmoc/tBu protocol
standardized in our laboratory. Fmoc group removal was carried
out by treatment of resin or resin-peptide with 2 mL of removal
reagent, i.e. 4-methylpiperidine 20% in DMF v/v or hydrazine
hydrate 16% in DMF v/v. For each removal reagent, three
experimental conditions were evaluated: method 1, reaction time
1
3
h at rt; method 2, reaction time 10 min at rt (twice); and method
, reaction time 30 s, assisted by microwave (twice).
Figure 2. Chromatographic profile and MALDI-TOF mass
spectrum of peptide LfcinB (21-25)Pal obtained by means of the
SPPS-Fmoc/tBu strategy, using as a Fmoc removal reagent
hydrazine hydrate 16% in DMF (A) and 4-methylpiperidine 20%
Rink amide resin (150 mg, 0.46 meq/g) was used as a solid
support. The coupling reaction was carried out as follows: Fmoc-
amino acid (0.21 mmol), DCC (0.20 mmol), and (6-Cl-HOBt

3
in DMF (B). In both cases, treatment for Fmoc removal was 1
hour. Theoretical monoisotopic mass was 1485.75.
Peptides were obtained with high purity, with similar yields in
all cases. For method 1, 70.6 mg of crude peptide LfcinB (20-25)
was obtained when 4-methylpiperidine was used, while it was
83.1 mg for the hydrazine hydrate reagent. Similarly, the yield
for peptide LfcinB (21-25)Pal was 93.7 mg and 115.1 mg,
respectively. Figure 2 shows the chromatographic profile of the
crude peptide LfcinB (20-25)Pal obtained using hydrazine hydrate
16% in DMF (Panel A) and 4-methylpiperidine 20% in DMF
(Panel B), using the conditions described for method 1. Both
chromatograms show a main specie with the same retention time.
MALDI-TOF MS spectra present the m/z corresponding to the
+
specie [M+H] . For the peptide LfcinB (20-25), the
chromatographic profiles show a peak with the same retention
R
time (t = 4.4 min). The chromatographic purities were 98% and
94%, using 4-methypiperidine and hydrazine hydrate as the
Fmoc removal reagent, respectively (data not shown). These
results indicate that SPPS-Fmoc/tBu using hydrazine hydrate
allows obtaining peptides with high purity and good yields, a
result similar to conventional SPPS-Fmoc/tBu.
Fmoc group removal in SPPS-Fmoc/tBu with hydrazine
hydrate by method 1 requires a long reaction time at rt (60 min),
limiting its application in SPPS. Fmoc group removal with 4-
methylpipridine by method 2 is routinely used by us in SPPS-
Fmoc/tBu. Thus we decided to synthesize the peptide LfcinB
(
20-25) using hydrazine hydrate 16% or 4-methylpiperidine 20%
Figure 3. Peptide LfcinB (20-25): RRWQWR circular dichroism
CD) spectra, molecule obtained by SPPS-Fmoc/tBu using as Fmoc
removal reagent hydrazine hydrate 16% (A) or 4-Methylpiperidine 20%
B) in DMF. In both cases, the reaction was assisted by MW (method 3).
in DMF using method 2. The peptides obtained exhibited similar
yields, purity, chromatographic profiles, and m/z signals. These
results also are in agreement with the results obtained by [13].
Finally, the peptide LfcinB (20-25) was also synthetized
simultaneously using hydrazine hydrate or 4-methylpiperidine
(
(
Hydrazine hydrate has proved to be a very efficient for the
(method 3). The final products exhibited similar yields,
removal of Fmoc group due to its high nucleophilicity under very
mild conditions, which insures very low degradation of the
peptide fragments. Additionally, hydrazine hydroxide is a very
inexpensive and easy access reagent that makes this synthetic
strategy an advantage over the current methods used. The
concern over the reactivity and risk of explosion of the hydrazine
is controlled by the use of a dilute and low concentration used.
Our results indicate that hydrazine hydrate as an Fmoc group
removal reagent in SPPS-Fmoc/tBu could be routinely
implemented in SPPS.
chromatographic profiles, and purity, and both products had the
expected m/z (data not shown). The peptide LfcinB (20-25)
obtained by means of SPPS-Fmoc/tBu (method 2 or 3) using 4-
methylpiperidine or hydrazine hydrate exhibited similar CD
spectra, suggesting that Fmoc group removal using hydrazine
hydrate does not induce amino acid racemization. CD spectra
obtained in this paper were in agreement with CD spectra
reported for this peptide synthesized via SPPS-Fmoc/tBu using 4-
methylpiperidine [14].
7
8
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N. Zinieris, L. Leondiadis, N. Ferderigos, J. Comb. Chem. 7
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J. Hachmann, M. Lebl, J. Comb. Chem. 8 (2006), 149.
C.F. Vergel; Z.J. Rivera; J.E. Rosas, J.E. García J. Mex. Chem.
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Acknowledgments
(
This research was conducted with the financial support of
División de Investigación y Extensión sede Bogotá (DIEB),
Universidad Nacional de Colombia through the Project code
9.
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4
Tetrahedron
Hydrazine hydrate as an Fmoc group removal reagent in SPPS-Fmoc/tBu
could be routinely implemented in SPPS
SPPS-Fmoc/tBu using hydrazine hydrate allows obtaining peptides with high
purity and good yields
Hydrazine hydrate a new Fmoc group removal reagent in SPPS-Fmoc/tBu
1
5.