Kinetics and mechanism of the peptide synthesis in solution

Article abstract of DOI:10.1023/A:1013993900972

The kinetics of the reaction of Boc-Xaa fluorophenyl esters (where Xaa = Ala, Val, Phe, Ser, Leu, Gly, Met, Pro, or Ile) with leucinamide was studied in order to measure changes in fluorescence emission at 375 nm of the fluorophenyl chromophore accompanying the reaction. It was found that the experimental kinetic data could not be described by a simple scheme of the second order reaction. Measurements of the kinetic parameters of the reaction at various initial concentrations of reagents indicated that the reaction rate can be expressed as: v = kC_N^aC_AE^b, where k is the reaction rate constant, C_N is the concentration of leucinamide, and C_AE is the concentration of fluorophenyl ester. The a and b reaction orders were close to 1/2 and 3/2 for Xaa = Ala, Val, Phe, Ser, or Leu, 1/2 and 1 for Gly, Met, or Pro, and 1 and 2 for Ile. The experimental equations for the reaction rate can theoretically be derived from a single scheme of chain reactions with various deactivation ways for active intermediates.

Full text of DOI:10.1023/A:1013993900972

Russian Journal of Bioorganic Chemistry, Vol. 28, No. 1, 2002, pp. 9–13. Translated from Bioorganicheskaya Khimiya, Vol. 28, No. 1, 2002, pp. 11–15.
Original Russian Text Copyright © 2002 by E. Permyakov, S. Permyakov, Medvedkin.
Kinetics and Mechanism of the Peptide Synthesis in Solution
1
E. A. Permyakov , S. E. Permyakov, and V. N. Medvedkin
Institute of Biological Instrumentation, Russian Academy of Sciences, Pushchino, Moscow oblast, 142290 Russia
Received February 16, 2001; in final form, June 20, 2001
Abstract—The kinetics of the reaction of Boc-Xaa fluorophenyl esters (where Xaa = Ala, Val, Phe, Ser, Leu,
Gly, Met, Pro, or Ile) with leucinamide was studied in order to measure changes in fluorescence emission at
3
75 nm of the fluorophenyl chromophore accompanying the reaction. It was found that the experimental kinetic
data could not be described by a simple scheme of the second order reaction. Measurements of the kinetic
parameters of the reaction at various initial concentrations of reagents indicated that the reaction rate can be
a
b
expressed as: v= kC C , where k is the reaction rate constant, C is the concentration of leucinamide, and
N
AE
N
CAE is the concentration of fluorophenyl ester. The a and b reaction orders were close to 1/2 and 3/2 for Xaa =
Ala, Val, Phe, Ser, or Leu, 1/2 and 1 for Gly, Met, or Pro, and 1 and 2 for Ile. The experimental equations for
the reaction rate can theoretically be derived from a single scheme of chain reactions with various deactivation
ways for active intermediates.
Key words: fluorescence, mechanism of peptide bond formation, peptide fluorophenyl esters, peptide synthesis,
reaction kinetics
1
INTRODUCTION
Emphasis is placed on the solid phase peptide synthesis
and, in this connection, on the development of instru-
mental methods, such as the short-wavelength IR spec-
troscopy [4].
Formation of the peptide bond between two amino
acids is one of the fundamental chemical reactions
2
studied in life sciences. Nowadays, the mechanism of
peptide bond formation during the ribosomal protein
Here, we studied the mechanism of peptide bond
synthesis is the subject of particularly intensive studies. formation in organic solvents. The use of organic sol-
Recent advances in the area of ribosome spatial struc- vents is connected with the chosen method of the amino
ture would undoubtedly rekindle interest in the more acid activation (Pfp esters). In future studies, we plan to
comprehensive studies of the mechanism of peptide carry out similar experiments in aqueous medium using
bond formation [1].
the hydrophilic p-sulfotetrafluorophenyl esters, which
we proposed previously [5, 6].
The mechanisms of peptide bond formation, other
than the ribosomal mechanism, are also studied. For
example, Huber and Wachterschauser have recently
demonstrated that amino acids can be activated for pep-
tide bond formation under geochemically acceptable
conditions (aqueous medium, 100°ë, pH 7–10) with
the use of carbon monoxide in the presence of a catalyst
Although active esters of protected amino acids are
used widely in peptide synthesis, the mechanism of
peptide bond formation by this method is not com-
pletely understood [7, 8]. Most kinetic studies in this
area were performed on the basis of the presumption
that this reaction proceeds the second order mechanism
[
2]. They directly associated the mechanism of peptide
[
7]. This approach was applied to most of the compar-
bond formation with the possibility of abiogenous pro-
tein synthesis and with the origin of life on Earth.
ative studies when the solution required no changes in
the starting reagent concentrations. For example,
Knowledge of kinetics and the mechanism of the Kovacs et al. [9–11] obtained important information on
reaction of peptide bond formation is also very impor- the dependence of racemization on the relative param-
tant from the practical point of view, especially for the eter of the ester activation using the proposal about the
combinatorial peptide chemistry, since an insufficient second order of the reaction.
information in this area results in failures in formation
Possible mechanisms of peptide bond formation
of peptide libraries [3].
were comprehensively discussed by Kemp et al. [12].
Unfortunately, there are few publications devoted to
The rate constants for 41 reactions of aminolysis of p-
the kinetics of the peptide synthesis in solution.
nitrophenyl esters of N-protected amino acids in DMF
were obtained. All rate constants, except those for the
1
Please address correspondence to: phone: +7 (095) 924-5749,
fax: +7 (827) 79-0522, e-mail: permyakov@ibp.serpukhov.su.
Abbreviations: CHA, cyclohexylamine; Pfp, pentafluorophenyl;
Tfc, p-chlorotetrafluorophenyl; and Tfp, tetraflorophenyl.
reaction of proline derivatives used as nucleophiles,
2
were approximated by the product of two partial rate
constants. The aminolysis in DMF was shown to follow
1
068-1620/02/2801-0009$27.00 © 2002 MAIK “Nauka /Interperiodica”

1
0
PERMYAKOV et al.
Fluorescence intensity, relative units
presence of traces of dimethylamine in the reaction
mixture.
Horiki and Murakami [13] studied the catalytic
4
3
00
action of potassium salt of 1-hydroxybenzotriazole on
the formation of peptide bond in THF when dimethy-
lamine was evidently absent. They found a substantial
deviation from the mechanism of the second order reac-
tion for the coupling of 2,3,5-Tfp ester of Z-phenylala-
nine and alanine p-nitroanilide (without any catalyst)
and concluded that this reaction follows a more compli-
cated mechanism. Earlier, Menger and Smith [14] stud-
ied the reactivity of various aromatic esters and
obtained similar results. The reaction orders of each of
the components were not determined in this study. The
complexity of the mechanism of amide bond formation
and closely related reactions is well documented in
organic chemistry (see review [15]).
00
2
1
00
00
0
3
00
350
400
450
500 nm
When studying relative reactivities of fluoroaro-
matic esters with various levels of fluorine substitution,
we failed to measure the rates of all reactions with the
same reagent concentrations [6, 16], because we had to
change the initial concentrations of both nucleophile
and active ester to increase the reaction rate or to gen-
erate an appropriate level of fluorescent signal. A sub-
stantial dependence of the rate constant of the second
order on the initial concentrations of reagents was
observed. This circumstance required the measurement
of the reaction orders for both components of the reac-
tion [16]. We found that the reaction rate of the standard
coupling of fluoroaromatic esters of Boc-Ala with
leucinamide or valine methyl ester corresponds to the
following equation:
Fig. 1. Fluorescence spectrum of Boc-Ala-OPfp in DMF
after excitation at 280.4 nm.
0
.5
0
–
0.5
1
00
8
6
4
2
0
0
0
0
0
1
N
/2 3/2
v = kC C ,
(1)
AE
where k is the reaction rate constant, C is the nucleo-
N
phile concentration, and CAE is the concentration of
active ester. This equation suggests the complicated
chain-reaction mechanism.
In this work, we present the results of our further
studies in this area. These demonstrate that not only the
rates but also the reaction orders depend on definite
pairs of amino acid derivatives. Probably, several ver-
sions of peptide bond formation exist, and their choice
depends on the nature of interacting amino acids.
0
20
40
60
Time, s
Fig. 2. Time dependence of fluorescence intensity at 375 nm
in the course of the reaction between Boc-Ala-OTfp
(
1.5 mM) and LeuNH (10 mM) in DMF in the presence of
2
triethylamine (40 mM) at 20°ë. Experimental data are
marked by points. The theoretical curve was calculated
using equations (1). The distribution of the differences
between the experimental and theoretical values is given in
the upper part of the figure.
RESULTS AND DISCUSSION
Active fluorophenyl esters have similar fluorescence
emission spectra. The example of the emission spec-
trum of Boc-Ala-OPfp is given in Fig. 1. The reaction
the kinetics of the first order relative to amine. A linear of an active fluorophenyl ester with amino acid deriva-
dependence of the rate constant of the pseudofirst order tives causes a decrease in the fluorescence intensity at
on the amine concentration was observed in all cases, 375 nm. This fact was used for the kinetic study of the
which suggests that the rates of these reactions depend reaction [16]. The change in the fluorescence during the
on concentrations of both amine and active ester. A reaction between Boc-Ala-OTfp and LeuNH
0% increase in the rate constants of the second order trated in Fig. 2. Pfp esters demonstrate the same
is illus-
2
1
was found when low concentrations of a nucleophile changes, but their fluorescent signal is weaker. The the-
were used. This effect was explained by the possible oretical curve calculated using equation (1) was fitted
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 28 No. 1 2002

KINETICS AND MECHANISM OF THE PEPTIDE SYNTHESIS IN SOLUTION
11
logv, M s–1
to experimental points by modifying the k rate constant.
The reaction orders in equation (1) (1/2 and 3/2) were
obtained from logarithmic dependencies of the initial
rate (v) on the reagent concentrations (Fig. 3) as
described in our previous paper [16]. The following are
the criteria for equation (1) correctness for the descrip-
tion of experimental data: a close correlation between
the experimental data and the calculated curve (Fig. 2,
upper panel) and the independence of the k rate con-
stant and the reagent concentrations (Fig. 4).
1
–
–
–
4.0
4.5
5.0
2
Similar measurements were performed for the reac-
tion of leucinamide with another type of active esters,
Boc-Xaa-OPfp, i.e., Pfp esters of the following Boc-
protected amino acids: alanine, glycine, methionine,
phenylalanine, isoleucine, proline, serine, and leucine.
PfpOAc was also used. The reaction orders and rate
constants for these reactions (measured using the
method described above) are given in the table. One can
see that the reaction orders are 1/2 and 3/2 in most cases
–
3.0
–2.5
–2.0
–1.5
logC, M
Fig. 3. The logarithmic dependence of the initial rate of the
peptide bond formation in the reaction of Boc-Ala-OTfc
with LeuNH on the concentration of (1) LeuNH (C
2
2
ÄÖ
0
.5 mM) and (2) Boc-Ala-OTfc (C 20 mM). The reaction
N
(
Xaa = Ala, Val, Phe, Ser, Leu, or Ac). In some cases,
was carried out in DMF in the presence of triethylamine
the reaction orders can be 1/2 and 1 (Xaa = Gly, Met,
and Pro) and even 1 and 2 (Ile). Usual reaction orders
(40 mM) at 20°ë.
(1 and 1) were found when leucinamide was replaced
by stronger nucleophile such as CHA, which can be
regarded, to a first approximation, as a topological ana-
logue of most amino acids (see the two examples in the
lower part of the table).
k, M–1 s–1
50
4
3
0
0
Note that the regularities observed in our experi-
ments did not depend on the nature of fluoroaromatic
ester. For example, the Pfp, Tfp, and Tfc esters of Boc-
Ala display similar reaction orders in their reaction
0
.5
1.0
1.5
2.0
2.5
Boc-Ala-OTfc, mM
with LeuNH , although they had different reaction rates
2
due to the different electrophilicity of the leaving
Fig. 4. The dependence of the rate of the reaction of
group.
Boc-Ala-OTfc with LeuNH (20 mM) in DMF in the pres-
2
The half-integer orders of reaction are characteristic
of chain reaction processes with various mechanisms of
chain termination [17]. Such chain reactions usually
imply the participation of free radicals that cannot exist
in our systems. Previously, we proposed the following intermediate (AE × N)*, phenolate E , and free proton
mechanism for reactions with the 1/2 and 3/2 orders:
ence of triethylamine (20 mM) on the concentration of
Boc-Ala-OTfc.
–
+
H .
(d) The interaction of two triple activated complexes
N × AE × N)* results in their decomposition with the
formation of reaction product (AN), free amino acid
(a) N + AE
(AE × N)*,
(
(b) N + (AE × N)*
(N × AE × N)*,
(c) (N × AE × N)* + AE
(2)
–
+
(
N), E , and ç .
–
+
AN + (AE × N)* + E + H ,
This hypothetical reaction scheme leads to equation
1) for the reaction rate. The activated intermediate
states indicated by asterisks can be the charged dipole
activated complexes.
–
+
(
(d) 2(N × AE × N)*
2AN + 2N + 2(E + H ).
Here: (a) The formation of the activated tetrahedral
complex (AE × N)* between the active fluorophenyl
ester AE and amino acid N.
Various reaction orders can be derived when chang-
ing the last reaction in scheme 2 (the deactivation of the
intermediate state). For example, if the reaction (d)
were replaced by the reaction
(b) The interaction of N with the complex (AE × N)*
that follows the chain initiation and leads to the forma-
tion of the triple activated complex (N × AE × N)*; the
possibility of the formation of such a complex was
repeatedly discussed by Johnson [15].
–
+
(
e) 2(N × AE × N)* + AE
3AN + N + 3(E + H ),
the equation for the reaction rate would be:
(c) The interaction of the (N × AE × N)* triple acti-
vated complex with the active fluorophenyl ester (AE)
results in the formation of the reaction product (AN),
1
N
/2
v = kC C ,
(3)
AE
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 28 No. 1 2002

1
2
PERMYAKOV et al.
Orders and rate constants of the reactions of peptide synthesis in DMF in the presence of 40 mM triethylamine*
–
1 –1
AE
BocAlaOPfp
N
a
b
k, M s
LeuNH2
0.70 (1/2)
0.55 (1/2)
0.52 (1/2)
0.51 (1/2)
0.67 (1/2)
0.79 (1)
1.53 (3/2)
0.92 (1)
33.6 ± 8.3
0.86 ± 0.12**
0.36 ± 0.06**
0.32 ± 0.04
16.9 ± 3.0
28.2 ± 4.8
0.18 ± 0.01**
8.2 ± 1.0
BocGlyOPfp
BocMetOPfp
BocValOPfp
BocPheOPfp
BocIleOPfp
BocProOPfp
BocSerOPfp
BocLeuOPfp
AcOPfp
''
''
1.04 (1)
''
1.60 (3/2)
1.31 (3/2)
1.83 (2)
''
''
''
0.46 (1/2)
0.56 (1/2)
0.57 (1/2)
0.58 (1/2)
0.72 (1)
1.11 (1)
''
1.31 (3/2)
1.36 (3/2)
1.49 (3/2)
1.13 (1)
''
10.6 ± 1.6
7.7 ± 1.5
''
BocIleOPfp
AcOPfp
CHA
2.1 ± 2.0
''
0.90 (1)
0.89 (1)
19.3 ± 3.5
a
b
*
The experimental kinetic data were approximated using the following equation: v= kC C , where k is the reaction rate constant, CN
N AE
is the concentration of a nucleophilic reagent, and CAE is the concentration of active ester. The values of k, a, and b were determined by
the method described in [16].
–
1/2 –1
*
* M
s .
whereas the replacement of reaction (d) by the reaction
Fluorescence spectra were recorded at 20°ë on a
laboratory spectrofluorimeter whose construction was
described in [20]. The fluorescence of active esters was
excited at 280.4 or 296.7 nm and measured from the
front surface of a quartz cell. The fluorescence intensity
was measured at 375 nm as a time function after mixing
the solutions in order to study the kinetics of the reac-
tions. Virtual rate constants of the reactions of active
esters were determined according to the reaction mech-
anisms described above with the use of scheme of non-
linear regression [21].
–
+
(f) (N × AE × N)*
AN + N + (E + H ),
gave the equation of the reaction rate
2
AE
v = kC C .
(4)
N
Thus, the experimental results can be described by one
and the same kinetic scheme (2), to which at least two
additional reactions [(e) and (f)] of the deactivation of
activated intermediate states should be added. One can
expect that the reactions of the active esters Boc-Xaa-
OPfp with LeuNH mainly proceed via the deactivation
2
pathway (d) (for Ala, Val, Phe, Ser, Leu, and PfpOAc);
the pathway (e) is valid for Xaa = Gly, Met, and Pro;
and the (f) pathway for Xaa = Ile.
The important conclusion from this study is that the
direct comparison of various reactions of peptide syn-
thesis with the use of virtual rate constants is often
inadequate, because the reaction mechanism may be
unique in each special case. One possible solution to
this problem may be a comparison of the initial rates of
these reactions.
REFERENCES
. Nisen, P., Hansen, J., Ban, N., Moore, P.B., and
Steitz, T.A., Science, 2000, vol. 289, pp. 920–930.
. Huber, C. and Wachtershauser, G., Science, 1998,
vol. 281, pp. 670–672.
1
2
3
. Boutin, J.A., Gesson, I., Henlin, J.M., Bertin, S., Lam-
bert, P.H., Volland, J.P., and Fauchere, J.L., Mol. Divers.,
1
997, vol. 3, pp. 43–60.
4
5
. Fischer, M. and Tran, C.D., Anal. Chem., 1999, vol. 71,
pp. 2255–2261.
. Medvedkin, V.N., Zabolotskikh, V.F., Permyakov, E.A.,
Mitin, Yu.V., Sorokina, M.N., and Klimenko, L.V.,
Bioorg. Khim., 1995, vol. 21, pp. 686–690.
EXPERIMENTAL
Fluorophenols of 99% purity (Perm division of the
Institute of Applied Chemistry), Boc-protected amino
acids, and leucinamide hydrochloride (Reanal, Hun-
gary) were used in this study. Fluorophenyl esters of the
Boc-protected amino acids were synthesized according
to procedures described previously [6, 10, 18, 19].
DMF was distilled in a vacuum with the addition of nin-
hydrin (0.5 g per liter) and stored at 4°ë. Triethylamine
was distilled over potassium hydroxide.
6
. Medvedkin, V.N., Klimenko, L.N., Mitin, Y.V., Podgor-
nova, N.N., Bystrichenko, A.V., Zabolotskikh, V.F.,
Korobeinikova, L.I., and Pozdeeva, V.V., Int. J. Peptide
Protein Res., 1994, vol. 44, pp. 477–484.
. Gross, E. and Meienhofer, J., The Peptides: Analysis,
Synthesis, Biology, New York: Academic, 1979, vol. 1.
. Atherton, E. and Sheppard, R.C., Solid Phase Peptide
Synthesis: A Practical Approach, Oxford: IRL Press at
Oxford Univ., 1989.
7
8
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 28 No. 1 2002

KINETICS AND MECHANISM OF THE PEPTIDE SYNTHESIS IN SOLUTION
13
9
. Kovacs, J., Cover, R.E., Johnson, R.H., Kalas, T.J., May- 16. Permyakov, E.A., Medvedkin, V.N., Klimenko, L.V.,
ers, G.L., and Roberts, J.E., J. Org. Chem., 1973, vol. 38,
Mitin, Y.V., and Permyakov, S.E., Int. J. Peptide Protein
Res., 1994, vol. 44, pp. 472–476.
pp. 2518–2521.
1
7. Emanuel’, N.M. and Knorre, D.G., Khimicheskaya kine-
tika (Chemical Kinetics), Moscow: Vysshaya Shkola,
1
1
1
1
1
1
0. Kovacs, J., Holleran, E.M., and Hui, K.Y., J. Org. Chem.,
1
979, vol. 45, pp. 1060–1065.
1
969.
1. Kovacs, J., Kim, S., Holleran, E., and Gorycki, P., J. Org. 18. Kisfaludy, L., Cerpini, M.Q., Racoczy, B., and
Chem., 1985, vol. 50, pp. 1397–1502.
Kovacs, J., Peptides: Proceedings of the Eighth Peptide
Symposium, Amsterdam: North Holland Publishing Co.,
2. Kemp, D.S., Choong, S.L.H., and Pekaar, J., J. Org.
Chem., 1974, vol. 39, pp. 3841–3847.
1
967, pp. 25–27.
1
2
9. Kisfaludy, L., Roberts, J.E., Johnson, R.H., May-
uers, G.L., and Kovacs, J., J. Org. Chem., 1970, vol. 35,
pp. 3563–3565.
0. Permyakov, E.A., Burstein, E.A., Sawada, Y., and
Yamazaki, Y., Biochim. Biophys. Acta, 1977, vol. 491,
pp. 149–154.
3. Horiki, K. and Murakami, A., Heterocycles, 1989,
vol. 28, pp. 615–622.
4. Menger, F.M. and Smith, J.H., J. Am. Chem. Soc., 1972,
vol. 94, pp. 3824–3829.
5. Johnson, S.L., Adv. Phys. Org. Chem., 1967, vol. 5, 21. Reich, J.A., Wangerman, G., Falk, M., and Rohde, K.,
pp. 237–330. Eur. J. Biochem., 1972, vol. 26, pp. 368–379.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 28 No. 1 2002