Amino acid and peptide derivatives of the tylosin family of macrolide antibiotics modified by aldehyde function

Article abstract of DOI:10.1134/S1068162010020159

Fourteen new functionally active amino acid and peptide derivatives of the antibiotics tylosin, desmycosin, and 5-O-mycaminosyltylonolide were synthesized in order to study the interaction of the growing polypeptide chain with the ribosomal tunnel. The co

Full text of DOI:10.1134/S1068162010020159

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2010, Vol. 36, No. 2, pp. 245–256. © Pleiades Publishing, Ltd., 2010.
Original Russian Text © N.V. Sumbatyan, I.V. Kuznetsova, V.V. Karpenko, N.V. Fedorova, V.A. Chertkov, G.A. Korshunova, A.A. Bogdanov, 2010, published in Bioorganicheskaya
Khimiya, 2010, Vol. 36, No. 2, pp. 265–276.
Amino Acid and Peptide Derivatives of the Tylosin Family
of MacrolideAntibiotics Modified by Aldehyde Function
1
N. V. Sumbatyana, , I. V. Kuznetsovaa, V. V. Karpenkoa, N. V. Fedorovab, V. A. Chertkova,
G. A. Korshunovab, and A. A. Bogdanova,b
a Faculty of Chemistry, Moscow State University, Moscow, 119991 Russia
b Belozersky Institute of Physicochemical Biology, Moscow, 119991 Russia
Received March 18, 2009; in final form, April 28, 2009
Fourteen new functionally active amino acid and peptide derivatives of the antibiotics tylosin, desmycosin,
and 5ꢀOꢀmycaminosyltylonolide were synthesized in order to study the interaction of the growing polypepꢀ
tide chain with the ribosomal tunnel. The conjugation of various amino acids and peptides with a macrolide
aldehyde group was carried out by two methods: direct reductive amination with the isolation of the intermeꢀ
diate Schiff bases or through binding via oxime using the preliminarily obtained derivatives of 2ꢀaminooxyꢀ
acetic acid.
Key words: macrolides, chemical modification, reductive amination, oximes, peptide derivatives, tylosin, desmyꢀ
cosin, 5ꢀOꢀmycaminosyltylonolide
DOI: 10.1134/S1068162010020159
1
INTRODUCTION
interactions between the growing peptide and RT walls
in turn actively affect the functioning of the ribosome
[4–6]. In some cases, such interactions lead to the
arrest of the translation that regulates the synthesis of
certain proteins [7, 8].
The ribosomal tunnel is the least studied functional
center of the ribosome [1]. The RT2 is mainly conꢀ
structed from nucleotide residues of ribosomal RNA
and its entrance part is formed from nucleotides
related to ribosomal PTC. During protein synthesis, a
segment of the growing polypeptide chain with a
length of 25–30 amino acid residues occupies the RT
[2], with its conformational state promoting the free
displacement of the growing polypeptide chain in the
RT, irrespective of its amino acid sequence. On one
hand, currently it has only been established that the
The RNA–protein interactions that take place in
the RT and, in particular, the mechanism of the pasꢀ
sage and the peculiarities of the conformational state
of the growing polypeptide in the RT, remain
unknown to date. Synthetic constructs could be used
as instruments of the investigation to clear up this
problem; on one hand, they could model the elements
of the growing polypeptide chain and, on the other
hand, could specifically bind with the RT regulatory
sites.
amino acid residues of the
the moment of its entrance to the RT are in a
Cꢀterminal dipeptide link at
β
ꢀconꢀ
formation stabilized by the specific contacts with the
RT walls [3], which provides the necessary orientation
of the polypeptide chain. On the other hand, recent
biochemical studies have shown that the RT is not a
passive channel for the passage of the growing peptide:
it is a dynamic functional unit in which the specific
Macrolide antibiotics inhibit protein synthesis due
to the specific interaction with the highꢀaffinity cenꢀ
ter, which is formed by the rRNA of the large ribosome
subunit and is located inside the RT at a distance of
approximately 20 Å from the PTC [9, 10]. Macrolides
are bound in this site either with the vacant or the
active ribosome at the early stages of translation, when
the length of the synthesized peptide chain does not
exceed 2–5 amino acid residues in dependence on the
size of the macrolide cycle and the length of its oliꢀ
gosaccharide chain [10]. A bound antibiotic blocks the
passage of the growing peptide into the channel, which
1
Corresponding author; phone: +7 (495) 939ꢀ5520; fax: +7 (495)
939ꢀ3181; eꢀmail: sumbtyan@belozersky.msu.ru
2
Abbreviations: Aoac, 2ꢀaminooxyacetyl; Des, desmycosin;
DIEA, N,Nꢀdiisopropylethylamine; HBTU, Oꢀ(benzotriazolyl)ꢀ
1,1,3,3ꢀtetramethyluronium hexafluorophosphate; HOBt, 1ꢀ
hydroxybenzotriazol; OMT, 5ꢀ ꢀmycaminosyltylonolide; PTC,
peptidyltransferase center; RT, ribosomal tunnel; Tyl, tylosin; and
Ual, 3ꢀ(uracilylꢀ1ꢀyl)alanine.
O
245

246
SUMBATYAN et al.
stops the translation and leads to the dissociation of anchor for their fixing to a specific RT site [13, 14]. In
the peptidyl–tRNA–ribosome complex [11, 12].
We previously suggested studying the conformation
of the synthetic polypeptide chain in the RT and
establishing the chemical nature of the contacts of the
this case, the main attention was focused on OMT
derivatives in which the peptide chain was directed to
the exit from the RT [14].
The goal of this work was obtaining the amino acid
polypeptide chains with the RT elements by the synꢀ and oligopeptide conjugates of Tyl, Des, and OMT
thesis of the model peptide derivatives of macrolide that differ in their hydrophobicity, charge, and conforꢀ
antibiotics with the peptide part mimicking the growꢀ mational preferences with the use of the most reactive
ing protein chain and the macrolide serving as an aldehyde group of macrolides.
O
20
21
CH3
7
O
22
H3C
11
9
12
19
6
8
10
H3C
H3C
CH3
H3C
2'
13
H3C
O
N
O
4'
23
18
5
14
3'
HO
O
1'''
O
4
O
OH
2'''
3'''
O
O
1''
5''
3''
CH3
2''
CH3
OH
15
1
2
3
HO
4H''' 3C
16
1'
O
OH
5'''
5'
O
CH3
O
4''
Mycaminose
CH3
17
Mycinose
(I)
Mycarose
O
21
CH3
20
O
9
22
H3C
11
12
19
7
6
10
8
H3C
O
H3C
CH3
4'
H3C
H3C
O
N
13
2'
23
5
18
3'
HO
O
1''
14
O
4
OH
3''
2''
O
O
CH3
3
1
2
HO
15
16
1'
O
OH
5'
5''
4H'' 3C
O
CH3
17
Mycaminose
Mycinose
(II)
O
20
21
O
9
22
H3C
CH3
11
19
12
7
6
10
8
CH3
2H' 3C
O
13
H3C
O
N
4'
23
5
18
14
15
3'
HO
O
4
OH
HO
CH3
1
2
3
1'
16
O
OH
5'
CH3
Mycaminose
17
(III)
Tyl [15] (I) contains in its structure a two carbohyꢀ the absence of mycarose and mycinose residues. All of
drate fragment: a disaccharide constructed from the macrolides contain a reactive acetaldehyde group
mycaminose and mycarose residues at position 5 of the at the 6 position.
16ꢀmembered lactone cycle and a residue of mycinose
at the 14 position. Des (II) [16] differs from Tyl by the
On one hand, an Xꢀray analysis of the Tyl complex
with a 50S subunit of ribosome [3, 9, 10] indicates that
absence of a mycarose residue, and OMT (III) [17] by the complex binding to a specific site of the RT leads
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AMINO ACID AND PEPTIDE DERIVATIVES
247
to the orientation of the disaccharide substituent at the acteristic conformational restrictions of the turn angle
5 position of the lactone ring along the PTC (arbiꢀ
trarily “up” the tunnel); the aldehyde group is also
directed up and contacts the adenine base of the
nucleotide residue A2062 and, probably, forms a covaꢀ
lent bond with it [10]. On the other hand, it has
recently been shown that the A2062 residue in 23S
rRNA interacts in a certain manner with the amino
acid residues of the regulatory peptide, the synthesis of
which in the ribosome results in the stop of translation
[18]. In this connection, it was interesting to syntheꢀ
size hybrid molecules by the conjugation of amino
acids, oligopeptides, or their derivatives directly with
the aldehyde group of tylosinꢀseries macrolides.
Moreover, a number of active Tyl derivatives and its
analogues were described in published data. They were
obtained by the modification of the aldehyde group, in
particular, one of the few commercially available antiꢀ
biotics, Tyl analogues, tilmycosin (20ꢀdeoxoꢀ20ꢀ(3,5ꢀ
dimethylꢀ1ꢀpiperidinyl)desmycosin [19–21].
We supposed that the obtainment of amino acid
and peptide derivatives of Tyl, Des, and OMT by the
aldehyde group would proceed so that the peptide
fragment would be directed from the macrolide ring up
the tunnel. To retain the direction, which the syntheꢀ
sized polypeptide chain adapts in the ribosome, it was
necessary to attach the model peptides to the macꢀ
rolide by their Nꢀtermini. We believed that such conꢀ
structs might be useful as instruments for studying the
mechanism of RT functioning.
of the peptide chain due to the presence of a C=N
double bond [23]. Oppositely, in the amino derivatives
of macrolides available based on the second approach
(reductive amination), an ordinary >C–N< bond
would provide for the greater mobility of the peptide
fragment.
We obtained the necessary derivatives of the antibiꢀ
otics by the conjugation of Tyl, Des, and OMT with a
set of amino acids and oligopeptides. First, we were
interested in the amino acids whose side chains bear
aromatic and heterocyclic radicals capable of both
hydrophobic interactions and the formation of hydroꢀ
gen bonds with the rRNA nucleic bases: phenylalaꢀ
nine, tyrosine, and 3ꢀ(uracilꢀ1ꢀyl)alanine.
Despite the aforementioned fact that the spatial
structure of the growing polypeptide chain has not
been established by direct methods, there are weighty
arguments in published data that its short sites, prone
to the formation of αꢀhelices within proteins, are also
in a helical conformation near the macrolideꢀbinding
site [24, 25]. This concept and the accounting for the
conformational characteristics of single amino acid
residues [26] determined our choice of model
sequences (IV)–(VI) used in the synthesis of the Tyl
and Des peptide derivatives. In particular, the HꢀAlaꢀ
AlaꢀPheꢀAlaꢀAlaꢀPheꢀLysꢀOMe
characterized by a high inclination toward the formaꢀ
tion of an ꢀhelix, whereas neither ordered structure is
(V) sequence is
RESULTS AND DISCUSSION
α
possible for HꢀGlyꢀProꢀGlyꢀProꢀGlyꢀProꢀOX (VI
(X = Me) and (VIa) (X = Bzl).
)
We chose two methods of the modification of the
macrolide aldehyde group for the solution of the task.
The first method is the attachment of a substituent
through the =NOCH2CO– linker prepared by the
interaction of the antibiotic aldehyde group with the
amino group of 2ꢀaminooxyacetic acid preliminarily
attached to the αꢀamino group of an amino acid or
peptide [13]. The second method implies the direct
interaction of the macrolide aldehyde group with the
The synthesis of model peptides was carried out as
follows (Scheme 1). The peptides BocꢀAlaꢀAlaꢀPheꢀ
OEt [14] and Bocꢀ(Ala)5ꢀOMe (IVa) [27, 28] were
obtained in a solution starting from HꢀPheꢀOEt and
HꢀAlaꢀOMe by a series of successive couplings with
BocꢀAlaꢀOH with the use of DCC in the presence of
amino acid αꢀamino group leading to a Schiff base folꢀ
lowed by its reduction.
HOBt. The heptapeptide Bocꢀ(GlyꢀPro)3ꢀOMe (VIb
)
was synthesized by the solidꢀphase method; we
described it in [14]. The peptide BocꢀAlaꢀAlaꢀPheꢀ
AlaꢀAlaꢀPheꢀLys(Z)ꢀOMe (Va ) was obtained by a
block method using the scheme [3 + (3 + 1)] starting
from HꢀLys(Z)ꢀOMe and BocꢀAlaꢀAlaꢀPheꢀOH with
the use of HBTU for the coupling of fragments [29].
The inevitable partial racemization of the Phe residues
at these stages was not studied specially; however, all of
the side products, including diastereomers, were sucꢀ
cessfully removed by column chromatography, and the
target heptapeptide was obtained in a chromatographꢀ
In published data, the synthesis of C20ꢀaminoꢀ
derivatives of Tylꢀgroup antibiotics by the reductive
amination of the macrolide aldehyde function with the
use of various primary and secondary amines was
described [19, 20, 22]. However, similar macrolide
conjugates of amino acids and peptides have not yet
been obtained. The modification by these two
methods appears to allow for the preparation of conꢀ
structs with different spatial characteristics and rigidꢀ
ity of the polypeptide chain near the PTC. For examꢀ
ple, the substituted oximes on the basis of the peptide
derivatives of the αꢀamino acid would have the charꢀ ically homogeneous state.
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248
SUMBATYAN et al.
1. BocꢀAlaꢀOH
DCC/HOBt
2. TFA
1. BocꢀAlaꢀOH
DCC/HOBt
2. TFA
HꢀAlaꢀOMe
Hꢀ(Ala)2ꢀOMe
Hꢀ(Ala)3ꢀOMe
1. BocꢀAlaꢀOH
DCC/HOBt
2. TFA
BocꢀAlaꢀOH
DCC/HOBt
TFA
Bocꢀ(Ala)5ꢀOMe
IVa
Hꢀ(Ala)5ꢀOMe
IV
Hꢀ(Ala)4ꢀOMe
(
)
(
)
BocꢀAoacꢀOH
DCC/HOBt
TFA
BocꢀAoacꢀ(Ala)5ꢀOMe
IV
HꢀAoacꢀ(Ala)5ꢀOMe
IV
(
b
)
( c)
1. BocꢀAlaꢀOH
DCC/HOBt
2. TFA
BocꢀAlaꢀOH
DCC/HOBt
HꢀPheꢀOEt
HꢀAlaꢀPheꢀOEt
BocꢀAlaꢀAlaꢀPheꢀOEt
NaOH
1. HꢀLys(Z)ꢀOMe
HBTU
2. TFA
HꢀAlaꢀAlaꢀPheꢀLys(Z)ꢀOMe
BocꢀAlaꢀAlaꢀPheꢀOH
BocꢀAlaꢀAlaꢀPheꢀOH
HBTU
1. NaOH
2. TFA
BocꢀAlaꢀAlaꢀPheꢀAlaꢀAlaꢀPheꢀLys(Z)ꢀOMe
HꢀAlaꢀAlaꢀPheꢀAlaꢀAlaꢀPheꢀLys(Z)ꢀOH
(Vа
)
(Vf)
TFA
BocꢀAoacꢀOH
DCC/HOBt
HꢀAlaꢀAlaꢀPheꢀAlaꢀAlaꢀPheꢀLys(Z)ꢀOH
BocꢀAoacꢀAlaꢀAlaꢀPheꢀAlaꢀAlaꢀPheꢀLys(Z)ꢀOMe
(Vc)
(Vb)
1. H2/Pd
2. TFA
TFA
HꢀAoacꢀAlaꢀAlaꢀPheꢀAlaꢀAlaꢀPheꢀLys(Z)ꢀOMe
HꢀAoacꢀAlaꢀAlaꢀPheꢀAlaꢀAlaꢀPheꢀLysꢀOMe
(Vd)
(Ve)
1. BocꢀGlyꢀOH
DCC
2. TFA
1. BocꢀProꢀOH
DCC
2. TFA
ꢀ
ꢀ
ꢀ
HꢀProꢀGlyꢀPro
P
HꢀPro
HꢀGlyꢀPro
P
P
1. BocꢀGlyꢀOH
DCC
2. TFA
1. BocꢀProꢀOH
DCC
2. TFA
ꢀ
ꢀ
P
HꢀProꢀGlyꢀProꢀGlyꢀPro
HꢀGlyꢀProꢀGlyꢀPro
P
BocꢀGlyꢀOH
DCC
ꢀ
HꢀGlyꢀProꢀGlyꢀProꢀGlyꢀProꢀOMe
BocꢀGlyꢀProꢀGlyꢀProꢀGlyꢀPro
P
(VI)
DIEA/MeOH
TFA
1. NaOH
2. TFA
BocꢀGlyꢀProꢀGlyꢀProꢀGlyꢀProꢀOMe
VI
HꢀGlyꢀProꢀGlyꢀProꢀGlyꢀProꢀOH
VI
(
b
)
( c)
BocꢀAoacꢀOH
DCC/HOBt
TFA
TFA
Bocꢀ(GlyꢀPro)3ꢀOBzl
Hꢀ(GlyꢀPro)3ꢀOBzl
VI
BocꢀAoacꢀ(GlyꢀPro)3ꢀOBzl
VI
HꢀAoacꢀ(GlyꢀPro)3ꢀOBzl
VI
(VId
)
(
а
)
(
e
)
( f)
Scheme 1.
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AMINO ACID AND PEPTIDE DERIVATIVES
249
Compounds (IV), (VIa), and (VIb) were condensed 1.5 equiv of aminooxyacetic acid or aminooxyꢀ
with Bocꢀ2ꢀaminooxyacetic acid to obtain the peptide acetylpeptide were incubated at 50°C for 12 h in a
and amino acid derivatives of Tyl, Des, and OMT by
the first method. The Z and Boc groups were then
consecutively removed, and the peptides with a Nꢀterꢀ
0.4ꢀM sodium acetate buffer (pH 4.7) or, in the case of
the insufficient solubility of the peptide, in a mixture
of this buffer and DMSO (1 : 1). The purification of
minal aminooxy group that can selectively conjugate
with aldehyde carbonyl were obtained.
the resulting derivatives of Tyl (VII)–(IX), Des (
XIII), and OMT (XIV) was carried out by preparative
TLC on silica gel in systems containing chloroform
X)–
(
The reaction of the formation of the macrolide
oximes was carried out under the following conditions and methanol; the yields were 20–25% after the puriꢀ
(Scheme 2): 1 equiv of Tyl (Des or OMT) and fication.
O
O
N
O
O
O
R3
CH3
O
H2N
R3
N
H3C
O
CH3
H3C
O
O
H3C
O
(HꢀAoacꢀPheꢀOMe,
(IVb), (Vd), (Ve), (VIf))
R1
N
CH3
HO
H3C
O
R2
O
H3C
O
O
CH3
CH3
H3C
O
R1
HO
O
OH
)–(III
R2
O
O
CH3
CH3
O
OH
(I
)
CH3
H3C
(
VII)–(XIV
)
H3C
HO
OH
CH3
OH
O
O
O
(I
): R1 =
;
R2 =
O
O
CH3
H3C
H3C
H3C
O
HO
(
II): R1 = H;
III): R1 = H;
R2 =
R2 = H
O
H3C
(
H3C
H3C
OH
O
O
2
VII): R1 =
; R =
; R3 = OH
CH3
(
HO
H3C
OH
O
O
CH3
H3C
H3C
OH
CH3
OH
O
O
2
(
(
(
(
(
(
VIII): R1 =
; R =
; R3 = ꢀPheꢀOEt
HO
O
O
CH3
OH
H3C
H3C
H3C
O
O
IX): R1 =
;
R2 =
R2 =
R2 =
R2 =
R2 =
R2 =
; R3 = ꢀAlaꢀAlaꢀAlaꢀAlaꢀAlaꢀOMe
; R3 = ꢀPheꢀOEt
CH3
OH
HO
O
O
CH3
H3C
H3C
H3C
O
O
O
O
O
O
X
): R1 = H;
HO
O
H3C
H3C
H3C
O
XI): R1 = H;
XII): R1 = H;
; R3 = ꢀAlaꢀAlaꢀAlaꢀAlaꢀAlaꢀOMe
HO
O
H3C
H3C
H3C
O
; R3 = ꢀAlaꢀAlaꢀPheꢀAlaꢀAlaꢀPheꢀLysꢀOMe
; R3 = ꢀAlaꢀAlaꢀPheꢀAlaꢀAlaꢀPheꢀLys(Z)ꢀOMe
HO
O
H3C
H3C
H3C
O
XIIa): R1 = H;
HO
O
H3C
H3C
H3C
O
(
XIII): R1 = H;
XIV): R1 = H;
; R3 = ꢀGlyꢀProꢀGlyꢀProꢀGlyꢀProꢀOBzl
R3 = ꢀPheꢀOEt
HO
O
H3C
R2 = H;
(
Scheme 2.
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250
SUMBATYAN et al.
The homogeneity of the compounds isolated was 3ꢀÅ molecular sieves at room temperature. The resultꢀ
assessed by TLC and HPLC, quantitative amino acid
analysis showed the expected ratio of amino acid resiꢀ
dues in products (VIII)–(XIV), and NMR and massꢀ
spectrometric data were in complete agreement with
the expected structures (Scheme 2).
ing Schiff bases (XV)–(XVIII) were sufficiently stable
in alcohol solutions and in neutral water solutions,
which allowed for their isolation in a homogeneous
state by preparative TLC on silica gel plates. The Des
derivatives (conjugates (XIX) and (XX)) with peptides
(
Vf) and (VIc) were obtained by this method. However,
We applied a procedure similar to that described in
[30] to obtain by the second method the Tyl and Des
derivatives with aromatic amino acids (tyrosine and
phenylalanine) and DLꢀ3ꢀ(uracilꢀ1ꢀyl)alanine, a
nucleoamino acid (Scheme 3). The reaction was carꢀ
ried out in absolute methanol with a twofold excess of
the yields of these compounds were extremely low due
to the formation of a great number of side products in
the reaction mixtures, which was first the consequence
of the hydrolysis of the lactone function by water split
off during the formation of imine on the background
amino acids in the presence of sodium methylate and of methylate.
O
O
CH3
H3C
H3C
H3C
O
CH3
CH3
H3C
H3C
O
N
O
R
HO
O
O
O
O
HO
O
OH
O
H3C
CH3
(
I), (II
)
NH2ꢀR1
(HꢀTyrꢀOH, HꢀPheꢀOH,
HꢀUalꢀOH, (Vf), (VI ))
b
N
R1
O
CH3
H3C
O
H3C
H3C
O
CH3
CH3
H3C
H3C
O
N
O
R
HO
O
OH
O
O
HO
O
(
O
H3C
XV)–(XX)
CH3
NaBH3CN
NH R1
O
CH3
H3C
O
H3C
H3C
O
CH3
CH3
H3C
H3C
O
N
O
R
HO
O
OH
O
O
HO
O
O
H3C
CH3
(XXI), (XXII)
Scheme 3.
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AMINO ACID AND PEPTIDE DERIVATIVES
251
OH
CH3
OH
(I): R =
O
CH3
(
II): R = H
XV): R =
OH
1
CH3
(
; R = –CH(CH2PhOH)COOH
CH
OH3
O
CH3
OH
1
CH3
OH
(
XVI): R =
; R = –CH(CH2Ph)COOH
O
O
CH3
5
6
4
3
2
HN
OH
1
CH3
OH
O
N1
(XVII): R =
; R =
7
O
8
CH3
9
OH
O
(
(
(
XVIII): R = H; R1 = –CH(CH2PhOH)COOH
XIX): R = H; R1 = –CH(CH3)COꢀAlaꢀPheꢀAlaꢀAlaꢀPheꢀLys(Z)ꢀOH
XX): R = H; R1 = –CH2COꢀProꢀGlyꢀProꢀGlyꢀProꢀOH
OH
1
CH3
OH
(XXI): R =
; R = –CH(CH2PhOH)COOH
O
CH3
(
XXII): R = H; R1 = –CH2COꢀProꢀGlyꢀProꢀGlyꢀProꢀOH
Scheme 3. (Contd.).
The Schiff bases (XV) and (XX) were then reduced 202–204 ppm corresponding to a
⎯
CH=O carbon
to the secondary amines (XXI) and (XXII) by sodium
atom and the appearance of a signal at 150–160 ppm
characteristic of the sp2 carbon in Schiff bases and
oximes. In this case, the signal at ~203 ppm correꢀ
sponding to C9 carbon was retained. Two sets of sigꢀ
cyanoborohydride. In parallel, by the example of
XX), the reduction was carried out without the isolaꢀ
tion of the corresponding imine by the direct addition
of sodium cyanoborohydride to the mixture of compoꢀ
nents in which imine was generated in situ as described
earlier [31].
(
nals corresponding to two (Z and E) isomers with a difꢀ
ferent orientation of the substituent are often observed
for oximes and Schiff bases related to Tyl in NMR
spectra [33]. Note that in the case of the solution of
our compounds in CDCl3, only one set of signals is
observed, which appears to be explained by the greater
conformational preferability of one of the isomers
We judged the substitution of the C20ꢀmacrolide
aldehyde group by a comparison of the NMR spectra
of the new compounds with the spectra of the starting
Tyl and Des macrolides. The complete assignment of
signals in the 1H and 13C NMR spectra of Tyl and Des
was made based on twoꢀdimensional NMR spectra
COSY, HSQC, and HMBC. According to our data
(see the Experimental section), the signal assignment
in earlier work [32] has substantial inaccuracies. Earꢀ
(
antiꢀisomer).
A preliminary study of the biological properties of
the series of new derivatives of the macrolide antibioꢀ
tics, the synthesis of which is described in this work,
showed that many of them is specifically bound to the
ribosomal RT of E. coli and inhibit protein synthesis in
the cellꢀfree system of transcription–translation [34,
35] with an efficiency comparable and, in some cases,
exceeding that of the starting macrolides. These data
imply that the more comprehensive structural and
functional analysis of the series of amino acid and pepꢀ
tide derivatives of macrolides we are carrying out now
could be a source of valuable information regarding
the peculiarities of the interaction of the growing
1
lier, we described the H and 13C NMR spectra of
OMT [14].
We chose the derivatives (VIII), (X), and (XIV)
obtained by the first method and (XVII) prepared by
the second method for the comprehensive structural
analysis. The proton spectra of the modified macꢀ
rolides differed from the spectra of the starting comꢀ
pounds by the absence of a signal in the area of 9.6–
9.7 ppm characteristic of an aldehyde proton and the
appearance of a ⎯CH=N proton in the area of 7.4–
7.5 ppm. Moreover, the modification of, namely, the
C20ꢀaldehyde group was confirmed by the disappearꢀ
ance from the 13C spectrum of the signal in the area of polypeptide chain with the RT.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 36
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2010

252
SUMBATYAN et al.
EXPERIMENTAL
a J filter was used, optimal for the constants 13CꢀH
through one bond (of the order of 135 Hz). In the case
of HMBC, a transmitting J filter was used for longꢀ
range constants 13CꢀH (of a 7ꢀHz order) and the refoꢀ
cusing of the 13CꢀH constants through one bond. The
registration of the spectra was carried out in a CDCl3
solution at 303 K. The chemical shifts are given in
ppm, and spin–spin coupling constants are given in
Hz. The data of 13C NMR and twoꢀdimensional
experiments COSY, HSQC, and HMBC were used for
signal assignment.
In this work, we used amino acids and reagents
from Fluka and Bachem (Switzerland), Merck (Gerꢀ
many), and Reanal (Hungary). The peptide Bocꢀ
(GlyꢀPro)3ꢀOBzl (VId) was a kind gift of Academician
N.F. Myasoedov and his coworkers (Institute of
Molecular Genetics, Russian Academy of Sciences).
TLC was carried out on Kieselgel 60F254 plates
(Merck, Germany) with the use of the following solꢀ
vent systems: (A) 9 : 1 chloroform–methanol,
(B) 100 : 50 : 2 benzene–acetone–acetic acid; (C)
4 : 1 chloroform–methanol; (D) 85 : 10 : 5 chloroꢀ
form–ethanol–acetic acid; (E) 14 : 1 : 5 isopropanolꢀ
25% ammonia–water; (F) 65 : 4 : 25 chloroform–
water–methanol; (G) 15 : 1 chloroform–methanol;
(H) 7 : 3 chloroform–methanol; (I) 6 : 1 chloroform–
methanol; (J) 10 : 1 chloroform–methanol; (K) 8 : 1 :
0.1 dichloromethane–methanol–25% ammonia; (L)
65 : 4 : 25 chloroform–25% ammonia–methanol; and
(M) 1 : 2 chloroform–methanol. The compounds
absorbing UV light were detected by a Brumberg cheꢀ
miscope, and the amino acids and peptides containing
free amino groups were detected with a standard ninꢀ
hydrin reagent. The Bocꢀprotected amino derivatives
were similarly detected after the keeping of the chroꢀ
matogram in vapors of trifluoroacetic acid. Des, Tyl,
and their derivatives were detected with a 0.4% soluꢀ
tion of 2,4ꢀdinitrophenylhydrazine in 2 M HCl.
UV absorption spectra were registered on a Cary 50
Bio spectrophotometer (Varian, United States).
Amino acid analysis was carried out on a Hitachi
835 analyzer (Japan).
Starting antibiotics. In this work, we used tylosin
(I) produced by ZAO Mosagrogen (Russia); desmyꢀ
cosin (II) and OMT (III) were obtained by the acidic
hydrolysis of tylosin ( ) [13, 14].
Tylosin (I): 1H NMR (600 MHz): 0.93 (3 H, t,
H17), 0.99 (3 H, d, 6.9 H18), 1.21 (3 H, d, J6.8,
H5'CH3), 1.22 (3 H, s, H3''CH3), 1.25 (3 H, d, H21),
1.26 (3 H, d, 6.3, H5'CH3), 1.28 (3 H, d, 6.2,
H5'''CH3), 1.48 (1 H, m, H7), 1.62 (3 H, m, H4, H7,
H16), 1.76 (1 H, dd, 3.9 and 3.8, H2''), 1.78 (3 H, s,
H22), 1.88 (1 H, m, H16), 1.93 (1 H, d, 16.4, H2),
2.03 (1 H, d, 13.6, H2''), 2.16 (2 H, m, H6, H19),
I
J
7.3,
J
J
J
J
J
J
2.48 (6 H, 2 H, s, m, N(CH3)2, H2, H3'), 2.60 (1 H,
m, H8), 2.87 (1 H, m, H19), 2.95 (1 H, m, H14), 3.02
(1 H, dd, H2'''), 3.17 (1 H, m, H4'''), 3.26 (2 H, m,
H4', H5'), 3.48 (3 H, s, H2''' OCH3), 3.52 (3 H, m,
H2', H23, H5'''), 3.60 (3 H, s, H3''' OCH3), 3.70 (1 H,
d,
10.4, H3), 3.91 (1 H, dd, H23), 4.04 (1 H, m, H4''),
4.21 (1 H, d, 7.4, H1'), 4.55 (1 H, d, 7.8, H1'''), 4.90
(1 H, m, H15), 5.06 (1 H, d, 3.4, H1''), 5.90 (1 H, d,
15.5, H10), 7.30 (1 H, d,
J 9.0, H5), 3.74 (1 H, t, J 3.03, H3'''), 3.82 (1 H, d,
J
J
J
J
J
J
10.5, H13), 6.30 (1 H, d, J
15.3, H11), 9.67 (1 H, m, H20).
13C NMR (150.92 MHz): 9.12 (C18), 9.66 (C17),
The reversedꢀphase HPLC was performed on a
Milichrom Aꢀ02 (EKONOVA, Russia) on a Prontoꢀ
SILꢀ120ꢀ5ꢀC18 AQ column (2.0
× 75 mm, 5 μm)
12.98 (C22), 17.36 (C21), 17.75 ( C5''' CH3), 18.24
(C5' CH3), 19.02 (C5'' CH3), 25.47 (C16), 30.00 (C6),
32.50 (C7), 39.36 (C2), 40.16 (C4), 40.90 (C3'
N(CH3)2), 40.91 (C2''), 41.96 (C3'' CH3), 43.75
(C14), 44.70 (C8), 45.06 (C19), 59.71 (C2''' OCH3),
61.75 (C3''' OCH3), 68.76 (C3'), 69.06 (C3, C23,
C4''), 70.63 (C5'''), 72.67 (C2', C4'''), 73.19 (C5''),
75.18 (C15, C4', C5'), 76.41 (C3''), 79.81 (C3'''), 81.93
(C5), 81.94 (C2'''), 96.50 (C1''), 101.08 (C1'''), 103.96
(C1'), 119.31 (C10), 134.88 (C12), 142.31 (C13),
(MachereyꢀNagel, Germany) in an acetonitrile gradiꢀ
ent in 0.1% trifluoroacetic acid; the elution rate was
100 μl/min and the detection was at 214 and 280 nm.
Silica gel 60 (0.063–0.2 mm, Fluka) was used for
column chromatography.
The massꢀspectrometric analyses with the use of
matrixꢀassisted ionization (MALDI–TOF) were carꢀ
ried out on Ultraflex and Autoflex mass spectrometers
from Bruker Daltonik (Germany).
The values of the optical rotation of substances 148.07 (C11), 173.93 (C1), 202.47 (C20), 203.03
were measured on a VNIEKIPRODMASH EPO 1A (C9).
Desmycosin (II): 1H NMR (600 MHz): 0.98 (3 H,
instrument at a wavelength of 589 nm.
1
The H and 13C NMR spectra of the compounds t,
J
7.4 H17), 1.04 (3 H, d,
J
6.8, H18), 1.22 (3 H, d,
under study were registered on Bruker AMꢀ300, H21), 1.28 (6 H, m, H5'CH3, H5'''CH3), 1.32 (1 H, d,
Bruker DRXꢀ500, and Bruker AVꢀ600 spectrometers 5.4, H3''CH3), 1.50 (1 H, m, H7), 1.63 (3 H, m, H4,
with working frequencies of 300, 500, and 600 MHz, H7, H16), 1.80 (3 H, s, H22), 1.88 (1 H, m, H16),
J
1
respectively, for the H nuclei. The multiplicities of 1.95 (1 H, d,
J 16.5, H2), 2.11 (1 H, m, H6), 2.39
signals in the 13C spectra were determined with the use (3 H, m, H2, H3', H19), 2.50 (6 H, m, N(CH3)2), 2.61
of INEPT experiments. The twoꢀdimensional NMR (1 H, m, H8), 2.95 (2 H, m, H14, H19), 3.05 (1 H, m,
COSY spectra were registered in a magnitude repreꢀ H2''', H4'), 3.19 (1 H, m, H4'''), 3.28 (1 H, m, H5'),
sentation; the transformation of the twoꢀdimensional 3.50 (3 H, s, H2'''OCH3), 3.55 (3 H, m, H2', H23,
HSQC and HMBC spectra was carried out in a phaseꢀ H5'''), 3.63 (3 H, s, H3'''OCH3), 3.75 (2 H, m, H3''',
sensitive regime. When registering the HSQC spectra H5), 3.85 (1 H, d, J 10.6, H3), 4.00 (1 H, dd, H23),
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 36
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2010

AMINO ACID AND PEPTIDE DERIVATIVES
253
4.26 (1 H, d,
(1 H, m, H15), 5.95 (1 H, d,
d, 15.3, H10), 7.33 (1 H, d,
s, H20).
J 7.3, H1'), 4.57 (1 H, d, J 7.8, H1'''), 4.90 perature, TFA was removed by evaporation on a rotary
J
J
10.3, H13), 6.27 (1 H, evaporator, and the residue was dried in a vacuum
15.0, H11), 9.70 (1 H, desiccator over NaOH. A white crystalline substance
J
was obtained; yield 99%; mp 158–159
050 (F).
BocꢀAlaꢀAlaꢀPheꢀAlaꢀAlaꢀPheꢀLys(Z)ꢀOMe (Va).
) and
°C; Rf 0.85 (E),
13C NMR (150.92 MHz): 8.97 (C18), 9.66 (C17),
12.97 (C22), 15.30 (C21), 15.70 (C5''' CH3), 15.70
(C5' CH3), 25.45 (C16), 29.65 (C6), 31.54 (C7), 39.37 HBTU (1 equiv) were added under cooling (
0°C
(C2), 40.16 (C4), 41.67 (C3'N(CH3)2), 43.76 (C14), stirring to a solution of BocꢀAlaꢀAlaꢀPheꢀOH
44.66 (C8), 45.05 (C19), 59.72 (C2'''OCH3), 61.79 (1 equiv) in DMF. The mixture was stirred for 5 min
(C3'''ꢀOCH3), 69.05 (C23), 70.11 (C3'), 70.54 (C5'''), and then treated with TFA·HꢀАlaꢀАlaꢀPheꢀLys(Z)ꢀ
70.77 (C2'), 70.99 (C3), 72.64 (C4'''), 73.34 (C5'), OMe (1.1 equiv) dissolved in DMF and neutralized
73.43 (C4'), 75.09 (C15), 79.83 (C3'''), 81.84 (C5), with 1.1 equiv of N,Nꢀdiisopropylethylamine. The
81.89 (C2'''),101.06 (C1'''), 103.96 (C1'),118.52 mixture was stirred for 5.5 h at 0°C, tenꢀtimes diluted
(C10), 134.86 (C12), 142.24 (C13), 148.00 (C11), with water, and fourꢀtimes extracted with chloroform.
173.86 (C1), 202.90 (C20), 203.09 (C9).
The extract was washed with 5% Na2CO3 (three
times), water (once), 0.05 M H2SO4 (three times), and
saturated solution of NaCl (once). Chloroform was
removed in a vacuum, and the residue was precipitated
with 10 volumes of petroleum ether from 1 volume of
ethyl acetate. The precipitated solid (75%) was then
purified by column chromatography on silica gel using
the chromatographic system G as an eluent. Comꢀ
Synthesis of Peptides
Bocꢀ(Ala)5ꢀOMe (IVa). Bocꢀ(Ala)nꢀOMe were obtained
by coupling BocꢀAlaꢀOH with Hꢀ(Ala)n – 1ꢀOMe (n =
2–5) using DCC in the presence of HOBt by the proꢀ
cedure described for Bocꢀ(Ala)2ꢀOMe in [14]. In this
manner, we obtained:
pound (Va ) resulted; yield 41%;
(gradient from 15 to 80% for 20 min); MS, m/z
(found/calc.): 973.5/973.12, 873.4/873.0 ( – Вoc);
839.4/839.0 ( – Z); f 0.71 (A), 0.66 (D), 0.52 (G).
τ (HPLC) 19 min
Bocꢀ(Ala)3ꢀOMe (65%), mp 182–184°C (from isoꢀ
М
propyl ether, dec.);
20 –20° (
[α]D
c
1, DMF), Rf 0.70 (А)
М
R
and 0.45 (B) (published data: mp 185 [36] and 193–
°
C
Amino acid analysis: Ala 3.78 (4), Phe 1.68 (2), Lys
1.00 (1).
20
20
194
(1,1,1,3,3,3ꢀhexafluoropropanꢀ2ꢀol)[27]);
ocꢀ(Ala)4ꢀOMe (48%), mp 148–150°C (from ethyl
°
C [27];
–48° (
c
1, CH Cl ) [36],
–8.48°
[α]D
[α]D
2
2
Preparation of
Nꢀaminooxyacetylpeptides. Peptides
(
IVa), (Va ), and VIb) were treated with TFA (20 equiv)
В
for 1 h at room temperature. TFA was removed in a
vacuum, and a solution of N,Nꢀdiisopropylethylamine
(1 equiv) in DMF was added to the residue.
HOBT (1.3 equiv) and DCC (1.3 equiv) were
added to a solution of Bocꢀ2ꢀaminooxyacetic acid
(BocꢀAoacꢀOH, 1 equiv) in DMF under cooling
acetate, dec.); 20 –35° (
c
1, DMF),Rf 0.62 (А),
Rf 0.19
[α]D
(B) (published data: mp 246°C [37],
20 –39.5
°
(c
1,
[α]D
20
[α]D
DMF) [36],
–8.4° (1,1,1,3,3,3ꢀhexafluoroproꢀ
panꢀ2ꢀol)[27]);
ocꢀ(Ala)5ꢀOMe (IVа)
(from ethyl acetate, dec.);
В
, (28%), mp 266–268°C
(0°C) and stirring. After 1 h, phenylalanine ethyl ester
20
or a peptide (IV), (Vb), or (VIa) (1 equiv) in DMF was
added to the solution. The mixture was stirred for 4 h
at room temperature, tenꢀtimes diluted with water,
and extracted with ethyl acetate. The extract was
washed with 0.1 M HCl (three times), 5% Na2CO3
(three times) and water, dried with MgSO4, and evapꢀ
orated. We obtained:
–45° (
c
1, DMF),
τ
[α]D
15 min (gradient 0–60% acetonitrile in 0.1% TFA
20
for 20 min); Rf 0.10 (A), 0 (B) (published data:
[α]D
–8.31° (1,1,1,3,3,3ꢀhexafluoropropanꢀ2ꢀol)[27]).
BocꢀAlaꢀAlaꢀPheꢀAlaꢀAlaꢀPheꢀLys(Z)ꢀOMe (Va).
BocꢀAlaꢀAlaꢀPheꢀLys(Z)ꢀOMe. HBTU (1 equiv) and,
after 5 min, HꢀLys(Z)ꢀOMe (1.1 equiv) were added to
a solution of BocꢀAlaꢀAlaꢀPheꢀOH [14] in DMF
BocꢀAoacꢀPheꢀOEt, yield 77%,
τ (HPLC) 8 min
(gradient 0–60% for 20 min), Rf 0.87 (A), 0.75 (B),
under cooling (0°C) and stirring. The reaction mixture
0.92 (C), 0.95 (D);
was kept for 2 h, ten times diluted with water, and
threeꢀtimes extracted with chloroform. The extract
was threeꢀtimes washed with 5% Na2CO3, water (one
time), 0.05 M H2SO4 (three times), a saturated soluꢀ
tion of NaCl (one time), dried by MgSO4, and evapoꢀ
BocꢀAoacꢀ(Ala)5ꢀOMe (IVb), yield 60%; Rf 0.75 (D);
BocꢀAoacꢀAlaꢀAlaꢀPheꢀAlaꢀAlaꢀPheꢀLys(Z)ꢀOMe (Vc),
yield 73%, Rf 0.66 (A), 0.57 (D);
BocꢀAoacꢀ(GlyꢀPro)3ꢀOBzl (VIe), yield 35%,
rated on a rotary evaporator. The residue was recrystalꢀ Rf 0.50 (A), 0.65 (C).
lized from ethyl acetate. Yield 86% of white crystals,
mp 165–169 C; f 0.40 (B), 0.85 (C), 0.90 (D); amino
acid analysis: Ala 1.68 (2), Phe 1.00 (1), Lys 1.05 (1).
TFAHꢀАlaꢀАlaꢀPheꢀLys(Z)ꢀOMe
BocꢀAoacꢀAlaꢀAlaꢀPheꢀAlaꢀAlaꢀPheꢀLysꢀOMe.
10% Pd/C (50% of the mass of the starting peptide)
and 4 equiv of ammonium formate were added to a
°
R
.
TFA (20 equiv) solution of BocꢀAoacꢀAlaꢀAlaꢀPheꢀAlaꢀAlaꢀPheꢀ
were added to BocꢀAlaꢀAlaꢀPheꢀLys(Z)ꢀOMe Lys(Z)ꢀOMe (1 equiv) in DMF, and the mixture was
(1 equiv), the solution was stirred for 1 h at room temꢀ stirred at room temperature for 24 h. The catalyst was
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 36
No. 2
2010

254
SUMBATYAN et al.
filtered off, the filtrate was evaporated in a vacuum,
DesꢀAoacꢀ(Ala)5ꢀOMe (XI); yield 18%; τ (HPLC)
and the residue was distributed between butanol and a 16.8 min (gradient 0–60% for 25 min); m/z
saturated solution of NaCl (5 : 1). Butanol was (found/calc.): 1214.0/1214.3; Rf 0.50 (H).
removed on a rotary evaporator, and the residue was
dried in a vacuum over CaCl2; yield 85%; Rf 0.42 (B),
0.71 (D).
DesꢀAoacꢀAlaꢀAlaꢀPheꢀAlaꢀAlaꢀPheꢀLys (Z)ꢀOMe
(XIIa); yield 21%; m/z (found/calc.): 1698.9/1699.9
(М
+ H), 1685.0/1685.8 (М – CH3); Rf 0.64 (C),
0.89 (H), 0.40 (I).
Preparation of the substituted oximes of macrolides.
Bocꢀaminooxyacetylpeptides were treated with TFA
(20 equiv) for 1 h at room temperature, and TFA was
removed in a vacuum.
DesꢀAoacꢀAlaꢀAlaꢀPheꢀAlaꢀAlaꢀPheꢀLysꢀOMe
(XII); yield 23%; m/z (found/calc.): 1551.2/1550.7
(М
– CH3); Rf 0.10 (H).
DesꢀAoacꢀGlyꢀProꢀGlyꢀProꢀGlyꢀProꢀOBzl (XIII);
A solution of Tyl, Des, or OMT (1 equiv) in a
0.4ꢀM sodium acetate buffer (pH 4.7) was mixed with
yield 26%;
τ (HPLC) 18.5 min (gradient 0–60% for
25 min); m/z (found/calc.): 1397.6/1397.5; Rf 0.80 (H).
OMTꢀAoacꢀPheꢀOEt (XIV); yield 23%; (HPLC)
13.9 min (gradient 20–80% for 25 min); m/z
(found/calc.): 846.5/846.0; Rf 0.83 (A), 0.98 (H),
0.82 (J), 0.8/2; The H NMR differs from the OMT
spectrum by the absence of an aldehyde proton (at
9.64 ppm), the appearance of an oxime proton H20
signal (br s at 7.40 ppm), and the presence of signals of
the AoacꢀPheꢀOEt residue.
Preparation of substituted imines of tylosin and desꢀ
mycosin. A 3.5ꢀM solution of sodium methylate in
absolute methanol (4 equiv) and 3ꢀÅ molecular sieves
N
ꢀaminooxyacetylpeptide (IVc), Vd), (Ve), or (VIf) in
τ
an equal volume of DMSO or 2ꢀaminooxyacetic acid
in an equal volume of a 0.4ꢀM sodium acetate buffer
(pH 4.7). The reaction mixture was incubated at 50°C
1
for 12 h, diluted with water, adjusted to pH 9, and
extracted with chloroform. The target compound was
isolated by preparative TLC in a chromatographic sysꢀ
tem A, C, or H, eluting from silica gel with methanol.
There were obtained in this manner:
TylꢀAoacꢀOH (VII), yield 28%,
τ (HPLC)
12.5 min (gradient 20–80% of acetonitrile in 0.1%
TFA for 25 min); m/z (found/calc.): 988.7/988.5; Rf
0.77 (C), 0.73 (H).
were added to an amino acid or a peptide (Vf) or (VIc
)
(2 equiv) to solution of 1 equiv tylosin (desmycosin) in
absolute methanol. The mixture was stirred for 12 h at
room temperature, the solvent was removed in a vacꢀ
uum, and the residue was separated on a glass plate
coated with a silica gel layer in system H, L, or M. The
resulting compound was eluted from silica gel by
methanol.
TylꢀAoacꢀPheꢀOEt (VIII), yield 21%;
19.5 min (gradient 0–60% of acetonitrile in 0.1% TFA
for 25 min; m/z (found/calc.): 1164.7/1164.4; f 0.45
τ (HPLC)
R
(A), 0.67 (C); The 1H NMR, COSY, and HSQC specꢀ
tra (600 MHz, 303 K, CDCl3) differ from the spectra
of tylosin by the absence of a signal of the aldehyde
proton (9.67 ppm), the appearance of oxime proton
H20 (br. s at 7.42 ppm), and the signals of the Aoacꢀ
Tyl=TyrꢀOH3 (XV); yield 25%; m/z (found/calc.):
1079.7/1079.3; Rf 0.25 (C), 0.38 (H); λmax (UV) 285
nm (ε285 = 1.9
×
104 М– cm–1), 340–345 nm (shoulꢀ
PheꢀOEt residue: 1.20 (3H, t,
Phe, 3.15 (3 H,
OCH2CH3
1
der), 385 nm (ε385 = 1.3
×
103 М–1cm–1); H NMR
m,
Phe , C4'''), 4.17 (2H, m,
Phe), 4.52 (1 H,
СН2
OCH 2CH3
(600 MHz, 303 K, CDCl3) differs from the spectrum
of tylosin by the absence of an aldehyde proton signal
(at 9.70 ppm) and the appearance of the proton H20 of
the Schiff base (7.45 ppm), and also the presence of
the signals of tyrosine residue: 2.16 (2 H, m, H6, H19),
2.87 (1 H, m, H19), 3.78 (1 H, m, CH2Tyr), 4.74 (1 H,
dd,
Aoac ), 4.88 (1 H, m,
Phe), 6.81 (1 H, br s,
CHCH2
CH2
NHPhe), 7.27 (6 H, m, Phe, H11), 7.42 (1 H, t,
J
5.75,
H20).
TylꢀAoacꢀ(Ala)5ꢀOMe (IX), yield 20%;
τ (HPLC)
m, CHα Tyr), 7.37 (5 H, m, H11, Tyr), 7.45 (1 H, br m,
H20).
18.5 min (gradient 0–60% for 25 min); m/z
(found/calc.): 1358.2/1358.4; Rf 0.76 (H).
DesꢀAoacꢀPheꢀOEt (X); yield 20%; RT (HPLC)
Tyl=PheꢀOH (XVI)
; yield 16%; m/z (found/calc.):
14.1 min (gradient 20–80% for 25 min; m/z 1063.7/1063.1;
Rf 0.30 (C), 0.63 (H); λmax (UV) 285,
(found/calc.): 1020.7/1020.2;
Rf 0.79 (A), 0.76 (J), 385 nm.
1
Tyl=UalꢀOH
1097.7/1097.1;
ε275 = 6.1
104 М–1cm–1; 1H NMR (600 MHz, 303 K,
(
XVII
) yield 21%; m/z (found/calc.)
0.80 (K); The H NMR, COSY, HSQC (600 MHz,
303 K, CDCl3) differ from the desmycosin spectra by
the absence of the signal of the aldehyde proton
(9.70 ppm) and the appearance of an oxime proton
H20 (br s at 7.45 ppm) and signals of the residue Aoacꢀ
Rf0.67 (E), 0.69 (L); λmax (UV) 275 nm
(
×
CDCl3): differs from the tylosin spectrum by the
absence of an aldehyde proton signal (9.70 ppm) and
the presence of an H20 signal of the Schiff base (br m
at 7.45 ppm) and the signals of Ual residue: 3.75 (1 H,
PheꢀOEt: 1.20 (3 H, t,
Phe), 3.16 (5 H, m,
Phe), 4.52
OCH 2CH3
OCH2CH3
CHP2he
(1 H, dd,
,
C4''', C4'), 4.17 (2 H, m,
3
Aoac ), 4.90 (1 H, m,
Phe), 6.85 (1 H,
A double hyphen between the abbreviated name of the antibiotic
CH2
CHCH2
and amino acid or peptide substituent designates the binding by
the Schiff base formation.
br s, NHPhe), 7.29 (5 H, m, Phe), 7.45 (1 H, br s, H20).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 36
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2010

AMINO ACID AND PEPTIDE DERIVATIVES
255
6. Gong, F. and Yanofsky, C., Science, 2002, vol. 297,
pp. 1864–1867.
7. Yonath, A., Annu. Rev. Biochem., 2005, vol. 74, pp.
m, H8 of
), 5.63 (1 H, m, H5Ual), 7.35 (2 H, H11, H6Ual),
CH
Ual), 4.35 (7 H, m, H15, H1''', H1', H7
CHU2 al
7.45 (1 H, br s, H20).
Des=TyrꢀOH XVIII)
(found/calc.): 933.6/935.0; Rf 0.15 (F), 0.30 (M).
Des=AlaꢀAlaꢀPheꢀAlaꢀAlaꢀPheꢀLys(Z)ꢀOH XIX),
649–679.
(
;
yield
18%;
m/z
8. Mankin, A.S., Trends Biochem. Sci., 2006, vol. 31,
pp. 11–13.
(
9. Schlunzen, Zarivach, R., F., Harms, J., Bashan, A.,
Tocilj, A., Albrecht, R., Yonath, A., and Franceschi, F.,
Nature, 2001, vol. 413, pp. 814–821.
m/z (found/calc.): 1477.2/1477.6 (M – Z); Rf 0.15
(M).
10. Hansen, J., Ippolito, J.A., Ban, N., Nissen, P., Moore,
P.B., and Steitz, T.A., Moll. Cell, 2002, vol. 10, pp. 117–
128.
11. Tenson, T., Lovmar, M., and Ehrenberg, M., J. Mol.
Biol., 2003, vol. 330, pp. 1005⎯1014.
12. Tenson, T. and Ehrenberg, M., Cell, 2002, vol. 108,
Des=GlyꢀProꢀGlyꢀProꢀGlyꢀProꢀOH
(found/calc.): 1231.0/1234.3; Rf 0.20 (M).
(
XX
)
;
m/z
Preparation of C20ꢀaminoderivatives of tylosin and
desmycosin. Sodium cyanoborohydride (4 equiv) was
added to imminoderivative (XV) or (XX) (1 equiv) in
methanol. The mixture was stirred for 12 h, and the
solvent was removed in a vacuum. The residue was disꢀ
solved in water, adjusted in the cold to pH 3 with 0.1 N
HCl and then to pH 7 with saturated sodium bicarꢀ
bonate and extracted with chloroform. The combined
chloroform extracts were washed with water, dried
with sodium sulfate, evaporated in a vacuum, and the
residue was subjected to chromatographic separation
on a silica gel plate in system C for (XXI) or M for
pp. 591–594.
13. Sumbatyan, N.V., Korshunova, G.A., and Bogdanov, A.A.,
Biokhimiya, 2003, vol. 68, pp. 1436⎯1438.
14. Korshunova, G.A., Sumbatyan, N.V., Fedorova, N.V.,
Kuznetsova, I.V., Shishkina, A.V., and Bogdanov, A.A.,
Bioorg. Khim., 2007, vol. 33, pp. 235–244 [Russ. J.
Bioorg. Chem. (Engl. Tranl.), 2007, vol. 33, pp. 218–
226].
15. McGuire, J., Bonience, W., Higgens, C., Hoehn, M.,
Stark, W., Westhead, J., and Wolfe, R., Antibiot.
Chemother, 1961, vol. 11, pp. 320–327.
(
XXII).
TylꢀTyrꢀOH (XXI); yield 23%; m/z (found/calc.):
1081.9/1081.0; f 0.56 (C), 0.68 (H); λmax (UV) 225,
16. Hamill, R.L., Haney, M.E., McGuire, J.M., and
Stamper, M.C., Antibiotics Tylosin and Desmycosin and
Derivatives Thereof, US Patent No. 3 178 341, 1965.
R
285 nm; 1H NMR (600 MHz, 303 K, CDCl3): unlike
the spectrum of Tyl=Tyr, the signals at 7.45 and 4.74
characteristic of Schiff base are absent, the signals of
the reduced Schiff base appear, signals H19 of tylosin
are shifted to a stronger field (2.16–2.87 ppm in a nonꢀ
reduced Schiff base; 1.5–1.7 ppm in a reduced Schiff
base): 1.5–1.7 (1 H, m, H19), 3.51 (1 H, m, H20),
3.63 (1 H, m, CHα Tyr), 3.74 (1 H, s, NHTyr), 3.76 (1 H,
17. Morin, R. and Gorman, M., OꢀMicaminosyl Tylonoꢀ
lide and Process for the Preparation Thereof, US
Patent No. 3459853, 1969.
18. VasquezꢀLaslop, N., Thum, C., and Mankin, A.S.,
Mol. Cell, 2008, vol. 30, pp. 190–202.
19. Debono, M., Willard, K.E., Kirst, H.A., Wind, J.A.,
Crouse, G.D., Tao, E.V., Vicenzi, J.T., Counter, F.T.,
Ott, J.L., and Ose, E.E., J. Antibiot., 1989, vol. 42,
pp. 1253–1267.
20. Kirst, H.A., Willard, K.E., Debono, M., Toth, J.E.,
Truedell, B.A., Leeds, J.P., Ott, J.L., FeltyꢀDuckꢀ
worth, A.M., Counter, F.T., Ose, E.E., Crouse, G.D.,
Tustin, J.M., and Omura, S., J. Antibiot., 1989, vol. 42,
pp. 1673–1683.
m,
Tyr), 7.37 (5 H, m, H11, Tyr).
CH2
DesꢀGlyꢀProꢀGlyꢀProꢀGlyꢀProꢀOH
(XXII); m/z
(found/calc.): 1233.0/1235.3; f 0.38 (M).
R
ACKNOWLEDGMENTS
We are grateful to the Russian Foundation for Basic
Research (project no. 07ꢀ04ꢀ00902ꢀa) for support.
A.A. Bogdanov is grateful to the Alexander Humboldt
Foundation for support.
21. Mutak, S., Marsik, N., Kramaric, M.D., and Pavlovic, D.,
J. Med. Chem., 2004, vol. 47, pp. 411–431.
22. Lundi, K.M. and Vu, C.B., Amide derivatives of 16ꢀ
Membered Ring Antibiotic Macrolides, US Patent
No. 5716939, 1998.
23. Yang, D., Ng, F.F., and Li, Z.J., J. Am. Chem. Soc.,
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