The two pathways for effective orthogonal protection of L-ornithine, for amino acylation of 5'-O-Pivaloyl nucleosides, describe the general and important role for the successful imitation, during the synthesis of the model substrates for the ribosomal mimic reaction

Article abstract of DOI:10.2174/092986610791306625

Bz(NO₂)-Orn(Boc)-OCH₂CN was synthesized as an amino acid component with effective and successful orthogonal protection for amino acylation of 5'-O-Pivaloyl nucleosides and preparation of substrates for model ribosome reactions. The synthesis was carried out using suitable combinations of the methods of peptide synthesis and modification of amino acids.

Full text of DOI:10.2174/092986610791306625

Protein & Peptide Letters, 2010, 17, 889-898
889
The Two Pathways for Effective Orthogonal Protection of L-Ornithine, for
Amino Acylation of 5’-O-Pivaloyl Nucleosides, Describe the General and
Important Role for the Successful Imitation, During the Synthesis of the
Model Substrates for the Ribosomal Mimic Reaction
1,
*
2
1
Stanislav G. Bayryamov , Nikolay G. Vassilev and Dimiter D. Petkov
1Laboratory of Biocatalysis, Institute of Organic Chemistry, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria;
2Laboratory of Nuclear Magnetic Resonance, Institute of Organic Chemistry, Bulgarian Academy of Sciences, Sofia
1113, Bulgaria
Abstract: Bz(NO2)-Orn(Boc)-OCH2CN was synthesized as an amino acid component with effective and successful or-
thogonal protection for amino acylation of 5’-O-Pivaloyl nucleosides and preparation of substrates for model ribosome re-
actions. The synthesis was carried out using suitable combinations of the methods of peptide synthesis and modification of
amino acids.
Keywords: Conventional peptide synthesis, orthogonal protection, ornithine.
1. INTRODUCTION
respectively. Recent studies of ribosome-substrate crystal
structures [6] reveal that the 2’-OH of the peptidyl tRNA 3’-
terminal adenosine hydrogen bonds to aminoacyl tRNA alfa-
NH2 and is the only functional group positioned to act as a
general base. The deleterious effect of its removal on ribo-
some catalysis [2, 10] and the dramatic change in the ami-
nolysis rate after deletion of the adenosine 2’-OH observed
before, strongly support its significant catalytic role in sub-
strate-assisted ribosome catalysis.
The dramatic difference in the aminolysis rate of the
adenosine and deoxyadenosine esters approaches the large
difference in reactivity of peptidyl tRNA and its 2’-deoxy
mutant in the ribosome fragment reaction [1]. ꢀhe inactiva-
tion of the peptidyl tRNA as substrate when its 2’-OH in 3’-
terminal adenosine A76 is deleted, presumes its crucial par-
ticipation in the catalysis by the ribosome in the process of
aminolysis reaction, during the protein biosynthesis [2], but
the real mechanism still remain obscure.
The main goal of the previous article [7] was to prove the
crucial role of 2’-OH-group of the ribose ring in the rate ac-
celeration of adenosine ester aminolysis, and the essential
role of 2’-OH group of peptidyl tRNA A76 in the reaction of
peptide bond formation. For this purpose 2’/3’-O-ornithinyl
adenosine and its 2’-deoxy analogue were synthesized. The
role of 2’-OH group in the transfer of acyl group to amine
was studied by kinetic experiments. The results revealed that
of 2’-OH group effectively catalyzes adenosine esters ami-
nolysis. The proper model substrates were synthesized, using
combination of methods, employing in peptide chemistry,
amino acid modification, nucleoside and nucleotide synthesis
and modification. Because of the strong requirements of
adequate imitation of substrates for intracomplex ribosome
reaction, the amino acyl and nucleoside component had to be
designed and prepared with maximum appropriation.
Understanding of the nature of the ribosome catalysis is
still a challenge of both chemists and chemical/molecular
biologists [3, 4]. This high catalytic proficiency of the ribo-
some has been recently attributed to the proton shuttling role
of the crucial peptidyl tRNA A76 2’-OH [5-8]. The latter is
thought to provide a proton shuttle from the attacking alpha-
NH2 to the P-site 3’OH of A76 [7]. In recent computational
studies [7, 9] of the proton shuttle mechanism we predicted
that syn-oriented 2-OH provided favourable proton transfer
geometry in 2-hydroxyalkyl ester aminolysis, a reaction
naturally catalyzed by the ribosome. This is a necessary but
not sufficient condition that can account for the ribosome
catalytic efficiency.
After the formation of the ribosome-substrate complex,
each of the ribosome substrates becomes an integral part of
the ribosome and the participation of substrate acid-base
groups in the catalytic process is possible. This reminds us of
the formation of a holoenzyme from an apoenzyme and co-
enzyme, the latter being the ribosome and peptidyl tRNA,
For this purpose Bz(NO2)-Orn(Boc)-OCH2CN was syn-
thesized, using our originally developed strategy for or-
thogonal functional group protection, which was used as an
amino acid component for amino acylation of 5’-O-Pivaloyl
nucleosides and preparation of substrates for model ribosome
reactions, using two possible pathways. Two strategies were
employed - the orthoformate strategy was employed, leading
to high purity, good yields and shortening of the reaction
time for the synthesis of the final products. The second
pathway employs the Fmoc-strategy. For the successful
choice of reaction conditions, we compared two of them
*Address correspondence to this author at the Laboratory of BioCatalysis,
Institute of Organic Chemistry, Bulgrian Academy of Sciences, Acad. Bon-
chev Str., bld. 9, 1113 Sofia, Bulgaria; Tel: +359-2-9606-191; Fax: +359-2-
8700-225; E-mails: stanislav_bayryamov@abv.bg;
stanly_chem2002@abv.bg
Contract/Grant Sponsor: National Research Fund; Contract/grand num-
ber: X-1517 and X-1408.
0929-8665/10 $55.00+.00
© 2010 Bentham Science Publishers Ltd.

890 Protein & Peptide Letters, 2010, Vol. 17, No. 7
Bayryamov et al.
(two strategies). The synthesis was carried out using suitable
combinations of the methods of peptide synthesis and modi-
fication of amino acids.
J=6.4 Hz, 2H, 5-CH2), 3.12(bs, 1H, 2-CH), 6.864(t, J=5.4
Hz, 1H, 5-CH2NH).
13C NMR (150 MHz, DMSO-d6, 25oC):
ꢀ =
Bz(NO2)-Orn(Boc)-OCH2CN was synthesized using two
main pathways for orthogonal amino acid protection, having
side functionalities. The design of the amino acid component
was carefully made, because of the strong requirements for
the stability of the final substrates and appropriate imitation
of the model intramolecular aminolysis reactions. Although
the orthoformate strategy spares some steps, because of the
lower yields, we also decided to synthesize Bz(NO2)-
Orn(Boc)-OCH2CN using Fmoc-strategy and to compare
everyone of them.
23.78(CH2), 24.94(CH2), 25.87(4-CH2), 28.24(CH3),
28.79(3-CH2), 39.97(5-CH2), 45.35(CH2), 54.03(2-CH),
77.28(C), 155.52(NHCOO), 173.34(COOH).
3.1.2. Synthesis of H-Orn(Boc)-OH (II). [14, 15]
1.686g (10mmol) of L-Orn monohydrochloride were
dissolved in 3ml H2O containing 0.4g NaOH (10mmol). Af-
ter that 3ml MeOH and a solution of 2.4g (11mmol) of di-
tert butyl pyrocarbonate-(Boc)2O in 3ml MeOH were added
at room temperature. The mixture was evaporated after 24
hours and the crude product was dissolved in boiling water.
A precipitate appeared after cooling and turned the solution
into a mass of crystals. The mixture was cooled in an ice-
water bath, filtered and the product washed with ice-water in
several portions. Some Nꢁ-substituted product was formed
during the reaction but remained in solution. The Nꢀ-
substituted derivatives of ornithine are usually insoluble in
water while the Nꢁ-acyl derivatives are more readily soluble.
The crude material was again dissolved in boiling water and
the solution was diluted with hot ethyl alcohol. Under cool-
ing, the crystals were separated. They were collected on a
filter, washed with 60% ethyl alcohol and dried in the air.
The product was prepared as piperidinium salt to allow its
dissolution in DMSO-d6 for NMR analysis. Recrystalysed
from MeOH:Hexane. Yield of the pure H-Orn(Boc)-OH -
1.207g (52%). M.p. 218-2190C.
2. EXPERIMENTAL
2.1. General Procedures, Methods and Materials
All reagents and solvents were purchased and used with-
out further purification. Reverse phase HPLC analyses were
performed on a Waters Liquid Chromatograph equipped with
an absorbance detector model 441 set at 280 nm and a col-
umn Nucleosil 100-5C18 (12.5 cm x4.6 mm) for analytical
1
runs. H and 13C NMR spectra were recorded on a Bruker
Avance II+ 600MHz spectrometer using BBO or TBI probe-
heads. Chemical shifts are expressed in ppm and coupling
1
constants in Hz. The precise assignments of the H and 13C
NMR spectra were accomplished by measurement of 2D
homonuclear correlation (COSY), DEPT-135 and 2D inverse
detected heteronuclear (C–H) correlations (HSQC and
HMBC). Chemical shifts are reported in ꢀ (ppm). The analy-
1H NMR (600 MHz, DMSO-d6, 25oC): ꢀ = 1.365(s, 9H,
CH3), 1.42(bs, 2H, 4-CH2), 1.48(bs, 6H, CH2), 1.51(bs, 1H)
and 1.64(bs, 1H, 3-CH2), 2.74(bs, 4H, CH2), 2.871(pseudo-q,
J=6.4 Hz, 2H, 5-CH2), 3.12(bs, 1H, 2-CH), 6.864(t, J=5.4
Hz, 1H, 5-CH2NH).
1
sis of first order multiplets in H NMR spectra was speed up
by the use of FAFOMA program [11].
3. SYNTHESIS
3.1. Synthesis of H-Orn(Boc)-OH [12, 13]
3.1.1. Synthesis of H-Orn(Boc)-OH (I)
13C NMR (150 MHz, DMSO-d6, 25oC):
ꢀ =
23.78(CH2), 24.94(CH2), 25.87(4-CH2), 28.24(CH3),
28.79(3-CH2), 39.97(5-CH2), 45.35(CH2), 54.03(2-CH),
77.28(C), 155.52(NHCOO), 173.34(COOH).
1.686g (10 mmol) of L-Orn hydrochloride were dis-
solved in 10ml 2N NaOH and a solution of CuSO4.5H2O
(cupric sulfate pentahydrate, 1.25g, 5mmol=10meq) in 5ml
of water was added. A precipitate formed which soon dis-
solved. The mixture was heated to 600C, cooled to room
temperature, diluted with 35ml MeOH and made more alka-
line with 2N NaOH (about 1.5ml-2ml). This was followed
by the addition of 2.4g (11mmol) of di-tert butyl pyrocar-
bonate-(Boc)2O. The mixture was vigorously stirred at 25-
200C for about one and a half hour. The purple-blue precipi-
tate was collected on a filter, washed with a mixture of
MeOH (5ml) and water (17.5ml), then with MeOH (2.5ml)
and dried in the air. The well disintegrated copper complex
was triturated and washed with 0.2N HCl (5 times with about
5ml each time), twice with distilled water (with about 3ml
each time), 5 times with approximately 1.5N NH4OH (with
about 3ml each time) and finally with water. The product
was prepared as its piperidinium salts to allow its dissolution
in DMSO-d6 for NMR analysis. Yield of the pure H-
Orn(Boc)-OH 1.508g (65%). M.p. 218-2190C.
3.1.3. Synthesis of H-Orn(Boc)-OH (III). [16, 17]
A solution of 1.686g of L-ornitine monohydrochloride
(10 mmol ) in water (100 ml) was heated under reflux while
CuCO3 (cupric carbonate - basic, 3g) was added with cau-
tion. After two hours of boiling the undissolved CuCO3 was
removed from the hot mixture by filtration and washed with
hot water (10ml). The combined filtrate and washings were
cooled to room temperature and treated with NaHCO3 (3.2g).
The mixture was vigorously stirred and a solution of 3.272g
(15mmol) (Boc)2O in 100ml acetone were added. Stirring
was continued overnight and the precipitated light blue cop-
per complex was collected on a filter, thoroughly washed
with water, acetone and ether and dried in the air. It weights
about 3.680-3.943g (70-75%). The finally powdered copper
complex (3.68g, 7mmol) was suspended in boiling distilled
water (40-50ml) and a stream of H2S was passed through the
solution for ꢂ hour. Boiling was continued to remove excess
H2S. Then it was cooled and 1.2ml of 3N HCl were added at
00C and the mixture was quickly filtered from CuS (cupric
sulfide). The pH of the filtrate was adjusted to about 6 with
4N NaOH and the product-Nꢀ-Boc-L-ornithine was insoluble
1H NMR (600 MHz, DMSO-d6, 25oC): ꢀ = 1.365(s, 9H,
CH3), 1.42(bs, 2H, 4-CH2), 1.48(bs, 6H, CH2), 1.51(bs, 1H)
and 1.64(bs, 1H, 3-CH2), 2.74(bs, 4H, CH2), 2.871(pseudo-q,

The Two Pathways for Effective Orthogonal Protection
Protein & Peptide Letters, 2010, Vol. 17, No. 7 891
in water and separates from the solution. The mixture was
cooled to 00C, the product was collected on a filter, washed
first with water, then with ethanol and dried in the air. Yield
of H-Orn(Boc)-OH: 1.625g (70%). A sample was purified by
dissolution in dilute HCl at 00C and precipitation with dilute
NaOH. The product was prepared as its piperidinium salts to
allow its dissolution in DMSO-d6 for NMR analysis. M.p.
218-2190C. (Decomposition of the copper complex was pos-
sible also without the use of H2S [25]. To a suspension of the
copper complex (10mmol) in 50ml of water 1.12g (15mmol)
of thioacetamide were added, then 2N NaOH to bring the pH
of the suspension to 8. The mixture was stirred at room tem-
perature for one day. The pH was lowered to 1.6 by the addi-
tion of 2N HCl and the CuS (cupric sulfide) was removed by
filtration. The filtrate was neutralized to precipitate the prod-
uct) [12, 13].
Hz, 2H, 1-CH and 8-CH), 7.922(d, J=7.5 Hz, 2H, 4-CH and
5-CH).
13C NMR (150 MHz, DMSO-d6, 25oC):
ꢀ =
25.23(CH2), 45.90(9-CH), 53.60(2-CH), 71.81(1-CH2),
120.27(4-CH and 5-CH), 124.86(1-CH and 8-CH), 127.27(2-
CH and 7-CH), 127.93(3-CH and 6-CH), 140.78(4a-C and
4b-C), 142.59(8a-C and 9a-C), 151.06(COO), 169.70(NCO).
3.4. Synthesis of Fmoc-Orn(Boc)-OH
To a stirred solution of 2.32g (10mmol) H-Orn(Boc)-OH
and 0.84g (10mmol) of NaHCO3 in a mixture of water
(14ml) and acetone (14ml) were added 3.36g (10mmol) of 9-
fluorenylmethyl succinimidyl carbonate (Fmoc-ONSu). Af-
ter stirring overnight the mixture was diluted with 30ml
CH2Cl2, acidified to pH 2-3 with NaHSO4 and washed sev-
eral times with CH2Cl2. The organic phases were collected
and the combined organic layer was washed several times
with brine, dried over Na2SO4 and evaporated to dryness in
vacuo. Yield-3.723g (82%), Rf-0.352 (CH2Cl2 : CH3OH-
9:1), Rf-0.320 (CH2Cl2 : CH3OH-9.5:0.5). M.p. 113-1150C.
1H NMR (600 MHz, DMSO-d6, 25oC): ꢀ = 1.365(s, 9H,
CH3), 1.42(bs, 2H, 4-CH2), 1.48(bs, 6H, CH2), 1.51(bs, 1H)
and 1.64(bs, 1H, 3-CH2), 2.74(bs, 4H, CH2), 2.871(pseudo-q,
J=6.4 Hz, 2H, 5-CH2), 3.12(bs, 1H, 2-CH), 6.864(t, J=5.4
Hz, 1H, 5-CH2NH).
1H NMR (600 MHz, DMSO-d6, 25oC): ꢀ = 1.369(s, 9H,
CH3), 1.40-1.47(m, 2H, 4-CH2), 1.52-1.58(m, 1H) and 1.66-
1.72(m, 1H, 3-CH2), 2.906(q, J=6.5 Hz, 2H, 5-CH2),
3.907(ddd, J=4.6, 8.0, 13.0 Hz, 1H, 2-CH), 4.21-4.23(m, 1H,
9-CH), 4.25-4.30(m, 2H, OCH2), 6.816(t, J=5.5 Hz, 1H, 5-
CH2NH), 7.329(t, J=7.4 Hz, 2H, 2-CH and 7-CH), 7.416(t,
J=7.4 Hz, 2H, 3-CH and 6-CH), 7.643(d, J=8.0 Hz, 1H, 2-
CHNH), 7.724(d, J=7.5 Hz, 2H, 1-CH and 8-CH), 7.894(d,
J=7.6 Hz, 2H, 4-CH and 5-CH), 12.578(bs, 1H, COOH).
13C NMR (150 MHz, DMSO-d6, 25oC): ꢀ = 26.20(4-CH2),
28.08(3-CH2), 28.21(CH3), 39.97(5-CH2), 46.57(9-CH),
53.60(2-CH), 65.54(OCH2), 77.34(C), 120.06 and 120.08(4-
CH and 5-CH), 125.23 and 125.25(1-CH and 8-CH),
127.02(2-CH and 7-CH), 127.58(3-CH and 6-CH), 140.64
and 140.66(4a-C and 4b-C), 143.74 and 143.79(8a-C and 9a-
C), 155.53 and 156.07(NHCOO), 173.82(COOH).
13C NMR (150 MHz, DMSO-d6, 25oC):
23.78(CH2), 24.94(CH2), 25.87(4-CH2), 28.24(CH3), 28.79
(3-CH2), 39.97(5-CH2), 45.35(CH2), 54.03(2-CH), 77.28(C),
155.52(NHCOO), 173.34(COOH).
ꢀ =
3.2. Synthesis of N-Hydroxysuccinimide.DCHA (HONSu.
DCHA)
The synthesis of HONSu.DCHA was carried out accord-
ing to the procedure of A. Paquet, discussed in the Part: Dis-
cussion.
To 1.151g (0.01mol) of N-hydroxysuccinimide (HONSu)
in acetone was added 1.986ml (0.01mol) of dicyclohexy-
lamine. The mixture was stirred overnight and the precipitate
was isolated by filtration, washed with cold acetone and
thoroughly dried. Yield-2.845g (96%).
3.3. Synthesis of 9-Fluorenylmethyl Succinimidyl Car-
bonate (Fmoc-ONSu)
3.5. Synthesis of Fmoc-Orn(Boc)-OMe
3.5.1. Synthesis of Fmoc-Orn(Boc)-OMe (I)
The synthesis of Fmoc-ONSu was accomplished accord-
ing to the procedure for Fmoc protection, used by A. Paquet,
as well as by Sigler G. F.; Fuller, W. D.; Chaturvedi, N. C. et
al., described in: Discussion.
To a solution of 1g (2.2 mmol) of Fmoc-Orn(Boc)-OH in
diethyl ether (10 ml) was added dropwise a solution of
CH2N2 in diethyl ether until the solution remained lightly
yellow and the mixture was allowed to stand at room tem-
perature for about 0.5 hours. The reaction mixture was
evaporated under reduced pressure and coevaporated to dry-
ness 2-3 times with diethyl ether.Yield-0.99g (96%). Rf-
0.878 (CH2Cl2: CH3OH-9:1). Rf-0.652 (CH2Cl2: CH3OH-
9.5:0.5). M.p. 119-1200C.
1H NMR (600 MHz, DMSO-d6, 25oC): ꢀ = 1.369(s, 9H,
CH3), 1.39-1.47(m, 2H, 4-CH2), 1.54-1.60(m, 1H) and 1.65-
1.71(m, 2H, 3-CH2), 2.88-2.93(m, 2H, 5-CH2), 3.616(s, 3H,
OCH3), 4.009(ddd, J=4.9 Hz, J=7.9 Hz, J=9.4 Hz, 1H, 2-
CH), 4.224(t, J=7.1Hz, 1H, 9-CH), 4.295(d, J=7.1Hz, 2H,
OCH2), 6.810(t, J=5.7 Hz, 1H, 5-CH2NH), 7.333(dt, J=7.4
Hz, J=0.9 Hz, 2H, 2-CH and 7-CH), 7.418(t, J=7.4 Hz, 2H,
3-CH and 6-CH), 7.714(dd, J=7.4 Hz, J=2.8 Hz, 2H, 1-CH
and 8-CH), 7.790(d, J=7.8 Hz, 1H, 2-CHNH), 7.895(d, J=7.6
Hz, 2H, 4-CH and 5-CH).
To
a stirred solution of 1.4g of Fmoc-chloride
(5.44mmol) in 10ml chloroform was added 1.61g
(5.44mmol) of N-hydroxysuccinimide.DCHA (HONSu.
DCHA) in portions. The stirring was continued overnight,
the precipitate was filtered off and washed with chloroform.
The filtrate and washings were combined and washed with
1/3 of the volume each of 10% citric acid, 10% NaHCO3,
water and dried. The solvent was distilled off in vacuo and
the residue was recrystallized from chloroform-ether to give
1.65g (90%) of the title compound, Rf-0.910 (CH2Cl2 :
CH3OH-9.5:0.5). M.p. 1510C.
1H NMR (600 MHz, DMSO-d6, 25oC): ꢀ = 2.745(s, 4H,
CH2), 4.420(d, J=6.0 Hz, 1H, 8-CH), 4.824(t, J=6.0 Hz, 2H,
1-CH2), 7.362(dt, J=1.0, 7.5 Hz, 2H, 2-CH and 7-CH),
7.446(t, J=7.4 Hz, 2H, 3-CH and 6-CH), 7.692(dd, J=0.6, 7.5

892 Protein & Peptide Letters, 2010, Vol. 17, No. 7
Bayryamov et al.
13C NMR (150 MHz, DMSO-d6, 25oC): ꢀ = 25.98(4-
CH2), 27.91(3-CH2), 28.19(CH3), 39.97(5-CH2), 46.56(9-
CH), 51.78(OCH3), 53.56(2-CH), 65.55(OCH2), 77.37(C),
120.08(4-CH and 5-CH), 125.18(1-CH and 8-CH), 127.01(2-
CH and 7-CH), 127.58(3-CH and 6-CH), 140.67(4a-C and
4b-C), 143.69 and 143.74(8a-C and 9a-C), 155.53 and
156.03(NHCOO), 172.82(COOH).
1) After the reaction was completed, the solution was
evaporated under reduced pressure and the amorphous resi-
due was dissolved in MeOH. The methanol mixture was di-
luted with H2O, then was extracted several times with hex-
ane or petroleum ether and then it (MeOH-phase) was evapo-
rated in vacuo.
2) After the reaction was completed (TLC) the solution
was evaporated to dryness under reduced pressure and the
amorphous residue was dissolved in CH2Cl2 : H2O-1:1. The
mixture was slowly acidified to pH 5-6 in ice cold bath. The
water phase was extracted several times with CH2Cl2, then
with hexane. After it (H2O-phase) was slowly alkalized to
pH 9-9.5 again in ice cold bath and extracted several times
with CH2Cl2. The combined organic phases were collected
together and washed several times with 10% solution of
Na2CO3 in water, brine, dried over anhydrous Na2SO4 and
evaporated to dryness in vacuum.
3.5.2. Synthesis of Fmoc-Orn(Boc)-OMe (II)
To a solution of 1g (2.2 mmol) of Fmoc-Orn(Boc)-OH in
10ml MeOH were added 0.550 ml (5mmol) trimethyl ortho-
formate and 0.414g (3mmol) of NaHSO4. H2O at room tem-
perature. The reaction mixture was allowed to stand for 24
hours at ambient temperature, after which it was neutralized
with 10% NaHCO3 to pH-7. After that the solvent was
evaporated to dryness in vacuo and the crude product was
dissolved in CH2Cl2, extracted several times with 2.5% solu-
tion of NaHCO3, brine, 0.5N HCl, again brine, dried over
anhydrous Na2SO4 and evaporated to dryness in vacuo.
Yield-0.856g (83%). Rf–0.878 (CH2Cl2:CH3OH-9:1). Rf–
0.652 (CH2Cl2:CH3OH-9.5:0.5). M.p. 119-1200C. The other
possible way to purify the final product was the following:
after the reaction was completed and mildly alkalized to pH
7-7.5, the methanol mixture was extracted several times with
hexane or petroleum ether, the combined hydrophobic (hex-
ane or petroleum ether) phases were washed several times
with 0.5 N HCl, brine, 2,5% solution of NaHCO3, and again
with brine and then it (hexane or petroleum ether-phase) was
evaporated under reduced pressure in vacuo.
1H NMR (600 MHz, DMSO-d6, 25oC): ꢀ = 1.369(s,
9H, CH3), 1.39-1.47(m, 2H, 4-CH2), 1.54-1.60(m, 1H) and
1.65-1.71(m, 2H, 3-CH2), 2.88-2.93(m, 2H, 5-CH2), 3.616(s,
3H, OCH3), 4.009(ddd, J=4.9 Hz, J=7.9 Hz, J=9.4 Hz, 1H, 2-
CH), 4.224(t, J=7.1Hz, 1H, 9-CH), 4.295(d, J=7.1Hz, 2H,
OCH2), 6.810(t, J=5.7 Hz, 1H, 5-CH2NH), 7.333(dt, J=7.4
Hz, J=0.9 Hz, 2H, 2-CH and 7-CH), 7.418(t, J=7.4 Hz, 2H,
3-CH and 6-CH), 7.714(dd, J=7.4 Hz, J=2.8 Hz, 2H, 1-CH
and 8-CH), 7.790(d, J=7.8 Hz, 1H, 2-CHNH), 7.895(d, J=7.6
Hz, 2H, 4-CH and 5-CH).
(Also it was possible to remove Fmoc-protection with
piperidine at room temperature for 2-3 hours [TLC] In the
synthetic scheme, the reaction with this reagent also is de-
scribed. The procedure includes reaction of Fmoc-Orn(Boc)-
OMe deprotection in 3 ml. of piperidine. The workup proce-
dure was the same as in the case of deprotection with 10%
solution of Et2NH (diethylamine) in DMF).
1H NMR (600 MHz, DMSO-d6, 25oC): ꢀ = 1.364(s, 9H,
CH3), 1.39(bs, 2H, 4-CH2), 1.41(bs, 1H) and 1.53(bs, 1H, 5-
CH2), 2.890(pseudo-q, J=6.2 Hz, 2H, 3-CH2), 3.276(pseudo-
t, J=6.2 Hz, 1H, CH), 3.602(s, 3H, CH3), 6.864(t, J=5.2 Hz,
1H, 3-CH2NH).
13C NMR (150 MHz, DMSO-d6, 25oC): ꢀ = 25.80(4-
CH2),
28.19(CH3),
31.78(5-CH2),
49.57(3-CH2),
51.29(OCH3), 53.67(CH), 77.26(C), 155.51(NHCOO),
176.20(COOCH3).
3.6.2. Synthesis of H-Orn(Boc)-OMe(II)
To a solution of 1.16g (5mmol) of H-Orn(Boc)-OH in
10ml MeOH was added 1.1ml (10mmol) trimethyl orthofor-
mate and 0.828g (6mmol) of NaHSO4. H2O at ambient tem-
perature. The reaction mixture was allowed to stand for 24
hours at ambient temperature and neutralized and then alka-
lized with 10% NaHCO3 to pH-8. After that the solvent was
evaporated in vacuo and the crude product was dissolved in
CH2Cl2, extracted several times with 5% solution of Na2CO3,
brine, dried over anhydrous Na2SO4 and evaporated to dry-
ness in vacuo. Oil. (Gum). Yield-0.677g (55%). Rf–0.436
(CH2Cl2:CH3OH-8:2). Rf–0.263 (CH2Cl2:CH3OH-9:1).
13C NMR (150 MHz, DMSO-d6, 25oC): ꢀ = 25.98(4-
CH2), 27.91(3-CH2), 28.19(CH3), 39.97(5-CH2), 46.56(9-
CH), 51.78(OCH3), 53.56(2-CH), 65.55(OCH2), 77.37(C),
120.08(4-CH and 5-CH), 125.18(1-CH and 8-CH), 127.01(2-
CH and 7-CH), 127.58(3-CH and 6-CH), 140.67(4a-C and
4b-C), 143.69 and 143.74(8a-C and 9a-C), 155.53 and
156.03(NHCOO), 172.82(COOH).
1H NMR (600 MHz, DMSO-d6, 25oC): ꢀ = 1.364(s, 9H,
CH3), 1.39(bs, 2H, 4-CH2), 1.41(bs, 1H) and 1.53(bs, 1H, 5-
CH2), 2.890(pseudo-q, J=6.2 Hz, 2H, 3-CH2), 3.276(pseudo-
t, J=6.2 Hz, 1H, CH), 3.602(s, 3H, CH3), 6.864(t, J=5.2 Hz,
1H, 3-CH2NH).
13C NMR (150 MHz, DMSO-d6, 25oC): ꢀ = 25.80(4-
CH2), 28.19(CH3), 31.78(5-CH2), 49.57(3-CH2), 51.29
(OCH3), 53.67(CH), 77.26(C), 155.51(NHCOO), 176.20
(COOCH3).
3.6. Synthesis of H-Orn(Boc)-OMe
3.6.1. Synthesis of H-Orn(Boc)-OMe (I)
To a solution of 0.5g (1.067 mmol) of Fmoc-Orn(Boc)-
OMe in 3ml DMF were added 3ml 10% solution of Et2NH
(diethylamine) in DMF and the reaction mixture was allowed
to stand for 2 hours at room temperature. After the reaction
was completed (TLC) the solution was evaporated under
reduced pressure and the amorphous residue was recrystal-
lized in CH2Cl2/Hexane to avoid the unnecessary dibenzo-
fulvene. Oil. Yield-0.258g (98%). Rf – 0.436 (CH2Cl2:
CH3OH-8:2). Rf–0.263 (CH2Cl2:CH3OH-9:1). Two other
ways to purify the final product were possible:
3.7. Synthesis of Bz(NO2)-Orn(Boc)-OMeꢀ
Over an ice cold bath in a round bottom flask were added
0.246g (1mmol) of H-Orn(Boc)-OMe, 5ml CH2Cl2 and a

The Two Pathways for Effective Orthogonal Protection
Protein & Peptide Letters, 2010, Vol. 17, No. 7 893
solution of 0.204g (1.1mmol) of Bz(NO2)Cl (nitrobenzoyl
chloride) in 1ml CH2Cl2. After 5 min. 0.167 ml (1.2 mmol)
of triethylamine were added to the mixture and the reaction
was allowed to stand for 2-3 hours at room temperature. The
reaction was stopped by adding 3ml of CH3OH and the mix-
ture shaken for 1 hours. At the end it was evaporated to dry-
ness in vacuo and the residual amount dissolved in CH2Cl2.
The organic phase was washed 3 times with 0.5N HCl, brine,
saturated solution (10%)of NaHCO3 and again with brine.
Finally the CH2Cl2 phase was dried over sodium sulfate and
evaporated to dryness in vacuo. Yield-0.359g (91%). Rf–
0.523 (CH2Cl2:CH3OH-9:1). M.p. 88-890C.
1H NMR (600 MHz, DMSO-d6, 25oC): ꢀ = 1.362(s, 9H,
CH3), 1.43-1.53(m, 2H, 4-CH2), 1.69-1.86(m, 2H, 3-CH2),
2.92-2.96(m, 2H, 5-CH2), 4.34-4.38(m, 1H, 2-CH), 6.830(t,
J=5.8 Hz, 1H, 5-CH2NH), 8.105(d, J=8.8 Hz, 2H, 3-CH and
5-CH), 8.334(d, J=8.8 Hz, 2H, 2-CH and 6-CH), 8.972(d,
J=7.3 Hz, 1H, 2-CH2NH), 12.71(bs, 1H, COOH).
13C NMR (150 MHz, DMSO-d6, 25oC): ꢀ = 26.37(4-
CH2), 27.83(3-CH2), 28.20(CH3), 39.35(5-CH2), 52.68(2-
CH), 77.37(C), 123.48(3-CH and 5-CH), 128.92(2-CH and
6-CH), 139.54(1-C), 149.05(4-C), 155.54(NHCOO),
164.90(NHCO), 173.31(COOH).
1H NMR (600 MHz, DMSO-d6, 25oC): ꢀ = 1.362(s, 9H,
CH3), 1.43-1.53(m, 2H, 4-CH2), 1.72-1.78(m, 2H, 3-CH2),
2.92-2.96(m, 2H, 5-CH2), 3.651(s, 3H, CH3), 4.42-4.86(m,
1H, 2-CH), 6.829(t, J=5.6 Hz, 1H, 5-CH2NH), 8.106(d,
J=8.8 Hz, 2H, 3-CH and 5-CH), 8.337(d, J=8.8 Hz, 2H, 2-
CH and 6-CH), 9.105(d, J=7.3 Hz, 1H, 2-CH2NH).
13C NMR (150 MHz, DMSO-d6, 25oC): ꢀ = 26.14(4-
CH2), 27.67(3-CH2), 28.19(CH3), 39.23(5-CH2), 51.92
(OCH3), 52.68(2-CH), 77.39(C), 123.51(2-CH and 6-CH),
128.96(3-CH and 5-CH), 139.24(1-C), 149.13(4-C), 155.54
(NHCOO), 165.00(NHCO), 172.31(COOH).
3.8.2. Synthesis of Bz(NO2)-Orn(Boc)-OH (II)
Bz(NO2)-Orn(Boc)-OMe was hydrolyzed using lipase as
catalyst, with different reaction conditions, compared to the
first method.
To a solution of 0.3g (0.759mmol) Bz(NO2)-Orn(Boc)-
OMe in 3ml dioxane and 3ml 25 mM sodium phosphate
buffer (at pH 7 or 8) at 250C were added 0.01g of lipase
(Boehringer Mannheim) at room temperature. During the
reaction, the pH value was maintained constant using a pH-
stat Mettler Toledo DL50 graphic and the enzymatic activity
was defined as μmole of substrate hydrolized per minute per
milligram protein. After the reaction was completed* (TLC)
it was evaporated under reduced pressure in vacuo, dissolved
in a saturated solution of NaHCO3 and washed 3 times with
CH2Cl2. After that the water phase was acidified to pH 2-3
with a solution of NaHSO4 and it was washed 3-4 times with
CH2Cl2. The combined organic phase was extracted several
times with brine, dried over Na2SO4 and evaporated to dry-
ness in vacuo. Yield-0.289g (100%). Rf–0.320
(CH2Cl2:CH3OH-9:1). M.p. 97.5 0C.
ꢀ
The procedure of nitrobenzoylation of H-Orn(Boc)-
OMe* also was carried out, using NaHCO3 as base, as well
as using CHCl3 as reaction mixture, and also using p-
nitrobenzoyl chloride (Bz(NO2)Cl) as reagent for nitroben-
zoylation, under the same reaction conditions. Yield-0.296g
1
(75%). The melting point, Rf-value, as well as the H NMR
and 13C NMR spectra of the product (Bz(NO2)-Orn(Boc)-
OMe) were the same, as in the case of using CH2Cl2/Et3N as
reaction conditions for p-nitrobenzoylation.
1H NMR (600 MHz, DMSO-d6, 25oC): ꢀ = 1.362(s, 9H,
CH3), 1.43-1.53(m, 2H, 4-CH2), 1.69-1.86(m, 2H, 3-CH2),
2.92-2.96(m, 2H, 5-CH2), 4.34-4.38(m, 1H, 2-CH), 6.830(t,
J=5.8 Hz, 1H, 5-CH2NH), 8.105(d, J=8.8 Hz, 2H, 3-CH and
5-CH), 8.334(d, J=8.8 Hz, 2H, 2-CH and 6-CH), 8.972(d,
J=7.3 Hz, 1H, 2-CH2NH), 12.71(bs, 1H, COOH).
1H NMR (600 MHz, DMSO-d6, 25oC): ꢀ = 1.362(s, 9H,
CH3), 1.43-1.53(m, 2H, 4-CH2), 1.72-1.78(m, 2H, 3-CH2),
2.92-2.96(m, 2H, 5-CH2), 3.651(s, 3H, CH3), 4.42-4.86(m,
1H, 2-CH), 6.829(t, J=5.6 Hz, 1H, 5-CH2NH), 8.106(d,
J=8.8 Hz, 2H, 3-CH and 5-CH), 8.337(d, J=8.8 Hz, 2H, 2-
CH and 6-CH), 9.105(d, J=7.3 Hz, 1H, 2-CH2NH).
13C NMR (150 MHz, DMSO-d6, 25oC): ꢀ = 26.37(4-
CH2), 27.83(3-CH2), 28.20(CH3), 39.35(5-CH2), 52.68(2-
CH), 77.37(C), 123.48(3-CH and 5-CH), 128.92(2-CH and
6-CH), 139.54(1-C), 149.05(4-C), 155.54(NHCOO),
164.90(NHCO), 173.31(COOH).
13C NMR (150 MHz, DMSO-d6, 25oC): ꢀ = 26.14(4-
CH2),
27.67(3-CH2),
28.19(CH3),
39.23(5-CH2),
51.92(OCH3), 52.68(2-CH), 77.39(C), 123.51(2-CH and 6-
CH), 128.96(3-CH and 5-CH), 139.24(1-C), 149.13(4-C),
155.54(NHCOO), 165.00(NHCO), 172.31(COOH).
3.9. Synthesis of Bz(NO2)-Orn(Boc)-OCH2CN
3.8. Synthesis of Bz(NO2)-Orn(Boc)-OH
The procedure on cyanomethylation of Bz(NO2)-
Orn(Boc)-OH includes reaction of the aminoacyl derivative
with ClCH2CN at room temperature and different solutions,
which vary from polar aprotic to hydrophobic. To a solution
of 0.289g (0.759mmol) of Bz(NO2)-Orn(Boc)-OH in DMF
were added 2 equiv. - 0.211ml (1.518mmol) triethylamine in
ice cold bath. After 10 minutes 2 equiv. – 0.096 ml
(1.518mmol) of chloroacetonitrile were added to the reaction
mixture. The temperature of the reaction was left to reach
3.8.1. Synthesis of Bz(NO2)-Orn(Boc)-OH (I)
To a solution of 0.3g (0.759mmol) Bz(NO2)-Orn(Boc)-
OMe in 3ml CH3OH were added 3ml of 1N NaOH at room
temperature. After the reaction was completed (TLC) it was
evaporated under reduced pressure in vacuo, dissolved in a
saturated solution of NaHCO3 and washed 3 times with
CH2Cl2. After that the water phase was acidified to pH 2-3
with a solution of NaHSO4 and it was washed 3-4 times with
CH2Cl2. The combined organic phase was extracted several
times with brine, dried over Na2SO4 and evaporated to dry-
ness in vacuo. Yield-0.289g (100%). Rf–0.320 (CH2Cl2:
CH3OH-9:1). M.p.97-980C.
*
For the completion of the reaction, a more time was needed (12-24 h). Although this
reaction needs to more reaction prolonged time for completed hydrolysis (ester
[methyl] group deprotection) it proceeds under mild conditions, compared to the hydro-
lytic reaction, using N NaOH as reagent for deprotection.

894 Protein & Peptide Letters, 2010, Vol. 17, No. 7
Bayryamov et al.
room t0C and was left overnight. It was then evaporated un-
der reduced pressure and dissolved in ethylacetate. The or-
ganic phase was washed three times with 0.5N HCl, brine, a
saturated solution (10%) of NaHCO3 and again brine. Finally
the organic phase was dried over sodium sulfate and evapo-
rated to dryness in vacuo. Yield-0.262g (87%). Rf-0.545
(CH2Cl2:CH3OH-9:1). M.p.780C.
1H NMR (600 MHz, DMSO-d6, 25oC): ꢀ = 1.365(s, 9H,
CH3), 1.46-1.55(m, 2H, 4-CH2), 1.76-1.88(m, 2H, 3-CH2),
2.94-2.98(m, 2H, 5-CH2), 4.50-4.54(m, 1H, 2-CH), 5.046(s,
2H, OCH2), 6.844(t, J=5.7 Hz, 1H, 5-CH2NH), 8.109(d,
J=8.8 Hz, 2H, 3-CH and 5-CH), 8.351(d, J=8.8 Hz, 2H, 2-
CH and 6-CH), 9.230(d, J=7.1 Hz, 1H, 2-CHNH).
tion of complexes with copper [12, 13, 16, 17]. Despite the
higher yields obtained by these methods, the release of the
complexes in one of them requires passing a stream of H2S
through the solution, and trituration of the product with HCl
in both, which leads to cleavage of Boc-group in some ex-
tent. The third approach (second procedure) employs the
protection of ꢁ-NH2 group without cupric complexes, in a
mixture of methanol and using one equivalent of NaOH to
release the ꢁ-NH2 group from its HCl salt [14, 15]. Some Nꢀ-
Boc-ornithine and Nꢀ, Nꢁ-di-Boc-ornithine were formed dur-
ing the reaction, but remained in solution. The Nꢁ-Boc de-
rivative of ornithine was insoluble in MeOH/water and pre-
cipitated. It was easily purified from the co-products of the
reaction. Although the yields were lower this method ex-
cludes the passing of the toxic H2S for destroying of the cu-
pric complexes, and was our method of choice.
13C NMR (150 MHz, DMSO-d6, 25oC): ꢀ = 25.97(4-
CH2), 27.29(3-CH2), 28.19(CH3), 39.97(5-CH2), 49.44
(OCH2), 52.41(2-CH), 77.42(C), 115.72(CN), 123.57(3-CH
and 5-CH), 128.96(2-CH and 6-CH), 138.94(1-C), 149.21(4-
C), 155.56(NHCOO), 156.12(NHCO), 170.94(COOC).
O
O
H2N
H2N
(Boc)2O
OH
OH
1) CuSO4/KOH/MeOH (Yield - 65%)
2) N NaOH/MeOH (Yield - 52%)
3) CuCO3/NaHCO3/acetone (Yield - 70%)
4. DISCUSSION
For the successful synthesis of nucleosides: 2’/3’-O-
[Bz(NO2)-Orn(Boc)]-5’-O-Piv-Ado (1) and its deoxy analog
3’-O-[Bz(NO2)-Orn(Boc)]-5’-O-Piv-2’-dAdo (2) as sub-
strates for model ribosomal reactions, the reaction of nucleo-
side aminoacylation was carried out, using Bz(NO2)-
Orn(Boc)-OCH2CN as an amino acid component. The design
of the amino acid component was carefully made, because of
the strong requirements for the stability of the final sub-
strates and appropriate mimic (imitation) of the model reac-
tions.
H N
HN
HCl.
2
O
O
H3C
CH3
CH3
Fig. (1). Scheme of the synthesis of H-Orn(Boc)-OH.
Bz(NO2)-Orn(Boc)-OMe was synthesized as a precursor
of a mildly activated cyanomethyl ester. It was needed be-
cause of the very low yields and long reaction time of nitro-
benzoylation of H-Orn(Boc)-OH with Bz(NO2)-Cl under
standard Schotten-Baumann conditions, probably due to the
low solubility of the Boc-amino acid in CH2Cl2 (CHCl3).
Therefore the standard Schotten-Baumann conditions were
avoided and modified. Moreover, when we tried to obain
Bz(NO2)-Orn(Boc)-OH according to the previous procedure
of nitrobenzoylation of Orn(Boc)-OH with N-hydroxy-
succinimide or cyanomethyl activated esters of p-
nitrobenzoic acid, the yields were also unsatisfactory due to
the very low reactivity of this reagents (sterically hindered).
Also at the stage of nitrobenzoylation of H-Orn(Boc)-OMe,
using the above mentioned N-hydroxysuccinimide or cya-
nomethyl activated esters of p-nitrobenzoic acid, the reaction
time was long and the yields were unsatisfactory. Therefore
we employed H-Orn(Boc)-OMe as an aminoacyl reagent
(aminocomponent) and Bz(NO2)-Cl under modification pro-
cedure of Schotten-Baumann conditions, using our originally
developed strategy and methodology. H-Orn(Boc)-OMe is
much more soluble, the reaction with Bz(NO2)-Cl proceeds
within 2-3 hours and the yields were higher.
4.1. Design of the Aminoacyl Component
The aminoacyl component was protected employing or-
thogonal type of protection of amino acids, having side func-
tional groups. The protection was accomplished, employing
different protecting groups, which are stable under different
conditions and cleaves using various (a wide range of) rea-
gents. Protection of ꢀ-NH2 goup with p-nitrobenzoyl group
allows monitoring of the reaction by HPLC and survives all
procedures of the synthesis of the model substrates. ꢁ-NH2
group was protected with Boc-protecting group to avoid un-
wanted side reactions during the aminoacylation of the nu-
cleoside component under base conditions. It also had to be
an acid-labile group to allow the deprotection of Nꢁ-amino
group at the last stage of synthesis of the model substrates,
immediatelly before the kinetic measurement experiments,
and the carboxyl group of the amino acid was activated as a
cyanomethyl ester, allowing its coupling with 5’-O-protected
nucleosides by the reaction of transesterification for aminoa-
cylation of nucleosides [7, 8, 18]. The cyanomethyl esters of
N-protected amino acids are known from classical peptide
synthesis to be midly activated, requiring about (ca) 24-48
hours for complete coupling [19, 20], but they are suitable
reagents for the aminoacylation of nucleosides [8, 18].
Two different pathways for the synthesis of methyl ester
of ꢀ-N-p-nitrobenzoyl-ꢁ-N-(BOC)-L-ornithine were used.
4.2. Synthesis of the Aminoacyl Component
The first pathway employs the protection of H-Orn(Boc)-
OH [19, 20] with Fmoc-protecting group [21, 22] and methy-
lation of Fmoc-Orn(Boc)-OH to Fmoc-Orn(Boc)-OMe. The
reaction of methylation of Fmoc-Orn(Boc)-OH to Fmoc-
The first step was H-Orn(Boc)-OH to be synthesized. It
was synthesized using three different ways (Fig. 1) [12-17].
Two of them (procedure 1 and procedure 3) include prelimi-
nary protection of ꢀ-NH2 and ꢀ-COOH goups by the forma-

The Two Pathways for Effective Orthogonal Protection
Protein & Peptide Letters, 2010, Vol. 17, No. 7 895
Orn(Boc)-OMe was carried out using two different ways.
The first way employs diazomethane as methylating reagent.
Although the yields were high, it has been known that dia-
zomethane is a very toxic gas and can cause detonation,
which makes its handling more difficult. In the second way
we used trimethyl orthoformate as reagent for esterification,
in a mixture of methanol and NaHSO4 as a catalyst. Because
of the good solubility of the reaction substrate (Fmoc-
Orn(Boc)-OH), the yields were satisfactory (83%), using this
reaction procedure. After the reaction was completed, the
product was purified employing methanol extraction (see
experimental procedures).
Bz(NO2)-Orn(Boc)-OH which was cyanomethylated to
Bz(NO2)-Orn(Boc)-OCH2CN [3] (Fig. 2).
The second pathway avoids the preliminary protection of
ꢀ-NH2 group of H-Orn(Boc)-OH and includes direct methy-
lation of H-Orn(Boc)-OH with HC(OCH3)3, using NaHSO4
as a catalyst and MeOH as a reaction media to prepare H-
Orn(Boc)-OMe, and subsequent nitrobenzoylation of ꢀ-NH2
group of H-Orn(Boc)-OMe. The disadvantage of this strat-
egy is the lower overall yield (73%) because of the acid con-
ditions (NaHSO4) under which the methylation of H-
Orn(Boc)-OH with HC(OCH3)3, using NaHSO4 as a catalyst
and MeOH as a reaction media, was carried out and which
are detrimental to the Boc-protecting group in some extent
(yield 55% on the methylation stage) , but it spares two steps
as compared to the first pathway (Fig. 3). The second reason
for the lower yields, using this strategy may be explayned by
the low solubility of the starting substrate: H-Orn(Boc)-OH
in the reaction medium (MeOH), used in the synthetic pro-
cedure on the esterification stage. In the procedure of methy-
lation (esterification) of Fmoc-Orn(Boc)-OH, using the same
After removing the Fmoc-protection [23, 24] and nitro-
benzoylation of ꢀ-N-amino group of H-Orn(Boc)-OMe the
overall yeld of the product was 92%. In the step of the re-
moving of Fmoc-protection two possible ways of deprotec-
tion were used: with piperidine, and with 10% diethylamine
in DMF. Three different methods for the purification of the
product from dibenzofulvene were employed (see experi-
mental). The hydrolysis of Bz(NO2)-Orn(Boc)-OMe yields
O
O
O
O
O
H2N
CH3
H
H
OH
OH
N
O
1) CH2N2/Et2O
N
NaHCO3/Acetone
N
O
O
O
O
+
O
or 2) HC(OCH3)3/NaHSO4/MeOH
O
O
HN
HN
O
HN
O
O
O
O
O
H3C
CH3
CH3
CH3
(Yield-82%)
CH3
CH3
H3C
H3C
CH3
1) (Yield-96%)
2) (Yield-83%)
-O
O
O
O
CH3
CH3
N+
H
N
CH3
H
O
O
H2N
O
N
O
10% Et2NH in DMF
O
Bz(NO2)-Cl/CH2Cl2
O
or piperidine
O
HN
O
HN
O
HN
O
O
O
O
CH3
CH3
CH3
H3C
H3C
H3C
CH3
CH3
CH3
(Yield-91%)
(Yield-98%)Ø
N
-O
N+
-O
N+
-O
O
O
O
O
CH3
H
N
N
N+
H
N
H
N
O
OH
1N NaOH/MeOH
DMF/Et3N
O
O
O
+
O
O
O
Cl
HN
O
HN
O
CH3
CH3
(Yield-100%)
HN
O
CH3
CH3
(Yield-87%)
O
O
O
CH3
H3C
H3C
H3C
CH3
Fig. (2). First reaction pathway for the synthesis of Bz(NO2)-Orn(Boc)-OCH2CN.
Ø When piperidine was used instead of 10% solution of Et2NH (diethylamine) in DMF, the yield was 96%.

896 Protein & Peptide Letters, 2010, Vol. 17, No. 7
Bayryamov et al.
O-
N+
O
O
O
O
O
H
H2N
OH
CH3
Cl
H2N
N
CH3
O
O
-O
N+
O
HC(OCH3)3/NaHSO4/MeOH
O
HN
O
HN
O
HN
CHCl3/NaHCO3
O
O
O
O
CH3
CH3
CH3
CH3
H3C
CH3
H3C
H3C
CH3
(Yield-55%)
(Yield-75%)*
(Yield-91%)ꢀ
O-
N+
O-
N+
O-
N+
N
O
O
O
O
O
O
H
H
N
H
N
CH3
N
O
O
OH
N
1N NaOH/MeOH
or lipase
O
HN
O
DMF/Et3N
O
O
+
Cl
HN
O
HN
O
O
O
O
CH3
CH3
CH
3
CH3
CH3
H3C
H3C
H3C
CH3
(Yield-87%)
1) (Yield-100%)#
2) (Yield-100%)&
Fig. (3). Second reaction pathway for the synthesis of Bz(NO2)-Orn(Boc)-OCH2CN.
* When CHCl3/NaHCO3 were used as reaction conditions.
¤ Using the procedure for nitrobenzoylation with Bz(NO2)Cl, and CH2Cl2/Et3N as reaction conditions.
# When 1N NaOH/MeOH were used for hydrolysis of Bz(NO2)-Orn(Boc)-OMe.
& When lipase was used as catalyst in hydrolysis reaction.
Fig. (4). A general scheme for the strategy and the possible pathways for the preparation of Bz-Orn(Boc)-OCH2CN.

The Two Pathways for Effective Orthogonal Protection
Protein & Peptide Letters, 2010, Vol. 17, No. 7 897
reaction conditions, the yields of Fmoc-Orn(Boc)-OMe were
higher (83%), probably due to the better solubility of Fmoc-
Orn(Boc)-OH.
Et2NH
Et3N
=
=
=
=
=
=
=
=
Diethylamine
Triethylamine
HMBC
HPLC
HSQC
IR
Heteronuclear Multiple Bond Correlation
High Performance Liquid Chromatography
Heteronuclear Single Quantum Correlation
Infrared spectroscopy
After hydrolysis of the methyl ester of Bz(NO2)-
Orn(Boc)-OMe, the deprotected carboxyl group was cya-
nomethylated with chloracetonitrile according to Robertson
et al. [18].
The unique of the second pathway was the step of the
preparing of H-Orn(Boc)-OMe from H-Orn(Boc)-OH. The
reacton was carried out in a mixture of CH3OH, NaHSO4 as
catalyst, and trimethyl orthoformate as methylation reagent.
NMR
Orn
Nuclear Magnetic Resonance
Ornithine
peptidyl-tRNA-
peptidyl
Also at the stage of hydrolysis of the methyl ester of
Bz(NO2)-Orn(Boc)-OMe the reaction was carried out, em-
ploying lipase as catalyst and using mild conditions, thus
providing an elegance of the reaction.
=
=
=
=
=
=
=
Transfer Ribonucleic acid
Pivaloyl (trimethyl acetyl)
Ribonucleic acid
Piv
RNA
tRNA
TBI
Transfer Ribonucleic acid
Triple broadband inverse
Thin layer chromatography
Trifluoroacetic acid
CONCLUSION
Bz(NO2)-Orn(Boc)-OCH2CN was synthesized as an
amino acyl component for amino acylation of 5’-O-Pivaloyl
adenosine and 5’-O-Pivaloyl 2’-deoxy adenosine and prepa-
ration of substrates for model ribosome reactions. The syn-
thesis was carried out using suitable combinations of protect-
ing groups and the methods of peptide synthesis and modifi-
cation of amino acids (Fig. 4).
TLC
TFA
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ACKNOWLEDGEMENTS
Supported by grants from the National Research Fund of
Bulgaria (grants X-1517 and X-1408), and for the purchase
of Bruker Avance II+ 600 NMR spectrometer in the frame-
work of the Program “Promotion of the Research Potential
through Unique Scientific Equipment” – Project UNA-
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LIST OF ABBREVIATIONS
Ado
=
=
=
=
=
=
=
=
=
Adenosine
BBO
Broadband observe
Tret-butyloxycarbonyl
Benzoyl
[9]
Boc
Bz
[10]
[11]
[12]
[13]
Bz(NO2)
CH2CN
COSY
dAdo
p-nitro-Benzoyl
Cyanomethyl
Correlation Spectroscopy
2’-deoxy-Adenosine
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DEPT-135
Distortionless Enhanced Polarization
Transfer using a 135 degree decoupler
pulse
[14]
DMF
=
Dimethylformamide

898 Protein & Peptide Letters, 2010, Vol. 17, No. 7
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Received: November 13, 2009
Revised: January 19, 2010
Accepted: February 26, 2010