Synthesis of biosynthetic precursors of chromophores of red fluorescent proteins

Article abstract of DOI:10.1134/S1068162011040066

A method for the synthesis of 5-arylidene-3,5-dihydro-4H-imidazol-4-ones corresponding to the chromophore of the green fluorescent protein (GFP) with acylaminoalkyl substituents at position 2 of the imidazolone core has been developed. These biomimetic mo

Full text of DOI:10.1134/S1068162011040066

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2011, Vol. 37, No. 4, pp. 411–420. © Pleiades Publishing, Ltd., 2011.
Original Russian Text © P.E. Ivashkin, K.A. Lukyanov, I.V. Yampolsky, 2011, published in Bioorganicheskaya Khimiya, 2011, Vol. 37, No. 4, pp. 464–474.
Synthesis of Biosynthetic Precursors of Chromophores
of Red Fluorescent Proteins
P. E. Ivashkin, K. A. Lukyanov, and I. V. Yampolsky1
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
ul. MiklukhoꢀMaklaya 16/10, Moscow, 117997 Russia
Received November 19, 2010; in final form, November 22, 2010
Abstract—A method for the synthesis of 5ꢀarylideneꢀ3,5ꢀdihydroꢀ4Hꢀimidazolꢀ4ꢀones corresponding to the
chromophore of the green fluorescent protein (GFP) with acylaminoalkyl substituents at position 2 of the
imidazolone core has been developed. These biomimetic model compounds are the precursors of the chroꢀ
mophores of red fluorescent proteins. The method is based on the masking of the dehydrotyrosine fragment
of target compounds by the
tion of ꢀhydroxytyrosine with the appropriate
ꢀacylation with subsequent elimination, and the cyclization of the resulting 3ꢀacylaminocinnamic acid
derivatives in basic medium.
βꢀhydroxytyrosine moiety. The key stages of the synthesis include the condensaꢀ
β
N
ꢀacetylamino acid, the unmasking of dehydrotyrosine by
O
Keywords: chromophore, imidazolone, green fluorescent protein, GFP, DsRed
DOI: 10.1134/S1068162011040066
1
INTRODUCTION
lengths is formed (red, yellow, and purple proteins).
The nature and mechanism of these additional modiꢀ
fications are poorly understood presently.
The popularity of using the green fluorescent proꢀ
tein and other fluorescent proteins as genetically
encoded labels largely determines a close interest in
studies of the natural diversity, structure, and bioꢀ
chemical and biophysical properties of the proteins of
this family. As distinct from other proteins, GFPꢀlike
proteins are capable of forming the chromophore
group independently, without the participation of exterꢀ
nal cofactors and enzymes, by the posttranslational
modification of their own amino acid residues. It has
been established that the chromophores of the GFP
family have a common structural core, 5ꢀ(4ꢀhydroxyꢀ
Earlier, methods for the chemical synthesis of
model compounds, the analogues of the chromophore
of the GFP, and some other fluorescent proteins, have
been developed [1]. The synthesis of model chroꢀ
mophores made it possible to obtain independent
proof of the chemical structure of natural chroꢀ
mophores and clarify the details of their spectral propꢀ
erties and the effect of various environmental factors
on them [2–5].
The present study deals with the synthesis of 5ꢀ(4ꢀ
hydroxybenzylidene)ꢀ3,5ꢀdihydroꢀ4Hꢀimidazolꢀ4ꢀones
benzylidene)ꢀ3,5ꢀdihydroꢀ4Hꢀimidazolꢀ4ꢀone. This
fragment is formed by cyclization, dehydration, and
oxidation (each of the three stages, in turn, is divided
into elementary stages whose nature has not been preꢀ
cisely determined) of three neighboring amino acids at
positions 65–67 (numbering is that of GFP from the
jellyfish Aequorea victoria) with the involvement of
molecular oxygen. If no further transformations occur,
a GFP is formed. However, the amino acid residue at
position 65 can undergo further modification, with the
containing an acylaminoalkyl substituent at position 2
of the imidazolone core. These compounds are the
biomimetic precursors of red chromophores and will
be further used for studying the mechanism of the oxiꢀ
α
dation of the
C ꢀN bond of the residue at position 65,
which occurs in many red fluorescent proteins and
chromoproteins.
The known methods of the synthesis of substituted
imidazolones involve: (a) the cyclization of dehydroꢀ
tyrosine derivatives by the action of bases [6], (b) the
reaction of imidates [7, 8] and amidines [9] with
appropriate 1,2ꢀdinucleophiles, (c) the cyclization of
result that the conjugated system of ꢀbonds increases,
and a chromophore with absorption at longer waveꢀ
π
Abbreviations: AIBN, asobisisobutyronitrile; Bn, benzyl;
DsRed, the red fluorescent protein of the coral polyp Discoꢀ
soma; ESI, electrospray ionization; GFP, green fluorescent proꢀ
αꢀazidoimides by the action of triphenylphosphine
[10], and (d) the crossꢀcoupling of boronic acids with
thioimidazolones [11].
tein; HOBt,
laser desorption ionization; NBS,
ꢀmethoxybenzyl; TBS, tertꢀbutyldimethylsilyl.
Corresponding author: phone/fax: (499) 742ꢀ81ꢀ22; eꢀmail:
ivyamp@gmail.com.
N
ꢀhydroxybezotriazole; MALDI, matrixꢀassisted
N
ꢀbromosuccinimide; PMB,
The necessity of introducing the ꢀacylamino group
α
p
1
at position 2 of imidazolone substantially limits the set of
available synthetic methods. Methods (c) and (d) cannot
411

412
IVASHKIN et al.
be applied in this case, since there are no approaches to is characterized by extremely low yields (<10%) [7].
the synthesis of the corresponding ꢀacylaminoꢀsubstiꢀ Based on the aforementioned, it was decided to use the
α
tuted imides and boronic acids. Rigorous conditions of cyclization of dehydrotyrosine derivatives [method (a)]
the synthesis of iminoesters and amidines [method (b)] as a key stage of the assembly of the imidazolone core.
are applicable only to the most inert amino acid residues; Below four successive attempts of synthesizing the target
in addition, the cyclization of alkylꢀsubstituted amidines compounds are discussed (Scheme 1).
OTBS
OH
OBn
Pathway
1
Pathway
2
W
N
R
N
O
X
R
Y
N
N
N
N
O
O
O
R
O
O
OH
W = H, MeO(C6H4)
X = Br, N3
Y = O, NAlk
HN
R
N
N
OBn
O
OBn
O
O
HN
O
R
NH2
O
HN
R
HO
NH2
O
CO2Me
HO
Pathway
4
Pathway
3
CO2Me
HO
Scheme 1. Four approaches to the synthesis of 3ꢀmethylꢀ2ꢀ(
arylideneꢀ3,5ꢀdihydroꢀ4 ꢀimidazolꢀ4ꢀones: functionalization of the
assembly of the heterocyclic core using oxazolones (2), dehydrotyrosine (3), and hydroxytyrosine
α
ꢀacetylamino)alkylꢀ5ꢀ
H
α
ꢀposition of the heterocycle (1);
.
OH
OTBS
OTBS
1. TBSCl
i
Pr NEt
2
NaN
3
N3
Br
N
N
N
N
N
N
2. NBS
AlBN
O
O
O
(
I
)
(II)
NH , BnNH ,
2
PPh (AcOH),
3
Zn, Na/Hg
3
Bn NH
2
Scheme 2. Functionalization of the αꢀposition of 2,3ꢀdimethylꢀ5ꢀarylideneꢀ3,5ꢀdihydroꢀ4Hꢀimidazolꢀ4ꢀone.
RESULTS AND DISCUSSION
thesizing
[5]. However, numerous attempts to reduce the double
C=N bond and synthesize ꢀacylimino derivatives
based on the compounds obtained failed. In the present
work, ꢀsilylated 2ꢀbromomethylimidazolone ( ) was
ously developed in our laboratory a method for synꢀ successfully synthesized (Scheme 2); however, during
α
ꢀketoꢀ [3] and
α
ꢀalkylimino imidazolones
Synthetic approach no. 1. The most adequate
α
approach to the synthesis of
cles, including imidazolones, is the functionalization
of the ꢀposition of the heterocycle. We have previꢀ
αꢀsubstituted heterocyꢀ
О
I
α
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SYNTHESIS OF BIOSYNTHETIC PRECURSORS OF CHROMOPHORES
413
subsequent reactions with amines, ammonium, and
acetamide, a complete degradation of the imidazolone
cycle occurred. We also failed to convert 2ꢀazidomeꢀ
thyl imidazolone (II) to amino derivatives through the
reduction by triphenylphosphine (probably due to a
high nucleophilicity of the intermediate ilide) and
other reductants.
(
III) (Scheme 3). Compounds (III) can be obtained
through the azlactonization of appropriate ꢀacylglyꢀ
cines and subsequent condensation with 4ꢀhydroxyꢀ
benzaldehyde [3, 12, 13]. Despite numerous attempts
N
to condense ꢀacylaminoꢀsubstituted azlactones with
α
aldehydes under the conditions of base or acid catalyꢀ
sis, we were unable to synthesize target oxazolones of
type (III). The main process in the case of
Synthetic approach no. 2. The most universal pathꢀ
way of de novo assembling the heterocyclic core of
N
ꢀacetylphenylalanine (IV) is the isomerization [14]
of saturated azlactone (
5ꢀarylideneꢀ3,5ꢀdihydroꢀ4Hꢀimidazolꢀ4ꢀones involves
the use of 4ꢀarylideneꢀ4,5ꢀdihydrooxazolꢀ4,5ꢀones zine (VI) (Scheme 3).
V) to
Nꢀacetyldiketopiperaꢀ
OAc
OH
OH
OAc
O
R'NH
Δ
2
H
N
HN
N
Base
N
N
N
O
R
R
R
O
Bn
O
)
O
O
NH
R'
O
O
OAc
R'
(
III
(
IIIа
)
R, R' = Alk, Ar
O
O
H
N
O
O
O
O
1. GlyOEt,
DCC
2. NaOH
HN
N
DCC
or
Ac O
HO2C
NH
NH
HO2C
N
H
Base
O
(
O
N
Bn
Bn
O
2
Bn
IV
Bn
(
)
V)
O
(
VI
)
Scheme 3. General strategy of the synthesis of 5ꢀarylideneꢀ3,5ꢀdihydroꢀ4Hꢀimidazolꢀ4ꢀones based on
oxazolones (III). Synthesis with the use of ꢀacetylphenylalanine.
N
The problem of eliminating the isomerization at dative deprotection of PMB amide (VIII) by the
the condensation stage can be solved by the introducꢀ action of ceric ammonium nitrate leads to the degraꢀ
tion of a protecting group at the amide atom of nitroꢀ dation of the imidazolone core, including the case that
gen of acetyl amino acid. The 4ꢀmethoxybenzyl there is a methyl group on the oxygen atom of the pheꢀ
group, which provides the sufficient flexibility in the nol fragment.
choice of deprotection conditions (reduction, oxidaꢀ
As known, the methoxybenzyl group can be
tion, and acidolysis), served as the protecting group.
removed in some cases by strong acids (among other
The corresponding
NꢀPMBꢀNꢀacetylphenylalanine
things, in the presence of the scavenging nucleophiles:
thioanisole, pentamethylbenzene, etc.). In our case,
the acidolysis of the methoxybenzylamide fragment
occurred only by the action of such a strong acid as triꢀ
(
VII) was successfully synthesized and converted to
imidazolone (VIII) with the residue of
Nꢀprotected
phenylalanine (Scheme 4).
It was found in subsequent experiments that the fluoromethanesulfonic acid in a nonꢀbasic solvent (for
paraꢀmethoxybenzyl group in compound (VIII) canꢀ example, dichloromethane) with a low yield (<20%).
not be selectively removed during catalytic hydration After the addition of nucleophiles, the yield of the tarꢀ
and reduction by metals and other reagents. Simultaꢀ get product decreased to 5% and less. Unexpectedly,
neously with the deprotection, the hydration of the the severe deprotection conditions appeared to be
C=C double bond of the imidazolone core occurs. inappropriate for other amino acid residues (alanine
The reoxidation of dihydroimidazolone by the method and glycine). A complex mixture of degradation prodꢀ
described in [15] occurs with a very low yield and canꢀ ucts formed from the corresponding chromophores by
not be considered as the preparative reaction. The oxiꢀ the action of trifluoromethanesulfonic acid.
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414
IVASHKIN et al.
OMe
1.
H
N
HO2C
NH2
Bn
HO2C
O
Ac O, NaOH
2
PMB
2. NaBH
4
Bn
OH
OAc
DDC,
NEt
1.
3
PMB
O
O
O
1. GlyOEt,
DCC
2. NaOH
O
O
N
N
HO2C
N
)
N
2. MeNH
2
3. Cs CO , DMF
HO2C
N
H
PMB
PMB
2
3
N
Bn
O
Bn
Bn
(
VIII)
(
VII
Scheme 4. Synthesis of the PMBꢀprotected chromophore
(
VIII).
Synthetic approach no. 3. An alternative approach to esters of pentafluorophenol and
the synthesis of dehydrotyrosine derivatives, which are (R = 2ꢀphenylꢀ1ꢀaminoethyl). It is known that the
the immediate precursors of chromophores, involves the amino group of ꢀprotected dehydrotyrosine can be
acylation of the amino group of ꢀprotected dehydrotyꢀ deprotonated by a strong base [17]. However, this
rosine (IX) [16] (Scheme 5). However, compound (IX) in approach appeared to be inapplicable to ꢀacylamino
Nꢀhydroxybenzotriazole
О
О
N
the neutral state has nucleophilicity too low to be introꢀ acids that were not protected at the amide group, probaꢀ
duced into peptide coupling with activated amino acid bly due to the intramolecular azlactonization in a
derivatives such as acid chlorides, azlactones, and the strongly basic medium.
OBn
OBn
OBn
OBn
RCOCl
RCOOPfp
RCOOBt
N3
CO2Et
EtONa
AlꢀHg
H
N
N3
NH2
OEt
COR
O
O
OEt
O
O
OEt
R = AcNHCH2, AcNHCH(CH3),
AcNHCH(CH2Ph)
(IX)
Scheme 5. Synthesis of protected dehydrotyrosine (IX) and a study of peptide synthesis with its participation.
Synthetic approach no. 4. We used protected followed by treatment with aqueous methylamine
β
ꢀhydroxytyrosine (X) (Scheme 6) as a synthetic anaꢀ
leads to target dipeptides of
XIIa)–(XIIc) with a good yield.
βꢀhydroxytyrosine
logue of dehydrotyrosine (IX). This compound is easꢀ
(
ily obtained from glycine and 4ꢀbenzyloxybenzaldeꢀ
hyde [18] as a mixture of relꢀ(2S,3R)ꢀ and relꢀ(2S,3S)
isomers (3.2 : 1), which was subsequently used without
separation.
As opposed to the literature data on related comꢀ
pounds [19], the treatment of compounds (XIIa)–
XIIc) with acetyl chloride or tosyl chloride in the
(
presence of a wide range of bases does not lead to
the formation of target acetate or tosylate, as well as
the corresponding elimination product. The main
reaction product is 4ꢀbenzyloxybenzaldehyde
(Scheme 7).
The Nꢀacetylation of amine (XI) by Nꢀacetylamino
acids [AcGly, AcPhe, AcLeut in the presence of HOBt
2
Leut, tertꢀleucine.
2
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SYNTHESIS OF BIOSYNTHETIC PRECURSORS OF CHROMOPHORES
415
OBn
OBn
OBn
O
1)
2)
1а)
(EtCO) O
2
ZnCl
HN
2
R
CO2H
1b) Ac O
2
AcBr
R
HOBt, DCC
SOCl
2
H
N
MeOH
2)
MeNH
2
H
2
NH2
OH
NH2
OMe
NHAc
HO
HO
HO
68–88%
Pd/C
O
O
O
O
NH
(X)
(XI
)
(XIIа–в)
72–74%
OH
OH
(
XIIа), (XIIIа), (XIVа) R = H
XIIb), (XIIIb), (XIVb) R = Bn
XIIc), (XIIIc), (XIVc) R = But
(
(
K CO
2
R
3
H
DMF
70–86%
(
XIIIа), (XIIIc) X = EtCO
XIIIb) X = Ac
N
NHAc
R
N
NHAc
XO
(
O
N
O
O
NH
(XIIIа–c
)
(
XIVа–c)
Scheme 6. Synthesis of chromophores (XIVa)–(XIVc) from βꢀhydroxytyrosine derivatives.
The problem was solved by using stronger Lewis cases. Presumably, this is related to the sterical effect
acids. The best yield of acylation products for inactive
glycine and tertꢀleucine residues [compounds (XIIa
of the tertꢀbutyl group, which hinders the attack of the
terminal amide nitrogen at the carbonyl group.
)
and (XIIc)] was with the use of a combination of
reagents ZnCl2–(EtCO)2O. In the case of phenylalaꢀ
nine residue (XIIb), acetyl bromide in acetic anhyꢀ
dride was applied since, upon heating in the presence
of zinc chloride, the corresponding dihydroisoquinoꢀ
line (a product of the Friedel–Crafts reaction between
the acetylamino group and the benzene ring of phenyꢀ
lalanine) formed.
Compared with the acetyl group, the propionyl
group (X = EtCO) provides higher yields at the stage
of elimination–cyclization of compounds (XIIIa)–
(
XIIIc), probably due to a higher resistance to the
hydrolysis by water released during the reaction. The
addition of molecular sieves ( Å) makes it possible to
4
increase the yield by 5–10% with the use of acetate.
Previously, we have shown the advantage of using the
nonnucleophilic cesium carbonate in the cyclization
reaction [5]. However, in the case of the hydrophilic
glycine chromophore (XIVa), cesium carbonate was
substituted for by potassium carbonate due to the difꢀ
ficulties in separating the product after the reaction.
The elimination–cyclization stage should be carried
OBn
OBn
Base
R
H
N
NHAc
HO
O
O
out in the absence of oxygen since ꢀacylaminoꢀsubꢀ
α
O
NH
stituted chromophores in the base medium undergo
autooxidation. The glycineꢀcontaining chromophore
(
XIIа–c
)
(
XIVa) undergoes autooxidation to the greatest extent.
Scheme 7. Baseꢀcatalyzed fragmentation
ꢀacylated ꢀhydroxytyrosine (XIIa)–(XIIc).
During its synthesis, the inert conditions should be
observed till the stage of the neutralization of the reacꢀ
tion mixture (for more detail, see the Experimental
section). It should be noted that chromophores withꢀ
of
N
β
The hydration products (XIIIa)–(XIIIc) were used
in the elimination–cyclization reaction without sepaꢀ
rating the intermediate products of the carboxylate
out the
α
ꢀacylamino group (including 2,3ꢀdimethylꢀ
5ꢀarylideneꢀ3,5ꢀdihydroꢀ
4Hꢀimidazolꢀ4ꢀone) are
elimination. The intermediate dehydrotyrosine (XVc
)
prone to autooxidation to a much lesser extent, and
the cyclization is usually conducted without any preꢀ
formed only in the case of the tertꢀleucine residue
(Scheme 8, R = But, X = EtCO); in this process, the
cyclization stage required more time than in the other cautions.
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416
IVASHKIN et al.
OH
OK
OK
R = H, Bn, But
X = Ac, EtCO
R
K CO
K CO
2 3
2
–HOX
3
H
N
R
–H O
2
H
N
NHAc
NHAc
R
XO
N
N
NHAc
O
O
O
NH
O
O
NH
(XIIIа–c
)
(
XVа–c
)
(XIVа–c)
Scheme 8. Mechanism of elimination–cyclization of βꢀ(Oꢀacyloxy)tyrosine derivatives (XIIIa)–(XIIIc).
CONCLUSIONS
356 mg) was added to a solution of silylated chroꢀ
mophore (0.90 mmol, 335 mg) in CCl4 (9 mL), after
which AIBN was added over a period of 2.5 h under
boiling (in all 40 mg). After the termination of the
reaction (control by TLC, hexane–ethyl acetate 3 : 1),
the solution was separated and evaporated in vacuo. The
reaction product was purified by column chromatograꢀ
phy (hexane–ethyl acetate 4 : 1). Yield: 210 mg (50%);
1H NMR (800 MHz, CDCl3): 0.25 (6 H, s, CH3ꢀSi),
1.01 (9 H, s, TBS), 3.32 (3 H, s, NꢀCH3), 4.32 (2 H, s,
Studying the transformations of biomimetic preꢀ
cursor compounds into red chromophores under difꢀ
ferent conditions and by the action of various reagents
will make it possible in future to model processes
occurring in natural fluorescent proteins and elucidate
the mechanisms of the formation of chromophores.
EXPERIMENTAL
CH2), 6.91 (2 H, d,
J 8.5, Harom), 7.24 (1 H, s, Hvinyl),
NMR spectra were recorded on a Bruker Avance
700 and a Bruker Avance III 800 spectrometers (Gerꢀ
many, 700 and 800 MHz, respectively; chemical shifts,
8.08 (2 H, d, 8.5, Harom).
J
2ꢀ(Azidomethyl)ꢀ1ꢀmethylꢀ4ꢀ(4ꢀ((tertꢀbutyldimethylꢀ
ꢀimidazolꢀ5(4 )ꢀ
trometry analysis was carried out on timeꢀofꢀflight one (II). Sodium azide (0.23 mmol, 15 mg) was added
(TOF) spectrometers ISI RAS MXꢀ5311 (Russia) and to a solution of compound ( ) (0.13 mmol, 60 mg) in
Bruker Daltonics UltraFlex TOF/TOF (MALDI) absolute ethanol (6 mL), and the solution was stirred
δ
, ppm, spin coupling constant,
J, Hz). Mass specꢀ silyl)oxy)benzylidene)ꢀ4,5ꢀdihydroꢀ1
H
H
I
(Germany).
The following compounds were synthesized and
characterized by the methods described in the literaꢀ
ture: 4ꢀ(4ꢀhydroxybenzylidene)ꢀ1,2ꢀdimethylꢀ
imidazolꢀ5(4 ꢀone [3], ꢀacetylꢀtertꢀleucine
(3,3ꢀdimethylꢀ2ꢀ( ꢀacetylamino)butanoic acid)
for 15 min (full conversion by TLC: hexane–ethyl
acetate 3 : 1). The reaction mixture was evaporated
in vacuo at room temperature, and the product was
purified by column chromatography (hexane–ethyl
acetate 4 : 1). Yield: 47 mg (87%). The resulting azide
1Hꢀ
H)
N
(
II) decomposes slowly at room temperature both in
N
the pure form and in solution in EtOAc. With slight
[20], 4ꢀbenzyloxybenzaldehyde [21], ethyl ether of
2ꢀaminoꢀ3ꢀ(4ꢀ(benzyloxy)phenyl)acrylic acid [22],
heating (at about 50°C), the substance turns dark and
1
melts with the release of gas. H NMR (800 MHz,
and
2ꢀaminoꢀ3ꢀ(4ꢀ(benzyloxy)phenyl)ꢀ3ꢀhydroꢀ
CDCl3): 0.24 (6 H, s, CH3ꢀSi), 0.99 (12 H, s, TBS),
3.22 (3 H, s, NꢀCH3), 3.72 (2 H, s, CH2), 6.88 (2 H,
xypropanoic acid [18].
2ꢀ(Bromomethyl)ꢀ1ꢀmethylꢀ4ꢀ(4ꢀ((tertꢀbutyldimethylꢀ
d,
J
8.6, Harom), 7.15 (1 H, s, Hvinyl), 8.08 (2 H, d,
J 8.6,
silyl)oxy)benzylidene)ꢀ4,5ꢀdihydroꢀ1
H
ꢀimidazolꢀ5(4
l) and
TBSCl (2.1 mmol, 316 mg) were added to a suspenꢀ
sion of 4ꢀ(4ꢀhydroxybenzylidene)ꢀ1,2ꢀdimethylꢀ1
imidazolꢀ5(4
H)ꢀ
H
arom).
one (I). Diisopropylethylamine (2.1 mmol, 362
µ
(2 )ꢀ2ꢀ(
propionic acid (VII). Acetic anhydride (18 mmol,
)ꢀone (2.08 mmol, 450 mg) in dichloꢀ 1.70 mL) and triethylamine (25 mmol, 3.50 mL) were
romethane (20 mL). The mixture was stirred until the added to a suspension of ꢀ(4ꢀmethoxybenzyl)ꢀLꢀ
S
Nꢀ(4ꢀMethoxybenzyl)acetylamino)ꢀ3ꢀphenylꢀ
Hꢀ
H
N
complete dissolution of the starting substance and left phenylalanine (12 mmol, 3.46 g) in chloroform
to stand for 16 h at room temperature. The mixture (40 mL). The mixture was stirred until complete disꢀ
was diluted with dichloromethane (30 mL), washed solution. After 24 h, the transparent yellow solution
with water (20 mL) and a brine (20 mL), and dried was washed with water, evaporated, and redissolved in
over Na2SO4. The pale yellow powder obtained after 1 M NaOH. The resulting solution was washed with
evaporation was purified by column chromatography ethyl acetate (a colored contamination separated out),
(hexane–ethyl acetate 3 : 1) to yield 616 mg (89%) as acidified by dilute hydrochloric acid to pH 2, and
an almost colorless solid substance. NBS (2 mmol, extracted with ethyl acetate. The extract was washed
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SYNTHESIS OF BIOSYNTHETIC PRECURSORS OF CHROMOPHORES
417
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418
IVASHKIN et al.
with a brine, dried over sodium sulfate, and evapoꢀ colored nonpolar contaminations. The aqueous susꢀ
rated. The product was recrystallized from dioxane. pension was coated with a layer of ethyl acetate
Yield: 3.0 g (76%); 1H NMR (800 MHz, DMSOꢀd6
,
,
(150 mL) and neutralized by a 10% aqueous solution
of Na2CO3 (100 mL) under vigorous stirring. The
major rotamer): 1.92 (3 H, s, Ac), 3.02 (1 H, dd,
13.9, PheꢀCH2), 3.19 (1 H, dd,
3.70 (3 H, s, PMBꢀOMe), 3.88 (1 H, d,
PMBꢀCH2), 4.28 (1 H, dd, 5.9, 9.1, PheꢀCH), 4.31 a brine, dried over Na2SO4, and evaporated. The
(1 H, d, 16.4, PMBꢀCH2), 6.80 (2 H, d, 8.6, PMBꢀ resulting compound (XI) of about 90% purity was
8.6, PMBꢀCH), 7.09–7.25 (5 H, additionally purified by column chromatography
CHCl3–EtOH, 10 : 1). Yield: 11.1 g (78%; according
to NMR data, a mixture of relꢀ(2 ,3 and relꢀ(2 ,3
isomers 3.2 : 1 [18]) in the form of slowly crystallizing
oil; mp 119°C (decomposition); relꢀ(2 ,3 isomer:
1H NMR (800 MHz, DMSOꢀ
6): 1.8–2.6 (3 H, br,
H8 and H10), 3.60 (1 H, d, 4.9, H9), 3.66 (3 H, s,
H11), 4.83 (1 H, d, 4.9, H7), 5.06 (2 H, s, H4), 6.96
(2 H, d, 8.6, H5), 7.28 (2 H, d, 8.6, H6), 7.32–7.43
(5 H, m, H1, H2 and H3); relꢀ(2 ,3 ) isomer:
1H NMR (800 MHz, DMSOꢀ
6): 1.8–2.6 (3 H, br,
H8 and H10), 3.74 (3 H, s, H11), 4.90 (1 H, d, 6.1,
H7), 5.05 (2 H, s, H4), 6.94 (2 H, d, 8.7, H5), 7.20
8.7, H6), 7.32–7.43 (5 H, m, H1, H2,
J
9.1
J
5.9, 13.9, PheꢀCH2), aqueous phase was additionally extracted by ethyl aceꢀ
16.4, tate ( 60 mL). Combined extracts were washed with
J
2
×
J
J
J
CH), 7.05 (2 H, d,
m, Pheꢀarom).
J
(
S
R)
S S)
(4
aminoꢀ2ꢀphenylethyl]ꢀ3ꢀmethylꢀ4ꢀ(4ꢀhydroxybenꢀ
zylidene)ꢀ4,5ꢀdihydroꢀ1 ꢀimidazolꢀ5ꢀone (VIII). DCC
(3.4 mmol, 700 mg) was added to a solution of
ꢀPMBꢀ ꢀacetylphenylalanylglycine (3.31 mmol,
Z)ꢀ2ꢀ[(1R,S)ꢀ1ꢀ(NꢀAcetylꢀNꢀ(4ꢀmethoxybenzyl))
S
S)
H
d
J
N
N
J
1.27 g) in tetrahydrofuran (15 mL). The mixture was
stirred for 15 h and filtered. 4ꢀAcetoxybenzaldehyde
(3.31 mmol, 543 mg) and triethylamine (3.4 mmol,
0.48 mL) were added to the resulting solution. After
4 h, 40% aqueous methylamine (17 mmol, 1.43 mL)
was added. After additional stirring for 20 min, the
reaction mixture was diluted with ethyl acetate
(50 mL) and washed with 0.5 M hydrochloric acid,
0.5 M NaOH, and a brine. After evaporation, the pure
product (as indicated by TLC; chloroform–ethanol
15 : 1) was recrystallized from alcohol, ethyl acetate.
Yield: 315 mg (19%). The resulting dehydrotyrosine
(0.608 mmol, 305 mg) was dissolved in aqueous DMF
(40 mL), and NaOH (0.67 mmol, 27 mg) was added.
Argon was passed through the reaction mixture, after
which the mixture was refluxed for 2 h, neutralized by
hydrochloric acid, and evaporated in vacuo. The resiꢀ
due was dissolved in an ethyl acetate–water mixture.
The organic layer was washed with water and a brine
and dried over sodium sulfate. After evaporation, the
residue was chromatographed in the system chloroꢀ
form–ethanol 15 : 1. Yield: 150 mg (51%). Along with
the major product, brightly colored contaminations
were separated in trace amounts, which correspond,
according to the MALDIꢀMS data, to the dehydration
J
J
S
R
d
J
J
(2 H, d,
and H3).
J
NꢀMethylamide of relꢀ(2S,3R,S)ꢀ2ꢀ(2ꢀacetylamiꢀ
noacetylamino)ꢀ3ꢀ(4ꢀ(benzyloxy)phenyl)ꢀ3ꢀhydroxyproꢀ
pionic acid (XIIa). A 1 M solution of DCC in tetrahyꢀ
drofuran (12 mmol, 12 mL) was added at 0°C to a
solution of acetylglycine (12 mmol, 1.40 g), iPr2NEt
(12 mmol, 2.09 mL), and HOBt (12 mmol, 1.62 g) in
DMF (30 mL). The mixture was stirred for 1 h at room
temperature, a solution of compound (XI) (11 mmol,
3.31 g) in DMF (10 mL) was added, and the stirring
was continued for 12 h. One mL of AcOH was added
(to destroy DCC residues), and the mixture was stirred
for an additional 20 min and filtered. DMF was evapꢀ
orated in vacuo. The residue was dissolved in ethyl aceꢀ
tate (120 mL), washed with water, 0.5 M HCl
(to remove unreacted amine), a saturated NaHCO3
solution (to remove HOBt and acetylglycine), and a
brine, and dried over Na2SO4. After the evaporation of
ethyl acetate, the resulting methyl ester (pure, as indiꢀ
cated by TLC in EtOAc–EtOH 2 : 1) was dissolved in
MeCN (60 mL). Aqueous methylamine (60 mmol,
5.17 mL) was added to the solution, and the mixture
product (
the PMBꢀacetamide group (
321). 1H NMR (800 MHz, CDCl3): 2.09 (3 H, s,
H9), 2.98 (3 H, s, H5), 3.16 (1 H, dd, 6.1, 13.7, H7),
3.51 (1 H, dd, 8.8, 13.7, H7), 3.74 (3 H, s, H13), 4.65
(1 H, d, 16.9, H10), 4.69 (1 H, d, 16.9, H10), 6.17
(1 H, dd, 6.1, 8.8, H6), 6.76 (2 H, d, 8.6, H12),
6.92 (2 H, d, 8.6, H2), 6.95 (2 H, d, 8.6, H3), 7.01
(1 H, s, H4), 7.19–7.30 (5 H, m, H8), 8.12 (H, d,
8.6, H3); MALDIꢀMS, : 484 ([
+ H]+), 506
([
+ Na]+).
Methyl ester of relꢀ(2
zyloxy)phenyl)ꢀ3ꢀhydroxypropionic acid (XI). To a susꢀ yield for two stages was 3.79 g (88%, a mixture of steꢀ
pension of compound ( ) (47.3 mmol, 13.6 g) in reoisomers); mp 191°C
1H NMR (800 MHz,
MeOH (80 mL), SOCl2 (0.1 mol, 7.8 mL) was added DMSOꢀ 6, major isomer/rotamer): 1.84 (3 H, s,
dropwise at –10°C under vigorous stirring. The mixꢀ H13), 2.58 (3 H, d, 4.7, H15), 3.60 (1 H, dd, 5.6,
ture was boiled for 3 h and evaporated in vacuo. The 16.4, H11), 3.77 (1 H, dd, 5.6, 16.4, H11), 4.27 (1 H,
solid residue was immersed in 0.5 M HCl (200 mL) dd, 3.2, 8.8, H9), 5.02 (1 H, t, 3.2, 4.6, H7), 5.06
and shaken thoroughly with ethyl acetate to remove (2 H, s, H4), 5.60 (1 H, d, 4.6, H8), 6.91 (2 H, d,
m/z
482), the product of the elimination of
m/z
305), and ꢀketone
α
(m/z
J
J
was allowed to stand at 40 C until full conversion
°
J
J
according to TLC (about 3 h). A colorless fineꢀcrystalꢀ
line precipitate occurs, which, after washing with aceꢀ
tonitrile and ester, represents a spectrally pure comꢀ
pound (XIIa) (3.03 g, yield for two stages 69%). After
evaporation from mother liquor and column chromaꢀ
tography (EtOAc–EtOH 3 : 1), 0.76 g (19%) of comꢀ
J
J
J
J
J
m/z
M
M
S,3R,S)ꢀ2ꢀaminoꢀ3ꢀ(4ꢀ(benꢀ pound (XIIa) was additionally obtained. The total
X
;
d
J
J
J
J
J
J
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 37
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2011

SYNTHESIS OF BIOSYNTHETIC PRECURSORS OF CHROMOPHORES
419
J
8.6, H5), 7.25 (2 H, d,
3m, H1, H2, H3), 7.67 (1 H, q,
d, 8.8, H10), 8.07 (1 H, t, 5.6, H12); ESIꢀMS,
400 ([
+ H]+), 382 ([ – H2O + H]+), 422 ([
Na]+).
J 8.6, H6), 7.31–7.44 (5 H, purified by column chromatography (CHCl3–EtOH
1
J
4.6, H14), 7.74 (1 H, 85 : 15). Yield for two stages 0.91 g (72%); H NMR
(700 MHz, D2O, major isomer/rotamer): 1.24 (3 H, t,
7.13, H11), 2.18 (3 H, s, H9), 3.08 (3 H, s, H13),
3.71 (2 H, q, 7.13, H10), 4.34 (2 H, s, H7), 6.92 (2
H, d, 8.7, H2), 6.95 (1 H, s, H4), 7.86 (2 H, d, 8.7,
H3); ESIꢀMS, : 366 ([
+ H]+), 388 ([ + Na]+),
292 ([
– C2H5CO2H + H]+).
J
J
m/z:
M
M
M
+
J
J
J
J
NꢀMethylamide of relꢀ(2S,3R,S)ꢀ2ꢀ((2R,S)ꢀ2ꢀ
m
/z
M
M
acetylamino(benzyl)acetylamino)ꢀ3ꢀ(4ꢀ(benzyloxy)
phenyl)ꢀ3ꢀhydroxypropionic acid (XIIb). Compound
M
(
XIIb) was obtained in the same way as derivative
NꢀMethylamide of relꢀ(2S,3R,S)ꢀ2ꢀ((2R,S)ꢀ2ꢀ
(
XIIa). The final product was purified by column acetylamino(benzyl)acetylamino)ꢀ3ꢀ(4ꢀ(hydroxyphenyl)ꢀ
chromatography (CHCl3–EtOH 85 : 15). Yield for 3ꢀ(acetoxy)propionic acid (XIIIb). A solution of AcBr
two stages 68% (a mixture of stereoisomers); 1H NMR in Ac2O (2.0 mmol, 0.15 mL in 1.5 mL of Ac2O) was
(700 MHz, DMSOꢀ
(3 H, s, H13), 2.56 (3 H, d,
10.3 and 13.9, H16), 2.87 (1 H, dd,
4.24 (1 H, dd, 2.3, 8.6, H9), 4.59 (1 H, m, H11), 5.05 reaction mixture was diluted with ethyl acetate
(3 H, m, H4 and H7), 5.61 (1 H, d, 4.4, H8), 6.89 (50 mL) and washed with saturated NaHCO3 solution
(2 H, d, 8.8, H5), 7.05 (1 H, d, 7.1, H), 7.18–7.41 and a brine. After evaporation, the residue was dried at
(5 H, m, H1, H2, and H3), 7.22 (2 H, d, 8.8, H6), 80°C using an oil pump. The acylation product was
8.6, H10), suspended in methanol (30 mL), Pd/C (150 mg) and
trifluoroacetic acid (100 l) were added, and the
d
6, major isomer/rotamer): 1.72 added to a solution of compound (XIIb) (2.0 mmol,
4.7, H15), 2.66 (1 H, dd, 1.0 g) in methylene chloride (10 mL). After 1 h (100%
4.9, 13.9, H16), conversion by TLC, chloroform–ethanol 9 : 1), the
J
J
J
J
J
J
J
J
7.53 (1 H, q,
8.09 (1 H, d,
4.7, H14), 7.65 (1 H, d,
J
μ
resulting mixture was hydrated at room temperature
and atmospheric pressure (TLC, chloroform–ethanol
7 : 1). The solution was filtered through a paper filter
and evaporated. The product was purified by column
chromatography (CHCl3–EtOH 8 : 1). Yield for two
N
ꢀMethylamide of relꢀ(2
S
,3
R
,
S
)ꢀ2ꢀ((2R,S)ꢀ2ꢀ
acetylamino(tertꢀbutyl)acetylamino)ꢀ3ꢀ(4ꢀ(benzyloxy)
phenyl)ꢀ3ꢀhydroxypropionic acid (XIIc). Compound
(
(
XIIc) was obtained in the same way as derivative
XIIa), except that the reaction mixture was stirred for
2 h before, and for 24 h after, the addition of amine
XI). The final product was purified by column chroꢀ
matography (CHCl3–EtOH 10 : 1). Yield for two
stages 73% (a mixture of stereoisomers); mp 156°C;
1H NMR (700 MHz, CDCl3, major isomer/rotamer):
0.67 (9 H, s, H16), 1.89 (3 H, s, H13), 2.58 (3 H, d,
stages 0.65 g (74%); 1H NMR (800 MHz, DMSOꢀd6
,
major isomer/rotamer): 1.91 and 2.05 (6 H, 2s, H11
and H16), 2.63 (3 H, d, 4.9, H7), 2.85 and 3.00 (2 H,
2 m, H12), 4.63 (m, H9), 4.74 (1 H, dd, 6.1, 8.8,
H5), 6.15 (1 H, d, 6.1, H4), 6.71–7.26 (8 H, m, H3,
H10, H13, H14, and H15), 6.74 (2 H, d, 8.6, H2),
6.84 (1 H, q, 4.9, H6), 7.09 (1 H, d, 8.8, H8).
(
J
J
J
J
J
J
J
J
4.6, H15), 4.05 (1 H, d,
2.9, 8.8, H9), 5.07 (2 H, m, H4), 5.16 (1 H, dd,
5.3, H8), 6.87 (2 H, d,
4.6, H14), 7.27 (2 H, d,
7.30–7.43 (5 H, m, H1, H2, H3), 7.73 (1 H, d,
H12), 7.87 (1 H, d, 8.8, H10); ESIꢀMS,
([
+ H]+), 438 ([ – H2O + H]+), 478 ([
J 6.8, H11), 4.30 (1 H, dd,
J
J
2.9,
N
ꢀMethylamide of relꢀ(2 ,3 )ꢀ2ꢀ((2
S
R
,
S
R,S)ꢀ2ꢀ
5.3, H7), 5.51 (1 H, d,
H5), 7.21 (1 H, q,
J
8.6, acetylamino(tertꢀbutyl)acetylamino)ꢀ3ꢀ(4ꢀhydroxyꢀ
J
J
8.6, H6), phenyl)ꢀ3ꢀ(propionyloxy)propionic acid (XIIIc). This
6.8, compound was prepared by the aboveꢀdescribed
J
z
1
J
m/
: 456 scheme. Yield: 81%; H NMR (700 MHz, CDCl3,
M
M
M
+ Na]+). major isomer/rotamer): 0.86 (9 H, s, H15), 0.98 (3 H,
t,
J
J
J
2.4,
9.5,
N
ꢀMethylamide of relꢀ(2 ,3 )ꢀ2ꢀ(2ꢀacetylamiꢀ
S R,S
7.5, H8), 2.42 (3 H, d,
H12), 4.70 (1 H, dd,
noacetylamino)ꢀ3ꢀ(4ꢀ(hydroxyphenyl)ꢀ3ꢀ(propionyloxy)
6.9, 8.8, H10), 5.06 (2 H, s,
8.8, H5),
8.8, H6), 7.33–7.43 (5 H, m, H1, H2,
H3), 7.73 (1 H, d, 9.5, H13), 7.78 (1 H, q, 4.7,
H16), 8.07 (1 H, d, 8.8, H11); ESIꢀMS, : 512
([
+ H]+), 438 ([ – C2H5CO2H + H]+), 534 ([
Na]+).
propionic acid (XIIIa). A mixture of compound (XIIa
(3.45 mmol, 1.38 g), propionic anhydride (15 mL),
and ZnCl2 (1 mmol, 136 mg) was stirred at 90°C until
the complete dissolution of the starting compound
)
H4), 6.85 (1 H, d,
7.19 (2 H, d,
J
(
XIIa) and full conversion according to TLC (total
M
M +
time 4 h). An excess of propionic anhydride and proꢀ
pionic acid was evaporated in vacuo at 70°C. The resꢀ
idue was reevaporated twice with isopropanol, disꢀ
(4
ꢀAcetylamino)methylꢀ3ꢀmethylꢀ4ꢀ(4ꢀ
ꢀimidazolꢀ5ꢀ
through a silica gel layer. After the evaporation of solꢀ one (XIVa). Compound (XIIIa) (0.60 mmol, 220 mg),
vents, the solid colorless residue was recrystallized K2CO3 (1.28 mmol, 176 mg), MS Å, and DMF
solved in an ethanol–chloroform mixture, and passed hydroxybenzylidene)ꢀ4,5ꢀdihydroꢀ1
from isopropanol. To a suspension of the recrystallizaꢀ (20 mL) were placed in a flask equipped with a septum
tion product in methanol, 10% Pd/C (200 mg) and and magnetic stirrer. Argon was blown through the
TFA (50 l) were added, and the resulting mixture was mixture for 20 min, and then the mixture was stirred at
μ
hydrated at atmospheric pressure for 3 h (100% conꢀ 110°C for 35 min. After cooling to room temperature,
version according to TLC). The solution was filtered a solution of NH4Cl (1.5 mmol, 80 mg) in H2O (5 mL)
through a paper filter and evaporated. The product was was added. The resulting orange solution was evapoꢀ
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 37
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2011

420
IVASHKIN et al.
rated in vacuo at 60°C. The residue was reevaporated
with ethanol and a threefold (relative to the solid subꢀ
stance) volume of silica gel. The resulting powder was
chromatographed on a column of silica gel in the sysꢀ
tem EtOAc–EtOH 20 : 1–15 : 1. Yield: 121 mg (74%,
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1
a thermodynamic mixture of Z/E isomers 10 : 1); H
NMR (800 MHz, DMSOꢀd6
H8), 3.08 (3 H, s, H5), 4.32 (2 H, d,
(2 H, d, 8.8, H2), 6.99 (1 H, s, H4), 8.11 (2 H, d,
8.8, H3), 8.41 (1 H, t, 5.6, H7), 10.16 (1 H, br, H1);
ESIꢀMS, : 274 ([
+ H]+), 296 ([ + Na]+), 312
([
+ K]+).
,
Z
isomer): 1.94 (3 H, s,
J
5.6, H6), 6.85
J
J
J
m/z
M
M
M
(4 )ꢀ2ꢀ[(1
Z
R
,
S
)ꢀ(1ꢀAcetylaminoꢀ2ꢀphenyl)ethyl]ꢀ
3ꢀmethylꢀ4ꢀ(4ꢀhydroxybenzylidene)ꢀ4,5ꢀdihydroꢀ1
Hꢀ
5. Yampolsky, I.V., Balashova, T.A., and Lukyanov, K.A.,
imidazolꢀ5ꢀone (XIVb). Compound (XIVb) was synꢀ
thesized in the same way as derivative (XIVa). Yield
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70%; 1H NMR (700 MHz, DMSOꢀd6
,
Z
isomer): 1.78
(3 H, s, H8), 2.97 (3 H, s, H5), 3.07 (1 H, dd, 8.6,
13.7, H9), 3.29 (1 H, dd, 6.4, 13.9, H9), 5.09 (1 H,
dd, 6.4, 8.3, and 8.6, H6), 6.86 (2 H, d, 8.8, H2),
7.00 (1 H, s, H4), 7.19–7.31 (5 H, m, H10, H11, and
H12), 8.13 (2 H, d, 8.8, H3), 8.53 (1 H, d, 8.6, H7),
10.17 (1 H, s, H1); HSQCꢀ1Hꢀ13C (800 MHz
DMSOꢀd6 isomer): 22.7 (H8), 26.8 (H5), 37.7
J
J
7. Devasia, G.M., Tetrahedron Lett., 1976, vol. 17,
pp. 571–572.
J
J
8. Ekeley, J.B. and Ronzio, A.R., J. Am. Chem. Soc., 1935,
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9. Kidwai, A.R. and Devasia, G.M., J. Org. Chem., 1962,
J
J
,
,
Z
vol. 27, pp. 4527–4531.
(H9), 48.0 (H6), 116.1 (H2), 126.6 (H12), 127.8
(H4), 128.4, and 129.7 (H11 and H12), 135.0 (H3).
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guignon, J.ꢀJ., Synthesis, 2005, pp. 25–27.
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(4
Z)ꢀ2ꢀ[(1R,S)ꢀ(1ꢀAcetylaminoꢀ2ꢀdimethyl)propyl]ꢀ
3ꢀmethylꢀ4ꢀ(4ꢀhydroxybenzylidene)ꢀ4,5ꢀdihydroꢀ1
Hꢀ
imidazolꢀ5ꢀone (XIVc). Compound (XIVc) was syntheꢀ
sized in the same way as derivative (XIVa). The reacꢀ
tion temperature was maintained at 140°C, and the
reaction occurred in two stages according to TLC (the
product of the first stage is the substance easily oxidizꢀ
able by permanganate with a strong absorption in the
UV region, which is presumably the product of splitꢀ
ting off of propionate). Yield: 86%; 1H NMR
13. Tripathy, K.P. and Mukerjee, A.K., Synthesis, 1985,
pp. 285–288.
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(700 MHz, CDCl3,
(3 H, s, H8), 3.26 (3 H, s, H5), 4.94 (1 H, d,
H6), 6.66 (1 H, d, 9.25, H7), 6.86 (2 H, d, 8.6, H2),
7.12 (1 H, s, H4), 7.99 (2 H, 8.6, H3), 8.26 (1 H, br s,
H1). ESIꢀMS, : 330 ([
+ H]+), 352 ([ + Na]+),
368 ([
+ K]+).
Z
isomer): 1.10 (9 H, s, H9) 2.08
J
9.25,
J
J
J
m/z
M
M
18. Bolhofer, W.A., J. Am. Chem. Soc., 1954, vol. 76,
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ACKNOWLEDGMENTS
This work was supported by the Russian Foundaꢀ
tion for Basic Research (project no. 09ꢀ04ꢀ00356ꢀa),
the program of the Presidium of the Russian Academy
of Sciences “Molecular and Cellular Biology,” and the
Program for Supporting the Leading Scientific
Schools of Russia (NShꢀ5638.2010.4).
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RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 37
No. 4
2011