The synthesis of immunomodulating peptide alloferon, the active principle of antiviral drug allokine-alpha

Article abstract of DOI:10.1134/S1068162006020051

Two variants of the synthesis of tridecapeptide alloferon, the active principle of antiviral preparation allokine-alpha, were developed on the basis of fragment condensation in solution or on the Merrifield resin. The solid phase variant of the synthesis

Full text of DOI:10.1134/S1068162006020051

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2006, Vol. 32, No. 2, pp. 136–145. © Pleiades Publishing, Inc., 2006.
Original Russian Text © M.V. Sidorova, A.S. Molokoedov, E.V. Kudryavtseva, M.I. Baldin, D.A. Frid, M.V. Ovchinnikov, Zh.D. Bespalova, V.N. Bushuev, 2006, published in
Bioorganicheskaya Khimiya, 2006, Vol. 32, No. 2, pp. 151–160.
The Synthesis of Immunomodulating Peptide Alloferon,
the Active Principle of Antiviral Drug Allokine-alpha
M. V. Sidorovaa,1, A. S. Molokoedova, E. V. Kudryavtsevab, M. I. Baldinb, D. A. Fridb,
M. V. Ovchinnikova, Zh. D. Bespalovaa, and V. N. Bushueva
a Cardiological Research and Production Association, Federal Agency on Healthcare and Social Development,
Tret’ya Cherepkovskaya ul. 15a, Moscow, 121552 Russia
b ZAO Peptide Synthesis, Moscow, Russia
Received July 11, 2005; in final form, July 13, 2005
Abstract—Two variants of the synthesis of tridecapeptide alloferon, the active principle of antiviral preparation
allokine-alpha, were developed on the basis of fragment condensation in solution or on the Merrifield resin. The
solid phase variant of the synthesis was shown to be more technological; it allows the preparation of the product
at a higher total yield (40% vs. 17% for conventional synthesis in solution from the starting derivatives of the
C-terminal dipeptide). The by-products formed during the synthesis of alloferon were identified.
Key words: alloferon, peptide synthesis, fragment condensation
DOI: 10.1134/S1068162006020051
INTRODUCTION
The SPS scheme recently suggested for alloferon
uses the peptide chain elongation by one amino acid
residue using the Fmoc-technology and the subse-
quent two-step purification of product by HPLC [1].
Most likely, it is not interesting from the practical
point of view, because is expensive and, besides, can-
not provide the obtaining of a homogeneous product
in tens gram quantities. The purification of crude SPS
product by HPLC in the case of a very hydrophilic
alloferon represents an independent problem that
demands a careful development of the chromato-
graphic conditions. The synthesis of alloferon at gram
scale is now a topical task, because alloferon is
already applied in medicine.
Several tens of peptide preparations used in medi-
cine for therapeutic or diagnostic purposes are now
known.2 Tridecapeptide alloferon is a rather new pep-
tide preparation that possesses an immunomodulating
activity [1]. It increases the formation of endogenous
interferons and provides the development of a cascade
of protective reactions mediated by cytokines. Allof-
eron could find application in medical practice for the
correction of interferon system and the system of nat-
ural killers, as well as for the treatment of viral, fun-
gal, and oncological diseases connected with the defi-
ciency of interferons or cytotoxic lymphocytes. Cur-
rently, alloferon is the active principle of the antiviral
preparation allokine-alpha, which is used for the treat-
ment of diseases caused by viruses of herpes simplex
of I and II types and hepatitis B.
Our research is devoted to the development of a
convenient synthetic scheme of alloferon preparation
upon a 100 g scale. A summary of the results of this
work has been represented on the II Russian sympo-
sium on the chemistry and biology of peptides [2] and
in patent [3].
1
Corresponding author; phone: +7 (495) 414-6716; e-mail:
peptide@cardio.ru.
2
In addition to those recommended by the IUPAC–IUB commis-
sion, we used the following abbreviations: DCM, dichloro-
methane;
DIEA,
N,N'-diisopropylethylamine;
HOBt,
1-hydroxybenzotriazole; HONb, N-hydroxy-5-norbornene-2,3-
dicarboximide; HONp, p-nitrophenol; MALDI MS, matrix-
assisted laser desorption ionization mass spectrometry; NMM,
N-methylmorpholine; (P), styrene–1% divinylbenzene copoly-
mer; and SPS, solid phase synthesis.
RESULTS AND DISCUSSION
Alloferon represents tridecapeptide of the following
structure:
136

THE SYNTHESIS OF IMMUNOMODULATING PEPTIDE ALLOFERON
137
H-His-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH
where arrows show the breakdown of its amino acid a decisive significance in the synthesis of alloferon that
sequence to fragments during the synthesis.
has four His residues in its sequence. Therefore, we
used a temporary (only during condensation) protection
of the His imidazole rings, namely, the Boc group,
which was subsequently split off by trifluoroacetic
acid. The tert-butyl group was used for the protection of
Ser hydroxyl function.
The presence of five Gly residues in alloferon mol-
ecule together with a repeating fragment His-Gly gives
wide opportunities for the use in the synthesis of the
fragment condensation with the conventional break-
down of the amino acid chain to blocks at the Gly resi-
dues. It is known that the fragment condensation both
in conventional and in solid phase variant as a rule pro-
vides the obtaining of product of higher quality than the
step-by-step synthesis [4, 5]. In addition, the purifica-
tion of peptides obtained by block synthesis is substan-
tially easier. Therefore, we have separated the peptide
sequence to short fragments containing the C-terminal
Gly residue. The Val and Gln residues were not
included in the fragment structures. The fragments cho-
sen, the repeating dipeptide block His-Gly and the
N-terminal pentapeptide His-Gly-Val-Ser-Gly, were
presumed to be synthesized in solution.
The Nα-Boc protected fragments, peptides (IV) and
(XVII) were obtained using the method of active esters
(see the scheme). During the synthesis of these pep-
tides, the glycine carboxyl group was protected by salt
formation. Note that fragments (IV) and (XVII) and
intermediates of their synthesis are crystal substances;
they are obtained in sufficiently high yields practically
on any scale.
It was interesting to us to compare the results of con-
ventional and solid phase variants of the assembly of
alloferon molecule, as each of these approaches has
The cheaper Boc-methodology was substituted for their own advantages and drawbacks. The coupling of
the Fmoc-methodology [1]. The problem of optimum fragments in solution seems to be more economic,
protection of histidine imidazole remains disputable since it does not require the application of great vol-
until now [6, 7]. The majority of groups used for this umes of solvents and two- to threefold excesses of acy-
purpose have some restrictions, one of which is incom- lating agents. The coupling of fragments on polymer
pleteness of the splitting off of the corresponding pro- seems to be more technological due to the simplicity of
tective group after the end of synthesis. This factor has isolation of intermediates, a possibility of the process
His
Gly
Val
Ser
Gly
His
Gly
Gln
His
Gly
Val
His
Gly
(b) (a)
Boc
Boc
Boc ONp
ONa
Boc
(1)
ONSu H
(P) -OBzl (I)
(P) -OBzl (II)
H
Boc
Boc
H
Boc
(2)
(IV) Boc
(P) -OBzl (III)
(P) -OBzl (VI)
OH
Boc
Boc
(1)
(1)
(V) Boc
ONp
But
ONb H OH
But
(2)
(P) -OBzl (VII)
(P) -OBzl (VIII)
Z
Z
Boc
ONp
Boc
Boc
H
OH (XIII)
But
But
(3)
(2)
(P) -OBzl (IX)
(P) -OBzl (X)
Z
ONSuH
OH (XIV)
Boc (V)
Boc
(1)
ONp H
(2)
(XV) Z
OH
OH
OH
Boc
Boc
H
But
But
Boc
(XVI)
(V) Boc
ONp
H
(XVII)
(P) -OBzl (XI)
(P) -OBzl (XII)
Boc
Boc
(1)
Boc
But
(4)
Boc
H
(P) -OBzl (XVIII)
(P) -OBzl (XIX)
(1)
(5)
(XX)
OH
H
The scheme of alloferon synthesis according to (a) the conventional and (b) solid phase variant of fragment coupling. (P) the Mer-
rifield polymer. (1) TFA, (2) HOBt, (3) H /Pd, (4) HONp + DCC/HOBt, (5) H /Pd in the case of synthesis in solution or trifluo-
2
2
romethanesulfonic acid in TFA in the case of SPS.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 2 2006

138
SIDOROVA et al.
Table 1. Homogeneity of SPS intermediates of alloferon analyzed by HPLC*
Amino acid sequence of the SPS
product obtained from peptidyl
polymer
Target peptide
Impurities
Pepti-
dyl-(P)
Impurity
structure
RT, min
content, %
RT, min
content, %
(VIb) H-Val-His-Gly-OH
(VIIIb) H-His-Gly-Val-His-Gly-OH
7.5
10.6
12.2
98.8
98.7
95.0
–
–
<2
<2
2.5
Not determined
Not determined
(Xb)
H-Gln-His-Gly-Val-His-Gly-OH
12.9
Glp-His-Gly-Val-
His-Gly-OH
(XIIb) H-His-Gly-Gln-His-Gly-Val-His-
13.8
92.3
12.2
12.9
13.8
4.0
2.2
H-Gln-His-Gly-
Val-His-Gly-OH
Gly-OH
Glp-His-Gly-Val-
His-Gly-OH
(XIXb) H-His-Gly-Val-Ser-Gly-His-Gly-
15.5
64–85
24–8
H-His-Gly-Gln-
His-Gly-Val-His-
Gly-OH
Gln-His-Gly-Val-His-Gly-OH
* Conditions: Ultrasphere ODS 4.6 × 250 mm column. Buffer A, 0.1% TFA; buffer B, 80% acetonitrile in 0.1% TFA. Gradient of B in A
from 0 to 40% for 40 min. Flow rate 1 ml/min, detection at 226 nm.
automation, and also a possibility of regeneration of the greater amounts of alloferon (more than 50 g) seems to
excessive peptide fragments.
be problematic at the application of fragment conden-
sation in solution.
The carboxyl group of the ë-terminal Gly residue
was blocked with benzyl protection at the synthesis in
solution. The use of temporary Nim-Boc protection and
the presence unprotected (after the first treatment of the
peptide with TFA) imidazole ring of His in the growing
peptide chain limited the choice of the methods of frag-
ment condensation. The method of the active
p-nitrophenyl esters was applied to the creation of
amide bond or, in the case of coupling with pentapep-
tide (XVII), the adduct of DCC and pentafluorophenol
(complex F) was used, because it is known that, under
these conditions, the imidazole ring of histidine and
carboxamide function of glutamine do not react [8].
A chloromethyl anchor group was used for SPS on
the Merrifield polymer. The SPS scheme repeats that of
the peptide preparation in solution except for the pro-
tection of Gly carboxyl group with benzyl ester con-
nected with the polymer support. The C-terminal
dipeptide (IV) was attached to the support by the
method of cesium salts [9].
A trial synthesis of alloferon was carried out to
determine the advantages and drawbacks of the SPS
variant. We started from 11.0 g of dipeptidyl polymer
Boc-His(Boc)-Gly-(P) containing 7.5 mmol of the
starting dipeptide according to the standard protocol
described in the Experimental section. The homogene-
ity of the intermediates during the trial synthesis of
alloferon was monitored by HPLC. The corresponding
compounds were obtained after the coupling with two-
fold excess of acylating agent and deblocking of an ali-
quot of the obtained peptidyl polymer with the use of
trifluoromethanesulfonic acid. The results of these
experiments are given in Table 1. The impurities whose
contents exceeded 2% were isolated from the corre-
sponding reaction mixtures by HPLC and identified
We have met certain difficulties at the isolation of
intermediate benzyl esters of Nα-Boc-protected pep-
tides at the alloferon synthesis in solution. A good sol-
ubility of these intermediates in water (beginning from
the stage of hexapeptide) makes impossible their tradi-
tional isolation from aqueous reaction mixtures by
extraction. All the compounds are hygroscopic and
badly crystallize. Moreover, the Nim-Boc protection
turned out to be rather labile at heating, which compli-
cated the monitoring of the occurring transformations
by TLC. Despite the fact that the reactions of formation
of amide bond proceeded quickly and completely dur-
ing the synthesis and the target product was enough
homogeneous after the final splitting off of the protec-
tion (the content of alloferon was 92% by HPLC, see
the figure), the yield of the purified alloferon was only
17% from the starting C-terminal dipeptide. This rela-
tively low yield of alloferon is explained by the losses
at the isolation of intermediates. The main impurity
(4% by HPLC, see the figure) in the crude product of
conventional synthesis was octapeptide H-His-Gly-
Gln-His-Gly-Val-His-Gly-OH, C-deblocked (XIIa). In
1
with the help of H NMR spectroscopy, amino acid
analysis, or mass spectrometry.
The trial synthesis allowed us to reveal several “bot-
tlenecks.” For example, it was established that the prep-
aration of octapeptidyl polymer (XIb) by the coupling
of hexapeptidyl polymer (Xb) with twofold excess of
Boc-His(Boc)-Gly-ONp (V) proceeds insufficiently
full: about 4% of amino component remains in the reac-
tion mixture. Note that the qualitative test with ninhy-
drin [10] also shows the presence of residual amino
groups in octapeptidyl polymer. After repeating of the
connection with the aforesaid, for preparation of coupling at the presence of 1 equiv of HOBt, the resid-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 2 2006

THE SYNTHESIS OF IMMUNOMODULATING PEPTIDE ALLOFERON
A220 A220
(a) (b)
139
1 (92%)
1 (75%)
2
3
2
11 12 13 14 15 16 17
min
11 12 13 14 15 16 17
min
A220
1 (82%)
(c)
2
11 12 13 14 15 16 17
min
The HPLC of the crude products of (a) conventional and (b, c) solid phase synthesis of alloferon (a fragment with target fractions
is given). (b) The reaction mixture of SPS after the splitting off of the peptide from polymer by the action of trifluoromethane-
sulfonic acid in TFA and (c) the subsequent treatment with 10% solution of ammonium bicarbonate (pH 8.5) for 2 h. Peak 1, allof-
eron (XX); peak 2, octapeptide H-His-Gly-Gln-His-Gly-Val-His-Gly-OH; and peak 3, the product of N
O-acyl migration of
Ser residue. Column: Nucleosil 100 C-18, 5 µm, 4.6 × 250 mm. Buffer A, 0.05 M äç êé , ç 3.0; buffer B, 70% ëç ëN + 30%
2
4
3
of buffer A; a gradient elution with buffer B in buffer A from 0 to 30% for 30 min, detection at 220 nm.
ual amino groups were absent. In addition, the crude sometimes observed at the SPS of peptides with the use
of Boc-methodology [11].
SPS product at this stage contains an impurity (about
2%) of hexapeptide with N-terminal pyroglutamic acid
instead of glutamine (see Table 1). Most likely, this
impurity is a product of an acid-catalyzed side reaction
of the glutamine cyclization to the corresponding lac-
The final fragment condensation (5 + 8) on solid
phase also proceeded in insufficiently high yield of the
product (XVIIIb). In dependence on the coupling con-
ditions, the content of the target alloferon in the reac-
tam, i.e., pyroglutamic acid. This side reaction was tion mixture changed from 64 to 85%, and the content
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 2 2006

140
SIDOROVA et al.
Table 2. The effect of conditions of the fragment condensation of pentapeptide Boc-His(Boc)-Gly-Val-Ser(But)-Gly-OH
(XVII) with octapeptidyl polymer H-His-Gly-Gln-His-Gly-Val-His-Gly-OCH2-(P) (XIIb) on the yield of alloferon
Alloferon
content, %
(HPLC**)
Excess,
mol/mol
Reaction
time, h
Acylating agent
Solvent
1. Boc-His(Boc)-Gly-Val-Ser(But)-Gly-ONp* + HOBt
2.
2
1
2
2
2
DMF
DMF
16
16
16
16
16
64
68
75
80
85
DMF/NMP, 1 : 1
1.5 M urea in DMF
DMF/NMP, 1 : 1
Boc-His(Boc)-Gly-Val-Ser(But)-Gly-OH (XVII) + complex F
Notes: * p-Nitrophenyl ester of the pentapeptide was not isolated.
** Conditions: Ultrasphere ODS 4.6 × 250 mm column. Buffer A, 0.1% TFA; buffer B, 80% acetonitrile in 0.1% TFA. Gradient of
B in A from 0 to 40% for 40 min. Flow rate 1 ml/min, detection at 226 nm.
of the amino component (octapeptide) not entered into desalting by preparative HPLC. The chromatography
on CM-Sephadex in a gradient of ionic force of pyri-
dine-acetate buffer, pH 4.5 (from 0.1 up to 2.0 M)
allowed us to completely separate the impurities of the
truncated peptides and to transfer the target peptide in
the acetate form. The product of ion-exchange chroma-
tography contained 95–96% of alloferon. After HPLC,
the yield of alloferon synthesized by SPS was 40%
from the starting dipeptide attached to polymer. The
purity of product determined with the help of analytical
HPLC was 98%.
Thus, the schemes of conventional and solid phase
synthesis of alloferon were developed, which allow the
preparation of the substance of necessary purity on a
scale of 50–100 g. An advantage of the fragment con-
densation on polymer for the preparation of alloferon
was shown (a higher yield and a better technology). The
main impurities formed during the alloferon synthesis
were identified.
the reaction, from 24 up to 8%, respectively. These data
are listed in Table 2.
We did not managed to find the conditions that
would provide at this stage an yield of the target peptide
above 85%. Low yields of products at separate stages of
SPS of peptides are frequently caused by a decreased
swelling of peptidyl polymers in the used solvents and,
consequently, inaccessibility of amino group. Mixtures
of such solvents as DMF, NMP, and DMSO and addi-
tives of urea, salts, or other reagents [12] are frequently
used to increase the swelling of peptidyl polymers. At
the synthesis of alloferon, the decrease in the yield is
also most likely caused by the insufficient swelling
octapeptidyl polymer (XIIb). This presumption is con-
firmed by the fact that a repeated coupling of (XVII)
with (XIIb) in DMF practically did not improve the
yield, whereas the replacement of DMF with a DMF–
NMP mixture or 1.5 M urea solution in DMF leads to a
substantial increase in the yield of target product (up to
75–80%). The application at this stage of a more active
condensing agent, complex F, has allowed an increase
in the yield of target peptide by 5% (see Table 2).
EXPERIMENTAL
Derivatives of L-amino acids (Bachem, Switzer-
land); and DCC, HONSu, HONp, HONb, pentafluo-
rophenol, and NMM (Fluka, Switzerland) were used in
this work. Dichloromethane, TFA, and NMP (Fluka,
Switzerland) were used for synthesis. DMF was dis-
tilled over ninhydrin and barium oxide. Solvents of
pure and chemically pure grade were used for extrac-
tion and crystallization of peptides. Analytical HPLC
was carried out on a Gilson chromatograph (France)
equipped with Ultrasphere ODS column 5 µm (4.6 ×
250 mm) (Beckman, United States). Buffer A was
0.05 M KH2PO4, pH 3.0, and buffer B, 70% acetonitrile
in buffer A; the elution with a gradient of the buffer B
concentration in the buffer A from 0 to 40% for 40 min.
Flow rate was 1 ml/min, detection at 220 nm.
A solution of trifluoromethanesulfonic acid in TFA
was used for splitting off of alloferon from the poly-
meric support [13]. An N
O acyl migration of the
Ser residue was observed under these rigorous acidic
conditions. This side reaction was repeatedly described
at the treatment of Ser- and Thr-containing peptides by
hydrogen fluoride or trifluoromethanesulfonic acid [13,
14]. On can see from the figure that the crude SPS prod-
uct in comparison with a product of conventional syn-
thesis contains an additional impurity, the substance
corresponding to peak with RT 11.8 min. Most likely,
this is formed due to the N
O-acyl migration. This
impurity disappears after the treatment of the reaction
mixture with 10% solution of ammonium bicarbonate
with pH 8.5 for 2 h [14].
Preparative HPLC was achieved on a Beckman
(United States) instrument equipped with a column
The crude products of both conventional and solid (50 × 250 mm) packed with Diasorb-130T-C16, 10 µm
phase synthesis were purified in two stages: first, a (ZAO Biokhimmak NO, Russia). Peptides were eluted
chromatography on ion exchanger and, second, by at a flow rate of 50 ml/min and detected at a wavelength
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 2 2006

THE SYNTHESIS OF IMMUNOMODULATING PEPTIDE ALLOFERON
141
of 220 nm. Buffer A was 0.01 M ammonium acetate, ness of the reaction was checked with TLC. The reac-
buffer B, 80% aqueous acetonitrile, elution was carried
out with a 0.3%/min gradient of B in A beginning from
0%. Acetonitrile from Lecbiopharm (Russia) was used
for HPLC.
tion mixture was evaporated in a vacuum, the residue
was dissolved in ethyl acetate (1000 ml), and the solu-
tion was washed with water (3 × 350 ml) and evapo-
rated. The residue was triturated with 1 : 1 ether–hex-
ane mixture (3 × 300 ml), the solvent was decanted, and
the residue was triturated with hexane. The precipitate
was quickly filtered and dried in a vacuum to give pep-
tide (III‡); yield 38.8 g (89%); Rf 0.55 (A), 0.89 (D),
0.75 (E).
The homogeneity of the compounds obtained was
confirmed by TLC on pre-packed Kieselgel 60 plates
(Merck, Germany) in the following solvent systems:
(A) 15 : 4 : 1 chloroform–methanol–32% ëH3COOH,
(B) 6 : 3 : 1 ethyl acetate–chloroform–methanol, (C)
60 : 45 : 20 chloroform–methanol–32% ëH3COOH,
(D) 5 : 3 : 1 chloroform–methanol–32% ëH3COOH,
(E) 9 : 1 : 0.5 chloroform–methanol–32%ëç3ëééç,
(F) 7 : 3 isopropanol–25% ammonia, and (G) 8 : 8 : 8 : 1
chloroform–methanol–benzene–water. Substance spots
on chromatograms were detected by a chlorine–benzi-
dine or a ninhydrin reagent. Mps (not corrected) were
determined on Boetius hot plate. An amino acid analy-
sis of peptides hydrolyzed with 6 N hydrochloric acid
containing 2% phenol at 110°ë for 24 h was carried out
on a Biotronik LC 5001 analyzer (Germany). 1H NMR
spectra were measured on a Bruker WH-500 spectrom-
eter (Germany) at 500 MHz in DMSO-d6 at 300 K; the
concentrations of peptides were 2–3 mg/ml. Chemical
shifts (δ, ppm) were calculated relative to tetramethyl-
silane. Mass spectra were registered on a device
PC-Kompact MALDI (Kratos, UK).
Boc-His(Boc)-Gly-OH (IV). A solution of Boc-
His(Boc)-ONp (33.42 g, 70 mmol) in DMF (250 ml)
was cooled to –10°ë, and glycine (5.2 g, 70 mmol) in
2 N NaOH (35 ml) was added. After 4 h (TLC monitor-
ing in system C), the reaction mixture was evaporated,
and the oily residue was dissolved in water (400 ml)
and extracted with ether (3 × 150 ml). The water phase
was acidified with citric acid to pH 4, extracted with
ethyl acetate (3 × 200 ml), and the combined ethyl ace-
tate extract was washed with water (3 × 150 ml), evap-
orated in a vacuum, and crystallized from an ethyl ace-
tate–ether mixture to give (IV), yield 28.2 g (75%);
Rf 0.60 (A), 0.51 (E); mp 134–135°ë.
Boc-His(Boc)-Gly-ONp (V). A solution of DCC
(33.0 g, 157 mmol) in DMF (150 ml) was added to a
cooled to –20°ë solution of (IV) (53.0 g, 143 mmol)
and HONp (22.0 g, 157 mmol) in DMF (500 ml). The
mixture was kept for 16–24 h at 4°ë. The precipitated
dicyclohexylurea was filtered off, and the filtrate was
evaporated. The residual oil was triturated with hexane
(3 × 500 ml), hexane was decanted, and the residue was
crystallized from an isopropanol–hexane mixture to
give (V); yield 54 g (71%); Rf 0.72 (A), 0.38 (B),
0.64 (E); mp 128–130°ë.
Boc- and Z-derivatives of amino acids and their
active esters were obtained by the described proce-
dures [15].
Boc-His(Boc)-Gly-OBzl (Ia). NMM (12.5 ml) and
a solution of Boc-His(Boc)-ONp (54.0 g, 114 mmol) in
DMF (400 ml) were added to a solution of H-Gly-
OBzl · TosOH (36.4 g, 114 mmol) in DMF (200 ml).
The reaction mixture was kept at 20°ë for 16 h, while
monitoring the course of reaction by TLC in systems B
and E. DMF was evaporated, and the residue was dis-
solved in ethyl acetate (1200 ml) and washed with 0.1%
ammonia (3 × 400 ml), water (2 × 400 ml), 2% aqueous
solution of citric acid (300 ml), and water (3 × 300 ml).
The organic phase was evaporated, and the residue was
dissolved in a 5 : 1 ethyl acetate–benzene mixture, and
evaporated. The residue was dried in a vacuum to give
an oily product (I‡); yield 46.9 g (85%); Rf 0.80 (A),
0.32 (B), and 0.58 (E).
Boc-Val-His-Gly-OBzl (IIIa). A solution of (I‡)
(46.9 g, 96.8 mmol) in TFA (200 ml) was kept for 1 h
at 20°ë and evaporated. The residue was successively
triturated with fresh portions of a 1 : 1 ether–hexane
mixture (2 × 250 ml) and with ether (3 × 250 ml), the
solvents were decanted, and the residual oil was dried
in a vacuum over alkali. Hereinafter, the completeness
of splitting off of Boc protection and the homogeneity
of the deblocked product were monitored by TLC in
systems A and D. The resulting product (II‡) was dis-
solved in DMF (500 ml), NMM (19.8 ml, 180 mmol)
Boc-His(Boc)-Gly-Val-His-Gly-OBzl (VIIa). A
solution of 38.8 g (80 mmol) of protected tripeptide
(III‡) in TFA (200 ml) was kept for 1 h at 20°ë and
evaporated. Dry ether (1000 ml) was added to the resi-
due, and the resulting precipitate was filtered and dried
in a vacuum over alkali to give 50 g of deblocked trip-
eptide (VI‡) (99%). A solution of (VI‡) (25.2 g,
40 mmol) in DMF (300 ml) was successively treated
with NMM (8.8 ml, 80.0 mmol) and a solution of active
ester (V) (21.4 g, 40.0 mmol) in DMF (200 ml). The
reaction mixture was stirred for 16 h at 20°ë and evap-
orated in a vacuum, and the residue was dissolved in
ethyl acetate (700 ml) and washed with water
(3 × 350 ml). Ethyl acetate was evaporated, and the res-
idue was triturated with dry ether (4 × 150 ml). The sol-
vent was decanted, the residue was triturated with hex-
ane, and the precipitated solid was filtered and dried in
a vacuum. The yield of (VII‡) was 28.6 g (90%);
Rf 0.52 (A), 0.75 (D); mp 94–98°ë; amino acid analy-
sis: Gly 2.00 (2), Val 0.98 (1), His 2.05 (2).
Boc-Gln-His-Gly-Val-His-Gly-OBzl (IXa). Pro-
and Boc-Val-ONSu (28.6 g, 90 mmol) were added, and tected pentapeptide (VII‡) (28.6 g, 36 mmol) was
the mixture was stirred for 16 h at 20°ë. The complete- deblocked with TFA as described above for (II‡).
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SIDOROVA et al.
NMM (10.0 ml, 99.0 mmol) and a solution of Boc-Gln- solution of Z-Val-ONSu (40.7 g, 0.117 mol) in DMF
ONp (12.4 g, 32.8 mmol) in DMF (200 ml) were suc- (100 ml) was added. After the end of reaction, the solu-
cessively added to a solution of pentapeptide (VIII‡) tion was evaporated, ethyl acetate (500 ml) was added
trifluoroacetate (27.0 g, 32.1 mmol) in DMF (300 ml). to the residue, and the mixture was acidified with 1 N
The mixture was stirred for 16 h at 20°ë and evaporated H2SO4. The organic layer was separated, washed with
in a vacuum. Ethyl acetate (1500 ml) was added to the water (3 × 200 ml), and evaporated. Ether (200 ml) was
residue, the precipitated solid was filtered, washed with added to the residue; the precipitated solid was filtered,
ethyl acetate (3 × 150 ml) and ether (3 × 150 ml), and washed with ether (3 × 50 ml), and dried. The yield of
dried. The product was precipitated from isopropanol peptide (XV) was 40.9 g (98%); mp 105–107°ë.
with hexane and dried, to yield (IX‡) (19.6 g, 72%); Rf 0.71 (A), 0.28 (B), 0.88 (C).
Boc-His(Boc)-Gly-Val-Ser(But)-Gly-OH (XVII).
Compound (XV) (40.9 g, 91 mmol) was hydrogenated
in ethanol (500 ml) over 10% Pd/C (4 g) in the presence
of 1 N NaOH (91 ml, 1 equiv). The catalyst was filtered
off, and the filtrate was evaporated. The residue was
dissolved in DMF (150 ml), cooled to –10°ë and a
solution of active ester (V) (48.5 g, 40.9 mmol) in DMF
(100 ml) was added. After the end of reaction, DMF
was evaporated, and the residue was treated as
described above for (IV). The product was crystallized
from ether (500 ml). The yield of (XVII) was 56.5 g
(87%); Rf 0.59 (A), 0.94 (D), 0.57 (G); amino acid
analysis: Ser 0.95 (1), Gly 2.00 (2), Val 1.00 (1),
His 1.02 (1).
Boc-His(Boc)-Gly-Val-Ser(But)-Gly-His-Gly-Gln-
His-Gly-Val-His-Gly-OBzl (XVIIIa). A solution of
pentapeptide (XVII) (14.4 g, 20.2 mmol) in DMF
(250 ml) was added to a solution of octapeptide (XIIa)
(21.5 g, 18.3 mmol) in DMF (250 ml). The mixture was
cooled to –10°ë, and complex F (15.3 g, 20.2 mmol)
was added. The reaction mixture was stirred for 2 h at
0°ë and 20–30 h at 20°ë. The dicyclohexylurea precip-
itate was filtered off, the filtrate was evaporated to the
volume of 100 ml, and ethyl acetate (500 ml) was
added. The precipitated solid was filtered, washed with
ethyl acetate (3 × 150 ml) and ether (3 × 150 ml), and
dried. The yield of (XVIIIa) was 27.5 g (92%); Rf 0.52
(C), 0.38 (D); amino acid analysis: Ser 0.97 (1), Glx
1.02 (1), Gly 5.00 (5), Val 1.96 (2), His 4.00 (4).
H-His-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-
His-Gly-OH acetate (XX). The protected tridecapep-
tide (XVIIIa) (27.5 g, 16.9 mmol) was deblocked with
TFA as described above. The product was dissolved in
80% AcOH (500 ml), 10% Pd/C (3.0 g) in 50 ml of
water was added, and the reaction mixture was hydro-
genated until the disappearance of starting compounds.
The catalyst was filtered off, the filtrate was evaporated
to dryness, and the residue was dissolved in 98%AcOH
(100 ml) and evaporated to the consistence of liquid oil.
Isopropanol (800 ml) was added, and the precipitated
solid was filtered, washed with isopropanol and ether
and dried. The yield of crude (XX) trifluoroacetate was
22.4 g. The content of target substance according to
analytical HPLC was 90%. The product was dissolved
in 0.1 M pyridine–acetate buffer (pH 4.5, 100 ml) and
applied onto a column (2.6 × 60 cm) filled with
Rf 0.12 (A), 0.59 (D), 0.48 (G); amino acid analysis:
Glx 1.02 (1), Gly 2.00 (2), Val 0.96 (1), His 2.00 (2).
For 1H NMR spectrum, see Table 3.
Boc-His(Boc)-Gly-Gln-His-Gly-Val-His-Gly-OBzl
(XI‡). NMM (7.90 ml, 72.0 mmol) and a solution of
active ester (V) (13.36 g, 25.0 mmol) in DMF
(200 ml) were successively added to a solution of
hexapeptide trifluoroacetate (ï‡), prepared from 19.5 g
(23.8 mmol) of hexapeptide (IXa) in DMF (300 ml).
The mixture was kept for 16 h at 20°ë and evaporated,
and the residue was triturated with 8 : 2 ether–ethyl
acetate mixture (500 ml). The precipitate was filtered,
washed with 1 : 1 ethyl acetate–ether mixture (2 × 150 ml),
ethyl acetate (3 × 150 ml), and ether (3 × 150 ml), and
dried. The product was precipitated with hexane from
isopropanol and dried to yield (XIa) (20.5 g, 78%);
Rf 0.2 (A), 0.65 (C), 0.57 (D); amino acid analysis:
Glx 1.05 (1), Gly 3.00 (3), Val 0.99 (1), His 3.10 (3).
H-His-Gly-Gln-His-Gly-Val-His-Gly-OBzl tri-
fluoroacetate (XIIa). Protected octapeptide (XIa)
(20.5 g, 18.4 mmol) was deblocked with TFA as
described above to give (XIIa); yield 25.1 g (99%);
purity by HPLC (elution with 10 to 70% gradient of
buffer B in buffer A for 30 min) no less than 90%;
Rf 0.13 (C), 0.08 (D), 0.51 (F); amino acid analysis: Glx
1.00 (1), Gly 3.00 (3), Val 0.98 (1), His 3.05 (3). For
1H NMR spectrum, see Table 3.
Z-Ser(But)-Gly-OH (XIII). A solution of glycine
(9.3 g, 0.124 mol) in 2 N NaOH (61.5 ml) was added to
a solution of Z-Ser(But)-ONb (56.2 g, 0.123 mol) in
DMF (400 ml) cooled to –10°ë. The reaction mixture
was kept for 18 h and evaporated. Distilled water (500
ml) was added to the residue, and it was extracted with
ether (3 × 200 ml). The water layer was acidified with
20% ç2SO4 to pH 4 and the target product was
extracted with ethyl acetate (300 ml). The organic layer
was washed with water to neutral reaction, evaporated,
and the residue was crystallized from ether (300 ml). The
precipitate was filtered, washed with ether (3 × 50 ml),
and dried. The yield of (XIII) was 41.3 g (95%); Rf 0.64
(A), 0.87 (D), 0.52 (G); mp 98–100°ë.
Z-Val-Ser(But)-Gly-OH (XV). Compound (XIII)
(41.3 g, 0.117 mol) was hydrogenated in 100 ml of
methanol in the presence of 35%Triton B (66.7 ml) and
10% PdO/C catalyst (4.0 g). The catalyst was filtered
off, and the filtrate was evaporated. The residue was CM-Sephadex C-25 equilibrated with the same buffer.
dissolved in DMF (300 ml), cooled to –10°C, and a The peptides were eluted by a linear gradient of ionic
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THE SYNTHESIS OF IMMUNOMODULATING PEPTIDE ALLOFERON
143
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 2 2006

144
SIDOROVA et al.
Table 4. The standard protocol of the alloferon SPS (for 11 g, 7.5 mmol, of the starting dipeptidyl polymer)
Time of single Volume of single
Stage
Operation
Washing
Reagent
treatment, min*
treatment, ml
1
2
3 × CH2Cl2
3
10
30
3
100
100
100
100
100
100
100
100
60
Deprotection of α-amino 50% TFA/CH2Cl2
groups
3
4
Washing
3 × CH2Cl2
3 × DMF
10% DIEA/DMF
3
Neutralization
5
10
3
5
6
Washing
Coupling
3 × DMF
15 mmol of active derivative of Boc-amino acid
or Boc-peptide in DMF
120
7
Washing
3 × DMF
3 × CH2Cl2
3
3
100
100
8
9
Determination of residual Ninhydrin solution [10, 14]
amino groups
Repeated coupling
7.5 mmol of active derivative of Boc-amino acid
or Boc-peptide in DMF
120
60
* In the case of coupling of the active derivative of N-terminal pentapeptide (XVII) with octapeptidyl polymer (XIIb), the reaction time
was increased to 16 h.
strength of the same buffer [from 0.1 (1 l) to 2 M (1 l)]. the Merrifield polymer (10 g) was added. The suspension
The fractions containing the target product (by HPLC) was slowly stirred for 24 h at 50°ë. The polymer was fil-
tered and successively washed with DMF (3 × 50 ml), a
1 : 1 DMF–water mixture (5 × 50 ml), DMF (3 × 50 ml),
EtOH (3 × 50 ml), and ether (3 × 50 ml) and dried in a
vacuum. The content of peptide in the polymer deter-
mined by the increase in its mass and with the help of
spectrophotometric titration with picric acid [14] was
0.6–0.7 mmol/g.
SPS was carried out from 11.0 g of dipeptidyl poly-
mer containing 7.5 mmol of starting dipeptide accord-
ing to the standard protocol outlined in Table 4. After
the end of SPS, the tridecapeptidyl polymer (XVIIIb)
was deblocked by the action of 50% TFA in CH2ël2
(100 ml) for 30 min. The peptidyl polymer (XVIIIb)
was washed with CH2ël2 (3 × 100 ml) and dried in a
vacuum over ê2é5. The mass of tridecapeptidyl poly-
mer (XIXb) was 21.6 g; its additional mass after the
termination of synthesis was 11.6 g.
were combined, evaporated to dryness, and then twice
coevaporated with water. The final purification of the
product was achieved with the help of preparative
HPLC under the conditions described above. The yield
of alloferon (XX) was 11.5 g [46%, or 17% from the
starting dipeptide (I)]. The purity of the product deter-
mined by analytical HPLC was 98%; Rf 0.42 (F); amino
acid analysis: Ser 0.98 (1), Glx 1.02 (1), Gly 5.00 (5),
Val 1.98 (2), His 4.00 (4); MS, m/z: 1264.7 (calculated
1265.3). Elemental analysis: found, %: C 44.90, I 6.72,
N 20.59. ë52H76N22O16 · 2ÄÒéç · 6H2O. Calculated,
%: C 45.04, H 6.48, N 20.63. According to the elemen-
tal analysis, the content of water in the alloferon prepa-
ration was 7.2%; the content of acetic acid, 8.0%; and
1
the content of peptide material, 84.8%. For H NMR
spectrum, see Table 3.
Solid phase synthesis of alloferon. A chlorometh-
ylated styrene copolymer with 1% divinylbenzene (the
H-His-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-
Merrifield polymer) containing 1.07 equiv of chlorine Val-His-Gly-OH (XX). Trifluoromethanesulfonic
per g with the particle size of 200–400 mesh (Sigma, acid (20 ml) was added in portions to a cooled to 0°ë
United States) was used for SPS. Dipeptide (IV) was suspension of tridecapeptidyl polymer (XIXb) (21.6 g)
attached to the polymer support with the use of the cor- in TFA (200 ml) containing 2 ml of anisole. The stirring
responding cesium salt [9]. The cesium salt was was continued for 10 min at 0°ë and 1.5 h at 25°ë. The
obtained by the addition of a solution of Cs2CO3 reaction mixture was carefully poured out in 1 l of dry
(1.95 g, 6 mmol) in 6 ml of water to a solution of dipep- ether cooled to 0°ë. The precipitated solid was filtered
tide (IV) (4.95 g, 12 mmol) in 10 ml of ethanol. After and washed on filter with ether (5 × 100 ml). The precip-
15 min, the mixture was evaporated to dryness; the itate on the filter, consisting of a mixture of the poly-
residual water was removed by coevaporation with ben- meric support and the target peptide split off from it,
zene (2 × 20 ml) and drying in a vacuum over ê2é5. The was treated with three portions of TFA (50 ml each),
dried dipeptide salt was dissolved in DMF (30 ml), and and the solution peptide in TFA was filtered in a flask
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 2 2006

THE SYNTHESIS OF IMMUNOMODULATING PEPTIDE ALLOFERON
145
5. Sidorova, M.V., Molokoedov, A.S., Ovchinnikov, M.V.,
Bespalova, Zh.D., and Bushuev, V.N., Rus. J. Bioorg.
Chem., 1997, vol. 23, pp. 41–50.
6. Barany, G. and Merrifield, R.B., in The Peptides: Analy-
sis, Synthesis and Biology, vol. 2, Gross, E.. and Meien-
hofer, J.., Eds., New York: Academic, 1980, pp. 1–284.
7. Bodanszky, M. and Martinez, J., in The Peptides: Anal-
ysis, Synthesis and Biology, vol. 5, Gross, E.. and Meien-
hofer, J.., Eds., New York: Academic, 1983,
pp. 111−216.
8. Kisfaludi, L., Low, M., Schon, I., Szirtes, T.,
Sarkosi, M.Gz., Turan, A., Beks, R., Juhacz, A., Graf, L.,
Medzigradszky, K., and Szpozny, L., US Patent
3 953 415, 1976.
containing 1 l of dry ether. The precipitate was sepa-
rated, washed with ether (5 × 100 ml), and dried in a
vacuum over alkali. The resulting crude product
(12.1 g) was kept for 2 h in 10% solution of ammonium
bicarbonate (pH 8.5). The purification of the peptide
was carried out by ion exchange chromatography and
preparative HPLC under the above-described condi-
tions. The yield of (XX) was 4.2 g (40% from the start-
ing dipeptide, attached to the polymeric support). The
purity of the product determined by analytical HPLC
was 98%; Rf 0.42 (F); amino acid analysis: Ser 0.98 (1),
Glx 1.02 (1), Gly 5.00 (5), Val 1.98 (2), His 4.00 (4);
MS, m/z: 1266.2 (1265.3 as calculated for
1
ë52H76N22O16); H NMR spectrum was completely
9. Gisin, B.F., Helv. Chim. Acta, 1973, vol. 56,
pp. 1476−1482.
identical to that of the above-mentioned product of con-
ventional synthesis.
10. Kaiser, E., Colescott, R.L., Bossinger, C.D., and Cook, P.I.,
Anal. Biochem., 1970, vol. 34, pp. 595–598.
11. Dimarchi, R.D., Tam, J.P., Kent, S.B.H., and Merrifi-
eld, R.B., Int. J. Pept. Protein Res., 1982, vol. 19,
pp. 88–93.
12. Fields, C.G. and Fields, G.B, in Methods in Mol. Biol.,
vol. 35, Peptide Synthesis Protocols, Pennington, M.W.
and Dunn, B.M., Eds., Totowa, NJ: Humana, 1994,
pp. 29–40.
13. Yajima, H. and Fujii, N. in The Peptides: Analysis, Syn-
thesis and Biology, vol. 5, Gross, E.. and Meienhofer, J..,
Eds., New York: Academic, 1983, pp. 65–109.
14. Stewart, J.M. and Young, J.D., Solid Phase Peptide Syn-
thesis, 2nd ed., Rockford, USA: Pierce Chem. Co., 1984.
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