Kilogram-Scale Synthesis of Osteogenic Growth Peptide (10-14) Using a Fragment Coupling Approach

Article abstract of DOI:10.1021/acs.oprd.5b00004

Kilogram-scale synthesis of a bioactive pentapeptide in solution by "3 + 2" fragment coupling strategy has been successively accomplished in the development of OGP (10-14), a minimal OGP-derived sequence that retains the full proliferative activity of the osteogenic growth peptide. The synthetic scheme, coupling conditions, and scaling-up of the process are systematically studied; the epimerization of the tripeptide fragment and pentapeptide are also evaluated.

Full text of DOI:10.1021/acs.oprd.5b00004

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Full Paper
Kilogram-Scale Synthesis of Osteogenic Growth
Peptide (10–14) Using a Fragment Coupling Approach
Teng Zhang, Zhenxing Chen, Yan Tian, Bin Han, Ning
Zhang, Wei Song, Zhulan Liu, Jinli Zhao, and Jian-Li Liu
Org. Process Res. Dev., Just Accepted Manuscript • Publication Date (Web): 18 Aug 2015
Downloaded from http://pubs.acs.org on August 18, 2015
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KilogramꢀScale Synthesis of Osteogenic Growth
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Peptide (10–14) Using a Fragment Coupling Approach
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Teng Zhang,†,‡ Zhenxing Chen,† Yan Tian,† Bin Han,† Ning Zhang,† Wei Song,‡ Zhulan Liu,† Jinli
Zhao,‡ and Jianli Liu*,†
†College of Life Science, Northwest University, Xi’an 710069, P. R. China
‡ Department of Polypeptide Engineering, Active Protein & Polypeptide Engineering Center of Xi'an
Hua Ao Li Kang, Xi’an 710054, P. R. China
*Corresponding Author: jlliu@nwu.edu.cn
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Table of Contents Graphic:
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1) DCC, HOSu/HONp
H
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H
Fmoc
N
COOH
COOH
Fmoc
N
2) two-solvent,
H2N
COONa
Gly
Tyr
Gly Phe Gly
Gly
Tyr Gly Phe
Gly
+
+
+
+
+
OR
O
O
O
H
N
H
N
N
H
N
OH
H
O
NH2
O
HO
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Abstract: Kilogramꢀscale synthesis of a bioactive pentapeptide in solution by “3+2” fragment coupling
strategy has been successively accomplished in the development of OGP (10ꢀ14), a minimal
OGPꢀderived sequence that retains the full proliferative activity of the osteogenic growth peptide. The
synthetic scheme, coupling conditions and scaling up of the process are systematically studied, the
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epimerization of the tripeptide fragment and pentapeptide are also evaluated.
Keywords: Osteogenic growth peptide; OGP(10ꢀ14); Liquidꢀphase synthesis; Fragment coupling
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Introduction
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The osteogenic growth peptide (OGP) is an endogenous tetradecapeptide physiologically present in
the blood circulation at micromolar concentrations.1 It is identical to the Cꢀterminal sequence of histone
H4 spanning residues 89ꢀ102 (ALKRQGRTLYGFGG). Native and synthetic OGP are mitogenic to
both osteoblastic and fibroblastic cells and can enhance osteogenesis and hematopoiesis in vivo. The
structureꢀactivity relationship revealed that Cꢀterminal pentapeptide OGP (10ꢀ14), with the sequence of
TyrꢀGlyꢀPheꢀGlyꢀGly, is the minimal fragment that retains the full OGPꢀlike activity.2 The analogues of
OGP (10ꢀ14) are used as bone anabolic agents and stimulators of hematopoiesis and will be carried out
clinical trials soon. The promising activity makes it also applicable for a wide range of uses, including
pharmaceuticals, intermediates, food additives and cosmeceuticals. Therefore, the demand for this
product has increased significantly in recent years.
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Though there are considerable number of research on the medicinal activity of OGPꢀpeptides, few
studies have investigated the synthetic method of these oligopeptide mimetics. Most of the described
synthesis have been based on the solidꢀphase chemistry.3 However, it is not the most costꢀeffective
method to produce kilograms or even tens of kilograms of peptides due to limitation of resin carriers.4
In some cases, the synthesis of OGP (10ꢀ14) was performed by enzyme catalysis or photochemical
method,5 but both of them only produce a small batch of products and cannot meet practical demands.
An excellent example of enfuvirtide,6 which is the first peptideꢀdrug substance that is produced in
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multiꢀtons, gave us incentive to explore a facile and costꢀeffective approach for large scale synthesis of
OGP (10ꢀ14). Alternatively, a solution process could provide considerable freedom regarding the choice
of synthetic schemes, protecting groups, solvent systems, monitoring measures and scaling up,7 these
properties suggest that it is possible to get substantial amounts of OGP (10ꢀ14) to meet the commercial
demands. Therefore, we set the goal of synthesizing OGP (10ꢀ14) in solution and investigating the
effect of different coupling conditions on the yield.
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Results and Discussion
Initially, we had planned to prepare the OGP(10ꢀ14) by the stepwise construction of the peptide
chain starting from the protected tyrosine moiety. Except for protected tyrosine, free glycine and
phenylalanine are used directly in this approach, showed lower costs and is suitable for industrialꢀscale
production. In our published patent,8 this scheme has satisfactorily performed on tens of grams
synthesis of the pentapeptide (Scheme 1). However, when it was applied to hundreds of gramꢀscale
preparation, some impurities gradually accumulated and the solubility of peptide chain decreased
substantially, especially after four links of amino acids were completed (4').
Tyr10
tBu
Fmoc
Fmoc
Fmoc
Fmoc
Fmoc
Fmoc
Fmoc
OH
tBu
tBu
tBu
tBu
tBu
tBu
tBu
tBu
tBu
OSu
Gly11
OH (1')
OSu
ONp
Phe12
OH (2')
(3')
OSu
Gly13
(4')
OH
Fmoc
Fmoc
H
ONp
Gly14
(5)
(6)
(7)
OH
OH
OH
H
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Scheme 1. Stepꢀwise construction of OGP (10ꢀ14).
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Further analyses show that Oꢀacylisourea tends to be the primary source of impurities (Figure 1), it is
one of the most reactive active species and could be rearranged into the Nꢀacylurea, which is
irreversible and consumes starting amino acid without generating product.9 In addition, due to excess
carbodiimide is used, traces of DCU are accumulated gradually and are difficult to be removed.
Although some of impurities can be removed by using column chromatography, this process is
undesirable, time consuming and limiting amplification. What's more, the purity of crude pentapeptide
obtained after all sideꢀchain deprotection was about 40–60%, which led to the overall yield of product at
25–35% seemed too low to achieve commercial objectives. Therefore, it is necessary to separate the
peptide chain into different fragments to decrease interference from impurities.
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O
O
R
H
N-acylurea
N
C N
O
N
O
O
C
R
O
O
HOSu
HONp
O
C
C
R
H
H
O
H
N
N
+
+
R
COOH
N
C
N
N C
N
O
Carbodiimide
O-acylisourea
DCU
R
C
O
NO2
Figure 1. Mechanism of active ester formation from an Oꢀacylisourea intermediate.
In selecting the fragments, both “3+2” and “2+3” convergence would avoid the need to purify
tetrapeptide (4') and shorten the synthetic cycle. But based on the consideration of atom economy, the
protected phenylalanine, which had to used as the starting amino acid in “2+3” strategy, is
comparatively expensive, while Boc2O is much less expensive in “3+2” strategy, the synthesis of
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BocꢀGlyꢀOH could be carried out under moderate conditions and facilitate industrial production.
Therefore, we decided to perform the synthesis by “3+2” fragment coupling strategy (Scheme 2).
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Tyr10
tBu
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Fmoc
Fmoc
Fmoc
Fmoc
Fmoc
Fmoc
Fmoc
H
OH
tBu
tBu
tBu
tBu
tBu
tBu
tBu
OSu
Gly13
Boc
Gly11
OH (1)
OH (4)
OSu
ONp
OSu
ONp
Boc
Boc
H
Gly14
Phe12
OH (2)
(5)
OH
OH (6)
(3)
OSu
OH
OH
OH
(7)
(8)
(9)
H
Scheme 2. “3+2” fragment condensation of OGP (10ꢀ14).
Due to the aforementioned difficulties with impurity and material costs, the peptide chain was
finally divided into two different sequences: FmocꢀTyr(tBu)ꢀGlyꢀPheꢀOH (2) and HꢀGlyꢀGlyꢀOH (6).
The intermediates were obtained as follows. The Nꢀterminal amines of tyrosine and glycine13 were
protected by the Fmocꢀ and Bocꢀ groups, respectively. The phenolic hydroxyl group of tyrosine was
protected by formation of its tꢀbutyl ether. The peptide bonds were facilitated by the use of “activated
ester”, which was obtained in a nearly stoichiometric yield and used in the next step without further
purification. The carboxyl groups of glycine11, phenylalanine and Gly14 were converted into their salt
forms before reacting with the relevant activated ester. The Bocꢀ protecting group was easily removed
with 50% trifluoroacetic acid in dichloromethane (v/v). After condensation of the phenylalanine and
glycine13 residues of two fragments, the protected pentapeptide (7) was obtained with a satisfactory
yield. Finally, the Fmocꢀ group was deprotected with 10% piperidine in dioxane (v/v) and the tBuꢀ
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Group was removed by 50% trifluoroacetic acid in dichloromethane (v/v). The crude product was
purified on cation exchange chromatography to yield the pure pentapeptide (9).
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“3+2” condensation is a crucial step in the amplifying synthesis of the protected pentapeptide (7).
Because the tripeptide ester (3) is freely soluble in organic solvent, while glycylglycine (6) was
hydrophilic, undissolving in the organic layer, the fragment condensation must be carried out in two
miscible liquids. Consequently, the proportion of mixed solvents directly affects the yield of the product
(7). From figure 2, the expected yield was not obtained when a lower percentage of water was added to
the reaction system, because water was essential for dissolving glycylglycine to ensure the homogeneity
of twoꢀsolvent system. The yield was satisfactory when the proportion of water was between 23% and
38% (v/v), but it decreased when the water content was greater 40% due to the increase of hydrolysis of
the tripeptide ester (3). Therefore, the optimal water proportion for the synthesis of protected
pentapeptide (7) was approximately 30% (v/v).
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40
30
20
10
0
0
10 20 30 40 50 60 70
Water proportion (V/V%)
Figure 2. The effects of water proportion on the yield of protected pentapeptide (7) in “3+2”
condensation.a
aMole ratio of tripeptide ester (3), glycylglycine, and sodium bicarbonate was about 1:1.2:1.2. Solvent
system is a mixture of water and tetrahydrofuran. All reactions were performed at 25°C for 10 hours
and analyzed by HPLC using a Kromasil C18 column (4.6 mm diameter × 250 mm) with a linear
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gradient from 10% to 90% aqueous acetonitrile (0.1% trifluoroacetic acid) over 30 min at a flow rate of
1.0 mL/min and detected at 215 nm.
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The aminolysis of peptide ester has been demonstrated to be a general baseꢀcatalyzed reaction,
however, the hydrolysis of active esters is performed under the same condition.10 Therefore, the type
and the amount of base in the solvent system of “3+2” condensation were also necessary to be
investigated. Figure 3 shows that the amount of base had substantial effects on the yield of protected
pentapeptide (7). Compared with the 10% yield of the free base, the synthetic yields promoted by the
general base were measurably increased. When the amount of base was added to 1.0ꢀ1.5 molar quantity,
NaHCO3, TEA and DIEA resulted in a high yield, whereas Na2CO3 generated the lowest yield (30%)
followed by DEA (40%). This result was due to the strong basicity that makes the hydrolysis of the
ester faster than the coupling reaction, especially with a large excess of base. To be environmentally
friendly, sodium bicarbonate was often used as a additional base with 1.2 equivalents in our research.
NaHCO
3
TEA
DIEA
DEA
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70
60
50
40
30
20
10
Na2CO3
0.0
0.5
1.0
1.5
2.0
2.5
Base (eq)
Figure 3. The effect of the type and amount of base on the yield of protected pentapeptide (7).a
aMole ratio of tripeptide ester (3) and glycylglycine was about 1:1.2; Volume ratio of water and
tetrahydrofuran was about 2:5; The determination method was the same as described in Fig. 2.
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A major problem in the repetitive process is the separation of the peptide products from liquid
mixtures. We have envisioned that this problem could be easily solved by reacting the fullꢀprotected
peptide ester with excess free amino acid to produce hydrophobic peptide acids products (Figure 4),
which could be precipitated by addition of 10% aqueous solution of citric acid (pH 3) after remove the
organic phase by reduced pressure distillation. While excess amino acids, base and other hydrophilic
byproducts remain soluble in the aqueous solution and can be easily removed by filtration. Using this
protocol, a series of peptide intermediates were quickly prepared in high yields with excellent purity.
Additionally, a low coupling yield would be obtained if 1,4ꢀdioxane was used as a solvent during the
kilograms preparation of FmocꢀTyr(tBu)ꢀGlyꢀPheꢀOH (2). It is because 1,4ꢀdioxane can easily form
azeotropes with water which makes it hard to be distilled, result in the uncomplete precipitation of the
tripeptide and ultimately lead to the loss of product during the postꢀtreatment.
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Figure 4. Schematic representation of the separation of the hydrophobic products from two solvents.
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Under aboveꢀmentioned optimal conditions, the production time for each fragment is less than five
days. At present, pilot scale of operation provides each fragment in 2 to 5kg batches. The cycle time
from building the fragments to completion of the analytical and preparative scale purification on the
pentapeptide (9) is approximately half a month. The efficiency of fragment coupling is approximately
70ꢀ80% and the overall yield of pure product is about 45%. Therefore, “3+2” fragment coupling
approach could satisfy kilogramꢀscale needs of OGP(10ꢀ14) and support lateꢀstage sustained
development.
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A potential problem in solution phase fragment condensation could be the epimerization leading to
diastereomers. In our research, several positive parameters were applied to minimize racemization
during peptide coupling reactions. A key issue is the use of an appropriate additive and condensing
reagent. HOSu is a relatively stable additive to HONp, while HONp is more prone to peptideꢀcoupling
than other additives. Both of them are less reactive than the Oꢀacylisourea toward peptide bond
formation (Figure 1). Therefore, except the optically inactive glycine residues, which can be activated
as ꢀONp or ꢀOSu at room temperature, tyrosine and phenylalanine must be activated as ꢀOSu in ice bath.
Although such procedures could possibly reduce racemization during the activation reaction, it is
necessary to further ensure the absolute configuration of the peptide chain by HPLC chiral analysis.
Therefore after replace Lꢀamino acid with Dꢀamino acid, we successfully obtained various epimers of
the tripeptide and pentapeptide. By comparing the chromatogram of desired peptide with the
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resynthesized epimers for “3+2” fragment coupling, both tripeptide (2, 17.418min) and pentapeptide (9,
40.00min) show a single peak with the different retention time of their isomers in the chromatograms
(Figure 5). These results show that the epimerization at the carboxyl terminus of tripeptide during the
coupling reaction is not observed and the configuration of pentapeptide is maintained under the optimal
synthetic process, which also indicated that “3+2” condensation is an excellent method for successfully
obtaining OGP(10ꢀ14) with high optical purity.
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Figure 5. Chiral analysis of epimerization for “3+2” fragment coupling step.
Conclusions
An efficient process for kilogramꢀscale synthesis of OGP(10ꢀ14) has been developed in solution by
“3+2” fragment coupling approach using free amino acids for each coupling cycle. Compared with our
original stepwise protocol, the yield obtained by fragment condensation was significantly improved
(from 25–35% to 45%) and the postꢀprocessing was more convenient. Furthermore, some important
factors such as route selection, aqueous proportion, base quantity, postꢀtreatment and epimerism
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evaluation were optimized for “3+2” fragment coupling step. All results indicate that “3+2” route is
a reasonably appropriate way for the synthesis OGP(10ꢀ14) on kilogramꢀscale with good yield and high
optical purity. Our study not only provides a rapid, economic and environmental friendly method for the
synthesis of OGP (10ꢀ14) on a large scale but also gives some useful information for the liquid phase
synthesis of peptides, especially for oligopeptides.
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Experimental Section
Thinꢀlayer chromatograms are run on GF254 plates in the following systems:
a1, ethyl acetateꢀnꢀhexaneꢀacetic acid (10:10:1);
a2, ethyl acetateꢀnꢀhexaneꢀacetic acid (10:5:1)
a3, ethyl acetateꢀnꢀhexaneꢀacetic acid (15:5:1);
b, ethyl acetateꢀnꢀhexaneꢀmethanolꢀacetic acid (10:10:1:1);
c, nꢀbutyl alcoholꢀacetic acidꢀwaterꢀ0.05 g/ml Nin (8:3:1:1).
Analysis conditions of high performance liquid chromatography are as follows:
d: A(0.1% TFA/ACN), B(0.1% TFA/H2O), 30 min 10ꢀ90%;
e: A(0.1% TFA/ACN), B(0.1% TFA/H2O), 30 min 50ꢀ90%;
f: A(0.1% TFA/ACN), B(0.1% TFA/H2O), 30 min 55ꢀ90%.
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TLC measurements were performed on silica gel GF254 plates (Qingdao Haiyang Chemical Co., Ltd,
China). Melting points were determined using a XTꢀ4 melting point apparatus with temperature
uncorrected (Beijing Tech Instrument Co., LTD, China). Compounds were visualized by irradiation
with UV light or by treatment with 0.05 g/ml ninhydrin in ethanol or potassium iodide reagent. Optical
rotations were determined using an Autopol II automatic polarimeter for solution in methanol.
Elemental analysis was performed by Vario EL III CHNOS analyzer. 1HꢀNMR and FTꢀIR spectra were
recorded on a Bruker Avanceꢀ400 MHz and Perkin Elmerꢀspectrum instrument, respectively. MS were
obtained with a LTQꢀXL apparatus. HPLC was performed by Hitachi L2000 instrument equipped with
a diode array detector (Lꢀ2455). Chiral analysis of tripeptide and pentapeptide were carried out
respectively on CHIRALPAK ADꢀH (0.46cm×25cm× 5ꢁm) and CHIRALPAK ZWIX(+)
(0.40cm×15cm×3ꢁm) columns using Shimadzu LC 20 with UV detector SPDꢀ20A. The crude
pentapeptide was purified by cation exchange chromatography using a SP sepharose fast flow column
(GE Healthcare), and desalted by reversed phase liquid chromatography on a SBC MCI GEI column
(SciꢀBioꢀChem Co.Ltd.), finally lyophilized with a Biocool lyophilizer. Saline refers to a saturated
sodium chloride solution. The Lꢀamino acids (protected or free) were obtained from GL Biochem Ltd.
(Shanghai, China). Other reagents were provided by SigmaꢀAldrich. The organic solvents were
commercially available product (Sinopharm Chemical Reagent Co., Let, China) and were dried with
anhydrous sodium sulfate before application. During the coupling reaction, material ratio was strictly
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controlled and evaporation was performed in vacuo below 37°C. Most of the solvents after distillation
can be reused.
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Abbreviations: acetonitrile (ACN), tertꢀbutyloxycarbonyl (Bocꢀ), diꢀtertꢀbutylpyrocarbonate (Boc2O),
N, N'ꢀdicyclohexyꢀ carbodiimide (DCC), dichloromethane (DCM), N, N'ꢀdicyclohexylurea (DCU),
diethylamine (DEA), diisopropylethylamine (DIEA), dimethylformamide (DMF), Ethyl acetate
(EtOAc), 9ꢀFluorenylmethoxycarbonyl (Fmoc),
nꢀhexane (HEX), pꢀnitrophenol (HONp),
Nꢀhydroxysuccinimide (HOSu), tertꢀButyl (tBu), triethylamine (TEA), trifluoroacetic acid (TFA),
Tetrahydrofuran (THF), sodium carbonate (Na2CO3), sodium bicarbonate (NaHCO3), ninhydrin (Nin),
Nꢀmethylmorpholine (NMM).
Fmoc-Tyr(tBu)-Gly-OH (1)
FmocꢀTyr(tBu)ꢀOH (2.3 kg, 5mol) and HOSu (0.69 kg, 6mol, 1.2eq) were dissolved in dry 1,4ꢀdioxane
(20 L) and stirred in an iceꢀbath. A solution of DCC (1.24 kg, 6mol, 1.2eq) in dry 1,4ꢀdioxane (5 L) was
slowly added. The mixture stirred in ice bath for 4h, and then at ambient temperature for further 8h.
After cooling in refrigerator for 1h, the precipitate of DCU was removed to leave the FmocꢀTyr(tBu)ꢀ
OSu in 1,4ꢀdioxane as a colorless transparent liquid, TLC: a1, Rf=0.4, which was used without further
purification.
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Glycine (0.45 kg, 6mol, 1.2eq) and NaHCO3 (0.5 kg, 6mol, 1.2eq) was dissolved in H2O (10 L), The
aqueous solution was added to the solution of FmocꢀTyr(tBu)ꢀOSu (get by above step) in 1,4ꢀdioxane
(25 L) in 2h. The mixture was stirred at about 25°C until the active ester disappeared by TLC (about
8h). The solution was concentrated to a small volume (6 L) and treated with ice ether (8 L). At the same
time, the pH value was adjusted close to 2ꢀ3 by addition of 10% aqueous solution of citric acid. The
white solid was precipitated, filtered and washed with cold water. After dried under vacuum,
FmocꢀTyr(tBu)ꢀ GlyꢀOH (2.42 kg, 93.7%) was yield as white pulverous solid, m.p. 108.9～110.9°C,
TLC: a1, Rf=0.2, HPLC: d, 25.63min, purity 89.38% , Anal. Calc. for C30H32N2O6: C, 69.75; H, 6.24; N,
5.42. Found: C, 69.55; H, 6.31; N, 5.38. ESIꢀMS for [M] calcd: 516.58, found: 540.87 ([M+Na]+,
100%); 521.81 (70%). [α]D20.5ꢀ35.51 (c=1.0, CH3OH). IR (KBr): 3679.9, 3331.0, 2973.0, 1676.0,
1525.6, 1237.5, 1032.6, 738.1, 616.4 cmꢀ1. 1H NMR (400 MHz, MeOD) δ 7.81 (d, J = 7.5 Hz, 2H), 7.62
(t, J = 7.7 Hz, 2H), 7.41 (t, J = 7.4 Hz, 2H), 7.33 (t, J = 7.1 Hz, 2H), 7.19 (d, J = 8.3 Hz, 2H), 6.86 (t, J
= 9.8 Hz, 2H), 4.42 (dd, J = 10.1, 4.3 Hz, 1H), 4.30 (dd, J = 10.1, 7.0 Hz, 1H), 4.14 (dt, J = 13.7, 7.1
Hz, 2H), 3.95 – 3.81 (m, 2H), 3.49 (dd, J = 12.8, 5.8 Hz, 1H), 3.22 (dd, J = 13.7, 4.4 Hz, 1H), 2.88 –
2.73 (m, 2H), 1.40 – 1.09 (m, 9H).
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Fmoc-Tyr(tBu)-Gly-Phe-OH (2)
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FmocꢀTyr(tBu)ꢀGlyꢀOH (2.42 kg, 4.68mol) was dissolved in dry THF (25 L). HOSu (0.65kg, 5.62mol,
1.2eq) or HONp (0.78 kg, 5.62mol, 1.2eq) were added and stirred at ambient temperature. A solution of
DCC (1.25 kg, 6.05mol, 1.3eq) in dry THF (5 L) was added and the mixture stirred for 10h. After
cooling for 1h, the precipitate of DCU was filtered off and the solution was used to the next step
directly, TLC: a1, Rf=0.3/0.4.
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A suspension of Lꢀphenylalanine (1 kg, 6.08mol, 1.3eq) in H2O (15 L) was mixed with NaHCO3 (0.51
kg, 6.08mol, 1.3eq), the mixture was dropꢀwise added to the solution of FmocꢀTyr(tBu)ꢀGlyꢀOSu in
THF (30 L). The reaction mixture was stirred at about 25°C until the solution become clear and then for
further 5h. The solvent was evaporated, the residue was distributed by H2O (30 L), EtOAc (10L) and
light petroleum (10 L). After organic layer removed, the aqueous phase was acidified to a pH 2ꢀ3 with
10% citric acid aqueous solution and further extracted with EtOAc (3×15 L). The combined organic
extracts were washed with saline (2×10 L), dried with magnesium sulfate and evaporated. After
ultrasonic extractions with ether (10 L), FmocꢀTyr(tBu)ꢀGlyꢀPheꢀOH (2.76 kg, 89.12%) was obtained
as a white crystal, m.p. 136.7～140.1°C, TLC: a2, Rf=0.2, HPLC: e, 22.14min, purity 87.52%, Calc. for
C39H41N3O7: C, 70.57; H, 6.23; N, 6.33. Found: C, 70.33; H, 6.31; N, 6.27. ESIꢀMS for [M] calcd:
663.75, found: 686.48 ([M+Na]+, 40%); 686.41 ([M+Na]+, 40%); 413.53 (100%). [α]D20.5ꢀ20.46 (c=1.0,
1
CH3OH). IR (KBr): 3292.6, 3193.3, 2956.4, 1660.0, 1541.6, 1400.7, 1301.5, 616.43 cmꢀ1. H NMR
(400 MHz, DMSO) δ 8.52 (t, J = 5.7 Hz, 1H), 8.17 (dd, J = 17.4, 7.2 Hz, 2H), 7.87 (d, J = 7.5 Hz, 2H),
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7.65 (t, J = 7.6 Hz, 3H), 7.40 (t, J = 7.4 Hz, 3H), 7.34 – 7.27 (m, 3H), 7.25 (d, J = 4.1 Hz, 3H), 7.17 (d,
J = 8.5 Hz, 2H), 4.56 (s, 1H), 4.19 (d, J = 11.2 Hz, 1H), 4.16 – 4.06 (m, 3H), 3.89 – 3.76 (m, 3H), 3.05
(dd, J = 13.9, 4.2 Hz, 1H), 2.95 (d, J = 9.8 Hz, 1H), 2.79 – 2.68 (m, 2H), 1.41 – 0.85 (m, 9H).
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Fmoc-Tyr(tBu)-Gly-Phe-OSu (3)
To a stirred solution of FmocꢀTyr(tBu)ꢀGlyꢀPheꢀOH (2.76 kg, 4.16mol) and HOSu (0.67 kg, 5.83mol,
1.4eq) in dry THF (25 L), a solution of DCC (1.21 kg, 5.83mol, 1.4eq) in dry THF (5 L) was added
slowly under iceꢀcooling within 3h, then the solution was stirred overnight at ambient temperature.
After cooling for 1 h, the resulting precipitate was filtered and filtrate was used further reaction without
purification. TLC: a2, Rf=0.3.
Boc-Gly-OH (4)
The mixture of glycine (1 kg, 13.32mol) and NaHCO3 (1.12 kg, 13.32mmol) in H2O (15 L) was added
slowly to the solution of Boc2O (3.06 L, 13.32mol) in THF (10 L) and stirred at ambient temperature
for 5h. When TLC indicated the absence of starting material, the residue was evaporated and distributed
by H2O (20 L), EtOAc (7 L) and light petroleum (7 L). The organic layer was removed and the aqueous
phase adjusted to pH 2ꢀ3 with 10% citric acid solution. After extracted with EtOAc (3×10 L), the
aqueous layer was separated and the combined organic extracts were washed with saline (2×10 L), dried
with magnesium sulfate and evaporated to give a white crystal (1.98 kg, 84.85%). m.p.85.7～86.5°C,
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TLC: a1, Rf=0.2, HPLC: d, 10.40min, purity 94.7%, Calc. for C7H13NO4: C, 47.99; H, 7.48; N, 8.00.
Found: C, 47.40; H, 7.02; N, 8.31. ESIꢀMS for [M] calcd: 175.18, found: 198.78 ([M+Na]+, 100%);
339.24 (100%). IR (KBr): 3340.6, 3116.5, 2982.0, 2732.4, 2534.0, 1746.4, 1672.8, 1538.4, 1413.5,
1202.67, 1058.2, 958.97, 856.54, 587.65cmꢀ1.
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Boc-Gly-Gly-OH (5)
BocꢀGlyꢀOH (1.98 kg, 11.3mol) and HONp (1.73 kg, 12.43mol, 1.1eq) or HOSu (1.43 kg, 12.43mol,
1.1eq) were dissolved in dry 1,4ꢀdioxane (25 L), a solution of DCC (2.8 kg, 13.56mol, 1.2eq) in dry
1,4ꢀdioxane (10 L) was added and the mixture stirred for 8ꢀ10h. After cooling for 2h, the precipitate
was removed, the filtrate was used directly for next reaction without purification, TLC: a1, Rf=0.4, 0.3.
The mixture of glycine (0.94 kg, 12.43mol, 1.1eq) and NaHCO3 (1.05 kg, 12.43mol, 1.1eq) in H2O (10
L) was slowly added to the solution of BocꢀGlyꢀONp (get by above step) in 1,4ꢀdioxane (25 L). The
solution was stirred at about 25°C for 7 h, then evaporated under reduced pressure. The residue was
acidified to a pH 2ꢀ3 with 10% citric acid and extracted with EtOAc (3×20 L). The organic phase was
successively washed with saline (20 L), dried, and evaporated to give BocꢀGlyꢀGlyꢀOH (2.37 kg,
90.3%) as a white schistose crystals. m.p.123.5～125.5°C, TLC: a3, Rf=0.2, HPLC: e, 6.53min, purity
93.5%, Calc. for C7H13NO4: C, 46.55; H, 6.94; N, 12.06. Found: C, 45.40; H, 6.02; N, 14.13. ESIꢀMS
for [M] calcd: 232.23, found: 255.03 ([M+Na]+, 100%); 233.06 ([M+H], 20%).
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Glycylglycine (6)
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TFA (15 L) was added to a solution of BocꢀGlyꢀGlyꢀOH (2.37 kg, 10.2mol) in DCM (15 L) with
stirring. After starting material run out indicated by TLC (a3), the solution was evaporated to a small
volume (3 L) and treated with ice ether (15 L). The resulting solid was collected to give HꢀGlyꢀGlyꢀOH
(1.15 kg, 84.9%) as a white solid. m.p.220.7～222.1°C, TLC: c, Rf=0.2, HPLC: d, 4.35min, purity
95.7%, Calc. for C4H8N2O3: C, 36.36; H, 6.01; N, 21.2. Found: C, 36.01; H, 5.92; N, 21.31. ESIꢀMS for
[M] calcd: 132.05, found: 132.76(100%); 132.69 (85%). IR (KBr): 3267.0, 3199.7, 2937.2, 1455.1,
1099.2, 1023.0, 930.16, 718.9, 622.86 cmꢀ1. 1H NMR (400 MHz, D2O) δ 3.77 (d, J = 2.6 Hz, 1H), 3.74
(d, J = 2.5 Hz, 1H), 3.69 (d, J = 8.6 Hz, 2H), 3.67 – 3.62 (m, 2H), 3.56 (dd, J = 11.6, 5.9 Hz, 2H).
“3+2” condensation (7)
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The mixture of glycylglycine (0.66 kg, 4.99mol, 1.2eq) and NaHCO3 (0.42 kg, 4.99mol, 1.2eq) in H2O
(13 L) was added to a solution of FmocꢀTyr(tBu)ꢀGlyꢀPheꢀOSu (4.16mol) in THF (30 L) and stirred for
8h at ambient temperature. After the tripeptide ester could not be detected by TLC, the solution was
evaporated and the residue was adjusted to pH 2ꢀ3 with 10% citric acid. The precipitate was filtered and
washed with cold water. The crude product was triturated with ice ether to give the protected
pentapeptide (2.43 kg, 75.1%) as a white solid. m.p.105.0～107.8°C. TLC: b, Rf=0.2, HPLC: e,
15.46min, purity 95.3%, Calc. for C43H47N5O9: C, 66.39; H, 6.09; N, 9.00. Found: C, 66.34; H, 6.11; N,
9.02. ESIꢀMS for [M] calcd: 777.58, found: 777.92 (100%); 800.32 ([M+Na]+, 85%); 876.42 (80%).
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[α]D20.5ꢀ27.13 (c=1.0, CH3OH). IR (KBr): 3705.5, 3286.2, 3084.5, 1672.8, 1644.0, 1551.2, 1202.3,
1135.0, 699.68 cmꢀ1. 1H NMR (400 MHz, DMSO) δ 8.41 (s, 1H), 8.19 (d, J = 19.8 Hz, 2H), 7.87 (d, J =
7.1 Hz, 2H), 7.64 (d, J = 7.7 Hz, 3H), 7.50 – 7.34 (m, 3H), 7.35 – 7.26 (m, 3H), 7.17 (d, J = 8.3 Hz, 3H),
6.79 (d, J = 8.3 Hz, 2H), 4.53 (s, 1H), 4.21 (s, 1H), 4.17 – 4.01 (m, 3H), 3.94 – 3.78 (m, 2H), 3.75 (dd, J
= 13.6, 5.5 Hz, 2H), 3.62 (s, 1H), 3.17 (d, J = 5.1 Hz, 2H), 3.05 (d, J = 9.1 Hz, 2H), 2.95 (d, J = 10.2 Hz,
2H), 2.82 – 2.65 (m, 2H), 1.27 (dd, J = 39.1, 28.2 Hz, 9H).
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H-Tyr(tBu)-Gly-Phe-Gly-Gly-OH (8)
FmocꢀTyr(tBu)ꢀGlyꢀPheꢀGlyꢀGlyꢀOH (2.43 kg, 3.13mol) was added to a solution of piperidine in
1,4ꢀdioxan (10% v/v, 30L) and stirred for 1h. After TLC (b) indicated the absence of starting material,
the solution was evaporated to dryness. The residue was distributed by H2O (30 L) and DCM (3×10 L).
The organic layer was removed and the aqueous phase evaporated to give HꢀTyr(tBu)ꢀGlyꢀPheꢀGlyꢀGly
ꢀOH (1.68kg, 96.47%) as a gummy solid. TLC: c, Rf=0.3, HPLC: e, 14.32min, purity 87.5%, ESIꢀMS
for [M] calcd: 555.62, found: 555.72 (20%); 555.00 (10%); 496.23 ([Mꢀ59], 100%).
H-Tyr-Gly-Phe-Gly-Gly-OH (9)
HꢀTyr(tBu)ꢀGlyꢀPheꢀGlyꢀGlyꢀOH (1.68 kg, 3.02mol) was dissolved in a solution of TFA in H2O (95%
v/v, 30L) and stirred for 1h. The solution was concentrated to a small volume (2 L) and ice ether was
added (9 L), then the solvent decanted and the residue triturated with more ether (5 L). The resulting
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solid was collected and dried under vacuum to yield the crude pentapeptide with white solid (1.43 kg,
94.79%, purity 80ꢀ85%). The crude product was dissolved in the minimum volume of 0.02mol/L acetic
acidꢀsodium acetate aqueous buffer (pH 4, K=0.36) and purified by a column (10×50cm) of SP
sepharose which had been equilibrated previously with the same buffer. The column was eluted with
0.02mol/L acetic acidꢀsodium acetate with 1mol/L sodium chloride aqueous buffer (pH 4) from 40% to
60% for 60min at a flow rate of 100mL/min and the effluent monitored by U.V. absorption at 254nm.
Fractions were pooled and evaporated. Desalting is done on a SBC MCI GEI column (10×50cm) with
buffer A (0.25% TFA/ACN) and B (0.25% TFA/H2O). The column was eluted with buffer A from
15%ꢀ25% for 30min at a flow rate of 50mL/min and monitored as in the case of cation exchange
chromatography. The fractions containing the major component were pooled and evaporated, which
was further lyophylized at ꢀ50°C for 3 days to obtain chromatographical homogeneous TryꢀGlyꢀPheꢀ
GlyꢀGly (1.14 kg) as a white crystalline solid. The purity is more than 97%, the overall yield is 45.64%.
m.p.176.8～180.0°C. TLC: c, Rf=0.2. HPLC: d, 11.34min. Calc. for C22H29N5O7: C, 57.71; H, 5.85; N,
14.02. Found: C, 57.73; H, 5.80; N,14.01. ESIꢀMS for [M] calcd: 449.51, found: 500.51 ([M+H],
100%); 500.45 (90%). IR (KBr): 3293.4, 3080.8, 2933.3, 1666.3, 1638.8, 1519.1, 1448.1, 1241, 1202,
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1139.8, 839.38, 723.63, 699.77 cmꢀ1. [α]D20.5+24.13 (c =0.65, MOH). H NMR (400 MHz, CD3OD) δ
6.55 – 6.38 (m, 5H), 6.28 (d, J = 8.4 Hz, 2H), 5.97 (d, J = 8.4 Hz, 2H), 3.79 (dd, J = 8.9, 5.7 Hz, 1H),
3.19 (dd, J = 12.1, 7.5 Hz, 2H), 3.14 (d, J = 10.2 Hz, 3H), 3.02 (d, J = 16.8 Hz, 1H), 2.90 (d, J = 16.4
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Acknowledgment
We gratefully acknowledge the National Natural Science Foundation of China (No. 20872118,
30070905), Innovative Research Team in University (No. IRT1174), the Foundation of the Education
Department of Shaanxi Province (12JK1010) and Technology innovation fund of Xi'an City
(No.CX13120) for financial support. We also thank R & D Center of Shaanxi EastꢀStar Biochemical
Technology Co., Ltd. for technical assistance.
Supporting Information Available
Experimental details and spectroscopic data for all peptides described here.
References and Notes
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