Spontaneous and promoted association of linear oligoglycines

Article abstract of DOI:10.1134/S1068162006050049

Linear oligoglycines of various lengths bearing a carboxyl or an amide group at their C-termini and also their poly(acrylamide) conjugates were synthesized. No self-assembly into supramolecular structures was observed for free oligoglycines H-(Gly)_m-OH (m = 3-5). At the same time, oligoglycylamides H-(Gly)_m-NH₂ (m = 3-5) demonstrated ability for both self-assembly in aqueous solution and assembly promoted by an additional interaction with surface. In the case of polymer-bound oligoglycines (and their amides), no intramolecular clustering of peptide chains, as expected, was observed. This means that the presence of several oligoglycine chains bound to each other in one center is not a necessary prerequisite for polyglycine II-type association. Pleiades Publishing, Inc., 2006.

Full text of DOI:10.1134/S1068162006050049

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2006, Vol. 32, No. 5, pp. 420–428. © Pleiades Publishing, Inc., 2006.
Original Russian Text © I.V. Gorokhova, A.A. Chinarev, A.B. Tuzikov, S.V. Tsygankova, N.V. Bovin, 2006, published in Bioorganicheskaya Khimiya, 2006, Vol. 32, No. 5,
pp. 467−476.
Spontaneous and Promoted Association of Linear Oligoglycines
I. V. Gorokhova, A. A. Chinarev, A. B. Tuzikov, S. V. Tsygankova, and N. V. Bovin1
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10,
Moscow, 117997 Russia
Received December 5, 2005; in final form, March 6, 2006
Abstract—Linear oligoglycines of various lengths bearing a carboxyl or an amide group at their C-termini and
also their poly(acrylamide) conjugates were synthesized. No self-assembly into supramolecular structures was
observed for free oligoglycines H-(Gly) -OH (m = 3–5). At the same time, oligoglycylamides H-(Gly) -NH
m
m
2
(
m = 3–5) demonstrated ability for both self-assembly in aqueous solution and assembly promoted by an addi-
tional interaction with surface. In the case of polymer-bound oligoglycines (and their amides), no intramolec-
ular clustering of peptide chains, as expected, was observed. This means that the presence of several oligogly-
cine chains bound to each other in one center is not a necessary prerequisite for polyglycine II–type association.
Key words: peptides, poly(acryl amide) conjugates, polyglycine II, self-assembly
DOI: 10.1134/S1068162006050049
INTRODUCTION
raantennary oligoglycines form tectomers when the
number of Gly residues in the antenna is seven or more.
In this case, the cooperativity of the system of hydrogen
bonds is so high that water cannot destroy it at room
temperature. A raise of temperature to 60–70°ë results
in a reversible dissociation of dissolved tectomers. Dis-
sociation also occurs due to the protonation of terminal
amino groups or introduction into solution of chaotro-
pic agents, such as lithium chloride, which suppresses
the formation of hydrogen bonds [9]. It was further
shown that the plane tectomers with PGII structure can
be formed not only from tetraantennary, but also from
triantennary peptides (Fig. 2c) and some diantennary
oligoglycines (Fig. 2b). When the assembly of
branched peptides is promoted by surface, extended
GII tectomer layers are formed, the assembly proceed-
ing much faster than in aqueous solutions [11].
In the 1950s, the spiral polypeptide chains of one of
the crystal forms of glycine polymer, so-called, polyg-
lycine II, have been found to be packed in a noncanon-
ical structure in which all hydrogen bonds are inter-
chain and are directed perpendicularly to the chain
axes, each polypeptide chain being surrounded with
2
other six chains (Fig. 1) [1, 2]. : The chain packing in
PGII is so tight that such structures cannot be formed
by any other α-amino acid, including alanine. Later, the
packing analogous to that of PGII has been found in
crystalline nylons, i.e., polymers built up of ϖ-amino
acid residues or of dicarboxylic acids and diamines [3,
4
]. Some relatively short oligoglycine peptides
R(NHCH CO) R' are known to be capable of the for-
2
m
mation of two-dimensional crystals with PGII structure
(
Fig. 1‡) [5–7]. The PGII structure was also reported to
These data allowed us to presume that the antennary
architecture of oligoglycines is a factor favoring their
association. In this connection, it was of interest to syn-
thesize polymer conjugates of linear oligoglycines and
to compare their ability to self-assembly with that of
nonconjugated peptides. We supposed that the close-
ness of oligoglycine antennae and their linkage with the
polymer carrier should entropically favor association
be formed in the solid phase by bolaamphiphils con-
taining oligoglycine fragments [8].
We have recently synthesized symmetric branched
molecules with four similar oligoglycine fragments
attached to one center (tetraantennary oligoglycines,
Fig. 2d) and demonstrated their ability to be packed
into PGII-type structure not only in solid phase, but
also in aqueous solutions [9]. The associates formed by
these oligoglycine tectons (from Greek τεχτϖν, a con-
struction block [10]) were named as tectomers [9]. Tet-
(see Fig. 2e).
Oligoglycine fragments, up to hexaglycine, occur in
some proteins, in particular, in the structural proteins of
plant cell walls, with their glycine content reaching up
to 60% [12, 13]. These proteins have a unique ability to
be organized into suprahelical structures in response to
microbial attack or mechanic damage of cell wall, thus
supporting the cell tissue with flexible and resistant to
1
Corresponding author; phone: +7 (495) 330-7138;
e-mail: bovin@carb.ibch.ru.
2
Abbreviations: AFM, atomic force microscopy; Boc, tert-butylox-
ycarbonyl; EA, ethanolamine; ONp, 4-nitrophenyl ester; PGII,
polyglycine II, PHEAA; poly(2-hydroxyethylacrylamide); and
PNPA, poly(nitrophenyl acrylate).
4
20

SPONTANEOUS AND PROMOTED ASSOCIATION
421
(‡)
(b)
Parameters of polyglycine II structure
dihedral angles of the peptide backbone
Φ = –76.9°
Ψ = 145.3°
pitch of helix
number of residues per one coil
diameter of helix core
distances between the axes of neighboring helices
length of the interchain hydrogen bonds (NH…..O)
3.1 Å
3
1.8 Å
4.8 Å
1.73 Å
Fig. 1. The model of the polyglycine II structure. (a) View from above. (b) Side view. Only tree chains are shown. Hydrogen bonds
are shown with dashed lines.
rupture fibers as a construction material. There is no lit- aqueous LiCl (which destroys hydrogen bonds); i.e.,
erature information on the ability of proteins to associ- the features previously observed for the associating tet-
ate due to their oligoglycine moieties. Therefore, we raantennary peptides [9]. The peptides with a free car-
considered the polymeric derivatives of oligoglycines boxyl group H-(Gly) -OH (m = 4, 5) do not display
m
(
see above) as model compounds containing several such properties. Obviously, the electrostatic repulsion
oligoglycine fragments like the aforementioned pro- of the terminal COO-groups and lesser lengths of the
teins.
peptide chains hinder their association.
The dynamic light scattering is a direct method for
the size estimation of macromolecules, supramole-
cules, and particles [14]. We have demonstrated by this
method that the observed size of objects in aqueous
RESULTS AND DISCUSSION
Peptides
Two series of oligoglycine peptides, namely, those
bearing carboxyl and amide groups at their ë-termini,
were obtained by the method of active esters. The oli-
gomers were synthesized by step elongation of the pep-
tide chain at the N-end using N-Boc-glycine succinim-
ide ester method. Glycylglycine or glycylamide,
respectively, were used as the starting compounds (see
Scheme 1a).
solution of HCl × H-(Gly) -OH is independent of the
m
peptide lengths (the value of m), though it increases to
some extent with time (24 h and more). An addition of
LiBr to the peptide solution does not affect the size of
objects in the solution (Table 1). This indicates the
absence of association in HCl × H-(Gly) -OH (m =
m
3
−5) solutions. At the same time, the data on the light
scattering for amides TFA × H-(Gly) -NH (m = 3–5)
m
2
A number of features point out to the ability of oli- are interpreted as a sharp increase in the size of the dis-
goglycines to self-assembly. In particular, they are: a solved particles 24 h after the addition of base (the
low solubility of Boc-derivatives of peptides (m = 6–8) maxima of distribution curves correspond to the size of
in aprotic polar solvents (DMF and DMSO), a low sol- 2–7 µm). Moreover, an opalescence of the solution is
ubility of the corresponding trifluoroacetates (m = 7, 8) observed. The addition of LiCl or HCl, which were pre-
and a broadening of signals in their NMR spectra (m = viously shown to destroy tetraantennary tectomers [9],
7
, 8), and also the fact that the peptides practically resulted in a decrease in particle size and disappearance
insoluble in water with nonprotonated amino group of opalescence. As a whole, these observations indicate
H-(Gly) -NH (m = 7, 8) are dissolved in saturated an association of linear oligoglycinamides H-(Gly)m-
m
2
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 5 2006

4
22
GOROKHOVA et al.
COR
(a)
(b)
(c)
(a)
Glym
NHR
(d)
0
2
4
6 µm
nm
9
.5
(b)
1
2
0
(e)
–
9.5
0
0.5
1.0
1.5
µm
(c)
Fig. 2. The scheme of oligoglycine association. (a) Assem-
bly of the planar crystalline monolayer from a linear pep-
tide. (b) Assembly of a diantennary peptide with oligogly-
cine fragments bound by a hydrocarbon bridge. (c) Assem-
bly of a triantennary peptide (monolayer, all tree antennas
are oriented in one direction). (d) Assembly of a tetraanten-
nary peptide (two antennae are oriented in one direction and
two other, in opposite direction). (e) The presumed assem-
bly of a peptide cluster with linear oligoglycines bound with
a flexible polymer.
0
.5
1
.0
1.5
µm
Fig. 3. (a) AFM images of aggregates formed by triglycyla-
mide on the mica surface; (b) cross-section of the aggregate
(the distance 1–2 is 2.0 nm); and (c) 3D-image of the aggre-
gate.
NH (m = 3−5) in aqueous media. This conclusion lycyl amide (data not shown); i.e., their organization is
2
agree with the literature information on the appearance
of an ordered structure with PGII packing in the series
of Ac-(Gly) -NHC H starting from diamide (m = 1)
similar to that of tectomers formed from tetra- [9], tri-,
and diantennary [11] oligoglycines. In both cases, the
height of the associate corresponds to the double length
of peptide in the PGII conformation. Thus, one can sup-
m
2
5
[5].
The associates forming from H-(Gly) -NH (m = 3, pose that the associates are bilayer structures. Obvi-
m
2
4
) were also studied using AFM method. Sodium bicar-
ously, the mica surface characterized by a weak nega-
tive charge favors the formation of a two-dimensional
crystal of PGII peptide due to the interaction with pep-
tide amino groups, as observed for antennary oligogly-
cines [11]. Whether the associate is really packed as
bonate was added to the solution of the corresponding
trifluoroacetate, and the sample was immediately
applied onto mica. According to AFM data, the associ-
ates are characterized by planar dimensions varying in
the range of 50–1000 nm and a height of 2.0 nm for
triglycyl amide (Fig. 3) or 2.5 nm in the case of tetrag- PGII and in what way does the second layer connected
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 5 2006

SPONTANEOUS AND PROMOTED ASSOCIATION
423
(a)
O
O
O
O
H
N
H
H
1
. Boc-Gly-ONSu
. CF COOH
1. Boc-Gly-ONSu
H2N
N
…
N
2
N
OH
2. CF COOH
3
H N
2
OH
3
H
mOH
H
O
O
O
H-(Gly) -OH; m = 3–5
m
O
O
O
H
H
N
H N
2
1. Boc-Gly-Gly-ONSu H N
2
N
1. Boc-Gly-ONSu
2. CF3COOH
…
H
mNH2
NH2
2. CF COOH
N
NH2
3
H
O
H-(Gly) -NH ; m = 3–8
m
2
(b)
TFA × H-(Gly) -NH2
m
NEt , DMSO
3
(100 – X)
n
X
n
n
O
NH2(CH2)2OH
O
O
NH(CH ) OH
2 2
H N–(Gly)
2
m
O
PHEAA–(Gly) -NH (X); X = 20%, 50%
m
2
TFA × H-(Gly) -OH
m
NEt , DMSO
3
X
(100 – X)n
n
O
O
NH(CH ) OH
NO2
HO–(Gly)m
2 2
NH (CH ) OH
2
2 2
PHEAA–(Gly) -OH (X); X = 5%, 10%, 20%
m
Scheme 1. (a) Synthesis of linear oligoglycines and oligoglycylamides and (b) their polymeric conjugates.
with the first one will be later studied using the method
of Raman spectroscopy.
Polymeric Conjugates of Peptides
Polymeric conjugates were obtained by coupling of
oligoglycines with PNPA and subsequent amidation of
unreacted –COONp groups with ethanolamine
Table 1. Particle size for oligoglycines H-(Gly) -OH and
m
(
Scheme 1b) [3]. We attempted to prepare conjugates
conjugates PHEAA–(Gly) -OH according to light scattering
m
data
with various degree of substitution with oligoglycines
within the interval from 5 to 50 mol %. Inclusion of oli-
1
3
Particle size, nm*
goglycines in the polymer was demonstrated by
C
NMR. However, the data of the amino acid analysis
prove that we failed to obtain conjugates with the
degree of oligoglycine binding more than 30 mol %
Compound
fresh
aqueous
solution
solution after 24 h
water 5 M LiCl
even for short peptides (m = 3). Ahe real content of gly-
3
cine was 18 mol % in PHEAA–(Gly) -NH (20) and
HCl × H-(Gly) -OH
400
330
500
980
970
430
580
810
670
780
530
1030
1550
1350
620
670
530
570
610
620
630
370
830
1440
1280
1160
1060
980
3
2
3
3
3 mol % in PHEAA–(Gly) -NH (50), whereas a wide
3 2
HCl × H-(Gly) -OH
4
range of molecules with the primary amino group was
previously characterized by the qualitative attachment
to PNPA [15].
HCl × H-(Gly) -OH
5
PHEAA–(Gly) -OH (5)
3
Note a possibility of interaction of the residual
active esters of carboxyl groups retained in the polymer
after the attachment of oligoglycine to PNPA with NH
groups of peptides, which can result in N-diacyl
adducts [16]. Acylation of the NH group used for
attachment of the peptide to the polymer can result in
six-membered cyclic diketopiperidine structures. Fur-
thermore, one can suppose the formation of adducts as
PHEAA–(Gly) -OH (10)
3
PHEAA–(Gly) -OH (20)
3
PHEAA–(Gly) -OH (5)
870
4
PHEAA–(Gly) -OH (10)
960
4
PHEAA–(Gly) -OH (20)
1110
990
4
PHEAA–(Gly) -OH (10)
5
3
PHEAA
2480
PHEAA–(Gly) -OH (X) and PHEAA–(Gly) -NH2 (X) are the
m
m
abbreviations for poly(2-hydroxyethylacrylamide)–oligopeptide
conjugates, where X is the expected degree of substitution of the
peptide moieties for 2-hydroxyethyl groups, mol %.
*
The values corresponding to the maximum on the curve of parti-
cle distribution by size are given.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 5 2006

4
24
GOROKHOVA et al.
Table 2. Particle size for oligoglycines H-(Gly) -NH and conjugates PHEAA-(Gly) -NH according to light scattering data
m
2
m
2
Particle size, nm*
solution after 24 h
Compound
fresh aqueous solution
water
5 M LiCl
1 M NaHCO3
1 M HCl
TFA × H-(Gly) -NH
591
2320
4460
710
1440
3600
7890
450
1230
2230
2680
780
4480
4150
7850
486
2200
2540
3
2
2
2
TFA × H-(Gly) -NH
4
TFA × H-(Gly) -NH
5
PHEAA–(Gly) -NH (20)
3
2
PHEAA–(Gly) -NH (20)
950
370
1390
710
4
2
PHEAA–(Gly) -NH (20)
380
330
5
2
PHEAA–(Gly) -NH (20)
380
450
640
6
2
PHEAA–(Gly) -NH (20)
700
330
650
7
2
PHEAA–(Gly) -NH (20)
523
510
840
8
2
*
The values corresponding to the maximum on the curve of particle distribution by size are given.
a result of acylation of one or several amide NH groups (Tables 1, 2). The curves of size distribution for freshly
in the peptide chain. If this process does proceed, then prepared solutions of PHEAA–(Gly) -OH and
m
the subsequent interaction with ethanolamine should PHEAA–(Gly) -NH appeared to be wide and had a
m
2
result in shortening of the peptide, as shown in maximum <1000 nm. A comparison with the freshly
Scheme 2 and consequently, the Gly content in the con- prepared solutions indicated that the samples stored for
jugate turned out to be lower than that expected.
24 h at room temperature showed a narrower distribu-
tion curves, the distribution maximum being within
We changed the order of addition of the reagents
upon the synthesis of conjugates to avoid the side pro-
cesses. Namely, the first step included 30–80% substi-
tution of ethanolamine for p-nitrophenyl groups in
PNPA, and H-(Gly) -NH was introduced at the second
4
00–700 nm. An addition of lithium chloride, an agent
suppressing dissociation, resulted in a change in the
distribution curve toward the increase in particle size.
However, an aqueous solution of PHEAA exhibited the
same behavior in control experiments; i.e., we observed
the salt effect on the polymer support rather than on the
peptide chains.
3
2
step. However, in this case, there was no binding of the
peptide to the polymer at all. De novo synthesis of
poly(acrylamide) chain, i.e., the synthesis of N-acryloyl
derivative of the peptide and its subsequent radical
polymerization, is another way to avoid the side reac-
tions. However, polymerization of N-acryloyloligogly-
cines did not proceed either in DMSO upon treatment
Thus, the experiments on light scattering also prove
the absence of oligoglycine association in PHEAA–
(
Gly) -OH and PHEAA–(Gly) -NH . What is the
m m 2
explanation of the association of even short (m = 3–5)
free oligoglycine amides and, at the same time, the
absence of association of the similar or even longer
peptides in polymeric form? First, one molecule of a
conjugate contains only several tens of oligoglycine
with 2,2'-azo-bis-iso-butyronitrile, or in H O/isopro-
2
panol upon treatment with potassium persulfate. We
also failed to carry out copolymerization of N-acryloy-
loligoglycines with acrylamide, though other acryloyl
fragments containing no oligoglycine readily under-
went polymerization under these conditions [15].
chains [about forty for PHEAA–(Gly) -OH (20)]. This
m
amount may appear to be insufficient for the formation
of a stable peptide cluster, as, in the case of tectomers
studied previously, the amount of constituent tectons
was several orders of magnitude higher (a monolayer
polymer with a diameter of ~50 nm is known to contain
~6000 oligoglycine chains) [9]. Second, the dynamics
of association of polymer-bound chains seems to be
unfavorable, as the diffusion of the next chains to the
initial nucleus is insufficiently fast. Therefore, the
nucleus of critical size guaranteeing the irreversible
character of associate formation cannot form. Finally,
we should take into account the entropy factor. The for-
mation of the peptide cluster should result in a decrease
Next, we proceeded to the study of association on a
partially degraded conjugate whose assumed structure
is given in Scheme 2, because the attempts to synthe-
size the oligoglycine polymeric conjugates failed to
result in the ideal target structure PHEAA–(Gly) -NH
m
2
(
20) (Scheme 2). Analytical gel chromatography dem-
onstrated that the retention time of oligoglycine poly-
meric conjugates did not differ from those of poly(2-
hydroxyethylacrylamide) PEAA obtained by modifica-
tion of PNPA with ethanolamine. This proves the
absence of association of peptides in the conjugates
synthesized.
The association of peptide chains within the poly- in degrees of freedom for the poly(acrylamide) chain,
meric conjugate were also studied by light scattering which is usually flexible. Obviously, this loss of
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SPONTANEOUS AND PROMOTED ASSOCIATION
425
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 5 2006

4
26
GOROKHOVA et al.
entropy is not compensated by the gain in energy due to
crystallization of the peptide cluster.
Boc-(Gly) -OH was obtained by the above-
4
described procedure from HCl × H-(Gly) -OH; yield
3
4
6%; mp >220°ë (decomp.); R 0.22 (4 : 3 : 2 iPrOH–
f
Thus, even such short oligoglycines as H-(Gly)m-
1
EtOAc–H O); H NMR (DMSO-d ): 8.290, 8.206,
2
6
NH (m) are capable of self-association promoted by an
2
7
3
.365, and 7.024 (4 × 1 H, 4 t, 4 NH); 3.745, 3.680, and
additional interaction with surface. Moreover, the pres-
ence of several (two, three, or four) peptide chains
bound in one center is not the necessary prerequisite for
association. The association is an equilibrium process,
and, in the case of short peptides, the equilibrium
appear to be shifted to monomers. At the same time,
when the chain length reaches seven Gly residues [10],
the cooperativity of the process grows to the extent that
the monomer–tectomer equilibrium is almost entirely
shifted to the supramolecular compound due to its set-
ting apart to the separate phase or favorable (hydropho-
bic, polar, or biospecific) binding to surface.
NH
.592 (3 × 2 H, 3 d, J 5.8 Hz, 3 CH of Gly), 3.346 (2
2
4
H, br. s, CH Gly ), 1.381 [9 H, s, C(CH ) ]; MALDI
2
3 3
+
+
MS: 370 (M + Na) , 386 (M + K) .
Boc-(Gly) -OH was obtained by the above-
5
described procedure from HCl × H-(Gly) -OH; yield
4
2
0%; mp > 250°ë (decomp.); R 0.15 (4 : 3 : 2 iPrOH–
f
1
EtOAc–H O); H NMR (DMSO-d ): 8.290, 8.115,
2
6
8
3
.063, 8.022, and 6.979 (5 × 1 H, 5 t, 5 NH), 3.750–
NH
.725 (6 H, m, 3 × CH of Gly), 3.582 (2 H, d, J 5.8
2
2
5
Hz, CH Gly ), 3.346 (2 H, br. s, CH Gly ), 1.381 [9 H,
2
2
+
s, C(CH ) ]; MALDI MS: 427 (M + Na) , 443 (M +
3 3
+
K) . Found, %: C 44.38, H 6.28, N 17.29. Calc. for
1
7.29. ë H N O , %: C 44.66, H 6.25, N 17.36.
15 25 5 8
EXPERIMENTAL
Synthesis of oligoglycine hydrochlorides çël ×
H-(Gly) -OH. General procedure. Trifluoroacetic
m
Reagents and solvents were from Merck (Germany),
except for Boc-Gly-ONSu, Boc-Gly-Gly-ONSu, and
PNPA, which were synthesized according to the proce-
dures reported in [17, 18], respectively. Column chroma-
tography was carried out on silica gel 60 (Merck, Ger-
many); gel chromatography, on Sephadex LH-20 (Phar-
macia BioTech, Austria); and TLC, on silica gel 60
acid (5 ml) was added to Boc-(Gly) -OH (1 g), the
m
reaction mixture was kept for 2 h at room temperature
and coevaporated with toluene (20 ml). The residue was
dissolved in 1 M HCl (20 ml), the solution was evapo-
rated to dryness, and the residue was dried in a vacuum
to yield 90–95% of HCl × H-(Gly) -OH.
m
çël × H-(Gly) -OH: mp 240°ë (decomp.); for H-
(
Merck, Germany) precoated aluminium sheets.
3
(
Gly) -OH, mp 215°ë [20]; R 0.38 (3 : 1 : 1 : 1 EtOAc–
3
f
1H NMR spectra were registered on a WM-500
1
Py–ÄÒéç–H O); H NMR (D O): 4.079, 4.040, and
2
2
Bruker (United States) spectrometer at 303 K calibrated
with the chemical shifts of the residual protons (DMSO,
3
(
.920 (3 ×2 H, 3 s, 3 CH of Gly); MALDI MS: 189
2
+
+
+
M) , 212 (M + Na) , 228 (M + K) .
δ = 2.5 ppm; D O, δ = 4.75 ppm). Mass spectra were reg-
2
çël × H-(Gly) -OH: mp >300°ë (decomp.); for H-
4
istered on a Vision-2000 (Thermo Bioanalysis, UK)
time-of-flight mass spectrometer with a laser ionization
of the sample from the matrix surface (MALDI).
(
Gly) -OH, mp 220°ë [20]; R 0.36 (3 : 1 : 1 : 1 EtOAc–
4
f
1
Py–ÄÒéç–H O); H NMR (D O): 4.080, 4.031,
2
2
4
.025, and 3.925 (4 × 2 H, 4 s, 4 CH of Gly); MALDI
2
2
,6-Dihydrobenzoic acid was used as a matrix.
+
+
+
MS: 246 (M) , 269 (M + Na) , 285 (M + K) .
Amino acid analysis of the oligoglycine polymeric
çël × H-(Gly) -OH: mp >300°ë (decomp.); for H-
5
conjugates was carried out on a LC 300 (Biotronik, Ger-
many) instrument supplied with a BTC 2410 (Biotronik,
Germany) column (1 × 125 mm) according to the proce-
dure [19].
(
Gly) -OH, mp 240°ë [20]; R 0.35 (3 : 1 : 1 : 1 EtOAc–
5
f
1
Py–ÄÒéç–H O); H NMR (D O): 4.087, 4.034, 4.024,
2
2
4
.016, and 3.927 (5 × 2 H, 5 s, 5 CH of Gly); MALDI
MS: 303 (M) , 326 (M + Na) , 342 (M + K) .
2
+
+
+
M-(tert-Butyloxycarbonyl)oligoglycines
Synthesis of N-(tert-
butyloxycarbonyl)oligoglycylamides
Boc-(Gly) -NH
Boc-(Gly) -OH
m
m
2
Boc-(Gly) -OH. Boc-Gly-ONSu (14.8 g, 55 mmol)
3
was added to a solution of H-Gly-Gly-OH (6.54 g,
Boc-(Gly) -NH . Triethylamine (7.9 ml, 55 mmol)
3 2
5
0 mmol) in DMSO (25 ml). The reaction mixture was and Boc-Gly-Gly-ONSu (18.1 g, 55 mmol) were added
stirred at room temperature for 24 h, DMSO was to a solution of HCl × NH CH ëONH (5.5 g,
2
2
2
removed by lyophilization, and the residue was chro- 50 mmol) in DMSO (40 ml). The reaction mixture was
matographed on silica gel. Elution with 7 : 10 : 1 : 0.1 stirred at room temperature for 24 h; DMSO was
iPrOH–EtOAc–H O–AcOH gave Boc-(Gly) -OH; yield removed by lyophilization; and the residue was chro-
2
3
7
.15 g (50%); mp 205°ë; R 0.28 (4 : 3 : 2 iPrOH– matographed on silica gel, elution with 32 : 8 : 1
f
1
EtOAc–H O); H NMR (DMSO-d ): 8.085, 7.394, and CHCl –MeOH–H O to give Boc-(Gly) -NH ; yield
2
6
3
2
3
2
6
.983 (3 × 1 H, 3 t, 3 NH), 3.686 and 3.573 (2 × 2 H, 2 d, 11.7 g (82%); mp 215°ë; R 0.35 (32 : 8 : 1 CHCl –
f
3
NH
3
1
J 5.8 Hz, 2 CH of Gly), 3.344 (2 H, br. s, CH Gly ), MeOH–H O); H NMR (DMSO-d ): 8.038 (2 H, t,
2
2
2
6
+
1
.381 [9 H, s, C(CH ) ]; MALDI MS: 313 (M + Na) , CONH ), 7.185, 7.052, and 6.998 (3 × 1 H, 3 t, 3 NH),
3
3
2
+
NH
3
29 (M + K) .
3.729, 3.620, and 3.577 (3 × 2 H, 3 d, J 5.7 Hz, 3 ×
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 5 2006

SPONTANEOUS AND PROMOTED ASSOCIATION
427
CH of Gly), 1.385 [9 H, s, C(CH ) ]; MALDI MS: 312 (D O): 4.080, 4.026, 3.957, and 3.928 (4 × 2 H, 4 s, 4
2
3 3
2
+
+
+
+
(
M + Na) , 328 (M + K) .
Boc-(Gly) -NH was obtained by the above-
described procedure from TFA × H-(Gly) -NH and
CH of Gly); MALDI MS: 245 (M) , 268 (M + Na) ,
84 (M + K) .
2
+
2
4
2
3
2
TFA × H-(Gly) -NH : mp >300°ë (decomp.); R
5
2
f
1
Boc-Gly-ONSu; yield 76%; mp >230°ë (decomp.); R 0.20 (3 : 1 : 1 : 1 EtOAc–Py–ÄÒéç–H O); H NMR
f
2
1
0
(
.23 (32 : 8 : 1 CHCl –MeOH–H O); H NMR (D O): 4.075, 4.065, 4.057, 3.995, and 3.840 (5 × 2 H,
3 2
2
+
+
DMSO-d ): 8.156, 7.191, 7.043, and 6.998 (4 × 1 H, 4 5 s, 5 CH Gly); MALDI MS: 302 (M) , 325 (M + Na) ,
6
2
+
t, 4 NH), 8.050 (2 H, t, CONH ), 3.749, 3.726, 3.621, 341 (M + K) .
2
NH
and 3.585 (4 × 2 H, 4 d, J 5.7 Hz, 4 × CH of Gly),
2
TFA × H-(Gly) -NH : mp >300°ë (decomp.); R 0
+
6
2
f
1
3
.385 [9 H, s, C(CH ) ]; MALDI MS: 369 (M + Na) ,
1
3
3
(
4
3 : 1 : 1 : 1 EtOAc–Py–ÄÒéç–H O); H NMR (D O):
+
2 2
85 (M + K) .
Boc-(Gly) -NH was obtained by the above-
.087, 4.038, 4.030, 4.015, 3.953, and 3.928 (6 × 2 H,
+
+
5
2
6 s, 6 CH Gly); MALDI MS: 359 (M) , 382 (M + Na) ,
2
+
described procedure from TFA × H-(Gly) -NH and
4
2
398 (M + K) .
Boc-Gly-ONSu; yield 73%, mp >250°ë (decomp.); R
f
1
TFA × H-(Gly) -NH : mp >300°ë (decomp.); H
NMR (D O): 4.075 (br. s, CH of Gly); MALDI MS:
1
7
2
0
.20 (24 : 8 : 1 CHCl –MeOH–H O); H NMR
3 2
2
2
(
DMSO-d ): 8.156, 8.144, 7.191, 7.043, and 6.998 (5 ×
6
+
+
+
4
16 (M) , 439 (M + Na) , 455 (M + K) .
TFA × H-(Gly) -NH : mp > 300°ë (decomp.); H
1
H, 5 t, 5 NH), 8.050 (2 H, t, CONH ), 3.755–3.615 (8
2
1
NH
H, m, 4 × CH of Gly), 3.585 (2 H, d, J 5.7 Hz, CH
8
2
2
2
5
NMR (D O): 4.070 (br. s, CH of Gly); MALDI MS:
of Gly ), 1.385 [9 H, s, C(CH ) ]; MALDI MS: 426
2 2
3
3
+
+
+
+
+
473 (M) , 496 (M + Na) , 512 (M + K) .
(
M + Na) , 442 (M + K) . Found, %: C 44.73, H 6.55,
N 20.80. Calc. for C 20.80. C H N O , %: C 44.77, H
Oligoglycinamides with m = 5–8 were additionally
15
26
6
7
6
.51, N 20.88.
Boc-(Gly) -NH was obtained by the above-
described procedure from TFA × H-(Gly) -NH and
Boc-Gly-ONSu. During the reaction process, the prod-
uct formed a hardly soluble precipitate. The reaction
mixture was lyophilized; the residue was washed with
purified by the following procedure: TFA × H-(Gly) -
m
NH (10 mmol) was dissolved in H O (10 ml), 1 M
2
2
6
2
NaHëé (20 ml) was added to the resulting solution,
3
5
2
and, after 1 h, the precipitated H-(Gly) -NH was fil-
m
2
tered and dried in a vacuum; yield 70–75%.
Synthesis of oligoglycine polymeric conjugates
MeOH, filtered, and dried in a vacuum; yield 50%, PHEAA–(Gly) -OH and PHEAA–(Gly) -NH .
m
m
2
mp >250°ë (decomp.); R 0.17 (24 : 8 : 1 CHCl – General procedure. Calculated amounts of peptides
f
3
1
MeOH–H O); H NMR DMSO-d ): 7.060 (~7 H, br. m, HCl × H-(Gly) -OH (0.05, 0.1, or 0.2 mol/mol of
2
6
m
7
%
× NH), 3.610 (~12 H, br. m, 6 × CH of Gly). Found, PNPA) or TFA × H-(Gly) –NH (0.2 or 0.5 mol/mol of
2
m 2
: C 44.42, H 6.39, N 21.29. Calc. for C H N O , %: PNPA) and NEt (1 mol/mol of peptide) were added to
1
7
29
7
8
3
C 44.44, H 6.36, N 21.34.
a solution of PNPA in DMSO (10 mg/ml). The reaction
Boc-(Gly) -NH was obtained by the procedure mixture was kept at 40°ë, the reaction was monitored
7
2
described above for Boc-(Gly) -NH from TFA × by TLC. After the disappearance of free peptide, EA
6
2
H-(Gly) -NH and Boc-Gly-ONSu; yield 37%, (300 µl) was added, and the reaction mixture was incu-
6
2
mp >250°ë (decomp.). Found, %: C 44.09, H 6.34, N bated for 24 h at 50°ë. The product was isolated by
2
2
1.52. Calc. for C H N O , %: C 44.18, H 6.24, N exclusion chromatography on Sephadex LH-20, elution
19 32 8 9
1.69.
Boc-(Gly) -NH was obtained by the procedure
with 1 : 1 MeCN–H
O; yields of conjugates 75–90%.
2
1
3
PHEAA–(Gly) -NH (20): C NMR (D O): 183.0
8
2
3
2
2
described above for Boc-(Gly) -NH from TFA × H- (CONH ), 177.0 and 176.5 (CONH, PHEAA), 172.0
6
2
2
(
Gly) -NH
and Boc-Gly-ONSu; yield 35%, and 171.1 (CONH, Gly), 59.9 (CHOH, PHEAA), 45.4
7
2
mp >250°ë (decomp.). Found, %: C 43.93, H 6.22, N (CH, PHEAA), 43.3, 42.5, and 41.5 (HNCH CO, Gly),
2
2
2
1.87. Calc. for C H N O , %: C 43.98, H 6.15, N 41.3 (NCH , EA), 35.8 (CH , PHEAA). Amino acid
21 35 9 1
2 2
1.98.
Synthesis of trifluoroacetates of oligoglycine
analysis: 0.39 µmol Gly/mg of sample (93% of theory).
PHEAA–(Gly) -NH (50). Amino acid analysis:
3
2
amides TFA × H-(Gly) -NH . General procedure. 0.52 µmol Gly/mg of sample (61% of theory).
m
2
Trifluoroacetic acid (5 ml) was added to Boc-(Gly) -
m
Dynamic light scattering of the aqueous solutions
NH (1 g). The reaction mixture was kept for 2 h at
2
of oligoglycines, oligoglycylamides, and their poly-
room temperature and coevaporated with toluene
meric conjugates, PHEAA–(Gly) -OH and PHEAA–
m
(
(
20 ml). The residue was stirred for 2 h with Öt O
2
(
Gly) -NH , was studied by a Coulter N4MD (Beck-
m 2
10 ml), filtered, and dried in a vacuum; yield 90–95%.
man Instruments, United States) automatic submicron
particle analyzer (He-Ne laser, λ 632.8 nm, detection
angle 62.5°, measurement interval 3–3000 nm). Solu-
tions with a concentration of 4 mg/ml were analyzed.
The solutions were prepared using the water purified by
TFA × H-(Gly) -NH : mp 230°ë (decomp.); R 0.55
3
2
f
1
(
4
3 : 1 : 1 : 1 EtOAc–Py–ÄÒéç–H O); H NMR (D O):
2 2
.080 4.056, and 3.930 (3 × 2 H, 3 s, 3 CH of Gly);
2
+
+
+
MALDI MS: 188 (M) , 211 (M + Na) , 227 (M + K) .
TFA × H-(Gly) -NH : mp >250°ë (decomp.), R Milli-Q. The experiments were carried out immediately
.46 (3 : 1 : 1 : 1 EtOAc–Py–ÄÒéç–H O); H NMR after the preparation of the solutions and also after 24-h
4
2
f
1
0
2
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 5 2006

4
28
GOROKHOVA et al.
incubation of solutions at room temperature. LiCl,
LiBr, or conc. HCl were added to study the effect of
medium on the behavior of the peptides in solutions.
The concentrations of solutions and the data on the light
scattering are given in Tables 1, 2.
4. Franco, L., Subirana, J., and Puiggali, J., Polymer, 1998,
vol. 39, pp. 5553–5560.
5. Avignon, M. and Garrigou-Lavrange, C., Spectrochim.
Acta, Part A: Mol. Spectroscopy, 1971, vol. 27, pp. 297–
3
09.
6
7
. Okabayashi, H., Ohshima, K., Etori, H., Debnath, R.,
Taga, K., Yoshida, T., and Nishio, E., J. Chem. Soc.,
Faraday Trans., 1990, vol. 86, pp. 1561–1567.
. Etori, H., Taga, K., Watanabe, K., Okabayashi, H., and
Oshima, K., J. Chem. Soc., Faraday Trans., 1997,
vol. 93, pp. 313–319.
Analytical gel chromatography of the resulting
compounds was carried out using an LKB 2140 chro-
matograph equipped with a TSK G 4000SW column
(
LKB-Produkter AB, Bromma, Sweden, 7.5 × 300mm);
eluent 0.2 M NaCl, 1 ml/min; UV detection at 210 nm.
The values of retention times for PHEAA–(Gly) -OH
m
8. Shimizu, T., Kogiso, M., and Masuda, M., J. Am. Chem.
Soc., 1997, vol. 119, pp. 6209–6210.
and PHEAA–(Gly) -NH conjugates and for PHEAA
are within the range of 9.3–9.6 min.
m
2
9
. Tuzikov, A., Chinarev, A., Gambaryan, A., Oleinikov, V.,
Klinov, D., Matsko, N., Kadykov, V., Ermishov, M.,
Demin, I., Demin, V., Rye, P., and Bovin, N., ChemBio-
Chem, 2003, vol. 4, pp. 147–154.
Atomic force spectroscopy. Sodium bicarbonate
(
2 equiv per amino group of peptide) was added to an
aqueous solution of TFA × H-(Gly) -NH (1 mg/ml).
m
2
1
1
0. Simrad, M., Su, D., and Wuest, J., J. Am. Chem. Soc.,
991, vol. 113, pp. 4696–4698.
1. Tsygankova, S., Zaitsev, I., Chinarev, A., Tuzikov, A.,
Severin, N., Kalachev, A., Rabe, J., and Bovin, N., Nano-
Lett., 2005 (in press).
The resulting solution (10 µl) was applied on mica sur-
face (PLANO W/Plannet, Germany). After 10 min, the
unabsorbed material was removed by an argon stream.
The resulting samples were dried by a nitrogen flow.
The samples were imaged with a NanoScope II (Digital
Instruments, Santa Barbara, United States) instrument
with a scanning rate of 3–7 Hz and a scanning angle of
1
1
1
1
2. Keller, B., Sauer, N., and Lamb, C.J., EMBO J., 1988,
vol. 7, pp. 3625–3633.
3. Keller, B., Templeton, M.D., and Lamb, C.J., Proc. Natl.
Acad. Sci. USA, 1989, vol. 86, pp. 1529–1533.
4. Coulter Scientific Instruments, ATTN: Technical com-
munications, Florida: BUSINESS REPLY MAIL,
Hialeah, 1986.
1
80° or 270°.
ACKNOWLEDGMENTS
The work was supported by the Russian Foundation 15. Bovin, N., Glycoconjugate J., 1998, vol. 15, pp. 431–
for Basic Research, project no. 04-03-032683 and by
436.
the grant for Molecular and Cell Biology of the Presid- 16. Arshady, R., Adv. Polym. Sci., 1994, vol. 111, pp. 1–41.
ium of Russian Academy of Sciences.
1
7. Gershkovich, A.A. and Kibirev, V.Ch., Khimicheskii sin-
tez peptidov (Chemical Synthesis of Peptides), Kiev:
Naukova Dumka, 1992.
8. Bovin, N., Korchagina, E., Zemlyanukhina, T., Byram-
ova, N., Galanina, O., Zemlyakov, A., Ivanov, A., Zubov,
V., and Mochalova, L., Glycoconjugate J., 1993, vol. 10,
pp. 142–151.
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9. Tugit, A. and Scheffler, J.-J., Eur. J. Biochem., 1982,
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RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 5 2006