Peptide derivatives of tylosin-related macrolides

Article abstract of DOI:10.1134/S1068162007020033

Approaches to the synthesis of model compounds based on the tylosin-related macrolides desmycosin and O-mycaminosyltylonolide were developed to study the conformation and topography of the nascent peptide chain in the ribosome tunnel using specially designed peptide derivatives of macrolide antibiotics. A method for selective bromoacetylation of desmycosin at the hydroxyl group of mycinose was developed, which involves preliminary acetylation of mycaminose. The reaction of the 4″-bromoacetyl derivative of the antibiotic with cesium salts of the dipeptide Boc-Ala-Ala-OH and the hexapeptide MeOTr-Gly-Pro-Gly-Pro- Gly-Pro-OH led to the corresponding peptide derivatives of desmycosin. The protected peptides Boc-Ala-Ala-OH, Boc-Ala-Ala-Phe-OH, and Boc-Gly-Pro-Gly-Pro- Gly-Pro-OH were condensed with the C23-hydroxyl group of O- mycaminosyltylonolide.

Full text of DOI:10.1134/S1068162007020033

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2007, Vol. 33, No. 2, pp. 218–226. © Pleiades Publishing, Inc., 2007.
Original Russian Text © G.A. Korshunova, N.V. Sumbatyan, N.V. Fedorova, I.V. Kuznetsova, A.V. Shishkina, A.A. Bogdanov, 2007, published in Bioorganicheskaya Khimiya,
2007, Vol. 33, No. 2, pp. 235–244.
Peptide Derivatives of Tylosin-Related Macrolides
G. A. Korshunova1, N. V. Sumbatyan, N. V. Fedorova, I. V. Kuznetsova,
A. V. Shishkina, and A. A. Bogdanov
Belozersky Research Institute of Physicochemical Biology,
Faculty of Chemistry, Moscow State University, Vorob’evy Gory, Moscow, 119992 Russia
Received September 26, 2005; in final form, September 6, 2006
Abstract—Approaches to the synthesis of model compounds based on the tylosin-related macrolides desmy-
cosin and O-mycaminosyltylonolide were developed to study the conformation and topography of the nascent
peptide chain in the ribosome tunnel using specially designed peptide derivatives of macrolide antibiotics.
A method for selective bromoacetylation of desmycosin at the hydroxyl group of mycinose was developed,
which involves preliminary acetylation of mycaminose. The reaction of the 4''-bromoacetyl derivative of the
antibiotic with cesium salts of the dipeptide Boc-Ala-Ala-OH and the hexapeptide MeOTr-Gly-Pro-Gly-Pro-
Gly-Pro-OH led to the corresponding peptide derivatives of desmycosin. The protected peptides Boc-Ala-Ala-
OH, Boc-Ala-Ala-Phe-OH, and Boc-Gly-Pro-Gly-Pro-Gly-Pro-OH were condensed with the C23-hydroxyl
group of O-mycaminosyltylonolide.
Key words: tylosin, desmycosin, O-mycaminosyltylonolide, peptide derivatives of macrolides
DOI: 10.1134/S1068162007020033
INTRODUCTION
rial ribosome have recently been obtained [2, 3]. The
X-ray diffraction analysis of these complexes with a
resolution of about 3Å showed that antibiotics are
bound in the ribosomal tunnel near PTC [4, 5]. In this
case, the disaccharide substituent at C5 of Tyl extends
along the tunnel toward PTC, and the mycinose residue
at C23 of the lactone ring is directed toward the tunnel
exit.
The antibiotic tylosin belongs to the class of mac-
rolides, which can bind to the large subunit of bacterial
ribosome and inhibit protein biosynthesis [1].2 Tyl (I) is
a derivative of 16-membered lactone that has a
mycarosylmycaminose disaccharide residue attached
at C5 position and a mycinose monosaccharide residue,
at C23 position.
The Tyl-related antibiotics contain the same lactone
ring with a smaller number of carbohydrate residues:
Des (II) is devoid of a mycarose residue, and OMT
(III) lacks the residues of mycarose and mycinose.
The known spatial orientation of Tyl in the ribosome
tunnel allows one to construct peptide derivatives of the
antibiotic at positions C20 and C23 whose peptide frag-
ments extend, correspondingly, toward the PTC (arbi-
trarily designated as “upstream the tunnel”) and in the
opposite direction (“downstream the tunnel”). These
peptide fragments would modulate to some extent the
nascent peptide chain in the ribosomal tunnel both near
the PTC and nearby the tunnel exit.
Macrolide antibiotics are of interest not only as thera-
peuticals but also as convenient tools for studying the
functional aspects of protein biosynthesis. A structural
analysis of the complexes of ribosomal subunits with
antibiotics is also important for understanding the
molecular mechanisms of the binding of antibiotics and
the reasons for antibiotic resistance, as well as for the
efficient design of drugs. Crystalline complexes of Tyl
and other antibiotics with the 50S subunit of the bacte-
These peptide derivatives of macrolides may serve
as useful tools for studying the conformation and
topography of a polypeptide being synthesized in the
ribosomal tunnel. Previously we have synthesized pep-
tide derivatives of Tyl and Des using their aldehyde
groups in C19 position [6].
1
Corresponding author; phone: +7 (495) 939-5520; fax: +7 (495)
939-3181; e-mail: korsh@genebee.msu.ru
2
Abbreviations: βAla, β-alanine; DCC, N,N'-dicyclohexylcarbodi-
imide; Des, desmycosin; DIEA, diisopropylethylamine; DMAP,
4-dimethylaminopyridine; DNPH, 2,4-dinitrophenylhydrazine;
Glyc, glycolyl; HBTU, N-hydroxybenzotriazolyluronium
hexafluorophosphate; MeOTr, 4-monomethoxytrityl; OMT,
O-mycaminosyltylonolide; PTC, the peptidyl transferase center;
Tyl, tylosin. All amino acids of the L series.
Here we describe the synthesis of a number of pep-
tides and approaches to their conjugation with Des (II)
and OMT (III) at the hydroxy groups at ë4'' and C23,
respectively.
218

PEPTIDE DERIVATIVES OF TYLOSIN-RELATED MACROLIDES
219
O
20
21
CH
O
22
H C
3
3
11
9
19
12
H C
H C
7
3
3
6
10
8
O
3'''
H C
HO 2'
3
O 1'''
23
CH
13
3
H C
N
2'''
3
14
5
O
O
3'
18
4
HO
O
OH
3''
O
3
4'
CH
O
O
5'''
4H''' C
2''
1''
1'
1
3
3
15
5'
16
CH
3
O
OH
2
OH
O
5''
CH
4''
3
3
CH
17
Mycinose
Mycaminose
Mycarose
(I)
O
20
21
CH
O
22
11
9
3
12
H C
3
19
H C
H C
3
7
3
6
10
O
8
H C
23
3
O 1''
CH
3
13
H C
N
3''
HO
2''
3
2'
HO
5
14
3'
O 18
O
4
O
OH
4'CH
O
4''
O
5''
1'
H C
3
16
3
15
3
1
2
5'
O
OH
CH
3
Mycaminose
17
Mycinose
(II)
O
20
21
O
22
11
CH
9
3
12
H C
3
19
7
6
8
10
H C
23
3
CH
13
3
H C
N
3
2'
HO
14
5
3'
O 18
O
4
HO
OH
4'CH
5'
O
1'
3
16
3
15
1
2
O
OH
Mycaminose
CH
3
17
(III)
RESULTS AND DISCUSSION
confirmed by mass spectrometry and 1ç and 13C NMR
spectra.
The initial stage of the study involved the synthesis
of the starting antibiotics Des (II) and OMT (III),
which can arise during an acidic hydrolysis of Tyl
[7, 8].
We chose the strategy of the synthesis of Des and
OMT peptide derivatives on the basis of the known
structures of the antibiotics and the available literature
data on the reactivity of their functional groups. The
Des molecule has three reactive hydroxy groups: 2'-
and 4'-OH groups in the mycaminose residue and
4''-hydroxy group in the mycinose residue. In the con-
text of the problem under study, the 4''-hydroxy group
is well suited for the introduction of peptide fragments
The maintaining of Tyl for 72 h in a hydrochloric
acid solution under mild conditions (pH 2.8, 20°C) gave
chromatographically homogeneous Des (yield 92%),
which was identical in characteristics to that described
in literature [7].
The preparation of pure OMT (III) meets some dif- that would occupy the expected downstream-the-tunnel
ficulties. Acidic hydrolysis of Tyl under rigorous condi- position. A great number of biologically active Des
tions (pH 1.7, 100°ë, 50 h) [8] was accompanied by the derivatives at different functional groups, including the
formation of large amounts of by-products, which 4''-hydroxy group of mycinose, have been reported;
required a thorough separation of the mixture on a silica however, no peptide derivatives at this position are
gel column under the conditions of gradient elution. known. The 2'- and 4'-hydroxy groups of mycaminose
The homogeneous substance isolated had a higher are more readily acylated than the 4''-hydroxy group of
melting temperature (132–135°C) compared to that mycinose [9]. We used this fact when developing the
reported for OMT in literature (115–118°C). Therefore, approaches to the synthesis of 4''-é-peptidyldesmy-
the structure of the antibiotic obtained was additionally cosins: the introduction of 2',4'-diprotected Des into the
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220
KORSHUNOVA et al.
As mentioned above, peptide derivatives of mac-
Desmycosin (II)
rolides can serve as tools for studying the conformation
and functioning of the nascent peptide chain in the ribo-
somal tunnel. Therefore, peptides of different structures
and spatial organization, in particular, those tending to
form α helices, β structures, and unordered structures,
are of interest. In the choice of these peptides, we were
followed the general theoretical concepts on amino acid
residues that prefer a particular secondary structure [13].
2',4'-di-OAc-Des (VIII) 2',4'-di-O-(Boc-βAla)-Des (IX)
2'-O-(Boc-βAla)-Des (X)
4'-O-(Boc-βAla)-Des (XI)
The peptides (IV) and (V) were synthesized by the
classical method using DCC in the presence of HOBt.
The reactions were carried out in DMF under cooling
(0°C), which ensures only a negligible racemization
degree [14]. Carboxyl groups were protected by esteri-
fication. The synthesis of peptide (V) was carried out
from the C-terminus to obtain first Boc-Ala-Phe-OEt
(Va), which was then coupled with Boc-Ala-OH after
the removal of its Boc-group by TFA. The peptides
esters were saponified with alcoholic alkali. As a result,
peptidyl acids (IV) and (V) necessary for the conjuga-
tion with OMT were obtained. For the modification of
Des, dipeptide (IV) was converted into the correspond-
ing cesium salt [10, 15].
Scheme 1.
reaction with bromoacetyl bromide was considered
with the aim of subsequent acylation of the resulting
Des bromoderivative by cesium salts of carboxylic
acids [10].
OMT (III) has a primary hydroxy group at C23,
which can be selectively acylated by highly reactive
reagents both with the preliminary protection of the
mycaminose hydroxy groups and without it [11].
Dipeptide derivatives of the antibiotic in position 23
obtained by the acylation of the amino group preliminarily
introduced into this position have been reported [12]. We
considered this pathway of synthesis to be rather com-
plicated and, therefore, used another approach, which
involved the direct acylation of 23-hydroxyl with the
preliminary blocking of the 2',4'-hydroxy functions of
mycaminose.
We considered two possible ways when developing
the approaches to the synthesis of peptidyl macrolides.
One approach consists in the attachment of the first
from the C-end amino acid to the antibiotic followed by
a stepwise elongation of the chain by the methods of
classical peptide chemistry. Another way involves the
acylation of the antibiotic by the preliminarily synthe-
sized peptide. We thought the first way to be more
promising, since the activation of amino acids used in
this method, unlike that of peptides, would be accom-
panied by a lesser racemization. In addition, this
method enabled one to use a combinatorial approach to
obtain a great number of analogues.
Our preliminary studies showed that it is possible to
obtain peptidyl macrolides in this way using Boc-
amino acids; however, under standard conditions, with
the removal of the protection group, antibiotics under-
went a severe degradation and resinification, which
made us to abandon this approach and seek for other
suitable protecting groups.
The second approach, namely, the acylation of anti-
biotics by the ready model peptides, proved to be more
successful. To this end, we used short fragments (IV)–
(VII), which were obtained in the synthesis of more
long target peptide sequences:
Peptide (VI) was obtained by the solid-phase syn-
thesis using Merrifield resin [16], Boc-amino acids, and
DCC as an activating agent. Boc groups were elimi-
nated by a solution of hydrogen chloride in dioxane,
and the peptide was cleaved from the polymer by trans-
esterification with methanol in the presence of DIEA.
Purification by column chromatography on silica gel
gave peptide (VI) as a homogeneous substance (by
TLC and HPLC). The structure of the peptide was con-
firmed by amino acid analysis and mass spectrometry.
As mentioned above, the use of the Boc-group for
the synthesis of peptidyl macrolides is not optimal, and,
therefore, a 4-monomethoxytrityl group was in some
cases substituted for Boc group, which can be removed
under milder conditions [17]. For example, the treat-
ment of the deblocked Boc-hexapeptide (VI) with 4-
methoxytriphenylchloromethane in DMF for 16 h at
40°C led to the peptide (VII). It was purified on a silica
gel column; however, the mass spectrum of peptide
(VII) showed only the peak corresponding to the detri-
tylated derivative.
Peptide derivatives of Des were synthesized as fol-
lows. At the first step, the reaction of the antibiotic with
acetic anhydride resulted in a 2',4'-diacetyl derivative of
Des (VIII), whose molecular mass was confirmed by
mass spectrometry (Scheme 1). It has earlier been
shown that the 4''-hydroxyl group of mycinose is not
affected under the acetylation of diethyl acetal of the
antibiotic by acetic anhydride [9]. This fact was con-
firmed by the results of the Des acylation by anhydride
(ÇÓÒ-βAla)2O. It was shown by NMR spectroscopy that
the acylation proceeds strictly at 2'- and 4'-hydroxyl
groups of mycaminose without affecting the mycinose
4''-hydroxyl. Even with a twofold excess of the acylat-
Boc-Ala-Ala-OH (IV),
Boc-Ala-Ala-Phe-OH (V),
Boc-Gly-Pro-Gly-Pro-Gly-Pro-OH (VI), and
MeOTr-Gly-Pro-Gly-Pro-Gly-Pro-OH (VII).
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PEPTIDE DERIVATIVES OF TYLOSIN-RELATED MACROLIDES
221
2',4'-di-OAc-Des (VIII)
BrCH2C(O)Br
2',4'-di-OAc-4''-O-BrAc-Des (XII)
Boc-Ala-Ala-OCs (XIII)
MeOTr-(Gly-Pro)3-OCs (XIV)
[2',4'-di-OAc-4''-O-(Boc-Ala-Ala-Glyc)-Des] [2',4'-di-OAc-4''-O-(MeOTr-(Gly-Pro)3-Glyc)-Des]
(XV) (XVI)
MeOH
MeOH/H+
4''-O-(Boc-Ala-Ala-Glyc)-Des (XVII)
4''-O-((Gly-Pro)3-Glyc)-Des (XVIII)
Scheme 2.
ing agent, a mixture of 2'- and 4'-isomers (X) and (XI) TLC showed that these derivatives are homogeneous;
instead of the disubstituted derivative (IX) is formed.
they absorb in UV-light and are detected by DNPH and
ninhydrin after exposure in TFA vapors. HPLC indi-
cated that the compounds contained impurities. The
mass spectra of peptidyl desmycosins showed peaks
corresponding to structures (XVII) and (XVIII); i.e.,
deacetylated products and, in the case of (XVII), also a
detritylated derivative. The absence of peaks of com-
pounds (XV) and (XVI) in the mass spectra suggested
that, most probably, the O-acetyl groups in the mycam-
inose residue and the trityl function are very unstable
under the conditions of chromatography on silica gel in
methanol-containing systems. These facts are consis-
tent with the literature data on the lability of 2'- and 4'-
acetyl groups of mycaminose in the presence of metha-
nol [9, 18].
Peptide derivatives of OMT were synthesized by
Scheme 3. OMT was treated with acetic anhydride in
dichloromethane at room temperature as described in
[19] to give a 2',4'-diacetyl derivative of OMT (XIX).
In this case, the primary hydroxyl at C23 does not enter
the reaction [18, 19]. Compound (XIX) was obtained in
a good yield and was homogeneous by TLC and HPLC.
No constants of this compound, except for the chro-
matographic mobility in one system, were given in the
patent [19], and we measured its mass spectrum, which
confirmed the structure (XIX). This derivative was then
coupled with protected peptides by the peptide chemis-
try methods.
The positions of acyl groups in (IX) were confirmed
by the methods of two-dimensional correlation spec-
troscopy COSY and HMBC. The COSY spectra
allowed us to unambiguously assign the signals of
mycinose and two nonequivalently substituted mycam-
inose fragments. In this case, the chemical shifts of the
mycinose ring protons corresponded to the signals of
the starting compound. Two nonequivalent amino sugar
residues were characterized by a considerable low-field
shift of the signal of protons at C2' (δ 4.98 ppm, m) or
at C4' (δ 4.80 ppm, t) relative to the signals of the cor-
responding protons of the starting compound, indicat-
ing that the hydroxyl at C2' or C4' underwent acylation.
This conclusion is confirmed by the presence of cross-
peaks between the signals of carbonyl atoms of acetyl
groups and the protons at C2' or C4'.
The diacetyl derivative of Des (VIII) was then put in
reaction with bromoacetyl bromide at –15°C in the
presence of pyridine (Scheme 2). The mass spectrum of
the bromoacetyl derivative of Des (XII) showed a clear
peak of the target compound, which proves with a high
degree of probability that bromoacetylation occurs at
position 4'' of the mycinose residue of Des.
4''-Bromoacetyl derivative of Des (XII) was a con-
venient starting compound for the attachment of pep-
tide fragments. Dipeptide Boc-Ala-Ala-OH and
hexapeptide MeOTr-Gly-Pro-Gly-Pro-Gly-Pro-OH,
which were introduced into the reaction with the bro-
moacetyl derivative (XII) as Cs salts (XIII) and (XIV)
according to Scheme 2, were used as protected pep-
tides.
We first used DCC as the coupling reagent in the
presence of DMAP as a catalyst for dipeptide Boc-Ala-
Ala-OH (IV). It is known that the condensation by the
DCC method [20] proceeds in high yields but can be
accompanied by a substantial racemization, especially
The reaction was carried out in anhydrous DMF at in the presence of DMAP [21]. To suppress the racemi-
50–60°ë. The proposed reaction products (XV) and zation, the reaction was carried out in dichloromethane
(XVI) were purified by column chromatography on sil- at a low temperature (0°C) in the presence of a mini-
ica gel in systems containing chloroform and methanol. mum amount of base (0.1–0.2 equiv). The isolation and
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 2 2007

222
KORSHUNOVA et al.
OMT (III)
Ac2O
2',4'-di-OAc-OMT (XIX)
a. Boc-Ala-Ala-OH (IV)
b. Boc-Ala-Ala-Phe-OH (V)
c. Boc-Gly-Pro-Gly-Pro-Gly-Pro-OH (VI)
a. 2',4'-di-OAc-23-O-(Boc-Ala-Ala)-OMT (XX)
b. 2',4'-di-OAc-23-O-(Boc-Ala-Ala-Phe)-OMT (XXI)
c. 2',4'-di-OAc-23-O-(Boc-Gly-Pro-Gly-Pro-Gly-Pro)-OMT (XXII)
a. 23-O-(Boc-Ala-Ala)-OMT (XXIII)
b. 23-O-(Boc-Ala-Ala-Phe)-OMT (XXIV)
c. 23-O-(Boc-Gly-Pro-Gly-Pro-Gly-Pro)-OMT (XXV)
Scheme 3.
purification of the proposed reaction product (XX) on of the nascent polypeptide chain in the ribosome tun-
silica gel gave a chromatographically homogeneous nel.
substance, which corresponded to the deacetylated
derivative 23-O-(Boc-Ala-Ala)-OMT (XXIII) as con-
EXPERIMENTAL
firmed by its mass spectrum. We were unable to deter-
mine the optical purity of the compound (as well as of
other peptidyl macrolides) because of a low quantity of
the material. In addition, without depreciating the
importance of this aspect, we should emphasize that
most attention in this study was paid to the search for
approaches to the synthesis of peptidyl macrolides.
The following reagents were used: derivatives of
amino acids (Fluka and Bachem); DCC, bromoacetyl bro-
mide, and DMAP (Merck, Germany); and N-hydro-
xybenzotriazolyluronium hexafluorophosphate (Fluka,
Switzerland). Other reagents, including Tyl (OOO Mosa-
grogen), were of domestic manufacture.
Like the dipeptide (IV), diacetate (XIX) was con-
densed with hexapeptide (VI) for which the conditions
of the reaction (DCC, DMAP, –5°C, the solution in
dichloromethane and DMF) were more appropriate
since the C-terminal proline usually shows no tendency
to racemize [22]. Tripeptide (V) was coupled with diac-
etate (XIX) using the uronium reagent HBTU [23],
which reduces racemization to a minimum but requires
a particular precaution because of its high reactivity.
TLC of peptide derivatives was carried out on silica
gel 60 F254 plates (Merck, Germany), column chroma-
tography, on silica gel 60 (0.063–0.2 mm) (Fluka, Swit-
zerland).
The following solvents systems were used: (1)
14 : 1 : 5 isopropanol–25% ammonia–water; (2) 7 : 3
chloroform–methanol; (3) 9 : 1 chloroform–methanol;
(4) 4 : 1 chloroform–methanol; (5) 50 : 25 : 2 benzene–
acetone–acetic acid; (6) 65 : 4 : 25 chloroform–25%
ammonia–methanol; (7) 8 : 1 : 0.1 dichloromethane–
methanol–25% ammonia; (8) 4 : 1 : 0.06 dichlo-
romethane–methanol–25% ammonia; (9) 4 : 1 ben-
zene–acetone; and (10) 60 : 45 : 20 chloroform–metha-
nol–32% acetic acid.
The resulting OMT peptide derivatives (XX),
(XXI), and (XXII) were purified on a silica gel column.
The chromatographically homogeneous compounds
(by TLC) were analyzed by mass spectrometry. In all
cases, we registered the peaks corresponding to target
compounds (XXIII)–(XXV), which contain no acetyl
groups. Like Des, this can be explained by an increased
lability of the acetyl groups of 3-deoxy-3-dimethylami-
noglucose during the purification on silica gel columns
in methanol-containing systems [9, 18].
Compounds absorbing the UV-light were monitored
using a Brumberg chemiscope. Compounds with a free
α-amino group were detected by ninhydrin reagent
[ninhydrin (0.2 g) in a mixture of acetone (100 ml),
water (5 ml), and acetic acid (5 ml)], and compounds
Thus, using model compounds, we elaborated containing a Boc group were detected by the same
approaches to the synthesis of specially designed pep- method after heating the plate at 90°C. Tyl, Des, and
tide derivatives of macrolides, which can be used as OMT as well as their derivatives were detected using
tools in the study of the conformation and topography 0.4% DNPH solution in 2 M HCl.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 2 2007

PEPTIDE DERIVATIVES OF TYLOSIN-RELATED MACROLIDES
223
Acidic hydrolysis of peptides was executed under Rf 0.26 (2) and 0.3 (7); MS: [M + H]+ 598.7 (100%)
1
standard conditions (6 M HCl, 105°ë, 24 h) in sealed (calc. for C31H51NO10: M 597.8); H NMR, COSY,
ampoules. The content of amino acids in hydrolyzates HSQC (CDCl3, 500 MHz): 9.64 (1 H, s, H20), 7.28
was determined using an amino acid analyzer, model (1 H, d, J 15.0, H11), 6.29 (1 H, d, J 15.0, H10), 5.86
835 (Hitachi, Japan).
(1 H, d, J 10.7, H13), 4.93 (1 H, ddd, J 9.4, 9.4, and 1.7,
H15), 4.22 (1 H, d, J 7.3, H1'), 3.80 (1 H, d, J 10.5, H3),
3.70 (3 H, m, H5, H23), 3.44 (1 H, dd, J 10.0 and 7.7,
H2'), 3.23 (1 H, m, H5'), 3.03 (1 H, t, J 9.0, H4'), 2.90
(1 H, dd, J 17.7 and 9.6, H19), 2.84 (1 H, m, H14), 2.50
(1 H, br s, H8), 2.47 [3 H and 1 H, s and m, N(CH3)2,
H2], 2.34 (2 H, m, H3', H19), 2.10 (1 H, br s, H6), 1.91
(1 H, d, J 16.5, H2), 1.81 (1 H, m, H16), 1.58 (4 H, m,
H4, H7, H16), 1.40 (1 H, m, H7), 1.78 (3 H, s, H22),
1.22 (3 H, d, J 6.4, H6'), 1.17 (3 H, d, J 6.6, H22), 0.97
(3 H, d, J 6.6, H21), 0.90 (3 H, t, J 7.3, H14); 13ë NMR,
INEPT, HSQC (CDCl3, 500 MHz): 203.5 (C9), 203.0
(C20), 173.9 (C1), 148.0 (C11), 141.8 (C13), 135.8
(C12), 118.9 (C10), 104.0 (C1'), 81.2 (C5), 75.1 (C15),
73.3 (C5'), 70.9 (C2'), 70.8 (C4'), 70.2 (C3'), 67.0 (C3),
62.4 (C23), 47.2 (C14), 44.5 (C8), 43.7 (C19), 41.7
The optical rotation of peptides was measured on a
Perkin-Elmer polarimeter (model 341) at a wavelength
of 589 nm. Melting points were measured on a PHMK
instrument (VEB Wagetechnic Rapido).
Reversed-phase HPLC was carried out on a Mili-
chrom A-02 chromatograph (Econova) on a ProntoSIL-
120-5-C18 AQ column (2.0 × 75 mm, 5 µm) in a 15 to
70% gradient of B in A for 20 min: (A) 0.1% TFA in
water and (B) 0.1% TFA in acetonitrile; elution rate
100 µl/min; detection at 214 and 300 nm.
Molecular masses were determined by MALDI
TOF mass spectrometry on an Ultraflex instrument
(Bruker Daltonics, Germany) equipped with a laser
(λ 337 nm).
Proton and two-dimensional spectra for solutions of (N(CH3)2), 40.3 (C4), 39.5 (C2), 32.8 (C7), 32.6 (C6),
substances in CDCl3 were recorded at 303 K on a 25.6 (C16), 17.8 (C6'), 17.3 (C21), 13.1 (C22), 9.7
Bruker DRX-500 spectrometer with an operating fre- (C17), 9.0 (C18).
quency of 500.13 MHz for protons; and 13C spectra
(with uncoupling from protons), on a Bruker AM-300
tert-Butyloxycarbonyl-alanyl-alanine (IV)
spectrometer with an operating frequency of 75.43 MHz.
The multiplicities of signals in 13C spectra were deter-
mined by the INEPT procedure. The residual signals of
chloroform (δç 7.27 ppm, δë 77.0 ppm) were used as
internal standards. Two-dimensional spectra were
recorded using standard methods of the Bruker com-
pany (COSY, in the magnitude presentation; for HSQC
Methyl ester of tert-butyloxycarbonyl-alanyl-ala-
nine (IVa). A solution of DCC (3.0 g, 14.5mmol) in
DMF (10 ml) was added to a mixture of Ain-alanine
(1.89 g, 10 mmol) and IIAt (2.0 g, 14.8 mmol) in DMF
(15 ml) under cooling (0°ë) and stirring, and stirring
was continued for 2 h at 0°ë. Then a solution of alanine
methyl ester hydrochloride (1.5 g, 10.7 mmol) and
DIEA (1.82 ml, 10.7 mmol) in DMF (15 ml) was added
to the reaction mixture, and the mixture was kept for 12
h at room temperature. After the completion of reac-
tion, the reaction mixture was filtered, and the filtrate
was diluted with water tenfold and extracted with ethyl
acetate (3 × 100 ml). The ethyl acetate extract was
washed with 0.05 M ç2SO4 (3 × 60 ml), water (60 ml),
5% NaçCO3 (3 × 60 ml), and saturated NaCl solution
(50 ml); dried with anhydrous MgSO4; filtered; and
evaporated on a rotor evaporator. The residue was crys-
tallized with petroleum ether; yield of (IVa) 2.25 g
(82%); Rf (5) 0.80.
1
3
and HMBC, JCH 135 Hz and JCH 8 Hz, respectively,
were used). Chemical shifts are given in ppm, and spin–
spin coupling constants, in Hz.
Des (II). A solution of Tyl (5.0 g, 5.47 mmol) in
water (100 ml) and 1 N HCl (10 ml) (pH 3) was kept for
4 days; pH was adjusted to 8.5 by adding dry sodium
bicarbonate; and extracted with chloroform (4 × 20ml).
Combined extracts were dried by magnesium sulfate,
filtered, and evaporated to dryness to give 3.85 g
(91.6%) of (II); mp. 95–120°ë (literature data [7]:
mp 95–115° ë; Rf 0.45 (2) and Rf 0.5 (7). åë: [M + H]+
772.8 (100%) (calc. for C39H65NO14: 771.8).
O-Mycaminosyltylonolide (III). Concentrated sul-
furic acid (600 µl) was added to a solution of Tyl (10 g,
10.92 mmol) in water (500 ml), pH was adjusted to
1.68; the solution was refluxed for 50 h and extracted
with chloroform (3 × 130 ml). The aqueous phase was
adjusted to pH 9.5 with 25% ammonia and again
extracted with chloroform (4 × 200 ml). The combined
organic extracts were dried with anhydrous MgSO4 and
concentrated to one fifth of its volume. Diethyl ether
was added to achieve complete precipitation. After the
precipitate was filtered, washed with ether, and dried,
6.62 g of (III) was obtained. One gram of the product
was purified on a silica gel column (25 × 3 cm) in a gra-
dient of system ‹ 2 to ‹ 8; yield of (III) 380 mg
A solution of (IVa) (274 mg, 1.0 mmol) in metha-
nol (5 ml) was mixed with 2 N NaOH (0.6 ml), and the
mixture was stirred for 3 h. Methanol was evaporated,
the pH of the solution was adjusted to 3 by 0.05 M
ç2SO4, extracted with ethyl acetate (3 × 20 ml); the
organic phase was washed with water and a saturated
NaCl solution; dried by anhydrous MgSO4; filtered;
the filtrate was evaporated to a small volume; and (IV)
was precipitated with petroleum ether; yield 180 mg
(70%); Rf (5) 0.50; mp 95–97°C; [α]2D0 –31° (c 1;
methanol) (literature mp 97–98°C; [α]2D0 –33° (c 1;
(38%); mp 132–135°C (literature [8]: mp 115–118°C; methanol) [24]).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 2 2007

224
KORSHUNOVA et al.
tert-Butyloxycarbonyl-alanyl-alanyl-phenylalanine (V) that at condensation, 20 ml): deblocking with a 4 N HCl
solution in dioxane (30 min), washing with dioxane,
Ethyl ester of tert-butyloxycarbonyl-alanyl-phe-
washing with dichloromethane, neutralization with
nylalanine (Va). A solution of DCC (7.17 g,
10% solution of Et3N in dichloromethane (10 min),
34.8 mmol) in DMF (15 ml) was added to a solution of
washing with dichloromethane, addition of a solution
Boc-alanine (5.10 g, 27 mmol) and HOBt (4.72 g,
of Boc-amino acid in dichloromethane (5 min), addi-
34.8 mmol) in DMF (35 ml) at 0°ë under stirring. The
tion of a solution of DCC in dichloromethane (2 h), and
stirring was continued for 2 h under cooling. Then a
washing with dichloromethane. At each step of the syn-
solution of phenylalanine ethyl ester hydrochloride
thesis, the completeness of the coupling reaction was
(6.5 g, 28.4 mmol) and triethylamine (6.77 ml,
monitored using the Kaiser test [25]. After the comple-
48.3 mmol) in DMF (35 ml) was added to the reaction
tion of the synthesis, the peptidyl polymer was trans-
mixture, and the mixture was stirred for 12 h at room
ferred from the Merrifield flask to a round bottom flask,
temperature. The reaction mixture was treated as in the
and the solution (152 ml, 0.46 mM) was treated at room
case of (IVa). Yield of (Va) 8.43 g (90%); mp 105–
temperature with DIEA in methanol under stirring. The
106°ë (from diethyl ether); Rf 0.91 (4), 0.82 (5). Amino
polymeric support was then filtered, and the filtrate was
acid analysis: Ala 1 (1), Phe 1.15 (1).
evaporated on a rotor evaporator. The product was
Ethyl ester of alanyl-phenylalanine trifluoroace-
obtained as a yellow oil, which was purified by column
tate (Vb). A solution of (Va) (8.1 g, 22.3 mmol) in TFA
chromatography on silica gel (50 g) in system 3 to give
(15 ml) was kept for 1 h at room temperature, evapo-
660 mg (55%) of (VIa) as a white hard oil; mp 150°C;
rated, and the residue was three times coevaporated
Rf (3) 0.58 and (4) 0.85; HPLC: RT 18.2 min (gradient
with methanol and dissolved in methanol (3 ml).
0 to 50% of B for 25 min); [α]2D0 – 48° (c 1; DMF); MS:
The peptide was precipitated with diethyl ether to give
595.2 [å + ç]+ (calc. for C27H42N6O9: 594.7). Amino
7.50 g of a white crystalline (Vb); yield 90%; mp 157–
159°ë. Rf (1) 0.80 and (6) 0.79.
acid analysis: Gly 1.31 (1), Pro 1 (1).
Ethyl ester of tert-butyloxycarbonyl-alanyl-ala-
nyl-phenylalanine (Vc). A solution of DCC (3.83 g,
18.6 mmol) in DMF (8 ml) was added to a solution of
Boc-Ala (2.34 g, 12.4 mmol) and HOBt (2.53 g,
18.6 mmol) in DMF (16 ml) at 0°ë. The stirring was
continued for several hours under cooling. Then a solu-
tion of (Vb) trifluoroacetate (4.95 g, 13.1 mmol) and
triethylamine (3.32 ml, 23.6 mmol) in DMF (16 ml)
was added to the reaction mixture, and stirring was con-
tinued for 12 h at room temperature. The reaction mix-
ture was treated as described for (IVa) to give 5.09 g
(90%) of (Vc); mp 139–140°ë (ethyl acetate–petro-
leum ether); Rf (4) 0.85 and (5) 0.50.
(V) was synthesized as described for (IV) in metha-
nol starting from (Vc) (500 mg, 1.15 mmol) and 2 M
NaOH (690 µl, 1.38 mmol); yield 448 mg (95%);
mp 113–115°ë. Rf (4) 0.73 and (5) 0.21; HPLC:
RT 15.2 min (gradient 0 to 60% of B in A for 20 min);
[α]2D0 −34° (c 1; DMF); MS: 408.6 [å + ç]+ (calc. for
C20H29N3O6: M 407.5. Amino acid analysis: Ala 2.14 (2),
Phe 1 (1).
A solution of (VIa) (260 mg, 0.438 mmol) in meth-
anol (3.5 ml) was treated with 2 M NaOH (0.265 ml,
0.53 mmol). The reaction mixture was stirred for 3 h at
room temperature and then treated as described for (IV);
yield of (VI) 180 mg (precipitation from ethyl acetate
with hexane) (71%); Rf (6) 0.41 and (10) 0.79; HPLC:
RT 13.3 min (gradient 0 to 60% of B for 20 min); MS:
581.8 [å + ç]+ (calc. for C26H40N6O9: 580.6).
4-Monomethoxytritylglycyl-prolyl-glycyl-prolyl-
glycyl-proline (VII). TFA (0.5 ml) was added to
hexapeptide (VI) (50 mg, 0.086 mmol), the mixture
was kept for 1 h at room temperature, and excess TFA
was removed on a rotor evaporator. The residue was
several times coevaporated with absolute methanol,
dissolved in DMF (0.5 ml), after which DIEA (31 µl,
0.18 mmol) and MeOTrCl (50 mg, 0.162 mmol) were
added. The mixture was kept for 16 h at 40°C, evapo-
rated in a vacuum, and the residue was purified by col-
umn chromatography on silica gel in 7 : 3 chloroform–
methanol system containing 0.5% pyridine. Yield of
(VII) 47 mg (73%); Rf (2) 0.43 and (4) 0.35; MS:480.4
[å + ç – MeOTr]+; calc. for C21H31N6O7: M 479.4.
tert-Butyloxycarbonylglycyl-prolyl-glycyl-prolyl-
2',4'-Di-O-acetylDes (VIII). Ac2O (75 µl,
0.8 mmol) was added to a solution of Des (183 mg,
0.25 mmol) in dichloromethane (7 ml) under cooling
with ice and stirring. The stirring was continued for 1 h
at room temperature; the reaction mixture was poured
into a diluted ammonia solution (0°ë) and extracted
twice with dichloromethane. The organic extracts were
dried with anhydrous MgSO4 and evaporated, to give
(VIII); yield of 183 mg (85.5%); Rf 0.91 (2), Rf 0.65 (3),
glycyl-proline (VI)
Methyl ester of tert-butyloxycarbonylglycyl-pro-
lyl-glycyl-prolyl-glycyl-proline (VIa). Ain-Pro-poly-
mer (3.5 g; 0.75 mmol/g of resin; the total amount of
amino acid 2.625 mmol) was placed in a reaction flask,
and dioxane (30 ml) was added. Threefold excesses of
Boc-Gly-OH and Boc-Pro-OH derivatives, and DCC
were applied using a 10% solution of DCC in dichlo-
romethane. The following operations were successively and 0.11 (9); MS: [M + H]+ 856.9 (100%), [M + Na]+
carried out (volume of washing solution was 30 ml and 878.8 (36%); calc. for C43H69NO16: M 855.9.
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PEPTIDE DERIVATIVES OF TYLOSIN-RELATED MACROLIDES
225
2'- and 4'-O-[N-(tert-butyloxycarbonyl)-b-ala- roform. The organic layer was washed with water and
nyl]-Dess (X) and (XI). Boc-βAla-éç (282 mg, dried over anhydrous MgSO4. After the evaporation of
1.5 mmol) was dissolved in a mixture of DMF (50 µl) chloroform, the residue (2.2 mg) was examined by
and dichloromethane (0.5 ml), the solution was cooled chromatography: Rf 0.59 (2); the substance absorbs in
to 0°ë, and a cooled (0°ë) solution of DCC (154.5 mg, UV-light and is developed by ninhydrin and DNPH.
0.75 mmol) in dichloromethane (0.5 ml) was added. HPLC: RT 22.3 min; MS: [M + H]+ 1073.3; calc. for
The mixture was stirred for 1 h under cooling, and a C52H85N3O20: M 1072.13.
solution in anhydrous dichloromethane (0.4 ml) of Des
4''-O-(Glycyl-prolyl-glycyl-prolyl-glycyl-prolyl-
(270 mg, 0.35 mmol) dried over ê2é5 in a vacuum at
glycolyl)-Des (XVIII). Peptide (VII) (12 mg,
63°ë was added. After 10 min, DMAP (17 mg,
0.14 mmol) was added, and the mixture was stirred at
0.016 mmol) was mixed with cesium carbonate
(5.2 mg, 0.016 mmol) and dissolved in ethanol (1 ml).
room temperature for 12 h. The precipitate was filtered
The solution was evaporated to dryness and then twice
off and washed with dichloromethane. The filtrate was
evaporated with dry pyridine. 2',4'-Di-O-acetyl-4''-O-
twice washed with saturated sodium bicarbonate solu-
bromoacetyl-Des (XII) (14 mg, 0.014 mmol) and anhy-
tion and water and dried with anhydrous MgSO4. After
drous DMF (0.5 ml) were added to the residue and kept
evaporation, a chromatographically homogeneous mix-
for 24 h at 50°ë under stirring. The reaction mixture
ture (230 mg) of (X) and (XI) was obtained: TLC:
was evaporated, equal volumes of water and dichlo-
Rf 0.95 (2); HPLC: RT 18.1 min (RT of the starting Des
romethane were added, and the mixture was intensively
11.7 min); MS: [M] 943.4 (calc. for C47H78N2O17: M
shaken. The organic layer was separated, washed with
943.02; 1ç NMR (CDCl3, 500 MHz), δ, ppm: (X): 4.98
water, and dried with molecular sieves 4Å. Purification
(1 H, m, H2'), 4.56 (1 H, d, J 8.0, H1'), 4.31 (1 H, d,
of the product on a silica gel column in system 2
J 8.0, H1'), 3.75 (1 H, s, H3''), 3.55 (1 H, m, H5''), 3.29
yielded 3 mg (14.5%) of (XVIII); MS: [M + H]+ 1293.3;
(1 H, m, J 6.5, H5'), 3.22 (1 H, d, J 7.0, H4''), 3.10 (1 H,
calc. for C62H97N7O22: M 1292.36.
t, J 8.5, C4'), 3.05 (1 H, dd, J 8.0, <2.0, H2''), 2.55 (1 H,
2',4'-Di-O-acetyl-IIO (XIX) [19]. A solution of
OMT (0.5 g, 0.84 mmol) and acetic anhydride (0.63 ml,
6.68 mmol) in acetone (4.5 ml) was stirred for 2 h at
room temperature, evaporated, and then coevaporated
with toluene (2 × 5 ml). The residue was dried in a vacuum
desiccator to get (XIX); yield 0.56 g (98%); Rf 0.9 (2) and
0.3 (9); mp 127–133°C, HPLC: RT 10.4 min (gradient
20–80% B for 20 min); MS: [M + H]+ 682.7 (100%),
and m/z 640.5 [AcOMT] (54%), 598.5 [OMT] (23%);
calc. for C35H55NO12: M 681.8.
t, J 8.0, C3'); (XI): 4.80 (1 H, t, J 8.5, H4'), 4.56 (1 H,
d, J 8.0, H1'), 4.25 (1 H, d, J 8.0, H1'), 3.75 (1 H, s,
H3''), 3.55 (1 H, m, H5''), 3.35 (1 H, m, H2'), 3.29 (1 H,
m, J 6.5, H5'), 3.22 (1 H, d, J 7.0, H4''), 3.05 (1 H, dd,
J 8.0, <2.0, H2''), 2.56 (1 H, t, J 8.5, H3').
2',4'-Di-O-acetyl-4''-O-bromoacetyldesmycosin
(XII). 2',4'-Di-O-acetylDes (171 mg, 0.2 mmol) was
dried by twofold coevaporation with anhydrous pyri-
dine, dissolved in dry dichloromethane (3 ml), mixed
with anhydrous pyridine (50 µl), and cooled to –15°C.
Bromoacetyl bromide (50 µl, 0.95 mmol) was added to
the reaction mixture under stirring. A white solid was
precipitated, which dissolved after addition of dichlo-
romethane (3 ml). After 10 min of stirring at –10 to
−15°C, the reaction mixture was transferred to a mix-
ture of ice and dichloromethane. The organic layer was
separated, washed with 5% sodium bicarbonate and
saturated sodium chloride solution, dried with MgSO4,
and evaporated on a rotary evaporator. Dry ether was
added to the resulting oily residue, and the mixture was
evaporated several times to give (XII) as a light yellow
precipitate; yield: 110 mg (56.3%); Rf 0.31 (9); MS:
[M]+ 975.6 (100%), unidentified peaks m/z 1007.7
(15%) and 1027.7 (37%); calculated for C45H70BrNO17:
M 976.8.
23-O-(tert-Butyloxycarbonyl-alanyl-alanyl)-OMT
(XXIII). A solution of DCC (3.1 mg, 0.015 mmol) in
dichloromethane (50 µl) was added to a solution of
2',4'-di-O-acetyl-OMT (XIX) (7.8 mg, 0.01mmol),
DMAP (0.2 mg), and peptide (IV) (4 mg, 0.015 mmol)
in dichloromethane (100 µl) under stirring and cooling
(0°ë). The reaction mixture was stirred for additional
36 h at room temperature, washed with a diluted ammo-
nia, and evaporated. The residue was purified on a silica
gel column (3 × 0.8 cm) in system 2; Rf 0.95 (2). The
substance was detected with DNPH and ninhydrin; MS,
m/z: 840.4 [M + H]+ (100%), 598.4 [OMT] (68%); calc.
for C42H69N3O14: M 839.5.
23-O-(tert-Butyloxycarbonyl-alanyl-alanyl-phe-
nylalanyl)-OMT (XXIV). A solution of 2',4'-di-O-
acetyl-OMT (XIX) (5 mg, 0.07 mmol), Boc-Ala-Ala-
4''-O-(tert-Butyloxycarbonyl-alanyl-analyl-glyc-
olyl)-Des (XVII). Derivative (XII) (10 mg, Phe-OH (V) (7.4 mg, 0.019 mmol), and HBTU (5 mg,
0.010 mmol) and the preliminarily prepared cesium salt 0.013 mmol) in a mixture of dichloromethane and
of Boc-alanyl-alanine (a methanol solution of the pep- DMF (100 µl) was stirred for 12 h at room temperature
tide was mixed with a 2 M aqueous cesium carbonate and evaporated. Dichloromethane (1 ml) was added,
solution in the equivalent ratio, evaporated to dryness, and the mixture was washed with a diluted ammonia
and thoroughly dried) (3.74 mg, 0.010 mmol) were dis- and dried using molecular sieves 4 Å. After evapora-
solved in DMF (100 µl). The solution was kept in a tion, the residue was fractionated on a silica gel column
thermostat for 68 h at 45°ë. The reaction mixture was (20 × 5 mm) in system 2, collecting the substance with
then diluted with water and twice extracted with chlo- Rf 0.91. The substance was homogeneous by TLC; it
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 2 2007

226
KORSHUNOVA et al.
6. Sumbatyan, N.V., Korshunova, G.A., and Bogdanov, A.A.,
absorbs in UV-light and is detected by DNPH and nin-
hydrin; MS, m/z: 988.0 [M + H]+ 988.0; [M + H – Boc]+
887.9; calc. for C51H78N7O15: M 987.1.
Biokhimiya (Moscow), 2003, vol. 68, pp. 1436–1438.
7. Hamill, R.L., Haney, M.E., McGuire, J.M., and
Stamper, M.C., US Patent 3178341, 1965.
23-O-(tert-Butyloxycarbonyl-glycyl-prolyl-gly-
cyl-prolyl-glycyl-prolyl)-OMT (XXV). A solution of
(XIX) (11.2 mg, 0.015 mmol), DMAP (0.3 mg,
0.0025 mmol), and hexapeptide (VI) (11.6 mg,
0.02 mmol) in a mixture of dichloromethane (100 µl)
and DMF (50 µl) was cooled to –5°ë, and a solution of
DCC (4.1 mg, 0.02 mmol) in dichloromethane (100 µl)
was added under stirring. The reaction mixture was
stirred for additional 12 h at room temperature, washed
with a diluted ammonia over anhydrous MgSO4, and
evaporated. The residue was purified on a silica gel col-
umn (30 × 10 mm) in system 2.A substance with Rf 0.91
was isolated. It was homogeneous by TLC, absorbed
UV-light, and was detected by DNPH and ninhydrin
upon heating; MS, m/z: 1161.2 [M + H]+ (100%),
1183.2 [M + H]+ (54%), 682.7 [Ac2-OMT], 640.9
[OMT]; calc. for C57H89N7O15: M 1160.3.
8. Morin, R. and Gorman, M., US Patent 3 459 853, 1969.
9. Tanaka, A., Watanabe, A., Tsuchiya, T., and Umezawa, S.,
J. Antibiot., 1981, vol. 34, pp. 1381–1384.
10. Gisin, B.F., Helv. Chim. Acta, 1973, vol. 56, pp. 1476–
1482.
11. Kirst, H., GB Patent 2111497, 1983
12. Kirst, H. and Toth, J., US Patent 4459290, 1984.
13. Finkel’shtein, A.V. and Ptitsyn, O.B., Fizika belka: Kurs
lektsii (Physics of Proteins: A Course of Lectures),
3rd Ed., Moscow: Knizhnyi dom Universitet, 2005.
14. Konig, W. and Geiger, R., Chem. Ber., 1970, vol. 103,
pp. 788–798.
15. Wang, S.S., Gisin, B.F., Winter, D.P., Makofske, R.,
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ACKNOWLEDGMENTS
17. Kohli, V., Blocker, H., and Koster, H., Tetrahedron Lett.,
1980, vol. 21, pp. 2683–2686.
18. Jian, T., Phanly, T., Busuyek, M., Hou,Y., Or,Y., Qiu,Y.,
and Vo, N., US Patent 6753415 B2, 2003.
This work was supported by the Russian Foundation
for Basic Research, project nos. 04-04-49480a and
05-04-08131.
19. Fujiwara, T., Watanabe, H., Hirano, T., and Sakak-
ibara, H., GB Patent 2116170A, 1983.
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