Solution-phase-peptide synthesis via the group-assisted purification (GAP) chemistry without using chromatography and recrystallization

Article abstract of DOI:10.1039/c3cc48509a

The solution phase synthesis of N-protected amino acids and peptides has been achieved through the Group-Assisted Purification (GAP) chemistry by avoiding disadvantages of other methods in regard to the difficult scale-up, expenses of solid and soluble po

Full text of DOI:10.1039/c3cc48509a

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Solution-phase-peptide synthesis via the group-
assisted purification (GAP) chemistry without
using chromatography and recrystallization†
Cite this: Chem. Commun., 2014,
50, 1259
Received 7th November 2013,
Accepted 26th November 2013
Jianbin Wu,ab Guanghui An,b Siqi Lin,b Jianbo Xie,b Wei Zhou,ab Hao Sun,a Yi Pana
and Guigen Li*ab
DOI: 10.1039/c3cc48509a
www.rsc.org/chemcomm
The solution phase synthesis of N-protected amino acids and peptides the work-up in a solid-phase manner, or, through convenient
has been achieved through the Group-Assisted Purification (GAP) extractions. However, the latter method needs soluble polymers
chemistry by avoiding disadvantages of other methods in regard to of high molecular weights, which makes it inconvenient to
the difficult scale-up, expenses of solid and soluble polymers, etc. The produce large amounts of peptides of much lower molecular
GAP synthesis can reduce the use of solvents, silica gels, energy and weights than polymeric templates. In addition, it often needs
manpower. In addition, the GAP auxiliary can be conveniently recovered long periods to generate solid or crystalline products by care-
for re-use and is environmentally friendly and benign, and substantially fully controlling solidification/crystallization conditions.
reduces waste production in academic labs and industry.
Recently, our labs have established the GAP (Group-Assisted
Purification) chemistry concept through the design and synthesis of
Peptides and peptidomimetic drugs are involved in nearly all new reagents that were attached with unique achiral and chiral
disciplines of chemical and biomedical sciences.1–5 The search for auxiliaries.24–31 By using GAP technology, organic synthesis can be
environmentally friendly synthetic approaches to these products performed efficiently and stereochemically without the use of tradi-
for academic research and pharmaceutical production has been tional purification methods such as chromatography, recrystalliza-
pursued for over five decades.6–11 A notable accomplishment in tion, etc. In fact, the stereoisomeric products can be obtained simply
peptide synthesis is shown by the solid-phase-peptide synthesis by washing the crude mixtures with inexpensive petroleum solvents
(SPPS) invented by the late Nobel Laureate, Bruce Merrifield, in the or co-solvents. Therefore, it can substantially reduce the use of
1980’s.12–13 This milestone discovery can overcome shortcomings starting materials, silica gels, energy, manpower, etc. In addition,
of traditional solution-phase-peptide synthesis in regard to tedious achiral and chiral auxiliaries can be conveniently recovered
purification, consumption of large amounts of materials including for re-use, and they can often be quantitatively recovered via a
coupling reagents, solvents and silica gels, and can reduce waste one-time extraction with n-butanol. It is believed that the GAP
generation; it has also greatly accelerated peptide and protein chemistry concept will have a huge impact on chemical synthesis
research. SPPS has also been imposing a huge impact on general and medicinal fields. In this report, we would like to disclose
chemical synthesis for drug discovery and development, particu- that the GAP chemistry strategy can advance peptide synthesis,
larly, on combinatorial screening.13 Since then several synthetic which has advantages of both solid-phase-peptide synthesis
protocols have been developed to complement SPPS so as to (SPPS) and liquid-phase synthesis of peptides by avoiding their
minimize the difficulty of scale-up, the use of excess amounts of shortcomings in regard to the factors mentioned above.22,23
reagents and expensive resins for coupling reactions, particularly
Amino acid residues are basic building blocks for the
for synthesizing longer peptides.14–21 In this regard, an alternative synthesis of peptides and proteins. We first explored if individual
method was developed by using soluble polymers as the templates amino acids can be protected via the GAP chemistry protocol.
for coupling of amino acid residues.22,23 This method enables N,N0-Di-(1-naphthylmethylene)-1,2-cyclohexyldiamino N-phosphonyl
the peptide synthesis to be performed in the solution phase but chloride A-1 was employed for the reactions with L-Pro-OBnꢀHCl,
D-Ala-OMeꢀHCl and L-Phe-OMeꢀHCl. To a solution of N-phosphonyl
a Institute of Chemistry & BioMedical Sciences (ICBMS), Nanjing University,
Nanjing 210093, P. R. China
chloride in dichloromethane triethylamine (2.5 eq.), 4 Å MS and
amino ester were added; the resulting mixture was stirred at 90 1C
overnight for complete consumption of major starting materials.
The pure protected amino acid esters of the above three products
were obtained via the GAP work-up washing with hexane–DCM
co-solvents by avoiding the use of column chromatography or
b Department of Chemistry and Biochemistry, Texas Tech University, Lubbock,
TX 79409-1061, USA. E-mail: guigen.li@ttu.edu
† Electronic supplementary information (ESI) available: Spectroscopic spectra of
all pure products shown in Schemes S1–S3 and Fig. S1. See DOI: 10.1039/
c3cc48509a
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Scheme 1 GAP protection of L-proline benzyl ester and its deprotection.
recrystallization to give yields of 96%, 92% and 86%, respectively.
For this protection experiment, 4 Å MS was necessary for achieving
higher yields. Interestingly, the protection of secondary amino acid
ester (L-Pro) occurred more smoothly to give better yields than other
primary amino cases (Scheme 1). The following catalytic hydro-
genation afforded corresponding N-phosphonyl amino acids with
free carboxylic acid groups.32 Our attention was next turned to the
protection of the hydroxy group of Tyr which is among the most
important aromatic amino acids5,33 so as to explore if the GAP
chemistry strategy is suitable for peptide synthesis. The simpler
achiral N-phosphonyl chloride was utilized for the protection which
was followed by catalytic hydrogenation to afford corresponding
N-phosphonyl tyrosine (Scheme 2). At this initial stage, this
concise and achiral protection is being continued for peptide
synthesis, although the GAP case of using chiral ones is also
showing promising potential in this laboratory.
The new GAP strategy is presented by the synthesis of biphalin
peptides that belong to the opioid pain killer series.33–37 The original
biphalin is a highly potent analgesic candidate but shows low
selectivity between d and m receptors.35–39 Its structural modifica-
tions are anticipated to generate new pain killers of high potency
and selectivity so as to reduce unwanted side effects. The use of a
phosphoric acid moiety for modifying peptides would meet the
above purpose. In fact, this strategy has not been well documented
yet. The present GAP peptide synthesis will concisely generate a
series of new biphalin derivatives and greatly accelerate opioid
peptide research.
Scheme 3 GAP synthesis of dipeptides and tripeptides.
It should be pointed out that that the GAP purification was
successfully applied for the preparation of I-3a to 3d. In this
preparation, the crude bis-coupling products were washed with
a co-solvent of EtOAc–hexanes (1 : 2, v/v) to give pure I-3a to 3d
as white solids. Similarly, the crude products of I-4a to 4d
obtained via the GAP procedure were washed with a co-solvent
of dichloromethane–hexanes (1 : 100, v/v) to give pure I-4a to 4d
as white solids as well.
N,N0-Di-phenyl-1,2-ethyldiamino O-phosphonyl attached bipha-
lins, I-1, were synthesized by reacting tripeptide I-2 (1.0 eq.) and
dimeric dipeptide I-3 (0.5 eq.) in dry dichloromethane (DCM) in the
presence of triethyl amine (Et3N) (1.1 eq.) and O-(benzotriazol-1-yl)-
N,N,N0,N0-tetramethyluronium tetrafluoroborate (TBTU) (1.1 eq.)
as the coupling reagent.12 The reaction mixture was cooled to 0 1C
before TBTU was added and kept stirring for 2 hours at this
temperature. The coupling reaction was completed after being
stirred at room temperature overnight. The reaction mixture
was diluted to double volume by DCM and washed with H2O,
0.5 M HCl, aq NaHCO3(sat.) and brine in order (Scheme 4).
The new O-phosphoryl biphalin 1 was generated by treating I-1
with trifluoroacetic acid (TFA) in DCM at r.t. for 24 hours as
monitored by 31P NMR. After the cleavage was finished, the resulting
mixture was evaporated before H2O and DCM (1 : 1, v/v) were added.
The resulting mixture was extracted with DCM five times to com-
pletely remove the N,N0-biphenylethylenediamine auxiliary from the
aqueous phase. Evaporating the aqueous phase to dryness afforded
pure O-phosphoryl biphalin 1 as a white solid in 88% yield. It is
noteworthy that the N,N0-biphenylethylenediamine auxiliary can be
recovered from DCM at a 95% recovery rate.
The GAP synthesis of dipeptide and tripeptide precursors to
biphalins is represented by the example shown in Scheme 3. The
coupling reactions were conducted under standard conditions of
using TBTU and Et3N in DCM at room temperature.32 The catalytic
hydrogenation to cleave the benzyl group gives O-phosphonyl
amino acid for the next coupling reaction. The individual N-t-Boc
amino acid benzyl esters of D-Ala, Gly and Phe were prepared by
following literature procedures.32 The dipeptide bridge precursors,
I-3a to 3d and I-4a to 4d, were also synthesized by following known
methods in standard coupling systems.38
Other O-phosphoryl biphalins 2–8 were conveniently
obtained under the same conditions and worked-up by follow-
ing the GAP washing without the use of column chromato-
graphy or re-crystallization. They were obtained as white solids
in good to excellent yields of 78% to 92%.
After we successfully synthesized the new biphalin analogs
with the phosphoryl moiety attached on the tyrosine ring
that would lead to interesting binding and biological profiles
in future, we further performed mild cleavage to give free
biphalins.32 We found that the treatment of O-phosphonyl
biphalins with calf intestinal phosphatase (CIP) in a buffer
solution of pH 7–8 at room temperature afforded free biphalin
Scheme 2 GAP protection of L-tyrosine benzyl ester and its deprotection. peptide in 87% of chemical yield.
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