Basic techniques of working on a solid phase: From ABC of the peptide synthesis to libraries of non-natural amino acids

Article abstract of DOI:10.1134/S107036321012025X

Libraries of hardly available amino acids bearing a heteroaromatic ring (2-pyrimidyl, substituted 2-pyridyl or 2-thiazolyl) at the amino group were prepared using solid-phase synthesis on various resins. The synthesized compounds are structurally similar to some known antidiabetic drugs. The paper combines features of a review (elementary introduction to the solid-phase synthesis methodology and technique for beginners and selected methods from peptide chemistry) and step-by-step experimental protocols (tested by the authors) useful as a methodic tool. The presented protocols (immobilization and modification of amino acids, placing and removal of common protective groups) require no sophisticated equipment and may be useful as pictorial introductory tasks for students education. Pleiades Publishing, Ltd., 2010.

Full text of DOI:10.1134/S107036321012025X

ISSN 1070-3632, Russian Journal of General Chemistry, 2010, Vol. 80, No. 12, pp. 2572–2589. © Pleiades Publishing, Ltd., 2010.
Original Russian Text © E.V. Babaev, D.S. Ermolat’ev, 2009, published in Rossiiskii Khimicheskii Zhurnal, 2009, Vol. 53, No. 5, pp. 42–56.
Basic Techniques of Working on a Solid Phase:
From ABC of the Peptide Synthesis to Libraries
of Non-Natural Amino Acids
E. V. Babaev and D. S. Ermolat’ev
Lomonosov Moscow State University, Vorob’evy Gory 1, Moscow, 119991 Russia
e-mail babaev@org.chem.msu.ru
Received July 10, 2009
Abstract―Libraries of hardly available amino acids bearing a heteroaromatic ring (2-pyrimidyl, substituted 2-
pyridyl or 2-thiazolyl) at the amino group were prepared using solid-phase synthesis on various resins. The
synthesized compounds are structurally similar to some known antidiabetic drugs. The paper combines features
of a review (elementary introduction to the solid-phase synthesis methodology and technique for beginners and
selected methods from peptide chemistry) and step-by-step experimental protocols (tested by the authors)
useful as a methodic tool. The presented protocols (immobilization and modification of amino acids, placing
and removal of common protective groups) require no sophisticated equipment and may be useful as pictorial
introductory tasks for students education.
DOI: 10.1134/S107036321012025X
INTRODUCTION
hetarylation, a reaction fairly rarely used for prepara-
tion of non-natural amino acids [1, 2].
The present paper has a slightly unusual structure.
It is based on the experiments aimed at developing a
training task for the special student’s laboratory course
in combinatorial chemistry in the Moscow State
University (MSU) and combines features of a review
(carefully selected publications on the techniques of
peptide synthesis), step-by-step experimental protocols
(as procedures tested by the authors), and an original
research, specifically, synthesis of a previously
inaccessible family of non-natural amino acids with a
well-defined structural motif of medicines. In its
chemical essence, the chosen reaction sequence is a
classical (while slightly reduced) scheme of peptide
synthesis:
First we substantiate why the molecular structures
in the obtained library may suggest biological activity.
Then we analyze features of each of the four stages in
terms of methodology and published techniques. In
conclusion we give concrete practical recommendation
and provide experimental protocols, as well as
evidence for the structure and purity of the synthesized
compounds. The principal methodical feature of the
presented protocols consists in that they require no
special equipment and can be reproduced in usual
student laboratory works.
Problem Statement
At present the biological activity of 3-
guanidinopropionic acid attracts interest of medical
chemists due to its expressed antidiabetic activity and,
simultaneously, metabolic instability in human’s body.
Among drugs applied medicines applied, the most
known is Tiformin (I) which is 3-guanidinopro-
pionamide. Larsen and co-workers [3, 4], optimizing
the structure of 3-guanidinopropionic acid and
searching for its biologically active analogs, syn-
thesized and tested for biological activity a series of
homologs and bioisosteres of this acid:
(1) immobilization of an N-substituted amino acid
on a solid support;
(2) removal of the protective group;
(3) modification of the amino acid NH2 group;
(4) removal of the modified amino acid from the
support.
An original element of this task, its peculiar zest, is
the last stage in which the amino acid is modified by
2572

BASIC TECHNIQUES OF WORKING ON A SOLID PHASE
2573
approach makes use of amino acid derivatives and
involves aromatic nucleophilic substitution in hetero-
rings containing a readily leaving group in the α
position. It is well known that free amino acids are
difficult to apply, since they tend to enter side reac-
tions. Therefore, amino acids should be preliminarily
protected and their intermolecular interaction should
be minimized.
NH
O
NH
O
H2N
N
NH2
OH
H2N
N
NH2
H
H
Me
O
I
II
HN
H
O
N
H2N
N
N
OH
A radical approach to the problem of synthesis of a
series of derivatives V by a unified strategy might be
provided by solid-phase synthesis. As known, the
solid-phase synthetic approach allows, on the one
hand, to block the carboxylic function of the amino
acid by forming an ester or amide bond with a polymer
support and also to exclude the intermolecular
interaction of amino acids by forming diketopipe-
razines and oligopeptides. On the other hand, using a
polymer support one can prepared both free acids and
esters and amides, which much extends the potential of
this method.
H
III
IV
A
A
OH
N
N
H
O
V
It was shown that activity is largely associated with
the amidine fragment of 3-guanidinopropionic acid,
whereas point mutations and substitution of the
guanidine fragment resulted in partial or complete loss
of activity. By contrast, the authors noted that α-
alkylation (compound II), ring closure in the amidine
or guanidine fragment (compound III), as well as
enhancement of the conformational rigidity of the
alkyl chain (compound IV) sometimes enhanced the
antihyperglycemic activity. It follows from these
results that one of the approaches to structural
modification of 3-guanidinopropionic acid might
involve a systematic screening of heterocycles for
replacing the guanidine fragment, as well as the
bioisosteric replacement of the carboxy group with
wide variation of the structure of the alkyl chain. Thus
we can set the task to develop a method of synthesis of
a wide series of heterocyclic amino acids of the
general formula V, having the guanidine and amidine
fragments incorporated in an α-aminoheterocycle and
an amino acid residue (AA). This class of compounds
is well documented, but, even though the problem of
preparation of N-hetarylamino acids is quite urgent, no
universal synthetic approach to them is still available.
Analysis of the literature showed that the principal
methods of synthesis of such N-hetarylamino acids is
N-alkylation of α-aminoheterocycles and nucleophilic
substitutionin the heterocyclic nucleus with amino acid
derivatives.
As a result, we consider the most acceptable an
approach based of the aromatic nucleophilic substitu-
tion in heterocycles under the action of amino acids
immobilized on a polymer support. (This reaction can
also be treated as N-hetarylation of amino acids). As
the heterocycles for hetarylation we chose 2-halo-
genated pyrimidines, 5-nitropyridine, and 5-nitro-
thiazole. All these halo derivatives fairly readily react
with primary and secondary aliphatic amines, the
results are thoroughly examined and published.
Thus, the chosen strategy for synthesis of N-hetaryl-
amino acids can be represented by the following
scheme (here and hereinafter, АА and PP stand for
amino acid residue and protective group, respectively).
Stage 1. Immobilization of an N-protected amino
acid on a polymer support:
O
+
HO
AA−NH-PG
XH
(1)
O
X
AA−NH-PG
Stage 2. Deblocking of the amino acid:
Comparing these two methods, one should take into
account that amino acids and their protected deriva-
tives are commercially available, and, consequently,
the second method is a more expedient synthetic
approach to a wide range of hetarylamino acids. This
O
O
X
AA−NH-PG −PG
X
AA−NH2
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 80 No. 12 2010

2574
BABAEV, ERMOLAT’EV
in a solution. In an appropriate solvent, a polymer
Stage 3. Nucleophilic substitution in the heteroring:
swells (Fig. 1) and converts into a low viscous but
strongly structured gel (cross-linked polymers) or
dissolves (noncross-linked polymers), and synthesis
occurs at an ultramicroheterogeneous level, i.e. in an
almost homogeneous system.
O
X
AA−NH2
(2)
O
Y
N
Solid-phase organic synthesis requires the use of a
polymer support (resin S) and its attached linker L.
The first stage (stage 1) involves attachment of a
molecule of sub-strate А to the linker [5]. Molecule А
is immobilized but preserves its ability to react with
reagent В (stage 2).
Product АВ stays on the resin, which allows excess
reagent В (and by-products) to be simply washed out
from the product. (Further reagents can be added so
that the structure of substrate А is successively com-
plicated; the key point is that the linker in these
reactions remains untouched). A bifunctional linker L
is selected so that it is bonded to resin S stronger that
to substrate А. Then at the last stage (stage 3) the
target compound AB can be separated from the resin
by breaking its bond with the linker. It is clear that the
L–AB bond should be destroyed in mild conditions,
not damaging both the compound itself (bond А–В)
and the linker–resin contact (bond L–S).
X
AA−N
N
H
Stage 4. Removal of the product from the polymer
support:
O
O
,
X
AA−
N
H
N
HX
AA−
N
H
N
O2N
O2N
S
N
=
,
,
.
N
N
N
N
Solid-Phase Organic Synthesis
The methodology of modern solid-phase organic
synthesis is in many respects adopted from the solid-
phase synthesis of peptides. However, there is scarce
information in domestic literature on the specific
features of solid-phase organic synthesis and the
advantages it offers over the traditional liquid-phase
synthesis. Therefore, we consider it appropriate to give
a brief characteristic of solid-phase synthesis and
polymer supports used in this method [5].
1
S
S
+ A
L
L
A
2
B
3
S
AB
L
AB
The aim of solid-phase organic synthesis is to
accelerate the synthetic process due to facilitated isola-
tion and exclusion of additional purification of both
intermediate and target products. The term solid-phase
relates in essence to physical characteristics of a sub-
stance on a support, since chemical reaction on a
polymer support occurs in a single phase, specifically
Thus, in an ideal case, by washing the resin after
each stage and breaking the bond with the support, one
can obtain a pure compound. It is natural to suggest
that using a large excess of reagents with subsequent
separation from the resin can allow in many cases to
shift the chemical equilibrium to the target product and
shorten the synthesis time.
The disadvantages of solid-phase organic synthesis
include the necessity of using a fairly large excess (2–
30 equiv) of reagent, difficulties in the identification of
intermediate products, as well as a fairly high cost of
modified polymer supports, which depends on the cost
of linkers.
Polymer Supports [5]
Сhloromethylated polystyrene (cross-linked with a
little divinyl benzene) introduced in the practice of
organic synthesis by Merrifield (so-called Merrifield
resin) is the most accessible of polymer supports.
Fig. 1. Swelling of Merrifield resin in CH2Cl2 (as seen,
1 ml of a granular material increases its volume 3 times).
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 80 No. 12 2010

BASIC TECHNIQUES OF WORKING ON A SOLID PHASE
2575
tion, amino acids are preliminarily protected. As a rule,
N-substituted amino acids are used, and the resulting
amino acid–support bond is an amide or ester bond.
OH
O
Cl
In solid-phase organic synthesis, amino groups are
most commonly protected by carbamate-type groups,
like tert-butoxycarbonyl (Boc) [6] and 9H-
fluorenylmethoxycarbonyl (Fmoc) [7] (X stands for a
group to be protected):
Merrifield resin
Wang resin
NHFmoc
OMe
O
O
NH
O
OMe
O
Rink resin
O
X
Me
Me
Me
The chloromethyl group of the polymer allows easy
alkylation, say, of carboxylic acid derivatives and their
fixation on the resin. However, the resulting ester bond
(of the benzyl type) cleaves in fairly rigid conditions
(conc. HF, HBr, alkali metal alkoholates, boiling with
amines, etc.). In the peptide synthesis, the peptide bond
with the Merrifield resin can be cleaved with HF in
thioanisole. Such conditions are unfavorable for many
compounds, and, therefore, the Merrifield resin is
modified to facilitate removal of products from the
support. For example, the functional group can be
“elongated” by replacing the chlorobenzyl group with
a p-benzyloxybenzyl (Wang linker) or benzhydryl
group (Rink linker).
O
X
Boc
Fmoc
It should be noted that the choice of protective
group is determined by the type of the polymer
support. Immobilization conditions for protected
amino acids vary from one polymer support to another.
Boc-amino acids are immobilized on the Merrifield
resin (chloromethylated polystyrene) in situ as cesium
salts, by adding a suspension of cesium carbonate in
DMF and a potassium iodide catalyst. The reagent-to-
support ratio is chosen individually in each case and
spans the range 1.5–4.
Such benzyl esters are cleaved with acids in milder
conditions. Additional introduction of the electron-
donor methoxy groups in the benzene ring further
enhances the stability of the benzyl and benzhydryl
cations formed by acid hydrolysis of the linker. This
allows the Wang linker to be cleaved with 50%
CF3COOH (TFA) and the Rink linker, with 20% TFA.
Moreover, modification of the support extends the
range of potential reaction products. Thus, with the
Wang resin one can obtain acids and alcohols and with
the Rink resin, primary and secondary amides.
O
H
N
Me
Me
O
Cl
+ HO
A
A
O
Me
O
O
H
Me
Me
Cs2CO3/KI
N
O
A
A
DMF, 80°C
O
Me
Immobilization of Fmoc-amino acids on the Wang
support (X=O) to form a benzyl ester linker is
performed by the carbodiimide method, by treatment
with diisopropylcarbodiimide (DIC) in the presence of
a 4-(dimethylamino)pyridine (DMAP) catalyst. The
immobilization reaction with sterically uncongested
amino acids occurs at room temperature. Sterically
congested amino acids are immobilized at 40–60°С for
2 days (two immobilization cycles) (Scheme 1).
Methodology and Principal Stages
of Solid-Phase Peptide Synthesis
The task we set ourselves required a polymer
support with a grafted amino acid to be reacted with a
heterocycle activated to substitution. Let us consider in
more detail the methodical issues of how to prepare
amino acids immobilized on polymer supports.
The immobilization of Fmoc-amino acids on the
Rink resin (X=NH) to form a benzhydryl amide linker
is performed in the presence of the Castro reagent
{(1H-1,2,3-benzotriazole-1-yloxy)tris(dimethylamino)-
phosphonium (BOP)}, diisopropylethylamine (DIEA)
as a base and 1-hydroxybenzotriazole (HOBt) as a
catalyst. The reaction occurs at room temperature for
Stage 1. Immobilization of N-Substituted Amino Acids
on Polymer Supports
The first stage of our scheme involves immobiliza-
tion of amino acids on polymer supports (reaction (1)].
To avoid such side processes as oligopeptide forma-
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 80 No. 12 2010

2576
BABAEV, ERMOLAT’EV
Scheme 1.
N
N
N
Me
Me
Me
NMe2
NMe2
N
C
N
NHFmoc
AA
NHFmoc
-
PF6
Me2N
X = NH
O
NHFmoc
AA
PH
XH
O
AA
O
Me
O
+
NH
X=O
O
OH
2 or 4–6 h with sterically uncongested and congested
amino acids, respectively.
tion of protected amino acids at the first stage of solid-
phase synthesis.
Stage 2. Deblocking Substituted Amino Acids
Consecutive treatment of the resin with a solution
of piperidine in dimethyl phthalate is recommended:
first for 5–10 min and then for 30 min in a fresh
solution. After deprotection the resin is washed no less
than 4 times with dimethyl phthalate to remove Fmoc
decomposition products. The progress of acylation on
supports or removal of protective functions from amino
acids can be followed by means of the Kaiser test.
on Polymer Supports
The second stage of our synthetic scheme (after
immobilization of a protected amino acid) involves
removal of the protective group to activate the amino
group. The Boc and Fmoc protections are removed in
different ways.
Removal of the Boc protection in amino acids on
the Merrifield resin is performed by treatment with
50% trifluoroacetic acid in dichloromethane for 30 min:
Under these conditions the Merrifield linker remains
intact.
Kaiser test for amino group. Analysis of resins after
a reaction which results in disappearance or appe-
arance of a free amino group is readily accomplished
by means of the ninhydrin (Kaiser) test [8]. This test
can be both qualitative and quantitative, and it is a
fairly sensitive color reaction for amino group.
O
H
N
Me
Me
O
O
A
A
A deep dark blue color of the resin and solution
provide clear evidence for the presence of a primary
amine function on the resin. If the color remains
yellow, one can conclude that such function is absent.
With secondary (proline) or sterically congested
(phenylalanine and β-phenylalanine) amines, the resin
and solution acquire a dark red color, which is typical
in such cases.
O
Me
O
(1) 50% TFA/DCM
(2) 5% Et3N/DCM
NH2
O
A
A
After deprotection the resin is washed with a
triethylamine solution to remove trifluoroaaetic acid.
The Fmoc protection in amino acids on the Wang
(X=O) and Rink (X=NH) supports is removed with a
20% piperidine solution in DMF for 40–50 min.
Stage 3. Nucleophilic Substitution in Heterocycles,
Involving Polymer-Immobilized Amino Acids
The next stage in our planned protocol involves
aromatic nucleophilic substitution; the nucleophile is
the grafted amino acid and the activated heterocycle is
present in solution {reaction (2)}. Most nucleophilic
substitutions on supports are accomplished similarly to
liquid-phase reactions. However, the reaction tem-
perature should be not above 120°С, since at higher
temperatures the polystyrene base of the carrier starts
to degrade. Conditions of supported reactions should
also preserve the linker.
O
NHFmoc
X
A
A
O
(1) 20% Pip/DMF
NH2
X
A
A
A considerable weight loss of the resin after
removal of the Fmoc protection can form a basis for
gravimetric assessment of the degree of immobiliza-
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 80 No. 12 2010

BASIC TECHNIQUES OF WORKING ON A SOLID PHASE
2577
O
O
O
O
OH
−H2O
N
NH2
+
OH
R
R
O
O
O
O
OH
OH
O
O
O
O
O
O
N
N
R
OH
In selecting appropriate activated heterocyclic
substrates one should take into account the nature of
the leaving group in the heterocycle. Tables 1 and 2
list the activities of pyrimidines with different leaving
groups in the 2 and 4 positions.
We would like to remind that the nature of the
linker determines the type of the terminal function in
the molecule removed from the support: The Wang
resin allows production of acids, and the Rink resin
allows production of amides.
As seen from Tables 1 and 2, the rate of substit-
ution in 2-halopyrimidines is the highest for the
fluorine derivative, the 4 position is more active than
the 2 position, and sulfonyl groups are preferred over
chlo-rine. The listed data are helpful in selecting other
hetarylating substrates.
Equipment and Materials
Experimental implementation of the above-
described four-stage sequence requires no specific
laboratory equipment. Reactions requiring no heating
are convenient to perform in capped vials or plastic
syringes with a porous partition (Fig. 2а), minimizing
manipulations at the stages and filtering and resin
washing. If heating is required, glass vials (desirably,
from a heat-resistant glass and hermetic screw caps)
can be fixed in a temperature-controlled shaker (we
used a low-cost domestic device shown in Fig. 2b).
Stage 4. Removal of Target Compounds
from Polymer Supports [11]
Most linkers in solid-phase organic synthesis are
cleaved in an acid medium. The resistance of linkers to
acids is sharply decreased in going from the Merrifield
to Wang and Rink resins. The Rink linker is cleaved in
milder conditions (10–20% CF3COOH) than the Wang
linker (50% CF3COOH). The Merrifield resin is passive
in such conditions, and it is cleaved by transesteri-
fication forming an acid ester [12].
The set of reagents includes three basic components:
a “school” set of amino acids, Merrifield and/or Rink
resin, and simple substituted heterocycles (vide infra).
Commercial immobilized amino acids can be used. In
our case (with account for protection/immobilization
of amino acids) such reagents as di-tert-butyl dicar-
bonate and 9-fluorenylmethyl chloroformate (introduc-
tion of Boc and Fmoc protections) and reagents for
O
O
NaOMe
Me
O
R
O
R
THF−MeOH
Table 2. Relative nucleophilic substitution rate constants in
activated pyrimidines under the action of cyclohexylamine
(29°С, EtOH)
Table 1. Reaction reaction rates of halopyrimidines with
piperidine in ethanol
Relative reaction rates [9]
Pyrimidine
Pyrimidine
Relative rate constant [10]
20°С
30°С
40°С
2-Cl
1.0
2.2
2-MeSO-
2-MeSO2-
4-MeSO-
4-MeSO2-
2-PhSO2-
2-Br
2-Cl
2-F
1
0.49
66.23
0.3
2.05
1.03
4.1
2.11
236
4.7
1.1×103
6.7×103
4.5
117.7
0.64
2-I
1.35
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 80 No. 12 2010

2578
BABAEV, ERMOLAT’EV
(b)
(a)
Fig. 2. Equipment for solid-phase synthesis: (a) plastic syringe with a porous partition and (b) temperature-controlled shaker.
immobilization (Cs2CO3/KI, HOBt, or BOP) are
needed. Ninhydrin is required for the Kaiser test and
CF3COOH and piperidine for removal of protective
groups. At certain stages we took DIEA. A large
consumption of solvents, specifically DMF and
dichloromethane (DCM), for washing resins should
also be taken into account.
stirred for 16–40 h until di-tert-butyl dicarbonate no
longer detected by TLC. The reaction mixture was
poured into 100 ml of ice water, and the pH of the
solution was brought to 3 by adding 10% HCl in small
portions. After chloroform extraction (2×100 ml) the
organic phases were combined, and the solution was
removed on a rotary evaporator. The remaining
colorless oil was crystallized by trituration in hexane in
an ultrasonic bath. The products were dried in a
vacuum oven and purified, if required, by recrystal-
lization from hexane/ethyl acetate. TLC control:
chloroform–methanol–acetic acid 9:1:0.1.
PRACTICAL IMPLEMENTATION OF THE TASK
Stage 1: Immobilization of Amino Acids on Supports
The starting materials for stage 1 are N-protected
amino acids. Boc- and Fmoc-amino acids are com-
mercially available but fairly expensive. By this
reason, we considered it reasonable to prepare Boc and
Fmoc derivatives of the selected amino acids.
In this way we obtained compounds Ia–Ih: Boc-
glycine, Boc-L-alanine, Boc-β-alanine, Boc-L-phenyl-
alanine, Boc-β-phenylalanine, Boc-L-proline, Boc-5-
aminovaleric acid
и Boc-4-(aminomethyl)benzoic
acid. The purity of the products was assessed by NMR
spectroscopy.
Preparation of Boc-amino acids I. Amino acids are
heated with di-tert-butyl dicarbonate in aqueous di-
oxane in the presence of NaOH [13].
Preparation of Fmoc-amino acids II. The Fmoc
protection was introduced using 9-fluorenylmethyl chloro-
formate [7] in the presence of sodium carbonate. By
replacing sodium carbonate with DIEA [14] we could
decrease the yield of 9-fluorenylmethyl chloroformate
hydrolysis products (dibenzofulvene and its polymeriza-
tion products, as well as 9-fluorenyl-methanol).
O
O
O
+
O
HO
AA−NH2
O
O
O
NaOH
40−50°C
HO
AA−NH
O
O
O
O
O
Ia−Ih
+
Cl
HO
AA−NH2
Amino acid, 0.04 mol, was added to a solution of
1.6 g of NaOH (0.04 mol) in 40 ml of water. The
solution was shaken for 10 min, cooled to 10°С, and a
solution of 6.55 g (0.03 mol) di-tert-butyl dicarbonate
in 30 ml of dioxane was added over the course of
15 min. After 20 min the solution was heated with
caution to 40–60°С (CO2 evolution is initiated) and
O
Na2CO3
HO
AA−NH
O
O
IIa−IIk
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 80 No. 12 2010

BASIC TECHNIQUES OF WORKING ON A SOLID PHASE
2579
Method A. Amino acid (20 mmol) was dissolved (or
partially suspended), with stirring and cooling on an
ice bath, in 50 ml of 10% Na2CO3. After that dioxane,
20 ml, was added and then, slowly, a solution of 5.7 g
(22 mmol) of 9-fluorenylmethyl chloroformate in 50
ml of dioxane. The mixture was shaken for 1 h at 0°С
and then at room temperature from 3 to 8 h, depending
on the amino acid. The reaction mixture was diluted to
1 l with ice water and extracted with ether (3×200 ml)
to remove chloroformate decomposition products:
dibenzofulvene and its polymers (Rf 0.85–0.9; ethyl
acetate/hexane 1:1). The aqueous layer was cooled on
an ice bath, acidified with conc. HCl to pH 2.0, and
extracted with ethyl acetate (4×200 ml). The combined
organic phases were washed with 0.1 N HCl and
water, dried over Na2SO4, and evaporated on a rotary
evaporator to obtain a viscous colorless oil which
almost always could be crystallized in n-heptane on an
ultrasound bath.
The Merrifield resin (1 g, 1.6 mmol) was placed in
a 30-ml vial, poured with 3 ml of DMF, and left to
swell for 10–15 min. A solution containing V mmol of
a Boc-amino acid, 1.6 g (5 mmol) of Cs2CO3, and a
catalytic amount of NaI (1 mmol, 0.17 g) was then
added to the swollen polymer so that the total volume
of the reaction mixture was no more than 10 ml.
Tightly capped vials were heated in an oil bath at 80–
85°С for 12 h with intermittent shaking. The resin was
then separated on a glass frit, washed successively
with DMF (20 ml), 10% NH4Cl (3×30 ml), DMF
(20 ml), DCM (2×30 ml), and MeOH (2×15 ml), dried
a in vacuum oven, and weighed.
The yields at the stage of immobilization of
protected amino acids on the Merrifield resin were as
follows, %:
Glycine (Gly)
94
β-Alanine (β-Ala)
95
92
96
5-Aminovaleric (5-Ava)
L-Alanine (L-Ala)
The resulting Fmoc-amino acids were finally
purified by recrystallization from ethyl acetate/hexane.
TLC control: chloroform/ethanol 3:1 (system I) and
chloroform/methanol/acetic acid 9:1:0.1 (system II).
L-Phenylalanine (L-Phe)
DL-β-Phenylalanine (DL-β-Phe)
L-Proline (L-Pro)
82
76
95
97
Method B. Here we used DIEA as a base and at
least a 20% excess of the base and amino acid with
respect to chloroformate, which allowed us to increase
the total reaction yield and much decrease by-product
formation.
4-(Aminomethyl)benzoic (Amb)
The Fmoc-substituted amino acids were immob-
ilized on the Rink resin by treatment with DIC in the
presence of 1-hydroxybenzotriazole:
Thus, we could prepare, in fairly high yields, the
Fmoc derivatives of glycine, β-alanine, 4-aminobutyric
acid, 5-aminovaleric acid, L-alanine, L-phenylalanine,
β-phenylalanine, L-asparagine, DL-valine, L-proline,
and 4-(aminomethyl)benzoic acid. The resulting pro-
ducts were characterized and their purity was assessed
by NMR spectroscopy.
O
+
HO
AA−NHFmoc
NH2
O
DIC/HOBt
N
H
AA−NHFmoc
Immobilization of Boc- and Fmoc-protected amino
acids was performed by standard procedures.
Carbodiimide method. A dry Rink-NH2 resin (1 g)
was placed in a 30-ml vial, poured with 3 ml of a
solution of 0.46 g (3 mmol) of 1-hydroxy-1H-1,2,3-
benzotriazole in 1:1 DMF/DCM, and left to swell for
10 min. After that 3–4 of a 1:1 ml DMF/DCM solution
containing 3 mmol of a Fmoc-amino acid and 3 mmol
(0.38 g) of DIC. If a precipitate formed, more DMF
was added. Tightly capped vials were agitated on an
orbital shaker for 15–30 h. The reaction progress was
followed by the Kaiser test. As a rule, the reaction time
was 4–5 h with unbranched amino acids and 6–9 h
with sterically congested amino acids. After reaction
Boc-substituted amino acids were immobilized on
the Merrifield acid in the presence of cesium carbonate
and a potassium iodide catalyst at 80–85°С:
O
+
HO
AA−NHBoc
Cl
O
Cs2CO3/KI
O
AA−NHBoc
DMF, 80°C
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 80 No. 12 2010

2580
BABAEV, ERMOLAT’EV
completion the resin was washed in succession with
DMF (3×20 ml), DCM (2×20 ml), and MeOH (2×10
ml) and dried in a vacuum oven. The resin weight gain
always nicely correlated was the calculated reaction
yield.
The yields at the immobilization stage were
determined by gravimetry. As a rule, the carbodiimide
method results in almost quantitative immobilization
yields on the Rink resin. With sterically congested
amino acids, the yields are slightly lower.
Sterically congensted amino acids, such as L-
phenylalanine, L-asparagine, DL-valine, and β-phenyl-
alanine were immobilized on the Rink resin by means
of (1H-1,2,3-benzotriazol-1-yloxy)tris (dimethylamino)-
phosphonium hexafluorophosphate (BOP) and diiso-
propylethylamine (DIEA) in the presence of 1-hyd-
roxybenzotriazole (HOBt).
Determination of the immobilization degree of
Fmoc-amino acids. To a resin obtained by coupling 1 g
of the Rink-NH2 resin with Fmoc-amino acids in
standard conditions, 14 ml of a 25% solution of
piperidine in DMF was added. The vial was shaken for
40–45 min, and the resin was separated on a glass filter
and washed with DMF (3×10 ml). The mother liquor
was diluted with ice water to 250 ml. 1-(9H-Fluoren-9-
ylmethyl)piperidine which precipitated as white flakes
was filtered off, washed with water, and dried in a
O
+
HO
AA−NHFmoc
NH2
1
vacuum, mp 119–120°С. H NMR spectrum (DMSO-
O
d6), δ, ppm: 7.77 m (H16, H20, 2H), 7.33 d (H13, H17,
2H), 7.15 m (H14, H15 H18, H19, 4H), 5.21 t (CH, J =
5.5 Hz, 1H), 3.71 d (CH2, J = 5.15 Hz, 2H), 2.50 m
(2CH2, 4H), 1.68 m (3CH2, 6H). The yield of the
immobilization reaction of Fmoc-amino acids on the
Rink resin was calculated by the formula:
BOP/DIEA/HOBt
N
H
AA−NHFmoc
A solution (6–7 ml) of 1.33 g (3 mmol) of BOP,
3 mmol Fmoc-amino acid, and 0.78 g (6 mmol) of
DIEA in 1:1 DMF/DCM was added to deblocked Rink
resin (1 g). The mixture was agitated on an orbital
shaker for 5 h. The resin was separated, washed with
DMF (2×20 ml), DCM (2×30 ml), and MeOH (1×
15 ml), and dried in a vacuum oven. The reaction
progress was followed by the Kaiser test.
m(precipitate)×1000
×100%,
(3)
Yield =
MC
where m is the weight of the precipitate, М = 263.4,
molecular weight of 1-(9H-fluoren-9-ylmethyl)pipe-
ridine, and C = 0.73, capacity of the Rink resin, mmol/g.
The yields at the stage of immobilization of
protected amino acids on the Rink resin were as
follows, %:
Stage 2: Removal of Protective Groups
Glycine (Gly)
99
100
98
99
98
89
94
90
98
87
95
β-Alanine (β-Ala)
The stage of immobilization is followed by the
stage of protection removal. Free amino groups on the
resin are readily determined by the Kaiser test (vide
supra).
4-Aminobutyric (GABA)
5-Aminovaleric (5-Ava)
L-Alanine (L-Ala)
Kaiser test. In 3 vials we prepared by 50 ml of the
following solutions: ~80% of phenol in ethanol; a
mixture of water and pyridine (1:4), containing 1 ml of
002 M NaCN; and 6% of ninhydrin in ethanol. A little
resin from the reaction vessel was transferred on a
Schott microfilter (0.5 ml), the resin was washed with
three portions of MeOH, transferred to a 3-ml tube,
each of the above solution (by 3 drops) was added, and
the tube was heated at 120°С with shaking for 3–
5 min.
L-Phenylalanine (L-Phe)
DL-β-Phenylalanine (DL-β-Phe)
DL-Valine (DL-Val)
L-Proline (L-Pro)
L-Asparagine (L-Asp)
4-(Aminomethyl)benzoic (Amb)
In total, we obtained at the first stage 19 supports
(1–4 g) containing N-substituted amino acids: 8 amino
acids on the Merrifield resin and 11 amino acids on the
Rink resin.
The Boc protection was removed by treatment with
50% TFA for 0.5 h. During reaction the resin sus-
pension foams because of vigorous gas evolution:
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 80 No. 12 2010

BASIC TECHNIQUES OF WORKING ON A SOLID PHASE
2581
O
The yields of immobilization of Fmoc-amino acids
on the Rink resin were as follows, %:
O
AA−NHBoc
Glycine (Gly)
89
90
O
+ CO2
β-Alanine (β-Ala)
(1) 50% TFA/DCM
(2) 5% Et3N/DCM
5-Aminovaleric (5-Ava)
L-Alanine (L-Ala)
84
94
O
AA−NH2
+
L-Phenylalanine (L-Phe)
L-Proline (L-Pro)
82
86
Removal of the Boc protection from amino acids on
the Merrifield resin. A 55% solution of TFA in DCM
(20 ml) was poured onto a 1-g sample of the resin with
an immobilized amino acid placed in a high 30-ml vial.
Gas evolution was observed, and the resin acquired a
characteristic pinkish color. The vial was shaken for
0.5 h, and the resin was separated on a glass filter and
washed with DCM (2×20 ml), 10% DIEA in DCM
(2×30 ml), DMF (2×10 ml), DCM (15 ml), and MeOH
(15 ml). The resin was dried in a vacuum oven and
weighed. Amino groups were detected by the Kaiser test.
Removal of the Fmoc protection from amino groups
on resins. A 20% solution of piperidine in DMF (20 ml)
was poured on a resin containing a Fmoc-protected
amino group or Fmoc-protected amino acid, placed in
a 30-ml standard vial with a porous bottom. The resin
was separated from the solution, and the procedure
was repeated; therewith, additional 30-min shaking
was applied. The resin was washed in succession with
DMF (3×30 ml), DCM (2×25 ml), and MeOH. (2×
15 ml). Amino groups were detected by the Kaiser test.
The Fmoc protection from amino acids im-
mobilized on the Rink support were selectively re-
moved with a 20% solution of piperidine in DMF. The
intermediate dibenzofulvene (DBF) rapidly reacts with
excess piperidine to form 1-(9H-fluoren-9-ylmethyl)-
piperidine which is water-insoluble and precipitates
when the reaction mixture is diluted with water
(Scheme 2).
Synthesis of Heterocycles
with a Readily Leaving Group
Experiments on hetarylation of amino acids were
performed with four fairly active heterocycles: 2-
(methylsulfonyl)pyrimidine (IV), 2-bromo-5-nitro-1,3-
thiazole (V), 2-fluoropyrimidine (VI), and 2-chloro-5-
nitropyridine (VII).
With the weight of a dried 1-(9H-fluoren-9-
ylmethyl)piperidine precipitate, the yield of
immobilization of Fmoc-amino acids on the Rink resin
is calculated by formula (3).
Pyrimidine IV was prepared in a moderate yield by
oxidation of 2-(methylsulfanyl)pyrimidine (III) with
m-chloroperbenzoic acid [15]. The starting pyrimidine
III is readily prepared by methylation of commercial
Scheme 2.
O
O
NH
H
N
+
H2N
H
-CO2
O
R
R
O
DBF
NH
O
HN
N
R
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 80 No. 12 2010

2582
BABAEV, ERMOLAT’EV
pyrimidine-2-thione with dimethyl sulfate in the
presence of NaOH [16].
cooled to –3°С and diazotized with a solution of 25 g of
sodium nitrite in 35 ml of water under vigorous stirring
for 1 h, after which it was diluted with water to 500 ml
and extracted with ether (3×200 ml). The combined
organic phases were dried over calcined Na2SO4. The
ether was removed on a rotary evaporated, and the
residue was subjected to steam distillation. After
drying, 15.89 g (38%) of compound V was obtained as
light weight crystals. TLC control: Rf 0.51 (hexane–
N
(MeO)2SO2
NaOH
N
Me
N
H
S
N
S
III
mCPBA
N
O
N
S
1
ethyl acetate, 5:1). mp 88–91°С. H NMR spectrum
O
Me
(360 MHz, CDCl3), δ, ppm: 8.30 s (CH, 1H).
IV
1
It should be added that the H NMR spectrum is
A solution of 32 g (0.13 mol) of 3-chloroperbenzoic
acid in 300 ml of chloroform was added dropwise with
cooling (–25°С) and vigorous stirring over the course
of 1.5 h to a solution of 8.2 (0.065 mol) of compound
III in 100 ml of chloroform. The mixture was stirred at
20°С for a day. After the reaction had been complete,
the reaction mixture was washed with a saturated
solution of sodium sulfite (2×300 ml) and then with a
saturated solution of sodium hydrocarbonate (2×400
ml). The combined organic phases were dried over
sodium sulfate. The solvents were distilled off, the dry
residue was recrystallized from ethanol to obtain, after
drying, 4.75 g (46%) of compound 4 as colorless
crystals, mp 73–75°С. TLC control: Rf 0.6 (methanol–
chloroform, 1:9. 1H NMR spectrum (360 MHz, CDCl3)
δ, ppm: 8.94 d (2CH, J = 4.3 Hz, 2H); 7.58 t (CH, J =
5.6Hz, 1H); 3.36 s (CH3, 3H).
poorly informative and contains only one signal at δ
8.3 ppm. The IR spectrum (KBr) is more informative:
3084 v (C–H), 1520 as, 1344 s (NO2), 740 (C–Br). For
comparison, the IR spectrum of compound V from the
site of Acros (www.acros.com) can be used.
2-Fluoropyrimidine (VI) was prepared by
diazotization of 2-aminopyrimidine under the action of
sodium nitrite in 48% HBF4 [10]. 2-Fluoropyrimidine
is so reactive that on attempted neutralization of the
reaction mixture with 1N NaOH by the procedure [10]
we always obtained a hydrolysis product, i.e.
pyrimidin-2-one. It was found that the yield of com-
pound VI can be improved by using a suspension of
sodium hydrocarbonate instead of a solution of NaOH
and cooling (to –10°С) during neutralization.
N
NaNO2/HBF4
N
2-Bromo-5-nitro-1,3-thiazole (V) was prepared by
nitration of 2-amino-1,3-thiazole followed by
diazotization and introduction of bromine in position 2
by the Sandmeyer reaction [17]:
−10°C
N
NH2
N
F
VI
A solution of 15.3 g of 2-aminopyrimidine
(0.16 mol) in 500 ml of 48% HBF4 was poured into a
high 1-l porcelain glass, cooled to –12ºС, and sub-
jected to diazotization under vigorous stirring by
treatment with a solution of 22.8 g of sodium nitrite
(0.32 mol) in 35 ml of water for 1 h. After adding
sodium nitrite, the mixture was stirred for an additional
1 h at –10ºС and then neutralized with a saturated
solution of sodium hydrocarbonate to pH 6–7 under
cooling and extracted with ether (4×250 ml). The
combined organic layers were washed with a 2%
solution of potassium carbonate (2×100 ml) and dried
over calcinated Na2SO4. The solvent was distilled off
at the water bath temperature of no higher than 20ºС.
The oily residue was distilled in a vacuum to obtain
6.4 g (41%) of a transparent mobile liquid, bp 72–73ºС
(20 mm). The product can be stored at 0ºС with no
appreciable decomposition signs for 10 days. TLC
control: Rf 0.61 (ethyl acetate, 1:1). 1H NMR spectrum
N
N
HNO3/H2SO4
O2N
N
NH2
NH2
S
S
NaNO2
CuBr
O2N
Br
S
V
2-Amino-1,3-thiazole, 20 g (0.2 mol), was added
with caution, in small portions, over the course of 0.5 h,
to 55 ml of a cold (to –5°С) conc. H2SO4 placed in a
0.5-l high glass. The mixture was then cooled to –10°С,
and 10 ml of fuming nitric acid (0.23 mol) was added
dropwise over the course of 25 min under vigorous
stirring. The solution was left to stand for three days at
room temperature in an uncovered glass. The bright
orange mixture was poured into 150 ml of ice water,
and a solution of 100 g of NaBr and 100 g of CuSO4 in
200 ml of water was added to it. The mixture was
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 80 No. 12 2010

BASIC TECHNIQUES OF WORKING ON A SOLID PHASE
Table 3. Elemental analyses of amides VIII
2583
(360 MHz, CDCl3) δ, ppm: 8.66 q (2CH, JС4(6),5 6 Hz,
С4,F 1.7, 2H); 7.38 q (CH, JС4,5 6 Hz, JС5,F 1.7, 1H).
J
Element content, %
Compound
2-Chloro-5-nitropyridine VII.
A
commercial
reagent was used. (Synthesis of compound VII is
C
N
described in detail in [18].)
Theor. 5.26 47.32
Exp. 5.74 46.88
Theor. 6.02 50.54
Exp. 6.73 49.48
Theor. 6.66 53.27
Exp. 6.38 52.51
Theor. 7.2 55.6
Exp. 6.89 53.75
36.84
36.57
33.69
32.6
N
N
Realization of Stage 3 (Solid-Phase Nucleophilic
Substitution) and Stage 4 (Product Removal from
Polymer Support)
N
N
NHCH2CONH2
Synthesis of N-(pyrimidin-2-yl)amino Acid Derivatives
NH(CH2)2CONH2
The nucleophilic substitution reactions with amino
acids immobilized on the Rink resin were performed
using 2-fluoropyrimidine (VI) and DIEA as the base.
During reaction the resin volume decreased 1.5–2
times, which, too, was evidence for reaction progress.
For control of reaction progress we used the Kaiser test
for amino group, taking from the reaction mixture
small resin portions.
31.07
N
N
30.95
29.86
N
N
NH(CH2)3CONH2
NH(CH2)4CONH2
29.32
10 min, when the resin gained weight, 0.39 g (4 mmol)
of 2-fluoropyrimidine was added. The viald were
tightly closed and heated at 50–55°С with intermittent
shaking. The Kaiser test showed that the reaction was
almost complete in 7–12 h. The reacted resin was
successively washed on a filter with DMF (15 ml),
DMF/DCM (15 ml 50%), DCM (2×25 ml), and MeOH
(2×15 ml) and dried in a vacuum oven, after which it
was transferred into a 30-ml flask, poured with 15 ml
of 60% TFA/DCM, and agitated on an orbital shaker
for 1 h. The resin was separated on a glass filter and
washed with absolute DCM (15 ml) and MeOH
(15 ml). The filtrate was evaporated on a rotary
evaporator. The oily residue was poured with absolute
MeOH (5 ml) and evaporated again. The procedure
was repeated until TFA was removed completely. The
product was purified, if necessary, by column chromato-
graphy (MeOH–CHCl3, 4:1) on silica. TLC was per-
formed in MeOH–CHCl3, 4:1. The yield (Table 4) was
calculated on the basis of the standard resin capacity.
O
N
+
N
H
AA−NH2
N
F
VI
DIEA/DMF
O
H
N
A
A
N
A
A
N
H
NH2
N
N
H
O
50% TFA/DCM
VIIIa−VIIIk
The resulting primary amides of N-(pyrimidin-2-
yl)-amino acids were removed from the support by
treatment with 50% TFA. Compounds VIIIa–VIIIk
were purified by column chromatography. Structural
1
assessment was performed by H NMR spectroscopy
and mass spectrometry. The elemental analyses and 1H
NMR spectral data are listed in Tables 3 and 4.
Synthesis of amides of N-(pyrimidin-2-yl)amino
acids. A solution of 0.52 (4 mmol) of DIEA in
absolute DMF was poured onto 0.5 g of the Rink resin
containing an unsubstituted amino acid. After 7–
For the activated pyrimidine for the reaction of ac-
tivated pyrimidines with amino acids on the Merrifield
resinе we chose 2-(methylsulfonyl)pyrimidine (IV):
O
N
O
H
N
N
DIEA/DMF
+
O
A
A
O
AA−NH2
N
N
N
SO2Me
IV
N
2 M NaOMe
N
HCl
A
A
A
MeOH−THF
OMe
A
OH
N
H
N
N
H
O
O
Xa−Xc
IXa−ΙXh
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 80 No. 12 2010

2584
BABAEV, ERMOLAT’EV
Table 4. Yields and chatacteristics of amide derivatives and N-(pyrimidin-2-yl)amino acid esters (1H NMR at 36-MHz,
DMSO-d6)
1H NMR spectrum, δ, ppm
Mass spectrum
Comp. no.
Compound
X
Yield, %
M+, m/z
H4,6
H5
NH
MeO
NH2
8.22
8.32
8.19
6.55
6.54
6.46
7.14
7.09
6.75
167
152
181
85
96
94
IXa
N
N
VIIIa
N
N
N
NHCH2COX
MeO
IXb
VIIIb
NH(CH2)2COX
NH2
NH2
8.31
8.36
6.63
6.75
7.31
6.64
166
180
98
87
N
VIIIc
NH(CH2)3COX
NH(CH2)4COX
MeO
NH2
MeO
NH2
8.26
8.28
8.22
8.33
6.53
6.56
6.54
6.54
6.85
6.47
7.14
7.25
209
194
182
166
34
65
79
88
N
IXc
N
VIIId
IXd
N
N
NHCH(CH3)COX
NHCH(CH2Ph)COX
NHCH(Ph)CH2COX
VIIIe
MeO
8.25
6.53
6.66
217
32
IXe
N
N
VIIIf
NH2
MeO
NH2
8.20
8.31
8.19
6.53
6.68
6.48
7.45
6.58
6.58
242
218
242
49
11
23
IXf
N
N
VIIIg
N
NH2
NH2
8.34
8.25
6.74
6.57
7.27
6.81
209
195
28
35
VIIIh
VIIIi
N
NHCH(CH2CONH2)COX
N
N
NHCH(i-Pr)COX
O
MeO
NH2
8.25
8.27
6.71
6.57
–
–
208
192
52
72
X
N
IXg
N
N
VIIIj
MeO
NH2
8.48
8.18
6.81
6.59
7.46
7.07
244
228
74
83
IXh
N
N
NHCH2C6H4COX
VIIIk
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 80 No. 12 2010

BASIC TECHNIQUES OF WORKING ON A SOLID PHASE
2585
Reaction progress was followed by the Kaiser test
for amino group. Removal of products from the resin
was performed in the presence of sodium methylate.
The resulting N-(pyrimidin-2-yl)amino acid esters
IXa–IXh were characterized by the mass and 1H NMR
spectra (Table 4). Three examples were used to
demonstrate the possibility to prepare free amino acids,
viz. N-(pyrimidin-2-yl)amino acids Xa–Xc, by hyd-
rolysis of the corresponding esters.
separated on a glass filter and washed with MeOH and
THF. The filtrate was neutralized with 10% HCl and
evaporated on a rotary evaporator. The dry residue was
treated with methanol to extract the resulting ester, the
extract was filtered off, and evaporated on a rotary
evaporator. The product is purified, if required, by
column chromatography (MeOH–CHCl3, 2:9) on silica.
The yield (Table 4) was calculated on the basis of the
resin capacity. TLC was performed in MeOH–CHCl3, 2:9.
Synthesis of methyl esters of N-(pyrimidin-2-yl)
amino acids (X). A solution (6 ml) of 0.65 (5 mmol) of
DIEA in absolute DMF was poured onto 1 g of the
Merrifield resin containing an unprotected amino acid.
After V min, 0.79 g (5 mmol) of pyrimidine IV was
added to the resin. The vials were tightly closed and
heated at 80–90°С with intermittent shaking. Reaction
progress was followed by the Kaiser test. The reacted
resin was successively washed on a filter with DMF
(25 ml), DCM (2×25 ml), and MeOH (2×15 ml), after
which it was transferred to a standard 30-ml vial and
poured with absolute THF (3 ml) and 2M NaOMe in
MeOH (1 ml). The vials were tightly closed and
shaken at room temperature for 6 h. The resin was
Synthesis of amino acids X from esters IX. Ester IX
(50–200 mg) was dissolved in 5 ml of 40% aqueous
MeOH, after which 1 ml of conc. HCl was added. The
solution was heated at 60°С for 10–15 min and eva-
porated. The acid was recrystallized was aqueous
methanol. TLC was performed in MeOH–CHCl3–
AcOH, 1:3:0.2. Acids X were prepared on the scale of
30–150 mg.
Synthesis of N-(5-nitrothiazol-2-yl)amino Acid
Derivatives
Amino acids immobilized on the Rink and
Merrifield resins were reacted with 2-bromo-
5-nitrothiazole V in the presence of triethylamine:
O
HN
O
+
AA
NH
S
N
H
AA−NH2
N
A
50% TEA/DCM
TEA/THF
N
A
NH2
O2N
N
H
S
N
O
XIa−XIg
NO2
O2N
Br
S
V
O
AA
O
O
NH
O
AA−NH2
N
A
TEA/THF
2 M NaOMe
N
A
N
OMe
O2N
+
N
MeOH−THF
S
S
H
O
NO2
XIIa−XIIg
O2N
Br
S
V
Synthesis of N-(5-nitrothiazol-2-yl)amino acid
amides. A solution (4 ml) of 0.2 (2 mmol) of triethyl-
amine in absolute DMF was added to 0.5 g of the Rink
resin containing an unprotected amino acid. After 10 min,
0.42 g (2 mmol) of 2-bromo-5-nitrothiazole was
added. The vial was tightly closed and cooled to 5°С
with constant shaking. The Kaiser test showed that the
reaction is almost complete in 1–2 h. The reacted resin
was successively washed on the filter with DMF (4×
10 ml), DCM (2×15 ml), and MeOH (2×15 ml). After
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 80 No. 12 2010

2586
BABAEV, ERMOLAT’EV
Table 5. Yields and characteristics of N-(5-nitrothiazol-2-yl)amino acid amides XI and esters XII
1H NMR spectrum, δ, ppm
Mass-spectrum
Comp. no.
Compound
X
Yield, %
[M+ – NO2], m/z
H4
NH
N
MeO
NH2
MeO
NH2
8.07
8.09
7.95
7.94
7.25
7.53
7.32
7.56
172
157
186
171
81
85
89
76
XIIa
XIa
O2N
NHCH2COX
S
N
XIIb
XIb
O2N
O2N
NH(CH2)2COX
NH(CH2)3COX
S
S
N
N
NH2
8.XII
7.37
184
79
XIc
MeO
NH2
7.91
–
7.34
–
214
198
186
92
–
XIIc
O2N
O2N
NH(CH2)4COX
S
MeO
7.99
7.41
84
XIId
XId
N
NHCH(CH3)COX
S
NH2
8.07
7.45
171
88
N
N
MeO
NH2
7.90
8.08
7.92
–
7.82
–
262
247
262
38
27
52
XIIe
XIe
O2N
O2N
NHCH(CH2Ph)COX
NHCH(Ph)CH2COX
S
S
MeO
XIIf
NH2
–
–
248
—
—
N
NH2
8.01
7.89
185
22
XIf
O2N
O2N
NHCH(i-Pr)COX
S
N
MeO
NH2
7.95
8.11
7.37
7.65
248
233
64
55
XIIg
XIg
NHCH2C6H4COX
S
drying in a vacuum oven and placed in a 30-ml vial,
poured with 55% TFA/DCM (10 ml), and agitated on
an orbital shaker for 1 h. The resin was separated on a
glass filter, washed with absolute DCM (5 ml) and
absolute MeOH (5 ml). The filtrate was evaporated on
a rotary evaporator. The oily residue was poured with
absolute MeOH (5 ml) and evaporated again. The
procedure was repeated until TFA was removed
completely. The product was purified, if necessary, by
column chromatography (MeOH–CHCl3, 1:2) on
silica. The yield was calculated on the basis of the
average resin capacity. Thus we obtained amides XIa–
XIe, XIf, and XIg (50–150 mg).
the Merrifield resin containing immobilized unpro-
tected amino acids. After 10 min, a solution (3 ml) of
0.65 g (3 mmol) of 2-bromo-5-nitrothiazole in THF
was added to the suspended resin. The reaction was
performed in cold with intermittent shaking. The resin
got intense red. After 10 h, the resin was separated on
a glass filter, washed with DMF (2×20 ml), DCM
(2×30 ml), and MeOH (2×15 ml), dried in a vacuum
oven, and weighed. The dry resin was placed in a
standard 30-ml vial and poured with absolute THF (3 ml)
and 2M NaOMe in MeOH (1 ml). The vial was tightly
closed and shaken at room temperature for 6–12 h. The
resin was separated on a glass filter and washed with
30% MeOH in THF. The filtrate was neutralized with
10% HCl and evaporated on a rotary evaporator. The dry
residue was treated with methanol to extract the resulting
ester, and the extract was evaporated on a rotary
Synthesis of methyl esters of N-(5-nitrothiazol-2-yl)
amino acids. A solution (3 ml) of 0.3 g (3 mmol) of
triethylamine in absolute THF was added to 0.5 g of
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 80 No. 12 2010

BASIC TECHNIQUES OF WORKING ON A SOLID PHASE
2587
Table 6. 1H NMR data (360 MHz, DMSO-d6) and characteristics of N-(5-nitropyridin-2-yl)amino acid derivatives
1H NMR spectrum, δ, ppm
Comp. no.
Formula
X
Puritya
Yield, %
H6
H4
H3
O2N
O2N
O2N
MeO
NH2
8.87
8.15
6.53
B
B
57
72
XIVa
8.87
8.16
6.77
N
NHCH2COX
MeO
NH2
8.84
8.82
7.96
7.98
6.57
6.55
C
C
68
60
XIVb
XIIIc
N
NH(CH2)2COX
NH(CH2)3COX
NH2
8.83
8.82
7.98
8.09
6.51
6.61
C
C
57
70
N
O2N
MeO
XIVc
XIVd
NH2
8.83
8.80
7.97
8.14
6.54
6.65
C
B
47
52
N
NH(CH2)4COX
MeO
O2N
NH2
8.83
7.97
6.62
C
84
N
NHCH(CH3)COX
O2N
MeO
NH2
–
–
–
–
–
–
–
N
8.80
8.16
6.67
B
28
NHCH(CH2Ph)COX
NHCH(Ph)CH2COX
O2N
O2N
MeO
NH2
–
–
–
–
–
N
8.81
7.97
6.58
C
18
NH2
8.83
8.03
6.81
B
12
XIIIh
N
NHCH(i-Pr)COX
O2N
MeO
NH2
–
–
–
–
–
–
O
X
–
N
N
8.88
8.12
6.78
32
O2N
MeO
NH2
8.85
8.84
8.14
8.02
6.55
6.59
A
A
69
39
XIVe
N
NHCH2C6H4COX
a
1
The purity of products XIIIa–XIIIg and XIVa–XIVe was estimated by the H NMR spectra as follows: A (<5% of admixtures), B
(<10% of admixtures), and C(>20% of admixtures).
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 80 No. 12 2010

2588
BABAEV, ERMOLAT’EV
evaporator. The product was recrystallized from hexane–
ethyl acetate, 3:1, and dried in a vacuum oven. Thus we
obtained esters XIIa–XIIg (20–170 mg).
O
HN
O
+
AA
NH
N
N
H
AA−NH2
With both the resins, an intense yellowish red
coloration of the resin was observed. The Kaiser test
showed that the reaction with 2-bromo-5-nitrothiazole
occurs at room temperature for 3–6 h.
DIEA/DMF
O2N
NO2
The resulting primary amides and methyl esters of
N
VII
Cl
N-(thiazol-2-yl)amino acids were characterized the
1
mass and H NMR spectra. The characteristics of
O
H2N
compounds XIa–XIg and XIIa–XIIg are listed in
50% TEA/DCM
AA
Table 5.
NH
N
The yields of chromatographic purity of the ob-
tained N-(thiazol-2-yl)amino acid derivatives proved to
be lower than those of N-(pyrimidin-2-yl)amino acid
derivatives. We can suggest that, along the main
nucleophilic substitution reaction, 2-bromo-5-nitro-
thiazole undergoes the side reaction involving thiazole
ring opening, which was mentioned in [19]:
NO2
XIIIa−XIIIj
Synthesis of N-(5-nitropyridin-2-yl)amino acid
amides. A solution (4 ml) of 0.39 (3 mmol) of DIEA in
absolute DMF was added to 0.5 g of the Rink resin
containing an unprotected amino acid. After 10 min,
0.73 g (3 mmol) of 2-chloro-5-nitropyridine was added
to the resin. The vial was tightly closed and heated at
95°С for 12 h with intermittent shaking. The reacted
resin was successively washed on a filter with DMF
(4×10 ml), DCM (2×15 ml), and MeOH (2×15 ml) and
dried in a vacuum oven. The dry resin was placed in a
30-ml vial, poured with 10 ml of 50% TFA/DCM, and
agitated on an orbital shaker for 1 h. The resin was
separated on a glass filter and washed with absolute
DCM (10 ml) and absolute MeOH (5 ml). The filtrate
was evaporated on a rotary evaporator. The oily
residue was poured with 5 ml of absolute MeOH and
evaporated again. The procedure was repeated until
TFA was removed completely. The product was
purified, if necessary, by recrystallization from
hexane–ethyl acetate. The yield was calculated on the
basis of the average resin capacity. Thus we prepared
amides XIIIa–XIIIg (30–160 mg).
O2N
O
+
O
AA−NH2
N
Cl
VII
NO2
O
O
DIEA/DMF
N
AA N
90−95°C
H
NO2
O
N
O
AA N
H
O2N
2 M NaOMe
A
A
OMe
MeOH−THF
N
N
H
O
XIVa−XIVe
Synthesis of N-(5-nitropyridin-2-yl)amino Acid
Esters XIV were prepared in the same way on the
Merrifield resin (see cheme bellow).
Derivatives
N-(Pyridin-2-yl)amino acid derivatives on the Rink
resin were prepared using commercial 2-chloro-5-
nitropyridine (VII) and DIEA as the base. Darkening of
the support was observed during reaction. Reaction
progress was followed by the Kaiser test for amino
group. It was shown that the reaction occurs at 90–95°С
for 5–6 h, on average.
Synthesis of methyl esters of N-(5-nitropyridin-2-
ya)amino acids. A solution of (4 ml) of 0.39 (3 mmol)
of DIEA in absolute DMF was added to 0.5 g of the
Rink resin containing an unprotected amino acid. After
10 min, 0.73 g (3 mmol) of 2-chloro-5-nitropyridine
was added to the resin. The vial was tightly closed and
heated at 95°С for 10–12 h with intermittent shaking.
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 80 No. 12 2010

BASIC TECHNIQUES OF WORKING ON A SOLID PHASE
2589
O2N
+
pensive. In this connection, the total number of stages
in a training task should be determined by the pos-
sibilities of the training laboratory. We set ourselves
the task to work out the total cycle (including
protection and immobilization stages).
O
O
AA−NH2
N
Cl
VII
NO2
O
O
REFERENCES
DIEA/DMF
N
AA N
H
90−95°C
1. Ermolat’ev, D.S. and Babaev, E.V., Molecules, 2003,
vol. 8, pp. 467–471; http://www.mdpi.org/molecules/
papers/80600467.pdf.
2. Ermolat’ev, D.S. and Babaev, E.V., ARKIVOC, 2005,
pp. 172–178; http://www.arkivoc.com/ark/journal/2005/
I04_Zefirov/1364/1364.pdf.
NO2
O
N
O
O2N
AA N
H
3. Larsen, S.D., Connell, M.A., Cudahy, M.M., et al.,
J. Med. Chem., 2001, vol. 44, pp. 1217–1230.
2 M NaOMe
A
A
OMe
4. Vailancourt, V.A., Larsen, S.D., Tanis, S.P., et al., Ibid.,
MeOH−THF
N
N
2001, vol. 44, pp. 1231–1248.
H
O
5. Nicolau, K.C., Hanko, R., and Hartwig, W., Handbook
of Combinatorial Chemistry, Weinheim: Wiley–VCH,
2002, vol. 1.
XIVa−XIVe
The reacted resin was successively washed on a filter
with DMF (4×10 ml), DCM (2×15 ml), and MeOH
(2×15 ml), dried in a vacuum oven, and weighed. The
dry resin was place in a standard 30-ml vial and poured
with absolute THF (3 ml) and 2M NaOMe in MeOH
(1 ml). The vial was tightly closed and shaken at room
temperature for 5–7 h. The resin was separated on a
glass filter, washed with 30% MeOH in THF, the
filtrate was neutralized with 10% HCl, and evaporated
on a rotary evaporator. The residue was treated with
methanol to extract the product, and the extract was
evaporated on a rotary evaporator. Thus we obtained
esters XIVa–XIVe (65–100 mg).
6. Berger, G.H., Synth. Commun., 1993, vol. 23, pp. 1443–
1145.
7. Carpino, L.A. and Grace, Y.H., J. Org. Chem., 1972,
vol. 37, pp. 3404–3409.
8. Kaiser, E., Colescott, R.L., Bossinger, C.D., and Cook, P.I.,
Anal. Biochem., 1970, vol. 34, pp. 595–611.
9. Brown, D.J. and Waring, P.J., J. Chem. Soc. Perkin.
Trans., 1974, vol. 2, pp. 204–208.
10. Brown, D.J. and Ford P.W., J. Chem. Soc. C, 1967,
pp. 568–572.
11. James, I.W., Tetrahedron, 1999, vol. 55, pp. 4855–
4946.
Compounds XIIIa–XIIIg and XIVa–XIVe were
12. Kobayashi, S., Akiyama, R., and Kitagawa, H.J., Comb.
Chem., 2000, vol. 2, pp. 4238–440.
characterized by the 1H NMR spectra (Table 6).
13. Tarbell, D.S., Yamomoto, Y., and Pope, B.M., Proc.
Natl. Acad. Sci. USA, 1972, vol. 69, pp. 780–786.
CONCLUSIONS
14. Greene, T.W. and Peter, G.M., Protective Groups in
Organic Synthesis, Weinheim: Wiley–VCH, 1999, ch. 6.
15. Adlington, R.M., Baldwin, J.E., Catterick, D., and
Pritchard, G.J., J. Chem. Soc. Perkin Trans. 1, 1999,
pp. 855–866.
The 4-stage solid-phase reaction procedure for the
preparation of non-natural amino acids, optimized by
us in a student’s laboratory course, is quite convenient
from the methodical viewpoint. No special equipment
is necessary, the yields of the target products are high,
and the isolated quantities are sufficient for full-scale
characterization.
16. Boarland, M.P.V. and McOmie, J. F. W., J. Chem. Soc.,
1952, pp. 3716–3722.
17. Deshpande, R.N. and Nargund, K.S., J. Pract. Chem.,
1974, vol. 316, no. 2, pp. 349–352.
The reactions can be accomplished concurrently
with variable number of amino acids and hetarylating
agents (for example, in the 2×2, 2×3, or 3×3 formats).
Amino acids themselves (unlike amino acids immob-
ilized on resins) are commercially available and inex-
18. Chichibabin, A.E., Zh. Ross. Fiz.-Khim. O–va, 1914,
vol. 46 (2), pp. 1236–1296.
19. Ilvespaa, A.O., Helv. Chim. Acta, 1968, vol. 51, no. 7,
pp. 1723–1733.
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 80 No. 12 2010