Synthesis of Met-enkephalin by solution-phase peptide synthesis methodology utilizing para-toluene sulfonic acid as N-terminal masking of l-methionine amino acid

Article abstract of DOI:10.1111/cbdd.12821

The Met-enkephalin, Tyr-Gly-Gly-Phe-Met, was synthesized by the solution-phase synthesis (SPS) methodology employing -OBzl group as carboxyls' protection, while the t-Boc groups were employed for the N-terminal α-amines' protection for the majority of the amino acids of the pentapeptide sequence. The l-methionine (l-Met) amino acid was used as PTSA.Met-OBzl obtained from the simultaneous protection of the α-amino, and carboxyl group with para-toluene sulfonic acid (PTSA) and as-OBzl ester, respectively in a C-terminal start of the 2?+?2?+?1 fragments condensation convergent synthetic approach. The protection strategy provided a short, single-step, simultaneous, orthogonal, nearly quantitative, robust, and stable process to carry through the protected l-methionine and l-phenylalanine coupling without any structural deformities during coupling and workups. The structurally confirmed final pentapeptide product was feasibly obtained in good yields through the current approach.

Full text of DOI:10.1111/cbdd.12821

Received: 27 February 2016
DOI: 10.1111/cbdd.12821
Revised: 24 May 2016
Accepted: 6 July 2016
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R E S E A R C H A R T I C L E
Synthesis of Met-enkephalin by solution-phase peptide synthesis
methodology utilizing para-toluene sulfonic acid as N-terminal
masking of l-methionine amino acid
Riaz A. Khan
Department of Medicinal Chemistry and
The Met-enkephalin, Tyr-Gly-Gly-Phe-Met, was synthesized by the solution-phase
synthesis (SPS) methodology employing -OBzl group as carboxyls’ protection, while
Pharmacognosy, College of Pharmacy, Qassim
University, Qassim, Saudi Arabia
the t-Boc groups were employed for the N-terminal α-amines’ protection for the
majority of the amino acids of the pentapeptide sequence. The l-methionine (l-Met)
amino acid was used as PTSA.Met-OBzl obtained from the simultaneous protection
of the α-amino, and carboxyl group with para-toluene sulfonic acid (PTSA) and
Correspondence
Riaz A. Khan, Department of Medicinal
Chemistry and Pharmacognosy, College
of Pharmacy, Qassim University, Qassim,
Saudi Arabia.
Email: kahnriaz@gmail.com
as-OBzl ester, respectively in a C-terminal start of the 2 + 2 + 1 fragments condensa-
tion convergent synthetic approach. The protection strategy provided a short, single-
step, simultaneous, orthogonal, nearly quantitative, robust, and stable process to carry
through the protected l-methionine and l-phenylalanine coupling without any struc-
tural deformities during coupling and workups. The structurally confirmed final pen-
tapeptide product was feasibly obtained in good yields through the current approach.
K E Y W O R D S
Bulk preparation, enkephalin, Met-enkephalin, para-toluene sulfonic acid, peptide synthesis, PTSA.
Met-OBzl, simultaneous amine and carboxyl protections, solution-phase synthesis
The discovery of enkephalin by Hughes in 1975 generated
a great deal of interest in its biological activity and develop-
ment of synthetic routes.[1] Both the solution[2], and solid-
phase preparation methods[3,4] for the l-methionine (l-Met)
and l-leucine (l-Leu) analogs of the enkephalin considering
their structure–activity relationship (SAR), involved receptor
subtypes,[5] peptidomimetics,[6] structural-turn mimetics,[7]
and the amino acid mutant analogs[8] have been utilized over
the years.[9–13] The recent spurt in synthesis[14] and molecular
modeling[15] including mathematical modeling[16] of enkeph-
alin rests on the biological activity relationship and requisite
structural and topological features.[17] Moreover, toward devel-
oping a more detailed understanding of the SAR for desir-
able structural features vis-a-vis pharmacophore modeling
and biological activity elicitation, structure-based bioactivity
modulation, and designing of opiate-behavior molecular tem-
plates, a number of different analogs and radiolabeled enkeph-
alin[18,19] have also been synthesized. Reports on the advances
in the enkephalin receptor interactions details through various
conjugates[20,21] and probe molecules toward modeling the
enkephalin structures[22] are some of the recent additions. The
current study is an attempt to synthesize the Met-enkephalin
for biological activity studies, and for finding a feasible route
for synthesis towards preparing a synthetic platform for more
diversified synthetic transformations to be enable to synthesize
designed peptides on preparative and scale-up levels.
1
METHODS AND MATERIALS
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All amino acids (AA) were of l-configuration and coupling
reactions were carried out with anhydrous reactants, and in
freshly prepared dry solvents, wherever required. The evapo-
ration of solvents during workup was done in vacuo below
40 °C. The homogeneity of all the amino acid derivatives
and peptide fragments were established by UV-active TLC
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Chem Biol Drug Des 2016; 1–5
wileyonlinelibrary.com/journal/cbdd
© 2016 John Wiley & Sons A/S.
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on precoated silica gel plates in specified solvents. Ninhydrin
and HBr-AcOH were used as spraying reagents. The melting
points were determined in a sulfuric acid bath and are uncor-
rected. All the single AA derivatives were compared with the
known samples as otherwise stated and were used only after
through comparison and identity establishment.
confirm the completion of the reaction and the mixture was
concentrated to dryness in vacuo and later at an elevated tem-
perature at 45 °C. Methanol (dry) was added (30 mL), and
the residue was again concentrated to dryness. The procedure
was repeated three times to remove excess of HCl. Tyrosine
methyl ester hydrochloride crystals were collected. The prod-
uct was again recrystallized from dry methanol–dry diethyl
ether to yield 4.2 g of the final product. TLC (BuOH-AcOH-
H2O: 4:1:5, upper layer), Rf 3.5, single spot, Ninhydrin, and
m.p. 188–189 °C confirmed the identity of the product on
comparison with the in-house reference sample.
1.1
Preparation of Boc-Gly
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l-Gly (1.5 g, 20 mm) was taken in a mixture of water–dioxane
(1:1, 20 mL) and cooled to 0 °C followed by the addition of
2N aq. NaOH to maintain highly basic condition (pH 11–12)
with stirring for 15 min. Di-tert-butyl-dicarbonate (DCC)
(22 mm) was added over a period of 30 min in portions with
pH maintaining at the higher basic range with 2N aq. NaOH
solution additions with the temperature maintained at 0 °C
under continued stirring for 2 h followed by stirring at RT for
another 1 h. The solvents were removed in vacuo below 40 °C
and the resultant residue was taken in 20 mL of water, acidi-
fied with solid citric acid to pH 2. The mixture was saturated
with brine, extracted with diethyl ether, and combined organ-
ic layers were dried over anhydrous Na2SO4 and evaporated
to give the product as a viscous oil, which was crystallized
with dry diethyl ether, yields 2.2 g (72%). TLC, co-TLC, and
m.p. were compared with the known sample and product was
found matching with the in-house reference standard.
1.4
Preparation of Boc-Tyr-OMe
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HCl.Tyr-OMe (3.71 g, 15 mm) was dissolved in dry DMF,
and trimethylamine (4.5 mL, 30 mm) was added to free
the salt. DCC (di-tert-butyl-dicarbonate) (3.27 g, 15 mm)
was added to the reaction mixture in about 30 min in small
portions at 0 °C under vigorous stirring, and stirring was con-
tinued at RT for another 2 h. The solvent was evaporated in
vacuo and residue took in EtOAc, washed with brine (3×),
dried over anhydrous Na2SO4, and combined organic lay-
ers were evaporated in vacuo below 40 °C to give a white
crude product as a solid, 3.7 g, which was recrystallized
to single spot product, 3.7 g. TLC Rf 6 (CHCl3: MeOH ::
95:5), Ninhydrin, single spot, dark orange in color. Co-TLC
and m.p. were compared with the known sample and found
matching with the in-house reference standard.
1.2
Preparation of Boc-Gly-Gly-OH
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Boc-Gly (1.75 g, 10 mm) was taken into dry THF (25 mL);
N-methyl morpholine (NMM) (1.15 mL, 10 mm) and isobutyl
chloroformate (1.35 mL, 10 mm) were added. After 2–3 min,
a mixture of Boc-Gly (1.125 g, 15 mm) and 1 N aq. NaOH
(16.5 mL, 11 mm) was added in one instance and the reaction
mixture stirred for 3 h. The solvents were evaporated in vacuo
below 45 °C and the resultant thick oil was crystallized with
vigorous scratching in dry diethyl ether to yield the crude sol-
id product, which was recrystallized with dry diethyl ether.
Yields 1.6 g, TLC single spot Rf 5 (MeOH: CHCl3: AcOH:
5:93:2, upper layer), HBr-AcOH and Ninhydrin. TLC, co-
TLC, and m.p. were compared with the known sample and
were found matching with the in-house reference standard.
1.5
Preparation of Boc-Tyr-OH
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Boc-Try-OMe (3.1 g, 10 mm) in methanol (75 mL) was
stirred at 0 °C and 2 N aq. NaOH (20 mm) was added drop-
wise with continued stirring for another 1 h. The solvents
were evaporated in vacuo, and the residue was washed with
ether, acidified with (solid) citric acid to neutral pH 7 at 0 °C.
EtOAc was added and the organic layers were extracted and
combined, washed with brine, and dried over anhydrous
Na2SO4, followed by drying in vacuo to give a TLC pure
single spot product. Yields 2.0 g, TLC: Rf 4 (CHCl3: MeOH:
95:5), Ninhydrin, single spot, light orange in color. Co-TLC
and m.p. were compared with the known sample and found
matching with the in-house reference standard.
1.3
Preparation of HCl. Tyr-OMe
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1.6
Preparation of Boc-Phe-OH
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Thionyl chloride (1.6 mL, 20 mm) was added dropwise over
a period of 10 min with vigorous stirring to precooled abso-
lute methanol (35 mL) in an RB flask fitted with calcium
chloride guard tube and acetone: ethyl acetate (1:1) dry-
ice mixture cold bath. To this solution, tyrosine was added
(3.6 g), and the reaction mixture was further stirred for 1 h at
4 °C on crushed ice: NaCl bath, and then at RT. The RM was
left in dark overnight. Next morning, TLC was monitored to
l-Phe (2.30 g, 20 mm) was taken in water (90 mL), and 2 N aq.
NaOH (10 mL) was added. After the dissolution of the start-
ing material, dioxane (100 mL) was added, followed by DCC
(4.36 g) in small portions in about 30 min with vigorous stir-
ring at 0 °C. Stirring was continued at 0 °C for another 1.5 h
and at RT for another 1 h. The solvents were concentrated
up to 1/4th of the volume, water (50 mL) was added, and the

Khan
3
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RM was acidified to pH 2 by the addition of solid citric acid.
The organic layers were extracted with EtOAc (3 × 50 mL),
washed with brine (3×), and dried over anhydrous Na2SO4.
The combined organic layers were dried in vacuo below
40 °C to give an oily product, which was recrystallized to
give the solid product, yields 3.4 g. TLC, co-TLC, and m.p.
were compared with the known sample and the product was
found matching with the in-house reference standard.
N-methyl morpholine (NMM) was added to neutralize the
mixture (pH 7) and Boc-Gly-Gly-OH in dry DMF followed
by HOBt (225 mg) were added under stirring at 0 °C. The
reaction mixture was kept stirring and chilled at 0 °C, and
DCC (350 mg) in dry CH2Cl2 was added. After 2 h of stir-
ring at 0 °C, which was followed by another 2 h of stirring at
RT, solvents were evaporated in vacuo below 40 °C to give
the solid product, which was recrystallized from dry MeOH-
dry diethyl ether, 2.6 g. TLC: Rf 3.5 (MeOH:CHCl3 :: 5:95),
single spot, HBr-AcOH, Ninhydrin.
1.7
Preparation of PTSA.Met-OBzl
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To a Dean-Stark assembly’s fitted round-bottom flask, l-Met
(2.98 g, 20 mm), para-toluene sulfonic acid (PTSA) (4.18 g,
22 mm), and benzyl alcohol (13 mL, 120 mm) were charged
in dry benzene as solvent (30 mL). The reaction mixture was
refluxed at 100 °C for 22 h and the collected water in lower
portion of DS assembly’s arm was removed intermittently
with filling of dry benzene to facilitate the reflux and distil-
lation off of the azeotrope formed. The RM was monitored
over a period of time but at first after 8 h of the reaction.
After completion of the reaction (22 h), as monitored by TLC
(BuOH-AcOH-H2O: 4:1:5, upper layer, Rf 6, Ninhydrin), a
solid product was obtained (6.1 g) after solvent removal in
vacuo below 40 °C. The crude product was recrystallized
from dry MeOH-diethyl ether, yields: 6.03 g.
1.10
Deblocking of Boc-Gly-Gly-Phe-Met-
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OBzl
5N HCl.dioxane was added to Boc-Gly-Gly-Phe-Met-OBzl
(2 g) followed by thioanisole and mercaptoethanol (0.2 mL
each), and the reaction mixture was kept at room temperature
for 1 h. The solvents were evaporated in vacuo, and the resi-
due was triturated with dry ether to give the HCl.Gly-Gly-
Phe-Met-OBzl as an amorphous solid, which was processed
further as such after drying in vacuo over P2O5 and NaOH
pellets at RT in a vacuum desiccator.
1.11
Preparation of Boc-Tyr-Gly-Gly-Phe-
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Met-OBzl
HCl.Gly-Gly-Phe-Met-OBzl (600 mg) was dissolved in dry
DMF and cooled to 0 °C followed by addition of NMM to
free the hydrochloride salt. Boc-Tyr-OH (0.25 g) was dis-
solved in dry DMF and HOBt was added (0.08 g), followed
by mixing of free amine H2N-Gly-Gly-Phe-Met-OBzl and
Boc-Tyr-OH in one instance. DCC was added as a CH2Cl2
solution, and stirring was continued at 0 °C for 2 h followed
by further stirring at room temperature for 2 h. The solvents
were removed in vacuo and residue was taken in water,
extracted with EtOAc, and the combined organic layers were
dried over anhydrous Na2SO4. The dried organic layer was
removed in vacuo below 40 °C to give the amorphous solid
(610 mg), which was recrystallized from dry MeOH to give
white crystalline material, 560 mg, Rf 6.5 (MeOH:CHCl3 ::
10:1), single spot, Ninhydrin.
1.8
Preparation of Boc-Phe-Met-OBzl
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Boc-Phe (2.65 g, 10 mm) was dissolved in dry DMF (30 mL),
and HOBt (1.53 g, 10 mm) was added at room temperature.
The mixture was kept separate under dry conditions protected
by a guard tube and away from moisture. The PTSA.Met-
OBzl (4.12 g) was dissolved in dry DMF (25 mL) at 0 °C,
and N-methyl morpholine (NMM) (1.3 mL, 11 mm) was
added to make pH 7. The Boc-Phe-HOBt and the deprotected
PTSA.Met-OBzl were mixed together over 5 min period at
0 °C temperature under stirring. The reaction mixture was
allowed to cool to 0 °C, and DCC (2.26 g, 11 mm) dissolved
in CH2Cl2 was added. Stirring was continued for another 2 h
under cool conditions and 2 h at RT. The solvents were evap-
orated in vacuo to give a residue, which was crystallized with
dry MeOH to give a crude solid, 4.5 g, which was processed
further as such after vacuum drying of the product over P2O5
and NaOH pellets at RT in a vacuum desiccator.
1.12
OH
Preparation of Tyr-Gly-Gly-Phe-Met-
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Boc-Tyr-Gly-Gly-Phe-Met-OBzl (250 mg) was dissolved
in 5% formic acid in methanol (50 mL), and 10% Pd-C was
added under stirring at RT for 20 min. After completion of
the reaction as monitored by TLC, the catalyst was filtered off
and solvents removed in vacuo below 40 °C. The residue was
treated with 5N HCl-dioxane at 0 °C under stirring for 30 min
in the presence of thioanisole and mercaptoethanol (0.2 mL
each). The solvents were again evaporated in vacuo below
1.9
Preparation of Boc-Gly-Gly-Phe-Met-
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OBzl
Boc-Phe-Met-OBzl was deprotected using 5N HCl in
dioxane in the presence of thioanisole and mercaptoethanol
under stirring at RT for 15 min. The solvents were evaporated
below 40 °C in vacuo to give the product, HCl.Phe-Met-
OBzl, which was dissolved in dry DMF and chilled to 0 °C.

4
Khan
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40 °C, and the residue was treated with NMM in MeOH to
get solution’s pH slightly above neutral. The reaction mixture
was again evaporated in vacuo at 40 °C. The solid residue,
so obtained, was washed (3 × 50 mL) with dry ether, and
the crude product was recrystallized with dry MeOH–dry
diethyl ether, yields 187 mg (66%). m.p., 197–98 °C, TLC,
and co-TLC, were compared with the known in-house stand-
ard sample. TLC: Rf 6 (n-BuOH: AcOH: H2O: 4:1:4), ([α]D
−22° (c = 1.1, DMF), elemental analysis: found C56.45 H6.22
N12.3%, required. C56.55 H6.15 N12.2%. PMR (DMSO-d6):
δ 1.51–2.02 (m, 7H, Met-CH3, 2xCH2), 3.01–3.40 (m, 4H,
Tyr, Phe 2xCH2), 3.71(brs, 4H, Gly2,3-CH), 3.78–4.56 (m,
3H, Tyr, Phe, Met-CH), 6.84 (dd, 4H, TyraromaticH), 7.30 (brs,
5H, PhearomaticH), 8.1-8.7(NH), unequivocally confirmed the
identity of the product.
comparatively simple, synthetically effective, convenient in
handling, robust, and cost-reducing method utilizing milder
synthetic conditions with convergent procedure requiring
shorter time span with improved optical purity and high
yields for preparative scale synthesis of the pentapeptide
product. Toward this effect, the synthesis was started with
suitably protected amino acids. The t-butyl-oxy-carbonyl
(t-Boc) group was employed (Scheme 1) for the protection of
the α-amino groups of the majority of the amino acids dur-
ing the peptide synthesis owing to its established and facili-
tated protection–deprotection state.[23–25] The carboxylic
ends were selectively masked with a neutral -OBzl group,[26]
which again proved to be a versatile and efficient carboxylic
protection strategy with its easy removal in catalytic transfer
hydrogenation employing low concentrations of formic acid
as the hydrogen donor.[27]
The approach to 2 + 2 + 1 fragments’ coupling plan fol-
lowed with the preparation of Boc-Tyr-OMe, Boc-Gly-Gly-
OH, and Boc-Phe-Met-OBzl as the protected, independent
fragments. The Boc-Phe-Met-OBzl was prepared from Boc-
Phe-OH and its coupling with the H2N-Met-OBzl. An in situ
coupling of the Boc-Phe-OH and PTSA.Met-OBzl was also
tried but with purity and yields issues present, was not pursued
further.However,thefreeamine,H2N-Met-OBzl,wasobtained
from the PTSA.Met-OBzl deprotection (Scheme 1), while
the PTSA.Met-OBzl itself was prepared (three batches) from
L-Met treatment with para-toluene sulfonic acid (PTSA) and
its simultaneous condensation in heating conditions (100 °C)
with the benzyl alcohol to mask the carboxyl group of the
L-Met amino acid. The coupling was mediated by dicyclohex-
yl carbodiimide (DCC) in dry CH2Cl2 at 0 °C for 30 min with
monitoring by TLC. The Boc-Gly-Gly-OH dipeptide frag-
ment was approached through mixed anhydride method[28,29]
2
RESULTS
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The pentapeptide, Tyr-Gly-Gly-Phe-Met, was synthesized in
goodyields(66%)followingthesolution-phasepeptidesynthesis
methodology using the PTSA-Met-OBzl as the coupling AA
component in the sequence assembly involving t-Boc and -OBzl
groups as the amino and carboxyl protections for the other four
amino acids (AA) in the sequence. The final product’s identity
was confirmed by spectro-analytical techniques and a through
comparison with an in-house reference sample.
3
DISCUSSION
|
The quest for the development of a new method for synthe-
sis of Met-enkephalin was driven by the need to establish a
SCHEME 1 Synthetic strategy for the
preparation of Met-enkephalin, Tyr-Gly-Gly-
Phe-Met-OH

Khan
5
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of peptide synthesis protocol through the coupling of the Boc-
Gly-OH and H2N-Gly-OH. A 2 + 2 DCC-HOBt (hydroxy
benzotriazole)-mediated coupling[30,31] of the H2N-Phe-
Met-OBzl and Boc-Gly-Gly-OH yielded the tetrapeptide, Boc-
Gly-Gly-Phe-Met-OBzl, which was deprotected to free amine
using 5N HCl-dioxane followed by resulting hydrochloride salt
neutralization with N-methyl morpholine (NMM) and con-
densing the obtained free amine to Boc-Tyr-OH, again mediat-
ed by DCC-HOBt, at cooled conditions. The Boc-Tyr-OH was
prepared from Boc-Tyr-OMe, which itself was obtained from
HCl.H2N-Tyr-OMe starting from the L-Tyr amino acid. The
protected pentapeptide sequence, Boc-Tyr-Gly-Gly-Phe-Met-
OBzl, was hydrogenated for 20 min over 10% Pd-C with for-
mic acid in a transfer hydrogenation step.[32–34] The obtained
formate salt, a highly hygroscopic product, was converted
to HCl.Tyr-Gly-Gly-Phe-Met-OH by 5N HCl-dioxane treat-
ment in the presence of thioanisole and mercaptoethanol. The
hydrochloride salt was neutralized, worked up, and recrystal-
lized to give the desired pentapeptide sequence, H2N-Tyr-Gly-
Gly-Phe-Met-OH, in 66% yields with its identity confirmed
through comparison with an in-house reference standard and
spectro-analytical techniques. Thus, the desired pentapeptide
was synthesized in a stepwise and linear elongation of the pep-
tide chain starting from C-terminus (Scheme 1) in a 2 + 2 + 1
fragments convergent approach.
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4
CONCLUSION
|
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The current approach in protection and deprotection strategy
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and carboxyl groups’ protections for Met AA. The method
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without the presence of harsh preparation or deprotection
conditions including many workups. The transformation
also provided near quantitative yields and is capable of easy
scale-up with cost-viable protection strategy for the sulfur-
containing l-Met amino group in lieu of the t-Boc protec-
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sequence assembly. The strategy may also have the potential
for polymer-based different SPPS strategies involving l-Met
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synthetic conditions.
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CONFLICT OF INTEREST
There is no conflict of interest.
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